AU2016323088A1 - IL-8-binding antibodies and uses thereof - Google Patents

Abstract

One nonexclusive aspect provides novel IL-8 antibodies that are superior as pharmaceuticals.

Description

Description

Title of Invention: IL-8-BINDING ANTIBODIES AND USES THEREOF

Technical Field [0001] Cross-Reference to Related Applications

This application is related to and claims priority to Japanese Priority Patent Application No. 2015-185254, filed in Japan on September 18, 2015. The content thereof is incorporated by reference in their entirety.

[0002] TECHNICAL FIELD

In one nonexclusive aspect, the disclosure provides anti-IL-8 antibodies, pharmaceutical compositions containing the antibodies, nucleic acids encoding the antibodies, and host cells containing the nucleic acids. Production methods and uses of the IL-8 antibodies and pharmaceutical composition in the treatment of for example, IL8-associated disorders, are also provided.

Background Art [0003] Antibodies attract attention as pharmaceuticals because they are highly stable in plasma and have few side effects. A number of IgG-type therapeutic antibodies are on the market, and even now many therapeutic antibodies are under development (Reichert et al., Nat. Biotechnol. 23:1073-1078 (2005)(NPLl); Pavlou et al., Eur. J. Pharm. Biopharm. 59(3):389-396 (2005)(NPL2)). Meanwhile, various techniques are being developed for second-generation therapeutic antibodies; including technologies for improving effector function, antigen-binding ability, pharmacokinetics or stability, and reducing the risk of immunogenicity (Kim et ah, Mol. Cells. 20 (1):17-29 (2005)(NPL3)). The dosage for therapeutic antibodies is generally very high, and consequently the development of therapeutic antibodies confronts issues such as difficulty in producing subcutaneous formulations and high production costs. Methods for improving therapeutic antibody pharmacokinetics, pharmacodynamics, and antigen binding properties provide ways to reduce the dosage and production costs associated with therapeutic antibodies.

[0004] The substitution of amino acid residues in the constant region provides one method for improving antibody pharmacokinetics (Hinton et ah, J. Immunol. 176 (1):346-356 (2006)(NPL4); Ghetie et ah, Nat. Biotechnol. 15(7):637-640 (1997))(NPL5). The technique of affinity maturation provides a method for enhancing antigen-neutralizing ability of an antibody (Rajpal et ah, Proc. Nath Acad. Sci. USA 102(24):8466-8471 (2005)(NPL6); Wu et ah, J. Mol. Biol. 368:652 (2007)(NPL7)), and may increase the antigen-binding activity by introducing mutation(s) into amino acid residue(s) in the

WO 2017/046994

PCT/JP2016/003616

CDRs and/or framework regions of an antibody variable domain. Improving the antigen-binding properties of an antibody may improve the biological activity of the antibody in vitro or reduce the dosage, and may further improve the efficacy in vivo (in the body) (Wu et al„ J. Mol. Biol. 368:652-665 (2007)(NPL8)).

[0005] The amount of antigen that can be neutralized by one antibody molecule depends on the affinity of the antibody for the antigen; and thus, it is possible to neutralize an antigen with a small amount of antibody by increasing affinity. Antibody affinity for an antigen may routinely be increased using various known methods (see, e.g., Rajpal et al., Proc. Nath Acad. Sci. USA 102(24):8466-8471 (2005)(NPL6)). Further, it is theoretically possible to neutralize one antigen molecule (2 antigens when an antibody is bivalent) with one antibody molecule, if it can bind covalently to the antigen to make the affinity infinite. Nevertheless, one limitation for therapeutic antibody development thus far is that one antibody molecule typically only binds to and neutralizes one antigen molecule (2 antigens when an antibody is bivalent). Recently it has been reported that the use of an antibody that binds to an antigen in a pH-dependent manner (herein below also referred to as pH-dependent antibody or pH-dependent-binding antibody) enables one antibody molecule to bind to and neutralize multiple antigen molecules (see, e.g., W02009/125825(PTL1); Igawa et ah, Nat. Biotechnol. 28:1203-1207 (2010)(NPL9)). A pH-dependent antibody binds to an antigen strongly under the neutral pH conditions in the plasma, and dissociates from the antigen under the acidic pH condition within the endosome of a cell. After dissociation from the antigen, the antibody is recycled to the plasma by FcRn and is then free to bind to and neutralize another antigen molecule; and thus one pH-dependent antibody may repeatedly bind to and neutralize multiple antigen molecules.

[0006] It has recently been reported that antibody recycling properties can be achieved by focusing on the difference of calcium (Ca) ion concentration between plasma and endosome, and using an antibody with an antigen-antibody interaction that demonstrates calcium dependency (herein below also referred to as calcium ion concentration-dependent antibody)(WO2012/073992(PTL2)). (Herein below, a pHdependent antibody and a calcium ion concentration-dependent antibody are collectively referred to as a pH/Ca concentration-dependent antibody.) [0007] By binding to FcRn, IgG antibodies have long retention in plasma. The binding between an IgG antibody and FcRn is strong under an acidic pH conditions (for example, pH 5.8), but there is almost no binding under a neutral pH condition (for example, pH 7.4). An IgG antibody is taken up into cells non-specifically, and returned to cell surface by binding to FcRn in the endosome under the acidic pH conditions in the endosome. The IgG then dissociates from the FcRn under the neutral pH conditions in the plasma.

WO 2017/046994 PCT/JP2016/003616 [0008] It is reported that a pH-dependent antibody that has been modified to increase its FcRn binding under neutral pH conditions has the ability to repeatedly bind to and eliminate antigen molecules from plasma; and thus administration of such an antibody allows antigen elimination from plasma (WO2011/122011(PTL3)). According to this report, a pH-dependent antibody that has been modified to increase its FcRn binding under neutral pH conditions (for example, pH 7.4) can further accelerate the elimination of the antigen compared to a pH-dependent antibody that comprises the Fc region of a native IgG antibody (WO2011/122011(PTL3)).

[0009] Meanwhile, when mutations are introduced into the Fc region of an IgG antibody to eliminate its binding to FcRn under acidic pH conditions, it can no longer be recycled from the endosome into the plasma, which significantly compromises the antibody's retention in the plasma. With that, a method of increasing FcRn binding under acidic pH conditions is reported as a method for improving the plasma retention of an IgG antibody. Introducing amino acid modifications into the Fc region of an IgG antibody to increase its FcRn binding under acidic pH conditions can enhance the efficacy of recycling from the endosome to plasma, which as a result leads to an improvement in plasma retention. For instance, the modifications M252Y/S254T/T256E (YTE; Dall'Acqua et al„ J. Biol. Chem. 281:23514-235249 (2006)(NPL10)), M428L/N434S (LS; Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010))(NPLll), and N434H (Zheng et ah, Clin. Pharm. & Ther. 89(2):283-290 (2011)(NPL12)), have been reported to result in increased antibody half-life relative to native IgGl.

[0010] However, in addition to the concern that the immunogenicity or occurrence rate of aggregates may worsen in an antibody that comprises such an Fc region variant whose FcRn binding is increased under a neutral pH condition or an acidic pH condition, an increase in the binding against an anti-drug antibody (herein below also referred to as Pre-existing ADA) (for example, rheumatoid factor) present in a patient before administration of a therapeutic antibody has been further reported (WO2013/046722(PTL4), W02013/046704(PTL5)). W02013/046704(PTL5) reports that an Fc region variant containing specific mutations (represented by two residue modifications of Q438R/S440E according to EU numbering) increase the binding to FcRn under acidic pH conditions and also showed a significant reduction in binding to rheumatoid factor compared to unmodified native Fc. However,

W02013/046704(PTL5) does not specifically demonstrate that this Fc region variant has superior plasma retention to an antibody with native Fc region.

[0011] Accordingly, safe and more favorable Fc region variants with further improved plasma retention that do not show binding to pre-existing ADA are desired.

[0012] Antibody-dependent cellular cytotoxicity (herein below noted as ADCC), complement-dependent cytotoxicity (herein below noted as CDC), antibody4

WO 2017/046994

PCT/JP2016/003616 dependent cellular phagocytosis (ADCP) which is phagocytosis of target cells mediated by an IgG antibody are reported as effector functions of an IgG antibody. In order for an IgG antibody to mediate ADCC activity or ADCP activity, the Fc region of the IgG antibody must bind to an antibody receptor present on the surface of an effector cell such as a killer cell, natural killer cell or activated macrophage (noted as Fey receptor, FcgR, Fc gamma receptor or FcyR within the scope of Disclosure A described herein). In human, FcyRIa, FcyRIIa, FcyRIIb, FcyRIIIa and FcyRIIIb isoforms are reported as FcyR family proteins, and their respective allotypes have also been reported (Jefferis et al., Immunol. Lett. 82:57-65 (2002)(NPL13)). The balance of the respective affinity of an antibody for an activating receptor comprising FcyRIa, FcyRIIa, FcyRIIIa or FcyRIIIb, and an inhibitory receptor comprising FcyRIIb is an important element in optimizing the antibody effector functions.

[0013] Various techniques that increase or improve the activity of a therapeutic antibody against an antigen have been reported so far. For instance, the activity of an antibody to bind to an activating FcyR(s) plays an important role in the cytotoxicity of the antibody, and consequently, antibodies that target a membrane-type antigen and that have increased cytotoxicity resulting from enhanced activating FcyR(s) binding have been developed. See, e.g., W02000/042072(PTL6); W02006/019447(PTL7); Lazar et al., Proc. Nat. Acad. Sci. USA. 103:4005-4010 (2006)(NPL14); Shinkawa et al., J.

Biol. Chem. 278, 3466-3473 (2003)(NPL15); Clynes et al., Proc. Natl. Acad. Sci. U SA 95:652-656 (1998)(NPL16); Clynes et al., Nat. Med. 6:443-446 (2000)(NPL17)). Similarly, the binding activity towards an inhibitory FcyR (FcyRIIb in human) plays an important role in the immunosuppressive activity, agonist activity, and thus there has been research on antibodies with increased inhibitory FcyR-binding activity that target a membrane-type antigen (Li et al., Proc. Nat. Acad. Sci. USA. 109 (27):10966-10971 (2012)(NPL18)). Further, the influence of FcyR binding of an antibody that binds to a soluble antigen has been examined mainly from the viewpoint of side effects (Scappaticci et al., J. Natl. Cancer Inst. 99 (16):1232-1239 (2007)(NPL19)). For instance, when an antibody with increased FcyRIIb binding is used as a drug, one can expect reduced risk from the generation of anti-drug antibodies (Desai et al., J. Immunol. 178(10):6217-6226 (2007)(NPL20)).

[0014] More recently, it has been reported that introducing amino acid modifications into the Fc region of an IgG antibody to increase the activity of an antibody that targets a soluble antigen to bind to an activating and/or inhibitory FcyR(s) can further accelerate elimination of the antigen from serum (WO2012/115241(PTL8),

WO2013/047752(PTL9), WO2013/125667(PTL10), W02014/030728(PTL11)). Also, an Fc region variant has been identified, which shows almost no change in its FcyRIIbbinding activity from a native IgG antibody Fc region, but has reduced activity to other

WO 2017/046994 PCT/JP2016/003616 activating FcyRs (W02014/163101(PTL12)).

[0015] The plasma retention of a soluble antigen is very short compared to an antibody that has an FcRn-mediated recycling mechanism, and thus a soluble antigen may display increased plasma retention and plasma concentration by binding to an antibody that has such a recycling mechanism (for example, an antibody that does not have the characteristics of a pH/Ca concentration-dependent antibody). Accordingly, for example, when a soluble antigen in plasma has multiple types of physiological functions, even if one type of physiological functions is blocked as a result of antibody binding, the plasma concentration of the antigen may worsen the pathogenic symptoms caused by the other physiological functions as a result of the increased plasma retention and/or plasma concentration of the antigen resulting from the antibody binding. In this case, in addition to a method of applying the above-mentioned exemplified modifications to an antibody to accelerate antigen elimination, for example, a method of utilizing the formation of a multivalent immune complex from multiple pH/Ca concentrationdependent antibodies and multiple antigens, and increasing the binding to FcRn, FcyR(s), a complement receptor, has been reported (W02013/081143(PTL13)).

[0016] Even when the Fc region is not modified, it is reported that by modifying amino acid residue(s) so as to change the charge of such amino acid residue(s) which may be exposed on the surface of an antibody variable region to increase or decrease the isoelectric point (pi) of the antibody, it is possible to control the half-life of the antibody in blood regardless of the type of target antigen or antibody, and without substantially reducing the antigen-binding activity of the antibody (WO2007/114319(PTL14): techniques of substituting amino acids mainly in the FR; W02009/041643(PTL15): techniques of substituting amino acids mainly in CDR). These documents show that it is possible to prolong the plasma half-life of an antibody by reducing the antibody's pi, and conversely shorten the plasma half-life of an antibody by increasing the antibody's pi.

[0017] With regard to modification of the charge of amino acid residues in the constant region of an antibody, it has been reported that the uptake of an antigen into cells can be promoted by modifying the charge of specific amino acid residue(s), particularly in its CH3 domain, to increase the antibody's pi, and it is also described that this modification preferably does not interfere with the binding to FcRn (WO2014/145159(PTL16)). It has also been reported that modifying the charge of amino acid residues in the constant region (mainly CHI domain) of an antibody to reduce pi can prolong the half-life of the antibody in plasma, and in combination with mutations of amino acid residues to increase FcRn binding, can enhance its binding to FcRn and prolong the plasma half-life of the antibody (WO2012/016227(PTL17)).

[0018] Meanwhile, when such modification techniques designed for increasing or reducing

WO 2017/046994

PCT/JP2016/003616 the pi of an antibody are combined with techniques other than the modification technique to increase or reduce the binding to FcRn or FcyR(s), it is unclear whether there is an effect in promoting the plasma retention of the antibody or elimination of the antigen from plasma.

[0019] The extracellular matrix (ECM) is a structure that covers cells in vivo, and is mainly constituted by glycoproteins such as collagen, proteoglycan, fibronectin, and laminin. The role of the ECM in vivo is to create a microenvironment for cells to survive, and the ECM is important in various functions carried out by cells such as, cell proliferation and cell adhesion.

[0020] The ECM has been reported to be involved in the in vivo kinetics of proteins administered to a living body. Blood concentration of the VEGF-Trap molecule, which is a fusion protein between the VEGF receptor and Fc, when subcutaneously administered was examined (Holash et al., Proc. Natl. Acad. Sci., 99(17):11393-11398 (2002)(NPL21)). Plasma concentration of the subcutaneously administered VEGFTrap molecule which has a high pi, was low, and therefore its bioavailability was low. A modified VEGF-Trap molecule whose pi was reduced by amino acid substitutions has a higher plasma concentration, and its bioavailability could be improved. Further, change in the bioavailability correlates with the strength of binding to the ECM, and thus it became evident that the bioavailability of the VEGF-Trap molecule when subcutaneously administered depends on the strength of its binding to the ECM at the subcutaneous site.

[0021] W02012/093704(PTL18) reports that there is an inverse correlation between antibody binding to the ECM and plasma retention, and consequently, antibody molecules that do not bind to the ECM have better plasma retention when compared to antibodies that bind to the ECM.

[0022] As such, techniques for reducing extracellular matrix binding with the objective of improving protein bioavailability in vivo and plasma retention have been reported. By contrast, the advantages of increasing antibody binding to the ECM have not been identified so far.

[0023] Human IL-8 (Interleukin 8) is a chemokine family member that is 72 or 77 amino acid residues in length. The term chemokine is a collective term for a family of proteins with a molecular weight of 8-12 kDa and contain 4 cysteine residues that form intermolecular disulfide bonds. Chemokines are categorized into CC chemokine, CXC chemokine, C chemokine, CA3C chemokine according to the characteristics of the cysteine arrangement. IL-8 is classified as a CXC chemokine, and is also referred to as CXCL8.

[0024] IL-8 exists in solution in monomeric and homodimeric form. The IL-8 monomer contains antiparallel β sheets, and has a structure in which a C-terminal a helix

WO 2017/046994

PCT/JP2016/003616 traverses and covers the β sheets. An IL-8 monomer, in the case of the 72 amino acid form of IL-8, comprises two disulfide crosslinks between cysteine 7 and cysteine 34, and between cysteine 9 and cysteine 50. IL-8 homodimers are stabilized by noncovalent interactions between the β sheets of the two monomers, as there is no covalent binding between molecules in homodimers.

[0025] IL-8 expression is induced in various cells such as peripheral blood monocytes, tissue macrophages, NK cells, fibroblasts, and vascular endothelial cells in response to stimulation by inflammatory cytokines (Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014)(NPL22)).

[0026] Chemokines are generally not detectable, or only weakly detectable, in normal tissue, but are strongly detected at inflamed sites, and are involved in eliciting inflammation by facilitating infiltration of leukocyte into inflamed tissue sites. IL-8 is a proinflammatory chemokine that is known to activate neutrophils, promote expression of cell adhesion molecules, and enhance neutrophil adhesion to vascular endothelial cells. IL-8 also has neutrophil chemotactic capacity and IL-8 produced at a damaged tissue facilitates chemotaxis of neutrophils adhered to vascular endothelial cells into the tissue, and induces inflammation along with neutrophil infiltration. IL-8 is also known to be a potent angiogenic factor for endothelial cells and is involved in promoting tumor angiogenesis.

[0027] Inflammatory diseases associated with elevated (e.g., excess) IL-8 levels include, inflammatory diseases of the skin such as inflammatory keratosis (e.g., psoriasis), atopic dermatitis, contact dermatitis; chronic inflammatory disorders which are autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and Behcet's disease; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, drug-induced allergic hepatitis; inflammatory renal diseases such as glomerulonephritis; inflammatory respiratory diseases such as bronchitis and asthma; inflammatory chronic vascular diseases such as atherosclerosis; multiple sclerosis, oral ulcer, chorditis, and inflammation associated with using artificial organs and/or artificial blood vessels. Elevated (e.g., excess) IL-8 levels are also associated with malignant tumors such as ovarian cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, melanoma, head and neck cancers, and kidney cancer; sepsis due to infection; cystic fibrosis; and pulmonary fibrosis. (See, e.g., Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014)(NPL22), which is herein incorporated by reference in its entirety).

[0028] For several of these diseases, human anti-IL-8 antibodies with high affinity have been developed as pharmaceutical compositions (Desai et al., J. Immunol. 178(10):6217-6226 (2007)(NPL23)), however, they have not been launched yet. So

WO 2017/046994

PCT/JP2016/003616 far, only one pharmaceutical composition comprising IL-8 antibody is available, which is a murine anti-IL-8 antibody for psoriasis as external medicine. New anti-IL-8 antibodies for treatment diseases are expected.

Citation List Patent Literature [0029] [PTL1] WO2009/125825 [PTL2] WO2012/073992 [PTL3] WO2011/122011 [PTL4] WO2013/046722 [PTL5] WO2013/046704 [PTL6] W02000/042072 [PTL7] W02006/019447 [PTL8] WO2012/115241 [PTL9] WO2013/047752 [PTL10] WO2013/125667 [PTL11] W02014/030728 [PTL12] W02014/163101 [PTL13] W02013/081143 [PTL14] W02007/114319 [PTL15] W02009/041643 [PTL16] WO2014/145159 [PTL17] WO2012/016227 [PTL18] W02012/093704

Non Patent Literature [0030] [NPL1] Reichert et al., Nat. Biotechnol. 23:1073-1078 (2005) [NPL2] Pavlou et al., Eur. J. Pharm. Biopharm. 59(3):389-396 (2005) [NPL3] Kim et al., Mol. Cells. 20 (1):17-29 (2005) [NPL4] Hinton et al., J. Immunol. 176 (1):346-356 (2006) [NPL5] Ghetie et al., Nat. Biotechnol. 15(7):637-640 (1997)) [NPL6] Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471 (2005) [NPL7] Wu et al., J. Mol. Biol. 368:652 (2007) [NPL8] Wu et al., J. Mol. Biol. 368:652-665 (2007) [NPL9] Igawa et al., Nat. Biotechnol. 28:1203-1207 (2010) [NPL10] Dall'Acqua et al., J. Biol. Chem. 281:23514-235249 (2006) [NPL11] Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)) [NPL12] Zheng et al., Clin. Pharm. & Ther. 89(2):283-290 (2011) [NPL13] Jefferis et al., Immunol. Lett. 82:57-65 (2002)

WO 2017/046994

PCT/JP2016/003616 [NPL14] Lazar et al., Proc. Nat. Acad. Sci. USA. 103:4005-4010 (2006) [NPL15] Shinkawa et al., J. Biol. Chem. 278, 3466-3473 (2003) [NPL16] Clynes et al., Proc. Natl. Acad. Sci. U SA 95:652-656 (1998) [NPL17] Clynes et al., Nat. Med. 6:443-446 (2000) [NPL18] Li et al., Proc. Nat. Acad. Sci. USA. 109 (27):10966-10971 (2012) [NPL19] Scappaticci et al., J. Natl. Cancer Inst. 99 (16):1232-1239 (2007) [NPL20] Desai et al., J. Immunol. 178(10):6217-6226 (2007) [NPL21] Holash et al., Proc. Natl. Acad. Sci., 99(17):11393-11398 (2002) [NPL22] Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619 (2014) [NPL23] Desai et al., J. Immunol. 178(10):6217-6226 (2007)

Summary of Invention [0031] In one nonexclusive aspect, a non-limited objective of embodiments of Disclosure A is to provide molecules with improved pharmacokinetic properties over antibodies, such as ion concentration-dependent antigen binding properties that improve antibody half-life and/or antigen clearance from the plasma.

[0032] In one nonexclusive aspect, a non-limited objective of embodiments of Disclosure B is to provide, safe and more favorable Fc region variants that have increased half-life and decreased binding to pre-existing anti-drug antibodies (ADAs).

[0033] In one nonexclusive aspect, a non-limited objective of embodiments of Disclosure C is to provide anti-IL-8 antibodies that have pH-dependent binding affinity towards IL8. An additional embodiment relates to anti-IL-8 antibodies that have an effect of rapidly eliminating IL-8 compared to a reference antibody when administered to an individual. In another embodiment, Disclosure C relates to anti-IL-8 antibodies that can stably maintain their IL-8-neutralizing activity when administered to an individual. In some embodiments, the anti-IL-8 antibodies display reduced immunogenicity. In additional embodiments, Disclosure C relates to a method of producing and using the above-mentioned anti-IL-8 antibodies. Another alternative non-limited objective of Disclosure C is, to provide novel anti-IL-8 antibodies that can be included in a pharmaceutical composition.

[0034] In one nonexclusive aspect, within the scope of Disclosure A as provided herein, the inventors have surprisingly discovered that the ability of an ion concentrationdependent antibody (which is an antibody comprising an ion concentration-dependent antigen-binding domain (an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions)) to eliminate antigen from plasma can be accelerated by modifying at least one of the amino acid residues exposed on the surface of the antibody to increase its isoelectric point (pi). In another nonexclusive aspect, the inventors discovered that an ion concentration-dependent antibody with

WO 2017/046994

PCT/JP2016/003616 increased pi can further increase the extracellular matrix-binding of the antibody.

Thus, without being confined to a particular theory, the inventors have discovered that antigen elimination from plasma can be increased, by increasing the binding of the antibody towards extracellular matrix.

[0035] In one nonexclusive aspect, within the scope of Disclosure B as provided herein, the inventors conducted dedicated research on safe and more favorable Fc region variants that do not show binding to anti-drug antibodies (pre-existing ADA) and that can further improve plasma retention. As a result, the inventors have surprisingly discovered that Fc region variants comprising a substitution of position 434 amino acid according to EU numbering with Ala (A) and two specific residue mutations (represented by Q438R/S440E according to EU numbering) as a combination of amino acid residue mutations, are preferred for maintaining significant reduction in the binding to rheumatoid factor, along with achieving a plasma retention of an antibody.

[0036] In one nonexclusive aspect, within the scope of Disclosure C as provided herein, the inventors generated a number of pH-dependent anti-IL-8 antibodies (anti-IL-8 antibodies that bind to IL-8 in a pH-dependent manner). From the results of various validations, the inventors identified pH-dependent anti-IL-8 antibodies that have an effect of rapidly eliminating IL-8 compared to a reference antibody when administered to an individual. In some embodiments the Disclosure C relates to pH-dependent anti-IL-8 antibodies that can stably maintain their IL-8-neutralizing activity. In additional nonlimiting embodiments, the pH-dependent anti-IL-8 antibodies have reduced immunogenicity and excellent expression levels.

[0037] Further, within the scope of Disclosure C, the inventors successfully obtained antiIL-8 antibodies that comprise an Fc region whose FcRn-binding affinity at acidic pH is increased relative to the FcRn-binding affinity of a native Fc region. In an alternative aspect, the inventors successfully obtained anti-IL-8 antibodies that comprise an Fc region whose binding affinity towards pre-existing ADA is reduced relative to the binding affinity of a native Fc region for the pre-existing ADA. In an alternative aspect, the inventors successfully obtained anti-IL-8 antibodies comprising an Fc region whose plasma half-life is increased relative to the plasma half-life of a native Fc region. In an alternative aspect, the inventors successfully obtained pH-dependent antiIL-8 antibodies that comprise an Fc region whose binding affinity towards effector receptors is reduced relative to the binding affinity of a native Fc region for the effector receptors. In a different aspect, the inventors identified nucleic acids encoding the above-mentioned anti-IL-8 antibodies. In another aspect, the inventors also obtained hosts comprising the above-mentioned nucleic acids. In another aspect, the inventors developed a method for producing the above-mentioned anti-IL-8 antibodies, which comprises culturing the above-mentioned host. In another aspect, the inventors

WO 2017/046994

PCT/JP2016/003616 developed a method for facilitating the elimination of IL-8 from an individual relative to a reference antibody, which comprises administering the above-mentioned anti-IL-8 antibodies to the individual.

[0038] In one embodiment, Disclosure A, relates without limitation to, [1] an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, wherein its isoelectric point (pi) is increased by the modification of at least one amino acid residue that may be exposed on the surface of the antibody;

[2] the antibody of [1], wherein the antigen is a soluble antigen;

[3] the antibody of [1] or [2], wherein the antigen-binding domain is a domain whose antigen-binding activity under a high ion concentration condition is higher than that under a low ion concentration condition;

[4] the antibody of any one of [1] to [3], wherein the ion concentration is a hydrogen ion concentration (pH) or a calcium ion concentration;

[5] the antibody of [4], wherein the ratio of its KD in an acidic pH range to that in a neutral pH range, KD (acidic pH range) / KD (neutral pH range), for the antigen, is 2 or higher;

[6] the antibody of any one of [1] to [5], wherein in the antigen-binding domain, at least one amino acid residue is substituted with histidine, or at least one histidine is inserted;

[7] the antibody of any one of [1] to [6], which can promote elimination of the antigen from plasma as compared to an antibody before the modification;

[8] the antibody of any one of [1] to [7], wherein its extracellular matrix-binding activity is enhanced as compared to an antibody before the modification;

[9] the antibody of any one of [1] to [8], wherein the amino acid residue modification is amino acid residue substitution;

[10] the antibody of any one of [1] to [9], wherein the amino acid residue modification is selected from the group consisting of:

(a) substitution of a negatively charged amino acid residue with an uncharged amino acid residue;

(b) substitution of a negatively charged amino acid residue with a positively charged amino acid residue; and (c) substitution of an uncharged amino acid residue with a positively charged amino acid residue;

[11] the antibody of any one of [1] to [10], wherein the antibody comprises a variable region and/or a constant region, and the amino acid residue modification is amino acid residue modification in the variable region and/or the constant region;

[12] the antibody of [11], wherein the variable region comprises complementarity-de

WO 2017/046994

PCT/JP2016/003616 termining region(s) (CDR(s)) and/or framework region(s) (FR(s));

[13] the antibody of [12], wherein the variable region comprises a heavy chain variable region and/or a light chain variable region, and at least one amino acid residue is modified in a position in a CDR or a FR selected from the group consisting of:

(a) position 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in a FR of the heavy chain variable region;

(b) position 31,61, 62, 63, 64, 65, and 97 in a CDR of the heavy chain variable region;

(c) position 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49,

57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in a FR of the light chain variable region; and (d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDR of the light chain variable region, according to Rabat numbering;

[14] the antibody of [13], wherein at least one amino acid residue is modified in a position in a CDR or a FR selected from the group consisting of:

(a) position 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105 in a FR of the heavy chain variable region;

(b) position 31,61, 62, 63, 64, 65, and 97 in a CDR of the heavy chain variable region;

(c) position 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 in a FR of the light chain variable region; and (d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDR of the light chain variable region;

[15] the antibody of any one of [11] to [14], wherein at least one amino acid residue is modified in a position in the constant region selected from the group consisting of position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU numbering;

[16] the antibody of [15], wherein at least one amino acid residue is modified in a position in the constant region selected from the group consisting of position 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434, and 443;

[17] the antibody of [16], wherein at least one amino acid residue is modified in a position in the constant region selected from the group consisting of position 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413, according to EU numbering;

[18] the antibody of any one of [1] to [17], wherein the constant region has Fc gamma receptor (FcyR)-binding activity, and wherein the FcyR-binding activity under a neutral pH condition is enhanced as compared to that of a reference antibody

WO 2017/046994

PCT/JP2016/003616 comprising a constant region of a native IgG;

[19] the antibody of [18], wherein the FcyR is FcyRIIb;

[20] the antibody of any one of [1] to [17], wherein the constant region has binding activity towards one or more activating FcyR selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb and FcyRIIa, and towards FcyRIIb, and the FcyRIIb-binding activity is maintained or enhanced and the binding activity to the activating FcyRs is decreased, as compared to those of a reference antibody which differs only in that its constant region is that of a native IgG;

[21] the antibody of any one of [1] to [20], wherein the constant region has FcRnbinding activity, and wherein the FcRn-binding activity under a neutral pH condition (e.g., pH 7.4) is enhanced as compared to that of a reference antibody which differs only in that its constant region is that of a native IgG;

[22] the antibody of any one of [1] to [21], which is a multispecific antibody that binds to at least two antigens;

[23] the antibody of any one of [1] to [22], wherein the antibody is an IgG antibody;

[24] a pharmaceutical composition comprising the antibody of any one of [1] to [23];

[25] the pharmaceutical composition of [24], which is for promoting the elimination of an antigen from plasma;

[26] the pharmaceutical composition of [24] or [25], which is for enhancing the antibody binding to an extracellular matrix;

[27] a nucleic acid encoding the antibody of any one of [1] to [23];

[28] a vector comprising the nucleic acid of [27];

[29] a host cell comprising the vector of [28];

[30] a method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, wherein the method comprises culturing the host cell of [29] and collecting the antibody from the cell culture;

[30A] a method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, wherein the method comprises modifying at least one amino acid residue that may be exposed on the surface of the antibody so as to increase the isoelectric point (pi);

[30B] the method of [30A], wherein at least one amino acid residue is modified (I) in a position in a CDR or FR selected from the group consisting of: (a) position 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in a FR of the heavy chain variable region; (b) position 31,61, 62, 63, 64, 65, and 97 in a CDR of the heavy chain variable region; (c) position 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105,

WO 2017/046994

PCT/JP2016/003616

106, 107, and 108 in a FR of the light chain variable region; and (d) position 24, 25,

26, 27, 52, 53, 54, 55, and 56 in a CDR of the light chain variable region, according to Rabat numbering; or (II) in a position in a constant region selected from the group consisting of position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330,

342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400,

401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU numbering;

[31] the method of [30A] or [30B], wherein the amino acid residue modification comprises a modification selected from the group consisting of:

(a) substitution of a negatively charged amino acid residue with an uncharged amino acid residue;

(b) substitution of a negatively charged amino acid residue with a positively charged amino acid residue;

(c) substitution of an uncharged amino acid residue with a positively charged amino acid residue; and (d) substitution or insertion with histidine in a CDR or FR.

[32] the method of any one of [30], or [30A] to [30C] which further optionally comprises any one or more of:

(a) selecting an antibody which can promote elimination of an antigen from plasma;

(b) selecting an antibody with enhanced binding activity to an extracellular matrix;

(c) selecting an antibody with enhanced FcyR-binding activity under a neutral pH condition (e.g., pH 7.4);

(d) selecting an antibody with enhanced FcyRIIb-binding activity under a neutral pH condition (e.g., pH 7.4);

(e) selecting an antibody with maintained or enhanced FcyRIIb-binding activity and decreased binding activity to one or more activating FcyR, preferably selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb and FcyRIIa;

(f) selecting an antibody with enhanced FcRn-binding activity under a neutral pH condition (e.g., pH 7.4);

(g) selecting an antibody with an increased isoelectric point (pi);

(h) confirming the isoelectric point (pi) of the collected antibody, and then selecting an antibody with an increased isoelectric point (pi); and (i) selecting an antibody whose antigen-binding activity is changed or increased according to ion concentration conditions;

as compared to a reference antibody;

[0039] In an alternative embodiment, Disclosure A relates without limitation to:

[Al] an antibody having a constant region, wherein at least one amino acid residue

WO 2017/046994

PCT/JP2016/003616 selected from the group of modification sites identical to the modification sites in the group defined in [15] or [16] is modified in the constant region;

[A2] the antibody of [Al], which further has a heavy-chain variable region and/or a light-chain variable region, wherein the variable region has CDR(s) and/or FR(s), and wherein at least one amino acid residue selected from the group of modification sites identical to the modification sites in the group defined in [13] or [14] is modified in a CDR and/or a FR;

[A3] an antibody having a constant region, wherein at least one amino acid residue selected from the group of modification sites identical to the modification sites in the group defined in [15] or [16] is modified in the constant region so as to increase its pi; [A4] the antibody of [A3], which further has a heavy-chain variable region and/or a light-chain variable region, wherein the variable region has CDR(s) and/or FR(s), and wherein at least one amino acid residue selected from the group of modification sites identical to the modification sites in the group defined in [13] or [14] is modified in a CDR and/or a FR;

[A5] an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, wherein the antibody has a constant region, and wherein at least one amino acid residue selected from the group of modification sites identical to the modification sites in the group defined in [15] or [16] is modified in the constant region;

[A6] the antibody of [A5], which further has a heavy-chain variable region and/or a light-chain variable region, wherein the variable region has CDR(s) and/or FR(s), and wherein at least one amino acid residue selected from the group of modification sites identical to the modification sites in the group defined in [13] or [14] is modified in a CDR and/or a FR;

[A7] use of the antibody of any one of [1] to [23] and [Al] to [A6] in the manufacture of a medicament for promoting antigen elimination from plasma;

[A8] use of the antibody of any one of [1] to [23] and [Al] to [A6] in the manufacture of a medicament for increasing extracellular matrix binding;

[A9] use of the antibody of any one of [1] to [23] and [Al] to [A6] for eliminating an antigen from plasma; and [A10] use of the antibody of any one of [1] to [23] and [Al] to [A6] for increasing extracellular matrix binding.

[All] an antibody obtained by the method of any one of [30], [30A], [30B], [31], [32]. [0040] According to various embodiments, Disclosure A encompasses combinations of one or multiple elements described in any of [1] to [30], [30A], [30B], [31], [32] and [Al] to [All] mentioned above, in part or as a whole, as long as such a combination is not technically inconsistent with the common technical knowledge in the art. For example,

WO 2017/046994

PCT/JP2016/003616 in some embodiments, Disclosure A encompasses a method for producing a modified antibody comprising an antigen-binding domain which promotes elimination of an antigen from plasma as compared to that before the antibody modification, wherein the method comprises:

(a) modifying at least one amino acid residue that may be exposed on the surface of an antibody, which is:

(I) in a position in a CDR or FR selected from the group consisting of: (a) position 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in a FR of the heavy chain variable region; (b) position 31,61, 62, 63, 64, 65, and 97 in a CDR of the heavy chain variable region; (c) position 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in a FR of the light chain variable region; and (d) position 24, 25,

26, 27, 52, 53, 54, 55, and 56 in a CDR of the light chain variable region, according to Rabat numbering; or (II) in a position in a constant region selected from the group consisting of position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU numbering;

(b) modifying the antigen-binding domain in a way such that the resulting antigenbinding activity changes according to ion concentration conditions, wherein said (a) and (b) can be carried out simultaneously or sequentially;

(c) culturing a host cell to express the nucleic acid encoding the modified antibody; and (d) collecting the modified antibody from the host cell culture.

In further embodiments, the method optionally further comprises any one or more of:

(e) selecting an antibody which can promote elimination of an antigen from plasma;

(f) selecting an antibody with enhanced binding activity to an extracellular matrix;

(g) selecting an antibody with enhanced FcyR-binding activity under a neutral pH condition (e.g., pH 7.4);

(h) selecting an antibody with enhanced FcyRIIb-binding activity under a neutral pH condition (e.g., pH 7.4);

(i) selecting an antibody with maintained or enhanced FcyRIIb-binding activity and decreased binding activity to one or more activating FcyR, preferably selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb and FcyRIIa;

(j) selecting an antibody with enhanced FcRn-binding activity under a neutral pH condition (e.g., pH 7.4);

WO 2017/046994

PCT/JP2016/003616 (k) selecting an antibody with an increased isoelectric point (pi);

(l) confirming the isoelectric point (pi) of the collected antibody, and then selecting an antibody with an increased isoelectric point (pi); and (m) selecting an antibody whose antigen-binding activity is changed or increased according to ion concentration conditions;

as compared to the antibody before the modification.

[0041] Another embodiment of Disclosure A relates to, for example, without limitation:

[DI] a method for producing a modified antibody, whose half-life in plasma is prolonged or reduced, as compared to that before the modification of the antibody, wherein the method comprises:

(a) modifying a nucleic acid encoding the antibody before the modification to change the charge of at least one amino acid residue at a position selected from the group consisting of position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU numbering;

(b) culturing a host cell to express the nucleic acid; and (c) collecting the antibody from the host cell culture; or [D2] a method for prolonging or reducing the half-life of an antibody in plasma wherein the method comprises modifying at least one amino acid residue at a position selected from the group consisting of position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU numbering.

[0042] In one embodiment, Disclosure B relates to, for example, without limitation:

[33] an Fc region variant comprising an FcRn-binding domain, wherein the FcRnbinding domain comprises Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gin at position 440, according to EU numbering;

[34] the Fc region variant of [33], wherein the FcRn-binding domain comprises Ala at position 434; Arg or Fys at position 438; and Glu or Asp at position 440, according to EU numbering;

[35] the Fc region variant of [33] or [34], wherein the FcRn-binding domain further comprises He or Feu at position 428; and/or He, Feu, Val, Thr, or Phe at position 436, according to EU numbering;

[36] the Fc region variant of [35], wherein the FcRn-binding domain comprises Feu at position 428; and/or Val or Thr at position 436, according to EU numbering;

[37] the Fc region variant of any one of [33] to [36], wherein the FcRn-binding domain comprises a combination of amino acid substitutions selected from the group

WO 2017/046994

PCT/JP2016/003616 consisting of: N434A/Q438R/S440E; N434A/Q438R/S440D; N434A/Q438K/S440E; N434A/Q438K/S440D; N434A/Y436T/Q438R/S440E;

N434A/Y436T/Q438R/S440D; N434A/Y436T/Q438K/S440E; N434A/Y436T/Q438K/S440D; N434A/Y436V/Q438R/ S440E; N434A/Y436V/Q438R/S440D; N434A/Y436V/Q438K/S440E; N434A/Y436V/Q438K/S440D; N434A/R435H/F436T/Q438R/S440E; N434A/R435H/F436T/Q438R/S440D; N434A/R435H/F436T/Q438K/S440E; N434A/R435H/F436T/Q438K/S440D; N434A/R435H/F436V/Q438R/S440E; N434A/R435H/F436V/Q438R/S440D; N434A/R435H/F436V/Q438K/S440E; N434A/R435H/F436V/Q438K/S440D; M428F/N434A/Q438R/S440E; M428F/N434A/Q438R/S440D; M428F/N434A/Q438K/S440E; M428F/N434A/Q438K/S440D; M428F/N434A/Y436T/Q438R/S440E; M428F/N434A/Y436T/Q438R/S440D; M428F/N434A/Y436T/Q438K/S440E; M428F/N434A/Y436T/Q438K/S440D; M428F/N434A/Y436V/Q438R/S440E; M428F/N434A/Y436V/Q438R/S440D; M428F/N434A/Y436V/Q438K/S440E; M428F/N434A/Y436V/Q438K/S440D;

F235R/G236R/S239K/M428F/N434A/Y436T/Q438R/S440E; and

F235R/G236R/A327G/A330S/P331S/M428F/N434A/Y436T/Q438R/S440E, according to EU numbering;

[38] the Fc region variant of [37], wherein the FcRn-binding domain comprises a com bination of amino acid substitutions selected from the group consisting of: N434A/Q438R/S440E; N434A/Y436T/Q438R/S440E; N434A/Y436V/Q438R/S440E; M428L/N434A/Q438R/S440E; M428L/N434A/Y436T/Q438R/S440E; M428L/N434A/Y436V/Q438R/S440E;

L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; and

L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E, according to EFT numbering;

[39] the Fc region variant of any one of [33] to [38], wherein its FcRn-binding activity under an acidic pH condition (e.g., pH 5.8) is enhanced as compared to that of an Fc region of a native IgG;

[40] the Fc region variant of any one of [33] to [39], wherein its binding activity to an anti-drug antibody (ADA) is not significantly enhanced under a neutral pH condition as compared to that of an Fc region of a native IgG;

[41] the Fc region variant of [40], wherein the anti-drug antibody (ADA) is a rheumatoid factor (RF);

[42] the Fc region variant of any one of [33] to [41], wherein its plasma clearance (CF) is decreased, plasma retention time is increased, or plasma half-life (t 1/2) is increased, as compared to that of an Fc region of a native IgG;

WO 2017/046994

PCT/JP2016/003616 [43] the Fc region variant of any one of [33] to [42], wherein its plasma retention is increased as compared to a reference Fc region variant comprising a combination of amino acid substitutions N434Y/Y436V/Q438R/S440E, according to EU numbering;

[44] an antibody comprising the Fc region variant of any one of [33] to [43];

[45] the antibody of [44], wherein the antibody is an IgG antibody;

[46] a pharmaceutical composition comprising the antibody of [44] or [45];

[47] the pharmaceutical composition of [46], which is for increasing retention of the antibody in plasma;

[48] a nucleic acid encoding the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45];

[49] a vector comprising the nucleic acid of [48];

[50] a host cell comprising the vector of [49];

[51] a method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the variant, which comprises culturing the host cell of [50], and then collecting the Fc region variant or the antibody comprising the variant from the cell culture;

[52] the method of [51], which further optionally comprises any one or more steps selected from the group consisting of:

(a) selecting an Fc region variant with enhanced FcRn-binding activity under an acidic pH condition as compared to that of an Fc region of a native IgG;

(b) selecting an Fc region variant whose binding activity to an anti-drug antibody (ADA) is not significantly enhanced under a neutral pH condition as compared to that of an Fc region of a native IgG;

(c) selecting an Fc region variant with increased plasma retention as compared to that of an Fc region of a native IgG; and (d) selecting an antibody comprising an Fc region variant that can promote elimination of an antigen from plasma as compared to a reference antibody comprising an Fc region of a native IgG; and [53] a method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the variant, wherein the method comprises substituting amino acids in a way such that the resulting Fc region variant or the antibody comprising the variant comprises Ala at position 434; Glu, Arg, Ser, or Fys at position 438; and Glu, Asp, or Gin at position 440, according to EU numbering.

[0043] In one embodiment, Disclosure B relates to, for example, without limitation:

[BI] use of the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45] in the manufacture of a medicament for increasing retention in plasma;

[B2] use of the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45] in the manufacture of a medicament for not significantly increasing the binding

WO 2017/046994

PCT/JP2016/003616 activity for an anti-drug antibody (ADA) under a neutral pH condition compared to the Fc region of a native IgG;

[B3] use of the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45] for increasing retention in plasma;

[B4] use of the Fc region variant of any one of [33] to [43] or the antibody of [44] or [45] for not significantly increasing the binding activity for an anti-drug antibody (ADA) under a neutral pH condition compared to the Fc region of a native IgG; and [B5] an Fc region variant or an antibody comprising the variant, which is obtained by the method of any one of [51], [52], and [53].

[0044] According to various embodiments, Disclosure B encompasses combinations of one or multiple elements described in any of [33] to [53] and [BI] to [B5] mentioned above, in part or as a whole, as long as such a combination is not technically inconsistent with the common technical knowledge in the art. For example, in some embodiments, Disclosure B encompasses an Fc region variant comprising an FcRnbinding domain, wherein the FcRn-binding domain can comprise:

(a) Ala at position 434; Glu, Arg, Ser, or Fys at position 438; and Glu, Asp, or Gin at position 440, according to EU numbering;

(b) Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering;

(c) lie or Leu at position 428; Ala at position 434; He, Leu, Val, Thr, or Phe at position 436; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gin at position 440, according to EU numbering;

(d) lie or Leu at position 428; Ala at position 434; He, Leu, Val, Thr, or Phe at position 436; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering;

(e) Leu at position 428; Ala at position 434; Val or Thr at position 436; Glu, Arg,

Ser, or Lys at position 438; and Glu, Asp, or Gin at position 440, according to EU numbering; or (f) Leu at position 428; Ala at position 434; Val or Thr at position 436; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering.

[0045] In one embodiment, Disclosure C relates to, for example, without limitation:

[54] an isolated anti-IL-8 antibody that binds to human IL-8, which comprises at least one amino acid substitution(s) in at least one of (a) to (f) below, and binds to IL-8 in a pH-dependent manner:

(a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:67;

(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:68;

(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:69;

(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:70;

WO 2017/046994

PCT/JP2016/003616 (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO :71; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:72;

[55] the anti-IL-8 antibody of [54], which comprises an amino acid substitutions of tyrosine at position 9 of the amino acid sequence of SEQ ID NO:68, arginine at position 11 of the amino acid sequence of SEQ ID NO:68, and tyrosine at position 3 of the amino acid sequence of SEQ ID NO:69;

[56] the anti-IL-8 antibody of [54] or [55], which further comprises an amino acid substitutions of alanine at position 6 of the amino acid sequence of SEQ ID NO:68 and glycine at position 8 of the amino acid sequence of SEQ ID NO:68;

[57] the anti-IL-8 antibody of any one of [54] to [56], which comprises an amino acid substitutions of asparagine at position 1 of the amino acid sequence of SEQ ID NO:71, leucine at position 5 of the amino acid sequence of SEQ ID NO:71, and glutamine at position 1 of the amino acid sequence of SEQ ID NO:72;

[58] the anti-IL-8 antibody of any one of [54] to [57], which comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:73, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:74;

[59] the anti-IL-8 antibody of any one of [54] to [58], which comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76;

[60] the anti-IL-8 antibody of any one of [54] to [59], which comprises the heavy chain variable region of SEQ ID NO:78 and the light chain variable region of SEQ ID NO:79;

[61] the anti-IL-8 antibody of any one of [54] to [60], which comprises an Fc region having at least one property selected from the properties of (a) to (f) below:

(a) increased binding affinity for FcRn of the Fc region relative to the binding affinity for FcRn of a native Fc region at acidic pH;

(b) reduced binding affinity of the Fc region for pre-existing ADA relative to the binding affinity of a native Fc region for the pre-existing ADA;

(c) increased plasma half-life of the Fc region relative to the plasma half-life of a native Fc region;

(d) reduced plasma clearance of the Fc region relative to the plasma clearance of a native Fc region; and (e) reduced binding affinity of the Fc region for an effector receptor relative to the binding affinity of a native Fc region for the effector receptor; and (f) increased binding to extracellular matrix.

[62] the anti-IL-8 antibody of [61], wherein the Fc region comprises amino acid sub22

WO 2017/046994

PCT/JP2016/003616 stitution(s) at one or more positions selected from the group consisting of position 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440, according to EU numbering;

[63] the anti-IL-8 antibody of [62], which comprises an Fc region comprising one or more amino acid substitutions selected from the group consisting of L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E;

[64] the anti-IL-8 antibody of [63], wherein the Fc region comprises the amino acid substitutions of L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E;

[65] the anti-IL-8 antibody of [63], wherein the Fc region comprises the amino acid substitution of L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E;

[66] an anti-IL-8 antibody that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:81 and a light chain comprising the amino acid sequence of SEQ ID NO:82;

[67] an anti-IL-8 antibody that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:80 and a light chain comprising the amino acid sequence of SEQ ID NO:82;

[68] an isolated nucleic acid encoding the anti-IL-8 antibody of any one of [54] to [67];

[69] a vector comprising the nucleic acid of [68];

[70] a host cell comprising the vector of [69];

[71] a method for producing an anti-IL-8 antibody, which comprises culturing the host of [70];

[72] the method for producing an anti-IL-8 antibody of [71], which comprises isolating the antibody from a culture supernatant;

[73] a pharmaceutical composition comprising the anti-IL-8 antibody of any one of [54] to [67], and a pharmaceutically acceptable carrier;

[74] the anti-IL-8 antibody of any one of [54] to [67] for use in a pharmaceutical composition;

[75] the anti-IL-8 antibody of any one of [54] to [67] for use in the treatment of a disorder with the presence of excess IL-8;

[76] use of the anti-IL-8 antibody of any one of [54] to [67] in the manufacture of a pharmaceutical composition for a disorder with the presence of excess IL-8;

[77] a method for treating a patient that has a disorder with the presence of excess IL8, which comprises administering the anti-IL-8 antibody of any one of [54] to [67] to the individual;

[78] a method for promoting elimination of IL-8 from an individual, which comprises administering the anti-IL-8 antibody of any one of [54] to [67] to the individual;

[79] a pharmaceutical composition comprising the anti-IL-8 antibody of any one of [54] to [67], wherein the antibody binds to IL-8 and binds to extracellular matrix; and

WO 2017/046994

PCT/JP2016/003616 [80] a method for producing an anti-IL-8 antibody comprising a variable region with a pH-dependent IL-8-binding activity, wherein the method comprises:

(a) evaluating binding of an anti-IL-8 antibody with extracellular matrix, (b) selecting an anti-IL-8 antibody with strong binding to the extracellular matrix, (c) culturing a host that comprises a vector comprising a nucleic acid encoding the antibody, and (d) isolating the antibody from the culture solution.

[0046] In an alternative embodiment, Disclosure C relates to:

[Cl] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for suppressing accumulation of IL-8 which has a biological activity;

[C2] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for suppressing accumulation of IL-8 which has a biological activity;

[C3] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for inhibiting angiogenesis;

[C4] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for inhibiting angiogenesis;

[C5] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for inhibiting facilitation of neutrophil migration;

[C6] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for inhibiting facilitation of neutrophil migration;

[C7] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for use in suppressing accumulation of IL-8 which has a biological activity;

[C8] a method for suppressing accumulation of IL-8 which has a biological activity, wherein the method comprises administering the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] to an individual;

[C9] a pharmaceutical composition for suppressing accumulation of IL-8 which has a biological activity, comprising the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31];

[CIO] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for use in inhibiting angiogenesis;

[Cl 1] a method for inhibiting angiogenesis in an individual, wherein the method comprises administering the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] to the individual;

[C12] a pharmaceutical composition for inhibiting angiogenesis, which comprises the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31];

[Cl3] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for use in

WO 2017/046994

PCT/JP2016/003616 inhibiting facilitation of neutrophil migration;

[Cl4] a method for inhibiting facilitation of neutrophil migration in an individual, wherein the method comprises administering the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] to the individual;

[Cl5] a pharmaceutical composition for inhibiting facilitation of neutrophil migration, which comprises the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31]; [C16] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for use in the treatment of a disorder with the presence of excess IL-8;

[C17] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for treating a disorder with the presence of excess IL-8;

[Cl8] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for treating a disorder with the presence of excess IL-8;

[C19] a method for treating a disorder with the presence of excess IL-8 in an individual, wherein the method comprises administering the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] to the individual;

[C20] a pharmaceutical composition for treating a disorder with the presence of excess IL-8, which comprises the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31];

[C21] the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for use in promoting elimination of IL-8;

[C22] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] in the manufacture of a pharmaceutical composition for promoting elimination of IL-8;

[C23] use of the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] for promoting elimination of IL-8;

[C24] a method for promoting elimination of IL-8 in an individual, wherein the method comprises administering the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] to the individual; and [C25] a pharmaceutical composition for promoting elimination of IL-8, which comprises the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31].

[C26] An anti-IL-8 antibody, which comprises an Lc region comprising amino acid substitution(s) at one or more positions selected from the group consisting of position 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440, according to EU numbering. [C27] The anti-IL-8 antibody of [C26], which comprises an Lc region having at least one property selected from the properties of (a) to (f) below:

(a) increased binding affinity for LcRn of the Lc region relative to the binding affinity for LcRn of a native Lc region at acidic pH;

(b) reduced binding affinity of the Lc region for pre-existing ADA relative to the

WO 2017/046994

PCT/JP2016/003616 binding affinity of a native Fc region for the pre-existing ADA;

(c) increased plasma half-life of the Fc region relative to the plasma half-life of a native Fc region;

(d) reduced plasma clearance of the Fc region relative to the plasma clearance of a native Fc region;

(e) reduced binding affinity of the Fc region for an effector receptor relative to the binding affinity of a native Fc region for the effector receptor; and (f) increased binding to extracellular matrix.

[C28] The anti-IL-8 antibody of [C26] or [C27], which comprises an Fc region comprising one or more amino acid substitution(s) selected from the group consisting of L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E, according to EU numbering.

[C29] The anti-IL-8 antibody of [C28], which comprises an Fc region comprising one or more amino acid substitutions selected from the group consisting of (a) L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E; or (b) L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E, according to EU numbering.

[C30] The anti-IL-8 antibody of [C26] that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:81 and a light chain comprising the amino acid sequence of SEQ ID NO:82.

[C31] The anti-IL-8 antibody of [C26] that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:80 and a light chain comprising the amino acid sequence of SEQ ID NO:82.

[C32] An isolated nucleic acid encoding the anti-IL-8 antibody of any one of [C26] to [C31], [C33] A vector comprising the nucleic acid of [C32].

[C34] A host cell comprising the vector of [C33].

[C35] A method for producing an anti-IL-8 antibody, which comprises culturing the host cell of [C34].

[C36] The method for producing an anti-IL-8 antibody of any one of [C26] to [C31], which further comprises isolating the antibody from the host cell culture.

[C37] A pharmaceutical composition comprising the anti-IL-8 antibody of any one of [C26] to [C31] and a pharmaceutically acceptable carrier.

[C38] A method for treating a patient that has a disorder with the presence of excess IL-8, which comprises administering the anti-IL-8 antibody of any one of [C26] to [C31] to the individual.

[C39] A method for promoting elimination of IL-8 from an individual, which comprises administering the anti-IL-8 antibody of any one of [C26] to [C31] to the in26

WO 2017/046994 PCT/JP2016/003616 dividual.

[C40] A method for inhibiting IL-8, wherein the method comprises contacting the antiIL 8 antibody of any one of [54] to [67] and [C26] to [C31] with IL-8.

[C41] The method of [C40], wherein the method inhibits a biological activity of IL-8.

[0047] According to various embodiments, Disclosure C encompasses combinations of one or multiple elements described in any of [54] to [80] and [Cl] to [C41] mentioned above, in part or as a whole, as long as such a combination is not technically inconsistent with the common technical knowledge in the art.

Brief Description of Drawings [0048] [fig. l]Fig. 1 shows changes in the plasma concentration of human IF-6 receptor in human FcRn transgenic mice administered with a human IF-6 receptor-binding antibody that binds to human IF-6 receptor in a pH-dependent manner and whose constant region is that of a native IgGl (Fow_pI-IgGl), or an antibody that has increased the pi of the variable region in the antibody (High_pI-IgGl).

[0049] [fig.2]Fig. 2 shows changes in the plasma concentration of human IF-6 receptor in human FcRn transgenic mice administered individually with a human IF-6 receptorbinding antibody that binds to human IF-6 receptor in a pH-dependent manner and has been conferred with binding to FcRn under a neutral pH condition (Fow_pl-F939), and antibodies that have increased the pi of the variable region in the antibody (Middle_pl-F939, High_pl-F939).

[0050] [fig.3]Fig. 3 shows changes in the plasma concentration of human IF-6 receptor in human FcRn transgenic mice administered individually with a human IF-6 receptorbinding antibody that binds to human IF-6 receptor in a pH-dependent manner and whose FcyR binding under a neutral pH condition is increased (Fow_pl-Fl 180), and antibodies that have increased the pi of the variable region in the antibody (Middle_pl-Fl 180, High_pl-Fl 180).

[0051] [fig.4]Fig. 4 shows changes in the plasma concentration of human IF-6 receptor in human FcRn transgenic mice whose soluble human IF-6 receptor concentration in plasma is maintained at a steady state, which have been administered individually with a human IF-6 receptor-binding antibody that binds to human IF-6 receptor in a pHdependent manner and whose constant region is that of a native IgGl (Fow_pI-IgGl), an antibody that comprises an Fc region variant in which the Fc region in the antibody has increased FcRn binding under a neutral pH condition (Fow_pl-Fl 1), and antibodies that have increased the pi of the variable region in these antibodies (High_pI-IgGl, High_pl-Fll).

[0052] [fig.5]Fig. 5 shows the extent of extracellular matrix binding of each of the three types of antibodies with different pis that bind to human IF-6 receptor in a pH-dependent

WO 2017/046994

PCT/JP2016/003616 manner (Low_pI-IgGl, Middle_pI-IgGl and High_pI-IgGl) and the two types of antibodies with different pis that do not bind to human IL-6 receptor in a pH-dependent manner (Low_pI(NPH)-IgGl and High_pI(NPH)-IgGl). NPH means pH independent within the scope of Disclosure A described herein.

[0053] [fig.6]Fig. 6 shows relative values of the extent of soluble human FcyRIIb binding (measured by BIACORE(registered trademark)) of antibodies that comprise an Fc region variant each of whose pi has been increased by modifying one amino acid residue in the constant region of the AMH-P600 antibody which binds to IgE in a pH dependent manner, by setting the value of AMH-P600 to 1.00.

[0054] [fig.7]Fig. 7 shows relative values of the rate at which antibodies that comprise an Fc region variant each of whose pi has been increased by modifying one amino acid residue in the constant region of AMH-P600 are taken up into cells of an hFcYRIIbexpressing cell line, respectively, evaluated with the value of AMH-P600 set to 1.00.

[0055] [fig.8]Fig. 8 shows the extent of binding of Fv4-IgGl, which has the Fc region of a native human IgGl, to rheumatoid factor in the serum of each RA patient.

[0056] [fig.9]Fig. 9 shows the extent of binding of Fv4-YTE, which has an Fc region variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0057] [fig. 10]Fig. 10 shows the extent of binding of Fv4-LS, which has an Fc region variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0058] [fig. 1 l]Fig. 11 shows the extent of binding of Fv4-N434H, which has an Fc region variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0059] [fig. 12]Fig. 12 shows the extent of binding of Fv4-F1847m, which has an Fc region variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0060] [fig. 13]Fig. 13 shows the extent of binding of Fv4-F1848m, which has an Fc region variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0061] [fig. 14]Fig. 14 shows the extent of binding of Fv4-F1886m, which has an Fc region variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0062] [fig.l5]Fig. 15 shows the extent of binding of Fv4-F1889m, which has an Fc region variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0063] [fig. 16]Fig. 16 shows the extent of binding of Fv4-F1927m, which has an Fc region variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0064] [fig. 17]Fig. 17 shows the extent of binding of Fv4-Fl 168m, which has an Fc region

WO 2017/046994

PCT/JP2016/003616 variant with increased FcRn binding, to rheumatoid factor in the serum of each RA patient.

[0065] [fig.l8]Fig. 18 shows average values of the binding of Fv4-IgGl, which has the Fc region of a native human IgGl, and each of the antibodies comprising a novel Fc region variant in which the Fc region has an Fc region variant with increased binding to each FcRn, to rheumatoid factor in the serum of RA patients.

[0066] [fig.l9]Fig. 19 shows changes in the plasma concentration of each anti-human IgE antibody in cynomolgus when administered with OHB-IgGl which is an anti-human IgE antibody and has the Fc region of a native human IgGl, and each of the antibodies comprising a novel Fc region variant in which each the Fc region has an Fc region variant with increased binding to FcRn (OHB-LS, OHB-N434A, OHB-F1847m, OHBF1848m, OHB-F1886m, OHB-F1889m and OHB-F1927m).

[0067] [fig.20]Fig. 20 shows changes in the plasma concentration of an anti-human IL-6 receptor antibody in human FcRn transgenic mouse when administered with Fv4-IgGl which is an anti-human IL-6 receptor antibody and has the Fc region of a native human IgGl, or Fv4-F1718 which has increased FcRn binding of the antibody at the acidic pH condition.

[0068] [fig.21]Fig. 21 shows sensorgrams obtained for IL-8 binding of H998/L63 and Hr9 at pH 7.4 and pH 5.8 measured with Biacore.

[0069] [fig.22]Fig. 22 shows changes of the human IL-8 concentration in mouse plasma when H998/L63 or H89/L118 was administered to mice at 2 mg/kg in a mixture with human IL-8.

[0070] [fig.23]Fig. 23 shows changes of the human IL-8 concentration in mouse plasma when H89/L118 was administered to mice at 2 mg/kg or 8 mg/kg in a mixture with human IL-8.

[0071] [fig.24]Fig. 24 shows changes of the human IL-8 concentration in mouse plasma when H89/L118 or H553/L118 was administered to mice at 2 mg/kg or 8 mg/kg in a mixture with human IL-8.

[0072] [fig.25A]Fig. 25A shows changes in the relative values of antibody concentrationdependent chemiluminescence with antibody Hr9, H89/L118 or H553/L118 before preservation in plasma.

[0073] [fig.25B]Fig. 25B shows changes in the relative values of antibody concentrationdependent chemiluminescence with antibody Hr9, H89/L118 or H553/L118 after one week of preservation in plasma.

[0074] [fig.25C]Fig. 25C shows changes in the relative values of antibody concentrationdependent chemiluminescence with antibody Hr9, H89/L118 or H553/L118 after two weeks of preservation in plasma.

[0075] [fig.26]Fig. 26 shows the predicted frequency of ADA occurrence for each anti-IL-8

WO 2017/046994 PCT/JP2016/003616 antibody (hWS4, Hr9, H89/L118, H496/L118 or H553/L118) and the predicted frequency of ADA occurrence for other pre-existing therapeutic antibodies predicted by the EpiMatrix.

[0076] [fig.27]Fig. 27 shows the predicted frequency of ADA occurrence for each anti-IL-8 antibody (H496/L118, H496vl/L118, H496v2/L118, H496v3/L118, H1004/L118 or H1004/L395) and the predicted frequency of ADA occurrence for other pre-existing therapeutic antibodies predicted by EpiMatrix.

[0077] [fig.28A]Fig. 28A shows changes in the relative values of antibody concentrationdependent chemiluminescence with antibody Hr9, H89/L118 or H1009/L395-F1886s before preservation in plasma.

[0078] [fig.28B]Fig. 28B shows changes in the relative values of antibody concentrationdependent chemiluminescence with antibody Hr9, H89/L118 or H1009/L395-F1886s after one week of preservation in plasma.

[0079] [fig.28C]Fig. 28C shows changes in the relative values of antibody concentrationdependent chemiluminescence with antibody Hr9, H89/L118 or H1009/L395-F1886s after two weeks of preservation in plasma.

[0080] [fig.29]Fig. 29 shows changes of the human IL-8 concentration in mouse plasma when mice were administered with each of H1009/L395, H553/L118 and H998/L63 in a mixture with human IL-8.

[0081] [fig.30]Fig. 30 shows the extent of extracellular matrix binding when Hr9, H89/L118 or H1009/L395 was added alone to extracellular matrix, and when they were added in a mixture with human IL-8.

[0082] [fig.31]Fig. 31 shows changes of antibody concentration in mouse plasma when an antibody that has the variable region of H1009/L395 and the Fc region that does not bind to FcRn (FI942m) was administered alone or in a mixture with human IL-8 to human FcRn transgenic mice.

[0083] [fig.32]Fig. 32 shows the predicted frequency of ADA occurrence for H1009/L395 and H1004/L395 and the predicted frequency of ADA occurrence for other pre-existing therapeutic antibodies predicted by EpiMatrix.

[0084] [fig.33]Fig. 33 shows changes in the concentration of the respective anti-human IL-8 antibody in the plasma of cynomolgus when administered with H89/L118-IgGl, which has the variable region of H89/L118 and the Fc region of a native human IgGl, and each antibody that has an Fc region variant with increased binding to FcRn (H89/L118-F1168m, H89/L118-F1847m, H89/L118-F 1848m, H89/L118-F1886m, H89/L118-F1889m and H89/L118-F1927m).

[0085] [fig.34]Fig. 34 shows the binding of antibodies that have the variable region of

H1009/L395 and whose Fc region is a variant (F1886m, F1886s, or F1974m) to each FcyR.

WO 2017/046994 PCT/JP2016/003616 [0086] [fig.35]Fig. 35 shows changes of the human IL-8 concentration in mouse plasma when an anti-IL-8 antibody was administered to human FcRn transgenic mice in a mixture with human IL-8. In this case, the anti-IL-8 antibody was H1009/L395-IgGl (2 mg/kg) which comprises the variable region of H1009/L395 and the Fc region of a native human IgGl, or H1009/L395-F1886s (2, 5 or 10 mg/kg) which comprises the variable region of H1009/L395 and the modified Fc region.

[0087] [fig.36]Fig. 36 shows changes in the antibody concentration in the plasma of cynomolgus when administered with Hr9-IgGl or H89/L118-IgGl, both of which comprise the Fc region of a native human IgGl, or H1009/L395-F1886s or H1009/L395-F 1974m, both of which comprise a modified Fc region.

[0088] [fig.37]Fig. 37 shows the IgE plasma concentration time profile of some anti-IgE antibodies in C57BL6J mice in terms of the antibody variable region modification.

[0089] [fig.38A]Fig. 38 (Figs. 38A-38D) shows Octet sensorgrams of selected 25 [twenty five] pH-dependent and/or calcium-dependent antigen binding clones.

[0090] [fig.38B]Fig. 38B is continuation of Fig. 38A.

[0091] [fig.38C]Fig. 38C is continuation of Fig. 38B.

[0092] [fig.38D]Fig. 38D is continuation of Fig. 38C.

[0093] [fig.39]Fig. 39 shows the C5 plasma concentration time profile of some anti-C5 bispecific antibodies in C57BL6J mice in terms of the antibody variable region modification.

[0094] [fig.40]Fig. 40 shows the IgE plasma concentration time profile of some anti-IgE antibodies in C57BL6J mice in terms of the antibody constant region modification.

Description of Embodiments [0095] DETAILED DESCRIPTION

Non-limiting embodiments of Disclosure A, B or C are described hereinbelow. All embodiments described in the Examples hereinbelow are described with the intention to be rightfully understood to be also described in the section on DETAILED DESCRIPTION, without constraints by any patent practices, ordinance, regulations, or others that may be attempted to narrowly interpret the contents described in the Examples in countries where acquisition of patent right from the present patent application is intended.

[0096] Disclosure A or Disclosure B

In some embodiments, Disclosure A relates to antibodies comprising an antigenbinding domain whose antigen-binding activity changes according to ion concentration conditions, in which the isoelectric point (pi) is increased by modification of at least one amino acid residue that may be exposed on the antibody surface (herein, also referred to as ion concentration-dependent antibodies with increased pi within the

WO 2017/046994

PCT/JP2016/003616 scope of Disclosure A; and the antigen-binding domains of the antibodies are also referred to as ion concentration-dependent antigen-binding domains with increased pi). The invention is partly based on the surprising discovery of the inventors that antigen elimination from plasma can be facilitated with an ion concentration-dependent antibody whose isoelectric point (pi) has been increased by the modification of at least one amino acid residue that can be exposed on the antibody surface (for example, when the antibody is administered in vivo); and that binding of an antibody to the extracellular matrix can be increased with an ion concentration-dependent antibody with increased (elevated) pi. The invention is also partly based on the surprising discovery of the inventors that this beneficial effect is brought about by combining two entirely different concepts of: an ion concentration-dependent antigen-binding domain or ion concentration-dependent antibody; and an antibody whose pi is increased by modification of at least one amino acid residue that can be exposed on the surface (herein, also referred to as an antibody with increased pi within the scope of Disclosure A; and an antibody whose pi is decreased (reduced) by modification of at least one amino acid residue that can be exposed on the surface is referred to as an antibody with decreased pi within the scope of Disclosure A). The invention is thus categorized as a type of pioneer invention which can lead to remarkable technological innovation in the field (e.g., medical field) to which Disclosure A belongs.

[0097] As a matter of course, for example, an antibody comprising an antigen-binding domain and whose pi is increased by modification of at least one amino acid residue that can be exposed on the antibody surface, which has been further modified so that the antigen-binding activity of the antigen-binding domain changes according to ion concentration conditions, are also included within the scope of Disclosure A described herein (herein, such antibody is also referred to as an ion concentration-dependent antibody with increased pi within the scope of the Disclosure A).

[0098] As a matter of course, for example, an antibody containing an ion concentrationdependent antigen-binding domain in which at least one amino acid residue that can be exposed on the antibody surface has a charge different from that of the at least one amino acid residue at the corresponding position(s) in an antibody before modification (native antibody (for example, native Ig antibody, preferably native IgG antibody), or reference or parent antibody (e.g., antibody before modification, or antibody prior to or during library construction, or the like)), and whose net antibody pi is increased is also included in Disclosure A described herein (such antibody is also referred to as an ion concentration-dependent antibody with increased pi within the scope of Disclosure A described herein).

[0099] As a matter of course, for example, an antibody containing an ion concentrationdependent antigen-binding domain, whose pi is increased by modification of at least

WO 2017/046994

PCT/JP2016/003616 one amino acid residue that can be exposed on the antibody surface in an antibody before the modification (native antibody (for example, native Ig antibody, preferably native IgG antibody, or reference or parent antibody (e.g., antibody before the modification, or antibody prior to or during library construction, or the like)) is also included in Disclosure A described herein (such antibody is also referred to as an ion concentration-dependent antibody with increased pi within the scope of Disclosure A described herein).

[0100] As a matter of course, for example, an antibody containing an ion concentrationdependent antigen-binding domain in which at least one amino acid residue that can be exposed on the antibody surface is modified for the purpose of increasing the pi of the antibody is also included in Disclosure A described herein (such antibody is also referred to as an ion concentration-dependent antibody with increased pi within the scope of Disclosure A described herein).

[0101] Within the scope of Disclosures A and B described herein, amino acids include not only natural amino acids but also unnatural amino acids. Within the scope of Disclosures A and B described herein, amino acids or amino acid residues may be represented by either one-letter (for example, A) or three-letter codes (for example, Ala), or both (for example, Ala(A)).

[0102] As used in the context of Disclosures A and B, modification of an amino acid, modification of an amino acid residue, or an equivalent phrase may be understood as, without being limited thereto, chemically modifying one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) specific amino acids (residues) in an antibody amino acid sequence with a molecule or adding, deleting, substituting or inserting one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) amino acids in an antibody amino acid sequence. Amino acid addition, deletion, substitution, or insertion can be carried out to a nucleic acid encoding an amino acid sequence, for example, by site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)) or overlap extension PCR; via affinity maturation of antibodies, or by using chain shuffling of antibody heavy chains or light chains; or by antigen panning-based selection using phage-display libraries (Smith et al., Methods Enzymol. 217:228-257 (1993)); and these can be performed alone or in appropriate combinations. Such amino acid modification is carried out preferably, without limitation, by substituting one or more amino acid residue in an antibody amino acid sequence with a different amino acid (individually). Amino acid addition, deletion, substitution, or insertion, and modification of an amino acid sequence by humanization or chimerization can be carried out by methods known in the art. Alteration or modification of an amino acid (residue), such as amino acid addition, deletion, substation, or insertion, may also be performed on an antibody variable region or an antibody constant region to be used in preparing

WO 2017/046994 PCT/JP2016/003616 recombinant antibodies for the antibodies of Disclosure A or B.

[0103] In one embodiment within the scope of Disclosures A and B described herein, substitution of amino acids (residues) refers to substitution with different amino acids (residues), and can be designed to modify, for example, matters such as in (a) to (c):

(a) the polypeptide backbone structure in a region of sheet or helical conformation; (b) charge or hydrophobicity at a target site; or (c) size of a side chain.

[0104] Amino acid residues are classified, based on properties of the side chains in the structure, for example, into the groups of: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, and lie; (2) neutral, hydrophilic: Cys, Ser, Thr, Asn, and Gin; (3) acidic: Asp and Glu; (4) basic: His, Lys, and Arg; (5) residues that affect the chain orientation: Gly and Pro; and (6) aromatic: Trp, Tyr, and Phe.

[0105] Substitution of amino acid residues within each group is referred to as conservative substitution, while substitution of amino acid residues between different groups is referred to as non-conservative substitution. Substitution of amino acid residues may be conservative substitution, non-conservative substitution, or a combination thereof. Several known appropriate methods may be used for substituting amino acids with those other than natural amino acids (Wang et al., Annu. Rev. Biophys. Biomol. Struct. 35:225-249 (2006); Forster et al., Proc. Natl. Acad. Sci. USA 100(11):6353-6357 (2003)). It is possible to use, for example, a cell-free translation system containing tRNA in which an unnatural amino acid is linked to amber suppressor tRNA complementary to UAG codon (amber codon) which is a stop codon (Clover Direct (Protein Express)).

[0106] Within the scope of Disclosures A and B described herein, it is understood that the structure of an antigen is not limited to a specific structure as long as the antigen includes an epitope that binds to an antibody. The antigen may be an inorganic or organic substance. Antigens may be any ligands, including various cytokines, for example, interleukins, chemokines, and cell growth factors. Alternatively, as a matter of course, for example, receptors that are present as in a soluble form or have been modified to be a soluble form in biological fluids such as plasma can also be used as antigens. Non-limiting examples of such soluble receptors include the soluble IL-6 receptor described in Mullberg et al., J. Immunol. 152(10):4958-4968 (1994). Furthermore, antigens may be monovalent (for example, soluble IL-6 receptor) or multivalent (for example, IgE).

[0107] In one embodiment, antigens that can be bound by an antibody of Disclosures A and B are preferably soluble antigens present in biological fluids (for example, biological fluids illustrated in WO2013/125667, preferably plasma, interstitial fluid, lymphatic fluid, ascitic fluid, or pleural fluid) of subjects (within the scope of Disclosures A and B described herein, subjects to be administered (applied) with the antibody, which can

WO 2017/046994

PCT/JP2016/003616 be virtually any animal, for example, a human, mouse, etc.,); however, the antigens may also be membrane antigens.

[0108] Within the scope of Disclosures A and B described herein, prolongation of the halflife in plasma or shortening of the half-life in plasma of a target molecule (which may be an antigen or antibody), or an equivalent phrase thereof can also be represented more specifically using in addition to the parameter of half-life in plasma (tl/2), any other parameter such as mean retention time in plasma, clearance (CL) in plasma, and area under the concentration curve (AUC) (Pharmacokinetics: Enshuniyoru Rikai (Understanding through practice) Nanzando). These parameters can be specifically assessed, for example, by carrying out noncompartmental analysis according to the protocol appended to the in vivo kinetics analysis software WinNonlin (Pharsight). It is known to those of ordinary skill in the art that these parameters normally correlate with one another.

[0109] Within the scope of Disclosures A and B described herein, an epitope refers to an antigenic determinant in an antigen and means a site on an antigen at which the antigen-binding domain of an antibody binds. Thus, an epitope can be defined, for example, based on its structure. Alternatively, the epitope may be defined by the antigen-binding activity of an antibody that recognizes the epitope. When an antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues that constitute the epitope. Alternatively, when an epitope is a sugar chain, the epitope can be specified based on its specific sugar chain structure. An antigen-binding domain of Disclosures A and B may bind to a single epitope or different epitopes on an antigen.

[0110] A linear epitope may be a primary amino acid sequence. Such a linear epitope typically contains at least three and commonly at least five, for example, 8 to 10 amino acids or 6 to 20 amino acids as a unique sequence.

[0111] In a conformational epitope, typically the amino acids that constitute the epitope are not present consecutively as a primary sequence. An antibody can recognize a conformational epitope in the three-dimensional structure of a peptide or protein. Methods for determining the conformation of an epitope include, but are not limited to, X ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance (Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.)).

[0112] Within the scope of Disclosures A and B described herein, an antibody is not particularly limited and used in the broadest sense, as long as it can bind to an antigen of target. Non-limiting examples of antibodies include widely known common antibodies (for example, native immunoglobulins (abbreviated as Ig)), and molecules and variants derived therefrom, for example, Fab, Fab', F(ab')₂, diabodies, ScFv (Holliger et al„ Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); EP404,097; WO93/11161; Peer

WO 2017/046994

PCT/JP2016/003616 et al., Nature Nanotechnology 2:751-760 (2007)), low molecular weight antibodies (minibodies) (Orita et al., Blood 105:562-566 (2005)), scaffold proteins, one-armed an tibodies (including all embodiments of one-armed antibodies described in

W02005/063816), multispecific antibodies (for example, bispecific antibodies: antibodies with specificity to two different epitopes, including antibodies that recognize different antigens and antibodies that recognize different epitopes on a same antigen). Within the scope of Disclosures A and B described herein, bispecific antibodies are not limited but may be prepared, for example, as antibody molecules having the common L chain described in W02005/035756, or by the method described in W02008/119353 where two general types of antibodies having an IgG4-like constant regions are mixed to cause an exchange reaction between the two types of such antibodies (known as the Fab-arm exchange method to those of ordinary skill in the art). In an alternative embodiment, they may be antibodies having a structure where the heavy-chain variable region and the light-chain variable region are linked together as a single chain (for example, sc(Fv)₂). Alternatively, they may be antibody-like molecules (for example, scFv-Fc) that result from linking the Fc region (a constant region that lacks the CHI domain) to scFv (or sc(Fv)₂) where the heavy-chain variable region (VH) is linked to the light-chain variable region (VF). Multispecific antibodies consisting of scFv-Fc have an (scFv)₂-Fc structure where the first and second polypeptides are VH1-linker-VFl-Fc and VH2-linker-VF2-Fc, respectively. Alternatively, they may be antibody-like molecules where a single-domain antibody is linked to an Fc region (Marvin et al., Curr. Opin. Drug Discov. Devel. 9(2):184-193 (2006)), Fc fusion proteins (for example, immunoadhesin) (US2013/0171138), functional fragments thereof, substances functionally equivalent thereto, and sugar chain-modified variants thereof. Herein, native IgG (e.g. native IgGl) refers to polypeptides that contain the same amino acid sequence as that of naturally occurring IgG (e.g. native IgGl) and belongs to the class of antibodies encoded substantially by the immunoglobulin gamma gene. Native IgG may be spontaneous mutants thereof and the like.

[0113] Typically, where an antibody has a structure that is substantially the same as or similar to that of native IgG, the Y-shaped structure of the four chains (two heavy chain polypeptides and two light chain polypeptides) can be the basic structure. Typically, the heavy chain and the light chain can be linked together via a disulfide bond (SS bond) and form a heterodimer. Such heterodimers may be linked together via a disulfide bond and form a Y-shaped heterotetramer. The two heavy chains or light chains may be identical or different from each other.

[0114] For example, an IgG antibody may be cleaved into two units of Fab (region) and a single unit of Fc (region) by papain digestion, which cleaves the hinge region (also

WO 2017/046994

PCT/JP2016/003616 referred to as the hinge within the scope of Disclosures A and B described herein) where the heavy-chain Fab region is linked to the Fc region. Typically, the Fab region contains an antigen-binding domain. Since phagocytic cells such as leukocytes and macrophages have receptors that are capable of binding to the Fc region (Fc receptors), and can recognize via the Fc receptors antibodies that are bound to an antigen and phagocytize the antigen (opsonization). Meanwhile, the Fc region is involved in the mediation of immune reactions such as ADCC or CDC, and has an effector function of inducing a reaction upon an antibody binding to antigens. The antibody effector function is known to vary according to the type of immunoglobulin (isotype). The Fc region of the IgG class would indicate a region that spans, for example, from cysteine of position 226 or from proline of position 230 (EU numbering) to the C terminus; however, the Fc region is not limited thereto. The Fc region can be appropriately obtained by partial digestion of a monoclonal IgGl, IgG2, IgG3, or IgG4 antibody, or others, with a protease such as pepsin, followed by elution of adsorbed fractions from a protein A or protein G column.

[0115] Within the scope of Disclosures A and B described herein, the positions of amino acid residues in the variable region (CDR(s) and/or FR(s)) of an antibody are shown according to Rabat, whereas the positions of amino acid residues in the constant region or Fc region are shown according to EU numbering based on Rabat's amino acid positions (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991).

[0116] Within the scope of Disclosures A and B described herein, library may refer to molecules (populations) such as multiple antibodies that have sequence variability, in which their respective sequences may be the same or different from one another; multiple fusion polypeptides containing the antibodies; or nucleic acids or polynucleotides encoding these amino acid sequences, as described in detail in

WO2013/125667 (for example, paragraphs 0121-0125). The library may, for example, contain at least 10⁴ antibody molecules, more preferably, at least 10⁵ antibody molecules, even more preferably, at least 10⁶ antibody molecules, particularly preferably, at least 10⁷ antibody molecules or more. The library may be phage libraries. The phrase primarily consist of means that antibodies which may have different antigen-binding activities account for a certain portion among the numerous independent clones with different sequences in the library. In one embodiment, immune libraries that are constructed based on antibody genes derived from lymphocytes of animals immunized with a specific antigen, patients with infection, humans with elevated antibody titer in blood due to vaccination, or patients with cancer or autoimmune disease can be appropriately used as randomized variable region libraries. In an alternative embodiment, naive libraries containing naive sequences (antibody

WO 2017/046994

PCT/JP2016/003616 sequences without bias in the repertoire), which are constructed from antibody genes derived from lymphocytes of healthy persons, can also be appropriately used as randomized variable region libraries (Gejima et al., Human Antibodies 11:121-129 (2002)); Cardoso et al., Scand. J. Immunol. 51:337-344 (2000)). Amino acid sequences containing naive sequences can refer to those obtained from such naive libraries. In an alternative embodiment, synthetic libraries in which the CDR sequence from a V gene of genomic DNA or a reconstructed functional V gene is substituted with a set of synthetic oligonucleotides containing a sequence encoding a codon set of appropriate length can also be appropriately used as randomized variable region libraries. In this case, it is also possible to substitute only the heavy chain CDR3 sequence, since sequence variations are observed in the CDR3 gene. A standard way to produce amino acid diversity in the antibody variable region may be to increase variations of amino acid residues at positions that can be exposed on the antibody surface.

[0117] In one embodiment, where antibodies of Disclosure A or B, for example, have a structure that is substantially the same as or similar to the structure of native Ig antibodies, they typically have variable regions (V regions) [heavy chain variable region (VH region) and light chain variable region (VL region)] and constant regions (C regions)[heavy chain constant region (CH region) and light chain constant region (CL region)]. The CH region is further divided into three: CHI to CH3. Typically, the Fab region of the heavy chain contains VH region and CHI, and typically the Fc region of the heavy chain contains CH2 and CH3. Typically, the hinge region is located between CHI and CH2. Furthermore, the variable region typically has complementarity determining regions (CDRs) and framework regions (FRs). Typically, the VH region and VL region each have three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1, FR2, FR3, and FR4). Typically, the six CDRs in the variable regions of the heavy chain and light chain interact and form the antigenbinding domain of the antibody. On the other hand, where there is only one single CDR, while the antigen-binding affinity is known to be lower as compared to where six CDRs are present, it has still the ability to recognize and bind to the antigen.

[0118] Ig antibodies are classified into several classes (isotypes) based on structural differences in their constant regions. In many mammals, they are categorized into five im munoglobulin classes based on structural differences in the constant region: IgG, IgA, IgM, IgD, and IgE. Furthermore, in the case of human, IgG has four subclasses: IgGl, IgG2, IgG3, and IgG4; and IgA has two subclasses: IgAl and IgA2. The heavy chain is classified into γ chain, μ chain, a chain, δ chain, and ε chain according to differences in the constant region, and based on these differences, there are five immunoglobulin classes (isotypes): IgG, IgM, IgA, IgD, and IgE. On the other hand, there are two types of light chains: λ chain and κ chain, and all immunoglobulins have either of these two.

WO 2017/046994 PCT/JP2016/003616 [0119] In one embodiment, where an antibody of Disclosure A or B has a heavy chain, for example, the heavy chain may be any one of γ chain, μ chain, a chain, δ chain, and ε chain, or may be derived from any one of them, and where an antibody of Disclosure A or B has a light chain, for example, the light chain may be either κ chain or λ chain, or may be derived from either. Furthermore, within the scope of Disclosures A and B described herein, the antibody may be of any isotype (for example, IgG, IgM, IgA,

IgD, or IgE) and of any subclass (for example, human IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2; mouse IgGl, IgG2a, IgG2b, and IgG3), or may be derived from any one of them, but is not limited thereto.

[0120] Within the scope of Disclosures A and B described herein, an antigen-binding domain may have any structure as long as it binds to an antigen of interest. Such domains may include, for example, the variable regions of antibody heavy chains and light chains (for example, 1 to 6 CDRs); a module of about 35 amino acids referred to as A domain, which is contained in Avimer, a cell membrane protein present in the body (W02004/044011 and W02005/040229); Adnectin containing the 10Fn3 domain which binds to the protein in the glycoprotein fibronectin expressed on cell membrane (W02002/032925); Affibody, having as scaffold the IgG-binding domain constituting a three-helix bundle of 58 amino acids of Protein A (WO1995/001937); Designed Ankyrin Repeat Proteins (DARPins) which are a region exposed on the molecular surface of an Ankyrin repeat (AR) having a structure in which a subunit with a turn containing 33 amino acid residues, two antiparallel helices, and a loop is repeatedly stacked (W02002/020565); Anticalins and others, which are a four loop region supporting one side of a centrally-twisted barrel structure of eight antiparallel strands that are highly conserved among lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (W02003/029462); and the concave region formed by the parallel-sheet structure inside the horseshoe-shaped structure formed by stacked repeats of the leucine-rich-repeat (LRR) module of the variable lymphocyte receptor (VLR) which does not have a immunoglobulin structure and is used in the system of acquired immunity in jawless vertebrates such as lamprey andhagfish (W02008/016854). Preferred antigen-binding domains of Disclosure A or B may include those having IgG antibody heavy-chain and light-chain variable regions, and more specifically, ScFv, single chain antibodies, Fv, scFv₂ (single chain Fv₂), Fab, and F(ab')₂.

[0121] In one embodiment of Disclosure A, ion concentration is not particularly limited and refers to hydrogen ion concentration (pH) or metal ion concentration. Herein, metal ions can be any one of ions of group I elements except hydrogen, such as alkaline metals and copper group elements, group II elements such as alkaline earth metals and zinc group elements, group III elements except boron, group IV elements

WO 2017/046994

PCT/JP2016/003616 except carbon and silicon, group VIII elements such as iron group and platinum group elements, elements belonging to subgroup A of groups V, VI, and VII, and metal elements such as antimony, bismuth, and polonium. Metal atoms have the property of releasing valence electrons to become cations. This is referred to as ionization tendency. Metals with strong ionization tendency are assumed to be chemically active.

[0122] In one embodiment of Disclosure A, preferred metal ions may be calcium ion, as described in detail in WO2012/073992 and WO2013/125667.

[0123] In one embodiment of Disclosure A, ion concentration condition(s) may be a condition that focuses on differences in the biological behavior of an ion concentration-dependent antibody between a low ion concentration and a high ion concentration. Furthermore, the antigen-binding activity changes according to the ion concentration condition can mean that the antigen-binding activity of an ion concentration-dependent antigen-binding domain or an ion concentration-dependent antibody of Disclosure A or B changes between a low ion concentration and a high ion concentration. Such cases include, for example, those with higher (stronger) or lower (weaker) antigen-binding activity at a high ion concentration than at a low ion concentration, without being limited thereto.

[0124] In one embodiment of Disclosure A, the ion concentration can be hydrogen ion concentration (pH) or calcium ion concentration. Where the ion concentration is hydrogen ion concentration (pH), the ion concentration-dependent antigen-binding domain may also be referred to as a pH-dependent antigen-binding domain; and where the ion concentration is calcium ion concentration, it may also be referred to as a calcium ion concentration-dependent antigen-binding domain.

[0125] In one embodiment in the context of Disclosure A, the ion concentration-dependent antigen-binding domains, ion concentration-dependent antibodies, ion concentrationdependent antigen-binding domains with increased pi, and ion concentrationdependent antibodies with increased pi can be obtained from libraries primarily consisting of antibodies that differ in sequence (have variability) and whose antigenbinding domains contain at least one amino acid residue that causes a change in the antigen-binding activity of the antigen-binding domain or antibody according to the ion concentration condition. The antigen-binding domains may be preferably located within the light chain variable region (which may be modified) and/or the heavy chain variable region (which may be modified). Furthermore, to construct a library, such light-chain or heavy-chain variable regions may be combined with heavy-chain or light-chain variable regions constructed as a randomized variable region sequence library. Where the ion concentration is hydrogen or calcium ion concentration, nonlimiting examples of the library include, for example, libraries in which heavy chain variable regions constructed as a randomized variable region sequence library are

WO 2017/046994

PCT/JP2016/003616 combined with light chain variable region sequences in which amino acid residue(s) in a germ line sequence such as SEQ ID NO:1 (Vkl), SEQ ID NO:2 (Vk2), SEQ ID NO:3 (Vk3), or SEQ ID NO:4 (Vk4) has been substituted with at least one amino acid residue that can alter the antigen-binding activity depending on ion concentrations. Furthermore, where the ion concentration is calcium ion concentration, the library includes, for example, those in which the heavy chain variable region sequence of SEQ ID NO:5 (6RL#9-IgGl) or SEQ ID NO:6 (6KC4-l#85-IgGl) is combined with light chain variable regions constructed as a randomized variable region sequence library or light chain variable regions having a germ line sequence.

[0126] In one embodiment, where the ion concentration is calcium ion concentration, the high calcium ion concentration is not particularly limited to a specific value; however, the concentration may be selected between 100 pM and 10 mM, between 200 μΜ and 5 mM, between 400 pM and 3 mM, between 200 pM and 2 mM, or between 400 μΜ and 1 mM. A concentration selected between 500 pM and 2.5 mM, which is close to the plasma (blood) concentration of calcium ion in vivo, may be also preferred. The low calcium ion concentration is not particularly limited to a specific value; however, the concentration may be selected between 0.1 pM and 30 pM, between 0.2 pM and 20 pM, between 0.5 pM and 10 pM, or between 1 pM and 5 pM, or between 2 pM and 4 pM. A concentration selected between 1 pM and 5 pM, which is close to the concentration of calcium ion in early endosomes in vivo, may be also preferred.

[0127] Whether the antigen-binding activity of an antigen-binding domain or antibody containing the domain changes according to the metal ion concentration (for example, calcium ion concentration) condition can be readily determined by known methods, for example, by the methods described herein in the context of Disclosure A, or described in WO2012/073992. For example, the antigen-binding activity of an antigen-binding domain or antibody containing the domain can be measured at low and high calcium ion concentrations and compared. In this case, conditions other than the calcium ion concentration may be preferably the same. Furthermore, conditions other than the calcium ion concentration in determining the antigen-binding activity can be appropriately selected by those of ordinary skill in the art. The antigen-binding activity can be determined, for example, under the conditions of HEPES buffer at 37°C, or using the BIACORE (GE Healthcare) or others.

[0128] In one embodiment in the context of Disclosure A, it is preferable that the antigenbinding activity of the ion concentration-dependent antigen-binding domain, ion concentration-dependent antibody, ion concentration-dependent antigen-binding domain with increased pi, or ion concentration-dependent antibody with increased pi is higher under a high calcium ion concentration condition than under a low calcium ion concentration condition. In this case, the ratio between the antigen-binding activity under a

WO 2017/046994

PCT/JP2016/003616 low calcium ion concentration condition and the antigen-binding activity under a high calcium ion concentration condition is not limited; however, the value of the ratio of the KD (dissociation constant) for an antigen under a low calcium ion concentration condition to the KD under a high calcium ion concentration condition, i.e., KD (3 μΜ Ca)/KD (2 mM Ca), may be preferably 2 or more, more preferably 10 or more, and still more preferably 40 or more. The upper limit of the KD (3 μΜ Ca)/KD (2 mM Ca) value is not limited, and may be any value such as 400, 1000, or 10000.

[0129] Where the antigen is a soluble antigen, the dissociation constant (KD) can be used as the value for antigen-binding activity. Meanwhile, where the antigen is a membrane antigen, the apparent dissociation constant (KD) can be used. The dissociation constant (KD) and apparent dissociation constant (KD) can be determined by known methods, for example, by BIACORE (GE healthcare), Scatchard plot, or flow cytometer.

[0130] Alternatively, for example, the dissociation rate constant (kd) can also be used as another indicator to represent the binding activity ratio. Where the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an indicator to represent the antigen binding activity ratio, the value of the ratio of the lowcalcium-ion-concentration-condition dissociation rate constant (kd) to the highcalcium-ion-concentration-condition dissociation rate constant (kd), i.e., kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition), may be preferably 2 or more, more preferably 5 or more, still more preferably 10 or more, and yet more preferably 30 or more. The upper limit of the kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) value is not limited, and may be any value such as 50, 100, or 200.

[0131] Where the antigen is a soluble antigen, the dissociation rate constant (kd) can be used as the value for antigen-binding activity. Meanwhile, where the antigen is a membrane antigen, the apparent dissociation rate constant (kd) can be used. The dissociation rate constant (kd) and the apparent dissociation rate constant (kd) can be determined by known methods, for example, by BIACORE (GE healthcare) or flow cytometer.

[0132] In one embodiment, methods for producing or screening for calcium ion concentration-dependent antigen-binding domains or calcium ion concentration-dependent antibodies whose antigen-binding activity is higher at a high calcium ion concentration condition than at a low calcium ion concentration condition, or libraries thereof, are not limited. The methods include, for example, those described in WO2012/073992 (for example, paragraphs 0200-0213).

[0133] Such a method may comprise, for example:

(a) determining the antigen-binding activity of an antigen-binding domain or antibody at a low calcium ion concentration condition;

(b) determining the antigen-binding activity of an antigen-binding domain or

WO 2017/046994

PCT/JP2016/003616 antibody at a high calcium ion concentration condition; and (c) selecting an antigen-binding domain or antibody whose antigen-binding activity at a low calcium ion concentration condition is lower than the antigen-binding activity at a high calcium ion concentration condition.

[0134] Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen-binding domain or antibody, or a library thereof, at a high calcium ion concentration condition;

(b) incubating an antigen-binding domain or antibody that bound to the antigen in step (a) at a low calcium ion concentration condition; and (c) isolating an antigen-binding domain or antibody that dissociated in step (b).

[0135] Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen-binding domain or antibody, or a library thereof at a low calcium ion concentration condition;

(b) selecting an antigen-binding domain or antibody that does not bind to the antigen or has a low antigen-binding ability in step (a);

(c) allowing the antigen-binding domain or antibody selected in step (b) to bind to the antigen at a high calcium ion concentration condition; and (d) isolating the antigen-binding domain or antibody that bound to the antigen in step (c).

[0136] Alternatively, the method may comprise, for example:

(a) contacting an antigen-binding domain or antibody, or a library thereof with an antigen-immobilized column at a high calcium ion concentration condition;

(b) eluting an antigen-binding domain or antibody bound to the column in step (a) from the column at a low calcium ion concentration condition; and (c) isolating an antigen-binding domain or antibody eluted in step (b).

[0137] Alternatively, the method may comprise, for example:

(a) allowing an antigen-binding domain or antibody, or a library thereof to pass through an antigen-immobilized column at a low calcium ion concentration condition to collect an antigen-binding domain or antibody eluted without binding to the column;

(b) allowing an antigen-binding domain or antibody collected in step (a) to bind to the antigen at a high calcium ion concentration condition; and (c) isolating an antigen-binding domain or antibody bound to the antigen in step (b). [0138] Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen-binding domain or antibody, or a library thereof at a high calcium ion concentration condition;

(b) obtaining an antigen-binding domain or antibody bound to the antigen in step (a);

(c) incubating an antigen-binding domain or antibody obtained in step (b) at a low calcium ion concentration; and

WO 2017/046994

PCT/JP2016/003616 (d) isolating an antigen-binding domain or antibody whose antigen-binding activity in step (c) is weaker than the criterion selected in step (b).

[0139] Each step of these various screening methods may be repeated several times, or the steps may be combined appropriately to obtain the most suitable molecules. The aforementioned conditions may be suitably selected for the low and high calcium ion concentration conditions. Desired calcium ion concentration-dependent antigen-binding domains or calcium ion concentration-dependent antibodies can be obtained thereby.

[0140] In the context of Disclosure A, in one embodiment, the antigen-binding domains or antibodies as a starting material may be, for example, modified antigen-binding domains or antibodies that have an increased pi as a result of modifying the charge of at least one amino acid residue that can be exposed on their surface. In an alternative embodiment, where amino acids that change the binding activity of an ion concentration-dependent antigen-binding domain are introduced into the sequence, they may be introduced in conjunction with a charge modification of at least one amino acid residue that can be exposed on the surface of the antigen-binding domain or antibody so as to increase the pi.

[0141] Alternatively, in the context of present disclosure A, for example, it is possible to use pre-existing antigen-binding domains or antibodies, preexisting libraries (phage library, etc.); antibodies prepared from hybridomas obtained by immunizing animals or from B cells of immunized animals, or libraries thereof; or antigen-binding domains, antibodies, or libraries obtained by introducing natural or unnatural amino acid mutations capable of chelating calcium (described below) thereinto (for example, libraries with an increased content of calcium-chelatable amino acids, or libraries introduced with calcium-chelatable amino acids at specific sites).

[0142] In one embodiment in the context of Disclosure A, where the ion concentration is calcium ion concentration, there is no limitation as to the type of amino acids that change the binding activity of ion concentration-dependent antigen-binding domains or ion concentration-dependent antigen-binding domains with increased pi, as long as they can form a calcium-binding motif. For example, calcium-binding motifs are known to those of ordinary skill in the art (for example, Springer et al. (Cell 102:275-277 (2000)); Kawasaki et al. (Protein Prof. 2:305-490 (1995)); Moncrief et al. (J. Mol. Evol. 30:522-562 (1990)); Chauvaux et al. (Biochem. J. 265:261-265 (1990)); Bairoch et al. (FEBS Lett. 269:454-456 (1990)); Davis (New Biol. 2:410-419 (1990)); Schaefer et al. (Genomics 25:638-643 (1995)); Economou et al. (EMBO J. 9:349-354 (1990)); Wurzburg et al. (Structure. 14(6):1049-1058 (2006)). Thus, where an antigenbinding domain has an arbitrary calcium-binding motif such as of a C-type lectin, for example, ASGPR, CD23, MBR, or DC-SIGN, the antigen-binding activity of the domain can be changed according to the calcium ion concentration condition. Such

WO 2017/046994

PCT/JP2016/003616 calcium-binding motifs may include, for example, in addition to those described above, the calcium-binding motif included in the antigen-binding domain shown in SEQ ID NO:7 (which corresponds to Vk5-2).

[0143] In one embodiment in the context of Disclosure A, where the ion concentration is calcium ion concentration, amino acids having a metal-chelating activity may be used as amino acids that change the binding activity of ion concentration-dependent antigen-binding domains or ion concentration-dependent antigen-binding domains of with increased pi. For example, any amino acids can be appropriately used as amino acids having a metal-chelating activity, as long as they can form a calcium-binding motif. Specifically, such amino acids include those having an electron-donating property. The amino acids preferably include, but are not limited to, Ser (S), Thr (T), Asn (N), Gin (Q), Asp (D), and Glu (E).

[0144] The location of such amino acids having a metal-chelating activity in an antigenbinding domain is not limited to specific positions. In one embodiment, the amino acids may be located at any positions in the heavy chain variable region and/or light chain variable region that may form an antigen-binding domain. At least one amino acid residue that causes calcium ion concentration-dependent changes in the antigenbinding activity of an antibody may be contained, for example, in CDR (one or more of CDR1, CDR2, and CDR3) and/or FR (one or more of FR1, FR2, FR3, and FR4) of the heavy chain and/or light chain. The amino acid residue(s) may be placed, for example, at one or more of positions 95, 96, 100a, and 101 according to Rabat numbering in heavy-chain CDR3; at one or more of positions 30, 31, and 32 according to Rabat numbering in light-chain CDR1; at position 50 according to Rabat numbering in light-chain CDR2; and/or at position 92 according to Rabat numbering in light-chain CDR3. Those amino acid residues may be placed alone or in combination.

[0145] Troponin C, calmodulin, parvalbumin, myosin light chain, and others are known to have multiple calcium-binding sites and assumed to be derived from a common origin in molecular evolution, and in one embodiment, one or more of light chain CDR1, CDR2, and CDR3 can be designed to contain binding motifs thereof. For the purpose described above, for example, the cadherin domain; the EF hand contained in calmodulin; the C2 domain contained in Protein kinase C; the Gia domain contained in blood-clotting protein Factor IX; C-type lectin of the asialoglycoprotein receptor or mannose-binding receptor; the A domain contained in the LDL receptor; Annexin; thrombospondin type-3 domain; and EGF-like domain may be suitably used.

[0146] In one embodiment, where the ion concentration is hydrogen ion concentration (pH), the concentration condition of proton, i.e., nucleus of a hydrogen atom, is used synonymously with the condition of hydrogen index (pH). Where the amount of activity of hydrogen ion in an aqueous solution is represented by aH⁺, pH is defined as -log 10aH⁺.

WO 2017/046994

PCT/JP2016/003616

Where the ionic strength of the aqueous solution is low (for example, less than 10³), aH⁺ is nearly equal to the hydrogen ion strength. For example, the ionic product for water at 25°C and 1 atmosphere is Kw = aH⁺ * aOH = 10 ¹⁴; thus, for pure water, aH⁺ = aOH = 10⁷. In this case, pH = 7 is neutral, and an aqueous solution with a pH of less than 7 is acidic, and an aqueous solution with a pH of greater than 7 is alkaline. Thus, the hydrogen ion concentration condition may be conditions that focus on differences in the biological behavior of a pH-dependent antibody at a high hydrogen ion concentration (acidic pH range) and at a low hydrogen ion concentration (neutral pH range) for the hydrogen ion concentration condition or pH condition. For example, in the context of Disclosure A, the antigen-binding activity at a high hydrogen ion concentration (acidic pH range) condition is lower than the antigen-binding activity at a low hydrogen ion concentration (neutral pH range) condition can mean that the antigen-binding activity of an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain with increased pi, or an ion concentration-dependent antibody with increased pi is weaker at a pH selected from pH 4.0 to pH 6.5, preferably from pH 4.5 to pH 6.5, more preferably from pH 5.0 to pH 6.5, and still more preferably from pH 5.5 to pH 6.5, than at a pH selected from pH 6.7 to pH 10.0, preferably from pH 6.7 to pH 9.5, more preferably from pH 7.0 to pH 9.0, and still more preferably from pH 7.0 to pH 8.0. Preferably, the above expression can mean that the antigen-binding activity at the pH within early endosomes in vivo is weaker than that at the plasma pH in vivo; and specifically can mean that the antigen-binding activity of an antibody, for example, at pH 5.8 is weaker than that, for example, at pH 7.4.

[0147] Whether the antigen-binding activity of an antigen-binding domain or an antibody containing the domain changes according to the hydrogen ion concentration condition can be readily assessed by known methods, for example, by the assay methods described herein in the context of Disclosure A, or described in WO2009/125825. For example, the antigen-binding activity of an antigen-binding domain or an antibody containing the domain toward an antigen of interest may be measured at low and high hydrogen ion concentrations and compared. In this case, it is preferable that conditions other than the hydrogen ion concentration are the same. Where determining the antigen-binding activity, those of ordinary skill in the art can suitably select conditions other than the hydrogen ion concentration, and for example, measurements can be carried out under the condition of HEPES buffer at 37°C, or using the BIACORE (GE Healthcare), or the like.

[0148] Within the scope of Disclosure A described herein, unless particularly specified otherwise in the context, neutral pH range (also referred to as low hydrogen ion concentration, high pH, neutral pH condition, or neutral pH) is not particularly

WO 2017/046994

PCT/JP2016/003616 limited to a specific value; however, it may be preferably selected from pH 6.7 to pH 10.0, from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0. The neutral pH range may be preferably pH 7.4 which is close to the in vivo pH in plasma (blood), but for the convenience of measurement, for example, pH 7.0 may be used.

[0149] Within the scope of Disclosure A described herein, unless particularly specified otherwise in the context, acidic pH range (also referred to as high hydrogen ion con centration, low pH, acidic pH condition, or acidic pH) is not particularly limited to a specific value; however, it may be preferably selected from pH 4.0 to pH 6.5, from pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. The acidic pH range may be preferably pH 5.8 which is close to the in vivo hydrogen ion concentration in the early endosome, but for the convenience of measurement, for example, pH 6.0 may be used.

[0150] In one embodiment in the context of Disclosure A, where the ion concentration is hydrogen ion concentration, it is preferable that the antigen-binding activity of the ion concentration-dependent antigen-binding domain, ion concentration-dependent antibody, ion concentration-dependent antigen-binding domain with increased pi, or ion concentration-dependent antibody with increased pi is higher under a neutral pH condition than under an acidic pH condition. In this case, the ratio of the antigenbinding activity under a neutral pH condition to the antigen-binding activity under an acidic pH condition is not limited; however, the value of the ratio of the dissociation constant (KD) for an antigen at an acidic pH condition to the KD at a neutral pH condition, i.e., KD (acidic pH range)/KD (neutral pH range), (for example, KD (pH 5.8)/KD (pH 7.4)) may be 2 or more; 10 or more; or 40 or more. The upper limit of KD (acidic pH range)/KD (neutral pH range) value is not limited, and may be any value such as 400, 1000, or 10000.

[0151] In an alternative embodiment, it is also possible to use, for example, the dissociation rate constant (kd) as an indicator to represent the above binding activity ratio. Where the dissociation rate constant (kd) is used instead of the dissociation constant (KD) as an indicator to represent the binding activity ratio, the value of the ratio of the dissociation rate constant (kd) for an antigen at a high hydrogen ion concentration condition to that at a low hydrogen ion concentration condition, i.e., kd (acidic pH range)/kd (neutral pH range) may be 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the kd (acidic pH range)/kd (neutral pH range) value is not limited, and may be any value such as 50, 100, or 200.

[0152] Where the antigen is a soluble antigen, the value of the antigen-binding activity can be represented by the dissociation rate constant (kd), whereas where the antigen is a membrane antigen, such value can be represented by the apparent dissociation rate constant (apparent kd). The dissociation rate constant (kd) and apparent dissociation rate constant (apparent kd) can be determined by known methods, for example, by

WO 2017/046994

PCT/JP2016/003616 using the BIACORE (GE healthcare) or a flow cytometer.

[0153] In one embodiment, methods for producing or screening for pH-dependent antigenbinding domains or pH-dependent antibodies whose antigen-binding activity is higher under a neutral pH condition than under an acidic pH condition, or libraries thereof, are not limited. Such methods include, for example, those described in WO2009/125825 (for example, paragraphs 0158-0190).

[0154] Such a method may comprise, for example:

(a) determining the antigen-binding activity of an antigen-binding domain or antibody in an acidic pH condition;

(b) determining the antigen-binding activity of an antigen-binding domain or antibody in a neutral pH condition; and (c) selecting an antigen-binding domain or antibody whose antigen-binding activity is lower in the acidic pH condition than in the neutral pH condition.

[0155] Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen-binding domain or antibody, or a library thereof, in a neutral pH condition;

(b) incubating an antigen-binding domain or antibody bound to the antigen in step (a) in an acidic pH condition; and (c) isolating an antigen-binding domain or antibody that dissociated in step (b).

[0156] Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen-binding domain or antibody, or a library thereof in an acidic pH condition;

(b) selecting an antigen-binding domain or antibody that does not bind to the antigen or has a low antigen-binding ability in step (a);

(c) allowing the antigen to bind to the antigen-binding domain or antibody selected in step (b) in a neutral pH condition; and (d) isolating an antigen-binding domain or antibody that bound to the antigen in step (c).

[0157] Alternatively, the method may comprise, for example:

(a) contacting an antigen-binding domain or antibody, or a library thereof with an antigen-immobilized column in a neutral pH condition;

(b) eluting an antigen-binding domain or antibody bound to the column in step (a) from the column in an acidic pH condition; and (c) isolating an antigen-binding domain or antibody eluted in step (b).

[0158] Alternatively, the method may comprise, for example:

(a) allowing an antigen-binding domain or antibody, or a library thereof to pass through an antigen-immobilized column in an acidic pH condition to collect an antigen-binding domain or antibody eluted without binding to the column;

WO 2017/046994

PCT/JP2016/003616 (b) allowing an antigen-binding domain or antibody collected in step (a) to bind to the antigen in a neutral pH condition; and (c) isolating an antigen-binding domain or antibody bound to the antigen in step (b).

[0159] Alternatively, the method may comprise, for example:

(a) contacting an antigen with an antigen-binding domain or antibody, or a library thereof in a neutral pH condition;

(b) obtaining an antigen-binding domain or antibody bound to the antigen in step (a);

(c) incubating an antigen-binding domain or antibody obtained in step (b) in an acidic pH condition; and (d) isolating an antigen-binding domain or antibody whose antigen-binding activity in step (c) is weaker than the criterion selected in step (b).

[0160] Each step in these various screening methods may be repeated several times, or the steps may be combined. The aforementioned conditions may be suitably selected for the acidic and neutral pH conditions. Desired pH-dependent antigen-binding domains or pH-dependent antibodies can be obtained thereby.

[0161] In the context of Disclosure A, in one embodiment, the antigen-binding domains or antibodies as a starting material may be, for example, modified antigen-binding domains or antibodies that have an increased pi as a result of modifying the charge of at least one amino acid residue that can be exposed on their surface. In an alternative embodiment, where amino acids that change the binding activity of an ion concentration-dependent antigen-binding domain are introduced into the sequence, they may be introduced in conjunction with a charge modification of at least one amino acid residue that can be exposed on the surface of the antigen-binding domain or antibody so as to increase the pi.

[0162] Alternatively, in the context of present disclosure A, for example, it is possible to use pre-existing antigen-binding domains or antibodies, pre-existing libraries (phage library, etc.); antibodies prepared from hybridomas obtained by immunizing animals or from B cells of immunized animals, or libraries thereof; or antigen-binding domains, antibodies, or libraries obtained by introducing natural or unnatural amino acid mutations having a side-chain with a pKa of 4.0-8.0 (described below) thereinto (for example, libraries with an increased content of natural or unnatural amino acid mutations with a side-chain pKa of 4.0-8.0, or libraries introduced at specific sites with natural or unnatural amino acid mutations with a side-chain pKa of 4.0-8.0). Such a preferred antigen-binding domain can have, for example, an amino acid sequence in which at least one amino acid residue has been substituted with an amino acid(s) with a side-chain pKa of 4.0-8.0 and/or which has been inserted with amino acid(s) with a side-chain pKa of 4.0-8.0, as described in WO2009/125825.

[0163] In one embodiment in the context of Disclosure A, the site at which the mutation of

WO 2017/046994

PCT/JP2016/003616 amino acids with a side-chain pKa of 4.0-8.0 is introduced is not limited, and the mutation may be introduced at any site as long as the antigen-binding activity becomes weaker in an acidic pH range than in a neutral pH range (the KD (acidic pH range)/KD (neutral pH range) value is increased or the kd (acidic pH range)/kd (neutral pH range) value is increased) as compared to before substitution or insertion. Where the antibody has a variable region or CDR(s), the site may be within the variable region or CDR(s). The number of amino acids that are substituted or inserted can be appropriately determined by those of ordinary skill in the art; and the number may be one or more. Furthermore, it is possible to delete, add, insert, and/or substitute, or modify other amino acids in addition to the substitution or insertion described above. Substitution with or insertion of amino acids with a side-chain pKa of 4.0-8.0 may be carried out in a random fashion by scanning methods such as histidine scanning, in which histidine is used instead of alanine in alanine scanning known to those of ordinary skill in the art, and/or antibodies whose KD (acidic pH range)/KD (neutral pH range) value or kd (acidic pH range)/kd (neutral pH range) value has increased as compared to before mutation may be selected from among the antigen-binding domains or antibodies that result from random substitution with or insertion mutations of these amino acids, or libraries thereof.

[0164] Furthermore, the antigen-binding domains or antibodies may be preferably those whose antigen-binding activity in a neutral pH range before and after these mutations is not significantly reduced, is not substantially reduced, is substantial identical, or is increased; and in other words, those whose activity may be maintained at at least 10% or higher, preferably 50% or higher, still more preferably 80% or higher, and yet more preferably 90% or higher, or even higher. Where the binding activity of an antigenbinding domain or antibody is decreased due to substitution with or insertion of amino acids with a pKa of 4.0-8.0, the binding activity may be recovered or increased by e.g., substituting, deleting, adding, or inserting one or more amino acids at sites other than the substitution or insertion sites described above.

[0165] In an alternative embodiment, amino acids with a side chain pKa of 4.0-8.0 may be placed at any location within the heavy-chain and/or light-chain variable regions that may form an antigen-binding domain. At least one amino acid residue with a sidechain pKa of 4.0-8.0 may be located, for example, in the CDR (one or more of CDR1, CDR2, and CDR3) and/or FR (one or more of FR1, FR2, FR3, and FR4) of the heavy chain and/or light chain. Such amino acid residues include, but are not limited to, amino acid residues at one or more of positions 24, 27, 28, 31, 32, and 34 according to Rabat numbering in the light-chain variable region CDR1; amino acid residues at one or more of positions 50, 51, 52, 53, 54, 55, and 56 according to Rabat numbering in the light-chain variable region CDR2; and/or amino acid residues at one or more of

WO 2017/046994

PCT/JP2016/003616 positions 89, 90, 91, 92, 93, 94, and 95A according to Kabat numbering in the lightchain variable region CDR3. Those amino acid residues may be included alone or in combination, as long as the antigen-binding activity of the antibody changes according to the hydrogen ion concentration condition.

[0166] In one embodiment within the scope of Disclosure A, an arbitrary amino acid residue can be suitably used as the amino acid residue that changes the antigen-binding activity of the antigen-binding domain or antibody according to the hydrogen ion concentration condition. Specifically, such amino acid residues can include those with a side-chain pKa of 4.0-8.0. Such amino acids having an electron-donating property may include, for example, natural amino acids such as His (H) and Glu (E), and unnatural amino acids such as histidine analogs (US2009/0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11(17):3761-3768 (2003)). The amino acid residues may preferably include, for example, amino acids with a side-chain pKa of 6.0-7.0, and in particular His (H).

[0167] Within the scope of Disclosure A described herein, unless otherwise specified and unless there are inconsistencies in the context, it is understood that the isoelectric point (pi) may be either a theoretical or an experimentally determined isoelectric point, and it is also referred to as pi.

[0168] The pi value can be determined experimentally, for example, by isoelectric focusing electrophoresis. Meanwhile, the theoretical pi value can be calculated using gene and amino acid sequence analysis software (Genetyx, etc.).

[0169] In one embodiment, whether the pi of an antibody with increased pi or an antibody of Disclosure A has been increased as compared to the antibody before modification (a native antibody (for example, a native Ig antibody, preferably a native IgG antibody) or reference antibody (e.g., an antibody before antibody modification, or prior to or during library construction)) can be determined by carrying out, in addition to or instead of the above-described methods, antibody pharmacokinetics test using plasma, for example, from mice, rats, rabbits, dogs, monkeys, or humans, in combination with methods such as BIACORE, cell proliferation assay, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA), or fluorescent immunoassay.

[0170] Within the scope of Disclosure A described herein, an amino acid residue that can be exposed on the surface generally can refer to an amino acid residue located on the surface of a polypeptide constituting an antibody. An amino acid residue located on the surface of a polypeptide can refer to an amino acid residue whose side chain can be in contact with solvent molecules (which in general may be mostly water molecules). However, the side chain does not necessarily have to be wholly in contact with solvent molecules, and when even a portion of the side chain is in contact with the solvent molecules, the amino acid residue is defined as an amino acid located on the

WO 2017/046994

PCT/JP2016/003616 surface. The amino acid residues located on the surface of a polypeptide can also include amino acid residues located close to the antibody surface and thereby can have a mutual electric charge influence from other amino acid residue(s) whose side chain, even partly, is in contact with the solvent molecules. Those of ordinary skill in the art can prepare a homology model of a polypeptide or antibody by for example homology modeling using commercially available softwares. Alternatively, it is possible to use methods such as X-ray crystallography. The amino acid residues that may be exposed on the surface can be determined, for example, using coordinates from a threedimensional model of an antibody using a computer program such as Insightll program (Accelrys). Surface-exposed sites may be determined using algorithms known in the technical field (for example, Lee and Richards (J. Mol. Biol. 55:379-400 (1971)); Connolly (J. Appl. Cryst. 16:548-558(1983)). Surface-exposable sites can be determined using software suitable for protein modeling and three-dimensional structure information obtained from the antibody. Software available for such purposes includes, for example, the SYBYL Biopolymer Module software (Tripos Associates). When an algorithm requires a user input size parameter, the size of a probe used in the calculation may be set to about 1.4 Angstrom or less in radius. Furthermore, methods for determining surface-exposed regions and areas using software for personal computers have been described by Pacios (Pacios, Comput. Cheml8(4):377-386 (1994); J. Mol. Model. 1:46-53 (1995)). Based on such information as described above, appropriate amino acid residues located on the surface of a polypeptide that constitutes an antibody can be selected.

[0171] A method for increasing the pi of a protein is, for example, to reduce the number of amino acids with a negatively charged side chain at a neutral pH condition (for example, aspartic acid and glutamic acid) and/or to increase the number of amino acids with a positively charged side chain (for example, arginine, lysine and histidine). Amino acid residues with a negatively charged side chain have a negative charge represented as -1 at a pH condition that is sufficiently higher than their side chain pKa, which is a theory well known to those of ordinary skill in the art. For example, the theoretical pKa for the side chain of aspartic acid is 3.9, and the side chain has a negative charge represented as -1 at a neutral pH condition (for example, in a solution of pH 7.0). Conversely, amino acid residues with a positively charged side chain have a positive charge represented as +1 at a pH condition that is sufficiently lower than their side chain pKa. For example, the theoretical pKa for the side chain of arginine is 12.5, and the side chain has a positive charge represented as +1 at a neutral pH condition (for example, in a solution of pH 7.0). Amino acid residues whose side chain has no charge at a neutral pH condition (for example, in a solution of pH 7.0) are known to include 15 types of natural amino acids, i.e., alanine, cysteine, phenylalanine, glycine,

WO 2017/046994

PCT/JP2016/003616 isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. As a matter of course, it is understood that amino acids for changing the pi may be unnatural amino acids.

[0172] From the above, as a method for increasing the pi of a protein at a neutral pH condition (for example, in a solution of pH 7.0), a charge alteration of + 1 can be conferred to a protein of interest, for example, by substituting amino acids (residues) with non-charged side chains for aspartic acid (residue) or glutamic acid (residue) (whose side chain has a negative charge of -1) in the amino acid sequence of the protein. Furthermore, a charge alteration of +1 can be conferred to the protein, for example, by substituting arginine or lysine (whose side chain has a positive charge of + 1) for amino acid (residue) whose side chain has no charge. Moreover, a charge alteration of +2 can be conferred at a time to the protein by substituting arginine or lysine (whose side chain has a positive charge of +1) for aspartic acid or glutamic acid (whose side chain has a negative charge of -1). Alternatively, to increase the pi of a protein, amino acids with a side chain having no charge and/or amino acids having a positively charged side chain can be added or inserted into the amino acid sequence of the protein, or amino acids with a side chain having no charge and/or amino acids with a negatively charged side chain present in the amino acid sequence of the protein can be deleted. It is understood that, for example, the N-terminal and C-terminal amino acid residues of a protein have a main chain-derived charge (NH³⁺ of the amino group at the N-terminus and COO of the carbonyl group at the C-terminus) in addition to their side chain-derived charges. Thus, the pi of a protein can also be increased by performing to the main chain-derived functional groups some addition, deletion, substitution, or insertion.

[0173] Those of ordinary skill in the art would appreciate that the effect of changing the net charge or pi of a protein, which is obtained by modifying one or more amino acids (residues) in the amino acid sequence with a focus on the presence or magnitude of electrical charges of the amino acids (residues), does not exclusively (or substantially) depend on the antibody-constituting amino acid sequences per se or the type of target antigen, but rather depends on the type and number of amino acid residues that are added, deleted, substituted, or inserted.

[0174] Antibodies which have been modified to have an increased pi by modification on at least one amino acid residue that can be exposed on the antibody surface (antibodies with increased pi or pi-increased antibodies) can be taken up more rapidly into cells or can promote antigen elimination from the plasma, as described or suggested in, for example, W02007/114319, W02009/041643, WO2014/145159, or WO2012/016227.

[0175] Of the several antibody isotypes, for example, the IgG antibody has a sufficiently large molecular weight, and thus its major metabolic pathway is not through renal

WO 2017/046994

PCT/JP2016/003616 excretion. The IgG antibody, which has an Fc region as a part of the molecule, is known to be recycled through a salvage pathway via FcRn, and thus has a long in vivo half-life. The IgG antibody is assumed to be mainly metabolized via a metabolic pathway in endothelial cells (He et al., J. Immunol. 160(2):1029-1035 (1998)). Specifically, it is believed that when taken up into endothelial cells nonspecifically,

IgG antibodies are recycled by binding to FcRn, while IgG antibodies that could not bind are metabolized. The plasma half-life of an IgG antibody may be shortened when its Fc region is modified such that its FcRn-binding activity is reduced. On the other hand, the plasma half-life of an antibody with an increased pi has been demonstrated to depend on the pi in a highly correlated manner, as described in e.g., W02007/114319 and W02009/041643. Specifically, the plasma half-life of the pi-increased antibodies described in the above documents was reduced without modifying the amino acid sequence constituting Fc which could potentially lead to acquisition of immunogenicity, and this result suggests that the pi-increasing technology is widely applicable even to any types of antibody molecules whose main metabolic pathway is renal excretion, such as scFv, Fab, or Fc fusion proteins.

[0176] The pH concentration in biological fluids (for example, plasma) is in a neutral pH range. Without being bound by a particular theory, it is believed that in biological fluids, the net positive charge of a pi-increased antibody is increased due to the increased pi, and as a result the antibody is more strongly attracted by physicochemical Coulomb interaction to the endothelial cell surface whose net charge is negative, when compared to antibodies whose pi has not been increased; via non-specific binding, the antibody binds thereto and is taken up into cells, which results in shortening of the antibody half-life in plasma or enhancement of antigen elimination from plasma. Furthermore, increasing the pi of an antibody enhances uptake into cells of the antibody (or antigen/antibody complex) and/or intracellular permeability, which is considered to result in reducing the antibody concentration in plasma, reducing the antibody bioavailability, and/or shortening the antibody half-life in plasma; and these phenomena are expected to occur commonly in vivo, regardless of cell type, tissue type, organ type, etc. Furthermore, where an antibody forms a complex with an antigen and is taken up into cells, not only the antibody's pi but also the antigen's pi can have an influence on the decrease or increase of the uptake into cells.

[0177] In one embodiment, methods for producing or screening for antibodies with an increased pi may include, for example, those described in W02007/114319 (for example, paragraphs 0060-0087), W02009/041643 (for example, paragraphs 0115-), WO2014/145159, and WO2012/016227. Such a method may comprise, for example:

(a) modifying a nucleic acid that encodes an antibody comprising at least one amino acid residue that can be exposed on the antibody surface such that the charge of the

WO 2017/046994

PCT/JP2016/003616 amino acid residue(s) is modified so as to increase the pi of the antibody;

(b) culturing a host cell such that the nucleic acid is expressed; and (c) collecting an antibody from the host cell culture.

[0178] Alternatively, the method may comprise, for example:

(a¹) modifying a nucleic acid that encodes an antibody comprising at least one amino acid residue that can be exposed on the antibody surface such that the charge of the amino acid residue(s) is modified;

(b¹) culturing a host cell such that the nucleic acid is expressed;

(c¹) collecting an antibody from the host cell culture; and (d¹) (optionally confirming or measuring and,) selecting an antibody with a pi increased as compared to an antibody before the modification. Here, the antibody as a starting material or the antibody before the modification or the reference antibody may be, for example, an ion concentration-dependent antibody. Alternatively, when modifying the amino acid residue(s), amino acid(s) that change the binding activity of the ion concentration-dependent antigen-binding domain may also be included in the sequence.

[0179] Alternatively, the method may simply be a method that comprises culturing the host cells obtained in step (b) or (b¹) and collecting an antibody from the cell culture.

[0180] In an alternative embodiment, the method may be, for example, a method for producing a multispecific antibody that comprises a first polypeptide and a second polypeptide, and optionally a third polypeptide and a fourth polypeptide, which comprises:

(A) modifying nucleic acid(s) that encodes the first polypeptide and/or the second polypeptide, and optionally the third polypeptide and/or the fourth polypeptide, any one or more of which comprises at least one amino acid residue that can be exposed on the polypeptide surface such that the charge of the amino acid residue(s) is modified so as to increase the antibody's pi;

(B) culturing a host cell such that the nucleic acid is expressed; and (C) collecting a multispecific antibody from the host cell culture.

[0181] Alternatively, the method may comprise, for example:

(A¹) modifying nucleic acid(s) that encodes the first polypeptide and/or the second polypeptide, and optionally the third polypeptide and/or the fourth polypeptide, any one or more of which comprises at least one amino acid residue that can be exposed on the polypeptide surface such that the charge of the amino acid residue(s) is altered;

(B¹) culturing a host cell such that the nucleic acid is expressed;

(C¹) collecting a multispecific antibody from the host cell culture; and (D¹) (optionally confirming and) selecting an antibody whose pi is increased as compared to an antibody before the modification.

WO 2017/046994 PCT/JP2016/003616 [0182] Here, the antibody as a starting material or the antibody before the modification or the reference antibody may be, for example, an ion concentration-dependent antibody. Alternatively, when modifying the amino acid residue(s), amino acid(s) that change the binding activity of the ion concentration-dependent antigen-binding domain may also be included in the sequence.

[0183] Alternatively, the method may simply be a method that comprises culturing the host cells obtained in step (B) or (B¹) and collecting an antibody from the cell culture. In this case, the polypeptides whose nucleic acid(s) is to be modified may be preferably a homomultimer of the first polypeptide, a homomultimer of the second polypeptide, or a heteromultimer of the first and second polypeptides (and optionally, a homomultimer of the third polypeptide, a homomultimer of the fourth polypeptide, or a heteromultimer of the third and fourth polypeptides).

[0184] In an alternative embodiment, the method may be, for example, a method for producing a humanized or human antibody with shortened half-life in plasma, which comprises: in an antibody which comprises CDR(s) selected from the group consisting of human-derived CDR(s), CDR(s) derived from an animal other than human, and synthetic CDR(s); human-derived FR(s); and a human constant region, (I) modifying at least one amino acid residue that can be exposed on the surface of at least one region selected from the group consisting of the CDR(s), FR(s), and constant region into amino acid residue(s) that has a different charge from the amino acid residue(s) present at the corresponding position(s) before the modification such that the pi of the antibody is increased.

[0185] Alternatively, the method may comprise, for example, in an antibody which comprises CDR(s) selected from the group consisting of human-derived CDR(s), CDR(s) derived from an animal other than human, and synthetic CDR(s); humanderived FR(s); and a human constant region, (Γ) modifying at least one amino acid residue that can be exposed on the surface of at least one region selected from the group consisting of the CDR(s), FR(s), and constant region into amino acid residue(s) that has a different charge from the amino acid residue(s) present at the corresponding position(s) before the modification; and (ΙΓ) (optionally confirming and) selecting an antibody whose pi is increased as compared to an antibody before the modification.

[0186] Here, the antibody as a starting material or the antibody before the modification or the reference antibody may be, for example, an ion concentration-dependent antibody. Alternatively, when modifying the amino acid residue(s), amino acid(s) that change the binding activity of the ion concentration-dependent antigen-binding domain may also be included in the sequence.

[0187] Alternatively, for example, it is possible to use pre-existing antigen-binding domains

WO 2017/046994

PCT/JP2016/003616 or antibodies, pre-existing libraries (phage library, etc.); antibodies prepared from hybridomas obtained by immunizing animals or from B cells of immunized animals, or libraries thereof; or antigen-binding domains or antibodies or libraries thereof with increased pi, prepared by modifying, in the above-described antigen-binding domains, antibodies, or libraries thereof, at least one amino acid residue that can be exposed on the surface according to for example any one of the above-described embodiments.

[0188] In one embodiment of the antibodies of Disclosure A, the pi value may be preferably increased, for example, at least by 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or more, or at least by 0.6, 0.7, 0.8, 0.9, or more, and to significantly shorten the antibody half-life in plasma, the pi value may be increased, for example, by at least by 1.0, 1.1, 1.2, 1.3,

1.4, 1.5, or more, or at least by 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or more, or by 3.0 or more, as compared to the antibodies before modification or alteration (native antibodies (for example, native Ig antibodies, preferably native IgG antibodies), or reference or parent antibodies (e.g., antibodies before antibody modification, or prior to or during library construction)). Those of ordinary skill in the art can appropriately routinely determine the optimal pi value for the antibodies of Disclosure A, in consideration of the balance between their pharmacological effect and toxicity, and for example, the number of antigen-binding domains of the antibodies or the pi of the antigen according to the purpose. Without being bound by a particular theory, it is believed that antibodies of Disclosure A, in one embodiment, are beneficial because, in addition to the characteristic of being shuttled between plasma and cellular endosomes and repeated binding to multiple antigens with one single antibody molecule due to the presence of an ion concentration-dependent antigen-binding domain, the antibody's net positive charge is increased as a result of increase in pi and this allows rapid cellular uptake of the antibody. These characteristics would shorten the antibody half-life in plasma, increase the extracellular matrix-binding activity of the antibodies, or enhance antigen elimination from plasma. One may decide on the optimal pi value to take advantage of these characteristics.

[0189] In one embodiment in the context of Disclosure A, when compared to the antibodies before modification or alteration of at least one amino acid residue to increase the pi (native antibodies (for example, native Ig antibodies, preferably native IgG antibodies), or reference or parent antibodies (e.g., antibodies before antibody modification, or prior to or during library construction), which can be ion concentration-dependent antibodies), the ion concentration-dependent antibodies of Disclosure A with increased pi may preferably enhance antigen elimination from plasma, for example, by at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold or more

WO 2017/046994

PCT/JP2016/003616 (when the antibodies are administered in vivo), or their extracellular matrix-binding activity may be preferably increased, for example, by at least 1.1-fold, 1.25-fold,

1.5- fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold,

3.75- fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, or 5-fold or more.

[0190] In one embodiment in the context of Disclosure A, when compared to the antibodies before introduction of an ion concentration-dependent antigen-binding domain (native antibodies (for example, native Ig antibodies, preferably native IgG antibodies), or reference or parent antibodies (e.g., antibodies before antibody modification, or prior to or during library construction), which can be antibodies with an increased pi), the ion concentration-dependent antibodies of Disclosure A with increased pi may preferably enhance antigen elimination from plasma, for example, by at least 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold,

3.5- fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, 5-fold, 5.5-fold, 6-fold,

6.5- fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold or more (when the antibodies are administered in vivo), or their extracellular matrix-binding activity may be preferably increased, for example, by at least 1.1-fold, 1.25-fold, 1.5-fold,

1.75- fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.25-fold, 3.5-fold, 3.75-fold, 4-fold, 4.25-fold, 4.5-fold, 4.75-fold, or 5-fold or more.

[0191] In one embodiment, assay methods for assessing whether the extracellular matrixbinding activity of antibodies of Disclosure A has been increased as compared to the antibodies before modification or alteration (native antibodies (for example, native Ig antibodies, which can be native IgG antibodies), or reference or parent antibodies (e.g., antibodies before antibody modification, or prior to or during library construction), which can be ion concentration-dependent antibodies or antibodies with an increased pi) are not limited. For example, the assay can be carried out using an EFISA system which detects the binding between an antibody and an extracellular matrix, where an antibody is added to an extracellular matrix-immobilized plate, and a labeled antibody against the antibody is added thereto. Alternatively, as described in Examples 1 to 4 herein and in WO2012/093704, it is also possible to use electrochemiluminescence (ECF) which enables high sensitivity detection of the extracellular matrix-binding ability. This method can be performed, for example, using an ECF system in which a mixture of an antibody and a ruthenium antibody is added to an extracellular matriximmobilized plate and the binding between the antibodies and the extracellular matrix is measured based on the electrochemiluminescence of ruthenium. The concentration of the antibody to be added can be set appropriately; the added concentration can be high in order to increase the sensitivity for detecting extracellular matrix binding. Such extracellular matrices may be derived from animals or plants, as long as they contain glycoproteins such as collagen, proteoglycan, fibronectin, laminin, entactin, fibrin, and

WO 2017/046994

PCT/JP2016/003616 perlecan; and animal-derived extracellular matrices may be preferred. For example, it is possible to use extracellular matrices derived from animals such as humans, mice, rats, monkeys, rabbits, or dogs. For example, a human-derived native extracellular matrix may be used as an indicator of antibody pharmacodynamics in human plasma. The condition for assessing extracellular matrix-binding of an antibody may be preferably a neutral pH range around pH 7.4, which is the physiological condition; however, the condition does not necessarily have to be a neutral range, and the binding may also be assessed in an acidic pH range (for example, around pH 6.0). Alternatively, when assessing the extracellular matrix-binding of an antibody, the assay can be performed in the co-presence of an antigen molecule to which the antibody binds and by assessing the binding activity of the antigen-antibody complex toward the extracellular matrix.

[0192] In one embodiment, antibodies of Disclosure A (substantially) can retain the antigenbinding activity when compared to the antibodies before modification or alteration of at least one amino acid residue to increase pi (native antibodies (for example, native Ig antibodies, preferably native IgG antibodies) or reference antibodies (e.g., antibodies before antibody modification, or prior to or during library construction)). In this case, to (substantially) retain the antigen-binding activity can mean to have an activity of at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and still more preferably 80%, 85%, 90%, or 95% or more as compared to the binding activity of the antibodies before modification or alteration. Alternatively, antibodies of Disclosure A only need to retain binding activity to a degree that allows them to retain their functions when they bind to antigens; thus, the affinity determined at 37°C under the physiological conditions may be, for example, 100 nM or less, preferably 50 nM or less, more preferably 10 nM or less, and still more preferably 1 nM or less.

[0193] In one embodiment of Disclosure A, the expression of modification of at least one amino acid residue that can be exposed on the antibody surface or an equivalent expression can mean that one or more of addition, deletion, substitution and insertion are performed on at least one amino acid residue that can be exposed on the surface of an antibody. Such modification may preferably include substitution of at least one amino acid residue.

[0194] The substitution of amino acid residues can include, for example, substitution of amino acid residues whose side chain has no charge for amino acid residues having a negatively charged side chain, substitution of amino acid residues having a positively charged side chain for amino acid residues whose side chain has no charge, and substitution of amino acid residues having a positively charged side chain for amino acid residues having a negatively charged side chain in the amino acid sequence of an antibody of interest, which can be performed alone or in appropriate combinations. The

WO 2017/046994

PCT/JP2016/003616 insertion or addition of amino acid residues can include, for example, insertion or addition of amino acids whose side chain has no charge and/or insertion or addition of amino acids having a positively charged side chain in the amino acid sequence of an antibody of interest, which can be performed alone or in appropriate combinations. The deletion of amino acid residues can include, for example, deletion of amino acid residues whose side chain has no charge and/or deletion of amino acid residues having a negatively charged side chain in the amino acid sequence of an antibody of interest, which can be performed alone or in appropriate combinations.

[0195] Those of ordinary skill in the art can appropriately combine one of more of these addition, deletion, substitution, and insertion in the amino acid sequence of an antibody of interest. Modification that causes a reduction in the local charge of amino acid residues is also acceptable since the net pi of an antibody of Disclosure A only has to be increased. For example, if desired, antibodies whose pi has been increased (too much) may be modified to decrease the pi (slightly). It is also acceptable that the local charge of amino acid residues is decreased as a result of modification of at least one amino acid residue carried out simultaneously or at a different time for other purposes (for example, to increase antibody stability or to reduce immunogenicity). Such antibodies include antibodies from libraries constructed for specific purposes.

[0196] In one embodiment, among amino acids (residues) used for modifying at least one amino acid residue that can be exposed on the antibody surface, natural amino acids are as follows: an amino acid with a negatively charged side chain can be Glu (E) or Asp (D); an amino acid whose side chain has no charge can be Ala (A), Asn (N), Cys (C), Gin (Q), Gly (G), His (H), He (I), Leu (L), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), or Val (V); and an amino acid with a positively charged side chain can be His (H), Lys (K), or Arg (R).

[0197] As described in detail in Examples 1 to 4 herein, in a solution of neutral pH (for example, pH 7.0), lysine and arginine have a positive charge in almost 100% when present as a residue in an antibody, while histidine has a positive charge in only about 9% when present as a residue in an antibody and the remaining major portion is assumed not to have any charge. Thus, Lys (K) or Arg(R) is preferably selected as an amino acid with a positively charged side chain.

[0198] In one embodiment, antibodies of Disclosure A preferably have a variable region and/or a constant region. Furthermore, the variable region may preferably have a heavy chain variable region and/or a light chain variable region, and/or may preferably have CDR(s) (for example, one or more of CDR1, CDR2, and CDR3) and/or FR(s) (for example, one or more of FR1, FR2, FR3, and FR4). The constant region may preferably have a heavy chain constant region and/or a light chain constant region, and in terms of the sequence and type, it may be, for example, an IgG-type constant region

WO 2017/046994

PCT/JP2016/003616 (preferably, human IgGl, human IgG2, human IgG3, or human IgG4-type constant region, human κ chain constant region, and human λ chain constant region). It is also possible to use modified variants of these constant regions.

[0199] In one embodiment, the modification of at least one amino acid residue that can be exposed on the antibody surface may be either a modification of a single amino acid or a combination of modifications of multiple amino acids. A preferred method can be to introduce a combination of multiple amino acid substitutions at sites where amino acids can be exposed on the antibody surface. Furthermore, without limitations, such multiple amino acid substitutions are preferably introduced at positions that are three dimensionally close to one another. When amino acids with a positively charged side chain (for example, Lys (K) or Arg (R)) have been substituted for amino acids that can be exposed on the surface of an antibody molecule (which are preferably, but are not limited to, amino acids with a negatively charged side chain (for example, Glu (E) or Asp (D)); or when pre-existing amino acids having a positively charge (for example, Lys (K) or Arg (R)) are used, for example, one or more amino acids (which may include amino acids embedded inside the antibody molecule depending on the situation) that are three-dimensionally close to the amino acids may also be substituted with amino acids having a positively charge to consequently create a dense state of local positive charge in a three-dimensionally proximal location. Herein, the definition of a three-dimensionally proximal location is not particularly limited; but it can mean a state where one or more amino acid substitution is introduced, for example, within 20 Angstroms, preferably within 15 Angstroms, and more preferably within 10 Angstroms. Whether an amino acid substitution site of interest is exposed on the surface of an antibody molecule or whether an amino acid substitution site is close to other amino acid substitution site(s) or the above pre-existing amino acids can be assessed by known methods such as X-ray crystallography.

[0200] In addition to those described above, methods for giving multiple positive charges at sites three-dimensionally close to one another can include those that use amino acids that originally have a positive charge in the native IgG constant region. Such amino acids include, for example: arginine at positions 255, 292, 301, 344, 355, and 416, according to EU numbering; and lysine at positions 121, 133, 147, 205, 210, 213, 214, 218, 222, 246, 248, 274, 288, 290, 317, 320, 322, 326, 334, 338, 340, 360, 370, 392, 409, 414, and 439, according to EU numbering. Multiple positive charges can be given into a three-dimensionally proximal location by substituting with positively charged amino acid(s) at sites three-dimensionally close to these positively charged amino acids.

[0201] Where antibodies of Disclosure A have a variable region (that may be modified), amino acid residues that are not masked by antigen binding (i.e., that still can be

WO 2017/046994

PCT/JP2016/003616 exposed on the surface) may be modified, and/or amino acid modification may not be introduced at sites that are masked by antigen binding or amino acid modification that does not (substantially) inhibit antigen binding may be carried out. Where amino acid residues that can be exposed on the surface of an antibody molecule present in the ion concentration-dependent binding domain are modified, amino acids of the antigenbinding domain may be modified in such a way that the modification does not (substantially) reduce the binding activity of amino acid residues that can change the antigen-binding activity of the antibody according to the ion concentration condition (for example, those in a calcium-binding motif, or a histidine insertion site and/or a histidine substituted site), or amino acid residues may be modified at sites other than of the amino acid residues that can change the antigen-binding activity of the antibody according to the ion concentration condition. On the other hand, where amino acid residues that can be exposed on the surface of an antibody molecule present in the ion concentration-dependent binding domain have already been modified, the type or the position of amino acid residues that can change the antigen-binding activity of the antibody according to the ion concentration condition may be selected such that the pi of the antibody is not reduced below an acceptable level. Where the pi of an antibody is reduced below an acceptable level, the pi of the overall antibody can be increased by modifying at least one amino acid residue that can be exposed on the surface of the antibody molecule.

[0202] Without limitations, FR sequences with a high pi may be preferably selected from human germline FR sequences or sequences of regions that are equivalent thereto, whose amino acid may be modified in some cases.

[0203] Where antibodies of Disclosure A have a constant region (that may be modified) having an FcyR-binding domain (which may be a binding domain to any of the FcyR isoforms and allotypes described below) and/or an FcRn-binding domain, sites for modification of at least one amino acid residue that can be exposed on the surface of the constant region can be amino acid residues other than those in the FcyR-binding domain and/or those in the FcRn-binding domain, if desired. Alternatively, where the modification sites are selected from amino acid residues in the FcyR-binding domain and/or in the FcRn-binding domain, it may be preferable to select sites that do not (substantially) affect the binding activity or binding affinity for FcyR and/or FcRn, or if they would affect, sites which is biologically or pharmacologically acceptable.

[0204] In one embodiment, the site of the at least one amino acid residue that is modified to produce an antibody of Disclosure A whose pi is increased by modification of at least one amino acid residue that can be exposed on the surface of the variable region (that may be modified) is not limited; however, such a site can be selected from the group consisting of, according to Rabat numbering: (a) position 1, 3, 5, 8, 10, 12, 13, 15, 16,

WO 2017/046994

PCT/JP2016/003616

18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b,

83, 84, 85, 86, 105, 108, 110, and 112 in a FR of the heavy chain variable region; (b) position 31,61, 62, 63, 64, 65, and 97 in a CDR of the heavy chain variable region; (c) position 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57,

60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in a FR of the light chain variable region; and (d) position 24, 25, 26, 27, 52, 53, 54,

55, and 56 in a CDR of the light chain variable region, wherein an amino acid at each position after modification can be selected from any of the amino acids described above in terms of the side-chain charge such as Lys (K), Arg (R), Gin (Q), Gly (G),

Ser (S), or Asn (N), but is not limited thereto. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 of the above amino acid positions are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of the above amino acid positions are modified.

[0205] In one embodiment, among the position(s) to be modified, the following position(s) can be used for aiding in pi increase of an antibody of Disclosure A, in combination with other position(s) which themselves can have sufficient effect of increasing pi of an antibody. Such position(s) for aiding in the pi increase can be, for example, as for a light chain variable region, selected from a group consisting of positions 27, 52, 56, 65, and 69, according to Rabat numbering.

[0206] Furthermore, the site of at least one amino acid residue that is modified in the CDR and/or FR is not limited; however, such a site can be selected from the group consisting of: (a) position 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82, 82a, 82b, 83, 84, 85, and 105 in the FR of the heavy chain variable region; (b) position 31,61,

62, 63, 64, 65, and 97 in the CDR of the heavy chain variable region; (c) position 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 in the FR of the light chain variable region; and(d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in the CDR of the light chain variable region. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 of the above amino acid positions are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of the above amino acid positions are modified.

[0207] Where the modification site of at least one amino acid residue is selected, for example, from a group comprising the above-described groups, the type of amino acid after modification in the heavy-chain variable region is, for example:

(a) 8K, 8R, 8Q, 8G, 8S, or 8N for position 8; (b) 13K, 13R, 13Q, 13G, 13S, or 13N for position 13; (c) 15K, 15R, 15Q, 15G, 15S, or 15N for position 15; (d) 16K, 16R, 16Q, 16G, 16S, or 16N for position 16; (e) 18K, 18R, 18Q, 18G, 18S, or 18N for position 18; (f) 39K, 39R, 39Q, 39G, 39S, or 39N for position 39; (g) 4IK, 41R, 41Q, 41G, 41S, or 41N for position 41; (h) 43K, 43R, 43Q, 43G, 43S, or 43N for position 43; (i) 44K, 44R, 44Q, 44G, 44S, or 44N for position 44; (j) 63K, 63R, 63Q, 63G, 63S, or 63N for position 63; (k) 64K, 64R, 64Q, 64G, 64S, or 64N for position 64; (1) 77K,

WO 2017/046994

PCT/JP2016/003616

77R, 77Q, 77G, 77S, or 77N for position 77; (m) 82K, 82R, 82Q, 82G, 82S, or 82N for position 82; (n) 82aK, 82aR, 82aQ, 82aG, 82aS, or 82aN for position 82a; (o) 82bK, 82bR, 82bQ, 82bG, 82bS, or 82bN for position 82b; (p) 83K, 83R, 83Q, 83G, 83S, or 83N for position 83; (q) 84K, 84R, 84Q, 84G, 84S, or 84N for position 84; (r) 85K, 85R, 85Q, 85G, 85S, or 85N for position 85; or (s) 105K, 105R, 105Q, 105G, 105S, or 105N for position 105. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 of any combination of the above amino acid positions are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of any combination of the above amino acid positions are modified.

[0208] Non-limiting examples of a combination of modified amino acids positions in the heavy-chain variable region is, for example:

any two or more of positions selected from the group consisting of positions 16, 43, 64, and 105; any two or more of positions selected from the group consisting of positions 77, 82a, and 82b; positions 77 and 85; positions 41 and 44; positions 82a and 82b; positions 82 and 82b; positions 82b and 83; or positions 63 and 64, according to Rabat numbering, wherein an amino acid at each position after modification can be selected from any of the amino acids described above in terms of the side-chain charge such as Lys (K), Arg (R), Gin (Q), Gly (G), Ser (S), or Asn (N), but is not limited thereto.

[0209] A specific combination can be, for example, 16Q/43R/64K/105Q; 77R/82aN/82bR; 77R/82aG/82bR; 77R/82aS/82bR; 77R/85G; 41R/44R; 82aN/82bR; 82aG/82bR; 82aS/82bR; 82K/82bR; 82bR/83R; 77R/85R; or 63R/64K.

[0210] Likewise, the type of amino acid after modification in the light-chain variable region is, for example: (a) 16K, 16R, 16Q, 16G, 16S, or 16N for position 16; (b) 18K, 18R, 18Q, 18G, 18S, or 18N for position 18; (c) 24K, 24R, 24Q, 24G, 24S, or 24N for position 24; (d) 25K, 25R, 25Q, 25G, 25S, or 25N for position 25; (e) 26K, 26R, 26Q, 26G, 26S, or 26N for position 26; (f) 27K, 27R, 27Q, 27G, 27S, or 27N for position 27; (g) 37K, 37R, 37Q, 37G, 37S, or 37N for position 37; (h) 41K, 41R, 41Q, 41G, 41S, or 41N for position 41; (i) 42K, 42R, 42Q, 42G, 42S, or 42N for position 42; (j) 45K, 45R, 45Q, 45G, 45S, or 45N for position 45; (k) 52K, 52R, 52Q, 52G, 52S, or 52N for position 52; (1) 53K, 53R, 53Q, 53G, 53S, or 53N for position 53; (m) 54K, 54R, 54Q, 54G, 54S, or 54N for position 54; (η) 55K, 55R, 55Q, 55G, 55S, or 55N for position 55; (ο) 56K, 56R, 56Q, 56G, 56S, or 56N for position 56; (p) 65K, 65R, 65Q, 65G, 65S, or 65N for position 65; (q) 69K, 69R, 69Q, 69G, 69S, or 69N for position 69; (r) 74K, 74R, 74Q, 74G, 74S, or 74N for position 74; (s) 76K, 76R, 76Q, 76G,

76S, or 76N for position 76; (t) 77K, 77R, 77Q, 77G, 77S, or 77N for position 77; (u) 79K, 79R, 79Q, 79G, 79S, or 79N for position 79; and (v) 107K, 107R, 107Q, 107G, 107S, or 107N for position 107. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10

WO 2017/046994

PCT/JP2016/003616 or more than 10 of any combination of the above amino acid positions are modified. In some embodiments, 1-20, 1-15, 1-10, or 1-5 of any combination of the above amino acid positions are modified.

[0211] Non-limiting examples of a combination of modified amino acids positions in the light-chain variable region is, for example: positions 24 and 27; positions 25 and 26; positions 41 and 42; positions 42 and 76; positions 52 and 56; positions 65 and 79; positions 74 and 77; positions 76 and 79; any two or more of positions selected from the group consisting of positions 16, 24, and 27; any two or more of positions selected from the group consisting of positions 24, 27, and 37; any two or more of positions selected from the group consisting of positions 25, 26, and 37; any two or more of positions selected from the group consisting of positions 27, 76, and 79; any two or more of positions selected from the group consisting of positions 41, 74, and 77; any two or more of positions selected from the group consisting of positions 41, 76, and 79; any two or more of positions selected from the group consisting of positions 24,

27, 41, and 42; any two or more of positions selected from the group consisting of positions 24, 27, 52, and 56; any two or more of positions selected from the group consisting of positions 24, 27, 65, and 69; any two or more of positions selected from the group consisting of positions 24, 27, 74, and 77; any two or more of positions selected from the group consisting of positions 24, 27, 76, and 79; any two or more of positions selected from the group consisting of positions 25, 26, 52, and 56; any two or more of positions selected from the group consisting of positions 25, 26, 65, and 69; any two or more of positions selected from the group consisting of positions 25, 26, 76, and 79; any two or more of positions selected from the group consisting of positions 27, 41, 74, and 77; any two or more of positions selected from the group consisting of positions 27, 41, 76, and 79; any two or more of positions selected from the group consisting of positions 52, 56, 74, and 77; any two or more of positions selected from the group consisting of positions 52, 56, 76, and 79; any two or more of positions selected from the group consisting of positions 65, 69, 76, and 79; any two or more of positions selected from the group consisting of positions 65, 69, 74, and 77; any two or more of positions selected from the group consisting of positions 18, 24, 45, 79, and 107; any two or more of positions selected from the group consisting of positions 27, 52, 56, 74, and 77; any two or more of positions selected from the group consisting of positions 27, 52, 56, 76, and 79; any two or more of positions selected from the group consisting of positions 27, 65, 69, 74, and 77; any two or more of positions selected from the group consisting of positions 27, 65, 69, 76, and 79; any two or more of positions selected from the group consisting of positions 41, 52, 56, 74, and 77; any two or more of positions selected from the group consisting of positions 41, 52, 56, 76, and 79; any two or more of positions selected from the group consisting of positions

WO 2017/046994

PCT/JP2016/003616

41, 65, 69, 74, and 77; any two or more of positions selected from the group consisting of positions 41, 65, 69, 76, and 79; any two or more of positions selected from the group consisting of positions 24, 27, 41, 42, 65, and 69; any two or more of positions selected from the group consisting of positions 24, 27, 52, 56, 65, and 69; any two or more of positions selected from the group consisting of positions 24, 27, 65, 69, 74, and 77; any two or more of positions selected from the group consisting of positions 24, 27, 65, 69, 76, and 79; any two or more of positions selected from the group consisting of positions 24, 27, 41, 42, 74, and 77; any two or more of positions selected from the group consisting of positions 24, 27, 52, 56, 74, and 77; any two or more of positions selected from the group consisting of positions 24, 27, 41, 42, 76, and 79; any two or more of positions selected from the group consisting of positions 24, 27, 52, 56, 76, and 79; any two or more of positions selected from the group consisting of positions 24, 27, 74, 76, 77, and 79; any two or more of positions selected from the group consisting of positions 52, 56, 65, 69, 74, and 77; or any two or more of positions selected from the group consisting of positions 52, 56, 65, 69, 76, and 79, according to Kabat numbering, wherein an amino acid at each position after modification can be selected from any of the amino acids described above in terms of the side-chain charge such as Lys (K), Arg (R), Gin (Q), Gly (G), Ser (S), or Asn (N), but is not limited thereto.

[0212] A specific combination can be, for example, 24R/27Q; 24R/27R; 24K/27K;

25R/26R; 25K/26K; 41R/42K; 42K/76R; 52R/56R; 65R/79K; 74K/77R; 76R/79K; 16K/24R/27R; 24R/27R/37R; 25R/26R/37R; 27R/76R/79K; 41R/74K/77R; 41R/76R/79K; 24R/27R/41R/42K; 24R/27R/52R/56R; 24R/27R/52K/56K; 24R/27R/65R/69R; 24R/27R/74K/77R; 24R/27R/76R/79K; 25R/26R/52R/56R; 25R/26R/52K/56K; 25R/26R/65R/69R; 25R/26R/76R/79K; 27R/41R/74K/77R; 27R/41R/76R/79K; 52R/56R/74K/77R; 52R/56R/76R/79K; 65R/69R/76R/79K; 65R/69R/74K/77R; 18R/24R/45K/79Q/107K; 27R/52R/56R/74K/77R; 27R/52R/56R/76R/79K; 27R/65R/69R/74K/77R; 27R/65R/69R/76R/79K; 41R/52R/56R/74K/77R; 41R/52R/56R/76R/79K; 41R/65R/69R/74K/77R; 41R/65R/69R/76R/79K; 24R/27R/41R/42K/65R/69R; 24R/27R/52R/56R/65R/69R; 24R/27R/65R/69R/74K/77R; 24R/27R/65R/69R/76R/79K;

24R/27R/41R/42K/74K/77R; 24R/27R/52R/56R/74K/77R; 24R/27R/41R/42K/76R/79K; 24R/27R/52R/56R/76R/79K; 24R/27R/74K/76R/77R/79K; 52R/56R/65R/69R/74K/77R; or 52R/56R/65R/69R/76R/79K.

[0213] In W02007/114319 or W02009/041643, it has already been explained or demonstrated based on theoretical evidence, homology modeling, or experimental techniques that the effect of increasing the pi via modification of some amino acid

WO 2017/046994

PCT/JP2016/003616 residues in the variable region does not exclusively (or substantially) depend on the antibody-constituting amino acid sequences per se or the type of target antigen, but rather it depends on the type and number of amino acid residues that are substituted. It has been also demonstrated that even after modification of some amino acids, the antigen-binding activity for several types of antigens is (substantially) maintained, or at least can be expected to be maintained with high possibility by those of ordinary skill in the art.

[0214] For example, W02009/041643 specifically shows that in the heavy-chain FR of a humanized glypican 3 antibody as shown in SEQ ID NO:8, preferred modification sites of amino acid residues that can be exposed on the surface are positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 69, 72, 73, 74, 76, 77, 82, 85, 87, 89, 90, 107, 110, 112, and 114 according to Rabat numbering. It also reports that the amino acid residue at position 97 according to Rabat numbering is preferred because it is exposed on the surface of almost all antibodies. W02009/041643 also shows that the amino acid residues of positions 52, 54, 62, 63, 65, and 66 in the heavy-chain CDR of the antibody are preferred. It also shows that the amino acid residues of positions 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 43, 44, 45, 46, 48, 49, 50, 54, 62, 65, 68, 70, 71, 73, 74, 75, 79, 81, 82, 84, 85, 86, 90, 105, 108, 110, 111, and 112 according to Rabat numbering in the light-chain FR of a humanized glypican 3 antibody as shown in SEQ ID NO:9 are preferred. It also shows that the amino acid residues of positions 24, 27, 33, 55, 59 in the light-chain CDR of this antibody are preferred. Furthermore, W02009/041643 specifically shows that the amino acid residues of positions 31, 64, and 65 according to Rabat numbering in the heavy-chain CDR of an anti-human IL-6 receptor antibody as shown in SEQ ID NO: 10 are preferred sites that allow modification of amino acid residues that can be exposed on the surface while maintaining the antigen-binding activity. It also shows that the amino acid residues of positions 24, 27, 53, and 55 according to Rabat numbering in the light chain CDR of an anti-human IL-6 receptor antibody as shown in SEQ ID NO: 11 are preferred. It also specifically shows that the amino acid residue of position 31 according to Rabat numbering in the heavy-chain CDR of an anti-human IL-6 receptor antibody as shown in SEQ ID NO: 12 is a preferred site that allows modification of amino acid residue that can be exposed on the surface while maintaining the antigen-binding activity. It also shows that the amino acid residues of positions 24, 53, 54, and 55 according to Rabat numbering in the light-chain CDR of an anti-human IL-6 receptor antibody as shown in SEQ ID NO: 13 are preferred. W02009/041643 also shows that the amino acid residues of positions 61, 62, 64, and 65 according to Rabat numbering in the heavy-chain CDR of an anti-human glypican 3 antibody as shown in SEQ ID NO: 14 are preferred sites that allow modification of amino acid residues that can be exposed on the surface while

WO 2017/046994

PCT/JP2016/003616 maintaining the antigen-binding activity. It also shows that the amino acid residues of positions 24 and 27 according to Rabat numbering in the light-chain CDR of an antihuman glypican 3 antibody as shown in SEQ ID NO: 15 are preferred. It also shows that the amino acid residues of positions 61, 62, 64, and 65 according to Rabat numbering in the heavy-chain CDR of an anti-human IL-31 receptor antibody as shown in SEQ ID NO: 16 are preferred sites that allow modification of amino acid residues that can be exposed on the surface while maintaining the antigen-binding activity. W02009/041643 also shows that the amino acid residues of positions 24 and 54 according to Rabat numbering in the light-chain CDR of an anti-human IL-31 receptor antibody as shown in SEQ ID NO: 17 are preferred. Similarly,

W02007/114319 reports that antibodies hA69-PF, hA69-pl8, hA69-N97R, hB26-F123e4, hB26-pl5, and hB26-PF, which were produced by modifying the charge of one or more amino acid residues that can be exposed on the surface, showed changes in pi as demonstrated by isoelectric focusing, and had an equivalent binding activity to Factor IXa or Factor X, which are their antigens, compared with that of the antibodies before modification or alteration. It also reports that when these antibodies were administered to mice, the pi of each antibody showed high correlation with their clearance (CL) in plasma, retention time in plasma, and half-life in plasma (Tl/2). W02007/114319 also demonstrates that amino acid residues of positions 10, 12, 23,

39, 43, 97, and 105 in the variable region are preferred as sites for modification of amino acid residues that can be exposed on the surface.

[0215] In an alternative or further embodiment, for example, using known methods such as X-ray crystallography or a homology model constructed by homology modeling from an antibody constant region (which is preferably a human constant region, more preferably a human Ig-type constant region, and still more preferably a human IgGtype constant region, but is not limited thereto), amino acid residues that can be exposed on the surface of an antibody constant region may be identified to determine the modification sites of at least one amino acid residue for producing an antibody of Disclosure A whose pi has been increased. The modification site of at least one amino acid residue that can be exposed on the surface of the constant region is not limited; however, the site can be preferably selected from the group consisting of: position 196,

253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315,

327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388,

389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and position 443, according to EU numbering, and may be preferably selected from the group consisting of: position 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434, and 443, and may be also preferably selected from the group consisting of:

WO 2017/046994

PCT/JP2016/003616 positions 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413, whose amino acid at each position after modification can be selected from the amino acids described above in terms of the side-chain charge such as Lys (K), Arg (R), Gin (Q), or Asn (N), but is not limited thereto. When the modification site of at least one amino acid residue is selected, for example, from a group comprising the above-described groups, for example, the type of amino acid after modification at each site can be as follows:

254K, 254R, 254Q, or 254N at position 254; 258K, 258R, 258Q, or 258N at position 258;

28IK, 281R, 28IQ, or 28IN at position 281; 282K, 282R, 282Q, or 282N at position 282;

285K, 285R, 285Q, or 285N at position 285; 309K, 309R, 309Q, or 309N at position 309;

IK, 3HR, 31 IQ, or 3UN at position 311; 315K, 315R, 315Q, or 315N at position 315;

327K, 327R, 327Q, or 327N at position 327; 330K, 330R, 330Q, or 330N at position 330;

342K, 342R, 342Q, or 342N at position 342; 343K, 343R, 343Q, or 343N at position 311;

345K, 345R, 345Q, or 345N at position 345; 356K, 356R, 356Q, or 356N at position 356;

358K, 358R, 358Q, or 358N at position 358; 359K, 359R, 359Q, or 359N at position 359;

361K, 361R, 361Q, or 361N at position 361; 362K, 362R, 362Q, or 362N at position 362;

384K, 384R, 384Q, or 384N at position 384; 385K, 385R, 385Q, or 385N at position 385;

386K, 386R, 386Q, or 386N at position 386; 387K, 387R, 387Q, or 387N at position 387;

389K, 389R, 389Q, or 389N at position 389; 399K, 399R, 399Q, or 399N at position 399;

400K, 400R, 400Q, or 400N at position 400; 401K, 401R, 401Q, or 401N at position 401;

402K, 402R, 402Q, or 402N at position 402; 413K, 413R, 413Q, or 413N at position 413;

418K, 418R, 418Q, or 418N at position 418; 419K, 419R, 419Q, or 419N at position 419;

421K, 421R, 421Q, or 421N at position 421; 433K, 433R, 433Q, or 433N at position 433;

WO 2017/046994

PCT/JP2016/003616

434K, 434R, 434Q, or 434N at position 434; and 443K, 443R, 443Q, or 443N at position 443.

[0216] In an alternative embodiment, the modification site of at least one amino acid residue and the type of amino acid after modification may include 345R or 345K, and/or 430R, 430K, 430G, or 435T, according to EU numbering.

[0217] In one embodiment of the antibodies of Disclosure A, the antibody's net pi may be increased by modifying at least one amino acid residue that can be exposed on the surface of the variable region (which may be modified) as described above and at least one amino acid residue that can be exposed on the surface of the constant region (which may be modified) as described above.

[0218] Within the scope of Disclosures A and B described herein, where an antibody of Disclosure A or B is an IgG-type antibody or a molecule derived therefrom, the antibody heavy-chain constant region may contain a constant region of the IgGl type, IgG2 type, IgG3 type, or IgG4 type. In Disclosure A or B, the heavy-chain constant region may be a human heavy-chain constant region, but is not limited thereto. Several allotypes are known to exist for human IgG. Specifically, it has been reported that there are some differences in the amino acid sequence of the human IgG constant region among individuals (Methods Mol. Biol. 882:635-80 (2012); Sequences of proteins of immunological interest, NIH Publication No.91-3242). Examples include human IgGl constant region (SEQ ID NO: 18), human IgG2 constant region (SEQ ID NO: 19), human IgG3 constant region (SEQ ID NO:20), and human IgG4 constant region (SEQ ID NO:21).

[0219] Of these, for example, allotypes called Glml,17 and Glm3 are known for human

IgGl. The allotypes differ in their amino acid sequences: Glml,17 has aspartic acid at position 356 and leucine at position 358 according to EU numbering, while Glm3 has glutamic acid at position 356 and methionine at position 358 according to EU numbering. There is, however, no report suggesting the presence of significant differences in essential antibody functions and properties among the reported allotypes. Thus, those of ordinary skill in the art can readily predict that various assessments were performed using specific allotypes, and the results are not limited to the allotypes used to obtain the Examples and the same effects are expected with any allotypes. Within the scope of Disclosures A and B described herein, when noted as human IgGl, human IgG2, human IgG3, or human IgG4, the allotypes are not limited to specific allotypes and can include all reported allotypes.

[0220] In an alternative or further embodiment of Disclosure A or B, the light-chain constant region of an antibody can include any constant region of the κ chain (IgK) type or λ chain (IgLl, IgL2, IgL3, IgL6, or IgL7) type. A light-chain constant region may be preferably a human light-chain constant region, but is not limited thereto. There are

WO 2017/046994

PCT/JP2016/003616 reports, such as in Sequences of proteins of immunological interest, NIH Publication No.91-3242, on several allotype sequences that result from gene polymorphism for the human κ chain constant region and human λ chain constant region. Such allotypes include, for example, human κ chain constant region (SEQ ID NO:22) and human λ chain constant region (SEQ ID NO:23). There is, however, no report suggesting the presence of significant differences in essential antibody functions and properties among the reported allotypes. Thus, those of ordinary skill in the art can readily understand that when reference is made to specific allotypes within the scope of Disclosures A and B described herein, the same effects are expected with any allotypes (hereinafter, also collectively referred to as native (human) IgG (type) constant region).

[0221] Moreover, since the Fc region of a native IgG antibody constitutes a part of the constant region of the native IgG antibody, when antibodies of Disclosure A or B are, for example, IgG type antibodies or molecules derived therefrom, the antibodies may have an Fc region contained in the constant region of a native IgG (IgGl, IgG2, IgG3, or IgG4 type) (hereinafter, also collectively referred to as a native (human) IgG (type) Fc region). The Fc region of a native IgG can refer to an Fc region consisting of the same amino acid sequence as an Fc region originating from a naturally occurring IgG. Specific examples of the Fc region of a native human IgG can include the Fc regions contained in the human IgGl constant region (SEQ ID NO: 18), human IgG2 constant region (SEQ ID NO: 19), human IgG3 constant region (SEQ ID NO:20), or human IgG4 constant region (SEQ ID NO:21) described above (an Fc region of the IgG class can refer to, for example, from cysteine of position 226 according to EU numbering to the C terminus, or from proline of position 230 according to EU numbering to the C terminus.).

[0222] In one embodiment, antibodies of Disclosures A and B may include variants in which one or more modifications selected from amino acid substitution, addition, deletion, or insertion have been made to the constant region of a native (preferably human) IgG (the heavy-chain constant region and/or the light-chain constant region) or in the Fc region of a native (preferably human) IgG.

[0223] Within the scope of Disclosure A described herein, W02013/081143 reports that for example, ion concentration-dependent antibodies capable of forming multivalent immune complexes with a multimeric antigen (multivalent antigen-antibody complexes) and multispecific ion concentration-dependent antibodies or multiparatopic ion concentration-dependent antibodies that can form multivalent immune complexes (multivalent antigen-antibody complexes) by recognizing two or more epitopes on monomeric antigens can bind more strongly to FcyR, FcRn, complement receptor, due to the avidity (sum of the strength of binding between multiple epitopes and multiple paratopes) via at least two or more multivalent constant regions (that may be modified)

WO 2017/046994

PCT/JP2016/003616 or Fc regions (that may be modified) contained in the antibody molecules, and as a result the antibodies are more rapidly taken up into cells. Thus, when modified to have an increased pi via modification of at least one amino acid residue that can be exposed on the antibody surface, the ion concentration-dependent antibodies described above, which are capable of forming multivalent immune complexes with a multimeric antigen or monomeric antigens, can also be used as antibodies of Disclosure A (ion concentration-dependent antibodies with increased pi). Those of ordinary skill in the art will appreciate that the ion concentration-dependent antibodies with increased pi that can form multivalent immune complexes with a multimeric antigen or monomeric antigens can be more rapidly taken up into cells, as compared to ion concentrationdependent antibodies with increased pi that are incapable of forming multivalent immune complexes. Those of ordinary skill in the art can also understand that in one embodiment, the activity of antibodies of Disclosure A to bind to FcRn and/or FcyR may be increased under a neutral pH condition and in this case, the ion concentrationdependent antibodies with increased pi that can form multivalent immune complexes with a multimeric antigen or monomeric antigens may be even more rapidly taken up into cells.

[0224] In one embodiment, antibodies of Disclosure A may be one-armed antibodies (including all embodiments of the one-armed antibodies described in W02005/063816). Typically, one-armed antibodies are antibodies that lack one of the two Fab regions an ordinary IgG antibody has, and can be produced, without limitations, for example, by the methods described in W02005/063816. Without limitations, in an IgG-type antibody that has a heavy chain whose structure is, for example, VH-CH1-Hinge-CH2-CH3, when one of the Fab regions is cleaved at a site more to the N terminus than the Hinge (for example, VH or CHI), the antibody will be expressed in a form containing an extra sequence, and when one of the Fab regions is cleaved at a site more to the C terminus than the Hinge (for example, CH2), the Fc region will have an incomplete form. Thus, without limitations, it is preferable from the viewpoint of antibody molecule stability that one-armed antibodies are produced by cleavage in the hinge region (Hinge) of one of the two Fab regions of an IgG antibody. It is more preferable that the heavy chain after cleavage is linked to the uncleaved heavy chain via intramolecular disulfide bond. W02005/063816 has reported that such one-armed antibodies have an increased stability as compared to Fab molecules. Antibodies with an increased or decreased pi can also be generated by preparing such one-armed antibodies. Furthermore, when an ion concentrationdependent antigen-binding domain is introduced into antibodies with an increased pi that are one-armed antibodies, the antibody half-life in plasma can be further shortened, cellular uptake of the antibody can be further enhanced, antigen elimination

WO 2017/046994

PCT/JP2016/003616 from plasma can be further enhanced, or the antibody's affinity for the extracellular matrix can be further increased, as compared to antibodies with increased pi that do not have an ion concentration-dependent antigen-binding domain.

[0225] Without being bound by a particular theory, an embodiment where the cellular uptake-accelerating effect of one-armed antibodies is expected can be envisaged to be, but is not limited to, a case in which the pi of a soluble antigen is lower than that of the antibodies. The net pi of a complex consisting of antibodies and antigens can be calculated by known methods by considering that the complex is a single molecule. In this case, the lower the pi of the soluble antigen is, the lower the net pi of the complex is; and the greater the pi of the soluble antigen is, the greater the net pi of the complex is. When an ordinary-type IgG antibody molecule (having two Fabs) is bound to a single low-pi soluble antigen versus to two low-pi soluble antigens, the net pi of the complex is lower in the latter case. When such an ordinary-type antibody is converted into a one-armed antibody, only one antigen can bind to a single molecule of the antibody; reduction of the pi of the complex resulting from the binding of the second antigen can thereby be suppressed. In other words, it is believed that when the pi of the soluble antigen is lower than that of the antibody, the conversion into a one-armed antibody allows the pi of the complex to increase as compared to an ordinary antibody, and thereby accelerates uptake into cells.

[0226] Furthermore, without limitations, when the Fab of an ordinary IgG-type antibody molecule (having two Fabs) has a lower pi than that of the Fc, conversion into a onearmed antibody increases the net pi of the complex consisting of the one-armed antibody and antigen. Moreover, when such conversion into a one-armed antibody is performed, it is preferable from the viewpoint of the stability of the one-armed antibody that one of the Fabs is cleaved in the Hinge region located at the junction between Fab and Fc. In this case, the pi can be expected to be effectively increased by selecting a site which would increase the pi of the one-armed antibody to the desired extent.

[0227] Thus, those of ordinary skill in the art can understand that without exclusively (or substantially) depending on the antibody amino acid sequence itself and the type of the soluble antigen, the pi of an antibody can be increased and the accompanying cellular uptake of the antigen may be accelerated by converting the antibody into a one-armed antibody by calculating the theoretical pi of the antibody (theoretical pi of Fc and theoretical pi of Fab) and the theoretical pi of the soluble antigen and predicting the relationship on the difference of their theoretical pi values.

[0228] In one embodiment, antibodies of Disclosure A or B may be multispecific antibodies, and the multispecific antibody may be, but is not limited to, a bispecific antibody. The multispecific antibody may be a multispecific antibody that contains a first polypeptide

WO 2017/046994

PCT/JP2016/003616 and a second polypeptide. Here, a multispecific antibody that contains a first polypeptide and a second polypeptide refers to an antibody that binds to at least two or more types of different antigens or at least two or more types of epitopes in a same antigen. The first polypeptide and second polypeptide preferably may contain a heavychain variable region, and more preferably the variable region contains CDR(s) and/or FR(s). In another embodiment, the first polypeptide and second polypeptide may preferably each contain a heavy-chain constant region. In still another embodiment, the multispecific antibody may contain a third polypeptide and a fourth polypeptide, each containing a light-chain variable region and preferably also a light-chain constant region. In this case, the first to the fourth polypeptides may assemble together to form a multispecific antibody.

[0229] In one embodiment, where antibodies of Disclosure A are multispecific antibodies and the multispecific antibodies contain a heavy-chain constant region, to reduce their pi, for example, the following sequences may be used: IgG2 or IgG4 sequence at position 137; IgGl, IgG2, or IgG4 sequence at position 196; IgG2 or IgG4 sequence at position 203; IgG2 sequence at position 214; IgGl, IgG3, or IgG4 sequence at position 217; IgGl, IgG3, or IgG4 sequence at position 233; IgG4 sequence at position 268; IgG2, IgG3, or IgG4 sequence at position 274; IgGl, IgG2, or IgG4 sequence at position 276; IgG4 sequence at position 355; IgG3 sequence at position 392; IgG4 sequence at position 419; or IgGl, IgG2, or IgG4 sequence at position 435.

Meanwhile, to increase their pi, for example, the following sequences may be used: IgGl or IgG3 sequence at position 137; IgG3 sequence at position 196; IgGl or IgG3 sequence at position 203; IgGl, IgG3, or IgG4 sequence at position 214; IgG2 sequence at position 217; IgG2 sequence at position 233; IgGl, IgG2, or IgG3 sequence at position 268; IgGl sequence at position 274; IgG3 sequence at position 276; IgGl, IgG2, or IgG3 sequence at position 355; IgGl, IgG2, or IgG4 sequence at position 392; IgGl, IgG2, or IgG3 sequence at position 419; or IgG3 sequence at position 435.

[0230] In one embodiment, where antibodies of Disclosure A have two heavy-chain constant regions, the pis of the two heavy chain constant regions may be the same or different from each other. Such heavy-chain constant regions may be IgGl, IgG2, IgG3 and IgG4 heavy-chain constant regions which originally have different pis. Alternatively, it is possible to introduce a pi difference between the two heavy-chain constant regions. Modification sites of at least one amino acid residue for introducing such a pi difference in the constant region may be the position(s) described above or position(s) selected, for example, from the group consisting of position 137, position 196, position 203, position 214, position 217, position 233, position 268, position 274, position 276, position 297, position 355, position 392, position 419, and position 435, according to

WO 2017/046994

PCT/JP2016/003616

EU numbering in the heavy-chain constant region as described in W02009/041643. Alternatively, the amino acid residue of position 297 which is a glycosylation site may be modified to remove the sugar chain, since the removal of a sugar chain from the heavy-chain constant region results in a pi difference.

[0231] In one embodiment, antibodies of Disclosure A or B may be polyclonal antibodies or monoclonal antibodies, and mammalian-derived monoclonal antibodies are preferred. Monoclonal antibodies include those produced by hybridomas or those produced by host cells transformed by genetic engineering techniques with expression vectors carrying antibody genes. The antibodies of Disclosure A or B may be, for example, antibodies such as chimeric antibodies, humanized antibodies, or antibodies generated by affinity maturation, or molecules derived therefrom.

[0232] In one embodiment, antibodies of Disclosure A or B may be derived, without limitations, from any animal species (for example, human; or nonhuman animals such as mouse, rat, hamster, rabbit, monkey, cynomolgus monkey, Rhesus monkey, hamadryas baboon, chimpanzee, goat, sheep, dog, bovine, or camel), or any birds; and the antibodies are preferably derived from human, monkey, or mouse.

[0233] In one embodiment, antibodies of Disclosure A or B may be Ig-type antibodies, and may be preferably IgG-type antibodies.

[0234] Within the scope of Disclosures A and B described herein, the Fc receptor (also referred to as FcR) refers to a receptor protein that can bind to the Fc region of an immunoglobulin (antibody) or a molecule derived therefrom, or an Fc region variant. For example, Fc receptors for IgG, IgA, IgE, and IgM are known as FcyR, FcaR,

FceR, and FcpR, respectively, within the scope of Disclosure A described herein. Fc receptors may also be, for example, FcRn (also referred to as neonatal Fc receptor), within the scope of Disclosures A and B described herein.

[0235] Within the scope of Disclosure A described herein, FcyR may refer to a receptor protein that can bind to the Fc region of an IgGl, IgG2, IgG3, or IgG4 antibody or a molecule derived therefrom, or an Fc region variant, and may include any one or more of, or all members of the family of proteins substantially encoded by the FcyR gene. In human, the family includes, but is not limited to, FcyRI (CD64) including isoforms FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32) including isoforms FcyRIIa (including allotypes H131 (type H) and R131 (type R)), FcyRIIb (including FcyRIIb-1 and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD 16) including isoforms FcyRIIIa (including allotypes VI58 and FI58) and FcyRIIIb (including allotypes FcyRIIIb-NA 1 and FcYRIIIb-NA2), as well as all unidentified human FcyRs and FcyR isoforms and allotypes. Furthermore, FcyRIIbl and FcYRIIb2 have been reported as splicing variants of human FcyRIIb (hFcYRIIb). There is also a report on a splicing variant called FcYRIIb3 (Brooks et al., J. Exp. Med, 170: 1369-1385 (1989)). In addition to those

WO 2017/046994

PCT/JP2016/003616 described above, hFcyRIIb includes all splicing variants such as those registered in NCBI under NP_001002273.1, NP_001002274.1, NP_001002275.1,

NP_001177757.1, and NP_003992.3. hFcyRIIb also includes all genetic polymorphisms already reported, for example, FcyRIIb (Fi et al., Arthritis Rheum. 48:3242-3252 (2003), Kono et al., Hum. Mol. Genet. 14:2881-2892 (2005); Kyogoku et al., Arthritis Rheum. 46(5):1242-1254 (2002)), as well as all genetic polymorphisms that will be reported in future.

[0236] FcyR may be derived from any organism, and may include those derived from humans, mice, rats, rabbits, or monkeys, without being limited thereto. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16) and FcyRIII-2 (CD 16-2), as well as all unidentified mouse FcyRs, and FcyR isoforms and allotypes. Such preferred FcyR includes, for example, human FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16), or FcyRIIIB (CD16). Since FcyR is present as a membrane form in vivo, it may be used in experimental systems after being artificially converted into an appropriate soluble form.

[0237] For example, as shown in W02014/163101, the polynucleotide sequence and amino acid sequence of FcyRI may be the sequences shown in NM_000566.3 and NP_000557.1, respectively; the polynucleotide sequence and amino acid sequence of FcyRIIA may be the sequences shown in BC020823.1 and AAH20823.1, respectively;

the polynucleotide sequence and amino acid sequence of FcyRIIB may be the sequences shown in BC146678.1 and AAI46679.1, respectively; the polynucleotide sequence and amino acid sequence of FcyRIIIA may be the sequences shown in BC033678.1 and AAH33678.1, respectively; the polynucleotide sequence and amino acid sequence of FcyRIIIB may be the sequences shown in BC128562.1 and AAI28563.1, respectively (RefSeq accession numbers are shown).

[0238] FcyRIIa has two genetic polymorphisms, in which the amino acid at position 131 of FcyRIIa is replaced with histidine (type H) or arginine (type R) (J. Exp. Med. 172:19-25, 1990).

[0239] In FcyRI (CD64) which includes FcyRIa, FcyRIb, and FcyRIc, and FcyRIII (CD 16) which includes FcyRIIIa (including allotypes V158 and F158), the a chain that binds to the Fc region of IgG is associated with a common γ chain having ITAM which transmits activation signals inside cells. FcyRIIIb (including allotypes FcyRIIIb-NA 1 and FcYRIIIb-NA2) is a GPI anchor protein. Meanwhile, the cytoplasmic domain of FcyRII (CD32) which includes the FcyRIIa (including allotypes H131 and R131) and FcyRIIc isoforms contains ITAM. These receptors are expressed on many immune cells such as macrophages, mast cells, and antigen-presenting cells. The activation signals transduced upon binding of these receptors to the Fc region of IgG promote the phagocytotic ability of macrophages, production of inflammatory cytokines, de76

WO 2017/046994

PCT/JP2016/003616 granulation of mast cells, and the increased function of antigen-presenting cells. An FcyR that has the ability to transduce activation signals as described above is also referred to as an activating FcyR within the scope of Disclosures A and B described here.

[0240] Meanwhile, the cytoplasmic domain of FcyRIIb (including FcyRIIb-1 and FcyRIIb2) contains ITIM which transmits inhibitory signals. In B cells, the crosslinking between FcyRIIb and B cell receptor (BCR) suppresses the activation signals from BCR, which results in suppression of antibody production by BCR. In macrophages, the crosslinking of FcyRIII and FcyRIIb suppresses the phagocytic ability and the ability to produce inflammatory cytokines. An FcyR that has the ability to transduce inhibitory signals as described above is also referred to as an inhibitory Fey Receptor within the scope of Disclosures A and B described herein.

[0241] Within the scope of Disclosure A described herein, whether the binding activity of an antibody or Fc region (variant) toward various FcyRs has been increased, (substantially) maintained, or reduced as compared to the antibody or Fc region (variant) before modification can be assessed by methods known to those of ordinary skill in the art. Such methods are not particularly limited and those described in the present Examples may be used, and for example, surface plasmon resonance (SPR) phenomenon-based BIACORE (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010) may be used. Alternatively, for example, ELISA and fluorescence activated cell sorting (FACS) as well as ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) may be used. In these assays, the extracellular domain of human FcyR may be used as a soluble antigen (for example, WO2013/047752).

[0242] For the pH condition for measuring the binding activity between an FcyR-binding domain contained in an antibody or Fc region (variant) and FcyR, an acidic or neutral pH condition may suitably be used. For the temperature used in the measurement conditions, the binding activity (binding affinity) between an FcyR-binding domain and FcyR may be assessed, for example, at any temperature between 10°C to 50°C. A preferred temperature for determining the binding activity (binding affinity) of a human FcyR-binding domain to FcyR is, for example, 15°C to 40°C. More preferably, to determine the binding activity (binding affinity) between an FcyR-binding domain and FcyR, any temperature from 20°C to 35°C, for example, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C may be used. A nonlimiting example of such temperature is 25°C.

[0243] In one embodiment, where an antibody of Disclosure A or B has a constant region (that may be modified), the constant region may have an Fc region or an Fc region variant (preferably, a human Fc region or a human Fc region variant), and preferably has an FcyR-binding domain within the scope of Disclosure A and an FcRn-binding

WO 2017/046994

PCT/JP2016/003616 domain within the scope of Disclosures A and B described herein.

[0244] In one embodiment, where an antibody of Disclosure A has FcyR-binding activity, it may have an FcyR-binding domain, preferably a human FcyR-binding domain. The FcyR-binding domain is not particularly limited as long as the antibody has binding activity to or affinity for FcyR at acidic pH and/or neutral pH, and it may be a domain that has an activity to directly or indirectly bind FcyR.

[0245] In one embodiment, where an antibody of Disclosure A has FcyR-binding activity, it is preferable that the FcyR-binding activity of the antibody under a neutral pH condition is increased as compared to that of a reference antibody which contains a native IgG constant region. From the perspective of comparing the FcyR-binding activity between the two, it is preferable, without limitations, that the antibody of Disclosure A and the reference antibody which contains a native IgG constant region have identical amino acid sequences in regions (for example, the variable region) other than, preferably, the constant region of the antibody of Disclosure A which has been modified at one or more amino acid residues.

[0246] In one embodiment, where an antibody of Disclosure A has an FcyR-binding activity or an increased FcyR-binding activity under a neutral pH condition (e.g., pH 7.4), without being bound by a theory, the antibody is thought to possess the following properties in combination: the property of being shuttled between plasma and cellular endosome and repeatedly binding to multiple antigens as a single antibody molecule by having an ion concentration-dependent antigen-binding domain; the property of being rapidly taken up into cells by having an increased pi and increased positive charge in the overall antibody; and the property of being rapidly taken up into cells by having an increased FcyR-binding activity under a neutral pH condition. As a result, the antibody half-life in plasma can be further shortened, or the binding activity of the antibody toward the extracellular matrix can be further increased, or antigen elimination from plasma can be further promoted; thus the antibody of Disclosure A is beneficial. Those of ordinary skill in the art can routinely determine an optimal pi value for the antibody to take advantage of these properties.

[0247] In one embodiment, an FcyR-binding domain whose FcyR-binding activity is higher than that of the Fc region or constant region of a native human IgG in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain can be produced by modifying amino acid residues in the Fc region or constant region of a native human IgG (see WO2013/047752). Furthermore, a domain of any structure that binds to FcyR can be used as an FcyR-binding domain. In this case, the FcyR-binding domain can be produced without the need to introduce an amino acid modification, and alternatively, its affinity for FcyR may be increased by introducing an additional modification. Such FcyR-binding domains can include Fab fragment an78

WO 2017/046994

PCT/JP2016/003616 tibodies that bind to FcyRIIIa, camel-derived single domain antibodies, and single chain Fv antibodies described in Schlapschly et al.(Protein Eng. Des. Sel. 22 (3):175-188 (2009), Behar et al. (Protein Eng. Des. Sel. 21(1):1-10 (2008)), and Kipriyanov et al., J Immunol. 169(1):137-144 (2002), and the FcyRI-binding cyclic peptide described in Bonetto et al., FASEB J. 23(2):575-585 (2009). Whether the FcyR-binding activity of an FcyR-binding domain is higher than that of the Fc region or constant region of a native human IgG in which the sugar chain linked at position 297 according to EU numbering is a fucose-containing sugar chain can be appropriately assessed using the methods described above.

[0248] In one embodiment of Disclosure A, the starting FcyR-binding domain preferably includes, for example, (human) IgG Fc region or (human) IgG constant region. As long as a variant of the starting Fc region or the starting constant region can bind to human FcyR in a neutral pH range, any Fc region or constant region can be used as the starting Fc region or starting constant region. An Fc region or constant region obtained by further modifying a starting Fc region or starting constant region whose amino acid residue(s) has been already modified from an Fc region or constant region can also be appropriately used as the Fc region or constant region of Disclosure A. A starting Fc region or starting constant region may refer to the polypeptide itself, a composition containing the starting Fc region or starting constant region, or an amino acid sequence encoding the starting Fc region or starting constant region. The starting Fc region or starting constant region may include known Fc regions or known constant regions produced by recombination technologies. The origin of the starting Fc region or starting constant region is not limited, and it can be obtained from any organism of nonhuman animals or from a human. Furthermore, the starting FcyR-binding domain can be obtained from cynomolgus monkeys, marmosets, Rhesus monkeys, chimpanzees, or humans. The starting Fc region or starting constant region can be preferably obtained from human IgGl; however, it is not limited to a particular IgG class. This means that the Fc region of human IgGl, IgG2, IgG3, or IgG4 can be used as an appropriate starting FcyR-binding domain, and it also means that within the scope of Disclosure A described herein, an Fc region or constant region of an IgG class or subclass derived from any organism can be preferably used as the starting Fc region or starting constant region. Examples of a native IgG variant or modified form are described in publicly known literature such as Strohl, Curr. Opin. Biotechnol. 20(6):685-691 (2009); Presta, Curr. Opin. Immunol. 20(4):460-470 (2008); Davis et al., Protein Eng. Des. Sel. 23(4):195-202 (2010); W02009/086320, W02008/092117; W02007/041635; and W02006/105338, but not limited thereto.

[0249] In one embodiment, amino acid residues of the starting FcyR-binding domain, starting Fc region, or starting constant region may contain, for example, one or more

WO 2017/046994

PCT/JP2016/003616 mutations: for example, substitutions with amino acid residues that are different from those in the starting Fc region or starting constant region; insertions of one or more amino acid residues into the amino acid residues in the starting Fc region or starting constant region; or deletions of one or more amino acid residues from those of the starting Fc region or starting constant region. The amino acid sequences of Fc regions or constant regions after modifications are preferably amino acid sequences containing at least a portion of an Fc region or constant region that may not occur naturally. Such variants necessarily have a sequence identity or similarity of less than 100% to the starting Fc regions or starting constant regions. For example, the variants have an amino acid sequence identity or similarity of about 75% to less than 100%, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, still more preferably about 90% to less than 100%, and yet more preferably about 95% to less than 100% to the amino acid sequence of the starting Fc region or starting constant region. In a non-limiting example, at least one amino acid is different between a modified Fc region or constant region of Disclosure A and the starting Fc region or starting constant region.

[0250] In one embodiment, an Fc region or constant region that has FcyR-binding activity in an acidic pH range and/or in a neutral pH range, which may be contained in an antibody of Disclosure A, may be obtained by any method. Specifically, a variant of Fc region or constant region that has FcyR-binding activity in a neutral pH range may be obtained by modifying amino acids of a human IgG antibody which can be used as the starting Fc region or starting constant region. IgG antibody Fc regions or IgG antibody constant regions suitable for modification can include, for example, the Fc regions or constant regions of human IgG (IgGl, IgG2, IgG3, or IgG4, or variants thereof), and mutants spontaneously generated therefrom. For the Fc regions or constant regions of human IgGl, human IgG2, human IgG3, or human IgG4 antibodies, a number of allotype sequences due to genetic polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242, and any of them may be used in Disclosure A. In particular, for the human IgGl sequence, the amino acid sequence of positions 356 to 358 according to EU numbering may be DEF or EEM.

[0251] In a further embodiment within the scope of Disclosure A, the modification into other amino acids is not limited as long as the variants have an FcyR-binding activity in a neutral pH range. Amino acid position(s) of such modification are reported, for example, in: W02007/024249, W02007/021841, W02006/031370, W02000/042072, W02004/029207, W02004/099249, W02006/105338, W02007/041635, W02008/092117, W02005/070963, W02006/020114, W02006/116260, W02006/023403, WO2013/047752, W02006/019447, WO2012/115241,

WO 2017/046994 PCT/JP2016/003616

WO2013/125667, WO2014/030728, W02014/163101, WO2013/118858, and W02014/030750.

[0252] Sites of amino acid modification in the constant region or Fc region to increase the FcyR-binding activity in a neutral pH range can include, for example, one or more positions selected from the group consisting of position: 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246,

247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269,

270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288,

290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311,

313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,

334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428,

429, 434, 436, and 440, according to EU numbering in the Fc region or constant region of a human IgG antibody, as described in WO2013/047752. Modification of such amino acid residue may increase the FcyR binding of the Fc region or constant region of an IgG antibody under a neutral pH condition. WO2013/047752 describes, as preferred modifications in an IgG-type constant region or Fc region, for example, mod ification of one or more amino acid residues selected from the group consisting of: the amino acid at position 221 to either Fys or Tyr; the amino acid at position 222 to any one of Phe, Trp, Glu, and Tyr; the amino acid at position 223 to any one of Phe, Trp, Glu, and Fys; the amino acid at position 224 to any one of Phe, Trp, Glu, and Tyr; the amino acid at position 225 to any one of Glu, Fys, and Trp ; the amino acid at position 227 to any one of Glu, Gly, Fys, and Tyr; the amino acid at position 228 to any one of Glu, Gly, Fys, and Tyr; the amino acid at position 230 to any one of Ala, Glu, Gly, and Tyr; the amino acid at position 231 to any one of Glu, Gly, Fys, Pro, and Tyr; the amino acid at position 232 to any one of Glu, Gly, Fys, and Tyr; the amino acid at position 233 to any one of Ala, Asp, Phe, Gly, His, lie, Fys, Feu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 234 to any one of Ala, Asp, Glu, Phe, Gly, His, lie, Fys, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 235 to any one of Ala, Asp, Glu, Phe, Gly, His, He, Fys, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 236 to any one of Ala, Asp, Glu, Phe, His, He, Fys, Feu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 237 to any one of Asp, Glu, Phe, His, He, Fys, Feu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 238 to any one of Asp, Glu, Phe, Gly, His, He, Fys, Feu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 239 to any one of Asp, Glu, Phe, Gly, His, He, Fys, Feu, Met, Asn, Pro, Gin, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 240 to any one of Ala, He, Met, and Thr; the amino acid at position 241 to any one of Asp, Glu, Feu, Arg, Trp, and Tyr; the amino acid at position 243 to any one of Glu, Feu,

WO 2017/046994

PCT/JP2016/003616

Gin, Arg, Trp, and Tyr; the amino acid at position 244 to His; the amino acid at position 245 to Ala; the amino acid at position 246 to any one of Asp, Glu, His, and Tyr; the amino acid at position 247 to any one of Ala, Phe, Gly, His, lie, Leu, Met,

Thr, Val, and Tyr; the amino acid at position 249 to any one of Glu, His, Gin, and Tyr; the amino acid at position 250 to either Glu or Gin; the amino acid at position 251 to Phe; the amino acid at position 254 to any one of Phe, Met, and Tyr; the amino acid at position 255 to any one of Glu, Leu, and Tyr; the amino acid at position 256 to any one of Ala, Met, and Pro; the amino acid at position 258 to any one of Asp, Glu, His, Ser, and Tyr; the amino acid at position 260 to any one of Asp, Glu, His, and Tyr; the amino acid at position 262 to any one of Ala, Glu, Phe, He, and Thr; the amino acid at position 263 to any one of Ala, He, Met, and Thr; the amino acid at position 264 to any one of Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 265 to any one of Ala, Leu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 266 to any one of Ala, He, Met, and Thr; the amino acid at position 267 to any one of Asp, Glu, Phe, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 268 to any one of Asp, Glu, Phe, Gly, He, Lys, Leu, Met, Pro, Gin, Arg, Thr, Val, and Trp; the amino acid at position 269 to any one of Phe, Gly,

His, He, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 270 to any one of Glu, Phe, Gly, His, He, Leu, Met, Pro, Gin, Arg, Ser, Thr, Trp, and Tyr; the amino acid at position 271 to any one of Ala, Asp, Glu, Phe, Gly,

His, He, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 272 to any one of Asp, Phe, Gly, His, He, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 273 to either Phe or He; the amino acid at position 274 to any one of Asp, Glu, Phe, Gly, His, He, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 275 to either Leu or Trp; the amino acid at position 276 to any one of Asp, Glu, Phe, Gly, His, He, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 278 to any one of Asp, Glu, Gly,

His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, and Trp; the amino acid at position 279 to Ala; the amino acid at position 280 to any one of Ala, Gly, His, Lys, Leu, Pro, Gin, Trp, and Tyr; the amino acid at position 281 to any one of Asp, Lys,

Pro, and Tyr; the amino acid at position 282 to any one of Glu, Gly, Lys, Pro, and Tyr; the amino acid at position 283 to any one of Ala, Gly, His, He, Lys, Leu, Met, Pro,

Arg, and Tyr; the amino acid at position 284 to any one of Asp, Glu, Leu, Asn, Thr, and Tyr; the amino acid at position 285 to any one of Asp, Glu, Lys, Gin, Trp, and Tyr; the amino acid at position 286 to any one of Glu, Gly, Pro, and Tyr; the amino acid at position 288 to any one of Asn, Asp, Glu, and Tyr; the amino acid at position 290 to any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; the amino acid at position

WO 2017/046994

PCT/JP2016/003616

291 to any one of Asp, Glu, Gly, His, He, Gin, and Thr; the amino acid at position 292 to any one of Ala, Asp, Glu, Pro, Thr, and Tyr; the amino acid at position 293 to any one of Phe, Gly, His, He, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 294 to any one of Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 295 to any one of Asp,

Glu, Phe, Gly, His, He, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 296 to any one of Ala, Asp, Glu, Gly, His, lie, Lys, Leu, Met, Asn,

Gin, Arg, Ser, Thr, and Val; the amino acid at position 297 to any one of Asp, Glu,

Phe, Gly, His, He, Lys, Leu, Met, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 298 to any one of Ala, Asp, Glu, Phe, His, He, Lys, Met, Asn, Gin,

Arg, Thr, Val, Trp, and Tyr; the amino acid at position 299 to any one of Ala, Asp,

Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 300 to any one of Ala, Asp, Glu, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, and Trp; the amino acid at position 301 to any one of Asp, Glu, His, and Tyr; the amino acid at position 302 to He; the amino acid at position 303 to any one of Asp, Gly, and Tyr; the amino acid at position 304 to any one of Asp, His, Leu, Asn, and Thr; the amino acid at position 305 to any one of Glu, He, Thr, and Tyr; the amino acid at position 311 to any one of Ala, Asp, Asn, Thr, Val, and Tyr; the amino acid at position 313 to Phe; the amino acid at position 315 to Leu; the amino acid at position 317 to either Glu or Gin; the amino acid at position 318 to any one of His, Leu, Asn, Pro, Gin, Arg, Thr, Val, and Tyr; the amino acid at position 320 to any one of Asp, Phe, Gly, His, He, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 322 to any one of Ala, Asp, Phe, Gly, His, He, Pro, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 323 to He; the amino acid at position 324 to any one of Asp, Phe, Gly, His, He, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 325 to any one of Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 326 to any one of Ala, Asp, Glu, Gly, He, Leu, Met, Asn, Pro, Gin, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 327 to any one of Ala, Asp, Glu, Phe, Gly, His, He, Lys,

Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 328 to any one of Ala, Asp, Glu, Phe, Gly, His, He, Lys, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 329 to any one of Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 330 to any one of Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 331 to any one of Asp, Phe, His, He, Leu, Met, Gin, Arg, Thr, Val, Trp, and Tyr; the amino acid at position 332 to any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr; the amino acid at position 333 to any one of Ala, Asp, Glu, Phe, Gly, His, He,

WO 2017/046994

PCT/JP2016/003616

Leu, Met, Pro, Ser, Thr, Val, and Tyr; the amino acid at position 334 to any one of Ala, Glu, Phe, He, Leu, Pro, and Thr; the amino acid at position 335 to any one of Asp, Phe, Gly, His, lie, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acid at position 336 to any one of Glu, Lys, and Tyr; the amino acid at position 337 to any one of Glu, His, and Asn; the amino acid at position 339 to any one of Asp, Phe, Gly, lie, Lys, Met, Asn, Gin, Arg, Ser, and Thr; the amino acid at position 376 to either Ala or Val; the amino acid at position 377 to either Gly or

Lys; the amino acid at position 378 to Asp; the amino acid at position 379 to Asn; the amino acid at position 380 to any one of Ala, Asn, and Ser; the amino acid at position 382 to either Ala or He; the amino acid at position 385 to Glu; the amino acid at position 392 to Thr; the amino acid at position 396 to Leu; the amino acid at position 421 to Lys; the amino acid at position 427 to Asn; the amino acid at position 428 to either Phe or Leu; the amino acid at position 429 to Met; the amino acid at position 434 to Trp; the amino acid at position 436 to He; and the amino acid at position 440 to any one of Gly, His, He, Leu, and Tyr, according to EU numbering. The number of amino acids to be modified is not particularly limited, and it is possible to modify an amino acid at only one position or amino acids at two or more positions. Combinations of amino acid modifications at two or more positions are shown in Table 5 of WO2013/047752. Modification of these amino acid residues can also be appropriately introduced into the antibodies of Disclosure A.

[0253] In one embodiment, the binding activity of (the FcyR-binding domain of) the antibody of Disclosure A toward (human) FcyR(s), such as any one or more of FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, and FcyRIIIb, may be higher than that of (the Fc region or constant region of) a native IgG or that of a reference antibody containing the starting Fc region or starting constant region. For example, the FcyR-binding activity of (the FcyR-binding domain of) an antibody of Disclosure A may be 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 105% or more, preferably 110% or more, 115% or more, 120% or more, 125% or more, particularly preferably 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 165% or more, 170% or more, 175% or more, 180% or more, 185% or more, 190% or more, or 195% or more as compared to the FcyR-binding activity of the reference antibody, or 2-fold or more, 2.5-fold or more, 3-fold or more, 3.5-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 7.5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more, 90-fold or more, or 100-fold or more greater than the FcyRbinding activity of the reference antibody.

[0254] In a further embodiment, the level of increase in the binding activity to an inhibitory

WO 2017/046994

PCT/JP2016/003616

FcyR (FcyRIIb-1 and/or FcyRIIb-2) (in a neutral pH range) may be greater than the level of increase in the binding activity to an activating FcyR (FcyRIa: FcyRIb;

FcyRIc; FcyRIIIa including allotype VI58; FcyRIIIa including allotype FI58;

FcyRIIIb including allotype FcyRIIIb-NA 1; FcyRIIIb including allotype FcyRIIIbNA2; FcyRIIa including allotype H131; or FcyRIIa including allotype R131).

[0255] In one embodiment, an antibody of Disclosure A may have binding activity to FcyRIIb (including FcyRIIb-1 and FcYRIIb-2).

[0256] In one embodiment, preferred FcyR-binding domains of Disclosure A also include, for example, FcyR-binding domains whose binding activity to a specific FcyR is greater than the binding activity to other FcyR (FcyR-binding domains having a selective FcyR-binding activity). Where an antibody (or the Fc region as the FcyRbinding domain) is used, a single antibody molecule can bind only to a single FcyR molecule. Thus, a single antibody molecule in a state bound to an inhibitory FcyR cannot bind to other activating FcyRs, and a single antibody molecule in a state bound to an activating FcyR cannot bind to other activating FcyRs or inhibitory FcyRs.

[0257] As described above, an activating FcyR preferably includes, for example, FcyRI (CD64) such as FcyRIa, FcyRIb, or FcyRIc; and FcyRIII (CD16) such as FcyRIIIa (such as allotype V158 or F158) or FcyRIIIb (such as allotype FcyRIIIb-NA 1 or FcYRIIIb-NA2). Meanwhile, an inhibitory FcyR preferably includes, for example, FcyRIIb (such as FcyRIIb-1 or FcYRIIb-2).

[0258] In one embodiment, FcyR-binding domains that have a greater binding activity to inhibitory FcyR than to activating FcyR can be used as the selective FcyR-binding domain contained in an antibody of Disclosure A. Such selective FcyR-binding domains can include, for example, FcyR-binding domains that have a greater binding activity to FcyRIIb (such as FcyRIIb-1 and/or FcYRIIb-2) than to any one or more of activating FcyR selected from the group consisting of: FcyRI (CD64) such as FcyRIa, FcyRIb, or FcyRIc; FcyRIII (CD16) such as FcyRIIIa (such as allotype V158 or F158) or FcyRIIIb (such as FcyRIIIb-NA 1 or FcYRIIIb-NA2); FcyRII (CD32) such as FcyRIIa (including allotype H131 orR131); and FeyRlie.

[0259] Furthermore, whether an FcyR-binding domain has a selective binding activity can be assessed by comparing the binding activity to each FcyR determined by the methods described above, for example, by comparing the value (ratio) obtained by dividing the KD value for activating FcyR by the KD value for inhibitory FcyR, more specifically by comparing the FcyR selectivity index shown in Equation 1 below:

[Equation 1] FcyR selectivity index = KD value for activating FcyR/KD value for inhibitory FcyR [0260] In Equation 1, the KD value for activating FcyR refers to the KD value for one or more of: FcyRIa; FcyRIb; FcyRIc; FcyRIIIa including allotype V158 and/or F158;

WO 2017/046994

PCT/JP2016/003616

FcyRIIIb including FcyRIIIb-NA 1 and/or FcyRIIIb-NAl; FcyRIIa including allotype H131 and/or R131; and FcyRIIc; and the KD value for inhibitory FcyR refers to the KD value for FcyRIIb-l and/or FcYRIIb-2. The activating FcyR and inhibitory FcyR for use in determining the KD values may be selected in any combination. For example, it is possible to use a value (ratio) determined by dividing the KD value for FcyRIIa including allotype H131 by the KD value for FcyRIIb-l and/or FcYRIIb-2, without limitations thereto.

[0261] The FcyR selectivity index can be, for example: 1.2 or greater, 1.3 or greater, 1.4 or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 or greater, 1.9 or greater, 2 or greater, 3 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 35 or greater, 40 or greater, 45 or greater, 50 or greater, 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, 95 or greater, 100 or greater, 110 or greater, 120 or greater, 130 or greater, 140 or greater, 150 or greater,

160 or greater, 170 or greater, 180 or greater, 190 or greater, 200 or greater, 210 or greater, 220 or greater, 230 or greater, 240 or greater, 250 or greater, 260 or greater, 270 or greater, 280 or greater, 290 or greater, 300 or greater, 310 or greater, 320 or greater, 330 or greater, 340 or greater, 350 or greater, 360 or greater, 370 or greater, 380 or greater, 390 or greater, 400 or greater, 410 or greater, 420 or greater, 430 or greater, 440 or greater, 450 or greater, 460 or greater, 470 or greater, 480 or greater, 490 or greater, 500 or greater, 520 or greater, 540 or greater, 560 or greater, 580 or greater, 600 or greater, 620 or greater, 640 or greater, 660 or greater, 680 or greater, 700 or greater, 720 or greater, 740 or greater, 760 or greater, 780 or greater, 800 or greater, 820 or greater, 840 or greater, 860 or greater, 880 or greater, 900 or greater, 920 or greater, 940 or greater, 960 or greater, 980 or greater, 1000 or greater, 1500 or greater, 2000 or greater, 2500 or greater, 3000 or greater, 3500 or greater, 4000 or greater, 4500 or greater, 5000 or greater, 5500 or greater, 6000 or greater, 6500 or greater, 7000 or greater, 7500 or greater, 8000 or greater, 8500 or greater, 9000 or greater, 9500 or greater, 10000 or greater, or 100000 or greater; but it is not limited thereto.

[0262] In one embodiment, (an antibody containing) an Fc region variant or constant region variant in which the amino acid at position 238 or 328, according to EU numbering of human IgG (IgGl, IgG2, IgG3, or IgG4) is Asp or Glu, respectively, can be preferably used as antibodies of Disclosure A containing an Fc region variant or constant region variant, since as specifically described in WO2013/125667, WO2012/115241, and WO2013/047752, it has a greater binding activity to FcYRIIb-1 and/or FcYRIIb-2 than to FcyRIa, FcyRIb, FcyRIc, FcyRIIIa including allotype V158, FcyRIIIa including allotype FI58, FcYRIIIb including allotype FcYRIIIb-NAl, FcyRIIIb including allotype

WO 2017/046994

PCT/JP2016/003616

FcYRIIIb-NA2, FcyRIIa including allotype H131, FcyRIIa including allotype R131, and/or FcyRIIc. In such an embodiment, the antibodies of Disclosure A have binding activity to all activating FcyRs (herein, which are selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb, FcyRIIa) and FcyRIIb, and their FcyRIIb-binding activity is maintained or increased, and/or their binding activity to all activating FcyRs is reduced, as compared to the reference antibody that contains a native IgG constant region or a native IgG Fc region.

[0263] In one embodiment for the antibodies of Disclosure A containing an Fc region variant or constant region variant, their binding activity to FcyRIIb may be maintained or increased and their binding activity to FcyRIIa (type H) and FcyRIIa (type R) may be reduced as compared to those of a reference antibody having the constant region or Fc region of a native IgG. Such antibodies may have increased binding selectivity to FcyRIIb over FcyRIIa.

[0264] Within the scope of Disclosure A described herein, the extent that the binding activity to all activating FcyRs is reduced can be, but is not limited to, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91%or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

[0265] Within the scope of Disclosure A described herein, the extent that the FcyRIIbbinding activity is maintained or increased, the binding activity to FcyRIIb is maintained or increased, or the maintained or increased binding activity to FcyRIIb can be, but is not limited to, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, 100% or greater, 101% or greater, 102% or greater, 103% or greater, 104% or greater, 105% or greater, 106% or greater, 107% or greater, 108% or greater, 109% or greater, 110% or greater, 112% or greater, 114% or greater, 116% or greater, 118% or greater, 120% or greater, 122% or greater, 124% or greater, 126% or greater, 128% or greater, 130% or greater, 132% or greater, 134% or greater, 136% or greater, 138% or greater, 140% or greater, 142% or greater, 144% or greater, 146% or greater, 148% or greater, 150% or greater, 155% or greater, 160% or greater, 165% or greater, 170% or greater, 175% or greater, 180% or greater, 185% or

WO 2017/046994

PCT/JP2016/003616 greater, 190% or greater, 195% or greater, 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 7-fold or greater, 8-fold or greater, 9-fold or greater, 10-fold or greater, 20-fold or greater, 30-fold or greater, 40-fold or greater, 50-fold or greater, 60-fold or greater, 70-fold or greater, 80-fold or greater, 90-fold or greater, 100-fold or greater, 200-fold or greater, 300-fold or greater, 400-fold or greater, 500-fold or greater, 600-fold or greater, 700-fold or greater, 800-fold or greater, 900-fold or greater, 1000-fold or greater, 10000-fold or greater, or 100000-fold or greater.

[0266] Within the scope of Disclosure A described herein, the extent that the binding activity to FcyRIIa (type H) and FcyRIIa (type R) is reduced or the reduced binding activity to FcyRIIa (type H) and FcyRIIa (type R) can be, but is not limited to, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 94% or less, 93% or less, 92% or less, 91% or less, 90% or less, 88% or less, 86% or less, 84% or less, 82% or less, 80% or less, 78% or less, 76% or less, 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

[0267] Within the scope of Disclosure A described herein, modifications that increase binding selectivity to FcyRIIb over FcyRIIa (type R) may be preferred, and modifications that increase binding selectivity to FcyRIIb over FcyRIIa (type H) may be more preferred, and as reported in WO2013/047752, preferred amino acid substitutions for such modifications may include, for example, according to EU numbering: (a) modification by substituting Gly at position 237 with Trp; (b) modification by substituting Gly at position 237 with Phe; (c) modification by substituting Pro at position 238 with Phe; (d) modification by substituting Asn at position 325 with Met; (e) modification by substituting Ser at position 267 with He; (f) modification by substituting Leu at position 328 with Asp; (g) modification by substituting Ser at position 267 with Val; (h) modification by substituting Leu at position 328 with Trp; (i) modification by substituting Ser at position 267 with Gin; (j) modification by substituting Ser at position 267 with Met; (k) modification by substituting Gly at position 236 with Asp; (1) modification by substituting Ala at position 327 with Asn; (m) modification by substituting Asn at position 325 with Ser; (n) modification by substituting Leu at position 235 with Tyr; (o) modification by substituting Val at position 266 with Met; (p) modification by substituting Leu at position 328 with Tyr; (q) modification by substituting Leu at position 235 with Trp; (r) modification by substituting Leu at position 235 with Phe; (s) modification by substituting Ser at position 239 with Gly; (t) modification by

WO 2017/046994

PCT/JP2016/003616 substituting Ala at position 327 with Glu; (u) modification by substituting Ala at position 327 with Gly; (v) modification by substituting Pro at position 238 with Leu; (w) modification by substituting Ser at position 239 with Leu; (x) modification by substituting Leu at position 328 with Thr; (y) modification by substituting Leu at position 328 with Ser; (z) modification by substituting Leu at position 328 with Met; (aa) modification by substituting Pro at position 331 with Trp; (ab) modification by substituting Pro at position 331 with Tyr; (ac) modification by substituting Pro at position 331 with Phe; (ad) modification by substituting Ala at position 327 with Asp; (ae) modification by substituting Leu at position 328 with Phe; (af) modification by substituting Pro at position 271 with Leu; (ag) modification by substituting Ser at position 267 with Glu; (ah) modification by substituting Leu at position 328 with Ala; (ai) modification by substituting Leu at position 328 with lie; (aj) modification by substituting Leu at position 328 with Gin; (ak) modification by substituting Leu at position 328 with Val; (al) modification by substituting Lys at position 326 with Trp; (am) modification by substituting Lys at position 334 with Arg; (an) modification by substituting His at position 268 with Gly; (ao) modification by substituting His at position 268 with Asn; (ap) modification by substituting Ser at position 324 with Val; (aq) modification by substituting Val at position 266 with Leu; (ar) modification by substituting Pro at position 271 with Gly; (as) modification by substituting lie at position 332 with Phe; (at) modification by substituting Ser at position 324 with He; (au) modification by substituting Glu at position 333 with Pro; (av) modification by substituting Tyr at position 300 with Asp; (aw) modification by substituting Ser at position 337 with Asp; (ax) modification by substituting Tyr at position 300 with Gin; (ay) modification by substituting Thr at position 335 with Asp; (az) modification by substituting Ser at position 239 with Asn; (ba) modification by substituting Lys at position 326 with Leu; (bb) modification by substituting Lys at position 326 with He; (be) modification by substituting Ser at position 239 with Glu; (bd) modification by substituting Lys at position 326 with Phe; (be) modification by substituting Lys at position 326 with Val; (bf) mod ification by substituting Lys at position 326 with Tyr; (bg) modification by substituting Ser at position 267 with Asp; (bh) modification by substituting Lys at position 326 with Pro; (bi) modification by substituting Lys at position 326 with His; (bj) modification by substituting Lys at position 334 with Ala; (bk) modification by substituting Lys at position 334 with Trp; (bl) modification by substituting His at position 268 with Gin; (bm) modification by substituting Lys at position 326 with Gin; (bn) modification by substituting Lys at position 326 with Glu; (bo) modification by substituting Lys at position 326 with Met; (bp) modification by substituting Val at position 266 with He; (bq) modification by substituting Lys at position 334 with Glu; (br) modification by substituting Tyr at position 300 with Glu; (bs) modification by substituting Lys at

WO 2017/046994

PCT/JP2016/003616 position 334 with Met; (bt) modification by substituting Lys at position 334 with Val; (bu) modification by substituting Lys at position 334 with Thr; (bv) modification by substituting Lys at position 334 with Ser; (bw) modification by substituting Lys at position 334 with His; (bx) modification by substituting Lys at position 334 with Phe; (by) modification by substituting Lys at position 334 with Gin; (bz) modification by substituting Lys at position 334 with Pro; (ca) modification by substituting Lys at position 334 with Tyr; (cb) modification by substituting Lys at position 334 with lie; (cc) modification by substituting Gin at position 295 with Leu; (cd) modification by substituting Lys at position 334 with Leu; (ce) modification by substituting Lys at position 334 with Asn; (cf) modification by substituting His at position 268 with Ala; (eg) modification by substituting Ser at position 239 with Asp; (ch) modification by substituting Ser at position 267 with Ala; (ci) modification by substituting Leu at position 234 with Trp; (cj) modification by substituting Leu at position 234 with Tyr; (ck) modification by substituting Gly at position 237 with Ala; (cl) modification by substituting Gly at position 237 with Asp; (cm) modification by substituting Gly at position 237 with Glu; (cn) modification by substituting Gly at position 237 with Leu; (co) modification by substituting Gly at position 237 with Met; (cp) modification by substituting Gly at position 237 with Tyr; (cq) modification by substituting Ala at position 330 with Lys; (cr) modification by substituting Ala at position 330 with Arg; (cs) modification by substituting Glu at position 233 with Asp; (ct) modification by substituting His at position 268 with Asp; (cu) modification by substituting His at position 268 with Glu; (cv) modification by substituting Lys at position 326 with Asp; (cw) modification by substituting Lys at position 326 with Ser; (ex) modification by substituting Lys at position 326 with Thr; (cy) modification by substituting Val at position 323 with He; (cz) modification by substituting Val at position 323 with Leu; (da) modification by substituting Val at position 323 with Met; (db) modification by substituting Tyr at position 296 with Asp; (dc) modification by substituting Lys at position 326 with Ala; (dd) modification by substituting Lys at position 326 with Asn; and (de) modification by substituting Ala at position 330 with Met.

[0268] The modifications described above may be at a single position alone or at two or more positions in combination. Alternatively, such preferred modifications may include, for example, those shown in Tables 14 to 15, 17 to 24, and 26 to 28 of WO2013/047752, for example, variants of human constant region or human Lc region, in which the amino acid at position 238 according to EU numbering is Asp and the amino acid at position 271 according to EU numbering is Gly in human IgG (IgGl, IgG2, IgG3, or IgG4); in addition, one or more of position(s) 233, 234, 237, 264, 265, 266, 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and 396, according to EU numbering may be substituted. In this case, the variants may include, but are not

WO 2017/046994

PCT/JP2016/003616 limited to, variants of human constant region or human Fc region that contain one or more of:

Asp at position 233, Tyr at position 234, Asp at position 237, lie at position 264, Glu at position 265, any one of Phe, Met, and Leu at position 266, any one of Ala, Glu, Gly, and Gin at position 267, either Asp or Glu at position 268, Asp at position 269, any one of Asp, Phe, lie, Met, Asn, and Gin at position 272, Asp at position 296, either Ala or Asp at position 326, Gly at position 327, either Lys or Arg at position 330, Ser at position 331, Thr at position 332, any one of Thr, Lys, and Arg at position 333, and any one of Asp, Glu, Phe, He, Lys, Leu, Met, Gin, Arg, and Tyr at position 396, according to EU numbering.

[0269] In an alternative embodiment, antibodies of Disclosure A containing an Fc region variant or constant region variant may have maintained or increased binding activity to FcyRIIb and reduced binding activity to FcyRIIa (type H) and FcyRIIa (type R) as compared to a reference antibody containing the constant region or Fc region of a native IgG. Preferred sites of amino acid substitution for such variants may be, as reported in WO2014/030728, for example, the amino acid at position 238 according to EU numbering and at least one amino acid position selected from the group consisting of position 233, 234, 235, 237, 264, 265, 266, 267, 268, 269, 271, 272, 274, 296, 326, 327, 330, 331, 332, 333, 334, 355, 356, 358, 396, 409, and 419, according to EU numbering.

[0270] More preferably, the variants may have Asp at position 238 according to EU numbering, and at least one amino acid selected from the amino acid group of: Asp at position 233, Tyr at position 234, Phe at position 235, Asp at position 237, He at position 264, Glu at position 265, Phe, Leu, or Met at position 266, Ala, Glu, Gly, or Gin at position 267, Asp, Gin, or Glu at position 268, Asp at position 269, Gly at position 271, Asp, Phe, He, Met, Asn, Pro, or Gin at position 272, Gin at position 274, Asp or Phe at position 296, Ala or Asp at position 326, Gly at position 327, Lys, Arg, or Ser at position 330, Ser at position 331, Lys, Arg, Ser, or Thr at position 332, Lys, Arg, Ser, or Thr at position 333, Arg, Ser, or Thr at position 334, Ala or Gin at position 355,Glu at position 356, Met at position 358, Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, or Tyr at position 396, Arg at position 409, and Glu at position 419, according to EU numbering.

[0271] In an alternative embodiment, antibodies of Disclosure A containing an Fc region variant or constant region variant may have maintained binding activity to FcyRIIb and reduced binding activity to all activating FcyRs, FcyRIIa (type R) in particular, as compared to a reference antibody containing the constant region or Fc region of a native IgG. Preferred sites of amino acid substitution for such variants may be, as reported in W02014/163101, for example, in addition to the amino acid at position

WO 2017/046994

PCT/JP2016/003616

238 according to EU numbering), at least one amino acid position selected from positions 235, 237, 241, 268, 295, 296, 298, 323, 324, and 330, according to EU numbering. More preferably, the variants may have Asp at position 238 according to EU numbering, and at least one amino acid selected from the amino acid group of: Phe at position 235; Gin or Asp at position 237; Met or Leu at position 241; Pro at position 268; Met or Val at position 295; Glu, His, Asn, or Asp at position 296; Ala or Met at position 298; He at position 323; Asn or His at position 324; and His or Tyr at position 330, according to EU numbering.

[0272] Within the scope of Disclosure A described herein, the level of the maintained binding activity to FcyRIIb can be, but is not limited to, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, 100% or greater, 101% or greater, 102% or greater, 103% or greater, 104% or greater, 105% or greater, 106% or greater, 107% or greater, 108% or greater, 109% or greater, 110% or greater, 120% or greater, 130% or greater, 140% or greater, 150% or greater, 175% or greater, or 2-fold or greater.

[0273] Within the scope of Disclosure A described herein, the level of the aforementioned reduced binding activity to all activating FcyRs, FcyRIIa (type R) in particular can be, but is not limited to, 74% or less, 72% or less, 70% or less, 68% or less, 66% or less, 64% or less, 62% or less, 60% or less, 58% or less, 56% or less, 54% or less, 52% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.01% or less, or 0.005% or less.

[0274] W02014/030750 also reports variants of the mouse constant region and Fc region. In an embodiment, antibodies of Disclosure A or B may comprise such a variant.

[0275] Within the scope of Disclosures A and B described herein, unlike FcyR which belongs to the immunoglobulin superfamily, FcRn, in particular human FcRn, is structurally similar to polypeptides of major histocompatibility complex (MHC) class I, and exhibits 22% to 29% sequence identity with MHC class I molecules (Ghetie et al., Immunol. Today 18(12), 592-598 (1997)). FcRn is expressed as a heterodimer consisting of a soluble β or light chain (β2 microglobulin) complexed with a transmembrane a or heavy chain. Like MHC, the a chain of FcRn contains three extracellular domains (al, a2, and a3), and its short cytoplasmic domain tethers proteins to the cell surface, al and a2 domains interact with the FcRn-binding domain of the

WO 2017/046994

PCT/JP2016/003616 antibody Fc region (Raghavan et al., Immunity 1:303-315 (1994)).

[0276] FcRn is expressed in the maternal placenta and yolk sac of mammals, and is involved in mother-to-fetus IgG transfer. In addition, in the small intestines of neonatal rodents where FcRn is expressed, FcRn is involved in transfer of maternal IgG across brush border epithelium from ingested colostrum or milk. FcRn is expressed in a variety of other tissues and endothelial cell systems of various species. FcRn is also expressed in adult human vascular endothelia, muscle vascular system, and liver sinusoidal capillaries. FcRn is believed to play a role in maintaining the plasma IgG concentration by binding to IgG and recycling the IgG to serum. Typically, binding of FcRn to IgG molecules is strictly pH dependent. The optimal binding is observed in an acidic pH range below 7.0.

[0277] The polynucleotide and amino acid sequences of human FcRn may be derived, for example, from the precursors shown in NM_004107.4 and NP_004098.1 (containing the signal sequence), respectively (RefSeq accession numbers are shown in parentheses).

[0278] The precursors form complexes with human [>2-microglobulin in vivo. Thus, by using known recombinant expression techniques, soluble human FcRn capable of forming a complex with human β2-ιηΐοπ^ΚΛ>η1πι may be produced for appropriate use in various experimental systems. Such soluble human FcRn may be used to assess antibodies or Fc region variants for their FcRn-binding activity. In Disclosure A or B, FcRn is not particularly limited as long as it is in a form which can bind to the FcRnbinding domain; however, preferred FcRn may be human FcRn.

[0279] Within the scope of Disclosures A and B described herein, where an antibody or Fc region variant has FcRn-binding activity, it may have an FcRn-binding domain, preferably a human FcRn-binding domain. The FcRn-binding domain is not particularly limited as long as the antibody has binding activity to or affinity for FcRn at an acidic pH and/or at a neutral pH; or it may be a domain that has the activity to directly or indirectly bind to FcRn. Such domains include, but are not limited to, the Fc regions of IgG-type immunoglobulins, albumin, albumin domain 3, anti-FcRn antibodies, anti-FcRn peptides, and anti-FcRn Scaffold molecules, which have the activity of directly binding to FcRn, and molecules that bind to IgG or albumin, which have the activity of binding to FcRn indirectly. In Disclosure A or B, it is also possible to use domains that have FcRn-binding activity in an acidic pH range and/or in a neutral pH range. If the domains have FcRn-binding activity in an acidic pH range and/or in a neutral pH range originally, they can be used without further modification. If the domains have only a weak or no FcRn-binding activity in an acidic pH range and/or in a neutral pH range, amino acid residues in the FcRn-binding domain of the antibody or Fc region variant may be modified to have FcRn-binding activity in an

WO 2017/046994

PCT/JP2016/003616 acidic pH range and/or in a neutral pH range. Alternatively, amino acids of domains that originally have FcRn-binding activity in an acidic pH range and/or in a neutral pH range may be modified to further increase their FcRn-binding activity. The FcRnbinding activity in an acidic pH range and/or in a neutral pH range can be compared before and after amino acid modification to find amino acid modifications of interest for the FcRn-binding domains.

[0280] FcRn-binding domains may be preferably regions that directly bind to FcRn. Such preferred FcRn-binding domains include, for example, constant regions and Fc regions of antibodies. However, regions capable of binding to a polypeptide having FcRnbinding activity, such as albumin and IgG, can indirectly bind to FcRn via albumin, IgG. Thus, the FcRn-binding regions may be regions that bind to a polypeptide that has binding activity to albumin or IgG. Without limitations, to promote antigen elimination from plasma, FcRn-binding domains whose FcRn-binding activity is greater at a neutral pH are preferred, while to improve antibody retention in plasma, FcRn-binding domains whose FcRn-binding activity is greater at an acidic pH are preferred. For example, it is possible to select FcRn-binding domains whose FcRn-binding activity is originally greater at a neutral pH or acidic pH. Alternatively, amino acids of an antibody or Fc region variant may be modified to confer FcRn-binding activity at a neutral pH or acidic pH. Alternatively, it is possible to increase the pre-existing FcRnbinding activity at a neutral pH or acidic pH.

[0281] Within the scope of Disclosures A and B described herein, whether the FcRn-binding activity of an antibody or Fc region (variant) is increased, (substantially) maintained, or reduced as compared to that of the antibody or Fc region (variant) before modification can be assessed by known methods such as those described in the Examples herein, and for example, BIACORE, Scatchard plot and flow cytometer (see WO2013/046722). The extracellular domain of human FcRn may be used as a soluble antigen in these assays. Those of ordinary skill in the art can appropriately select the conditions besides pH in measuring the FcRn-binding activity of an antibody or Fc region (variant). The assay can be carried out, for example, under the conditions of MES buffer and 37 °C, as described in WO2009/125825. The FcRn-binding activity of an antibody or Fc region (variant) can be assessed, for example, by loading FcRn as an analyte on an antibody-immobilized chip.

[0282] The FcRn-binding activity of an antibody or Fc region (variant) can be assessed based on the dissociation constant (KD), apparent dissociation constant (apparent KD), dissociation rate (kd), apparent dissociation (apparent kd).

[0283] As for the pH conditions for measuring the binding activity between FcRn and the FcRn-binding domain contained in an antibody or Fc region (variant), acidic pH condition or neutral pH condition may be suitably used. As for the temperature

WO 2017/046994

PCT/JP2016/003616 conditions for measuring the binding activity (binding affinity) between FcRn and the FcRn-binding domain, any temperature of 10°C to 50°C may be used. To determine the binding activity (binding affinity) between FcRn and the human FcRn-binding domain, preferably a temperature of 15°C to 40°C may be used. More preferably, any temperature from 20°C to 35°C such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C may be used. A non-limiting example of such temperature can be 25°C.

[0284] In one embodiment, where antibodies of Disclosure A or B have FcRn-binding activity, they may have an FcRn-binding domain, preferably a human FcRn-binding domain. The FcRn-binding domain is not particularly limited as long as the antibodies have binding activity to or affinity for FcRn at an acidic pH and/or a neutral pH, and it may be a domain that has an activity of directly or indirectly binding to FcRn. In one specific embodiment, it may be preferable that the antibody of Disclosure A or B has, for example, an increased FcRn-binding activity under a neutral pH condition as compared to a reference antibody containing the constant region of a native IgG (see WO2013/046722). From the perspective of comparing the FcRn-binding activity between the two, it may be preferable that, without limitations, the antibody of Disclosure A or B and the reference antibody containing the constant region of a native IgG have identical amino acid sequences in the regions (for example, the variable region) other than, preferably, the constant region of the antibody of Disclosure A or B which has been modified at one or more amino acid residues.

[0285] In one embodiment, within the scope of Disclosure A described herein, where an antibody of Disclosure A has an increased FcRn-binding activity under a neutral pH condition, without being bound by a particular theory, the antibody of Disclosure A may possess any two or more of the following properties in combination: the property of being shuttled between plasma and cellular endosome and repeatedly binding to multiple antigens as a single antibody molecule by having an ion concentrationdependent antigen-binding domain; the property of being rapidly taken up into cells by having increased pi and increased positive charge in the overall antibody; and the property of being rapidly taken up into cells by having an increased FcRn-binding activity under a neutral pH condition. As a result, the antibody half-life in plasma can be further shortened, or the binding activity of the antibody toward the extracellular matrix can be further increased, or antigen elimination from plasma can be further promoted. Those of ordinary skill in the art can determine an optimal pi value for the antibody of Disclosure A to take advantage of these properties.

[0286] Within the scope of Disclosures A and B described herein, according to the Yeung et al. (J. Immunol. 182:7663-7671 (2009)), the activity of a native human IgGl to bind to human FcRn is KD 1.7 pM in an acidic pH range (pH 6.0), whereas in a neutral pH

WO 2017/046994

PCT/JP2016/003616 range the activity is almost undetectable. Thus, to increase the FcRn-binding activity in a neutral pH range, it may preferable to use, as an antibody of Disclosure A or B: an antibody or a constant region variant or Fc region variant whose human FcRn-binding activity in an acidic pH range is KD 20 pM or stronger and whose human FcRnbinding activity in a neutral pH range is comparable to or stronger than that of a native human IgG; preferably an antibody or a constant region variant or Fc region variant whose human FcRn-binding activity in an acidic pH range is KD 2.0 μΜ or stronger and whose human FcRn-binding activity in a neutral pH range is KD 40 μΜ or stronger; and more preferably an antibody or a constant region variant or Fc region variant whose human FcRn-binding activity in an acidic pH range is KD 0.5 μΜ or stronger and whose human FcRn-binding activity in a neutral pH range is KD 15 μΜ or stronger. The KD values are determined by the method described in Yeung et al. (J. Immunol. 182:7663-7671 (2009) (by immobilizing an antibody onto a chip and loading human FcRn as an analyte)).

[0287] Within the scope of Disclosures A and B described herein, a domain of any structure that binds to FcRn can be used as an FcRn-binding domain. In this case, the FcRnbinding domain can be produced without the need to introduce an amino acid modification, or the affinity for FcRn may be increased by introducing an additional modification.

[0288] Within the scope of Disclosures A and B described herein, the starting FcRn-binding domain can include for example, the Fc region or constant region of (human) IgG. As long as a variant of the starting Fc region or starting constant region can bind to FcRn in an acidic pH range and/or in a neutral pH range, any Fc region or constant region can be used as the starting Fc region or starting constant region. Or, an Fc region or constant region obtained by further modifying a starting Fc region or starting constant region whose amino acid residues have been already modified from an Fc region or constant region can also be appropriately used as the Fc region or constant region. The starting Fc region or starting constant region may include known Fc regions produced by recombination. A starting Fc region or starting constant region may refer to the polypeptide itself, a composition containing the starting Fc region or starting constant region, or an amino acid sequence encoding the starting Fc region or starting constant region, depending on the context. The origin of the starting Fc region or starting constant region is not limited, and it can be obtained from any organism of nonhuman animals or from a human. Furthermore, the starting FcRn-binding domain can be obtained from cynomolgus monkeys, marmosets, Rhesus monkeys, chimpanzees, and humans. Starting Fc regions or starting constant regions may be obtained from human IgGl, but are not limited to any particular IgG class. This means that an Fc region of human IgGl, IgG2, IgG3, or IgG4 can be used as an appropriate starting FcRn-binding

WO 2017/046994

PCT/JP2016/003616 domain, and an Fc region or constant region of an IgG class or subclass derived from any organism can be used as a starting Fc region or as a starting constant region. Examples of native IgG variants or modified forms are described in, for example, Strohl, Curr. Opin. Biotechnol. 20(6):685-691 (2009); Presta, Curr. Opin. Immunol. 20(4):460-470 (2008); Davis et al., Protein Eng. Des. Sel. 23(4):195-202 (2010), W02009/086320, W02008/092117; W02007/041635; and W02006/105338).

[0289] Within the scope of Disclosures A and B described herein, amino acid residues of the starting FcRn-binding domain, starting Fc region, or starting constant region may contain, for example, one or more mutations: for example, substitution mutations with amino acid residues that are different from the amino acid residues in the starting Fc region or starting constant region; insertions of one or more amino acid residues into the amino acid residues in the starting Fc region or starting constant region; or deletions of one or more amino acid residues from the amino acid residues of the starting Fc region or starting constant region. The amino acid sequences of Fc regions or constant regions after modifications may be preferably amino acid sequences containing at least a portion of an Fc region or constant region that does not occur naturally. Such variants necessarily have a sequence identity or similarity of less than 100% to the starting Fc regions or starting constant regions. For example, the variants have an amino acid sequence identity or similarity of about 75% to less than 100%, more preferably about 80% to less than 100%, even more preferably about 85% to less than 100%, still more preferably about 90% to less than 100%, and yet more preferably about 95% to less than 100% to the amino acid sequence of the starting Fc region or starting constant region. In a non-limiting example, at least one amino acid is different between a modified Fc region or constant region of Disclosure A or B and the starting Fc region or starting constant region.

[0290] Within the scope of Disclosures A and B described herein, an Fc region or constant region that has FcRn-binding activity in an acidic pH range and/or in a neutral pH range may be obtained by any method. Specifically, a variant of Fc region or constant region that has FcRn-binding activity in an acidic pH range and/or in a neutral pH range may be obtained by modifying amino acids of a human IgG-type antibody which can be used as the starting Fc region or starting constant region. IgG-type antibody Fc regions or constant regions suitable for modification include, for example, the Fc regions or constant regions of human IgG (IgGl, IgG2, IgG3, and IgG4, and variants thereof), and mutants spontaneously generated therefrom are also included in the IgG Fc regions or constant regions. For the Fc regions or constant regions of human IgGl, human IgG2, human IgG3, and human IgG4 antibodies, a number of allotype sequences due to genetic polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No.91-3242, and any of them may be used in

WO 2017/046994

PCT/JP2016/003616

Disclosure A or B. In particular, for the human IgGl sequence, the amino acid sequence of positions 356 to 358 according to EU numbering may be DEL or EEM.

[0291] In one embodiment of Disclosure A or B, the modification into other amino acids is not particularly limited, as long as the resulting variants have FcRn-binding activity in an acidic pH range and/or in a neutral pH range, and preferably in a neutral pH range. Sites of amino acid modification to increase the FcRn-binding activity under a neutral pH condition are described, for example, in WO2013/046722. Such modification sites include, for example, one or more positions selected from the group consisting of: position 221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, 291 to 312, 315 to 320, 324, 325, 327 to 339, 341, 343, 345, 360, 362, 370, 375 to 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442, according to EU numbering in the Fc region or constant region of a human IgG antibody, as described in WO2013/046722. WO2013/046722 also describes, as a part of the preferred modifications in the Fc region or constant region, for example, modification of one or more amino acids selected from the group consisting of: the amino acid at position 256 to Pro, the amino acid at position 280 to Lys, the amino acid at position 339 to Thr, the amino acid at position 385 to His, the amino acid at position 428 to Leu, and the amino acid at position 434 to Trp, Tyr, Phe, Ala, or His, according to EU numbering. The number of amino acids to be modified is not particularly limited, and modification may be performed at a single position alone or at two or more positions. Modification of these amino acid residues can enhance the FcRn binding of the Fc region or constant region of an IgG-type antibody under a neutral pH condition. Modification of these amino acid residues may also be introduced appropriately into antibodies of Disclosure A or B.

[0292] In a further or alternative embodiment, it is also possible to use appropriate amino acid modification sites for increasing the FcRn-binding activity under an acidic pH condition. Among such modification sites, one or more modification sites that allow an increase in the FcRn binding also in a neutral pH range can be appropriately used in Disclosure A or B. Such modification sites include, for example, those reported in WO2011/122011, WO2013/046722, WO2013/046704, and WO2013/046722. The sites of amino acids that allow such modification of the constant region or Fc region of a human IgG-type antibody and the types of amino acids after modification are reported in Table 1 of WO2013/046722. WO2013/046722 also describes, as particularly preferred, modification sites in the constant region or Fc region, for example, the location of one or more amino acid positions selected from the group consisting of position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436, according to EU

WO 2017/046994

PCT/JP2016/003616 numbering. Modification of these amino acid residue positions can also enhance the human FcRn binding of the FcRn-binding domain in a neutral pH range. WO2013/046722 also describes, as a part of the preferred modification in the IgG-type constant region or Fc region, for example, modification of one or more amino acid residues selected from the group consisting of: (a) the amino acid at position 237 to Met; (b) the amino acid at position 238 to Ala; (c) the amino acid at position 239 to Fys; (d) the amino acid at position 248 to lie; (e) the amino acid at position 250 to any one of Ala, Phe, He, Met, Gin, Ser, Val, Trp, and Tyr; (f) the amino acid at position 252 to any one of Phe, Trp, and Tyr; (g) the amino acid at position 254 to Thr; (h) the amino acid at position 255 to Glu; (i) the amino acid at position 256 to any one of Asp, Glu, and Gin; (j) the amino acid at position 257 to any one of Ala, Gly, He, Feu, Met, Asn, Ser, Thr, and Val; (k) the amino acid at position 258 to His; (1) the amino acid at position 265 to Ala; (m) the amino acid at position 270 to Phe; (n) the amino acid at position 286 to either Ala or Glu; (o) the amino acid at position 289 to His; (p) the amino acid at position 297 to Ala; (q) the amino acid at position 298 to Gly; (r) the amino acid at position 303 to Ala; (s) the amino acid at position 305 to Ala; (t) the amino acid at position 307 to any one of Ala, Asp, Phe, Gly, His, He, Fys, Feu, Met, Asn, Pro, Gin, Arg, Ser, Val, Trp, and Tyr; (u) the amino acid at position 308 to any one of Ala, Phe, He, Feu, Met, Pro, Gin, and Thr; (v) the amino acid at position 309 to any one of Ala, Asp, Glu, Pro, and Arg; (w) the amino acid at position 311 to any one of Ala, His, and lie; (x) the amino acid at position 312 to either Ala or His; (y) the amino acid at position 314 to either Fys or Arg; (z) the amino acid at position 315 to either Ala or His; (aa) the amino acid at position 317 to Ala; (ab) the amino acid at position 325 to Gly; (ac) the amino acid at position 332 to Val; (ad) the amino acid at position 334 to Feu; (ae) the amino acid at position 360 to His; (af) the amino acid at position 376 to Ala; (ag) the amino acid at position 380 to Ala; (ah) the amino acid at position 382 to Ala; (ai) the amino acid at position 384 to Ala; (aj) the amino acid at position 385 to either Asp or His; (ak) the amino acid at position 386 to Pro; (al) the amino acid at position 387 to Glu; (am) the amino acid at position 389 to either Ala or Ser; (an) the amino acid at position 424 to Ala; (ao) the amino acid at position 428 to any one of Ala, Asp, Phe, Gly, His, He, Fys, Feu, Asn, Pro, Gin, Ser, Thr, Val, Trp, and Tyr; (ap) the amino acid at position 433 to Fys; (aq) the amino acid at position 434 to Ala, Phe, His, Ser, Trp, and Tyr; and (ar) the amino acid at position 436 to His; according to EU numbering. The number of amino acids to be modified is not particularly limited, and modification may be performed at a single position alone or at two or more positions. Combinations of amino acid modifications at two or more positions include, for example, those shown in Table 2 of WO2013/046722. Modification of these amino acid residues may also be appropriately introduced into an99

WO 2017/046994 PCT/JP2016/003616 tibodies of Disclosures A and B.

[0293] In one embodiment, the FcRn-binding activity of the FcRn-binding domain of an antibody of Disclosure A or B has been increased when compared to that of a reference antibody containing an Fc region or constant region of a native IgG or that of a reference antibody containing a starting Fc region or starting constant region. Namely, the FcRn-binding activity of an Fc region variant or constant region variant of Disclosure A or B, or an antibody containing such variant is greater than that of the reference antibody). This can mean that when compared to the FcRn-binding activity of the reference antibody, that of an antibody of Disclosure A or B can be, for example: 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 100% or greater, 105% or greater, preferably 110% or greater, 115% or greater, 120% or greater, 125% or greater, more preferably 130% or greater, 135% or greater, 140% or greater, 145% or greater, 150% or greater, 155% or greater, 160% or greater, 165% or greater, 170% or greater, 175% or greater, 180% or greater, 185% or greater, 190% or greater, 195% or greater, 2-fold or greater, 2.5-fold or greater, 3-fold or greater, 3.5-fold or greater, 4-fold or greater, 4.5-fold or greater, or 5-fold or greater.

[0294] In one embodiment, amino acid sequences to be modified in an antibody of

Disclosure A or B can preferably contain human sequences (sequences found in native human-derived antibodies) in order to not increase the immunogenicity of the antibody when the antibody is administered in vivo (preferably, into a human body). Alternatively, after modification, mutations may be introduced at positions other than the sites of amino acid modification in such a way that one or more of the FRs (FR1, FR2, FR3, and FR4) is substituted with a human sequence. Methods for substituting FR(s) with a human sequence are known in the art and include, but are not limited to that reported in Ono et al., Mol. Immunol. 36(6):387-395 (1999). Humanization methods are known in the art and include, but are not limited to that reported in, Methods 36(1):43-60 (2005).

[0295] In one embodiment, the framework region sequences (also referred to as FR sequences) of the heavy chain and/or light chain variable region of an antibody of Disclosure A or B may contain human germ-line framework sequences. When the framework sequences are completely human germ-line sequences, the antibody is expected to induce little or no immunogenic reaction when administered to humans (for example, to treat or prevent a certain disease).

[0296] FR sequences preferably can include, for example, fully human FR sequences such as those shown in V-Base (vbase.mrc-cpe.cam.ac.uk/). These FR sequences can be appropriately used for Disclosure A or B. The germ-line sequences may be categorized based on their similarity (Tomlinson et al. (J. Mol. Biol. 227:776-798 (1992); Williams

100

WO 2017/046994

PCT/JP2016/003616 et al. (Eur. J. Immunol. 23:1456-1461 (1993); and Cox et al. (Nat. Genetics 7:162-168 (1994)). Preferred germ-line sequences can be appropriately selected from Vk , which is categorized into seven subgroups; VX , which is categorized into ten subgroups; and VH, which is categorized into seven subgroups.

[0297] Fully human VH sequences can preferably include, for example, VH sequences of: subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, and VH1-69); subgroup VH2 (for example, VH2-5, VH2-26, and VH2-70); subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, and VH3-74); subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, and VH4-61); subgroup VH5 (VH5-51); subgroup VH6 (VH6-1); or subgroup VH7 (VH7-4 and VH7-81). These are also described in, for example, Matsuda et al. (J. Exp. Med. 188:1973-1975 (1998)), and those of ordinary skill in the art can appropriately design antibodies based on information of these sequences. It can be also preferable to use other fully human FR sequence or sequences of regions that are equivalent thereto.

[0298] Fully human Vk sequences can preferably include, for example: A20, A30, LI, L4, L5, L8, L9, Lll, L12, L14, L15, L18, L19, L22, L23, L24, 02, 04, 08, 012, 014, or 018, which are classified as subgroup Vkl; Al, A2, A3, A5, A7, A17, A18, A19, A23, 01, and Oil, which are classified as subgroup Vk2; All, A27, L2, L6, L10, L16, L20, and L25, which are classified as subgroup Vk3; B3, classified as subgroup Vk4; B2 (also referred to as Vk5-2), classified as subgroup Vk5; or A10, A14, and A26, which are classified as subgroup Vk6 (Kawasaki et al. (Eur. J. Immunol. 31:1017-1028 (2001)); (Hoppe Seyler Biol. Chem. 374:1001-1022 (1993)); Brensing-Kuppers et al. (Gene 191:173-181 (1997)).

[0299] Fully human VX sequences can preferably include, for example: Vl-2, Vl-3, Vl-4, Vl-5, Vl-7, Vl-9, Vl-11, Vl-13, Vl-16, Vl-17, Vl-18, Vl-19, Vl-20, and Vl-22, which are classified as subgroup VL1; V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19, which are classified as subgroup VL2; V3-2, V3-3, and V3-4, which are classified as subgroup VL3; V4-1, V4-2, V4-3, V4-4, and V4-6, which are classified as subgroup VL4; or V5-1, V5-2, V5-4, and V5-6, which are classified as subgroup VL5 (Kawasaki et al. Genome Res. 7:250-261 (1997)).

[0300] Normally, these FR sequences are different from one another at one or more amino acid residues. These FR sequences can be used in the modification of antibody amino acid residues. Fully human FR sequences that may be used in the modification also include, for example, KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (see, for example, aforementioned Kabat et al. (1991); Wu et al. (J. Exp. Med. 132:211-250 (1970)).

101

WO 2017/046994 PCT/JP2016/003616 [0301] Within the scope of Disclosures A and B described herein, flexible residues can refer to amino acid residue variations that are present at positions showing high amino acid diversity at which the light chain or heavy chain variable regions have several different amino acids when the amino acid sequences of known and/or native antibodies or antigen-binding domains are compared. Positions showing high diversity are generally located in the CDRs. The data provided by Rabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991), can be effective in determining such positions with high diversity in known and/or native antibodies. Furthermore, several databases on the Internet (vbase.mrc-cpe.cam.ac.uk/, bioinf.org.uk/abs/index.html) provide a collection of numerous human light chain and heavy chain sequences and their locations. Information on these sequences and locations is useful to determine the locations of flexible residues. Without limitations, for example, when an amino acid residue at a particular position has a variability of, preferably, 2 to 20, 3 to 19, 4 to 18, 5 to 17, 6 to 16, 7 to 15, 8 to 14, 9 to 13, or 10 to 12 amino acid residues, the position can be judged to show (high) diversity.

[0302] In an embodiment, it can be understood that where an antibody of Disclosure A or B contains the whole or a portion of the light chain variable region and/or heavy chain variable region, the antibody may contain one or more appropriate flexible residues, if needed. For example, a heavy chain and/or light chain variable region sequence selected to have an FR sequence which originally contains amino acid residues that change the antigen-binding activity of an antibody according to the ion concentration (hydrogen ion concentration or calcium ion concentration) conditions can be designed to contain, other amino acid residues in addition to these amino acid residues. In this case, for example, the number and locations of the flexible residues can also be determined without being limited to a specific embodiment, as long as the antigenbinding activity of the antibody of Disclosure A or B changes according to the ion concentration condition. Specifically, the CDR sequence and/or FR sequence of a heavy chain and/or light chain may contain at least one flexible residue. For example, where the ion concentration is calcium ion concentration, flexible residues that can be introduced into the light-chain variable region sequence (aforementioned Vk5-2) include, but are not limited to, one or more amino acid residue positions shown in Table 1 or Table 2. Fikewise, appropriate flexible residues can be introduced, for example, into an ion concentration-dependent antibody or antibody without such ion concentration dependency, containing the whole or a portion of the light chain variable region and/or heavy chain variable region, in which at least one amino acid residue that may be exposed on the antibody surface has been modified such that the pi is increased.

[0303]

102

WO 2017/046994

PCT/JP2016/003616 [Table 1]

CDR	Kabat numbering	Amino acid in 70% of the total
CDR1	28	8:100%
29	1:100%
30	E:72%	N 14%	S.14%
31	D:100%
32	D:100%
33	L: 100%
34	A; 70%	λ 30%
CDR2	50	E 100%
51	A: 100%
52	8.100% II 5% I 100% Q 100% 8:100%
53	N 25%	S 45%	T 25%
54
55
56
CDR3	90	Q:10G%
91	H:25%	S: 15%	R:15%	Y:45%
92	D:80%	N: 10%	S:10%
93	D:5%	G: 10%	N:25%	S:50%	R: 10%
94	S;50%	Y-50%
95	p-100%
96	L:50%	Y:50%

(Positions are shown according to Kabat numbering.) [0304]

103

WO 2017/046994

PCT/JP2016/003616 [Table 2]

CDR	Kabat numbering	Amino acid in 30% of the total
CDR1	28	S:100%
29	1 100%
30	E:83%	S. 17%
31	D 100%
32	D:100%
33	L; 100%
34	A; 70%	N;30%
CDR2 CDR3	50	H: 100%
51	A 100%
52	S: 100%
S3	H:5%	N 25%	S:45%	T:25%
54	L :1()0%
55 56	Q:100% S S 100%	---------------------------	----------------------—......	---------------------------
90	Q:100%
9.1	H: 25%	S: 15%	R: 15%	Y: 45%
92	D:80%	N:10%	S:10%
93	D: 5%	G:10%	N:25%	S:50%	R:10%
94	S:50%	Y:50%
95	P: 100%
96	L:50%	Y:50%

(Positions are shown according to Rabat numbering.) [0305] In one embodiment, when humanizing a chimeric antibody, the pi of the chimeric antibody is increased by modifying one or more amino acid residues that can be exposed on the antibody surface as to produce a humanized antibody of Disclosure A or B with a shortened plasma half-life as compared to the chimeric antibody absent such modification. The modification of amino acid residues that can be exposed on the surface of the humanized antibody can be carried out before or concurrently with humanization of the antibody. Alternatively, by using the humanized antibody as a starting material, amino acid residues that can be exposed on the surface may be modified to further alter the pi of the humanized antibody.

[0306] Adams et al. (Cancer Immunol. Immunother. 55(6):717-727 (2006)) reports that the humanized antibodies, trastuzumab (antigen: HER2), bevacizumab (antigen: VEGF), and pertuzumab (antigen: HER2), which were humanized using the same human antibody FR sequences, were almost comparable in plasma pharmacokinetics. Specifically, it can be understood that the plasma pharmacokinetics is almost comparable when humanization is performed using the same FR sequences. According

104

WO 2017/046994

PCT/JP2016/003616 to one embodiment of Disclosure A, the antigen concentration in plasma is reduced by increasing the antibody's pi by modifying amino acid residues that can be exposed on the antibody surface, in addition to the humanization step. In an alternative embodiment for Disclosure A or B, human antibodies can be used. By modifying amino acid residues that can be exposed on the surface of a human antibody produced from a human antibody library, a human antibody-producing mouse, a recombinant cell, etc., and increasing the pi of the human antibody, the ability of the originally-produced human antibody to eliminate antigen from plasma can be increased.

[0307] In one embodiment, antibodies of Disclosure A may contain modified sugar chains.

Antibodies with modified sugar chains include, for example, antibodies with modified glycosylation (WO99/54342), antibodies that lack fucose (WOOO/61739;

W002/31140, W02006/067847; W02006/067913), and antibodies having sugar chains with bisecting GlcNAc (WO02/79255).

[0308] In one embodiment, antibodies of Disclosure A or B can be used, for example, in techniques for exhibiting increased antitumor activities against cancer cells or in techniques for promoting elimination of antigens that are harmful to the organism from the plasma.

[0309] In an alternative embodiment, Disclosure A or B relate to libraries of the ion concentration-dependent antigen-binding domains with an increased pi or ion concentration-dependent antibodies with an increased pi, as described above.

[0310] In an alternative embodiment, Disclosure A or B relates to nucleic acids (polynucleotides) encoding the above-described ion concentration-dependent antigenbinding domains with an increased pi or ion concentration-dependent antibodies with an increased pi. In a specific embodiment, the nucleic acids can be obtained using appropriate known methods. For specific embodiments, for example, WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704,

W02000/042072, W02006/019447, WO2012/115241, WO2013/047752, WO2013/125667, WO2014/030728, W02014/163101, WO2013/081143, W02007/114319, W02009/041643, WO2014/145159, WO2012/016227, and WO2012/093704 can be referred to, each of these are incorporated herein by reference in their entirety.

[0311] In one embodiment, nucleic acids of Disclosure A or B can be isolated or purified nucleic acids. Nucleic acids encoding the antibodies of Disclosure A or B may be any genes, and may be DNA or RNA, or other nucleic acid analogs.

[0312] Within Disclosures A and B described herein, when amino acids of an antibody are modified, the amino acid sequence of the antibody before modification may be a known sequence or the amino acid sequence of an antibody newly obtained. For example, antibodies can be obtained from antibody libraries, or by cloning nucleic

105

WO 2017/046994

PCT/JP2016/003616 acids encoding the antibody from hybridomas or B cells that produce monoclonal antibodies. The methods for obtaining nucleic acids encoding an antibody from hybridomas may use the techniques of: performing immunization by conventional immunization methods using an antigen of interest or cells expressing the antigen of interest as a sensitizing antigen; fusing the resulting immune cells with known parental cells by conventional cell fusion methods; screening for monoclonal antibody-producing cells (hybridomas) by conventional screening methods; synthesizing cDNAs of the variable region (V region) of the antibody using reverse transcriptase from mRNAs of the obtained hybridomas; and linking the cDNA to a DNA encoding an antibody constant region (C region) of interest.

[0313] Sensitizing antigens which are used to obtain nucleic acids encoding the abovedescribed heavy chain and light chain include, but are not limited to, both complete antigens with immunogenicity and incomplete antigens including haptens which exhibit no immunogenicity. For example, it is possible to use whole proteins of interest or partial peptides of the proteins. In addition, substances that are composed of polysaccharides, nucleic acids, lipids, and other compositions are known to be potential antigens. Thus, in some embodiments, antigens for the antibodies of Disclosure A or B are not particularly limited. The antigens can be prepared by, for example, baculovirus-based methods (see, e.g., WO98/46777). Hybridomas can be produced, for example, according to the method of G. Kohler and C. Milstein, Methods Enzymol. 73:3-46 (1981)). When the immunogenicity of an antigen is low, immunization may be performed by linking the antigen with a macromolecule having immunogenicity, such as albumin. Alternatively, if necessary, soluble antigens can be prepared by linking the antigen with other molecules. When a transmembrane molecule such as membrane antigens (for example, receptors) is used as an antigen, a portion of the extracellular region of the membrane antigen can be used as a fragment, or cells expressing the transmembrane molecule on their surface may be used as an immunogen.

[0314] In some embodiments, antibody-producing cells can be obtained by immunizing an animal with an appropriate sensitizing antigen described above. Alternatively, antibody-producing cells can be prepared by in vitro immunization of lymphocytes that are capable of producing antibodies. Various mammals can be used for immunization and other routine antibody producing procedures. Commonly used animals include rodents, lagomorphs, and primates. The animals may include, for example, rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates including monkeys such as cynomolgus monkeys, rhesus monkeys, baboons, and chimpanzees.

In addition, transgenic animals carrying a human antibody gene repertoire are also known, and these animals can be used to obtain human antibodies (see, e.g.,

106

WO 2017/046994

PCT/JP2016/003616

WO96/34096; Mendez et al., Nat. Genet. 15:146-156 (1997); WO93/12227, WO92/03918, W094/02602, WO96/34096, and WO96/33735). Instead of using such transgenic animals, it is also possible to obtain desired human antibodies having antigen-binding activity by, for example, sensitizing human lymphocytes in vitro with desired antigens or cells expressing the desired antigens and then fusing the sensitized lymphocytes with human myeloma cells such as U266 (JP Pat. Publ. No. HOI-59878).

[0315] Animal immunization can be carried out, for example, by appropriately diluting and suspending a sensitizing antigen in phosphate buffered saline (PBS), physiological saline, or others, and mixing it with an adjuvant to emulsify, if needed; and then injecting it intraperitoneally or subcutaneously into animals. Then, the sensitizing antigen mixed with Freund's incomplete adjuvant can be preferably administered several times every four to 21 days. Antibody production can be confirmed, for example, by measuring the titer of the antibody of interest in animal sera.

[0316] Antibody-producing cells obtained from lymphocytes or animals immunized with a desired antigen can be fused with myeloma cells to generate hybridomas using conventional fusing agents (for example, polyethylene glycol) (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, 59-103). If needed, hybridomas are cultured and expanded, and the binding specificity of antibodies produced by the hybridomas is assessed by, for instance, immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA). Then, if needed, antibody-producing hybridomas whose specificity, affinity, or activity of interest has been determined may also be subcloned by methods such as limiting dilution.

[0317] Nucleic acids encoding the selected antibody can be cloned from hybridomas or antibody-producing cells (sensitized lymphocytes, etc.) using probes that can specifically bind to the antibody (for example, oligonucleotides complementary to sequences encoding the antibody constant regions). Alternatively, the nucleic acids can be cloned from mRNA using RT-PCR. Heavy chains and light chains for use in producing antibodies of Disclosure A or B may be derived from antibodies that, for example, belong to any of Ig antibody classes and subclasses, and IgG may be preferred.

[0318] In one embodiment, nucleic acids encoding amino acid sequences that constitute the heavy chain (the whole or a portion thereof) and/or light chain (the whole or a portion thereof) of an antibody of Disclosure A or B, for example, are modified by genetic engineering techniques. Recombinant antibodies with artificial sequence modification to, for example, reduce heterologous antigenicity against humans, such as chimeric antibodies or humanized antibodies, may be appropriately generated by, for example, modifying nucleotide residues encoding amino acid sequences associated with

107

WO 2017/046994

PCT/JP2016/003616 components of antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies, or camel antibodies. Chimeric antibodies can be obtained, for example, by ligating a DNA encoding a mouse-derived antibody variable region with a DNA encoding a human antibody constant region and incorporating the ligated DNA coding sequence into an expression vector, then introducing the resulting recombinant vector into a host to express the genes. Humanized antibodies, which are also referred to as reshaped human antibodies, are antibodies in which human antibody FR(s) are linked in frame with antibody CDR(s) isolated from non-human mammals, such as mice, to form a coding sequence. A DNA sequence encoding such a humanized antibody can be synthesized by overlap extension PCR using a number of oligonucleotides as templates. Materials and experimental methods for overlap extension PCR are described in WO98/13388 and others. For example, a DNA encoding the amino acid sequence of, for example, an antibody variable region of Disclosure A or B may be obtained by overlap extension PCR using a number of oligonucleotides designed to have overlapping nucleotide sequences. The overlapping DNA is then linked in frame to a DNA encoding a constant region to form a coding sequence. The DNA linked as described above may be then inserted into an expression vector so that the DNA can be expressed, and the resulting vector may be introduced into a host or host cell. The antibody encoded by the DNA can be expressed by raising the host or culturing the host cells. The expressed antibody can be appropriately purified from culture media of the host or others (EP239400; WO96/02576). Furthermore, the FR(s) of a humanized antibody which are linked via CDR(s) may be selected, for example, to allow the CDRs to form an antigen-binding site suitable for the antigen. If necessary, amino acid residues that constitute FR(s) of a variable region of the selected antibody, for example, can be modified with appropriate substitution.

[0319] In one embodiment, to express antibodies of Disclosure A or B or fragments thereof, nucleic acid cassettes may be cloned into appropriate vectors. For such purposes, several types of vectors, such as phagemid vectors are available. In general, phagemid vectors can contain various elements including regulatory sequences such as promoters or signal sequences, phenotype selection genes, replication origins, and other necessary elements.

[0320] Methods for introducing desired amino acid modifications into antibodies have been established in the field of the art. For example, libraries can be constructed by introducing at least one modified amino acid residue that can be exposed on the surface of antibodies of Disclosure A or B and/or at least one amino acid that can change the antigen-binding activity of antibodies according to the ion concentration condition. Additionally, if needed, flexible residues can be added using the method of Kunkel et al. (Methods Enzymol. 154:367-382 (1987)).

108

WO 2017/046994 PCT/JP2016/003616 [0321] In an alternative embodiment, Disclosure A relates to vectors containing nucleic acids encoding an above-described ion concentration-dependent antigen-binding domain with increased pi or an above-described ion concentration-dependent antibody with increased pi. In a specific embodiment, the vectors can be obtained by, for example, vectors as described in WO2009/125825, WO2012/073992,

WO2011/122011, WO2013/046722, WO2013/046704, W02000/042072, W02006/019447, WO2012/115241, WO2013/047752, WO2013/125667, W02014/030728, W02014/163101, WO2013/081143, W02007/114319, W02009/041643, WO2014/145159, WO2012/016227, or WO2012/093704, each of which is incorporated herein by reference in their entirety.

[0322] In one embodiment, the nucleic acids encoding embodiments of Disclosure A or B may be operably cloned (inserted) into appropriate vectors and introduced into host cells. For example, when E. coli is used as a host, vectors include the cloning vector, pBluescript vector (Stratagene) or any of various other commercially available vectors.

[0323] In one embodiment, expression vectors are useful as vectors containing a nucleic acid for Disclosure A or B. Expression vectors can be used to allow polypeptide expression in vitro, in E. coli, in culture cells, or in vivo. For example, it is possible to use pBEST vector (Promega) for in vitro expression; pET vector (Invitrogen) for E. coli expression; pME18S-FL3 vector (GenBank Accession No. AB009864) for culture cell expression; and pME18S vector (Takebe et al., Mol. Cell Biol. 8:466-472 (1988)) for in vivo expression. DNAs can be inserted into vectors by conventional methods, for example, by ligase reaction using restriction enzyme sites (see, Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0324] In an alternative embodiment, Disclosure A relates to a host or host cells that comprise a vector containing a nucleic acid encoding an above-described ion concentration-dependent antigen-binding domain with increased pi or an above-described ion concentration-dependent antibody with increased pi. In a specific embodiment, the host or host cells can be prepared by, for example, methods described in WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722,

WO2013/046704, W02000/042072, W02006/019447, WO2012/115241,

WO2013/047752, WO2013/125667, WO2014/030728, W02014/163101, W02013/081143, W02007/114319, W02009/041643, WO2014/145159,

WO2012/016227, or WO2012/093704, each of which is incorporated herein by reference in their entirety.

[0325] The type of host cell of Disclosure A or B is not particularly limited, and the host cells include, for example, bacterial cells such as E. coli, as well as various animal cells. The host cells can be appropriately used as production systems for producing and

109

WO 2017/046994

PCT/JP2016/003616 expressing the antibodies. Both eukaryotic and prokaryotic cells can be used.

[0326] Eukaryotic cells for use as host cells include, for example, animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells, for example,

CHO (Puck et al., J. Exp. Med. 108:945-956 (1995)), COS, HEK293, 3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero; amphibian cells, for example, Xenopus oocyte (Valle et al., Nature 291:338-340 (1981)); and insect cells, for example, Sf9, Sf21, and Tn5. Recombinant vectors or others can be introduced into host cells, for example, using calcium phosphate methods, DEAE-dextran methods, methods using cationic liposome DOTAP (Boehringer-Mannheim), electroporation, and lipofection.

[0327] Plant cells that are known to serve as a protein production system include, for example, Nicotiana tabacum-derived cells and duckweed (Lemna minor)-derived cells. Calluses can be cultured from these cells to produce antibodies of Disclosure A or B. Fungal cell-based protein production systems include those using yeast cells, for example, cells of genus Saccharomyces such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; and cells of filamentous fungi, for example, genus Aspergillus such as Aspergillus niger. When prokaryotic cells are used, bacterial cellbased production systems can be used. Bacterial cell-based production systems include, for example, those using Bacillus subtilis as well as E. coli.

[0328] To produce an antibody of Disclosure A or B using host cells, the host cells are transformed with an expression vector containing a nucleic acid encoding an antibody of Disclosure A or B and cultured to express the nucleic acid. For example, when animal cells are used as a host, culture media may include, for example, DMEM,

MEM, RPMI1640, and IMDM, which may be appropriately used in combination with serum supplements such as FBS or fetal calf serum (FCS). Alternatively, the cells may be cultured serum free.

[0329] On the other hand, animals or plants can be used for in vivo production systems for producing antibodies of Disclosure A or B, For example, a nucleic acid(s) encoding an antibody of Disclosure A or B can be introduced into such animals or plants to produce the antibody in vivo, and the antibody can then be collected from the animals or plants.

[0330] When animals are used as a host, production systems using mammals or insects are available. Preferred mammals include, but are not limited to, goats, pigs, sheep, mice, and bovines (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Transgenic animals can also be used.

[0331] In one example, a nucleic acid encoding an antibody of Disclosure A or B is prepared as a fusion gene with a gene encoding a polypeptide that is specifically included in milk, such as goat β-casein. Then, goat embryos are injected with a polynucleotide fragment containing the fusion gene and transplanted into a female goat. The antibody of interest can be obtained from milk produced by the transgenic goats, which are bom

110

WO 2017/046994

PCT/JP2016/003616 from goats that received the embryos, or from their offspring. Hormones can be appropriately administered to the transgenic goats to increase the volume of milk containing the antibody produced by the goats (Ebert et al., Bio/Technology 12:699-702 (1994)).

[0332] Insects for use in producing antibodies of Disclosure A or B include, for example, silkworms. When silkworms are used, baculoviruses whose viral genome is inserted with a polynucleotide encoding an antibody of interest is used to infect the silkworm. The antibody of interest can be obtained from the body fluids of the infected silkworms (Susumu et al., Nature 315:592-594 (1985)).

[0333] When plants are used for producing antibodies of Disclosure A or B, tobacco may be used. When tobacco is used, a recombinant vector resulting from insertion of a polynucleotide encoding an antibody of interest into a plant expression vector, for example, pMON 530 may be introduced into bacteria such as Agrobacterium tumefaciens. The resulting bacteria can be used to infect tobacco, for example, Nicotiana tabacum (Ma et al., Eur. J. Immunol. 24:131-138 (1994)) and the desired antibody is obtained from the leaves of the infected tobacco. Such modified bacteria can be also used to infect duckweed (Lemna minor), and the desired antibody is obtained from cloned cells of the infected duckweed (Cox et al., Nat. Biotechnol. 24(12):1591-1597 (2006)).

[0334] In order to secrete the antibody which is expressed in the host cells into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, suitable secretion signals may be incorporated into the polypeptide of interest. Such signals may be endogenous to the antibody of interest or may be a heterogeneous signal known in the art.

[0335] The antibody of Disclosure A or B produced as described above may be isolated from the inside or outside (such as media and milk) of host cells or a host, and purified to a substantially pure and homogenous antibody. The antibodies can be suitably isolated and purified, for example, by appropriately selecting and combining chromatographic columns, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and others. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. Such chromatography can be performed, for example, by using liquid chromatography such as HPLC and FPLC. Columns for use in affinity chromatography may be Protein A column or Protein G column. Protein A column include, for example, Hyper D, POROS, Sepharose F.F. (Pharmacia).

[0336] The antibody can be modified or the peptide can be partially deleted by treating the antibody with appropriate protein modifying enzymes before or after antibody puIll

WO 2017/046994

PCT/JP2016/003616 rification, as necessary. For such protein modifying enzymes, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinases, and glucosidases can be used.

[0337] In an alternative embodiment, Disclosure A relates to methods for producing antibodies containing an antigen-binding domain whose antigen-binding activity changes according to the ion concentration condition, which may comprise culturing the host cells or raising the hosts and collecting antibodies from cultures of these cells, materials secreted from the hosts, or by other means known in the art.

[0338] In one embodiment, Disclosure A relates to a production method which comprises any one or more steps selected from the group consisting of: (a) selecting an antibody which can promote elimination of an antigen from plasma; (b) selecting an antibody with enhanced binding activity to an extracellular matrix; (c) selecting an antibody with enhanced FcyR-binding activity under a neutral pH condition; (d) selecting an antibody with enhanced FcyRIIb-binding activity under a neutral pH condition; (e) selecting an antibody with maintained or enhanced FcyRIIb-binding activity and decreased binding activity to one or more activating FcyR selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb, and FcyRIIa; (f) selecting an antibody with enhanced FcRn-binding activity under a neutral pH condition; (g) selecting an antibody with an increased pi; (h) confirming the pi of the collected antibody, and then selecting an antibody with an increased pi; and (i) selecting an antibody whose antigen-binding activity is changed or increased according to ion concentration conditions, as compared to a reference antibody.

[0339] Here, the reference antibody includes, but is not limited to, a native antibody (for example, a native Ig antibody, preferably a native IgG antibody) and an antibody before modification (an antibody prior to or during library construction, for example, an ion concentration-dependent antibody prior to increasing its pi, or an antibody with increased pi prior to conferring an ion concentration-dependent antigen-binding domain).

[0340] After producing antibodies of Disclosure A, the resulting antibodies may be assessed by antibody pharmacokinetic assay using plasma such as of mice, rats, rabbits, dogs, monkeys, humans, to select antibodies with enhanced antigen elimination from plasma as compared to the reference antibody.

[0341] Alternatively, after producing antibodies of Disclosure A, the resulting antibodies may be compared with a reference antibody in terms of the extracellular matrixbinding ability by electrochemiluminescence or others to select antibodies with increased binding to extracellular matrix.

[0342] Alternatively, after producing antibodies of Disclosure A, the resulting antibodies may be compared with a reference antibody in terms of the binding activity to various FcyRs under a neutral pH condition using BIACORE(registered trademark) or others

112

WO 2017/046994

PCT/JP2016/003616 to select antibodies with increased binding activity to various FcyRs under the neutral pH condition. In this case, the various FcyRs may be a type of FcyR of interest, for example, FcyRIIb. Similarly, it is also possible to select antibodies whose FcyRIIbbinding activity (under a neutral pH condition) has been maintained or increased and their binding activity to one or more activating FcyR selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb and FcyRIIa, and so on, has been reduced. In such cases, FcyR can be human FcyR.

[0343] Alternatively, after producing antibodies of Disclosure A, the resulting antibodies may be compared with a reference antibody in terms of the FcRn-binding activity under a neutral pH condition using BIACORE or other known techniques to select antibodies with increased FcRn-binding activity under the neutral pH condition. In this case, the FcRn can be human FcRn.

[0344] Alternatively, after producing antibodies of Disclosure A, the resulting antibodies may be evaluated for their pi by isoelectric focusing or others to select antibodies with increased pi as compared to the reference antibody. In this case, it is possible to select antibodies whose pi value has been increased, for example, by at least 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 or more, or at least 0.6, 0.7, 0.8, or 0.9 or more; or antibodies whose pi value has been increased by at least 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 or more, or at least 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 or more, or 3.0 or more.

[0345] Alternatively, after producing antibodies of Disclosure A, the resulting antibodies may be compared with a reference antibody in terms of binding activity to a desired antigen under low and high ion concentration conditions using BIACORE or others to select antibodies whose antigen-binding activity have been changed or increased according to the ion concentration condition. The ion concentration may be, for example, hydrogen ion concentration or metal ion concentration. When the ion concentration is a metal ion concentration, it can be, for example, calcium ion concentration. Whether the binding activity has been changed or increased may be assessed based on the presence of, for example: (a) an altered or enhanced antigen uptake by cells; (b) an altered or increased ability to bind to different antigen molecules multiple times; (c) an altered or enhanced reduction of antigen concentration in plasma; or (d) an altered plasma retention of the antibody. Alternatively, any two or more of these selection methods may be appropriately combined, if needed.

[0346] In an alternative embodiment, Disclosure A relates to methods for producing or screening for antibodies that contain an antigen-binding domain whose antigen-binding activity changes according to the ion concentration condition and whose pi has been increased by modifying at least one amino acid residue that can be exposed on the antibody surface (ion concentration-dependent antibodies with increased pi). The production methods can be performed, for example, by appropriately combining as

113

WO 2017/046994

PCT/JP2016/003616 needed, the related embodiments described within the scope of Disclosure A herein, for example, the embodiment of methods for producing or screening for antibodies with increased pi described above, as well as the embodiment of methods for producing or screening for calcium ion concentration-dependent antigen-binding domains or calcium ion concentration-dependent antibodies whose antigen-binding activity is higher under a high calcium ion concentration condition than under a low calcium ion concentration condition, or libraries thereof described above and/or the embodiment of methods for producing or screening for pH-dependent antigen-binding domains or pH-dependent antibodies whose antigen-binding activity is higher in a neutral pH condition than in an acidic pH condition, or libraries thereof described above.

[0347] In an alternative embodiment, Disclosure A provides a method for producing or screening for an antibody containing an antigen-binding domain whose extracellular matrix-binding activity has been increased, wherein its antigen-binding activity changes according to the ion concentration condition and its pi has been increased by modifying at least one amino acid residue that can be exposed on the antibody surface (an ion concentration-dependent antibody with increased pi). Increase in pi of an ion concentration-dependent antibody may be contemplated in the method. Such method can be performed, for example, by appropriately combining as needed, related embodiments described within the scope of Disclosure A herein, for example, the embodiment of method for producing or screening for antibodies with an increased pi described above, as well as the embodiment of methods for producing or screening for calcium ion concentration-dependent antigen-binding domains or calcium ion concentration-dependent antibodies whose antigen-binding activity is higher under a high calcium ion concentration condition than under a low calcium ion concentration condition, or libraries thereof described above and/or the embodiment of methods for producing or screening for pH-dependent antigen-binding domains or pH-dependent antibodies whose antigen-binding activity is higher under a neutral pH condition than under an acidic pH condition, or libraries thereof described above. For example, the resulting antibodies may be compared with a reference antibody in terms of the extracellular matrix-binding ability by electrochemiluminescence or other known techniques to select antibodies with increased extracellular matrix binding.

[0348] Here, the reference antibody may include, but is not limited to, a native antibody (for example, a native Ig antibody, preferably a native IgG antibody) and an antibody before modification (an antibody prior to or during library construction, for example, an ion concentration-dependent antibody before its pi is increased or an antibody with increased pi before it is conferred with an ion concentration-dependent antigen-binding domain).

114

WO 2017/046994 PCT/JP2016/003616 [0349] In an alternative embodiment, Disclosure A relates to a method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, wherein the method comprises modifying at least one amino acid residue that may be exposed on the surface of the antibody so as to increase the isoelectric point (pi). In some embodiments, the amino acid residue modification comprises a modification selected from the group consisting of: (a) substitution of a negatively charged amino acid residue with an uncharged amino acid residue; (b) substitution of a negatively charged amino acid residue with a positively charged amino acid residue; and (c) substitution of an uncharged amino acid residue with a positively charged amino acid residue. In some embodiments, at least one modified amino acid residue is substituted with histidine. In further embodiments, the antibody comprises a variable region and/or a constant region, and an amino acid residue is modified in the variable region and/or the constant region. In further embodiments, at least one amino acid residue modified according to the method is in a position in a CDR or FR selected from the group consisting of: (a) position 1, 3, 5, 8, 10, 12, 13, 15, 16, 18, 19, 23, 25, 26, 39, 41, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 77, 81, 82, 82a, 82b, 83, 84, 85, 86, 105, 108, 110, and 112 in a FR of the heavy chain variable region; (b) position 31,61, 62, 63, 64, 65, and 97 in a CDR of the heavy chain variable region; (c) position 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 37, 38, 39, 41, 42, 43, 45, 46, 49, 57, 60, 63, 65, 66, 68, 69, 70, 74, 76, 77, 79, 80, 81, 85, 100, 103, 105, 106, 107, and 108 in a FR of the light chain variable region; and (d) position 24, 25,

26, 27, 52, 53, 54, 55, and 56 in a CDR of the light chain variable region, according to Rabat numbering. In yet further embodiments, at least one amino acid residue modified according to the method is in a position in a CDR or FR selected from the group consisting of (a) position 8, 10, 12, 13, 15, 16, 18, 23, 39, 41, 43, 44, 77, 82,

82a, 82b, 83, 84, 85, and 105 in a FR of the heavy chain variable region; (b) position 31,61, 62, 63, 64, 65, and 97 in a CDR of the heavy chain variable region; (c) position 16, 18, 37, 41, 42, 45, 65, 69, 74, 76, 77, 79, and 107 in a FR of the light chain variable region; and (d) position 24, 25, 26, 27, 52, 53, 54, 55, and 56 in a CDR of the light chain variable region. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of an antibody produced according to the method for its corresponding antigen in an acidic pH (e.g., pH 5.8) and a neutral pH (e.g., pH 7.4). In further embodiments, the method comprises selecting an antibody that has a KD (acidic pH range (e.g., pH 5.8)) / KD (neutral pH range (e.g., pH 7.4)), for the antigen of 2 or higher. In some embodiments, the method further comprises comparing the antigen binding activity of an antibody produced according to the method under a high ion concentration (e.g., a hydrogen ion or calcium ion concentration) and a low ion concentration condition. In further em115

WO 2017/046994

PCT/JP2016/003616 bodiments the method further comprises selecting an antibody that has a higher antigen binding activity under a high ion concentration (e.g., 2-fold) than under a low ion concentration. In some embodiments, where the ion concentration is calcium ion concentration, the high calcium ion concentration may be selected between 100 pM and 10 mM, between 200 μΜ and 5 mM, between 400 μΜ and 3 mM, between 200 μΜ and 2 mM, or between 400 μΜ and 1 mM. A concentration selected between 500 uM and 2.5 mM, which is close to the plasma (blood) concentration of calcium ion in vivo, may be also preferred. In some embodiments, the low calcium ion concentration may be selected between 0.1 μΜ and 30 μΜ. between 0.2 pM and 20 pM, between 0.5 pM and 10 pM, or between 1 pM and 5 pM, or between 2 pM and 4 pM. A concentration selected between 1 pM and 5 pM, which is close to the concentration of calcium ion in early endosomes in vivo, may be also preferred. In some embodiments, the lower limit of the KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (e.g., KD (3 pM Ca)/KD (2 mM Ca)) value is 2 or more, 10 or more, or 40 or more, and the upper limit thereof is 400 or less, 1000 or less, or 10000 or less. In alternative some embodiments, the lower limit of the kd (low calcium ion concentration conditioned (high calcium ion concentration condition) (e.g., kd (3 pM Ca)/kd (2 mM Ca)) value is 2 or more, 5 or more, 10 or more, or 30 or more, and the upper limit thereof is 50 or less, 100 or less, or 200 or less. In some embodiments, where the ion concentration is hydrogen ion concentration, low hydrogen ion concentration (neutral pH range) may be selected from pH 6.7 to pH 10.0, from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0. The low hydrogen ion concentration may be preferably pH 7.4 which is close to the in vivo pH in plasma (blood), but for the convenience of measurement, for example, pH 7.0 may be used. In some embodiments, high hydrogen ion concentration (acidic pH range) may be selected from pH 4.0 to pH 6.5, from pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. The acidic pH range may be preferably pH 5.8 which is close to the in vivo hydrogen ion concentration in the early endosome, but for the convenience of measurement, for example, pH 6.0 may be used. In some embodiments, the lower limit of KD (acidic pH range)/KD (neutral pH range) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more, and the upper limit thereof is 400 or less, 1000 or less, or 10000 or less. In some embodiments, the method further comprises comparing the elimination of antigen from plasma after the administration of an antibody produced according to the method as compared to that when a reference antibody which differs only in that it does not include the modification(s) introduced according to the method, is administered. In further embodiments, the method further comprises selecting an antibody produced according to the method that promotes elimination of the antigen from plasma (e.g., 2-fold) as compared to an antibody that does not contain the modi116

WO 2017/046994

PCT/JP2016/003616 fications introduced according to the method. In some embodiments, the method further comprises comparing extracellular matrix-binding of the antibody produced according to the method as compared to the antibody which differs only in that it does not include the modification(s) introduced according to the method. In further embodiments the method further comprises selecting an antibody produced according to the method that has increased extracellular matrix-binding (e.g., 2-fold when bound to an antigen) as compared to an antibody which differs only in that it does not include the modification(s) introduced according to the method. In further embodiments, the antibodies produced according to the method (substantially) retain the antigen-binding activity when compared to the antibodies before modification or alteration of at least one amino acid residue to increase pi (native antibodies (for example, native Ig antibodies, preferably native IgG antibodies) or reference antibodies (e.g., antibodies before antibody modification, or prior to or during library construction)). In this case, to (substantially) retain the antigen-binding activity can mean to have an activity of at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and still more preferably 80%, 85%, 90%, or 95% or more as compared to the binding activity of the antibodies before modification or alteration.

[0350] In an additional embodiment, Disclosure A relates to a method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, wherein the method comprises modifying at least one amino acid residue that may be exposed on the surface of a constant region of an antibody so as to increase the isoelectric point (pi). In some embodiments, the amino acid residue modification comprises a modification selected from the group consisting of: (a) substitution of a negatively charged amino acid residue with an uncharged amino acid residue; (b) substitution of a negatively charged amino acid residue with a positively charged amino acid residue; and (c) substitution of an uncharged amino acid residue with a positively charged amino acid residue. In some embodiments, at least one modified amino acid residue is substituted with histidine. In further embodiments, the antibody comprises a variable region and/or a constant region, and an amino acid residue is modified in the variable region and/or the constant region. In further embodiments, at least one amino acid residue modified according to the method is in a position in a constant region selected from the group consisting of position 196, 253, 254, 256, 258, 278, 280, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU numbering. In further embodiments, at least one amino acid residue modified according to the method is in a position in a constant region selected from the group consisting of position 254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 342,

117

WO 2017/046994 PCT/JP2016/003616

343, 345, 356, 358, 359, 361, 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434, and 443, according to EU numbering. In yet further embodiments, at least one amino acid residue modified according to the method is in a position in a constant region selected from the group consisting of position 282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and 413, according to EU numbering. In some embodiments, the method further comprises comparing the antigen binding activity of an antibody produced according to the method under a high ion concentration (e.g., a hydrogen ion or calcium ion concentration) and a low ion concentration condition. In further embodiments the method further comprises selecting an antibody that has a higher antigen binding activity under a high ion concentration than under a low ion concentration. In some embodiments, the method comprises comparing the elimination of antigen from plasma after the administration of an antibody produced according to the method as compared to that when a reference antibody which differs only in that it does not include the modification(s) introduced according to the method, is administered. In further embodiments the method further comprises selecting an antibody produced according to the method that promotes elimination of the antigen from plasma (e.g., 2-fold) as compared to an antibody that does not contain the modifications introduced according to the method. In some embodiments, the method comprises comparing extracellular matrix-binding of the antibody produced according to the method as compared to the antibody which differs only in that it does not include the modification(s) introduced according to the method. In further embodiments the method further comprises selecting an antibody produced according to the method that has increased extracellular matrix-binding binding (e.g., 2-fold when bound to antigen) as compared to an antibody which differs only in that it does not include the modification(s) introduced according to the method. In some embodiments, the method comprises comparing the Fc gamma receptor (FcyR)-binding activity under neutral pH (e.g., pH 7.4) of an antibody produced according to the method with that of a reference antibody comprising a constant region of a native IgG. In further embodiments the method comprises selecting an antibody produced according to the method that has enhanced FcyR-binding activity under a neutral pH (e.g., pH 7.4) as compared to that of the reference antibody comprising a constant region of a native IgG. In some embodiments, the selected antibody produced according to the method has enhanced FcyRIIb binding activity under neutral pH. In some embodiments, the selected antibody produced according to the method has binding activity towards one or more activating FcyR, preferably selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb and FcyRIIa, and towards FcyRIIb, and optionally the FcyRIIb-binding activity is maintained or enhanced and the binding activity to the activating FcyRs is decreased, as compared to those of a reference antibody which differs

118

WO 2017/046994

PCT/JP2016/003616 only in that its constant region is that of a native IgG. In some embodiments, the method further comprises comparing FcRn-binding activity under a neutral pH condition of the antibody produced according to the method as compared to a reference antibody which differs only in that its constant region is that of a native IgG. In further embodiments, the method further comprises selecting an antibody produced according to the method that has increased FcRn-binding activity under a neutral pH condition as compared to that of a reference antibody which differs only in that its constant region is that of a native IgG (e.g., 2-fold). In some embodiments, the antibodies produced according to the method (substantially) retain the antigen-binding activity when compared to the antibodies before modification or alteration of at least one amino acid residue to increase pi (native antibodies (for example, native Ig antibodies, preferably native IgG antibodies) or reference antibodies (e.g., antibodies before antibody modification, or prior to or during library construction)). In this case, to (substantially) retain the antigen-binding activity can mean to have an activity of at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and still more preferably 80%, 85%, 90%, or 95% or more as compared to the binding activity of the antibodies before modification or alteration.

[0351] In additional embodiments, the method comprises modifying at least one amino acid residue that may be exposed on the surface of a variable region and constant region of an antibody so as to increase the isoelectric point (pi). In further embodiments, at least one amino acid residue modified according to the method is in a position in a constant region disclosed above. In further embodiments at least one amino acid residue modified according to the method is in a position in a variable region disclosed above. In further embodiments, at least one amino acid residue modified according to the method is in a position in a constant region disclosed above and at least one amino acid residue modified according to the method is in a position in a variable region disclosed above. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method further comprises comparing the KD of an antibody produced according to the method for its corresponding antigen in an acidic pH (e.g., pH 5.8) and a neutral pH (e.g., pH 7.4). In further embodiments, the method comprises selecting an antibody that has a KD (acidic pH range) / KD (neutral pH range), for the antigen of 2 or higher. In some embodiments, the method further comprises comparing the antigen binding activity of an antibody produced according to the method under a high ion concentration (e.g., a hydrogen ion or calcium ion concentration) condition and a low ion concentration condition. In further embodiments, the method further comprises selecting an antibody that has a higher antigen binding activity under a high ion concentration than under a low ion concentration. In some embodiments, where the ion concentration is calcium ion concentration, the high calcium ion concentration may be

119

WO 2017/046994

PCT/JP2016/003616 selected between 100 μΜ and 10 mM, between 200 μΜ and 5 mM, between 400 μΜ and 3 mM, between 200 μΜ and 2 mM, or between 400 μΜ and 1 mM. A concentration selected between 500 μΜ and 2.5 mM may be also preferred. In some embodiments, the low calcium ion concentration may be selected between 0.1 μΜ and 30 μΜ, between 0.2 μΜ and 20 μΜ, between 0.5 μΜ and 10 μΜ, or between 1 μΜ and 5 μΜ, or between 2 μΜ and 4 μΜ. A concentration selected between 1 μΜ and 5 μΜ may be also preferred. In some embodiments, the lower limit of the KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (e.g., KD (3 μΜ Ca)/KD (2 mM Ca)) value is 2 or more, 10 or more, or 40 or more, and the upper limit thereof is 400 or less, 1000 or less, or 10000 or less. In alternative some embodiments, the lower limit of the kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) (e.g., kd (3 μΜ Ca)/kd (2 mM Ca)) value is 2 or more, 5 or more, 10 or more, or 30 or more, and the upper limit thereof is 50 or less, 100 or less, or 200 or less. In some embodiments, where the ion concentration is hydrogen ion concentration, low hydrogen ion concentration (neutral pH range) may be selected from pH 6.7 to pH 10.0, from pH 6.7 to pH 9.5, from pH 7.0 to pH 9.0, or from pH 7.0 to pH 8.0. The low hydrogen ion concentration may be preferably pH 7.4 which is close to the in vivo pH in plasma (blood), but for the convenience of measurement, for example, pH 7.0 may be used. In some embodiments, high hydrogen ion concentration (acidic pH range) may be selected from pH 4.0 to pH 6.5, from pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. The acidic pH range may be pH 5.8 or pH 6.0, for example. In some embodiments, the lower limit of KD (acidic pH range)/KD (neutral pH range) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more, and the upper limit thereof is 400 or less, 1000 or less, or 10000 or less.

[0352] In some embodiments, the method further comprises comparing the elimination of antigen from plasma after the administration of an antibody produced according to the method as compared to that when a reference antibody which differs only in that it does not include the modification(s) introduced according to the method is administered. In further embodiments, the method further comprises selecting an antibody produced according to the method that promotes elimination of the antigen from plasma (e.g., 2-fold) as compared to an antibody that does not contain the modifications introduced according to the method. In some embodiments, the method further comprises comparing extracellular matrix-binding of the antibody produced according to the method as compared to the antibody which differs only in that it does not include the modification(s) introduced according to the method. In further embodiments, the method further comprises selecting an antibody produced according to the method that has increased extracellular matrix-binding binding (e.g., 5-fold when

120

WO 2017/046994

PCT/JP2016/003616 complexed with antigen) as compared to an antibody which differs only in that it does not include the modification(s) introduced according to the method. In some embodiments, the method further comprises comparing the Fc gamma receptor (FcyR)-binding activity under neutral pH (e.g., pH 7.4) of an antibody produced according to the method with that of a reference antibody comprising a constant region of a native IgG. In further embodiments, the method comprises selecting an antibody produced according to the method that has enhanced FcyR-binding activity under a neutral pH (e.g., pH 7.4) as compared to that of the reference antibody comprising a constant region of a native IgG. In some embodiments, the selected antibody produced according to the method has enhanced FcyRIIb binding activity under neutral pH. In some embodiments, the selected antibody produced according to the method has binding activity towards one or more activating FcyR, preferably selected from the group consisting of FcyRIa, FcyRIb, FcyRIc, FcyRIIIa, FcyRIIIb and FcyRIIa, and towards FcyRIIb, and optionally the FcyRIIb-binding activity is maintained or enhanced and the binding activity to the activating FcyRs is decreased, as compared to those of a reference antibody which differs only in that its constant region is that of a native IgG. In some embodiments, the method further comprises comparing FcRnbinding activity under a neutral pH condition of the antibody produced according to the method as compared to a reference antibody which differs only in that its constant region is that of a native IgG. In further embodiments, the method further comprises selecting an antibody produced according to the method that has increased FcRnbinding activity under a neutral pH condition (e.g., 2-fold) as compared to that of a reference antibody which differs only in that its constant region is that of a native IgG. In further embodiments, the antibodies produced according to the method (substantially) retain the antigen-binding activity when compared to the antibodies before modification or alteration of at least one amino acid residue to increase pi (native antibodies (for example, native Ig antibodies, preferably native IgG antibodies) or reference antibodies (e.g., antibodies before antibody modification, or prior to or during library construction)). In this case, to (substantially) retain the antigen-binding activity can mean to have an activity of at least 50% or more, preferably 60% or more, more preferably 70% or 75% or more, and still more preferably 80%, 85%, 90%, or 95% or more as compared to the binding activity of the antibodies before modification or alteration.

[0353] In an alternative embodiment, Disclosure A relates to an antibody obtained by the above-described method of Disclosure A for producing or screening antibodies.

[0354] In an alternative embodiment, Disclosure A relates to a composition or pharmaceutical composition comprising an antibody of Disclosure A described above. In one embodiment, the pharmaceutical composition of Disclosure A may be a pharma121

WO 2017/046994

PCT/JP2016/003616 ceutical composition for accelerating antigen elimination from a biological fluid (preferably, plasma, etc.) of subjects and/or for increasing the extracellular matrix binding (when an antibody of Disclosure A is administered to (applied to) the subject (preferably, in vivo)). The pharmaceutical composition of Disclosure A may optionally contain a pharmaceutically acceptable carrier. Herein, pharmaceutical compositions may typically refer to agents for use in treatment, prevention, diagnosis, or examination of diseases.

[0355] The compositions or pharmaceutical compositions of Disclosure A can be suitably formulated. In some embodiments, they can be used parenterally, for example, in a form of a sterile solution or suspension for injection in water or any other pharmaceutically acceptable liquid. The compositions can be suitably formulated at a unit dose required for generally accepted pharmaceutical practice, by appropriately combining with pharmaceutically acceptable carriers or media. Such pharmaceutically acceptable carriers or media include, but are not limited to, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, and binding agents. The amount of active ingredient in the compositions may be adjusted in such a way that the dose falls within an appropriate pre-determined range.

[0356] In some embodiments, the compositions or pharmaceutical compositions of

Disclosure A can be administered parenterally. The compositions or pharmaceutical compositions may be appropriately prepared as, for example, an injectable, transnasal, transpulmonary, or transdermal composition. The compositions or pharmaceutical compositions may be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0357] In some embodiments, the disclosure provides antibodies whose pi is increased by modifying at least one amino acid residue that can be exposed on the surface (antibodies with increased pi); methods for producing these antibodies; or use of these antibodies to enhance antigen elimination from plasma (when the antibodies are administered to the subjects in vivo). It can be understood that the scope of Disclosure A described herein and the contents described in the counterpart Examples herein can be appropriately applied to such embodiments. In other embodiments, the disclosure provides antibodies whose pi is decreased by modifying at least one amino acid residue that can be exposed on the surface (antibodies with decreased pi); methods for producing these antibodies; or use of these antibodies to improve plasma retention (when the antibodies are administered to the subjects in vivo). The inventors have revealed that cellular internalization of an antibody can be enhanced by increasing its pi by introducing specific amino acid mutations into specific sites in the amino acid sequence of the constant region. Those of ordinary skill in the art can understand that

122

WO 2017/046994

PCT/JP2016/003616 antibody plasma retention can be prolonged as the pi has been reduced to suppress cellular internalization of the antibody by introducing amino acids with a different side-chain charge property into the sites described above. It can be understood that the scope of Disclosure A described herein and the contents described in the counterpart Examples herein can be appropriately applied to such embodiments.

[0358] In one embodiment, the disclosure provides a method for producing a modified antibody, whose half life in plasma is prolonged or reduced, as compared to that before the modification of the antibody, wherein the method comprises: (a) modifying a nucleic acid encoding the antibody before the modification to change the charge of at least one amino acid residue located at a position selected from the group consisting of position 196, 253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315, 327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU numbering; (b) culturing a host cell to express the modified nucleic acid and to produce the antibody; and (c) collecting the produced antibody from the host cell culture.

[0359] An additional embodiment provides a method for prolonging or reducing the half-life of an antibody in plasma wherein the method comprises modifying at least one amino acid residue located at a position selected from the group consisting of position 196,

253, 254, 256, 257, 258, 278, 280, 281, 282, 285, 286, 306, 307, 308, 309, 311, 315,

327, 330, 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388,

389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443, according to EU numbering.

[0360] These methods may further comprise determining that the half life in plasma of the collected and/or modified antibody is prolonged or reduced, as compared to that before the modification of the antibody.

[0361] The change of charge may be achieved by amino acid substitution(s). In some embodiments, the substituted amino acid residue(s) may be selected from the group consisting of the amino acid residues of group (a) and (b) below, but is not limited thereto: (a) Glu (E) and Asp (D); and (b) Lys (K), Arg (R) and His (H).

[0362] In some embodiments, the antibody may be an Ig-type antibody such as an IgG antibody. In some embodiments, the antibody may be a chimeric antibody, humanized antibody, or human antibody. In some embodiments, the antibody may be a multispecific antibody such as a bispecific antibody.

[0363] Disclosure B

In non-limited embodiments, Disclosure B relates to Fc region variants, uses thereof, and production methods thereof.

[0364] Within the scope of Disclosures A and B described herein, an Fc region variant

123

WO 2017/046994

PCT/JP2016/003616 may refer, for example, to an Fc region modified from the Fc region of a native IgG antibody by modifying at least one amino acid with another amino acid, or may refer to an Fc region modified from such an Fc region variant by additionally modifying at least one amino acid with another amino acid. Herein, such Fc region variants include not only Fc regions that have been introduced with the amino acid modification but also Fc regions containing the same amino acid sequence as an aforementioned Fc region.

[0365] In an alternative embodiment, Disclosure B relates to Fc region variants containing an FcRn-binding domain which contains Ala at position 434; any one of Glu, Arg, Ser, and Lys at position 438; and any one of Glu, Asp, and Gin at position 440, according to EU numbering (within the scope of Disclosure B described herein, such an Fc region variant is also referred to as a novel Fc region variant for descriptive purposes).

[0366] In practice, Fc region variants of Disclosure B can be incorporated into virtually any antibody (e.g., multispecific antibodies such as bispecific antibodies) regardless of the type of the target antigen. For example, Anti-factor IXa/factor X bispecific antibodies can be produced using such Fc region variants as shown in Example 20 (e.g., F8M-F1847mv [F8M-F1847mvl (SEQ ID NO:323) and F8M-F1847mv2 (SEQ ID NO:324) as the heavy chains and F8ML (SEQ ID NO:325) as the light chain]; F8M-F1868mv [ F8M-F1868mvl (SEQ ID NO:326) and F8M-F1868mv2 (SEQ ID NO:327) as the heavy chains and F8ML (SEQ ID NO:325) as the light chain]; and F8M-F1927mv [ F8M-F1927mvl (SEQ ID NO:328) and F8M-F1927mv2 (SEQ ID NO:329) as the heavy chains and F8ML (SEQ ID NO:325) as the light chain]).

[0367] As described above, W02013/046704 reports that Fc region variants that have been introduced with a mutation to increase their FcRn binding under acidic conditions in combination with a specific mutation (a representative example is dual-residue mutation Q438R/S440E according to EU numbering) exhibit significantly reduced binding to rheumatoid factor. However, WO2013/046704 does not describe that the Fc region variants whose rheumatoid factor binding has been reduced due to the Q438R/S440E modification are superior in plasma retention as compared to antibodies with a native Fc region. Thus, there is a demand for safe and more advantageous Fc region variants that allow improved plasma retention, but do not bind to pre-existing ADA. The inventors disclose herein safe and more advantageous Fc region variants that allow improved plasma retention, but do not bind to anti-drug antibodies (pre-existing ADA, etc.). In particular, it is first disclosed herein that surprisingly, Fc region variants that contain combined mutations of amino acid residues, which are a substitution of Ala (A) for the amino acid at position 434 according to EU numbering and a specific dual-residue mutation (a representative example is Q438R/S440E), are

124

WO 2017/046994

PCT/JP2016/003616 preferable for prolonging antibody retention in plasma while maintaining a significantly reduced binding to rheumatoid factor.

[0368] Thus, the novel Fc region variants of Disclosure B disclosed herein provides an advantageous and surprising improvement over the Fc region variants described in WO2013/046704, which is incorporated herein by reference in their entirety.

[0369] In one embodiment, Disclosure B provides novel combinations of amino acid substitutions in the FcRn-binding domain, which increase the FcRn-binding activity of antibodies in an acidic pH range and in a neutral pH range, in particular, in an acidic pH range.

[0370] In one embodiment, an Fc region variant of Disclosure B contains Ala at position 434; any one of Glu, Arg, Ser, and Fys at position 438; and any one of Glu, Asp, and Gin at position 440, according to EU numbering; and more preferably contain Ala at position 434; either Arg or Fys at position 438; and either Glu or Asp at position 440, according to EU numbering. Preferably, the Fc region variant of Disclosure B additionally contains either He or Feu at position 428, and/or any one of He, Feu, Val, Thr, and Phe at position 436, according to EU numbering. More preferably the Fc region variant contains Feu at position 428, and/or either Val or Thr at position 436, according to EU numbering.

[0371] In one embodiment, the Fc region variant of Disclosure B can be an Fc region variant of a native Ig antibody, and more preferably the Fc region variant of a native IgG (IgGl, IgG2, IgG3, or IgG4 type) antibody. The native Fc region is partly described within the scope of Disclosures A and B, herein. More specifically, in Disclosure B, the native Fc region can refer to an unmodified or naturally-occurring Fc region, and preferably, an unmodified or naturally-occurring Fc region of a native Ig antibody whose Fc region amino acid residues remain unmodified. The antibody origin of the Fc region can be an Ig such as IgM or IgG, for example, human IgGl, IgG2, IgG3, or IgG4. In one embodiment, it may be human IgGl. Meanwhile, a (reference) antibody comprising a native Fc region can refer to an antibody comprising an unmodified or naturally-occurring Fc region.

[0372] Positions 428, 434, 438, and 440 are common to Fc regions of all native human IgGl, IgG2, IgG3, and IgG4 antibodies. However, at position 436 in the Fc region, native human IgGl, IgG2, and IgG4 antibodies share Tyr (Y) whereas native human IgG3 antibody has Phe (F). On the other hand, Stapleton et al. (Nature Comm. 599 (2011) reported that human IgG3 allotypes containing the amino acid substitution of R435H according to EU numbering have a plasma half-life in human comparable to that of IgGl. Thus, the inventors also conceived that plasma retention could be improved by increasing FcRn binding under an acidic condition by introducing the R435H amino acid substitution in combination with the amino acid substitution at

125

WO 2017/046994

PCT/JP2016/003616 position 436.

[0373] W02013/046704 also specifically reported dual amino acid residue substitutions of

Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D according to EU numbering, which result in a significant reduction of the rheumatoid factor binding when combined with an amino acid substitution that can increase the FcRn binding under an acidic condition.

[0374] Thus, in a preferred embodiment, the FcRn-binding domain of an Fc region variant of Disclosure B may contain a combination of substituted amino acid positions selected from the group consisting of: (a) N434A/Q438R/S440E; (b) N434A/Q438R/ S440D; (c) N434A/Q438K/S440E; (d) N434A/Q438K/S440D; (e) N434A/Y436T/ Q438R/S440E; (f) N434A/Y436T/Q438R/S440D; (g) N434A/Y436T/Q438K/S440E; (h) N434A/Y436T/ Q438K/S440D; (i) N434A/Y436V/Q438R/S440E; (j) N434A/Y436V/ Q438R/S440D; (k) N434A/Y436V/Q438K/S440E; (1) N434A/Y436V/Q438K/S440D; (m) N434A/R435H/ F436T/Q438R/S440E; (n) N434A/R435H/F436T/Q438R/S440D; (o) N434A/R435H/ F436T/Q438K/S440E; (p) N434A/R435H/F436T/Q438K/S440D; (q) N434A/ R435H/F436V/Q438R/S440E; (r) N434A/R435H/F436V/Q438R/S440D; (s) N434A/ R435H/F436V/Q438K/S440E; (t) N434A/R435H/F436V/Q438K/S440D; (u) M428F/ N434A/Q438R/S440E; (v) M428F/N434A/Q438R/S440D; (w) M428F/N434A/ Q438K/ S440E; (x) M428F/N434A/Q438K/S440D; (y) M428F/N434A/Y436T/Q438R/ S440E; (z) M428F/N434A/Y436T/Q438R/S440D; (aa) M428F/N434A/Y436T/Q438K/ S440E; (ab) M428F/N434A/Y436T/Q438K/S440D; (ac) M428F/N434A/Y436V/Q438R/ S440E; (ad) M428F/N434A/Y436V/Q438R/S440D; (ae)

M428F/N434A/Y436V/Q438K/ S440E; (af) M428F/N434A/Y436V/Q438K/S440D; (ag) F235R/G236R/S239K/M428F/ N434A/ Y436T/Q438R/S440E; and (ah) F235R/G236R/A327G/A330S/P331S/M428F/ N434A/ Y436T/Q438R/S440E, according to EU numbering.

[0375] In a further preferred embodiment, the FcRn-binding domain of an Fc region variant of Disclosure B may contain a combination of substituted amino acids selected from the group consisting of: (a) N434A/Q438R/S440E; (b) N434A/Y436T/Q438R/ S440E; (c) N434A/Y436V/Q438R/S440E; (d) M428F/N434A/Q438R/S440E; (e) M428F/N434A/Y436T/Q438R/S440E; (f) M428F/N434A/ Y436V/Q438R/S440E; (g) F235R/G236R/S239K/M428F/N434A/Y436T/Q438R/S440E; and (h) F235R/G236R/ A327G/A330S/P331S/M428F/N434A/Y436T/Q438R/S440E, according to EU numbering.

[0376] In one embodiment, it is preferable that the FcRn-binding activity of an Fc region variant of Disclosure B has been increased under an acidic pH condition as compared to the Fc region of a native IgG.

126

WO 2017/046994 PCT/JP2016/003616 [0377] An increase in the FcRn-binding activity (binding affinity) of an FcRn-binding domain in a pH range may correspond to an increase of the measured FcRn-binding activity (binding affinity) when compared to the measured FcRn-binding activity (binding affinity) of a native FcRn-binding domain. In this case, KD (native Fc region)/KD (an Fc region variant of Disclosure B), which represents a difference in the binding activity (binding affinity), may be at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 70-fold, 80-fold, 100-fold, 500-fold, or 1000-fold. Such an increase may occur in an acidic pH range and/or in a neutral pH range; however, the increase in an acidic pH range can be preferred from the viewpoint of the action mechanism for Disclosure B.

[0378] In some embodiments, the FcRn-binding activity (for example, at pH 6.0 and 25°C) of an Fc region variant of Disclosure B whose FcRn-binding activity has been increased in an acidic pH range is greater than that of the Fc region of a native IgG, for example, by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more. In some embodiments, the increased FcRn-binding activity of an Fc region variant in an acidic pH range may be greater than the FcRn-binding activity of the Fc region of a native IgG by at least 5-fold or at least 10-fold.

[0379] Manipulation of the FcRn-binding domain by introducing amino acid substitutions can occasionally reduce antibody stability (W02007/092772). Proteins with poor stability tend to aggregate easily during storage, and the stability of pharmaceutical proteins is highly important in production of pharmaceutical agents. Thus, the decrease in stability caused by substitutions in the Fc region can lead to difficulty in developing stable antibody preparations (W02007/092772).

[0380] The purity of pharmaceutical proteins in terms of monomer and highmolecular-weight species is also important in developing pharmaceutical agents. After purification with Protein A, wild-type IgGl does not contain a significant amount of high-molecular-weight species, whereas manipulation of the FcRn-binding domain by introducing substitutions can produce a large amount of high-molecular-weight species. In this case, such high-molecular-weight species may have to be removed from the drug substance by purification steps.

[0381] Amino acid substitutions in antibodies can result in negative consequences, such as an increase in the immunogenicity of therapeutic antibodies which in turn can cause a cytokine storm and/or production of anti-drug antibodies (ADAs). The clinical utility and efficacy of therapeutic antibodies can be limited by ADAs, since they affect the efficacy and pharmacokinetics of therapeutic antibodies and sometimes lead to serious side effects. Many factors influence the immunogenicity of therapeutic antibodies, and the presence of effector T-cell epitopes is one of the factors. Fikewise, the presence of

127

WO 2017/046994

PCT/JP2016/003616 pre-existing antibodies against a therapeutic antibody can also be problematic. An example of such pre-existing antibody is rheumatoid factor (RF), an auto-antibody (an antibody directed against a self-protein) against the Fc portion of an antibody (i.e., IgG). Rheumatoid factor is found in particular in patients with systemic lupus erythematosus (SFE) or rheumatoid arthritis. In arthritis patients, RF and IgG join to form immune complexes that contribute to the disease process. Recently, a humanized antiCD4 IgGl antibody with a Asn434His mutation has been reported to elicit significant rheumatoid factor binding (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290 (2011)). Detailed studies have confirmed that the Asn434His mutation in human IgGl increases the binding of rheumatoid factor to the Fc region of the antibody as compared to the parental human IgGl.

[0382] RF is a polyclonal auto-antibody against human IgG. The RF epitope in the human IgG sequence varies among clones; however, the RF epitope seems to be located in the CH2/CH3 interface region as well as in the CH3 domain which may overlap with the FcRn-binding epitope. Thus, mutations to increase the FcRn-binding activity at a neutral pH may possibly increase the binding activity to specific RF clones as well.

[0383] In the context of Disclosure B, the term anti-drug antibody or ADA can refer to an endogenous antibody that has binding activity to an epitope located on a therapeutic antibody and thus can bind to the therapeutic antibody. The term pre-existing antidrug antibody or pre-existing ADA can refer to an anti-drug antibody that is present and detectable in the blood of a patient prior to administration of the therapeutic antibody to the patient. In some embodiments, the pre-existing ADA is a human antibody. In further embodiments, the pre-existing ADA is rheumatoid factor.

[0384] The binding activity of an antibody Fc region (variant) against a pre-existing ADA can be, for example, represented by electrochemiluminescence (ECF) response at an acidic pH and/or at a neutral pH. The ECF assay is described, for example, in Moxness et al. (Clin Chem. 51:1983-1985 (2005)) and in Example 6. Assays can be performed, for example, under the conditions of MES buffer and 37 °C. The antigen-binding activity of antibodies can be determined by, for example, BIACORE(registered trademark) analysis.

[0385] The binding activity to a pre-existing ADA can be assessed at any temperature from 10°C to 50°C. In some embodiments, the binding activity (binding affinity) of a human Fc region to a human pre-existing ADA is determined at a temperature of 15 °C to 40°C, for example, such as between 20°C to 25°C, or 25°C. In a further embodiment, the interaction between a human pre-existing ADA and a human Fc region is measured at pH 7.4 (or pH 7.0) and 25°C.

[0386] Within the scope of Disclosure B described herein, the binding activity to (pre-existing) ADA has been significantly increased or an equivalent expression may

128

WO 2017/046994

PCT/JP2016/003616 mean that the measured (pre-existing) ADA-binding activity (binding affinity) (i.e., KD) of an Fc region variant of Disclosure B or an antibody containing it has been increased, for example, by 0.55-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, or 2.3-fold or more, as compared to the measured (pre-existing) ADA-binding activity (binding affinity) of a reference Fc region variant or a reference antibody containing the reference Fc region variant. Such an increase in the binding activity to a pre-existing ADA can be observed in an individual patient or in a patient group.

[0387] In one embodiment, as used in the context of Disclosure B, the term patients or a patient is not limited, and can include all humans with a disease who are under treatment with a therapeutic antibody. The patients may be humans affected with autoimmune disease, such as an arthritic disease or systemic erythematosus (SFE). The arthritic disease can include rheumatoid arthritis.

[0388] In one embodiment of Disclosure B, the binding activity to a pre-existing ADA is significantly increased in an individual patient may mean that the binding activity of an antibody comprising an Fc region variant (e.g., therapeutic antibody) to a pre-existing ADA measured in a patient has been increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% or more when compared to the binding activity of a reference antibody to the pre-existing ADA. Alternatively, this may mean that the ECF reaction for the antibody is preferably above 250, or at least 500, or at least 1000, or even at least 2000. Preferably, this increase may be an increase relative to a reference antibody whose ECF reaction is less than 500 or 250. Specifically, between the binding activity of a reference antibody to a pre-existing ADA and such binding activity of an antibody having an Fc region variant, the ECF reaction preferably ranges from less than 250 to at least 250, less than 250 to at least 500, less than 500 to 500 or more, less than 500 to 1000 or more, or less than 500 to at least 2000, without being limited thereto.

[0389] In one embodiment, the binding activity to a pre-existing ADA is increased can mean that in a group of patients, the measured proportion of patients who have an ECF reaction of at least 500 (preferably, at least 250) for an antibody comprising an Fc region variant with (a) increased binding activity to FcRn at an acidic pH and (b) increased binding activity to a pre-existing ADA at a neutral pH is elevated as compared to the proportion of patients who have an ECF reaction of at least 500 (preferably, at least 250 or more) for a reference antibody, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50% when compared to the proportion of patients who have an ECF reaction for a reference antibody.

[0390] In one embodiment of Disclosure B, the binding activity to a pre-existing ADA

129

WO 2017/046994

PCT/JP2016/003616 decreases can mean that the measured binding activity (i.e., KD or ECL reaction) of an antibody comprising an Fc region variant decreases as compared to that of a reference antibody. Such decrease can be observed in an individual patient or in a group of patients. The affinity of an antibody comprising an Fc region variant for a pre-existing ADA at a neutral pH significantly decreases in each patient can mean that the measured binding activity to a pre-existing ADA at a neutral pH measured in the patient is decreased as compared to the binding activity of a reference antibody to the pre-existing ADA measured at the neutral pH, for example, by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0391] Alternatively, the binding activity of an antibody containing an Fc region variant to a pre-existing ADA significantly decreases in an individual patient can mean that the ECF reaction for the antibody that used to be 500 or more (preferably, 1000 or more, or 2000 or more) is measured to be less than 500, preferably less than 250 as compared to the ECF reaction for a reference antibody, for example.

[0392] In a preferred embodiment, the Fc region variants of Disclosure B and antibodies comprising them have low binding activity to a pre-existing ADA at a neutral pH. Specifically, it is preferable that the binding activity of antibodies containing the Fc region variants of Disclosure B to a pre-existing ADA at a neutral pH is lower than or has not significantly been increased, as compared to the binding activity of a reference antibody containing the Fc region of a native IgG to the pre-existing ADA at a neutral pH (e.g., pH 7.4). The binding activity (binding affinity) to a pre-existing ADA is low or the affinity is at the baseline level can mean an ECF reaction of less than 500, or less than 250 in an individual patient, but is not limited thereto. The binding activity to a pre-existing ADA is low in a group of patients can mean that the ECF reaction is less than 500 in 90%, preferably 95%, and more preferably 98% of the patients in the group, for example.

[0393] It can be preferable to select Fc region variants of Disclosure B or antibodies containing them, whose binding activity to a (pre-existing) ADA in plasma at a neutral pH is not significantly increased, and whose FcRn-binding activity at a neutral pH and/ or at an acidic pH is increased. Preferably the FcRn-binding activity at an acidic pH (e.g., pH 5.8) is increased. In one embodiment, the Fc region variants preferably do not have a significantly increased binding activity to ADA under a neutral pH condition (e.g., pH 7.4) as compared to the Fc region of a native IgG, and the ADA may be a preexisting ADA, preferably rheumatoid factor (RF).

[0394] In one embodiment, it can be preferable that the Fc region variants of Disclosure B have an increased FcRn-binding activity under an acidic pH condition as compared to the Fc region of a native IgG, and as a result they exhibit reduced clearance (CF) in plasma, prolonged retention time in plasma, or prolonged half-life in plasma (t 1/2).

130

WO 2017/046994 PCT/JP2016/003616

Their correlation is known in the art.

[0395] In one embodiment, it can be preferable that the Fc region variants of Disclosure B have an increased FcRn-binding activity under an acidic pH condition but do not have a significantly increased ADA-binding activity under a neutral pH condition as compared to the Fc region of a native IgG, and they exhibit reduced clearance (CF) in plasma, prolonged retention time in plasma, or prolonged half-life in plasma (t 1/2).

The ADA may be a pre-existing ADA, preferably rheumatoid factor (RF).

[0396] In one embodiment, the Fc region variants of Disclosure B are advantageous, since their plasma retention is improved as compared to a reference Fc region variant comprising a combination of amino acid substitutions N434Y/Y436V/Q438R/S440E according to EU numbering.

[0397] Examples 5 to 7 compare the plasma retention of two Fc region variants: Fc region variant F1718 (Fc region with mutations introduced at four sites: N434Y/Y436V/Q438R/S440E) described in W02013/046704 and novel Fc region variant FI848m (introduced with mutations at four sites:

N434A/Y436V/Q438R/S440E). Difference in amino acid mutation between the two Fc region variants is only at position 434 according to EU numbering, where the introduced amino acid mutation is Y (tyrosine) for F1718 and A (alanine) for FI848m. Nevertheless, when compared to a native IgGl, F1848m exhibited improved plasma retention while F1718 showed no such improvement in plasma retention (see Example (7-2)). Thus, the Fc region variants of Disclosure B can preferably have improved plasma retention as compared to reference Fc region variants containing the combination of amino acid substitutions N434Y/Y436V/Q438R/S440E. The experimental results described in Examples (5-2) and (7-3) herein demonstrate that among various Fc region variants, F1847m, F1886m, F1889m, and F1927m are further improved in plasma retention time than FI848m. Thus, those of ordinary skill in the art can appreciate that Fc region variants of Disclosure B comprising F1847m, F1886m,

FI889m, or FI927m, as well as FI848m have improved plasma retention as compared to reference Fc region variants containing the substitutions

N434Y/Y436V/Q438R/S440E.

[0398] The binding to FcyR or a complement protein can also have an unfavorable impact (for example, inappropriate platelet activation). Fc region variants that do not bind to effector receptors such as the FcyRIIa receptor can be safer and/or more advantageous. In some embodiments, the Fc region variants of Disclosure B have only a weak effector receptor-binding activity or do not bind to effector receptors. Examples of effector receptors include, activating FcyR, particularly FcyRI, FcyRII, and FcyRIII. FcyRI includes FcyRIa, FcyRIb, and FcyRIc, and subtypes thereof. FcyRII includes FcyRIIa (having two allotypes: R131 and H131) and FcyRIIb. FcyRIII includes

131

WO 2017/046994

PCT/JP2016/003616

FcyRIIIa (which has two allotypes: VI58 and FI58) and FcyRIIIb (which has two allotypes: FcyRIIIb-NAl and FcyRIIIb-NA2). Antibodies that have only a weak effector receptor-binding activity or do not bind to the receptors include, for example, antibodies containing a silent Fc region and antibodies that do not have an Fc region (for example, Fab, F(ab)'₂, scFv, sc(Fv)₂, and diabodies).

[0399] Examples of Fc regions which have only a weak or no effector receptor-binding activity are described, for example, in Strohl et al. (Curr. Op. Biotech. 20(6):685-691 (2009)), and specifically include, for example, deglycosylated Fc regions (N297A and N297Q), and silent Fc regions resulting from manipulation of Fc regions to silence their effector functions (or to suppress immunity) (IgGl-F234A/F235A,

IgG 1-H268Q/A330S/P331S, IgG 1-C226S/C229S,

IgG 1-C226S/C229S/E233P/F234V/F235A, IgG 1-F234F/F235E/P33IS, IgG2-V234A/G237A, IgG2-H268Q/V309F/A330S/A33IS,

IgG4-F235A/G237A/E318A, and IgG4-F236E). W02008/092117 describes antibodies comprising a silent Fc region that contains a substitution of G236R/F328R, F235G/G236R, N325A/F328R, or N325F/F328R, according to EU numbering. W02000/042072 describes antibodies comprising a silent Fc region that contains substitutions at one or more of positions EU233 (position 233 according to EU numbering), EU234, EU235, and EU237. W02009/011941 describes antibodies comprising a silent Fc region that lacks the residues of EU231 to EU238. Davis et al.

(J. Rheum. 34(11):2204-2210 (2007)) describes antibodies with a silent Fc region containing substitutions C220S/C226S/C229S/P238S. Shields et al. (J. Biol. Chem. 276(9):6591-6604 (2001)) describes antibodies comprising a silent Fc region containing substitution D265A. Modification of these amino acid residues may also be appropriately introduced into the Fc region variants of Disclosure B.

[0400] The expression weak binding to effector receptors can mean that the effector receptor-binding activity is, for example, 95% or less, preferably 90% or less, 85% or less, 80% or less, 75% or less, more preferably 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less than that of a native IgG or an antibody containing a native IgG Fc region.

[0401] The silent Fc region is an Fc region variant containing one or more amino acid sub stitutions, insertions, additions, deletions, and others that reduce binding to effector receptors as compared to a native Fc region. Since the effector receptor-binding activity can be reduced considerably, such silent Fc regions may no longer bind to the effector receptors. The silent Fc regions may include, for example, Fc regions containing amino acid substitutions at one or more positions selected from the group

132

WO 2017/046994 PCT/JP2016/003616 consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332. Modification of these amino acid positions may also be appropriately introduced into the Fc region variants of Disclosure B.

[0402] In further embodiments, the silent Fc region has a substitution at one or more positions selected from the group consisting of: EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327, EU328, EU329, EU331, and EU332, and preferably the group consisting of: EU235, EU237, EU238, EU239, EU270, EU298, EU325, and EU329, wherein the substitution is with an amino acid residue selected from the listing below:

[0403] The amino acid at position EU234 is preferably substituted with an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, Fys,

Met, Phe, Pro, Ser, and Thr.

[0404] The amino acid at position EU235 is preferably substituted with an amino acid selected from the group consisting of Ala, Asn, Asp, Gin, Glu, Gly, His, lie, Fys, Met, Pro, Ser, Thr, Val, and Arg.

[0405] The amino acid at position EU236 is preferably substituted with an amino acid selected from the group consisting of Arg, Asn, Gin, His, Feu, Fys, Met, Phe, Pro, and Tyr.

[0406] The amino acid at position EU237 is preferably substituted with an amino acid selected from the group consisting of Ala, Asn, Asp, Gin, Glu, His, lie, Feu, Fys, Met, Pro, Ser, Thr, Val, Tyr, and Arg.

[0407] The amino acid at position EU238 is preferably substituted with an amino acid selected from the group consisting of Ala, Asn, Gin, Glu, Gly, His, He, Fys, Thr, Trp, and Arg.

[0408] The amino acid at position EU239 is preferably substituted with an amino acid selected from the group consisting of Gin, His, Fys, Phe, Pro, Trp, Tyr, and Arg.

[0409] The amino acid at position EU265 is preferably substituted with an amino acid selected from the group consisting of Ala, Arg, Asn, Gin, Gly, His, He, Feu, Fys, Met, Phe, Ser, Thr, Trp, Tyr, and Val.

[0410] The amino acid at position EU266 is preferably substituted with an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, Fys, Phe, Pro, Ser, Thr, Trp, and Tyr.

[0411] The amino acid at position EU267 is preferably substituted with an amino acid selected from the group consisting of Arg, His, Fys, Phe, Pro, Trp, and Tyr.

[0412] The amino acid at position EU269 is preferably substituted with an amino acid

133

WO 2017/046994

PCT/JP2016/003616 selected from the group consisting of Ala, Arg, Asn, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0413] The amino acid at position EU270 is preferably substituted with an amino acid selected from the group consisting of Ala, Arg, Asn, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0414] The amino acid at position EU271 is preferably substituted with an amino acid selected from the group consisting of Arg, His, Phe, Ser, Thr, Trp, and Tyr.

[0415] The amino acid at position EU295 is preferably substituted with an amino acid selected from the group consisting of Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, and Tyr.

[0416] The amino acid at position EU296 is preferably substituted with an amino acid selected from the group consisting of Arg, Gly, Lys, and Pro.

[0417] The amino acid at position EU297 is preferably substituted with Ala.

[0418] The amino acid at position EU298 is preferably substituted with an amino acid selected from the group consisting of Arg, Gly, Lys, Pro, Trp, and Tyr.

[0419] The amino acid at position EU300 is preferably substituted with an amino acid selected from the group consisting of Arg, Lys, and Pro.

[0420] The amino acid at position EU324 is preferably substituted with either Lys or Pro.

[0421] The amino acid at position EU325 is preferably substituted with an amino acid selected from the group consisting of Ala, Arg, Gly, His, lie, Lys, Phe, Pro, Thr, Trp, Tyr, and Val.

[0422] The amino acid at position EU327 is preferably substituted with an amino acid selected from the group consisting of Arg, Gin, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

[0423] The amino acid at position EU328 is preferably substituted with an amino acid selected from the group consisting of Arg, Asn, Gly, His, Lys, and Pro.

[0424] The amino acid at position EU329 is preferably substituted with an amino acid selected from the group consisting of Asn, Asp, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, and Arg.

[0425] The amino acid at position EU330 is preferably substituted with either Pro or Ser.

[0426] The amino acid at position EU331 is preferably substituted with an amino acid selected from the group consisting of Arg, Gly, and Lys.

[0427] The amino acid at position EU332 is preferably substituted with an amino acid selected from the group consisting of Arg, Lys, and Pro.

[0428] The silent Fc region preferably may contain a substitution with either Lys or Arg at EU235, a substitution with either Lys or Arg at EU237, a substitution with either Lys or Arg at EU238, a substitution with either Lys or Arg at EU239, a substitution with Phe at EU270, a substitution with Gly at EU298, a substitution with Gly at EU325, or a substitution with either Lys or Arg at EU329. More preferably, the silent Fc region

134

WO 2017/046994

PCT/JP2016/003616 may contain a substitution with arginine at EU235 or a substitution with lysine at EU239. Even more preferably, the silent Fc region may contain F235R/S239K substitutions. Modification of these amino acid residues may also be appropriately introduced into the Fc region variants of Disclosure B.

[0429] In one embodiment, antibodies comprising an Fc region variant of Disclosure B have only a weak complement protein-binding activity or do not bind to complement proteins. In some embodiments, the complement protein is Clq. In some embodiments, the weak complement protein-binding activity refers to a complement protein-binding activity reduced by 10-fold or more, 50-fold or more, or 100-fold or more when compared to the complement protein-binding activity of a native IgG or an antibody containing a native IgG Fc region. The complement protein-binding activity of an Fc region can be reduced by amino acid modifications such as amino acid substitution, insertion, addition, or deletion.

[0430] In one embodiment, the Fc region variants of Disclosure B or antibodies comprising the Fc region variants can be assessed for their (human) FcRn-binding activity in a neutral pH range and/or in an acidic pH range in the same manner as described above.

[0431] In one embodiment, a method for modifying antibody constant regions to produce the Fc region variants of Disclosure B may be based, for example, on assessment of several constant region isotypes (IgGl, IgG2, IgG3, and IgG4) to select isotypes that have a reduced antigen-binding activity in an acidic pH range and/or have an increased dissociation rate in an acidic pH range. An alternative method may be based on introduction of amino acid substitutions into the amino acid sequence of a native IgG isotype to reduce the antigen-binding activity in an acidic pH range (e.g., pH 5.8) and/ or to increase the dissociation rate in an acidic pH range. The hinge region sequence of an antibody constant region varies greatly across isotypes (IgGl, IgG2, IgG3, and IgG4), and differences in the hinge-region amino acid sequence can have a significant impact on the antigen-binding activity. Therefore, isotypes with reduced antigenbinding activity in an acidic pH range and/or increased dissociation rate in an acidic pH range can be selected by selecting suitable isotypes depending on the type of antigen or epitope. Furthermore, since differences in the hinge-region amino acid sequence can have a significant impact on the antigen-binding activity, amino acid substitutions in the amino acid sequences of native isotypes can be located in the hinge region.

[0432] In an alternative embodiment, Disclosure B provides a use of an antibody containing the above-described Fc region variant of Disclosure B to accelerate the release of the antibody that has been internalized into cells in an antigen-bound form to the outside of the cells in an antigen-free form. Herein, release of an antibody that has been internalized into cells in an antigen-bound form to the outside of the cells in an antigen135

WO 2017/046994

PCT/JP2016/003616 free form does not necessarily mean that the antibody that has been internalized into cells in an antigen-bound form is completely released to the outside of the cells in an antigen-free form. It is acceptable that the proportion of the antibody released in an antigen-free form to the outside of the cells is increased as compared to that before modification of its FcRn-binding domain (for example, before increasing the FcRnbinding activity of the antibody in an acidic pH range). It is preferable that the antibody released to the outside of the cells maintains its antigen-binding activity.

[0433] The ability to eliminate antigen from plasma or an equivalent term can refer to the ability to eliminate antigen from plasma when an antibody is administered or secreted in vivo. Thus, the antibody's ability to eliminate antigen from plasma is increased can mean that when an antibody is administered, for example, the rate of antigen elimination from plasma is increased as compared to that before modification of its FcRn-binding domain. The increase in the antibody's activity of antigen elimination from plasma can be assessed, for example, by administering a soluble antigen and the antibody in vivo, and measuring the concentration of the soluble antigen in plasma after administration. The soluble antigen may be an antibody-bound or antibody-free antigen, and their concentrations can be determined as antibody-bound antigen concentration in plasma and antibody-free antigen concentration in plasma, respectively. The latter is synonymous with free antigen concentration in plasma. The total antigen concentration in plasma can refer to the sum of antibody-bound antigen concentration and antibody-free antigen concentration.

[0434] In an alternative embodiment, Disclosure B provides a method for prolonging the plasma retention time of an antibody containing the Fc region variant of Disclosure B. Native human IgG can bind to FcRn derived from nonhuman animals. For example, since native human IgG can bind to mouse FcRn more strongly than to human FcRn (Ober et al., Inti. Immunol. 13(12):1551-1559 (2001)), the antibodies can be administered to mice for assessing the properties of the antibodies. Alternatively, for example, mice with their own FcRn gene has been disrupted but instead have and express the human FcRn gene as a transgene (Roopenian et al., Meth.Mol. Biol. 602:93-104 (2010)) are also suitable for assessing the antibodies.

[0435] Within the scope of Disclosures A and B described herein, the plasma concentration of free antigen not bound to the antibody or the ratio of free antigen concentration to the total antigen concentration can be determined (e.g., Ng et al., Pharm. Res. 23(1):95-103 (2006)). Alternatively, when an antigen exhibits a particular function in vivo, whether the antigen is bound to an antibody that neutralizes the antigen function (antagonistic molecule) can be assessed by testing whether the antigen function is neu tralized. Whether the antigen function is neutralized can be evaluated by measuring a particular in vivo marker reflective of the antigen function. Whether an antigen is

136

WO 2017/046994

PCT/JP2016/003616 bound to an antibody that activates the antigen function (agonistic molecule) can be assessed by measuring a particular in vivo marker reflective of the antigen function.

[0436] There are no particular limitations on measurements such as determination of the free antigen concentration in plasma, determination of the ratio of the amount of free antigen in plasma to the amount of total antigen in plasma, and in vivo marker measurement; however, the measurements can be preferably carried out after a certain period of time following antibody administration. In the context of Disclosure B, after a certain period of time following antibody administration is not particularly limited, and the period can be appropriately determined by those of ordinary skill in the art depending on the properties of the administered antibody and others, and includes, for example, one day, three days, seven days, 14 days, or 28 days after antibody administration. Herein, the term plasma antigen concentration can refer to either total antigen concentration in plasma which is the sum of antibody-bound antigen concentration and antibody-free antigen concentration, or free antigen concentration in plasma which is antibody-free antigen concentration.

[0437] Molar ratio of antigen to antibody can be calculated using the formula: C = A/B, wherein value A is the molar concentration of antigen at each time point, value B is the molar concentration of antibody at each time point, and value C is the molar concentration of antigen per molar concentration of antibody (molar ratio of antigen/ antibody) at each time point.

[0438] A smaller C value indicates a higher efficiency of antigen elimination per antibody, and a larger C value indicates a lower efficiency of antigen elimination per antibody.

[0439] In some aspects, when an antibody of Disclosure B is administered, the molar ratio of antigen/antibody is reduced by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold or more, as compared to when a reference antibody containing a native human IgG Fc region as a human FcRn-binding domain is administered.

[0440] The reduction of total antigen concentration in plasma or molar ratio of antigen/ antibody can be assessed using methods know in the art, such as that described in Examples 6, 8, and 13 of WO2011/122011. More specifically, they can be assessed based on either an antigen-antibody co-injection model or a steady-state antigen infusion model using the human FcRn transgenic mouse line 32 or 276 (Jackson Faboratories, Methods Mol. Biol. 602:93-104 (2010)), when an antibody of interest in Disclosure B does not cross-react with the mouse counterpart antigen. When the antibody cross-reacts with the mouse counterpart, it can be assessed by simply injecting the antibody into the human FcRn transgenic mouse line 32 or 276 (Jackson Faboratories). In the co-injection model, a mixture of the antibody and antigen is administered to mice. In the steady-state antigen infusion model, an infusion pump filled

137

WO 2017/046994

PCT/JP2016/003616 with an antigen solution is implanted into mice to achieve a constant antigen concentration in plasma, and then the antibody is injected into the mice. All test antibodies are administered at the same dosage. The total antigen concentration in plasma, free antigen concentration in plasma, and antibody concentration in plasma can be measured at appropriate time points.

[0441] To assess the long-term effects of an antibody of Disclosure B, the total or free antigen concentration in plasma, or the molar ratio of antigen/antibody can be measured two days, four days, seven days, 14 days, 28 days, 56 days, or 84 days after administration. In other words, for assessing properties of the antibody, an antigen con centration in plasma in a long period of time can be determined by measuring the total or free antigen concentration in plasma, or the molar ratio of antigen/antibody two days, four days, seven days, 14 days, 28 days, 56 days, or 84 days after antibody administration. Whether the antigen concentration in plasma or the molar ratio of antigen/antibody is reduced with the antibody can be determined by assessing such reductions at one or more time points as described above.

[0442] To assess the short-term effects of an antibody of Disclosure B, the total or free antigen concentrations in plasma, or the molar ratio of antigen/antibody can be measured 15 minutes, one hour, two hours, four hours, eight hours, 12 hours, or 24 hours after administration. In other words, for assessing properties of the antibody, an antigen concentration in plasma in a short period of time can be determined by measuring the total or free antigen concentrations in plasma, or the molar ratio of antigen/antibody 15 minutes, one hour, two hours, four hours, eight hours, 12 hours, or 24 hours after administration. When the plasma retention in human is difficult to determine, it may be predicted based on the plasma retention in mice (for example, normal mice, human antigen-expressing transgenic mice, or human FcRn-expressing transgenic mice) or in monkeys (for example, cynomolgus monkeys).

[0443] In an alternative embodiment, Disclosure B relates to an antibody comprising the Fc region variant of Disclosure B described above. The various embodiments of the antibodies described within the scope of Disclosures A and B described herein can be applicable without opposing the common technical knowledge in the art and unless there are inconsistencies in the context.

[0444] In one embodiment, antibodies comprising an Fc region variant of Disclosure B are useful as therapeutic antibodies for treating human patients with auto-immune diseases, transplantation rejection (graft versus host disease), other inflammatory diseases, or allergy diseases, as described in WO2013/046704.

[0445] In one embodiment, antibodies comprising an Fc region variant of Disclosure B may have modified sugar chains. Antibodies with modified sugar chains can include, for example, antibodies with modified glycosylation (WO99/54342), antibodies that are

138

WO 2017/046994 PCT/JP2016/003616 deficient in fucose (WOOO/61739, W002/31140, W02006/067847, W02006/067913), and antibodies having sugar chains with bisecting GlcNAc (WO02/79255). In one embodiment, the antibodies may be deglycosylated. In some embodiments, the antibodies comprise, for example, mutations at the heavy-chain glycosylation site to inhibit glyco sylation at such location as described in W02005/03175. Such non-glycosylated antibodies can be prepared by modifying the heavy-chain glycosylation site, i.e., by introducing the N297Q or N297A substitution according to EU numbering, and expressing the proteins in appropriate host cells.

[0446] In an alternative embodiment, Disclosure B relates to a composition or a pharmaceutical composition comprising an antibody containing such an Fc region variant. The various embodiments of the compositions or pharmaceutical compositions described within the scope of Disclosures A and B herein can be applicable without opposing the common technological knowledge in the art and unless there are inconsistencies in the context. Such compositions can be used for enhancing the plasma retention (in subjects, when an antibody of the Disclosure B is administered (applied) to the subjects).

[0447] In an alternative embodiment, Disclosure B relates to nucleic acids encoding an Fc region variant or antibodies containing the Fc region variant. The various embodiments of the nucleic acids described within the scope of Disclosures A and B described herein can be applicable without opposing the common technical knowledge in the art and unless there are inconsistencies in the context. Alternatively, Disclosure B relates to vectors comprising the nucleic acids. The various embodiments thereof within the scope of Disclosures A and B described herein can be applicable without opposing the common technical knowledge in the art and unless there are inconsistencies in the context. Alternatively, Disclosure B relates to hosts or host cells comprising the vectors. The various embodiments thereof within the scope of Disclosures A and B described herein can be applicable without opposing the common technical knowledge in the art and unless there are inconsistencies in the context.

[0448] In an alternative embodiment, Disclosure B relates to methods for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the Fc region variant, which comprise culturing the host cells described above, or growing the hosts described above and collecting the Fc region variant or antibody comprising the Fc region variant from the cell culture, materials secreted from the hosts. In this case, Disclosure B may include production methods optionally further comprising any one or more of: (a) selecting an Fc region variant with enhanced FcRn-binding activity under an acidic pH condition as compared to that of an Fc region of a native IgG; (b) selecting an Fc region variant whose binding activity to a (pre-existing) ADA is not significantly enhanced under a neutral pH condition as compared to that of an Fc

139

WO 2017/046994

PCT/JP2016/003616 region of a native IgG; (c) selecting an Fc region variant with increased plasma retention as compared to that of an Fc region of a native IgG; and (d) selecting an antibody comprising an Fc region variant that can promote elimination of an antigen from plasma as compared to a reference antibody comprising an Fc region of a native IgG.

[0449] From the perspective of assessing the plasma retention of an Fc region variant of

Disclosure B, without limitations, it can be preferable that an antibody comprising the Fc region variant produced in Disclosure B and the reference antibody comprising the Fc region of a native IgG are identical to each other except for the Fc region to be compared. The FcRn can be human FcRn.

[0450] For example, after producing an antibody comprising an Fc region variant of

Disclosure B, the antibody may be compared with the reference antibody comprising a native IgG Fc region in terms of the FcRn-binding activity under an acidic pH condition (e.g., pH 5.8) using BIACORE(registered trademark) or other known techniques, to select an Fc region variant or an antibody comprising the Fc region variant whose FcRn-binding activity has been increased under the acidic pH condition.

[0451] Alternatively, for example, after producing an antibody comprising an Fc region variant of Disclosure B, the antibody may be compared with a reference antibody comprising a native IgG Fc region in terms of the ADA-binding activity under a neutral pH condition by electrochemiluminescence (ECF) or known techniques, to select an Fc region variant or antibodies comprising the Fc region variant whose ADA binding activity has not been significantly increased under the neutral pH condition.

[0452] Alternatively, for example, after producing an antibody comprising an Fc region variant of Disclosure B, the antibody may be compared with a reference antibody comprising a native IgG Fc region by conducting antibody pharmacokinetic tests using plasma, for example, from mice, rats, rabbits, dogs, monkeys, or humans, to select Fc region variants or antibodies comprising the Fc region variant which are demonstrated to have improved plasma retention in the subjects.

[0453] Alternatively, for example, after producing an antibody comprising an Fc region variant of Disclosure B, the antibody may be compared with a reference antibody comprising a native IgG Fc region by conducting antibody pharmacokinetic tests using plasma, for example, from mice, rats, rabbits, dogs, monkeys, or humans, to select Fc region variants or antibodies comprising the Fc region variant which have enhanced antigen elimination from plasma.

[0454] Alternatively, for example, the selection methods described above may be appropriately combined, if needed.

[0455] In one embodiment, Disclosure B relates to a method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the variant,

140

WO 2017/046994

PCT/JP2016/003616 wherein the method comprises substituting amino acids in a way such that the resulting Fc region variant or the antibody comprising the variant comprises Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gin at position 440, according to EU numbering. In an additional embodiment, such method comprises substituting the amino acids in a way such that the resulting Fc region variant or the antibody comprising the variant further comprises, lie or Leu at position 428 and/or lie, Leu,

Val, Thr, or Phe at position 436, according to EU numbering. In a further embodiment, the amino acids are substituted in a way such that the resulting Fc region variant or the antibody comprising the variant produced according to the method further comprises Leu at position 428 and/or Val or Thr at position 436, according to EU numbering.

[0456] In one embodiment, Disclosure B relates to a method for producing an Fc region variant comprising an FcRn-binding domain or an antibody comprising the variant, wherein the method comprises substituting amino acids in a way such that the resulting Fc region variant or the antibody comprising the variant comprises Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440, according to EU numbering. In an additional embodiment, such method comprises substituting the amino acids in a way such that the resulting Fc region variant or the antibody comprising the variant further comprises, He or Leu at position 428 and/or He, Leu,

Val, Thr, or Phe at position 436, according to EU numbering. In a further embodiment, the amino acids are substituted in a way such that the resulting Fc region variant or the antibody comprising the variant produced according to the method further comprises Leu at position 428 and/or Val or Thr at position 436, according to EU numbering.

[0457] In one embodiment, such method comprises substituting all amino acids at positions 434, 438, and 440 with Ala; Glu, Arg, Ser, or Lys; and Glu, Asp, or Gin, respectively. In an additional embodiment, such method comprises substituting the amino acids in a way such that the resulting Fc region variant or the antibody comprising the variant further comprises, He or Leu at position 428 and/or He, Leu, Val, Thr, or Phe at position 436, according to EU numbering. In a further embodiment, the amino acids are substituted in a way such that the resulting Fc region variant or the antibody comprising the variant produced according to the method further comprises Leu at position 428 and/or Val or Thr at position 436, according to EU numbering.

[0458] In an alternative embodiment, Disclosure B relates to an Fc region variant or an antibody comprising the Fc region variant obtained by any of the production methods of Disclosure B described above.

[0459] In an alternative embodiment, Disclosure B provides methods for reducing the (pre-existing) ADA-binding activity of antibodies comprising an Fc region variant with increased FcRn-binding activity at an acidic pH; and methods for producing Fc region variants with increased FcRn-binding activity at an acidic pH (e.g., pH 5.8) and

141

WO 2017/046994

PCT/JP2016/003616 reduced pre-existing ADA-binding activity, which comprise: (a) providing an antibody comprising an Fc region (variant) whose FcRn-binding activity at an acidic pH has been increased as compared to a reference antibody; and (b) introducing into the Fc region, according to EU numbering, (i) an amino acid substitution with Ala at position 434; (ii) an amino acid substitution with any one of Glu, Arg, Ser, and Fys at position 438; and (iii) an amino acid substitution with any one of Glu, Asp, and Gin at position 440, (iv) optionally, an amino acid substitution with He or Feu at position 428; and/or (v) optionally, an amino acid substitution with any one of He, Feu, Val, Thr, and Phe at position 436.

[0460] In some embodiments, the Fc domain (variant) in step (a) is preferably a human IgG Fc domain (variant). Furthermore, to increase the FcRn-binding activity at an acidic pH and to decrease the (pre-existing) ADA-binding activity in a neutral pH range (e.g., pH 7.4), the Fc region (variant) is to contain a combination of substituted amino acids selected from the group consisting of: (a) N434A/Q438R/S440E; (b)

N434A/Q438R/S440D; (c) N434A/Q438K/S440E; (d) N434A/ Q438K/S440D; (e) N434A/Y436T/Q438R/S440E; (f) N434A/Y436T/Q438R/S440D; (g) N434A/Y436T/Q438K/S440E; (h) N434A/Y436T/Q438K/S440D; (i) N434A/Y436V/ Q438R/S440E; (j) N434A/Y436V/Q438R/S440D; (k) N434A/Y436V/Q438K/S440E; (1) N434A/Y436V/Q438K/S440D; (m) N434A/R435H/F436T/Q438R/S440E; (n) N434A/ R435H/F436T/Q438R/S440D; (o) N434A/R435H/F436T/Q438K/S440E; (p) N434A/ R435H/F436T/Q438K/S440D; (q) N434A/R435H/F436V/Q438R/ S440E; (r) N434A/ R435H/F436V/Q438R/S440D; (s) N434A/R435H/F436V/Q438K/S440E; (t) N434A/R435H/ F436V/Q438K/S440D; (u) M428F/N434A/Q438R/S440E; (v) M428F/N434A/ Q438R/ S440D; (w) M428F/N434A/Q438K/S440E; (x) M428F/N434A/ Q438K/S440D; (y) M428F/ N434A/Y436T/Q438R/S440E; (z) M428F/N434A/Y436T/Q438R/S440D; (aa) M428F/ N434A/Y436T/Q438K/S440E; (ab) M428F/N434A/Y436T/Q438K/S440D; (ac) M428F/ N434A/Y436V/Q438R/S440E; (ad) M428F/N434A/Y436V/Q438R/S440D; (ae) M428F/ N434A/Y436V/Q438K/S440E; (af) M428F/N434A/Y436V/Q438K/S440D; (ag) F235R/ G236R/S239K/M428F/N434A/Y436T/Q438R/S440E; and (ah) F235R/G236R/A327G/A330S/P331S/M428F/N434A/Y436T/Q438R/S440E.

[0461] The methods may optionally further comprise: (c) assessing whether the (pre-existing) ADA-binding activity of an antibody comprising a produced Fc region variant is reduced as compared to the binding activity of the reference antibody.

[0462] Alternatively, the methods may be used as a method for enhancing the release of an antibody that has been internalized into cells in an antigen-bound form to the outside of the cells in an antigen-free form, without significantly increasing the (pre-existing) ADA-binding activity of the antibody at a neutral pH.

142

WO 2017/046994 PCT/JP2016/003616 [0463] Disclosure C

Disclosure C also relates to anti-IL-8 antibodies, nucleic acids encoding the antibodies, pharmaceutical compositions comprising the antibodies, methods for producing the antibodies, and uses of the antibodies in treating diseases related to IL-8, as described in detail hereinbelow. The meanings of the terms given hereinbelow apply throughout the description of Disclosure C herein, without being contrary to the common technical knowledge of those of ordinary skill in the art as well as embodiments known to those of ordinary skill in the art.

[0464] I, Definitions within the scope of Disclosure C

Within the scope of Disclosure C described herein, acidic pH refers to pH that may be selected, for example, from pH 4.0 to pH 6.5. In one embodiment, acidic pH refers to, but is not limited to, pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, or pH 6.5. In a specific embodiment, the term acidic pH refers to the pH 5.8.

[0465] Within the scope of Disclosure C described herein, neutral pH refers to pH that may be selected, for example, from pH 6.7 to pH 10.0. In one embodiment, neutral pH refers to, but is not limited to, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0, pH 9.5, or pH 10.0. In a specific embodiment, the term neutral pH refers to the pH 7.4.

[0466] The term IL-8, as used in Disclosure C, refers to any native IL-8 derived from any vertebrates, primates (e.g., humans, cynomolgus monkeys, rhesus monkeys) and other mammals (e.g., dogs and rabbits), unless otherwise indicated. The term IL-8 encompasses full-length IL-8, unprocessed IL-8 as well as any form of IL-8 that results from processing in the cell. The term IL-8 also encompasses derivatives of native IL 8, for example, splice variants or allelic variants. The amino acid sequence of an exemplary human IL-8 is shown in SEQ ID NO:66.

[0467] The terms anti-IL-8 antibody and an antibody that binds to IL-8 refer to an antibody that is capable of binding to IL-8 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting IL-8.

[0468] In one embodiment, the extent of binding of an anti-IL-8 antibody to an unrelated, non-IL-8 protein is, for example, less than about 10% of the binding of the antibody to IL-8.

[0469] Affinity within the scope of the description of Disclosure C herein generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used within the scope of the description of Disclosure C herein,

143

WO 2017/046994

PCT/JP2016/003616 binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Binding affinity can be measured using methods known in the art, including those described within the scope of the description of Disclosure C herein.

[0470] In certain embodiments, an antibody that binds to IL-8 may have a dissociation constant (KD) of, for example, < 1000 nM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10⁸ M or less, from 10⁸ M to 10 ¹³ M, from 10⁹ M to 10 ¹³ M).

[0471] The term antibody within the scope of the description of Disclosure C herein is used in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0472] An antibody that binds to the same epitope as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by, for example, 50%, 60%, 70%, or 80% or more; and conversely, the reference antibody blocks binding of the antibody to its antigen by, for example, 50%, 60%, 70%, or 80% or more. Here, an exemplary competition assay can be used without being limited thereto.

[0473] A chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remaining portion is derived from a different source or species.

[0474] A humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody may comprise substantially at least one, and typically two, variable regions, in which all (or substantially all) of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all (or substantially all) of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

[0475] The term monoclonal antibody as used within the scope of the description of

Disclosure C herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that constitute the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, those containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, which are generally present in minor amounts. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an

144

WO 2017/046994

PCT/JP2016/003616 antigen. Thus, the modifier monoclonal indicates the characteristics of an antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any specific method. For example, the monoclonal antibodies to be used in accordance with Disclosure C may be made by various techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals comprising all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0476] Within the scope of Disclosure C described herein, native antibody refers to immunoglobulin molecules with various naturally occurring structures. In an embodiment, a native IgG antibody, for example, is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. In the order from N- to C- terminus, each heavy chain has a variable region (VH), which is also referred to as a variable heavy-chain domain or heavy-chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Fikewise, in the order from N- to C- terminus, each light chain has a variable region (VF), which is also referred to as a variable light-chain domain or light-chain variable domain, followed by a constant light-chain (CF) domain. An antibody light chain may be assigned to one of the two types, called kappa (k) and lambda (λ), based on the amino acid sequence of its constant domain. Such constant domains for use in the Disclosure C include those of any reported allotype (allele) or any subclass/isotype. The heavy-chain constant region includes, but is not limited to, the constant region of a native IgG antibody (IgGl, IgG2, IgG3, and IgG4). Known IgGl alleles include, for example, IGHGDO1, IGHG1*O2, IGHGl*03, IGHG1*O4, and IGHGl*05 (see at imgt.org), and any of these can be used as a native human IgGl sequence. The constant domain sequence may be derived from a single allele or subclass/isotype, or from multiple alleles or subclasses/isotypes. Specifically, such antibodies include, but are not limited to, an antibody whose CHI is derived from IGHGl*01 and CH2 and CH3 are derived from IGHG1*O2 and IGHGl*01, respectively.

[0477] Effector functions within the scope of the description of Disclosure C herein refers to biological activities attributable to the Fc region of an antibody, which may vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity; Fc receptor binding; antibodydependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation, but are not limited thereto.

[0478] The term Fc region within the scope of the description of Disclosure C herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at

145

WO 2017/046994

PCT/JP2016/003616 least a portion of the constant region. The term includes native Fc regions and variant Fc regions. The native Fc region indicates the Fc region of a native antibody.

[0479] In one embodiment, a human IgG heavy-chain Fc region extends from the amino acid residue of Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Fys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified within the scope of the description of Disclosure C herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Rabat et al., Sequences of Proteins of Immunological Interest,

5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0480] Framework or FR within the scope of the description of Disclosure C herein refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence: FR1-H1(F1)-FR2-H2(F2)-FR3-H3(F3)-FR4 in VH (or VF).

[0481] A human consensus framework within the scope of the description of Disclosure C herein is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VF or VH framework sequences. Generally, the selection of human immunoglobulin VF or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup according to Rabat et al., Sequences of proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0482] In one embodiment, the subgroup for the VF is subgroup κΐ as in Rabat et al., supra. In one embodiment, the subgroup for the VH is subgroup III as in Rabat et al., supra.

[0483] An acceptor human framework for purposes within the scope of the description of Disclosure C herein is a framework comprising the amino acid sequence of a VF or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain existing amino acid sequence substitutions. In some embodiments, the number of existing amino acid substitutions are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In one embodiment, the VF acceptor human framework is identical to the VF human immunoglobulin framework sequence or human consensus framework sequence.

[0484] The term variable region or variable domain within the scope of the description of Disclosure C herein refers to the domain of an antibody heavy or light chain involved in binding of the antibody to an antigen. The variable regions of the heavy

146

WO 2017/046994

PCT/JP2016/003616 chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, Kindt et al., Kuby Immunology, 6 ^th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain is sufficient to confer antigen-binding specificity, but is not limited thereto. Furthermore, antibodies that bind to a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0485] The term hypervariable region or HVR as used within the scope of the description of Disclosure C herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (complementarity determining regions or CDRs) and/or form structurally defined loops (hypervariable loops) and/or contain the antigen-contacting residues (antigen contacts). Generally, antibodies comprise six hypervariable regions: three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3).

[0486] Without being limited thereto, exemplary HVRs herein include: (a) hypervariable loops in which amino acid residues are 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs in which amino acid residues are 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts in which amino acid residues are 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (Hl), 26-35b (Hl), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0487] Unless otherwise instructed, HVRs and other residues in variable regions (e.g., FR residues) are numbered as in Kabat et al., supra.

[0488] An individual within the scope of the description of Disclosure C herein is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual is a human.

[0489] An isolated antibody within the scope of the description of Disclosure C herein is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to be greater than 95% or 99% in purity as determined, for example, electrophoretically (e.g., SDS-PAGE, isoelectric focusing elec147

WO 2017/046994

PCT/JP2016/003616 trophoresis (IEF), capillary electrophoresis) or chromatographic ally (e.g., ion exchange or reverse phase HPFC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0490] An isolated nucleic acid within the scope of the description of Disclosure C herein refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0491] Isolated nucleic acid encoding an anti-IF-8 antibody within the scope of the description of Disclosure C herein refers to one or more nucleic acid molecules encoding anti-IF-8 antibody heavy and light chains (or fragments thereof), including such nucleic acid(s) in a single vector or separate vectors, nucleic acid(s) present at one or more locations in a host cell.

[0492] Within the scope of the description of Disclosure C herein, the terms host cell, host cell line, and host cell culture are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. A progeny may not be completely identical in its nucleic acid content to a parent cell, but may contain mutations. A mutant progeny that has the same function or biological activity as that screened or selected in the originally transformed cell are included herein.

[0493] The term vector, as used within the scope of the description of Disclosure C herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors in a self-replicating nucleic acid structure as well as vectors introduced into a host cell and become incorporated into its genome. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as expression vectors.

[0494] Within the scope of the description of Disclosure C herein, the term package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[0495] Within the scope of the description of Disclosure C herein, percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after sequence alignment,

148

WO 2017/046994

PCT/JP2016/003616 by introducing gaps if necessary and not considering any conservative substitutions as part of the sequence identity, in order to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways within the scope of the ability of those of ordinary skill in the art, for instance, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or

GENETYX(registered trademark) (Genetyx Co., Ltd.). Those of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, values of the % amino acid sequence identity are generated, for example, using the sequence comparison computer program ALIGN-2. The ALIGN-2 was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX (registered trademark) operating system, including digital UNIX (registered trademark) V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0496] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches in the program alignment of A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % values of amino acid sequence identity are obtained using the ALIGN-2 computer program as demonstrated under the scope of the description of Disclosure C herein.

[0497] Within the scope of Disclosure C described herein, a pharmaceutical composition generally refers to an agent for treating, preventing, examining, or diagnosing diseases. A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. Such pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, and preservatives.

149

WO 2017/046994 PCT/JP2016/003616 [0498] As used within the scope of Disclosure C described herein, treatment (and grammatical variations thereof such as treat or treating) refers to a clinical intervention in an attempt to alter the natural course of the individual being treated. Such a clinical intervention can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, decrease of the rate of disease progression, amelioration or palliation of the disease state, and remission or improvement of prognosis. In one embodiment, an antibody of Disclosure C can be used to slow down the progression of a disease or disorder.

[0499] Within the scope of Disclosure C described herein, an effective amount of an antibody or pharmaceutical composition refers to an amount that is effective when used at doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result.

[0500] II. Compositions and methods

In one embodiment, Disclosure C is based on the applicability of anti-IL-8 antibodies that have pH-dependent affinity for IL-8 as pharmaceutical compositions. The antibodies of Disclosure C are useful, for example, in diagnosing or treating diseases where IL-8 is present in an excessive amount.

[0501] A, Exemplary anti-IL-8 antibodies

In one embodiment, Disclosure C provides an anti-IL-8 antibody having pHdependent affinity for IL-8.

[0502] In one embodiment, Disclosure C provides an anti-IL-8 antibody having pHdependent affinity for IL-8, which comprises a sequence with at least one, two, three, four, five, six, seven, or eight amino acid substitution(s) within the amino acid sequences of: (a) HVR-H1 which comprises the amino acid sequence of SEQ ID NO:67; (b) HVR-H2 which comprises the amino acid sequence of SEQ ID NO:68; (c) HVR-H3 which comprises the amino acid sequence of SEQ ID NO:69; (d) HVR-L1 which comprises the amino acid sequence of SEQ ID NO:70; (e) HVR-L2 which comprises the amino acid sequence of SEQ ID NO:71; and (f) HVR-L3 which comprises the amino acid sequence of SEQ ID NO:72.

[0503] In another embodiment, Disclosure C provides an anti-IL-8 antibody having pHdependent affinity for IL-8, which comprises at least one amino acid substitution(s) in at least one of the amino acid sequences of: (a) HVR-H1 which comprises the amino acid sequence of SEQ ID NO:67; (b) HVR-H2 which comprises the amino acid sequence of SEQ ID NO:68; (c) HVR-H3 which comprises the amino acid sequence of SEQ ID NO:69; (d) HVR-L1 which comprises the amino acid sequence of SEQ ID

150

WO 2017/046994

PCT/JP2016/003616

NO:70; (e) HVR-L2 which comprises the amino acid sequence of SEQ ID NO:71; and (f) HVR-L3 which comprises the amino acid sequence of SEQ ID NO:72.

[0504] Unless otherwise specified, the amino acids may be substituted with any other amino acid. In one embodiment, an anti-IL-8 antibody of Disclosure C comprises one or more amino acid substitution(s) at position(s) selected from the group consisting of: (a) aspartic acid at position 1 in the sequence of SEQ ID NO:67; (b) tyrosine at position 2 in the sequence of SEQ ID NO:67; (c) tyrosine at position 3 in the sequence of SEQ ID NO:67; (d) leucine at position 4 in the sequence of SEQ ID NO:67; (e) serine at position 5 in the sequence of SEQ ID NO:67; (f) leucine at position 1 in the sequence of SEQ ID NO:68; (g) isoleucine at position 2 in the sequence of SEQ ID NO:68; (h) arginine at position 3 in the sequence of SEQ ID NO:68; (i) asparagine at position 4 in the sequence of SEQ ID NO:68; (j) lysine at position 5 in the sequence of SEQ ID NO:68; (k) alanine at position 6 in the sequence of SEQ ID NO:68; (1) asparagine at position 7 in the sequence of SEQ ID NO:68; (m) glycine at position 8 in the sequence of SEQ ID NO:68; (n) tyrosine at position 9 in the sequence of SEQ ID NO:68; (o) threonine at position 10 in the sequence of SEQ ID NO:68; (p) arginine at position 11 in the sequence of SEQ ID NO:68; (q) glutamic acid at position 12 in the sequence of SEQ ID NO:68; (r) tyrosine at position 13 in the sequence of SEQ ID NO:68; (s) serine at position 14 in the sequence of SEQ ID NO:68; (t) alanine at position 15 in the sequence of SEQ ID NO:68; (u) serine at position 16 in the sequence of SEQ ID NO:68; (v) valine at position 17 in the sequence of SEQ ID NO:68; (w) lysine at position 18 in the sequence of SEQ ID NO:68; (x) glycine at position 19 in the sequence of SEQ ID NO:68; (y) glutamic acid at position 1 in the sequence of SEQ ID NO:69; (z) asparagine at position 2 in the sequence of SEQ ID NO:69; (aa) tyrosine at position 3 in the sequence of SEQ ID NO:69; (ab) arginine at position 4 in the sequence of SEQ ID NO:69; (ac) tyrosine at position 5 in the sequence of SEQ ID NO:69; (ad) aspartic acid at position 6 in the sequence of SEQ ID NO:69; (ae) valine at position 7 in the sequence of SEQ ID NO:69; (af) glutamic acid at position 8 in the sequence of SEQ ID NO:69; (ag) leucine at position 9 in the sequence of SEQ ID NO:69; (ah) alanine at position 10 in the sequence of SEQ ID NO:69; (ai) tyrosine at position 11 in the sequence of SEQ ID NO:69; (aj) arginine at position 1 in the sequence of SEQ ID NO:70; (ak) alanine at position 2 in the sequence of SEQ ID NO:70; (al) serine at position 3 in the sequence of SEQ ID NO:70; (am) glutamic acid at position 4 in the sequence of SEQ ID NO:70; (an) isoleucine at position 5 in the sequence of SEQ ID NO:70; (ao) isoleucine at position 6 in the sequence of SEQ ID NO:70; (ap) tyrosine at position 7 in the sequence of SEQ ID NO:70; (aq) serine at position 8 in the sequence of SEQ ID NO:70; (ar) tyrosine at position 9 in the sequence of SEQ ID NO:70; (as) leucine at position 10 in the sequence of SEQ ID NO:70; (at)

151

WO 2017/046994

PCT/JP2016/003616 alanine at position 11 in the sequence of SEQ ID NO:70; (au) asparagine at position 1 in the sequence of SEQ ID NO:71; (av) alanine at position 2 in the sequence of SEQ ID NO:71; (aw) lysine at position 3 in the sequence of SEQ ID NO:71; (ax) threonine at position 4 in the sequence of SEQ ID NO:71; (ay) leucine at position 5 in the sequence of SEQ ID NO:71; (az) alanine at position 6 in the sequence of SEQ ID NO:71; (ba) aspartic acid at position 7 in the sequence of SEQ ID NO:71; (bb) glutamine at position 1 in the sequence of SEQ ID NO:72; (be) histidine at position 2 in the sequence of SEQ ID NO:72; (bd) histidine at position 3 in the sequence of SEQ ID NO:72; (be) phenylalanine at position 4 in the sequence of SEQ ID NO:72; (bf) glycine at position 5 in the sequence of SEQ ID NO:72; (bg) phenylalanine of position 6 in the sequence of SEQ ID NO:72; (bh) proline at position 7 in the sequence of SEQ ID NO:72; (bi) arginine at position 8 in the sequence of SEQ ID NO:72; and (bj) threonine at position 9 in the sequence of SEQ ID NO:72.

[0505] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises one or more amino acid substitution(s) at position(s) selected from the group consisting of: (a) alanine at position 6 in the sequence of SEQ ID NO:68; (b) glycine at position 8 in the sequence of SEQ ID NO:68; (c) tyrosine at position 9 in the sequence of SEQ ID NO:68; (d) arginine at position 11 in the sequence of SEQ ID NO:68; and (e) tyrosine at position 3 in the sequence of SEQ ID NO:69.

[0506] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises combination(s) of amino acid substitutions at positions selected from the group consisting of: (a) alanine at position 6 in the sequence of SEQ ID NO:68; (b) glycine at position 8 in the sequence of SEQ ID NO:68; (c) tyrosine at position 9 in the sequence of SEQ ID NO:68; (d) arginine at position 11 in the sequence of SEQ ID NO:68; and (e) tyrosine at position 3 in the sequence of SEQ ID NO:69.

[0507] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises amino acid substitutions at the following positions: (a) tyrosine at position 9 in the sequence of SEQ ID NO:68; (b) arginine at position 11 in the sequence of SEQ ID NO:68; and (c) tyrosine at position 3 in the sequence of SEQ ID NO:69.

[0508] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises amino acid substitutions at the following positions: (a) alanine at position 6 in the sequence of SEQ ID NO:68; (b) glycine at position 8 in the sequence of SEQ ID NO:68; (c) tyrosine at position 9 in the sequence of SEQ ID NO:68; (d) arginine at position 11 in the sequence of SEQ ID NO:68; and (e) tyrosine at position 3 in the sequence of SEQ ID NO:69.

[0509] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a) substitution of alanine with aspartic acid at position 6 in the sequence of SEQ ID NO:68; (b) substitution of arginine with proline at position 11 in the sequence of SEQ ID NO:68; and

152

WO 2017/046994

PCT/JP2016/003616 (c) substitution of tyrosine with histidine at position 3 in the sequence of SEQ ID NO:69.

[0510] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a) substitution of glycine with tyrosine at position 8 in the sequence of SEQ ID NO:68; and (b) substitution of tyrosine with histidine at position 9 in the sequence of SEQ ID NO:68.

[0511] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a) substitution of alanine with aspartic acid at position 6 in the sequence of SEQ ID NO:68; (b) substitution of glycine with tyrosine at position 8 in the sequence of SEQ ID NO:68; (c) substitution of tyrosine with histidine at position 9 in the sequence of SEQ ID NO:68;

(d) substitution of arginine with proline at position 11 in the sequence of SEQ ID NO:68; and (e) substitution of tyrosine with histidine at position 3 in the sequence of SEQ ID NO:69.

[0512] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises HVR-H2 which comprises the amino acid sequence of SEQ ID NO:73.

[0513] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises HVR-H3 which comprises the amino acid sequence of SEQ ID NO:74.

[0514] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, HVR-H2 comprising the amino acid sequence of SEQ ID NO:73, and HVR-H3 comprising the amino acid sequence of SEQ ID NO:74.

[0515] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises one or more amino acid substitution(s) at position(s) selected from the group consisting of: (a) serine at position 8 in the sequence of SEQ ID NO:70; (b) asparagine at position 1 in the sequence of SEQ ID NO:71; (c) leucine at position 5 in the sequence of SEQ ID NO:71; and (d) glutamine at position 1 in the sequence of SEQ ID NO:72. In a further embodiment, the anti-IL-8 antibody comprises a combination of any 2, 3, or all 4 of these substitutions.

[0516] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises combination(s) of amino acid substitutions at positions selected from the group consisting of: (a) serine at position 8 in the sequence of SEQ ID NO:70; (b) asparagine at position 1 in the sequence of SEQ ID NO:71; (c) leucine at position 5 in the sequence of SEQ ID NO:71; and (d) glutamine at position 1 in the sequence of SEQ ID NO:72. In a further embodiment, the anti-IL-8 antibody comprises a combination of any 2, 3, or all 4 of these substitutions.

[0517] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises amino acid substitutions at the following positions: (a) asparagine at position 1 in the sequence of SEQ ID NO:71; (b) leucine at position 5 in the sequence of SEQ ID NO:71; and (c) glutamine at position 1 in the sequence of SEQ ID NO:72.

153

WO 2017/046994 PCT/JP2016/003616 [0518] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises amino acid substitutions at the following positions: (a) serine at position 8 in the sequence of SEQ ID NO:70; (b) asparagine at position 1 in the sequence of SEQ ID NO:71; (c) leucine at position 5 in the sequence of SEQ ID NO:71; and (d) glutamine at position 1 in the sequence of SEQ ID NO:72.

[0519] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises:

(a) substitution of asparagine with lysine at position 1 in the sequence of SEQ ID

NO:71; (b) substitution of leucine with histidine at position 5 in the sequence of SEQ ID NO:71; and (c) substitution of glutamine with lysine at position 1 in the sequence of SEQ ID NO:72.

[0520] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a) substitution of serine with glutamic acid at position 8 in the sequence of SEQ ID NO:70; (b) substitution of asparagine with lysine at position 1 in the sequence of SEQ ID NO:71; (c) substitution of leucine with histidine at position 5 in the sequence of SEQ ID NO:71; and (d) substitution of glutamine with lysine at position 1 in the sequence of SEQ ID NO:72.

[0521] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises HVR-L2 comprising the amino acid sequence of SEQ ID NO:75.

[0522] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises HVR-L3 comprising the amino acid sequence of SEQ ID NO:76.

[0523] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, HVR-L2 comprising the amino acid sequence of SEQ ID NO:75, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:76.

[0524] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises amino acid substitutions at the following positions: (a) alanine at position 55 in the sequence of SEQ ID NO:77; (b) glycine at position 57 in the sequence of SEQ ID NO:77; (c) tyrosine at position 58 in the sequence of SEQ ID NO:77; (d) arginine at position 60 in the sequence of SEQ ID NO:77; (e) glutamine at position 84 in the sequence of SEQ ID NO:77; (f) serine at position 87 in the sequence of SEQ ID NO:77; and (g) tyrosine at position 103 in the sequence of SEQ ID NO:77.

[0525] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises: (a) substitution of alanine with aspartic acid at position 55 in the sequence of SEQ ID NO:77; (b) substitution of glycine with tyrosine at position 57 in the sequence of SEQ ID NO:77; (c) substitution of tyrosine with histidine at position 58 in the sequence of SEQ ID NO:77; (d) substitution of arginine with proline at position 60 in the sequence of SEQ ID NO:77; (e) substitution of glutamine with threonine at position 84 in the sequence of SEQ ID NO:77; (f) substitution of serine with aspartic acid at position 87 in the

154

WO 2017/046994

PCT/JP2016/003616 sequence of SEQ ID NO:77; (g) and substitution of tyrosine with histidine at position 103 in the sequence of SEQ ID NO:77.

[0526] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:78.

[0527] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:79.

[0528] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:79. The antiIL-8 antibody that comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:79 may be an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. The anti-IL-8 antibody that comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:79 may be an antiIL-8 antibody that maintains the IL-8-neutralizing activity stably in vivo (for example, in plasma). The anti-IL-8 antibody that comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:79 may be an antibody with low immunogenicity.

[0529] In an alternative aspect, anti-IL-8 antibodies of Disclosure C also include those that have pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least any one amino acid sequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 102; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 103; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:104; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:105; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 106; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 107.

[0530] In an alternative aspect, anti-IL-8 antibodies of Disclosure C also include those that have pH-dependent affinity for IL-8 and contain at least one amino acid substitution in an amino acid sequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 108; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 110; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:111; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 112; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 113.

[0531] In an alternative aspect, anti-IL-8 antibodies of Disclosure C also include those that have pH-dependent affinity for IL-8 and contain at least one amino acid substitution in

155

WO 2017/046994

PCT/JP2016/003616 at least any one amino acid sequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 114; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 115; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 116; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 117; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 118; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 119.

[0532] In an alternative aspect, anti-IL-8 antibodies of Disclosure C also include those that have pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least any one amino acid sequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 120; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 121; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 122; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 123;

(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and (f) HVRL3 comprising the amino acid sequence of SEQ ID NO: 125.

[0533] In an alternative aspect, anti-IL-8 antibodies of Disclosure C also include those that have pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least any one amino acid sequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 126; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 127; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 128; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 130; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:131.

[0534] In an alternative aspect, anti-IL-8 antibodies of Disclosure C also include those that have pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least any one amino acid sequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 132; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 133; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:134; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:135; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 136; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 137.

[0535] In an alternative aspect, anti-IL-8 antibodies of Disclosure C also include those that have pH-dependent affinity for IL-8 and contain at least one amino acid substitution in at least any one amino acid sequence of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 138; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 139; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 140; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 141; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 142; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 143.

156

WO 2017/046994 PCT/JP2016/003616 [0536] In one embodiment, an anti-IL-8 antibody of Disclosure C has IL-8-neutralizing activity. The IL-8-neutralizing activity refers to an activity of inhibiting the biological activity of IL-8, or may refer to an activity of inhibiting the receptor binding of IL-8.

[0537] In alternative aspect, an anti-IL-8 antibody of Disclosure C is an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. In the context of Disclosure C, an antiIL-8 antibody that binds to IL-8 in a pH-dependent manner refers to an antibody whose binding affinity for IL-8 at an acidic pH has been reduced as compared to the binding affinity for IL-8 at a neutral pH. For example, pH-dependent anti-IL-8 antibodies include antibodies that have a higher affinity for IL-8 at a neutral pH than at an acidic pH. In one embodiment, an anti-IL-8 antibody of Disclosure C has an IL-8 affinity at a neutral pH that is at least 2 times, 3 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 200 times, 400 times, 1000 times, 10000 times or more greater than the affinity at an acidic pH. The binding affinity can be measured using, without particular limitations, surface plasmon resonance methods (such as BIACORE(registered trademark)). The association rate constant (kon) and dissociation rate constant (koff) can be calculated using the BIACORE(registered trademark) T200 Evaluation Software (GE Healthcare) based on a simple one-to-one Langmuir binding model by fitting the association and dissociation sensorgrams simultaneously. The equilibrium dissociation constant (KD) is calculated as a ratio of koff/kon. To screen for antibodies whose binding affinity varies depending on pH, without particular limitations, ELISA, kinetic exclusion assay (KinExA™), and others as well as surface plasmon resonance methods (such as BIACORE(registered trademark)) can be used. The pH-dependent IL-8-binding ability refers to the property to bind to IL-8 in a pH-dependent manner. Meanwhile, whether an antibody is capable of binding to IL-8 multiple times can be assessed by the methods described in WO2009/125825.

[0538] In one embodiment, it is preferable that an anti-IL-8 antibody of Disclosure C has a small dissociation constant (KD) for IL-8 at a neutral pH. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at a neutral pH is, for example, 0.3 nM or less, but is not limited thereto. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at a neutral pH is, for example, 0.1 nM or less, but is not limited thereto. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at a neutral pH is, for example, 0.03 nM or less, but is not limited thereto.

[0539] In one embodiment, it is preferable that an anti-IL-8 antibody of Disclosure C has a small dissociation constant (KD) for IL-8 at pH 7.4. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at pH 7.4 is, for example,

157

WO 2017/046994

PCT/JP2016/003616

0.3 nM or less, but is not limited thereto. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at pH 7.4 is, for example, 0.1 nM or less, but is not limited thereto. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at pH 7.4 is, for example, 0.03 nM or less, but is not limited thereto.

[0540] In one embodiment, it is preferable that an anti-IL-8 antibody of Disclosure C has a large dissociation constant (KD) for IL-8 at an acidic pH. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at an acidic pH is, for example, 3 nM or more, but is not limited thereto. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at an acidic pH is, for example, 10 nM or more, but is not limited thereto. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at an acidic pH is, for example, 30 nM or more, but is not limited thereto.

[0541] In one embodiment, it is preferable that an anti-IL-8 antibody of Disclosure C has a large dissociation constant (KD) for IL-8 at pH 5.8. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at pH 5.8 is, for example, 3 nM or more, but is not limited thereto. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at pH 5.8 is, for example, 10 nM or more, but is not limited thereto. In one embodiment, the dissociation constant of an antibody of Disclosure C for IL-8 at pH 5.8 is, for example, 30 nM or more, but is not limited thereto.

[0542] In one embodiment, it is preferable that the binding affinity of an anti-IL-8 antibody of Disclosure C for IL-8 is greater at a neutral pH than at an acidic pH.

[0543] In one embodiment, the dissociation constant ratio between acidic pH and neutral pH, [KD (acidic pH)/KD (neutral pH)], of an anti-IL-8 antibody of Disclosure C is, for example, 30 or more, but is not limited thereto. In one embodiment, the dissociation constant ratio between acidic pH and neutral pH, [KD (acidic pH)/KD (neutral pH)], of an anti-IL-8 antibody of Disclosure C is, for example, 100 or more, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, but is not limited thereto.

[0544] In one embodiment, the dissociation constant ratio between pH 5.8 and pH 7.4, [KD (pH 5.8)/KD (pH 7.4)], of an anti-IL-8 antibody of Disclosure C is 30 or more, but is not limited thereto. In one embodiment, the dissociation constant ratio between pH 5.8 and pH 7.4, [KD (pH 5.8)/KD (pH 7.4)], of an antibody of Disclosure C is, for example, 100 or more, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000,

1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, but is not limited thereto.

158

WO 2017/046994 PCT/JP2016/003616 [0545] In one embodiment, it is preferable that an anti-IL-8 antibody of Disclosure C has a large dissociation rate constant (koff) at an acidic pH. In one embodiment, the dissociation rate constant of an antibody of Disclosure C at an acidic pH is, for example, 0.003 (1/s) or more, but is not limited thereto. In one embodiment, the dissociation rate constant of an antibody of Disclosure C at an acidic pH is, for example, 0.005 (1/s) or more, but is not limited thereto. In one embodiment, the dissociation rate constant of an antibody of Disclosure C at an acidic pH is, for example, 0.01 (1/s) or more, but is not limited thereto.

[0546] In one embodiment, it is preferable that an anti-IL-8 antibody of Disclosure C has a large dissociation rate constant (koff) at pH 5.8. In one embodiment, the dissociation rate constant of an antibody of Disclosure C at pH 5.8 is, for example, 0.003 (1/s) or more, but is not limited thereto. In one embodiment, the dissociation rate constant of an antibody of Disclosure C at pH 5.8 is, for example, 0.005 (1/s) or more, but is not limited thereto. In one embodiment, the dissociation rate constant of an antibody of Disclosure C at pH 5.8 is, for example, 0.01 (1/s) or more, but is not limited thereto.

[0547] In one embodiment, it is preferable that the anti-IL-8 antibody of Disclosure C maintains the IL-8-neutralizing activity stably in a solution (for example, in PBS). Whether the activity is maintained stably in a solution can be assessed by measuring whether the IL-8-neutralizing activity of the antibody of Disclosure C added to the solution changes before and after storage for a certain period of time at a certain temperature. In one embodiment, the storage period is, for example, one, two, three, or four weeks, but is not limited thereto. In one embodiment, the storage temperature is, for example, 25°C, 30°C, 35°C, 40°C, or 50°C, but is not limited thereto. In one embodiment, the storage temperature is, for example, 40°C, but is not limited thereto; and the storage period is, for example, two weeks, but is not limited thereto. In one embodiment, the storage temperature is, for example, 50°C, but is not limited thereto; and the storage period is, for example, one week, but is not limited thereto.

[0548] In one embodiment, it is preferable that the anti-IL-8 antibody of Disclosure C maintains the IL-8-neutralizing activity stably in vivo (for example, in plasma). Whether the activity is maintained stably in vivo can be assessed by measuring whether the IL-8-neutralizing activity of the antibody of Disclosure C added to plasma of an animal (for example, mouse) or human changes before and after storage for a certain period of time at a certain temperature. In one embodiment, the storage period is, for example, one, two, three, or four weeks, but is not limited thereto. In one embodiment, the storage temperature is, for example, 25°C, 30°C, 35°C, or 40°C, but is not limited thereto. In one embodiment, the storage temperature is, for example, 40°C, but is not limited thereto; and the storage period is, for example, two weeks, but is not limited thereto.

159

WO 2017/046994 PCT/JP2016/003616 [0549] In one embodiment, the rate of cellular uptake of an anti-IL-8 antibody of Disclosure C is greater when the antibody forms a complex with IL-8 than the antibody alone. The IL-8 antibody of Disclosure C is more easily taken up into cells when it is complexed with IL-8 outside of cells (for example, in plasma) than when not complexed with IL8.

[0550] In one embodiment, it is preferable that the predicted immunogenicity of an anti-IL-8 antibody of Disclosure C, which is predicted in human hosts, is reduced. Low immunogenicity may mean, without being limited thereto, for example, that the administered anti-IL-8 antibody does not induce immune response of a living body in at least half or more of the individuals administered with a sufficient amount of the antibody for a sufficient period of time to achieve therapeutic efficacy. The induction of immune response may include production of anti-drug antibodies. Low anti-drug antibody production is interchangeable with low immunogenicity. The immunogenicity level in human can be estimated with a T cell epitope prediction program.

Such T cell epitope prediction programs include Epibase (Lonza), iTope/TCED (Antitope), EpiMatrix (EpiVax), and so on. EpiMatrix is a system for predicting the immunogenicity of a protein of interest where sequences of peptide fragments are auto matically designed by partitioning the amino acid sequence of a protein being analyzed for its immunogenicity into nine amino acids each to predict their ability to bind to eight major MHC Class II alleles (DRBl*0101, DRBl*0301, DRBl*0401, DRBl*0701, DRBl*0801, DRBl*1101, DRBl*1301, and DRBl*1501) (De Groot et al., Clin. Immunol. 131(2):189-201 (2009)). Sequences in which amino acids of the amino acid sequence of an anti-IL-8 antibody have been modified can be analyzed using the above-described T cell epitope prediction programs to design sequences with reduced immunogenicity. Preferred sites of amino acid modification to reduce the immunogenicity of the anti-IL-8 antibody of Disclosure C include, but are not limited to, the amino acids at position 81 and/or position 82b according to Rabat numbering in the heavy-chain sequence of the anti-IL-8 antibody shown in SEQ ID NO:78.

[0551] In one embodiment, Disclosure C provides methods for enhancing elimination of IL8 from an individual as compared to when using a reference antibody, comprising administering an anti-IL-8 antibody of Disclosure C to the individual. In one embodiment, Disclosure C relates to the use of an anti-IL-8 antibody of Disclosure C in the enhancement of the elimination of IL-8 from an individual as compared to when using a reference antibody. In one embodiment, Disclosure C relates to an anti-IL-8 antibody of Disclosure C for use in the enhancement of the elimination of IL-8 from an individual as compared to when using a reference antibody. In one embodiment, Disclosure C relates to the use of an anti-IL-8 antibody of Disclosure C in the production of pharmaceutical compositions for enhancing the elimination of IL-8 in

160

WO 2017/046994

PCT/JP2016/003616 vivo as compared to when using a reference antibody. In one embodiment, Disclosure C relates to pharmaceutical compositions comprising an anti-IL-8 antibody of Disclosure C for enhancing the elimination of IL-8 as compared to when using a reference antibody. In one embodiment, Disclosure C relates to methods for enhancing the elimination of IL-8 as compared to when using a reference antibody, comprising administering an anti-IL-8 antibody of Disclosure C to a subject. In the embodiments of Disclosure C, the reference antibody refers to an anti-IL-8 antibody before modification to obtain the antibody of Disclosure C, or an antibody whose IL-8 binding affinity is strong at both acidic and neutral pHs. The reference antibody may be an antibody comprising the amino acid sequence of SEQ ID NOs:83 and 84, or SEQ ID NOs:89 and 87.

[0552] In one embodiment, Disclosure C provides pharmaceutical compositions comprising an anti-IL-8 antibody of Disclosure C, characterized that the anti-IL-8 antibody of Disclosure C binds to IL-8 and then to extracellular matrix. In one embodiment, Disclosure C relates to the use of an anti-IL-8 antibody of Disclosure C in producing pharmaceutical compositions characterized that the anti-IL-8 antibody of Disclosure C binds to IL-8 and then to extracellular matrix.

[0553] In any of the embodiments described above, the anti-IL-8 antibody may be a humanized antibody.

[0554] In one aspect, the antibody of Disclosure C comprises the heavy chain variable region of any one of the embodiments described above and the light chain variable region of any one of the embodiments described above. In one embodiment, the antibody of Disclosure C comprises each of the heavy-chain variable region of SEQ ID NO:78 and the light-chain variable region of SEQ ID NO:79, and also may comprise post-translational modifications in their sequences.

[0555] In a further aspect, an anti-IL-8 antibody according to any one of the embodiments described above may incorporate, singly or in combination, any of the features described in Sections 1 to 7 below.

[0556] 1, Chimeric Antibody and Humanized Antibody

In certain embodiments, an antibody provided in Disclosure C may be a chimeric antibody. Certain chimeric antibodies are described, for example, in US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984). In one example, a chimeric antibody may comprise a non-human variable region (e.g., a variable region derived from a mouse, a rat, a hamster, a rabbit, or a non-human primate such as a monkey) and a human constant region.

[0557] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a

161

WO 2017/046994

PCT/JP2016/003616 humanized antibody comprises one or more variable regions in which HVRs, e.g., CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody may be substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to retain or improve antibody specificity or affinity.

[0558] Humanized antibodies and methods of making them are reviewed, for example, in Almagro et al., Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the guided selection approach to FR shuffling).

[0559] Human framework regions that may be used for humanization include but are not limited to framework regions selected using the best-fit method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0560] 2, Antibody Fragments

In certain embodiments, an antibody provided in Disclosure C may be an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')₂, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and US Patent Nos. 5,571,894 and 5,587,458.

[0561] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA

162

WO 2017/046994

PCT/JP2016/003616

90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0562] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody may be a human singledomain antibody (Domantis, Inc., Waltham, MA; see, e.g., US Patent No. 6,248,516 BI). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described within the scope of the description of Disclosure C herein.

[0563] 3. Human Antibody

In certain embodiments, an antibody provided in Disclosure C may be a human antibody. Human antibodies can be prepared by various techniques known in the art. Human antibodies are described in general terms in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the immunoglobulin loci of the animal (non-human), or are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the immunoglobulin loci of the animal (non-human) have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also US Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology; US Patent No. 5,770,429 for HUMAB™ technology; US Patent No. 7,041,870 for K-M MOUSE™ technology, and US Patent Appl. Publ. No. US 2007/0061900 for VELOCIMOUSE™ technology.

[0564] Human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with a different human constant region. Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described, for example, in Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol. 147:86 (1991). Human antibodies generated via human B-cell hybridoma are described in Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006). Additional methods include, for example, the method described in US Patent No. 7,189,826, for the production of monoclonal human IgM antibodies from hybridoma cell lines, as well as,

163

WO 2017/046994

PCT/JP2016/003616 for example, the method described in Ni, Xiandai Mianyixue, 26 (4):265-268 (2006), for human-human hybridomas. Human hybridoma technology (trioma technology) is also described in Vollmers et al., Histol. and Histopath. 20(3):927-937 (2005) and Vollmers et al., Methods and Findings in Experimental and Clinical Pharmacology 27(3):185-91 (2005). Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0565] 4, Fibrary-derived antibodies

Antibodies of Disclosure C can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics. Such methods are reviewed in Hoogenboom et al., in Meth.Mol. Biol. 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), as wells as, for example, in McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Meth.Mol. Biol. 248:161-175 (Fo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Fee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.

USA 101(34):12467-12472 (2004); and Fee et al., J. Immunol. Meth. 284(1-2):119-132 (2004).

[0566] In certain phage display methods, repertoires of VH and VF coding sequences may be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries. The resulting phage libraries are screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.

[0567] Alternatively, the naive repertoire can be cloned (for example, from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J. 12:725-734 (1993).

[0568] Finally, naive libraries can also be constructed synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequences to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro (see below and Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992); patent publications that describe human antibody phage libraries include, for example: US Patent No. 5,750,373; and US Appl. Publ. Nos.

164

WO 2017/046994 PCT/JP2016/003616

2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360. Here, antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments.

[0569] 5. Multispecific antibody

In certain embodiments, an antibody provided according to Disclosure C may be, for example, a multispecific antibody such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites.

[0570] In certain embodiments, one of the binding specificities is for IL-8, and the others are for any other antigens.

[0571] In certain embodiments, bispecific antibodies may bind to two different epitopes on IL-8. Bispecific antibodies may also be used to localize cytotoxic agents to cells that express IL-8. Bispecific antibodies may be prepared as full length antibodies or as antibody fragments.

[0572] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983)); WO 93/08829; and Traunecker et al., EMBO J. 10:3655 (1991)) and the knob-in-hole method (see US Patent No. 5,731,168). Multispecific antibodies can be made by using electrostatic steering effects to prepare Fc-heterodimeric molecules (WO

2009/089004A1), by cross-linking two or more antibodies or fragments (US Patent No. 4,676,980; and Brennan et al., Science 229:81 (1985)), by using leucine zippers to produce bispecific antibodies (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)), by using diabody technology to make bispecific antibody fragments (Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), by using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol. 152:5368 (1994)), or other methods. Preparation of trispecific antibodies is described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0573] Engineered antibodies with three or more functional antigen binding sites, including Octopus antibodies, are also included herein (see, e.g., US 2006/0025576).

[0574] Within the scope of the description of Disclosure C herein, the antibody or antibody fragment also includes a Dual Acting Fab or DAF comprising an antigen binding site that binds to IL-8 as well as another, different antigen (see US 2008/0069820, for example).

[0575] 6. Antibody variants

Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into a nucleotide sequence encoding the antibody, or by

165

WO 2017/046994

PCT/JP2016/003616 peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues in the amino acid sequence of the antibody. A final construct can be attained with any combination of deletion, insertion, and substitution, as long as the final construct is an antibody that has the desired properties described in the context of Disclosure C.

[0576] In one embodiment, Disclosure C provides antibody variants having one or more amino acid substitutions. Such substitution sites may be any positions in an antibody. Amino acids for conservative substitutions are shown in Table 10 under the heading of conservative substitutions. Amino acids for typical substitutions that result in more substantial changes are shown in Table 10 under the heading of typical substitutions, and as further described in reference of amino acid side chain classes.

[0577] [Table 10]

Original Residue	Typical Substitution	Conservative Substitution
Ala (A)	Val; Leu; He	Val
Arg (R)	Lys; Gin; Asn	Lys
Asn (N)	Gin; His; Asp, Lys; Arg	Gin
Asp (D)	Glu; Asn	Glu
Cys(C)	Ser; Ala	Ser
Gln(Q)	Asn; Glu	Asn
Glu (E)	Asp; Gin	Asp
Gly (G)	Ala	Ala
His (H)	Asn; Gin; Lys; Arg	Arg
lie (I)	Leu; Val; Met; Ala; Phe; Norleucine	Leu
Leu (L)	Norleucine; He; Val; Met; Ala; Phe	He
Lys (K)	Arg; Gin; Asn	Arg
Met (M)	Leu; Phe; He	Leu
Phe(F)	Trp; Leu; Val; He; Ala; Tyr	Tyr
Pro {P)	Ala	Ala
Ser(S)	Thr	Thr
Thr (T)	Val; Ser	Ser
Trp (W)	Tyr; Phe	Tvr
Tyr(Y)	Trp; Phe; Thr; Ser	Phe
Val (V)	He; Leu; Met; Phe; Ala; Norleucine	Leu

[0578] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0579] Non-conservative substitutions will entail exchanging a member of one of these

166

WO 2017/046994 PCT/JP2016/003616 classes for another class.

[0580] Amino acid insertions include fusion of a polypeptide comprising one, two, or three to one hundred or more residues at the N terminus and/or C terminus, as well as insertion of one or more amino acid residues into a sequence. Antibodies with such terminal insertion include, for example, antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include those that result from N- or C-terminal fusion of the antibody to an enzyme (for example, ADEPT) or a polypeptide that increases plasma half-life of antibody.

[0581 ] 7. Glycosylated variants

In one embodiment, antibodies provided according to Disclosure C may be glycosylated antibodies. Glycosylation sites can be added to or deleted from an antibody by altering amino acid sequences in such a way as to create or remove glycosylation sites.

[0582] When an antibody comprises an Fc region, the sugar chain attached thereto can be altered. Naive antibodies produced by animal cell typically contain a branched, biantennary oligosaccharide, which is attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (see Wright et al. TIBTECH 15:26-32 (1997)). The oligosaccharide includes, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the stem of the biantennary oligosaccharide structure. In one embodiment, the oligosaccharide in the antibody of Disclosure C is modified to create antibody variants having certain improved properties.

[0583] 8. Fc region variants

In one embodiment, one or more amino acid modifications are introduced into the Fc region of an antibody provided according to Disclosure C, thereby generating an Fc region variant. Fc region variants include those that have a modification (for example, a substitution) of one, two, three, or more amino acids in a native human Fc region sequence (for example, the Fc region of human IgGl, IgG2, IgG3, or IgG4).

[0584] An anti-IF-8 antibody of Disclosure C may contain an Fc region having at least one of the following five properties, without being limited thereto: (a) increased binding affinity for FcRn of the Fc region relative to the binding affinity for FcRn of a native Fc region at acidic pH; (b) reduced binding affinity of the Fc region for pre-existing ADA relative to the binding affinity of a native Fc region for the pre-existing ADA; (c) increased plasma half-life of the Fc region relative to the plasma half-life of a native Fc region; (d) reduced plasma clearance of the Fc region relative to the plasma clearance of a native Fc region; and (e) reduced binding affinity of the Fc region for an effector receptor relative to the binding affinity of a native Fc region for the effector receptor.

In some embodiments, the Fc region has 2, 3 or 4 of the above-listed properties. In one

167

WO 2017/046994

PCT/JP2016/003616 embodiment, Fc region variants include those having an increased FcRn-binding affinity at an acidic pH. Fc region variants with increased FcRn-binding affinity include, but are not limited to, Fc region variants whose FcRn-binding affinity is increased up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold,

50-fold, or 100-fold as compared to the FcRn-binding affinity of an antibody comprising the native IgG Fc region.

[0585] In one embodiment, an Fc region variant includes a safe and advantageous Fc region variant that does not bind to pre-existing ADA, and at the same time has improved plasma retention. As used in the context of Disclosure C, the term ADA refers to an endogenous antibody having binding affinity for an epitope on a therapeutic antibody. As used in the context of Disclosure C, the term pre-existing ADA refers to a detectable anti-drug antibody present in a patient's blood prior to administration of a therapeutic antibody to the patient. Pre-existing ADA includes the rheumatoid factor. Fc region variants with low binding affinity for pre-existing ADA include, but are not limited to, Fc region variants whose ADA-binding affinity is reduced to 1/10 or less, 1/50 or less, or 1/100 or less as compared to the ADA-binding affinity of an antibody comprising the native IgG Fc region.

[0586] In one embodiment, an Fc region variant includes an Fc region variant whose binding affinity for complement proteins is low or that do not bind to complement proteins. Complement proteins include Clq. Fc region variants with low binding affinity for complement proteins include, but are not limited to, Fc region variants whose binding affinity for complement proteins is reduced to 1/10 or less, 1/50 or less, or 1/100 or less as compared to the complement protein-binding affinity of an antibody comprising a native IgG Fc region.

[0587] In one embodiment, an Fc region variant includes an Fc region variant whose binding affinity for effector receptors is low or that does not have the binding affinity for an effector receptor. The effector receptors include, but are not limited to, FcyRI, FcyRII, and FcyRIII. FcyRI includes, but is not limited to, FcyRIa, FcyRIb, and FcyRIc, as well as subtypes thereof. FcyRII includes, but is not limited to, FcyRIIa (which has two allotypes: R131 and H131) and FcYRIIb. FcyRIII includes, but is not limited to,

FcyRI I la (which has two allotypes: VI58 and FI58) and FcyRIIIb (which has two allotypes: FcyRIIIb-ΝΑΙ and FcyRIIIb-NA2). Fc region variants with low binding affinity for effector receptors include, but are not limited to, Fc region variants whose binding affinity for effector receptors is reduced to at least 1/10 or less, 1/50 or less, or 1/100 or less as compared to the binding affinity of an antibody comprising a native IgG Fc region.

[0588] In one embodiment, an Fc region variant includes an Fc region comprising one or more amino acid substitutions at any of the positions of the group consisting of

168

WO 2017/046994

PCT/JP2016/003616 positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438, and 440, according to EU numbering as compared to the native Fc region.

[0589] In one embodiment, an Fc region variant includes an Fc region comprising amino acid substitutions at positions 235, 236, 239, 428, 434, 436, 438, and 440, according to EU numbering as compared to the native Fc region.

[0590] In one embodiment, an Fc region variant includes an Fc region comprising amino acid substitutions at positions 235, 236, 327, 330, 331, 428, 434, 436, 438, and 440, according to EU numbering as compared to the native Fc region.

[0591] In one embodiment, an Fc region variant includes an Fc region comprising one or more amino acid substitutions selected from the group consisting of: L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

[0592] In one embodiment, an Fc region variant includes an Fc region comprising the amino acid substitutions of M428L, N434A, Y436T, Q438R, and S440E.

[0593] In one embodiment, an Fc region variant includes an Fc region comprising the amino acid substitutions of L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E.

[0594] In one embodiment, an Fc region variant includes an Fc region comprising the amino acid substitutions of L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

[0595] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may be an anti-IL-8 antibody that binds to IL8 in a pH-dependent manner. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may maintain IL-8-neutralizing activity stably in vivo (for example, in plasma). The antiIL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may be an antibody with low immunogenicity. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fc region whose FcRn-binding affinity at an acidic pH (e.g., pH 5.8) is increased as compared to the FcRn-binding affinity of a native Fc region at the acidic pH. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fc region whose binding affinity for pre-existing ADA is reduced as compared to the binding affinity of a native Fc region for pre-existing ADA. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fc region whose half-life in plasma is prolonged as compared to that of a native Fc region. The anti169

WO 2017/046994

PCT/JP2016/003616

IL-8 antibody comprising the amino acid sequence of SEQ ID NO:80 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fc region whose binding affinity for effector receptors is reduced as compared to that of a native Fc region. In a further embodiment, the anti-IL-8 antibody comprises a combination of any 2, 3, 4, 5, 6, or all 7 of above-listed properties.

[0596] In one embodiment, an anti-IL-8 antibody of Disclosure C comprises the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may be an anti-IL-8 antibody that binds to IL8 in a pH-dependent manner. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may maintain IL-8-neutralizing activity stably in vivo (for example, in plasma). The antiIL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may be an antibody with low immunogenicity. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fc region whose FcRn-binding affinity at an acidic pH is increased as compared to the FcRn-binding affinity of a native Fc region. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fc region whose binding affinity for pre-existing ADA is reduced as compared to the binding affinity of a native Fc region for pre-existing ADA. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fc region whose half-life in plasma is prolonged as compared to that of a native Fc region. The anti-IL-8 antibody comprising the amino acid sequence of SEQ ID NO:81 and/or the amino acid sequence of SEQ ID NO:82 may contain an Fc region whose binding affinity for effector receptors is reduced as compared to that of a native Fc region. In a further embodiment, the anti-IL-8 antibody comprises a combination of any 2, 3, 4, 5, 6, or all 7 of above-listed properties.

[0597] In certain embodiments, Disclosure C encompasses an antibody variant that possesses some but not all effector functions. The antibody variant can be a desirable candidate for cases in which certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays known in the art can routinely be conducted to confirm the reduction/complete loss of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to confirm that an antibody lacks FcyR binding (hence lacking ADCC activity), but retains FcRn binding ability.

[0598] The primary cultured cells for mediating ADCC and NK cells express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyR!!!. FcR expression on

170

WO 2017/046994

PCT/JP2016/003616 hematopoietic cells is summarized in Table 3 on page 464 of Ravetch et al., Annu.

Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in US Patent No. 5,500,362 (see, e.g. Hellstrom. et al., Proc. Natl Acad. Sci. USA 83:7059-7063 (1986), Hellstrom et al., Proc. Natl Acad. Sci. USA 82:1499-1502 (1985); US Patent No. 5,821,337, and Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive isotope assays are available for assessing effector cell function (see, for example, ACTI(TM) non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.

[0599] Alternatively or additionally, ADCC activity of an antibody variant of interest may be assessed in vivo, for example, in an animal model as disclosed in Clynes et al.,

Proc. Natl. Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, e.g., GazzanoSantoro et al., J. Immunol. Meth. 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can be performed using methods known in the art (see, e.g., Petkova et al., Inti. Immunol. 18(12):1759-1769 (2006)).

[0600] Antibodies with reduced effector functions include those with substitution of one or more of Fc region residues at position 238, 265, 269, 270, 297, 327 or 329 (US Patent No. 6,737,056). Such Fc region variants include Fc region variants with substitutions at two or more of residues at position 265, 269, 270, 297 or 327, including the so-called DANA Fc region variants with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).

[0601] Antibody variants with improved or diminished binding to FcR groups are described below. (See, e.g., US Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).) [0602] Antibodies with increased blood half lives and improved FcRn binding at an acidic pH are described in US2005/0014934. The described antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn. Such Fc region variants include those with substitutions at one or more of positions selected from 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 in an Fc region, for example, substitution of position 434 in an Fc region (US Patent No. 7,371,826).

[0603] See also Duncan et al., Nature 322:738-40 (1988); US Patent No. 5,648,260; US

171

WO 2017/046994

PCT/JP2016/003616

Patent No. 5,624,821; and WO 94/29351 for other examples of Fc region variants.

[0604] 9. Antibody derivatives

In certain embodiments, an antibody provided in Disclosure C may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include, but are not limited to, water soluble polymers. Examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol or propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolylpropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in industrialization due to its stability in water.

[0605] The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymers are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivertization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, when the antibody derivative is used in a defined therapy, etc.

[0606] In another embodiment, conjugates of an anti-IL-8 antibody of Disclosure C and nonproteinaceous moiety that may be selectively heated by exposure to radiation may be provided. In one embodiment, the nonproteinaceous moiety is, for example, a carbon nanotube (see, e.g., Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength and includes, without being limited thereto, wavelengths that are harmless to humans but can heat the nonproteinaceous moiety to a temperature so as to kill cells proximal to the antibody-nonproteinaceous moiety.

[0607] B, Recombination methods and compositions

Anti-IL-8 antibodies of Disclosure C may be produced using recombinant methods and compositions, for example, as described in US Patent No. 4,816,567. One embodiment provides isolated nucleic acid(s) encoding an anti-IL-8 antibody which are presented as Disclosure C. Such nucleic acid(s) may encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In one embodiment, a host cell comprising such nucleic acid(s) is

172

WO 2017/046994

PCT/JP2016/003616 provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes the VL of the antibody and the VH of an antibody, or (2) a first vector comprising a nucleic acid that encodes the VL of an antibody and a second vector comprising a nucleic acid that encodes the VH of the antibody.

[0608] In one embodiment, the host is eukaryotic (e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, SP20 cell)).

[0609] In one embodiment, a method of producing an anti-IL-8 antibody of Disclosure C is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-IL-8 antibody as provided above, under conditions suitable for expressing the antibody, and optionally recovering the antibody (e.g., from the host cell or host cell culture medium).

[0610] For recombinant production of an anti-IL-8 antibody, nucleic acid(s) encoding an antibody, for example, as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid(s) may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to nucleic acids encoding the heavy and light chains of the antibody).

[0611] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described within the scope of the description of Disclosure C herein. For example, antibodies may be produced in bacteria, in particular, when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see for example, US Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, for expression of antibody fragments in E. coli). After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0612] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for cloning or expression of antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been humanized, which enable production of antibodies with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0613] Suitable host cells for the expression of a glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Without particular limitations, baculovirus is used in conjunction with insect cells for transfection of Spodoptera frugiperda cells and numerous baculoviral strains have been identified.

173

WO 2017/046994 PCT/JP2016/003616 [0614] Plant cell cultures can also be utilized as hosts. See US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).

[0615] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension are useful. Other examples of useful mammalian host cells are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells.

[0616] Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp20, but are not limited thereto. For a review of other mammalian host cell lines suitable for antibody production, see Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0617] An antibody of Disclosure C produced by culturing such host cells as described above to carry nucleic acids that encode the antibody under conditions that are suitable for antibody expression may be isolated from inside or outside of the host cells (media, milk, etc.), and purified as a substantially pure homogeneous antibody. Isolation/purification methods that are generally used to purify polypeptides can be appropriately used to isolate and purify the antibody; however, the methods are not limited to the above example. The antibody can be appropriately separated and purified, for example, by appropriately selecting and combining column chromatography, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization, without being limited thereto. Chromatography includes, but is not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. Such chromatography can be performed using liquid chromatography, for example, HPLC and FPLC. Columns for use in affinity chromatography include, but are not limited to, Protein A column and Protein G column. Protein A columns include, but are not limited to, Hyper D, POROS, Sepharose F. F. (Pharmacia) and so on.

174

WO 2017/046994 PCT/JP2016/003616 [0618] Focusing on the characteristics of anti-IL-8 antibodies such as increased extracellular matrix-binding activity and enhanced cellular uptake of the complex described above, Disclosure C provides methods for selecting antibodies with increased extracellular matrix-binding and antibodies with enhanced cellular uptake. In one embodiment, Disclosure C provides methods for producing an anti-IL-8 antibody comprising variable region whose binding activity to IL-8 is in an pH-dependent manner, which comprise the steps of: (a) assessing the binding between anti-IL-8 antibody and extracellular matrix; (b) selecting an anti-IL-8 antibody that strongly binds to extracellular matrix; (c) culturing a host that comprises a vector carrying a nucleic acid encoding the antibody; and (d) isolating the antibody from the culture medium.

[0619] Binding with extracellular matrix can be assessed by any methods without particular limitations, as long as they are known to those of ordinary skill in the art. For example, assays can be carried out using an ELISA system for detecting the binding between an antibody and extracellular matrix, where the antibody is added to an extracellular matrix-immobilized plate and a labeled antibody against the antibody is added thereto. Alternatively, such assays can be performed, for example, using an electrochemiluminescence (ECL) method in which a mixture of the antibody and a ruthenium antibody is added to an extracellular matrix-immobilized plate and the binding between the antibody and extracellular matrix is assessed based on the electrochemiluminescence of ruthenium.

[0620] The anti-IL-8 antibody being assessed for extracellular matrix binding in step (a) above may be the antibody by itself or in contact with IL-8. Selecting an anti-IL-8 antibody that strongly binds to extracellular matrix in step (b) means that an anti-IL-8 antibody is selected based on the criterion that a value representing the binding between extracellular matrix and the anti-IL-8 antibody is higher than a value representing the binding between extracellular matrix and the control antibody in the assessment of extracellular matrix binding, and may be, for example, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more; however, the ratio is not particularly limited to the examples above. Other than the presence of IL-8, the conditions are preferably the same in the step of assessing the binding between an anti-IL-8 antibody and extracellular matrix. The control anti-IL-8 antibody for use in comparing several modified anti-IL-8 antibodies may be the unmodified anti-IL-8 antibody. In this case, the conditions are preferably the same other than the presence of IL-8. Specifically, in one embodiment, Disclosure C includes selecting an antibody with a higher value representing extracellular matrix binding from several anti-IL-8 antibodies that are not in contact with IL-8. In another embodiment, Disclosure C includes selecting an antibody with a higher value representing extracellular matrix binding from several anti-IL-8 antibodies that are in contact with

175

WO 2017/046994

PCT/JP2016/003616

IL-8. In an alternative embodiment, selecting an anti-IL-8 antibody that strongly binds to extracellular matrix in step (b) means that an antibody may be selected based on the criterion that the binding between an antibody and extracellular matrix varies depending on the presence of IL-8, when assessing extracellular matrix binding. The ratio of a value representing the extracellular matrix binding of an anti-IL-8 antibody in contact with IL-8 to a value representing the extracellular matrix binding of an antiIL-8 antibody not in contact with IL-8 may be, for example, 2 to 1000. Furthermore, the ratio between the values may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.

[0621] C. Assays

Anti-IL-8 antibodies provided within the scope of Disclosure C described herein can be identified, screened, or characterized in terms of their physical/chemical properties and/or biological activities by various methods known in the art.

[0622] 1, Binding assays and other assays

In one aspect, the antibodies of Disclosure C can be assessed for their antigenbinding activity by known methods, for example, ELISA, Western blotting, kinetic exclusion assay (KinExA™), and surface plasmon resonance using a device such as BIACORE (GE Healthcare).

[0623] In one embodiment, the binding affinity can be measured using BIACORE T200 (GE Healthcare) in the following manner. An appropriate amount of a trapping protein (for example, Protein A/G (PIERCE)) is immobilized onto a sensor chip CM4 (GE Healthcare) by the amine-coupling method, and an antibody of interest is allowed to be captured. Then, a diluted antigen solution and running buffer (as a reference solution: for example, 0.05% tween20, 20 mM ACES, 150 mM NaCl, pH 7.4) are injected to interact antigen molecules with the antibody trapped on the sensor chip. The sensor chip is regenerated using 10 mM glycine HC1 solution (pH 1.5). Measurements are performed at a pre-determined temperature (for example, 37°C, 25°C, or 20°C). The association rate constant kon (1/Ms) and dissociation rate constant koff (1/s), both of which are kinetic parameters, are calculated from sensorgrams obtained by measurement. The KD (M) of each antibody for the antigen is calculated based on these constants. Each parameter is calculated using the BIACORE T200 Evaluation Software (GE Healthcare).

[0624] In one embodiment, IL-8 can be quantitated as described below. An anti-human IL-8 antibody comprising the mouse IgG constant region is immobilized onto a plate. A solution comprising IL-8 bound to a humanized anti-IL-8 antibody, which does not compete with the above-described anti-human IL-8 antibody, is aliquoted to the immobilized plate. After stirring, a biotinylated anti-human IgK light chain antibody is added and allowed to react for a certain period of time. Then, SULFO-Tag-labeled

176

WO 2017/046994

PCT/JP2016/003616 streptavidin is further added and allowed to react for a certain period of time. Then, assay buffer is added and immediately measurement is performed with SECTOR Imager 2400 (Meso Scale Discovery).

[0625] 2, Activity assays

In one aspect, assays are provided to identify an anti-IL-8 antibody having a biological activity. The biological activity includes, for example, IL-8-neutralizing activity and the activity of blocking IL-8 signals. The Disclosure C also provides antibodies with such biological activity in vivo and/or ex vivo.

[0626] In one embodiment, the method for determining the level of IL-8 neutralization is not particularly limited and it can also be determined by the methods described below. PathHunter™ CHO-K1 CXCR2 β-Arrestin Cell Line (DiscoveRx, Cat.# 93-0202C2) is an artificial cell line created to express human CXCR2 known as a human IL-8 receptor and emit chemiluminescence when receiving signals by human IL-8. When human IL-8 is added to a culture medium of the cells, chemiluminescence is emitted from the cells in a manner that depends on the concentration of added human IL-8. When human IL-8 is added in combination with an anti-human IL-8 antibody to the culture medium, the chemiluminescence of the cells is reduced or undetectable as compared to when the antibody is not added, since the anti-human IL-8 antibody can block the IL-8 signal transduction. Specifically, the stronger the human IL8-neutralizing activity of the antibody is, the weaker the level of chemiluminescence is; and the weaker the human IL-8-neutralizing activity of the antibody is, the greater the level of chemiluminescence is. Thus, the human IL-8-neutralizing activity of the anti-human IL-8 antibody can be assessed by examining the difference described above.

[0627] D, Pharmaceutical formulations

Pharmaceutical formulations comprising an anti-IL-8 antibody as described within the scope of the description Disclosure C herein may be prepared by mixing an antiIL-8 antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized formulations or aqueous solution formulations.

[0628] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include without being limited to buffers such as phosphate, citrate, histidine, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularΥΠ

WO 2017/046994

PCT/JP2016/003616 weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS ™, or polyethylene glycol (PEG).

[0629] Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX™, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Appl. Publ. Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more glycosaminoglycanases such as chondroitinases.

[0630] Exemplary lyophilized antibody formulations are described in US Patent No.

6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and W02006/044908; and the W02006/044908 formulations include a histidine-acetate buffer.

[0631] The formulation within the scope of Disclosure C herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0632] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0633] Sustained-release preparations may be prepared. Suitable examples of sustainedrelease preparations include semipermeable matrices of solid hydrophobic polymers containing the anti-IL8 antibody of the Disclosure C, in which the matrices are in the form of shaped articles, for example, films or microcapsules.

[0634] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

178

WO 2017/046994 PCT/JP2016/003616 [0635] E, Therapeutic methods and compositions

In some embodiments, the anti-IL-8 antibodies provided according to Disclosure C are used in therapeutic methods.

[0636] In one aspect, an anti-IL-8 antibody for use as a pharmaceutical composition is provided. In an alternative aspect, an anti-IL-8 antibody for use in treating a disease where IL-8 is present in an excessive amount is provided. In one embodiment, an antiIL-8 antibody for use in methods for treating a disease where IL-8 is present in an excessive amount is provided. In one embodiment, Disclosure C provides methods for treating an individual with a disease where IL-8 is present in an excessive amount (for example, a disease caused by the presence of excessive IL-8), which comprises administering an effective amount of an anti-IL-8 antibody to the individual. In another embodiment, Disclosure C provides anti-IL-8 antibodies for use in such methods. In one embodiment, Disclosure C relates to a pharmaceutical composition comprising an effective amount of an anti-IL-8 antibody, which is used to treat a disease where IL-8 is present in an excessive amount. In one embodiment, Disclosure C relates to the use of an anti-IL-8 antibody in producing a pharmaceutical composition for a disease where IL-8 is present in an excessive amount. In one embodiment, Disclosure C relates to the use of an effective amount of an anti-IL-8 antibody in treating a disease where IL-8 is present in an excessive amount. Diseases where IL-8 is present in an excessive amount include, but are not limited to, inflammatory skin diseases such as inflammatory keratosis (psoriasis, etc.), atopic dermatitis, and contact dermatitis; autoimmune diseases such as chronic inflammatory diseases including chronic rheumatoid arthritis, systemic lupus erythematosus (SLE), and Behcet disease; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, and allergic hepatitis induced by drugs; inflammatory kidney diseases such as glomerular nephritis; inflammatory respiratory diseases such as bronchitis and asthma; chronic inflammatory vascular disease such as atherosclerosis; multiple sclerosis; oral ulcer; chorditis; inflammation induced by an artificial organ/artificial blood vessel; a malignant tumor such as ovarian cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, melanoma, head and neck cancer, and kidney cancer; sepsis caused by infection; cystic fibrosis; pulmonary fibrosis; and acute lung injury.

[0637] In an alternative embodiment, Disclosure C provides an anti-IL-8 antibody for use in suppressing the accumulation of IL-8 with biological activity. Suppressing the accumulation of IL-8 may be achieved by preventing the amount of pre-existing IL-8 in vivo from increasing or by reducing the amount of pre-existing IL-8 in vivo. In one embodiment, the Disclosure C provides an anti-IL-8 antibody for suppressing the accumulation of IL-8 in an individual to suppress the accumulation of IL-8 with biological

179

WO 2017/046994

PCT/JP2016/003616 activity. Here, IL-8 present in vivo may refer to IL-8 complexed with anti-IL-8 antibody or free IL-8; alternatively, it may refer to total IL-8 as its sum. Herein, present in vivo may mean secreted to the outside of the cells in vivo. In one embodiment, Disclosure C provides a method for suppressing the accumulation of IL-8 with biological activity, which comprises the step of administering an effective amount of an anti-IL-8 antibody to an individual. In one embodiment, Disclosure C relates to a pharmaceutical composition for suppressing the accumulation of IL-8 with biological activity, which comprises an effective amount of an anti-IL-8 antibody. In one embodiment, Disclosure C relates to the use of an anti-IL-8 antibody in producing a pharmaceutical composition for suppressing the accumulation of IL-8 with biological activity. In one embodiment, Disclosure C relates to the use of an effective amount of an anti-IL-8 antibody in suppressing the accumulation of IL-8 with biological activity. In one embodiment, an anti-IL-8 antibody of Disclosure C suppresses the accumulation of IL-8 as compared to an anti-IL-8 antibody that does not have pH-dependent binding activity. In the above-described embodiments, the individual is preferably a human.

[0638] In an alternative embodiment, Disclosure C provides an anti-IL-8 antibody for use in inhibiting angiogenesis (e.g., neoangiogenesis). In one embodiment, Disclosure C provides a method for inhibiting neoangiogenesis in an individual which comprises administering an effective amount of an anti-IL-8 antibody to the individual, and also provides an anti-IL-8 antibody for use in the method. In one embodiment, Disclosure C relates to a pharmaceutical composition for inhibiting neoangiogenesis which comprises an effective amount of an anti-IL-8 antibody. In one embodiment,

Disclosure C relates to the use of an anti-IL-8 antibody in producing a pharmaceutical composition for inhibiting neoangiogenesis. In one embodiment, Disclosure C relates to the use of an effective amount of an anti-IL-8 antibody in inhibiting neoangiogenesis. In the above-described embodiments, the individual is preferably a human.

[0639] In an alternative aspect, Disclosure C provides an anti-IL-8 antibody for use in inhibiting the facilitation of neutrophil migration. In one embodiment, Disclosure C provides a method for inhibiting the facilitation of neutrophil migration in an individual, which comprises administering an effective amount of an anti-IL-8 antibody to the individual; and also provides an anti-IL-8 antibody for use in the method. In one embodiment, Disclosure C relates to pharmaceutical compositions for inhibiting facilitation of neutrophil migration in an individual, which comprise an effective amount of an anti-IL-8 antibody. In one embodiment, Disclosure C relates to the use of an antiIL-8 antibody in producing a pharmaceutical composition for inhibiting facilitation of neutrophil migration in an individual. In one embodiment, Disclosure C relates to the use of an effective amount of an anti-IL-8 antibody in inhibiting facilitation of

180

WO 2017/046994

PCT/JP2016/003616 neutrophil migration in an individual. In the above-described embodiments, the individual is preferably a human.

[0640] In an alternative embodiment, Disclosure C provides a pharmaceutical composition comprising an anti-IL-8 antibody provided herein for example, for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical composition comprises an anti-IL-8 antibodies provided in Disclosure C and a pharmaceutically acceptable carrier.

[0641] An antibody of Disclosure C can be used either alone or in combination with other agents in a therapy. For instance, an antibody of Disclosure C may be co-administered with at least one additional therapeutic agent.

[0642] An antibody of Disclosure C (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injections such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules include, without being limited to, single or multiple administrations over various time-points, bolus administration, and pulse infusion may be contemplated herein.

[0643] Preferably an antibody of Disclosure C is formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question.

[0644] The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These may be generally used at the same dosages and via the same administration routes described within the scope of the description of Disclosure C herein, or from 1 to 99% of the dosages described within the scope of the description of Disclosure C herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

[0645] For the prevention or treatment of a disease, the appropriate dose of an antibody of Disclosure C (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody variant is administered for

181

WO 2017/046994

PCT/JP2016/003616 preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (for example, 0.1 mg/kg to 10 mg/kg) of an antibody can be an initial candidate dose for administration to the patient, for example, by a single administration or several separate administrations, or by continuous infusion. One typical daily dose may range from about 1 mg/kg to 100 mg/kg or more, depending on the factors described above. For repeated administrations over several days or longer, depending on the condition, the treatment may be generally sustained until a desired suppressive effect of disease symptoms is seen. A typical dose of an antibody may fall, for example, in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of, for example, about 0.5 mg/kg, for example, 2.0 mg/kg, for example, 4.0 mg/kg, or for example, 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (for example, in such a manner that the patient receives from about two to about twenty doses, or about six doses of the antibody). It is possible to administer an initial higher loading dose, followed by one or more lower doses; however, other dosage regimens may be useful. The progress of this therapy can be easily monitored by conventional techniques and assays.

[0646] F. Articles of manufacture

In another aspect of Disclosure C, the disclosure provides articles of manufacture comprising materials useful for the treatment, prevention and/or diagnosis of a disorder described above. Such an article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, and intravenous solution bags. The containers may be formed from various materials such as glass or plastic. Such a container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active ingredient in the composition is an antibody of Disclosure C. The label or package insert indicates that the composition is used for treating the condition of choice.

[0647] Moreover, the article of manufacture may include: (a) a first container that comprises a composition comprising an antibody of Disclosure C; and (b) a second container that comprises a composition comprising an additional cytotoxic agent or a different therapeutic agent. The article of manufacture in the embodiments of Disclosure C may further include a package insert indicating that the compositions can be used to treat a

182

WO 2017/046994

PCT/JP2016/003616 particular condition. Alternatively or additionally, the article of manufacture may further include, for example, a second (or third) container that comprises a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. The article of manufacture may further include other materials desirable from a commercial perspective or a user's standpoint, including other buffers, diluents, filters, needles, and syringes.

[0648] Those of ordinary skill in the art can appreciate based on the technological common knowledge in the art, that the Disclosure C also includes all combinations of the whole or part of one or more of the entire embodiments described herein, except where there is a technological inconsistency.

[0649] Disclosure A. B. or C

All technical background documents cited herein are incorporated herein by reference.

[0650] As used herein, the phrase and/or is understood to include the meaning of combinations of terms before and after the phrase and/or, which include all combinations of the terms appropriately linked by the phrase.

[0651] While various elements are described herein with terms such as first, second, third, fourth, etc., it is appreciated that the elements are not limited by such terms. These terms are used only to distinguish an element from other elements, and it is appreciated that, for example, a first element could be termed a second element, and similarly, a second element could be termed a first element, without departing from the scope of Disclosures A, B, and C.

[0652] Unless explicitly stated otherwise or unless there are inconsistencies in the context, any terms expressed in the singular form herein are meant to also include the plural form and any terms expressed in the plural form herein are meant to also include the singular form.

[0653] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure. Unless otherwise defined differently, all terms (including technical and scientific terms) used herein are interpreted to have the same meaning as commonly understood by those of ordinary skill in the art to which Disclosures A, B, and C pertain, and will not be interpreted in an idealized or overly formal sense.

[0654] As used herein, the term comprises is intended to specify the presence of described items (members, steps, elements, numbers, etc.), unless the context clearly indicates otherwise; and the term does not preclude the presence of other items (members, steps, elements, numbers, etc.).

[0655] Embodiments of the Disclosures A, B, and C are described with reference to schematic illustrations, which may be exaggerated for clarity.

183

WO 2017/046994 PCT/JP2016/003616 [0656] Unless there are inconsistencies in the context, numerical values used herein are understood to be values that represent a certain range based on the common technical knowledge of those of ordinary skill in the art. For example, the expression 1 mg is understood to be described as about 1 mg with certain variations. For example, the expression 1 to 5 items is understood to be described specifically and individually as 1 item, 2 items, 3 items, 4 items, 5 items, unless there are inconsistencies in the context.

Examples [0657] Hereinbelow, Disclosures A, B, and C will be specifically described by Examples 1 to 4 and 21 to 23, Examples 5 to 7, 19 and 20, and Examples 8 to 19, respectively, but they are not to be construed as being limited thereto. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1 [0658] Production of pH-dependent human IL-6 receptor-binding human antibodies with increased pi

Fv4-IgGl disclosed in WO2009/125825 is an antibody that binds to the human IL-6 receptor in a pH-dependent manner, and comprises VH3-IgGl (SEQ ID NO:24) as the heavy chain and VL3-CK (SEQ ID NO:32) as the light chain. To increase the pi of Fv4-IgGl, the variable region of Fv4-IgGl was introduced with amino acid substitutions that decrease the number of negatively charged amino acids (such as aspartic acid and glutamic acid), while increasing the positively charged amino acids (such as arginine and lysine). Specifically, VH3(High_pI)-IgGl (SEQ ID NO:25) was produced as a heavy chain with increased pi by substituting glutamic acid at position 16 with glutamine, glutamic acid at position 43 with arginine, glutamine at position 64 with lysine, and glutamic acid at position 105 with glutamine, according to Rabat numbering, in the heavy chain VH3-IgGl. Similarly, VL3(High_pI)-CR (SEQ ID NO:33) was produced as a light chain with increased pi by substituting serine at position 18 with arginine, glutamine at position 24 with arginine, glutamic acid at position 45 with lysine, glutamic acid at position 79 with glutamine, and glutamic acid at position 107 with lysine, according to Rabat numbering, in the light chain VL3-CR. When introducing the substitution at position 79 of VL3-CR, modifications that involve substituting alanine at position 80 with proline and alanine at position 83 with isoleucine were simultaneously introduced, although not with the aim to increase the pi.

[0659] The following antibodies were produced by the method of Reference Example 2: (a) Low_pI-IgGl comprising VH3-IgGl as the heavy chain and VL3-CR as the light chain; (b) Middle_pI-IgGl comprising VH3-IgGl as the heavy chain and

184

WO 2017/046994

PCT/JP2016/003616

VL3(High_pI)-CK as the light chain; and (c) High_pI-IgGl comprising VH3(High_pI)-IgGl as the heavy chain and VL3(High_pI)-CK as the light chain.

[0660] Next, the theoretical pi for each of the produced antibodies was calculated using

GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION) using methods known in the art (see, e.g., Skoog et al., Trends Analyt. Chem. 5(4): 82-83 (1986)). The side chains of all cysteines in the antibody molecule were assumed to form disulfide bonds, and the contribution of cysteine side chains to pKa was excluded from the calculation.

[0661] The calculated theoretical pi values are shown in Table 3. While the theoretical pi of Low_pI-IgGl was 6.39, those of Middle_pI-IgGl and High_pI-IgGlwere 8.70 and 9.30, respectively, showing that the theoretical pi values increased in a stepwise manner.

[0662] WO2011/122011 discloses Fv4-IgGl-Fl 1 (hereinafter, referred to as Low_pl-Fl 1) and Fv4-IgGl-F939 (hereinafter, referred to as Low_pl-F939) whose FcRn-mediated uptake into cells has been enhanced by introducing amino acid substitutions into the Fc region of Fv4-IgGl and conferring FcRn-binding ability under neutral pH conditions. Furthermore, WO2013/125667 discloses Fv4-IgGl-F1180 (hereinafter, referred to as Low_pl-Fl 180) whose Fey R-mediated uptake into cells has been enhanced by introducing amino acid substitutions into the Fc region of Fv4-IgGl to increase its FcyRbinding ability under neutral pH conditions. Simultaneously, amino acid modification for enhancing the plasma retention of the antibody by increasing its FcRn binding under the acidic pH condition in the endosomes was introduced into Fv4-IgGl-Fl 180. The antibodies shown below were produced by increasing the pi of antibodies containing these novel Fc region variants.

[0663] Specifically, VH3-IgGl-Fl 1 (SEQ ID NO:30) and VH3-IgGl-F939 (SEQ ID NO:26) in WO2011/122011, and VH3-IgGl-F1180 (SEQ ID NO:28) in WO2013/125667 were each subjected to substitutions of glutamic acid at position 16 with glutamine, glutamic acid at position 43 with arginine, glutamine at position 64 with lysine, and glutamic acid at position 105 with glutamine, according to Rabat numbering, to produce VH3(High_pI)-Fll (SEQ ID NO:31), VH3(High_pI)-F939 (SEQ ID NO:27), and VH3(High_pI)-F1180 (SEQ ID NO:29), respectively, as heavy chains with increased pi.

[0664] The following antibodies were produced by the method of Reference Example 2 using these heavy chains: (1) Low_pl-F939 comprising VH3-IgGl-F939 as the heavy chain and VL3-CK as the light chain; (2) Middle_pl-F939 comprising

VH3(High_pI)-F939 as the heavy chain and VL3-CK as the light chain; (3) High_pl-F939 comprising VH3(High_pI)-F939 as the heavy chain and VL3(High_pI)-CK as the light chain; (4) Low_pl-F1180 comprising VH3-IgGl-F1180 as the heavy chain and VL3-CK as the light chain; (5) Middle_pl-Fl 180 comprising

185

WO 2017/046994

PCT/JP2016/003616

VH3-IgGl-Fl 180 as the heavy chain and VL3(High_pI)-CK as the light chain; (6) High_pI-F1180 comprising VH3(High_pI)-F1180 as the heavy chain and VL3(High_pI)-CK as the light chain; (7) Low_pl-Fll comprising VH3-IgGl-Fll as the heavy chain and VL3-CK as the light chain; and (8) High_pl-Fl 1 comprising VH3(High_pI)-Fl 1 as the heavy chain and VL3(High_pI)-CK as the light chain.

[0665] Next, the theoretical pi for each of the produced antibodies was calculated using

GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION) by a method similar to that described previously. The calculated theoretical pi values are shown in Table 3. In all novel Fc region variant-containing antibodies, the theoretical pi values increased in a stepwise manner in the order of Low_pl, Middle_pl, and High_pl.

[0666] [Table 3]

Antibody Name	Theoretical pi
Low pl-lgG1	6.39
Middle pl-lgG1	8.70
High pl-lgG1	9.30
Low pl-F939	6.67
Middle pl-F939	8.70
High pl-F939	9.42
Low pl-F1180	6.39
Middie p!-F1180	8.70
High pl-F1180	9.29
Low pl-F11	6.39
High pl-F11	9.28

Example 2 [0667] Antigen eliminating effects of antibodies with increased pi that show pH-dependent binding

12-11 In vivo assay of pl-adiusted pH-dependent human IL-6 receptor-binding antibodies

As shown below, in vivo assays were performed using the various pH-dependent human IL-6 receptor-binding antibodies produced in Example 1: Low_pI-IgGl, High_pI-IgGl, Low_pl-F939, Middle_pl-F939, High_pl-F939, Low_pl-F1180, Middle_pl-Fl 180, and High_pl-Fl 180.

[0668] Soluble human IL-6 receptor (also called hsIL-6R) prepared by the method of Reference Example 3, the anti-human IL-6 receptor antibody, and human immunoglobulin preparation Sanglopor were administered simultaneously to human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories; Methods Mol. Biol. 602: 93-104 (2010)), and the subsequent in vivo kinetics of the soluble human IL-6 receptor were evaluated. A mixed solution containing the soluble human IL-6 receptor, the anti-human IL-6 receptor antibody, and Sanglopor (at

186

WO 2017/046994

PCT/JP2016/003616 concentrations of 5 ug/mL, 0.1 mg/mL, and 100 mg/mL, respectively) was administered once at 10 mL/kg through the tail vein. Since the anti-human IL-6 receptor antibody was present in sufficient excess of the soluble human IL-6 receptor, almost all of the soluble human IL-6 receptor was assumed to be bound by the antibody. Blood was collected 15 minutes, seven hours, one day, two days, three days, and seven days after the administration. The collected blood was immediately subjected to centrifugation at 4°C and 15,000 rpm for 15 minutes to obtain the plasma. The separated plasma was stored in a freezer set to -20°C or below until measurements were taken.

[0669] (2-2) Measurement of the soluble human IL-6 receptor concentration in plasma by the electrochemiluminescence method

The soluble human IL-6 receptor concentration in mouse plasma was measured by the electrochemiluminescence method. Samples of soluble human IL-6 receptor adjusted to concentrations of 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90 pg/mL for the calibration curve, and mouse plasma assay samples diluted 50-fold or more were prepared, respectively. The samples were mixed with a monoclonal anti-human IL-6R antibody (R&D) ruthenium-labeled with SULFO-TAG NHS Ester (Meso Scale Discovery), a biotinylated anti-human IL-6R Antibody (R&D), and Tocilizumab (CAS number: 375823-41-9) which is a human IL-6 receptor-binding antibody, and then they were allowed to react overnight at 37 °C. The final concentration of Tocilizumab was adjusted to 333 pg/mL. Then, the reaction solutions were dispensed into a Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery). After another hour of reaction at room temperature, the reaction solution was washed. Then, immediately after Read Buffer T(x4) (Meso Scale Discovery) was dispensed into the plate, measurement was carried out using the SECTOR Imager 2400 (Meso Scale Discovery). The soluble human IL-6 receptor concentration was calculated based on the response in the calibration curve using the analytical software, SOFTmax PRO (Molecular Devices).

[0670] The observed changes in the concentration of the soluble human IL-6 receptor in the plasma of human FcRn transgenic mice after the intravenous administration are shown in Figs. 1, 2, and 3. Fig. 1 shows the effect of enhancing antigen elimination where the pi of the variable region was increased in the case of a native IgGl constant region.

Fig. 2 shows the effect of enhancing antigen elimination where the pi of the variable region was increased in an antibody that has been conferred with the ability to bind to FcRn under a neutral pH condition (F939). Fig. 3 shows the effect of enhancing antigen elimination where the pi of the variable region was increased in an antibody whose FcyR-binding ability under a neutral pH condition has been enhanced (FI 180).

[0671] In all cases, it was shown that by increasing the pi of the antibodies, the rate of antigen elimination by the pH-dependent binding antibodies can be accelerated. It was also shown that by further conferring an increase in the binding ability toward FcRn or

187

WO 2017/046994

PCT/JP2016/003616

FcyR under the neutral pH conditions, the rate of antigen elimination can be further accelerated as compared to when only the pi was increased in the pH-dependent binding antibodies (comparison of Fig. 1 to Figs. 2 and 3).

[0672] (2-3) In vivo infusion assay of pi-adjusted pH-dependent human IL-6 receptorbinding antibodies

An in vivo assay was conducted below using the various pH-dependent human IL-6 receptor-binding antibodies produced in Example 1: Low_pI-IgGl, High_pI-IgGl, Low_pl-Fl 1, and High_pl-Fl 1.

[0673] An infusion pump (MINI-OSMOTIC PUMP MODEL 2004; alzet) containing a soluble human IL-6 receptor was implanted subcutaneously on the back of human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)) to produce model animals whose plasma concentration of the soluble human IL-6 receptor was kept constant. Antihuman IL-6 receptor antibodies were administered to the model animals, and the in vivo kinetics of the antibodies after the administration were assessed.

[0674] Specifically, a monoclonal anti-mouse CD4 antibody obtained by a method known in the art was administered once at 20 mg/kg into the tail vein to suppress the production of neutralizing antibodies potentially producible by the mouse itself against the soluble human IL-6 receptor. Then, an infusion pump containing 92.8 pg/ml of the soluble human IL-6 receptor was implanted subcutaneously on the back of the mice. Three days after implantation of the infusion pump, anti-human IL-6 receptor antibodies were administered once at 1 mg/kg into the tail vein. Blood was collected from the mice 15 minutes, seven hours, one day, two days, three or four days, six or seven days, 13 or 14 days, 20 or 21 days, and 27 or 28 days after the administration of the anti-human IL-6 receptor antibodies. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at -20°C or below until measurements were taken.

[0675] (2-4) Measurement of the plasma hsIL-6R concentration by the electrochemiluminescence method

The hsIL-6R concentration in mouse plasma was measured by the electrochemiluminescence method. Samples of hsIL-6R adjusted to 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90 pg/mL for the calibration curve and mouse plasma assay samples diluted 50-fold or more were mixed with a monoclonal anti-human IL-6R antibody (R&D) ruthenium-labeled with SULFO-TAG NHS Ester (Meso Scale Discovery), a biotinylated anti-human IL-6R Antibody (R&D), and Tocilizumab, and they were allowed to react overnight at 37°C. The final concentration of Tocilizumab was adjusted to 333 pg/mL. Then, the reaction solutions were dispensed into a Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery). After another hour of reaction at

188

WO 2017/046994

PCT/JP2016/003616 room temperature, the reaction solution was washed. Then, immediately after Read Buffer T(x4) (Meso Scale Discovery) was dispensed into the plate, measurement was carried out using the SECTOR Imager 2400 (Meso Scale Discovery). The hsIL-6R concentration was calculated based on the response in the calibration curve using the analytical software SOFT max PRO (Molecular Devices).

[0676] Changes in the measured human IF-6 receptor concentration are shown in Fig. 4. As for both the antibody whose Fc region is that of the native IgGl (High_pI-IgGl) and the antibody which contains the novel Fc region variant with enhanced binding toward FcRn under the neutral pH conditions (High_pl-Fl 1), the plasma concentration of the soluble human IF-6 receptor was decreased in the case a high-pi antibody (also called High_pl) is administered as compared to the case a low-pi antibody (also called Fow_pl) is administered.

[0677] Without being bound by a particular theory, results obtained from these experiments can also be explained as follows: when the Fc region of the administered antibody is that of a native IgG antibody, uptake into the cell is thought to take place mainly by non-specific uptake (pinocytosis). Here, since the cell membrane is negatively charged, the higher the pi of the administered antibody-antigen complex is (i.e., the charge of the molecule as a whole is inclined toward positive charge), the more readily the complex may approach the cell membrane, and the easier the nonspecific uptake may take place. When an antibody with increased pi forms a complex with an antigen, that complex as a whole also has an increased pi in comparison to a complex formed between the original antibody and the antigen; therefore, uptake into cells may be increased. Therefore, by increasing the pi of an antibody that shows pH-dependent antigen binding, the speed or rate of antigen elimination from the plasma can be further accelerated, and the antigen concentration in the plasma can be maintained at a lower level.

[0678] In these Examples, increase of the pi of the antibody was accomplished by introducing amino acid substitutions that decrease the number of negatively charged amino acids and/or increase the number of positively charged amino acids that may be exposed on the surface of the antibody molecule in the antibody variable region. Those of ordinary skill in the art will understand that effects obtained by such pi increase do not depend primarily (or substantially) on the type of the target antigen or the amino acid sequence that constitutes the antibody, but can be expected to depend on the pi. For example, W02007/114319 and W02009/041643 describe the following matters in general terms.

[0679] Since the molecular weight of an IgG antibody is sufficiently large, its major metabolic pathway does not involve renal excretion. IgG antibodies that have Fc are known to have long half-lives since they are recycled by the salvage pathway of FcRn

189

WO 2017/046994

PCT/JP2016/003616 expressed in cells including the endothelial cells of blood vessels, and IgG is considered to be mainly metabolized in endothelial cells. More specifically, it is thought that IgGs that are non-specifically taken up into endothelial cells are recycled by binding to FcRn, and the molecules that cannot bind FcRn are metabolized. IgGs whose FcRn-binding ability has been reduced have shorter blood half-lives, and conversely, the blood half-life can be prolonged by increasing their binding ability toward FcRn. This way, previous methods for controlling the kinetics of IgG in blood involve modifying Fc to change the binding ability toward FcRn; however, the Working Examples of W02007/114319 (mainly, techniques for substituting amino acids in the FR region) and W02009/041643 (mainly techniques for substituting amino acids in the CDR region) showed that regardless of the target antigen type, by modifying the pi of the variable region of an antibody, its blood half-life can be controlled without modifying the Fc. The rate of non-specific uptake of an IgG antibody into endothelial cells is thought to depend on the physicochemical Coulombic interaction between the negatively charged cell surface and the IgG antibody. Therefore, it is considered that lowering (increasing) the pi of the IgG antibody and thus reducing (increasing) Coulombic interactions decreases (increases) its nonspecific uptake into endothelial cells, and consequently decreases (increases) its metabolism in endothelial cells, thereby enabling the control of plasma pharmacokinetics. Since the Coulombic interaction between endothelial cells and the cell surface's negative charge is a physicochemical interaction, this interaction is considered not to depend primarily on the antibody-constituting amino acid sequence per se. Therefore, the methods for controlling plasma pharmacokinetics provided herein are not just applicable to specific antibodies, but they can be widely applied to any polypeptide containing an antibody variable region. Herein, a reduction (an increase) of Coulombic interactions means a decrease (an increase) of the Coulombic force represented as an attractive force and/or an increase (a decrease) of the Columbic force represented as a repulsive force.

[0680] The amino acid substitutions for accomplishing the above may be a single amino acid substitution or a combination of multiple amino acid substitutions. In some embodiments, a method is provided for introducing a single amino acid substitution or a combination of multiple amino acid substitutions into a position(s) exposed on the antibody molecule surface. Alternatively, the multiple amino acid substitutions introduced may be positioned conformationally close to each other. The inventors arrived at the idea that, for example, when substituting amino acids that may be exposed on the antibody molecule surface with positively charged amino acids (preferably arginine or lysine) or when using pre-existing positively charged amino acids (preferably arginine or lysine), it may be preferable to further substitute one or

190

WO 2017/046994

PCT/JP2016/003616 more amino acids that are conformationally proximal to those amino acids (in certain cases, even one or more amino acids buried within the antibody molecule) with positively charged amino acids to produce, as a result, a state of locally clustered positive charges at conformationally proximal positions. Here, the definition of confer mationally proximal position(s) is not particularly limited, but for example, it may mean a state where a single amino acid substitution or multiple amino acid substitutions are introduced within 20 Angstroms, preferably within 15 Angstroms, or more preferably within 10 Angstroms of one another. Whether the amino acid substitution of interest is at a position exposed on the antibody molecule surface, or whether the amino acid substitution is proximally positioned can be determined by known methods such as X-ray crystallography.

[0681] This way, by noting that the pi is one indicator representing the overall charge of the molecule, and that charges buried inside the antibody molecule and charges on the antibody molecule surface are treated without any distinction, the inventors also conceived that by producing an antibody molecule with broad and comprehensive consideration of the effects from charges, which include not only the pi but also the surface charges and local clustering of charges on antibody molecules, the speed of antigen elimination from the plasma can be further accelerated and the antigen concentration in the plasma can be maintained at even lower levels.

[0682] Receptors such as FcRn or FcyR are expressed on the cell membrane, and antibodies that have an enhanced affinity toward FcRn or FcyR under neutral pH conditions are thought to be taken up into cells mainly through these Fc receptors. Since the cell membrane is negatively charged, the administered antibody-antigen complex approaches the cell membrane more readily when its pi is high (the charge of the molecule as a whole is shifted toward positive charge), and uptake through the Fc receptor may take place more easily. Therefore, antibodies that have an enhanced affinity towards FcRn or FcyR under neutral pH conditions as well as an increased pi also show increased uptake into cells through Fc receptors when they form a complex with antigens. Accordingly, the speed of antigen elimination from the plasma by antibodies that bind to antigens in a pH-dependent manner and have an enhanced affinity toward FcRn or FcyR under neutral pH conditions can be hastened by increasing their pis, and the plasma antigen concentration can be maintained at lower levels.

Example 3 [0683] Evaluation of the extracellular matrix binding of pH-dependent binding antibodies with increased pis (3-1) Evaluation of the extracellular matrix-binding ability

The following experiment was carried out to evaluate the effects of conferring an191

WO 2017/046994

PCT/JP2016/003616 tibodies with the pH-dependent antigen-binding property and further modifying the pi on their extracellular matrix-binding ability.

[0684] In a manner similar to the method of Example 1, three types of antibodies with different pi were produced as antibodies that show pH-dependent binding toward the IL-6 receptor: Low_pI-IgGl, Middle_pI-IgGl, and High_pI-IgGl. As ordinary antibodies that do not show pH-dependent binding to the IL-6 receptor,

Low_pI(NPH)-IgGl comprising H54 (SEQ ID NO:34) and L28 (SEQ ID NO:35) and High_pI(NPH)-IgGl comprising H(WT) (SEQ ID NO:36) and L(WT) (SEQ ID NO:37) described in WO2009125825 were produced by the method of Reference Example 2, respectively.

[0685] In a manner similar to the method of Example 1, the theoretical pi was calculated for these antibodies and shown in Table 4. Antibodies that do not show pH-dependent binding to the IL-6 receptor were also shown to have an increased pi similarly to antibodies that show pH-dependent binding.

[0686] [Table 4]

Antibody Name	Theoretical pi
Low pl-IgG1	6.39
Mtddle pl-lgG1	8.70
High pl-IgG1	9,30
Low pl(NPH)-lgG1	6.10
High pl(NPH)-lgG1	9.35

[0687] f3-21 Evaluation of antibody binding to the extracellular matrix by the electrochemiluminescence fECLl method

The extracellular matrix (BD Matrigel Basement Membrane Matrix; manufactured by BD) was diluted to 2 mg/mL using TBS (Takara). The diluted extracellular matrix was dispensed into a MULTI-ARRAY 96well Plate, High bind, Bare (manufactured by Meso Scale Discovery:MSD) at 5 pL per well, and immobilized overnight at 4°C. Then, 20mM ACES buffer at pH 7.4 containing 150 mM NaCl, 0.05% Tween 20,

0.5% BSA, and 0.01% NaN₃ was dispensed into the plate for blocking. The antibodies to be evaluated were diluted to 30, 10, and 3 pg/mL using 20 mM ACES buffer at pH 7.4 (ACES-T buffer) containing 150 mM NaCl, 0.05% Tween 20, and 0.01% NaN₃, and then were further diluted using 20 mM ACES buffer at pH 7.4 containing 150 mM NaCl, 0.01% Tween 20, 0.1% BSA, and 0.01% NaN₃ (Dilution Buffer) to produce a final concentration of 10, 3.3, and 1 pg/mL, respectively. The diluted antibody solutions were added to the plate from which the blocking solution was removed, and this was shaken at room temperature for one hour. The antibody solutions were removed, ACES-T buffer containing 0.25% glutaraldehyde was added, and after letting this stand for 10 minutes, the plate was washed with DPBS (manufactured by Wako

192

WO 2017/046994

PCT/JP2016/003616

Pure Chemical Industries) containing 0.05% Tween 20. The antibodies for ECL detection were prepared by sulfo-tagging goat anti-human IgG (gamma) (manufactured by Zymed Laboratories) using Sulfo-Tag NHS Ester (manufactured by MSD). Antibodies for detection were diluted in a dilution buffer to be 1 pg/mL, added to the plate, and then shaken in the dark at room temperature for one hour. The antibodies for detection were removed, and a 2-fold diluted solution prepared by diluting MSD Read Buffer T (4x) (manufactured by MSD) with ultrapure water was added; and then the amount of luminescence was measured on SECTOR Imager 2400 (manufactured by MSD).

[0688] The results are shown in Fig. 5. Antibodies showing pH-dependent binding as well as antibodies that do not show pH-dependent binding both showed increased binding toward the extracellular matrix by increasing their pis. Furthermore, surprisingly, the effect of improving extracellular matrix binding by increasing the pi was significant in the antibodies with pH-dependent antigen binding. In other words, the antibody that binds to an antigen in a pH-dependent manner and has high pi (High_pI-IgGl) was found to have the strongest affinity toward the extracellular matrix.

[0689] Without being limited to a particular theory, results obtained from these experiments can also be explained as follows. The introduction of histidine modifications into the antibody variable region is known to be one method for conferring pH-dependent antigen-binding property to an antibody (see e.g., WO2009/125825). Histidine has an imidazoyl group on its side chain, and is uncharged under neutral pH to basic pH conditions, but it is known to be positively charged under acidic pH conditions. Using this property of histidine, by introducing histidine into the antibody variable region, particularly in the CDR(s) positioned close to the site of interaction with the antigen, one can change the charge environment and conformational environment at the site of interaction with the antigen between the neutral and acidic pH conditions. Such antibodies can be expected to have an antigen affinity that changes in a pH-dependent manner. Those of ordinary skill in the art will understand that the effects obtained by such introduction(s) of histidine do not primarily (or substantially) depend on the type of target antigen or the amino acid sequence that constitutes the antibody, but depend on the site of histidine introduction or the number of histidine residues introduced.

[0690] W02009/041643 describes in general terms as follows: protein-protein interactions consist of hydrophobic interactions, electrostatic interactions, and hydrogen bonds, and the strength of such binding can usually be represented using a binding constant (affinity) or an apparent binding constant (avidity). pH-dependent binding, where the strength of binding changes between a neutral pH condition (e.g., pH 7.4) and an acidic pH condition (for example, pH 5.5 to pH 6.0), depends on naturally-occurring proteinprotein interactions. For example, the aforementioned binding between an IgG

193

WO 2017/046994

PCT/JP2016/003616 molecule and FcRn, which is known to be a salvage receptor for the IgG molecule, shows strong binding under acidic pH conditions and very weak binding under neutral pH conditions. Histidine residues are involved in many of the protein-protein interactions that change in a pH-dependent manner. Since the pKa of a histidine residue is close to 6.0 to 6.5, the state of proton dissociation in the histidine residue changes between neutral and acidic pH conditions. More specifically, the histidine residue is uncharged and neutral under neutral pH conditions, and functions as a hydrogen atom acceptor; while under acidic pH conditions, it is positively charged and functions as a hydrogen atom donor. Also in the IgG molecule-FcRn interaction described above, histidine residues present on the IgG molecule have been reported to be involved in pH-dependent binding (Martin et al., Mol. Cell. 7(4):867-877 (2001)).

[0691] Therefore, substituting a histidine residue for an amino acid residue involved in the protein-protein interaction, or introducing a histidine at an interacting site can confer pH dependence to protein-protein interactions. A similar undertaking has been made for protein-protein interactions between an antibody and an antigen; and an antibody mutant with decreased antigen affinity under acidic pH conditions has been successfully obtained by introducing histidine into the CDR sequence of an anti-egg-white lysozyme antibody (Ito et al., FEBS Lett. 309(1):85-88 (1992)). Furthermore, antibodies have been reported which specifically bind to antigens under the low pH of cancer tissues and weakly bind the corresponding antigen under neutral pH conditions due to introduction of histidine in the CDR sequence (W02003/105757).

[0692] Meanwhile, an amino acid residue that is introduced to increase the pi is preferably lysine, arginine, or histidine which have positively charged side chains. The standard pKa for these amino acid side chains is 10.5 for lysine, 12.5 for arginine, and 6.0 for histidine (Skoog et al., Trends Anal. Chem.5(4):82-83 (1986)). Based on the acid-base equilibrium theory known in the art, these pKa values mean that in a solution of pH 10.5, 50% of the lysine side chains are positively charged and the remaining 50% are uncharged. As the pH of the solution increases, the positively charged proportion of lysine side chains decreases, and in a solution of pH 11.5 which is 1 pH value higher than the pKa for lysine, the positively charged proportion becomes approximately 9%. On the other hand, as the pH of the solution decreases, the positively charged proportion increases, and in a solution of pH 9.5 which is 1 pH value lower than the pKa of lysine, the positively charged proportion becomes approximately 91%. This theory works in a similar manner for arginine and histidine as well. More specifically, nearly 100% of lysine or arginine is positively charged in a solution at neutral pH (for example, pH 7.0), whereas approximately 9% of histidine is positively charged. Therefore, while histidine is positively charged under neutral pH conditions, since that level is low compared to lysine or arginine, lysine and arginine are considered as more

194

WO 2017/046994

PCT/JP2016/003616 favorable amino acids to be introduced for increasing the pi. Furthermore, according to Holash et al. (Proc. Natl. Acad. Sci. 99(17):11393-11398 (2002), while the introduction of modifications that increase the pi have been considered to be effective as modifications for increasing extracellular matrix binding, substitutions introducing lysine or arginine have been considered to be more favorable amino acid modifications than substitutions introducing histidine for the above-mentioned reasons.

[0693] As first disclosed herein, a surprising synergistic increase in the affinity of an antibody toward extracellular matrix is possible by combining the introduction of histidine to confer the pH-dependent antigen-binding property along with a plincreasing modification of the antibody. In fact, while the pi of High_pI-IgGl was 9.30, the pi of High_pI(NPH)-IgGl was 9.35, and therefore the value for High_pI-IgGl was slightly lower. Nevertheless, the actual affinity towards extracellular matrix was clearly stronger for High_pI-IgGl. This shows that the affinity towards extracellular matrix cannot be necessarily explained by just the pi level, and can be said to show a synergistic effect from introducing a combination of plincreasing modifications and histidine modifications. This result is a phenomenon that was both surprising and unexpected.

[0694] However, one must note that since the pKa of the amino acid side chains in a protein are greatly affected by the surrounding environment, they will not always match the above-mentioned theoretical pKa values. More specifically, the above description is presented herein based on general scientific theory; however, one can easily speculate that there may be many exceptions in actual proteins. For example, in Hayes et al., J. Biol. Chem. 250(18):7461-7472 (1975), when the pKa of histidine contained in myoglobin was determined experimentally, while the values centered around 6.0, they were reported to vary from 5.37 to 8.05. Naturally, histidine which has a high pKa will be mostly positively charged under neutral pH conditions. Therefore, the abovementioned theory does not negate the pi-increasing effect of introducing histidine and in the actual three-dimensional structure of the protein. It is sufficiently possible that an amino acid modification introducing histidine, as seen in the case with lysine or arginine, may also to exert an effect of pi increase.

Example 4 [0695] pi increase by one amino acid substitution in the constant region

Methods for increasing the pi of an antibody that binds to an antigen in a pHdependent manner by introducing amino acid substitutions into the antibody variable region have been described. In addition, methods for increasing the pi of an antibody can also be carried out by performing as few as one amino acid substitution in the antibody constant region.

195

WO 2017/046994 PCT/JP2016/003616 [0696] The method of adding one amino acid substitution to the antibody constant region to increase pi is not particularly limited, but for example, it can be performed by the method described in WO2014/145159. As in the case with the variable region, amino acid substitutions introduced into the constant region are preferably those that decrease the number of negatively charged amino acids (such as, aspartic acid or glutamic acid) while increasing the positively charged amino acids (such, as arginine or lysine).

[0697] Without limitation, the positions for introducing amino acid substitutions in the constant regions are preferably positions where amino acid side chains may be exposed on the antibody molecule surface. Preferable examples include the method of introducing a combination of multiple amino acid substitutions at such positions that may be exposed on the antibody molecule surface. Alternatively, the multiple amino acid substitutions introduced are preferably positioned so that they are conformationally close to each other. Furthermore, without limitation, the multiple amino acid substitutions introduced are preferably substitutions to positively charged amino acids, so that in certain cases they result in a state where multiple positive charges are present at conformationally proximal positions. The definition of a conformationally proximal position is not particularly limited, but for example, it may mean a state where a single amino acid substitution or multiple amino acid substitutions are introduced within 20 Angstroms, preferably within 15 Angstroms, or more preferably within 10 Angstroms of one another. Whether the amino acid substitution of interest is at a position exposed on the antibody molecule surface, or whether the multiple positions of amino acid substitutions are proximally positioned can be determined by known methods such as X-ray crystallography.

[0698] Furthermore, the method for conferring multiple positive charges at conformationally proximal positions includes, in addition to the above-mentioned methods, a method of using amino acids that are originally positively charged in an IgG constant region. Examples of such positively charged amino acid positions include (a) arginine at position 255, 292, 301, 344, 355, or 416, according to EU numbering; and (b) lysine at position 121, 133, 147, 205, 210, 213, 214, 218, 222, 246, 248, 274, 288, 290, 317,

320, 322, 326, 334, 338, 340, 360, 370, 392, 409, 414, or 439, according to EU numbering. By performing substitution with a positively charged amino acid at a position conformationally proximal to these positively charged amino acids, it is possible to confer multiple positive charges at conformationally proximal positions.

[0699] (4-1) Production of pH-dependent anti-IgE binding antibodies

The following three antibodies were produced by the method of Reference Example as pH-dependent anti-human IgE antibodies: (1) Abl, which is a conventional antibody comprising AblH (SEQ ID NO:38) as the heavy chain and AblL (SEQ ID NO:39) as the light chain; (2) Ab2, which is a conventional antibody comprising Ab2H

196

WO 2017/046994

PCT/JP2016/003616 (SEQ ID NO:40) as the heavy chain and Ab2L (SEQ ID N0:41) as the light chain; and (3) Ab3, which is a conventional antibody comprising Ab3H (SEQ ID NO:42) as the heavy chain and Ab3L (SEQ ID NO:43) as the light chain.

[0700] (4-2) Evaluation of pH dependence in human IgE binding

Affinities of Abl toward human IgE at pH 7.4 and pH 5.8 were evaluated as follows.

Kinetic analyses of human IgE and Abl were performed using BIACORE T100 (GE Healthcare). Measurements were carried out using the following two buffers as the running buffers: (1) 1.2 mM CaCl₂ /0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 1.2 mM CaCl₂ /0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 5.8.

[0701] An appropriate amount of Protein A/G (ACTIGEN) was fixed onto Sensor chip CM4 (GE Healthcare) by the amine coupling method to capture the antibodies of interest. Next, human IgE was made to interact with the antibodies captured onto the sensor chip by injecting a diluted IgE solution and a running buffer (used as a reference solution). For the running buffer, either of the buffers (1) and (2) above was used, and human IgE was diluted using the respective buffer. To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements were carried out at 25°C. KD (M) for human IgE was calculated for each antibody based on the association rate constant ka (1/Ms) and dissociation rate constant kd (1/s), which are kinetic parameters calculated from the sensorgrams obtained by the measurements. The BIACORE T100 Evaluation Software (GE Healthcare) was used to calculate each parameter.

[0702] Affinities of Ab2 and Ab3 for human IgE at pH 7.4 and pH 5.8 were evaluated as follows. The binding activity (dissociation constant KD (M)) of anti-hlgE antibodies toward hlgE were evaluated using BIACORE T200 (GE Healthcare). Measurements were carried out using the following two buffers as the running buffers: (1) 1.2 mM CaCl₂ /0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 1.2 mM CaCl₂ /0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 5.8.

[0703] An appropriate amount of a peptide produced by adding biotin to Lys present at the C terminus of a chemically synthesized human glypican 3 (a.k.a., GPC3) proteinderived peptide (having the amino acid sequence of (VDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO:44))(biotinylated GPC3 peptide) was added to Sensor chip SA (GE Healthcare) and immobilized onto the chip by utilizing the affinity between streptavidin and biotin. An appropriate concentration of hlgE was injected and immobilized onto the chip by capturing of the biotinylated GPC3 peptide. An appropriate concentration of an anti-hlgE antibody was injected as an analyte, and this was made to interact with hlgE on the sensor chip. Then, to regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was injected. All measurements were carried out at 37°C. Association rate constants ka (1/Ms) and dis197

WO 2017/046994

PCT/JP2016/003616 sociation rate constants kd (1/s) were calculated by analyzing the measurement results by curve-fitting using the BIACORE T200 Evaluation Software (GE Healthcare), and dissociation constants KD (M) were calculated based on those values.

[0704] The results are presented in Table 5. All antibodies, Abl, Ab2, and Ab3, showed pHdependent binding toward human IgE, and their affinity at an acidic pH condition (pH 5.8) was shown to be dramatically weakened when compared with their affinity at a neutral pH condition (pH 7.4). Accordingly, administration of these antibodies to a living animal is expected to show an effect of accelerating the elimination of human IgE which is the antigen.

[0705] [Table 5]

Antibody Name	Buffer pH Condition	ka (1/Ms)	kd (1/s)	KD (M)
AM	pH7.4	2.7E+06	6.0E-03	2.3E-09
AD I	pH5.8	1.6E+04	3.9E-02	2.4E-06
Ah')	pH7,4	2.9E+06	4.5E-03	1.5E-09
ADZ	pH5.8	5.5E+05	5.3E-02	9.7E-08
Ah'}	pH7.4	1.6E+06	7.9E-03	4.9E-09
	pH5.8	1.4E+05	3.3E-02	2.3E-07

[0706] The theoretical pi values (pis) for Abl-Ab3 calculated in a similar manner to the method of Example 1 are shown in Table 6.

[0707] [Table 6]

Antibody Name	Theoretical pi
Ab1	6.77
Ab2	6.48
Ab3	6.48

[0708] (4-3) Production of antibodies with increased-pl by a single amino acid modification in the constant region

Abl produced in Example (4-1) is an antibody having native human IgGl as the constant region. AblH-P600 was produced by modifying the Fc region of AblH, which is the heavy chain of Abl, through substituting the proline at position 238 according to EU numbering with aspartic acid and substituting the serine at position 298 according to EU numbering with alanine. Furthermore, various Fc variants were produced by the method of Reference Example 2 by introducing the various single amino acid substitutions indicated in Tables 7-1 and 7-2 into the Fc region of AblH-P600, respectively. For all of the Fc variants, AblL (SEQ ID NO:39) was used as the light chain. The affinity of these antibodies for hFcyRII2b was comparable to the P600 variant (data not shown).

[0709]

198

WO 2017/046994

PCT/JP2016/003616 [Table 7-1]

Variant Name	Amino Acid Mutation Added to P600	Biacore	Imaging
P600	None	1.00	1.00
P828	Q196K	1.27	0.98
P829	S337R	0.17	0.89
P830	L358K	1.24	2.35
P831	P387R	3.85	1.30
P836	E345Q	1.85	No Data
P837	E345R	1.88	No Data
P838	D356Q	1.87	No Data
P839	D356N	2.17	No Data
P840	T359K	2.25	No Data
P841	N361R	1.86	No Data
P842	Q362K	2.37	No Data
P843	E380R	-0.04	No Data
P844	E382Q	1.24	No Data
P845	E382K	1.38	No Data
P846	Q386K	1.71	No Data
P847	N389K	1.57	No Data
P848	S415R	1.38	No Data
P849	Q418R	2.21	No Data
P850	Q419K	2.22	No Data
P851	N421R	1.43	1.56
P852	S424K	1.40	No Data
P854	L443R	1.93	No Data
P905	N384R	1.34	2.36
P906	G385R	1.74	1.12
P907	H433R	0.09	3.55
P908	N434R	0.42	1.88
P909	H435R	0.73	0,77
P910	L309R	1.73	1.80
P912	T307R	0.24	1.30
P914	D399R	1.72	3.78
P915	S400R	0.85	2.01
P917	A327R	-0.05	2.49
P918	L328R	-0.06	0.00
P919	P329R	-0.06	0.00

[0710]

199

WO 2017/046994

PCT/JP2016/003616 [Table 7-2]

P920	A330R	0.01	1.67
P921	P331R	-0,07	0.02
P923	Q311R	1.63	2.88
P924	N315R	2.08	2.66
P925	Y296R	-0.05	0.34
P926	Q295R	-0.06	0.04
P927	E294R	0.25	0.29
P928	E293R	0.20	0.04
P929	P291R	0.47	0.53
P930	A287R	-0.07	0.00
P931	N286R	0.68	1.27
P932	H285R	1.38	1.96
P934	V282R	1.54	1.67
P935	G281R	-0.06	2.14
P937	E272R	0.21	0.56
P938	P271R	-0.06	0.04
P939	D270R	-0.07	0.01
P940	E269R	-0.05	0.01
P941	H268R	0.47	0,44
P942	E258R	No Data	3.07
P944	T256R	No Data	1.49
P945	S254R	No Data	5.70
P946	1253R	No Data	1.05
P947	M252R	No Data	0.94
P948	L251R	No Data	0.13

[0711] (4-4) Human FcvRIIb-binding assay by BIACORE using novel Fc region variantcontaining antibodies

Fc region variant-containing antibody binding assays between soluble human FcyRIIb (a.k.a. hFcYRIIb) and antigen-antibody complexes were performed using BIACORE(registered trademark) T200 (GE Healthcare). Soluble hFcyRIIb was produced in the form of a His-tagged molecule using methods known in the art. An ap propriate amount of an anti-His antibody was fixed onto Sensor chip CM5 (GE Healthcare) by the amine coupling method using a His capture kit (GE Healthcare) to capture hFcYRIIb. Next, an antibody-antigen complex and a running buffer (as a reference solution) was injected, and interaction was allowed to take place with the hFcYRIIb captured onto the sensor chip. 20 mM N(2-Acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl₂, and 0.05% (w/v) Tween 20 at pH 7.4 was used as the running buffer, and the respective buffer was also used to dilute the soluble hFcYRIIb. To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements were carried out at 25°C. Analyses were performed based on binding (RU) calculated from sensorgrams obtained by the measurements, and relative values when the binding amount of P600 was defined as 1.00 are shown. To calculate the parameters, the BIACORE(registered trademark)

200

WO 2017/046994

PCT/JP2016/003616

T100 Evaluation Software (GE Healthcare) was used. The results are shown in Tables 7-1 and 7-2 (see the BIACORE column in the Tables) and in Fig. 6. Several Fc variants were shown to have enhanced affinity toward hFcyRIIb fixed on the BIACORE(registered trademark) sensor chip.

[0712] While not being restricted to a particular theory, this result can be explained as follows. The BIACORE(registered trademark) sensor chip is known to be negatively charged, and this charged state can be considered to resemble the cell membrane surface. More specifically, the binding of an antigen-antibody complex for hFcyRIIb fixed onto the negatively charged BIACORE sensor chip is surmised to resemble the manner in which the antigen-antibody complex binds to hFcyRIIb present on a negatively charged cell membrane surface.

[0713] The antibodies produced by introducing the pi-increasing modification into the Fc region are antibodies in which the charge of the Fc region (constant region) is more positively charged when compared with those before introduction of the modification. Therefore, the Coulombic interaction between the Fc region (positive charge) and the sensor chip surface (negative charge) can be considered to have been strengthened by the pi-increasing amino acid modification. Furthermore, such effects are expected to take place similarly on the negatively charged cell membrane surface; therefore, they are also expected to show an effect of accelerating the speed or rate of uptake into cells in vivo.

[0714] From the above results, a ratio of above about 1.2 fold or more for the binding to hFcyRIIb of a variant when compared to the binding to hFcyRIIb of AMH-P600 was considered to have strong charge effect on binding of an antibody to hFcyRIIb on the sensor chip. Thus, a modification that is expected to yield a charge effect includes, for example, a modification at position 196, 282, 285, 309, 311, 315, 345, 356, 358, 359, 361, 362, 382, 384, 385, 386, 387, 389, 399, 415, 418, 419, 421, 424, or 443, according to EU numbering. Preferably the modification is at position 282, 309, 311, 315, 345, 356, 359, 361, 362, 385, 386, 387, 389, 399, 418, 419, or 443. The amino acid substitution introduced at such position is preferably arginine or lysine. Another example of an amino acid mutation position where such a charge effect can be expected includes the glutamic acid at position 430 according to EU numbering. The preferred amino acid substitution to be introduced at position 430 is arginine or lysine which is positively charged, or among uncharged residues, substitution to glycine or threonine is preferred.

[0715] 14-51 Uptake of Fc region variant-containing antibodies by hFcyRIIb-expressing cells

To evaluate the rate of intracellular uptake into an hFcyRIIb-expressing cell line using the produced novel Fc region variant-containing antibodies, the following assay was performed.

201

WO 2017/046994 PCT/JP2016/003616 [0716] An MDCK (Madin-Darby canine kidney) cell line that constitutively expresses hFcyRIIb was produced using known methods. Using these cells, intracellular uptake of antigen-antibody complexes was evaluated. Specifically, pHrodoRed (Fife Technologies) was used to label human IgE (antigen) according to an established protocol, and antigen-antibody complexes were formed in a culture solution with the antibody concentration being 10.8 mg/mF and the antigen concentration being 12.5 mg/mF. The culture solution containing the antigen-antibody complexes was added to culture plates of the above-mentioned MDCK cells which constitutively express hFcyRIIb and incubated for one hour, and then the fluorescence intensity of the antigen taken up into the cells was quantified using InCell Analyzer 6000 (GE healthcare). The amount of antigen taken up was presented as relative values to the P600 value which is taken as 1.00.

[0717] The results are shown in Tables 7-1 and 7-2 (see the Imaging column in the Tables) and in Fig. 7. Strong fluorescence derived from the antigen in the cells was observed in several Fc variants.

[0718] While not being restricted to a particular theory, this result can be explained as follows: the antigen and antibodies added to the cell culture solution form antigenantibody complexes in the culture solution. The antigen-antibody complexes bind to hFcyRIIb expressed on the cell membrane via the antibody Fc region, and are taken up into the cells in a receptor-dependent manner. Abl used in this experiment is an antibody that binds to the antigen in a pH-dependent manner; therefore, the antibody can dissociate from the antigen. Since the dissociated antigen is labeled with pHrodoRed as described earlier, it fluoresces in the endosomes. Thus, a stronger fluorescence intensity inside the cell compared to the control is thought to indicate that the uptake of the antigen-antibody complexes into the cells is taking place more quickly or more frequently.

[0719] Here, a ratio of above about 1.05 fold or more of the fluorescence intensity of the antigen taken up into the cells of the variants compared to the fluorescence intensity of AMH-P600 was considered to have charge effect on an antigen taken up into the cells. A ratio of above about 1.5 fold or more of the fluorescence intensity of the antigen taken up into the cells of the variants compared to the fluorescence intensity of AblH-P600 was considered to have a strong charge effect on an antigen taken up into the cells. Thus, the above results showed that by introducing the pi-increasing modification into the appropriate position in the Fc region, uptake into cells can be accelerated as compared to before introduction of the modification. An amino acid position modification that shows such effect is, for example, position 253, 254, 256, 258, 281, 282, 285, 286, 307, 309, 311, 315, 327, 330, 358, 384, 385, 387, 399, 400, 421, 433, or 434, according to EU numbering. Preferably, modification is at position

202

WO 2017/046994 PCT/JP2016/003616

254, 258, 281, 282, 285, 309, 311, 315, 327, 330, 358, 384, 399, 400, 421, 433, or 434, according to EU numbering. An amino acid substitution introduced at such a position is preferably arginine or lysine. Without limitation, the position where an amino acid substitution is introduced in the constant region with the objective of increasing the pi of the antibody may be, for example, the amino acid residue at position 285 according to EU numbering. Alternatively, other examples may include an amino acid substitution of the amino acid residue at position 399 according to EU numbering. Example 5 [0720] Production of Fc variants with enhanced FcRn binding under acidic pH conditions for improving retention in the plasma

Under the acidic pH condition in the endosomes, IgG antibodies taken up into cells are known to be returned to the plasma by binding to FcRn. Therefore, IgG antibodies generally have long plasma half-life compared to proteins that do not bind to FcRn. Methods that utilize this property to enhance plasma retention of antibodies by increasing their FcRn affinity under acidic pH conditions through the introduction of amino acid modifications in the antibody Fc region are known. Specifically, methods for improving plasma retention of an antibody by increasing its affinity for FcRn under acidic pH conditions through amino acid modifications, such as the

M252Y/S254T/T256E (YTE) modification (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)), M428L/N434S (LS) modification (Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)), and N434H modification (Zheng et al., Clinical Pharmacology & Therapeutics 89(2):283-290 (2011)) are known.

[0721] On the other hand, as described above, Fc variants with increased FcRn affinity under acidic pH conditions are also known to show undesired affinity towards the rheumatoid factor (RF) (WO2013/046704). Therefore, the following examinations were carried out with an objective of producing Fc variants that can improve plasma retention with decreased or substantially no binding to rheumatoid factor.

[0722] 15-11 Production of novel Fc region variant-containing antibodies

Fc variants with increased FcRn affinity under acidic pH conditions such as those including the known modifications, YTE, LS, or N434H, and several novel Fc variants (F1847m, F1848m, F1886m, F1889m, F1927m, and FI 168m) were produced as shown below.

[0723] Sequences encoding heavy chains to which amino acid modifications were introduced in the Fc region of the heavy chain (VH3-IgGlm) of Fv4-IgGl, which is an anti-human IL-6 receptor antibody, were produced by the method of Reference Example 1. These heavy chains were used to produce the following antibodies by the method of Reference Example 2: (a) Fv4-IgGl comprising VH3-IgGlm (SEQ ID

203

WO 2017/046994

PCT/JP2016/003616

NO:46) as the heavy chain and VL3-CK as the light chain; (b) Fv4-YTE comprising VH3-YTE (SEQ ID NO:47) as the heavy chain and VF3-CK as the light chain; (c) Fv4-FS comprising VH3-FS (SEQ ID NO:48) as the heavy chain and VF3-CK as the light chain; (d) Fv4-N434H comprising VH3-N434H (SEQ ID NO:49) as the heavy chain and VF3-CK as the light chain; (e) Fv4-F1847m comprising VH3-F1847m (SEQ ID NO:50) as the heavy chain and VF3-CK as the light chain; (f) Fv4-F1848m comprising VH3-F1848m (SEQ ID NO:51) as the heavy chain and VF3-CK as the light chain; (g) Fv4-F1886m comprising VH3-F1886m (SEQ ID NO:52) as the heavy chain and VF3-CK as the light chain; (h) Fv4-F1889m comprising VH3-F1889m (SEQ ID NO:53) as the heavy chain and VF3-CK as the light chain; (i) Fv4-F1927m comprising VH3-F1927m (SEQ ID NO:54) as the heavy chain and VF3-CK as the light chain; and (j) Fv4-F1168m comprising VH3-F1168m (SEQ ID NO:55) as the heavy chain and VF3-CK as the light chain.

[0724] (5-2) Kinetic analyses of binding toward human FcRn

Antibodies containing VH3-IgGlm or an above-mentioned variant as the heavy chain and F(WT) (SEQ ID NO:37) as the light chain were produced by the method of Reference Example 2, and the binding activity toward human FcRn was evaluated as follows.

[0725] Kinetic analyses of human FcRn and each of the antibodies were carried out using BIACORE T100 (GE Healthcare). An appropriate amount of Protein F (ACTIGEN) was fixed onto Sensor chip CM4 (GE Healthcare) by the amine coupling method to capture the antibodies of interest. Next, human FcRn was made to interact with the antibodies captured on the sensor chip by injecting a diluted FcRn solution and a running buffer (used as a reference solution). For the running buffer, 50 mM sodium phosphate, 150 mM NaCl, and 0.05% (w/v) Tween 20 at pH 6.0 was used, and the respective buffer was also used to dilute FcRn. To regenerate the sensor chip, 10 mM glycineHC1 at pH 1.5 was used. All measurements were carried out at 25°C. KD (M) for human FcRn was calculated for each antibody based on the association rate constant ka (1/Ms) and dissociation rate constant kd (1/s), which are kinetic parameters calculated from sensorgrams obtained by the measurements. The BIACORE T100 Evaluation Software (GE Healthcare) was used to calculate each parameter.

[0726] The results are shown in Table 8.

[0727]

204

WO 2017/046994

PCT/JP2016/003616 [Table 8]

Variant Name	Amino Acid Mutation(s)	KD Value (nM) for hFcRn at pH 6.0
lgG1		1382
LS	M428L/N434S	116
YTE	M252Y/S254T/T256E	148
F1847m	N434A/Y436T/Q438R/S440E	367
F1848m	N434A/Y436V/Q438R/S440E	295
F1886m	M428L/N434A/Y436T/Q438R/S440E	108
F1889m	M428L/N434A/Y436V/Q438R/S440E	103
Ft 927m	M428L/N434A/Q438R/S440E	125
FI 168m	N434A/Q438R/S440E	410

Example 6 [0728] Evaluation of the affinity of Fc region variant-containing antibodies with enhanced FcRn binding under acidic pH conditions toward the rheumatoid factor

Anti-drug antibodies (ADAs) affect the efficacy and pharmacokinetics of therapeutic antibodies, and lead to serious side-effects at times; therefore, clinical utility and efficacy of therapeutic antibodies may be limited by production of ADAs. Many factors influence the immunogenicity of therapeutic antibodies, and the presence of effector T cell epitopes is one factor. In addition, the presence of ADA in a patient before administration of the therapeutic antibody (also called Pre-existing ADA) may have similar problems. Specifically, in the case of therapeutic antibodies for patients with autoimmune diseases such as rheumatoid arthritis (RA), rheumatoid factor (RF) which is an autoantibody against human IgG may cause a pre-existing ADA problem. Recently, a humanized anti-CD4 IgGl antibody having an N434H (Asn434His) mutation was reported to induce significant rheumatoid factor binding (Zheng et al., Clinical Pharmacology & Therapeutics 89(2):283-290 (2011)). Detailed studies confirmed that the N434H mutation in human IgGl increases binding of the rheumatoid factor to the Fc region of antibodies as compared to that of the parent human IgGl.

[0729] The rheumatoid factor is a polyclonal autoantibody against human IgG, and its epitopes in human IgG differ depending on the clone and seem to be positioned in the CH2/CH3 interface region, and in the CH3 domain that may overlap with the FcRnbinding epitope. Therefore, mutations that increase the binding activity (binding affinity) towards FcRn may increase the binding activity (binding affinity) towards specific clones of the rheumatoid factor.

[0730] In fact, regarding Fc with increased affinity for FcRn at acidic pH or neutral pH, not only the N434H modification but many other amino acid modifications are also known to similarly increase the binding of the Fc to rheumatoid factor (WO2013/046704).

[0731] On the other hand, several amino acid modifications that selectively suppress the

205

WO 2017/046994

PCT/JP2016/003616 affinity toward the rheumatoid factor while not affecting affinity toward FcRn have been presented as examples in WO2013/046704, and among them, combinations of two amino acid mutations, namely Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D, have been indicated. Accordingly, Q438R/S440E was introduced to Fc with novel increased affinity under acidic pH conditions first disclosed herein to examine whether binding toward rheumatoid factors can be decreased.

[0732] (6-1) Rheumatoid factor binding assay of Fc region variant-containing antibodies

A binding assay toward rheumatoid factor was performed by utilizing electrochemiluminescence (ECL) at pH 7.4 using individual sera (Proteogenex) from 30 RA patients. A 50-fold diluted serum sample, a biotinylated test antibody (1 pg/mL), and a SULFO-TAG NHS Ester (Meso Scale Discovery)-labeled test antibody (1 pg/mL) was each mixed and incubated at room temperature for three hours. Thereafter, the mixture was added to a Streptavidin-coated MULTI-ARRAY 96-well plate (Meso Scale Discovery), and the plate was incubated at room temperature for two hours and then washed. After adding Read Buffer T(x4) (Meso Scale Discovery) to each well, the plate was immediately set on the SECTOR imager 2400 Reader (Meso Scale Discovery), and chemiluminescence was measured.

[0733] The results of this assay are shown in Figs. 8 to 17. Fv4-IgGl (Fig. 8) which has a native human IgGl only showed weak binding to the rheumatoid factor, whereas the existing Fc variants with increased FcRn binding, Fv4-YTE (Fig. 9), Fv4-LS (Fig. 10), and Fv4-N434H (Fig. 11), all showed significantly increased rheumatoid factor binding in a number of donors. On the other hand, all novel Fc region variants with increased FcRn binding, Fv4-F1847m (Fig. 12), Fv4-F1848m (Fig. 13), Fv4-F1886m (Fig. 14), Fv4-F1889m (Fig. 15), Fv4-F1927m (Fig. 16), and Fv4-F1168m (Fig. 17), showed only weak rheumatoid factor binding, and this showed that binding to the rheumatoid factor as a result of modifications to increase FcRn binding was significantly inhibited.

[0734] Fig. 18 shows the average values of rheumatoid factor-binding affinity in the serum of 30 RA patients for each of the variants. All of the six new variants showed a lower affinity than the three pre-existing variants (YTE, LS, and N434H), and they also showed a lower affinity toward the rheumatoid factor as compared with native human IgGl. As such, when considering clinical development of therapeutic antibodies with improved affinity towards FcRn for autoimmune diseases such as rheumatoid arthritis and the like, the risk associated with the rheumatoid factor, which is of concern in existing Fc variants, was suppressed in the Fc variants first disclosed herein, and accordingly they may be used more safely than existing known Fc variants.

Example 7

206

WO 2017/046994 PCT/JP2016/003616 [0735] PK evaluation of the Fc variants with increased FcRn binding under acidic pH conditions in cynomolgus monkeys

In Example 7, the effect of improving plasma retention in cynomolgus monkeys was evaluated by the following method using novel Fc region variant-containing antibodies provided herein whose binding to rheumatoid factor was confirmed to be suppressed.

[0736] (7-1) Production of novel Fc region variant-containing antibodies

The following anti-human IgE antibodies were produced: (a) OHB-IgGl comprising OHBH-IgGl (SEQ ID NO:56) as the heavy chain and OHBL-CK (SEQ ID NO:57) as the light chain; (b) OHB-LS comprising OHBH-LS (SEQ ID NO:58) as the heavy chain and OHBL-CK as the light chain; (c) OHB-N434A comprising OHBH-N434A (SEQ ID NO:59) as the heavy chain and OHBL-CK as the light chain;

(d) OHB-F1847m comprising OHBH-F1847m (SEQ ID NO:60) as the heavy chain and OHBL-CK as the light chain; (e) OHB-F1848m comprising OHBH-F1848m (SEQ ID NO:61) as the heavy chain and OHBL-CK as the light chain; (f) OHB-F1886m comprising OHBH-F1886m (SEQ ID NO:62) as the heavy chain and OHBL-CK as the light chain; (g) OHB-F1889m comprising OHBH-F1889m (SEQ ID NO:63) as the heavy chain and OHBL-CK as the light chain; and (h) OHB-F1927m comprising OHBH-F1927m (SEQ ID NO:64) as the heavy chain and OHBL-CK as the light chain.

[0737] 17-21 Monkey PK assay on novel Fc region variant-containing antibodies

The in vivo kinetics of anti-human IgE antibodies in the plasma after administration of the anti-human IgE antibodies to cynomolgus monkeys were evaluated. The antihuman IgE antibody solution was intravenously administered once at 2 mg/kg. Blood collection was performed five minutes, (two hours), seven hours, one day, two days, three days, (four days), seven days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately subjected to centrifugation at 4°C and 15,000 rpm for 5 minutes to obtain plasma. The separated plasma was stored in a freezer set to -80°C or lower until performing the measurements. Eight types of anti-human IgE antibodies, namely OHB-IgGl, OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m, were used.

[0738] (7-3) Measurement of the anti-human IgE antibody concentration in the plasma by

ELISA

The concentration of anti-human IgE antibodies in the plasma of cynomolgus monkeys was measured by ELISA. First, an anti-human IgG kappa chain antibody (Antibody Solution) was dispensed into a Nunc-Immuno Plate, MaxiSorp (Nalge Nunc International) and allowed to stand overnight at 4°C to produce an anti-human IgG kappa chain antibody-immobilized plate. Calibration curve samples having a plasma concentration of 640, 320, 160, 80, 40, 20 or 10 ng/mL, and cynomolgus monkey

207

WO 2017/046994

PCT/JP2016/003616 plasma measurement samples diluted 100-fold or more were prepared. These calibration curve samples and plasma measurement samples were produced such that cynomolgus monkey IgE (product prepared within the company) was added at a concentration of 1 pg/mL. Subsequently, the samples were dispensed into the anti-human IgG kappa chain antibody-immobilized plate, and allowed to stand at room temperature for two hours. Then, an HRP-anti human IgG gamma chain antibody (Southern Biotech) was dispensed, and allowed to stand at room temperature for one hour. Subsequently, a chromogenic reaction was carried out using the TMB Chromogen Solution (Life Technologies) as a substrate, and after stopping the reaction by adding IN sulfuric acid (Wako), the absorbance at 450 nm was measured by a microplate reader. The concentration of anti-human IgE antibody in the monkey plasma was calculated from absorbance of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices). The measured change in the concentration of anti-human IgE antibody in the monkey plasma is shown in Fig. 19. From the measured change in the concentration of anti-human IgE antibody in the monkey plasma, elimination clearance was calculated by moment analysis using Phoenix WinNonlin Ver. 6.2 (Pharsight Corporation). The calculated pharmacokinetic parameters are shown in Table 9. Samples from individuals who were positive for antibodies against the administered sample in plasma were excluded from the calculation of the change in the anti-human IgE antibody concentration and clearance in monkey plasma.

[0739] [Table 9]

Elimination Clearance of Administered Sample after Anti-Human IgE Antibody Administration

Sampte Name	EimhaBoo Clearance (mUdayftsg)
OHB-igGI	9.33
OHB-F1847m	2..83
OHB-F1848m	4.02
OHB-F1886m	1.92
OHB-F1889m	2.39
GHB-F1327m	1.51
OHB-LS	1.80
ΟΗΒ-Ν434Ά	4,36

[0740] (7-4) Measurement of antibodies against the administered samples in plasma by the electrochemiluminescence method

Antibodies in monkey plasma against the administered samples were measured by an electrochemiluminescence method. An administered sample that was rutheniumlabeled using SULFO-TAG NHS Ester (Meso Scale Discovery), an administered sample that was biotinylated using EZ-Link Micro Sulfo-NHS-Biotinylation Kit

208

WO 2017/046994

PCT/JP2016/003616 (Pierce), and a cynomolgus monkey plasma measurement sample were mixed in equal amounts, and were left to stand overnight at 4°C. The samples were added to a MULTI-ARRAY 96-well Streptavidin Gold Plate (Meso Scale Discovery), then allowed to react at room temperature for two hours, and washed. Then, immediately after Read Buffer T(x4) (Meso Scale Discovery) was dispensed into the plate, measurements were carried out using SECTOR Imager 2400 (Meso Scale Discovery).

[0741] As a result, all of the novel Fc variants were confirmed to show greatly improved plasma retention in comparison to the Fc region of native IgGl.

[0742] (7-5) Mouse PK assay on Fc variants

The following experiment was carried out to compare F1718, which is an Fc variant described in WO2013/046704, and F1848m, which is an Fc variant newly discovered this time, as Fc variants for increasing FcRn binding at acidic pH.

[0743] Sequences encoding heavy chains into which amino acid modifications were introduced into the Fc region of the heavy chain (VH3-IgGl) of Fv4-IgGl(an antihuman IL-6 receptor antibody), were produced by the method of Reference Example 1. Using these heavy chains, the following antibodies were produced by the method of Reference Example 2: (a) Fv4-IgGl comprising VH3-IgGl as the heavy chain and VL3-CK as the light chain; and (b) Fv4-F1718 comprising VH3-F1718 (SEQ ID NO:65) as the heavy chain and VL3-CK as the light chain.

[0744] The above-mentioned anti-human IL-6 receptor antibodies were administered once at 1 mg/kg into the tail vein of human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse; Jackson Laboratories, Methods Mol. Biol. 602:93-104 (2010). Blood was collected 15 minutes, seven hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after administration of the anti-human IL-6 receptor antibodies. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer at -20°C or below until measurements were taken.

[0745] (7-6) Measurement of the anti-human IL-6 receptor antibody concentration in plasma by ELISA

The concentration of anti-human IL-6 receptor antibodies in the mouse plasma was measured by ELISA. First, an Anti-Human IgG (gamma-chain specific) F(ab')₂ Fragment of Antibody (SIGMA) was dispensed into a Nunc-Immuno Plate, MaxiSorp (Nalge nunc International) and allowed to stand overnight at 4°C to produce an antihuman IgG immobilized plate. Calibration curve samples containing an anti-human IL 6 receptor antibody at a plasma concentration of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, or 0.0125 pg/mL and mouse plasma measurement samples diluted 100-fold or more were each prepared. 200 uL of 20 ng/mL soluble human IL-6 receptor was added to 100 uL of the calibration curve samples or the plasma measurement samples, and then the

209

WO 2017/046994

PCT/JP2016/003616 mixed solutions were allowed to stand for one hour at room temperature. Subsequently, the mixed solutions were dispensed into each well of the anti-human IgGimmobilized plate, and the plate was allowed to stand for one hour at room temperature. Then, a Biotinylated Anti-Human IL-6R Antibody (R&D) was added to react for one hour at room temperature. Subsequently, Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added to react for one hour at room temperature, and chromogenic reaction of this reaction solution was carried out using TMB One Component HRP Microwell Substrate (BioFX Faboratories) as a substrate. After stopping the reaction by adding 1 N sulfuric acid (Showa Chemical), the absorbance at 450 nm of the reaction solution in each well was measured on a microplate reader. The antibody concentration in mouse plasma was calculated from the absorbance of the calibration curve using the analytical software SOFTmax PRO (Molecular Devices).

[0746] The results are shown in Fig. 20. F1718, which is an Fc variant for increasing FcRn binding at acidic pH described in WO2013/046704, did not show any effect of prolonging antibody PK, but showed plasma retention equivalent to that of native IgGl.

[0747] F1718 has four mutations, namely N434Y/Y436V/Q438R/S440E, introduced in the

Fc region. By contrast, FI848m, first disclosed herein, has been introduced with four mutations, namely N434A/Y436V/Q438R/S440E. The only difference between the amino acid mutations introduced in these two types of Fc's is that the amino acid mutation introduced at position 434 according to EU numbering is Y (tyrosine) in F1718 and A (alanine) in FI848m. In Example (7-2), FI848m showed improved plasma retention compared to that of the native IgGl, whereas F1718 did not show any improvement in plasma retention. Therefore, without limitation, this suggests that A (alanine) is preferred as the amino acid mutation to be introduced at position 434 for improving plasma retention.

[0748] (7-7) Predicted immunogenicitv score of Fc variants

Generation of anti-drug antibodies (ADA) influences the efficacy and pharmacokinetics of therapeutic antibodies, and brings about serious side effects in some cases; and therefore, clinical utility and drug efficacy of therapeutic antibodies may be limited by the generation of ADA. The immunogenicity of therapeutic antibodies is known to be affected by many factors, and in particular, the importance of effector T cell epitopes carried by the therapeutic antibodies in particular has been reported many times.

[0749] In silico tools for predicting T cell epitopes such as Epibase (Fonza), iTope/TCED (Antitope), and EpiMatrix (EpiVax) have been developed. Using these in silico tools,

T cell epitopes in each of the amino acid sequences can be predicted (Walle et al.,

210

WO 2017/046994

PCT/JP2016/003616

Expert Opin. Biol. Ther. 7(3):405-418 (2007)), and the potential immunogenicity of therapeutic antibodies can be evaluated.

[0750] EpiMatrix was used to calculate the immunogenicity scores of evaluated Fc variants.

EpiMatrix is a system for predicting the immunogenicity of a protein of interest by automatically designing sequences of peptide fragments by sectioning the amino acid sequence of the protein to be predicted for its immunogenicity by nine amino acids, and then calculating their ability to bind eight major MHC Class II alleles (DRBl*0101, DRBl*0301, DRBl*0401, DRBl*0701, DRBl*0801, DRBl*1101, DRBl*1301, and DRBl*1501) (Clin. Immunol. 131(2): 189-201 (2009)).

[0751] F1718 and F1756 (N434Y/Y436T/Q438R/S440E) described in W02013/046704 contain a N434Y mutation. In contrast, newly disclosed FI848m and FI847m contain a N434A mutation.

[0752] The immunogenicity scores of these four variants, namely F1718, F1848m, F1756 and F1847m, which were calculated as described above, are shown in the EpiMatrix Score column of Table 31. Furthermore, regarding the EpiMatrix Scores, immunogenicity scores corrected for the Tregitope content are shown in the tReg Adjusted Epx Score column. Tregitope is a peptide fragment sequence present in large amounts mainly in naturally-occurring antibody sequences, and is a sequence considered to inhibit immunogenicity by activating regulatory T cells (Treg).

[0753] [Table 31]

Protein Sequence	Mutations	EpiMatrix Score	tReg Adjusted Epx Score
F1718	N434Y/Y436V/Q438R/S440E	-10.97	-32.64
F1848m	N434A/Y436V/Q438R/S440E	-15.38	-37.06
F1756	N434Y/Y436T/Q438R/S440E	-14.05	-35.73
F1847m	N434A/Y436T/Q438R/S440E	-18.4	-40.08

[0754] According to these results, both the EpiMatrix Score and the tReg Adjusted Epx Score showed that the immunogenicity scores of N434A variants F1848m and F1847m were decreased as compared to that of N434Y variants. This suggests that A (alanine) is preferred as the amino acid mutation to be introduced at position 434 for the lower immunogenicity scores.

Example 8 [0755] Production of humanized anti-human IL-8 antibodies

18-11 Production of the humanized anti-human IL-8 antibody hWS-4

Humanized anti IL-8 antibodies disclosed in US Patent No. 6,245,894 bind to human

IL-8 (hIL-8) and block its physiological function. Modified humanized anti-IL-8 antibodies can be produced by combining the variable region sequences of the heavy and light chains disclosed in US Patent No. 6,245,894 with virtually any of the various known human antibody constant region sequences. Thus, the human antibody constant

211

WO 2017/046994

PCT/JP2016/003616 region sequences of these modified antibodies are not particularly limited, but native human IgGl sequences or native human IgG4 sequences may be used as the heavy chain constant regions, and native human Kappa sequences can be used as the light chain constant region sequence.

[0756] From among the humanized IL-8 antibodies disclosed in US Patent No. 6,245,894, the coding sequence of hWS4H-IgGl (SEQ ID NO:83) which was combined the heavy chain variable region RVHg and the native human anti-IgGl sequence for the heavy chain constant region was produced by the method of Reference Example 1. Furthermore, the coding sequence of hWS4L-kOMT (SEQ ID NO:84) which was combined the light chain variable region RVLa and the native human Kappa sequence for the light chain constant region was produced by the method of Reference Example 1. An antibody which was combined the above heavy chain and light chain was produced, and was named the humanized WS-4 antibody (hereinafter, hWS-4).

[0757] (8-2) Production of humanized anti-human IL-8 antibody Hr9

A new humanized antibody was produced using human consensus framework sequences that are different from the FRs used in hWS-4.

[0758] Specifically, a hybrid sequence of VH3-23 and VH3-64 was used as the heavy chain FR1, a sequence seen in VH3-15 and VH3-49 was used as FR2, a sequence seen in VH3-72 was used as FR3 (provided that 82a according to Kabat numbering is excluded), and a sequence seen in JH1 was used as FR4. These sequences were linked to the CDR sequences of the hWS-4 heavy chain to produce Hr9-IgGl (SEQ ID NO:85), a novel humanized antibody heavy chain.

[0759] Next, two types of antibodies were produced, namely, hWS-4 having hWS4H-IgGl as the heavy chain and hWS4L-kOMT as the light chain, and Hr9 having Hr9-IgGl as the heavy chain and hWS4L-kOMT as the light chain. Within the scope of Disclosure C described herein, when referring to the light chain in particular, Hr9 is written as Hr9/hWS4L. The antibodies were expressed using FreeStyle 293F cells (Invitrogen) according to the protocol attached to the product. Antibodies were purified from the culture supernatant by the method of Reference Example 2. As a result, antibodies were obtained in the amounts shown in Table 11. Surprisingly, the expression level of Hr9 was approximately 8 times the expression level of hWS-4.

[Table 11]
	Antibody Yield per 1mL Medium fug)
hWS-4	6.4
Hr9	50

[0761] 18-31 Human IL-8-binding activities of hWS-4 and Hr9

Binding affinities of hWS-4 and Hr9 towards human IL-8 were determined as follows using BIACORE T200 (GE Healthcare).

212

WO 2017/046994 PCT/JP2016/003616 [0762] A running buffer having the composition of 0.05% tween 20, 20 mM ACES, and 150 mM NaCl (pH 7.4) was used. An appropriate amount of Protein A/G (PIERCE) was immobilized onto Sensor chip CM4 (GE Healthcare) by the amine coupling method and the antibody of interest was captured. Next, human IL-8 was made to interact with the antibody captured on the sensor chip by injecting a diluted human IL-8 solution and a running buffer (used as a reference solution). For the running buffer, the solution having the above-described composition was used, and this buffer was also used to dilute human IL-8. To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements were carried out at 37°C. KD (M) of each antibody for human IL-8 was calculated based on the association rate constant kon (1/Ms) and dissociation rate constant koff (1/s), which are kinetic parameters calculated from sensorgrams obtained by the measurements. The BIACORE T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

[0763] The results are shown in Table 12. hWS-4 and Hr9 were confirmed to have equivalent binding affinities toward human IL-8.

[0764] [Table 12]

Antfoody Name	kon (1/Ms)	koff (1/s)	KD (M)
hWS-4	9.74E+05	2.03E04	2.Q9E-10
Hr9	1.11E+06	2.17E-04	1.95E-10

[0765] For development of antibody pharmaceuticals, the production level of antibody molecules is an important factor, and generally, a high production level is desirable. It is particularly notable that from the above-mentioned examination, a more appropriate human consensus framework-derived sequence was selected for combination with the HVR sequence of hWS-4, and yielded Hr9 which had an improved production level while maintaining the binding affinity toward human IL-8.

Example 9 [0766] Generation of antibodies with pH-dependent IL-8 affinity

19-11 Production of Hr9-modified antibodies for conferring pH dependency Studies were carried out with the objective of conferring pH-dependent IL-8 affinity to the Hr9 antibody obtained in Example 8.

[0767] While not being bound by particular theory, antibodies having pH-dependent affinity towards IL-8 may show the following behavior in vivo. The antibodies administered to a living organism can bind strongly to IL-8 in an environment where neutral pH is maintained (for example, in plasma), and block its function. A portion of such IL8/antibody complexes are taken up into cells by nonspecific interaction with the cell membrane (pinocytosis) (hereinafter, referred to as non-specific uptake). Under the acidic pH conditions in the endosomes, the binding affinities of the aforementioned an tibodies toward IL-8 become weak, and therefore the antibodies dissociate from IL-8.

213

WO 2017/046994

PCT/JP2016/003616

Then, the antibodies that dissociated from IL-8 can return to the outside of the cell via FcRn. The aforementioned antibodies that returned to the outside of the cell (into the plasma) in this manner can bind again to another IL-8 and block its function. Antibodies having pH-dependent affinity towards IL-8 are thought to be capable of binding to IL-8 multiple times by the above-mentioned mechanism.

[0768] In contrast, in the case of an antibody that does not have the property possessed by the aforementioned antibody, an antibody molecule is capable of neutralizing an antigen only once, but cannot neutralize the antigen multiple times. Generally, since an IgG antibody has two Fabs, a single antibody molecule can neutralize two molecules of IL-8. On the other hand, antibodies which can bind to IL-8 multiple times could bind to IL-8 any number of times as long as they stay in the living body. For example, a single molecule of a pH-dependent IL-8-binding antibody that is taken up into cells ten times since being administered until being eliminated can neutralize a maximum of 20 molecules of IL-8. Therefore, an antibody that can bind multiple times to IL-8 has the advantage of being able to neutralize several IL-8 molecules even with a small amount of the antibody. From another viewpoint, an antibody that can bind multiple times to IL-8 has the advantage of being able to maintain a state of being able to neutralize IL-8 for a longer period of time than when the same amount of antibody which does not have the property possessed is administered. From yet another viewpoint, an antibody that can bind multiple times to IL-8 has the advantage of being able to block the biological activity of IL-8 more strongly than when the same amount of an antibody which does not have the property possessed is administered.

[0769] To achieve these advantages, amino acid modifications, mainly histidine, were introduced into the variable regions of Hr9-IgGl and WS4L-kOMT with the objective of producing antibodies that can bind to IL-8 multiple times. Specifically, the variants shown in Table 13 were produced by the methods of Reference Examples 1 and 2.

[0770] Notations such as Y97H indicated in Table 13 show the position where the mutation is introduced as defined by Rabat numbering, the amino acid before introduction of the mutation, and the amino acid after introduction of the mutation. Specifically, when denoted as Y97H, it shows that the amino acid residue at position 97 according to Rabat numbering has been substituted from Y (tyrosine) to H (histidine). Furthermore, when a combination of multiple amino acid substitutions is introduced, it is written in a manner such as N50H/L54H.

[0771]

214

WO 2017/046994

PCT/JP2016/003616 [Table 13]

Antibody Name	Mutation Introduced into Heavy Chain	Mutation Introduced into Light Chain
Hr9/WS4L	None	None
Hr9/L16	None	L54H
H89/WS4L	Y97H	None
H89/L12	Y97H	N50H
H89/L16	Y97H	L54H

[0772] (9-2) pH-dependent IL-8 affinity

The human IL-8-binding affinity of the antibodies produced in Example 9-1 was determined as described below using BIACORE T200 (GE Healthcare). The following two running buffers were used: (1) 0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 7.4; and (2) 0.05% tween 20, 20 mM ACES, 150 mM NaCl, pH 5.8.

[0773] An appropriate amount of Protein A/G (PIERCE) was immobilized onto Sensor chip CM4 (GE Healthcare) by the amine coupling method and the antibodies of interest was captured. Next, human IL-8 was made to interact with the antibodies captured on the sensor chip by injecting a diluted human IL-8 solution and a running buffer (used as a reference solution). For the running buffer, any of the above-mentioned solutions was used, and the respective buffers were also used to dilute human IL-8. To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements were carried out at 37°C. KD (M) of each antibody for human IL-8 was calculated based on the association rate constant kon (1/Ms) and dissociation rate constant koff (1/s), which are kinetic parameters calculated from sensorgrams obtained by the measurements. The BIACORE T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

[0774] The results are shown in Table 14-1. First, compared to Hr9, Hr9/L16 which contains a L54H modification in the light chain had a slightly enhanced human IL-8-binding affinity at neutral pH (pH 7.4) but a lowered human IL-8-binding affinity at acidic pH (pH 5.8). On the other hand, anti-IL-8 antibodies (H89/WS4L, H89/L12, and H89/L16) produced by combining various light chains with H89 containing the Y97H modification in the heavy chain all showed a decreased human IL-8-binding affinity at acidic pH as well as a decreased human IL-8-binding affinity at neutral pH.

[0775]

215

WO 2017/046994

PCT/JP2016/003616 [Table 14-1]

Antibody Name	pH	kon (1/Ms)	koff (1/8)	KD (M)	kon Ratio (pH7.4/pH5.8)	Koff Ratio (pH5.8/pH7.4)	KD Ratio (pH5.8/pH7.4)
Hr9 ......(Hr9/WS4L)......	pH 7,4 pH 5,8......	8.59E+05 ......3.23E+05.....	2.11 E-04 ‘ 4.69E-04	2.46E-10 1.45E-09	2.7	.......................22..........*...............	5.9
Hr9/L16	pH 7,4 ..............ΡΗΪ8.........*	8.90E+05 .....3/9IE+O4'	9.57E-05 ........t97E-04*'	1.08E-10 .......5.04E-09......	***....................	*.......................2T........................	.......“*.....46*8....................
H89/WS4L	pH 7.4 .............pH5*8..........	8.51E+05 ......1*62E+05	7.65E-04 .........7.27E-03......	8.99E-10 ........4.48E-08.....	..........................5.2..................*........	............................9.5...........................	......................49*8..........................
........H89/L12..........	pH 7.4 ......*pH5*8..........	5.95E+05 *'l7l9E+O5.....	2.48E-04 *' 3.52E-03	4.17E-10 *’Z96E-08~	5.0	......***...........1*4*2.........*.....**	.......................71*0.......*...........*
H89/L16	pH 7.4 ........ph” Ks.....	6.02E+05 1.20E+Q5	4.21 E-04 .....<22E-03	6.99E-10 3.51E-08	5.0	10.0	........ 50.3
H89/L63	pH 7.4 '*.........pHS.8...........	5.37E+05 .....O2E+05	1.13E-04 ' 2.1 GE-03	2.10E-10 ......&04E-Q9......	2.1	18,7	..................30....................*
H89/L118.....	pH 7.4 ............PH5J3..........	5.80E+05 ......Ϊ79Ε+05'	2.13E-05 .......a.84E/03'	3.67E-11 ........F5E-08......	........................§7F......*.......*	*...................180*3*...................	...................580.......***

[0776] (9-3) Production and evaluation of modified antibodies for conferring pH dependence

Combinations of promising modifications found in 9-2 and new amino acid mutations were evaluated, and the following combinations were found as a result.

[0777] [Table 14-2]

Antibody Name	Mutaion{s) Introduced into Heavy Chain	Mutation(s) Introduced into Light Chain
H89/L63	Y97H	N50H/L54H
H89/L118	Y97H	N50H/L54H/Q89K

[0778] The variants were produced by the methods of Reference Examples 1 and 2, and the binding affinity towards human IL-8 was evaluated by a method similar to that of Example 9-2.

[0779] The results are also shown in Table 14. H89/L63 which has H89-IgGl (SEQ ID

NO:86) as the heavy chain and L63-k0MT (SEQ ID NO:87) as the light chain showed a human IL-8-binding affinity at neutral pH (pH 7.4) equivalent to that of Hr9, and a decreased human IL-8-binding affinity at acidic pH (pH 5.8). Specifically, both the koff (dissociation rate constant) and KD (dissociation constant) of H89/L63 at pH5.8 were higher than those of Hr9. This means that under the acidic pH condition in the endosomes, H89/L63 has a property of readily releasing human IL-8.

[0780] Surprisingly H89/L118, which has H89-IgGl as the heavy chain and LI 18-k0MT (SEQ ID NO:88) as the light chain, was found to have an enhanced human IL8-binding affinity (KD) under neutral pH conditions as compared to that of Hr9, but a weakened human IL-8-binding affinity (KD) under acidic pH conditions as compared to that of Hr9. Without particular limitation, generally, when antibodies that can bind multiple times to antigens are used as a pharmaceutical product, the pH-dependent antigen-binding antibodies preferably have a strong binding affinity (small KD) so that they can strongly neutralize the antigens under neutral pH conditions (such as in

216

WO 2017/046994

PCT/JP2016/003616 plasma). On the other hand, the antibodies preferably have a large dissociation rate constant (koff) and/or a weak binding affinity (large KD) so that they can quickly release the antigens under acidic pH conditions (such as in the endosomes). In comparison to Hr9, H89/L118 had acquired favorable properties in both these neutral pH and acidic pH conditions.

[0781] Thus, useful amino acid modifications were identified for Hr9 such as Y97H for its heavy chain and N50H/L54H/Q89K for its light chain. While not being limited thereto, it has been shown that pH-dependent IL-8-binding antibodies that are superior as pharmaceuticals could be generated by introducing a single or a combination of multiple amino acid modifications selected from these modifications.

[0782] While not being bound by a particular theory, it is considered that an important factor when using a pH-dependent antigen-binding antibody as a pharmaceutical is whether or not the antibody administered to the body can release the antigen in the endosome.

In this regard, a sufficiently weak binding (large dissociation constant (KD)) under acidic pH conditions or a sufficiently fast dissociation rate (large dissociation rate constant (koff)) is thought to be important. Therefore, it was examined in the following experiment whether the KD or koff of H89/L118 obtained by BIACORE is sufficient for dissociating the antigen in the endosome in vivo.

Example 10 [0783] Production of high-affinity antibodies for mouse PK assay

Methods for confirming the effect of an antibody on the rate of human IL-8 elimination in mice are not particularly limited. In one instance, the method involves administering an antibody in a condition mixed with human IL-8 to mice and then comparing the rate of human IL-8 elimination from mouse plasma.

[0784] Here, the reference antibody to be used for the mouse PK assay desirably has a sufficiently strong binding affinity under both neutral pH and acidic pH conditions. Then, a search for modifications that confer Hr9 with high-affinity was conducted, and as a result H998/L63 having H998-IgGl (SEQ ID NO:89) as the heavy chain and L63-k0MT as the light chain was created.

[0785] H998/L63 was used to evaluate the human IL-8-binding affinity by a method similar to that of Example 9-2. The resulting sensorgrams are shown in Fig. 21.

[0786] H998/L63 showed a surprisingly slow dissociation rate under both neutral pH and acidic pH conditions, and was shown to have stronger IL-8-binding affinity than Hr9. However, it is known that, due to the mechanical limits of BIACORE, analytical values such as dissociation rate constant (koff) and dissociation constant (KD) cannot be calculated accurately in such cases where the protein-protein interaction has a slow dissociation rate. As accurate analytical values could not be obtained for H998/L63, its

217

WO 2017/046994

PCT/JP2016/003616 analytical values are not shown here. However, it is confirmed from the results of the experiment that H998/L63 has very strong binding affinity at both neutral pH and acidic pH, and is suitable as an antibody to be used for comparison in mouse PK assays.

Example 11 [0787] Mouse PK assay using the pH-dependent IL-8-binding antibody H89/L118 f 11-11 Mouse PK assay using H89/L118

The rate of human IL-8 elimination in vivo was evaluated using H89/L118 produced in Example 9 and H998/L63 produced in Example 10.

[0788] After simultaneous administration of human IL-8 and anti-human IL-8 antibodies to mice (C57BL/6J, Charles river), pharmacokinetics of human IL-8 were evaluated. A mixed solution of human IL-8 and an anti-human IL-8 antibody (10 ug/mL and 200 ug/mL, respectively) was administered in a single dose at 10 mL/kg to the tail vein. At this time, since a sufficiently excessive amount of the anti-human IL-8 antibody is present with respect to human IL-8, almost all the human IL-8 is considered to be bound to the antibody. Blood was collected five minutes, two hours, four hours, seven hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after the administration. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set at -20°C or below until measurements were taken.

[0789] (11-2) Measurement of the human IL-8 concentration in plasma

The human IL-8 concentration in mouse plasma was determined by an electrochemiluminescence method. First, an anti-human IL-8 antibody (prepared in-house) comprising a mouse IgG constant region was dispensed into a MULTI-ARRAY 96-well Plate (Meso Scale Discovery), and was allowed to stand at room temperature for one hour. Then, a PBS-Tween solution containing 5% BSA (w/v) was used for blocking at room temperature for two hours to prepare an anti-human IL-8 antibodyimmobilized plate. Calibration curve samples containing human IL-8 at a plasma concentration of 275, 91.7, 30.6, 10.2, 3.40, 1.13, or 0.377 ng/mL and mouse plasma measurement samples diluted 25-fold or more were prepared. The samples were mixed with hWS-4 and allowed to react overnight at 37°C. Subsequently, 50 pL of the mixed solutions were dispensed into each well of the anti-human IL-8 antibody-immobilized plate, and the solution was stirred at room temperature for one hour. The final concentration of hWS-4 was adjusted to 25 ug/mL. Then, after one hour of reaction with a Biotin Mouse Anti-Human IgK Light Chain (BD Pharmingen) at room temperature, and then one hour of reaction with SULFO-TAG Labeled Streptavidin (Meso Scale Discovery) at room temperature, Read Buffer T (xl) (Meso Scale Discovery) was

218

WO 2017/046994

PCT/JP2016/003616 dispensed, and measurements were performed immediately with SECTOR Imager 2400 (Meso Scale Discovery). The human IL-8 concentration was calculated based on the response in the calibration curve using the analytical software, SOFT Max PRO (Molecular Devices).

[0790] The resulting data on the concentration of human IL-8 in plasma is shown in Fig. 22, and the values of human IL-8 clearance (CL) from mouse plasma are shown in Table 15.

[0791] [Table 15]

Antibody Name	Human IL-8 CL (mL/d/kg)
H998/L63	H89/L118
#1	21.4	472.2
#2	27.5	447.2
#3	24.7	478.0
Average (N=3)	24.5	465.1
Standard Deviation	3.0	15.6

[0792] As clear from Fig. 22, in comparison to human IL-8 administered simultaneously with H998/L63, human IL-8 administered simultaneously with H89/L118 was shown to be eliminated surprisingly quickly from mouse plasma. Furthermore, CL values which quantitatively represent the rate of human IL-8 elimination from mouse plasma indicate that the rate of human IL-8 elimination was increased about 19-fold for H89/L118 as compared to H998/L63.

[0793] Without being bound by a particular theory, the following can be speculated from the obtained data. Most of the human IL-8 administered simultaneously with the antibody binds to the antibody in the plasma and exists in a complexed form. Human IL-8 bound to H998/L63 may exist in an antibody-bound state even under the acidic pH condition in the endosome, due to the antibody's strong affinity. Thereafter, H998/L63 may be returned to the plasma via FcRn while still in the human IL-8-complexed form; therefore, when this occurs, human IL-8 is also returned to the plasma at the same time. Therefore, most of the human IL-8 taken up into the cells again may be returned to the plasma. That is, the rate of elimination of human IL-8 from plasma decreases remarkably when H998/L63 is simultaneously administered. On the other hand, as described previously, human IL-8 taken up into cells in a form complexed with H89/L118, a pH-dependent IL-8-binding antibody, may dissociate from the antibody under the acidic pH condition in the endosome. Human IL-8 dissociated from the antibody would be degraded after being transferred to the lysosome. Therefore, pHdependent IL-8-binding antibodies can significantly accelerate the elimination of human IL-8 as compared to an IL-8-binding antibody such as H998/L63 which has strong binding affinity at both acidic pH and neutral pH.

[0794] (11-3) Mouse PK assay with increased dose of H89/L118

219

WO 2017/046994

PCT/JP2016/003616

Next, an experiment that verifies the effect of varying the dose of H89/L118 was carried out as follows. After simultaneous administration of human IL-8 and H89/L118 (2 mg/kg or 8 mg/kg) to mice (C57BL/6J, Charles river), pharmacokinetics of human IL-8 were evaluated. A mixed solution of human IL-8 (2.5 pg/mL) and an anti-human IL-8 antibody (200 iig/mL or 800 pg/mL) was administered to the tail vein in a single dose of 10 mL/kg. At this time, since a sufficiently excessive amount of the antihuman IL-8 antibody is present compared to human IL-8, almost all of the human IL-8 are considered to be bound to the antibody. Blood was collected five minutes, seven hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after the administration. The collected blood was immediately centrifuged at 15,000 rpm and 4°C for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set at -20°C or below until measurements were taken.

[0795] Measurement of the human IL-8 concentration in mouse plasma was carried out by a method similar to that of Example 11-2. The resulting data on the human IL-8 concentration in plasma is shown in Fig. 23, and the values for human IL-8 clearance (CL) from mouse plasma are shown in Table 16.

[0796] [Table 16]

	Human IL-8 CL (mL/d/kg) |
Antibody Name	H89/L118	H89/L118 |
Antibody Dose	2 mg/kg	8 mg/kg
#1	181.2	93.0
#2	237	101.6
#3	247	114.5
Average (N = 3)	221.8	103.0
Standard Deviation	35.6	10,8 j

[0797] As a result, it was confirmed that as compared to the group administered with 2 mg/ kg of H89/L118, the group administered with 8 mg/kg of the antibody had an approximately 2-fold slower rate of human IL-8 elimination.

[0798] Herein below, the inventors describe contents surmised as one of possible factors that bring about the aforementioned results based on the scientific background, but the contents of the Disclosure C are not limited to the contents of the following discussion.

[0799] Among the antibodies that are returned from inside the endosome into the plasma via FcRn, it is preferred that the proportion of human IL-8-bound antibodies is low. With the focus on human IL-8 present in the endosome, it is desirable to have a high proportion of the free form not bound by an antibody. When human IL-8 is administered together with an antibody that does not have pH-dependent IL-8-affinity, most (nearly 100%) of the human IL-8 in the endosome is considered to exist in a form complexed with the antibody, and a small amount (close to 0%) is considered to be in the free form. On the other hand, when administered together with the pH-dependent

220

WO 2017/046994

PCT/JP2016/003616

IL-8-binding antibody (for example H89/L118), a certain proportion of human IL-8 should exist in a free form in the endosome. Hypothetically, the proportion of free form in this case can be understood as follows: [proportion of free human IL-8 in the endosome (%)] = [free human IL-8 concentration in the endosome] / [total human IL-8 concentration in the endosome] x 100.

[0800] The proportion of free human IL-8 in the endosome as understood by the above equation is desirably higher, and for example, 20% is more preferable than 0%, 40% is more preferable than 20%, 60% is more preferable than 40%, 80% is more preferable than 60%, and 100% is more preferable than 80%.

[0801] Thus, there is a correlation between the proportion of free human IL-8 in the endosome described above and the binding affinity (KD) and/or dissociation rate constant (koff) for human IL-8 at acidic pH. That is, the weaker the binding affinity and/or the greater the dissociation rate for human IL-8 at acidic pH, the higher the proportion of free human IL-8 in the endosome. However, in the case of pH-dependent IL-8-binding antibodies which can make the proportion of free human IL-8 close to 100% in the endosome, further weakening the binding affinity and/or increasing the dissociation rate at acidic pH does not necessarily lead to an effective increase in the proportion of free human IL-8. One can easily understand that, for example, even if the proportion of free human IL-8 is improved from 99.9% to 99.99%, such a degree of improvement may not be significant.

[0802] Furthermore, according to the general chemical equilibrium theory, when an antiIL-8 antibody and human IL-8 coexist and their binding reaction and dissociation reaction have reached an equilibrium, the proportion of free human IL-8 is unambiguously determined by three parameters: antibody concentration, antigen concentration, and dissociation constant (KD). Here, when the antibody concentration is high, when the antigen concentration is high, or when the dissociation constant (KD) is small, complexes are readily formed and the proportion of free human IL-8 decreases. On the other hand, when the antibody concentration is low, when the antigen concentration is low, or when the dissociation constant (KD) is large, complex formation becomes difficult, and the proportion of free human IL-8 increases.

[0803] Meanwhile, in this experiment, the rate of elimination of human IL-8 when

H89/L118 was administered at 8 mg/kg was slower than when the antibody was administered at 2 mg/kg. This therefore suggests that in the endosome, the proportion of free human IL-8 was decreased when antibody was administered at 8 mg/kg compared to when the antibody was administered at 2 mg/kg. The reason for this decrease may be that increasing the antibody dosage by four-fold increased the antibody concentration in the endosome, and thereby facilitated formation of the IL-8-antibody complex in the endosome. That is, in the group administered with an increased dose of

221

WO 2017/046994

PCT/JP2016/003616 the antibody, the proportion of free human IL-8 in the endosome decreased, and therefore the rate of elimination of human IL-8 has been decreased. This also suggests that when the antibody is administered at 8 mg/kg, the degree of the dissociation constant (KD) of H89/L118 under acidic pH conditions is insufficient for bringing free human IL-8 to nearly 100%. More specifically, if it is an antibody that has a larger dissociation constant (KD) (weaker binding) under acidic pH conditions, it may achieve a state of nearly 100% free IL-8 even when the antibody is administered at 8 mg/kg, and a rate of human IL-8 elimination equivalent to that when the antibody is administered at 2 mg/kg.

[0804] Based on the above, to confirm whether the pH-dependent IL-8-binding antibody of interest can accomplish a proportion of nearly 100% free human IL-8 in the endosome, without being particularly limited, one can verify whether there is room for increasing the degree of the antigen-eliminating effect in vivo or not. For example, one method compares the rate of human IL-8 elimination when using a novel pH-dependent IL8-binding antibody to that when H89/L118 is used, where the novel antibody has a weaker binding affinity at acidic pH and/or an increased dissociation rate at acidic pH compared to that of H89/L118. In case that the aforementioned novel pH-dependent IL-8-binding antibody shows an equivalent rate of human IL-8 elimination to that for H89/L118, this suggests that the binding affinity and/or dissociation rate of H89/L118 at acidic pH is already at a level sufficient for achieving a proportion of nearly 100% free human IL-8 in the endosome. On the other hand, in instances where the aforementioned novel pH-dependent IL-8-binding antibody shows a higher rate of human IL-8 elimination, this suggests that the binding affinity and/or dissociation rate of H89/L118 at acidic pH has room for improvement.

Example 12 [0805] Production and evaluation of the pH-dependent IL-8-binding antibody H553/L118 112-11 Production of antibody H553/L118 having pH-dependent IL-8 binding ability

Here, the inventors aimed to generate antibodies that have an even weaker human IL 8-binding affinity under acidic pH conditions and/or a greater dissociation rate than those of H89/L118.

[0806] Amino acid modifications, mainly involving histidine, were introduced using H89/L118 as a base, to produce the modified antibodies shown in Table 17 by a method similar to that of Example 9. Furthermore, the human IL-8-binding affinity for these antibodies was determined by a method similar to that of Example 9-2.

[0807] Part of the results is shown in Table 17. The antibody H553/L118 comprising

H553-IgGl (SEQ ID NO:90) as the heavy chain and LI 18-k0MT as the light chain, and the antibody H496/L118 comprising H496-IgGl (SEQ ID NO: 101) as the heavy

222

WO 2017/046994

PCT/JP2016/003616 chain and LI 18-kOMT as the light chain were shown to have further increased pH dependency than H89/L118.

[0808] [Table 17]

Antibody Name	pH	kon (1/Ms)	koff(1/s)	KD(M)	ton Ratio (pH7.4/pH5.8)	toff Ratio (pH5.8/pH7.4)	KD Ratio (pH5,8/pH7.4)
H89/L118	pH 7.4 ' pH 5. 8.......	9.45E+05 .......T23E+05 /	1.14E-Q4 3.90E-03	1.21E-10 3.Ϊ8Ε-Ο8	7.7	.......................34?2..........................	.....................263^0..........................
H496/L118	pH 7.4 .........pH	1.29E+06 .....Ϊ78Ε+05’	5.03E-05 .......5X7E/03.....	3.91 E-11 Έθ7Ε438.....	’..... 7.2 ’	........ 108.6 ..............	....... 785.0 .............
.....H553/L118	pH 7.4 ...........pFfKes...........	1.15E+06 1T4E+Q5	1.13E-04	9.76E-11 .....497ΈΪΙ8	...............*..................................	'...........‘270.7...................	......................50i3......................

[0809] In the obtained H553/L118, two amino acid modifications, Y55H and R57P, were introduced into the heavy chain of H89/L118. On the other hand, H496/L118, in which only R57P was introduced into the heavy chain of H89/L118, has an enhanced binding affinity for human IL-8 at neutral pH but a hardly changed human IL-8-binding affinity at acidic pH, in comparison to H89/L118. More specifically, the R57P modification introduced into H89/L118 is a modification that enhances the human IL8-binding affinity only at neutral pH without changing the binding affinity at acidic pH. Furthermore, H553/L118 produced by introducing the Y55H modification into the heavy chain of H496/L118 has a maintained or slightly enhanced binding affinity at neutral pH, but on the other hand, a decreased binding affinity at acidic pH in comparison to those of H89/L118. That is, introducing a combination of the two amino acid modifications, Y55H and R57P, into H89/L118 enabled further enhancement of the property of decreasing the binding affinity at acidic pH, while maintaining or slightly enhancing the binding affinity at neutral pH.

[0810] (12-2) Mouse PK assay using H553/L118

Evaluation of the rate of human IL-8 elimination in mice using H553/L118 was carried out by a method similar to that of Example 11-2. The resulting data on the human IL-8 concentration in plasma is shown in Fig. 24, and the values of human IL-8 clearance (CL) from mouse plasma are shown in Table 18.

[0811] [Table 18]

	Human IL-8 CL (mL/d/kg)
Antibody Name	H89/L118	H89/L118	H553/L118	H553/L118
Antibody Dose	2 mg/kg	8 mg/kg	2 mg/kg	8 mg/kg
#1	181.2	93.0	250	256.6
#2	237	101.6	245	248.4
#3	247	114.5	249	244.1
Average (N = 3)	221,8	103.0	248	249.7
Standard Deviation	35.6	10.8	3	6.4

[0812] As a result, large differences were not observed between H553/L118 and H89/L118 when the data of mice administered with 2 mg/kg antibody were compared; however, it was confirmed that H553/L118 accelerates the elimination of human IL-8 by 2.5 fold or so in comparison to H89/L118 when the data of mice administered with 8 mg/kg

223

WO 2017/046994

PCT/JP2016/003616 antibody were compared. From another viewpoint, H553/F118 did not show difference in the rate of human IF-8 elimination between 2 mg/kg and 8 mg/kg, and a reduction of the antigen elimination rate due to increase of the antibody dose as with H89/F118 was not observed.

[0813] Without particular limitation, one reason why such results were obtained may be discussed as follows. H533/F118 showed an equivalent rate of human IF-8 elimination when the antibody was administered at 2 mg/kg and at 8 mg/kg. This can indicate that the proportion of free IF-8 in the endosome can reach a level close to 100%, since the IF-8 binding by H553/F118 at acidic pH is sufficiently weak even under the conditions of 8 mg/kg-administration. In other words, this suggests that while H89/F118 can achieve a maximum human IF-8 elimination effect at a dose of 2 mg/kg, its effects may be weakened at a high dose of around 8 mg/kg. On the other hand, H553/F118 can achieve a maximum effect of eliminating human IF-8 even at a high dose of 8 mg/ kg· [0814] 112-31 Stability evaluation using H553/F118

H553/F118 was shown to be an antibody that can accelerate the elimination of human IF-8 more remarkably than H89/F118 in mice. However, in order for this antibody to sustain this inhibitory effect on human IF-8 for a long period of time in vivo, it is also important that the IF-8-neutralizing activity is stably kept (stability in IF-8-neutralizing activity of this antibody) during the period when the administered antibody is present in vivo (for example, in plasma). Accordingly, the stability of these antibodies in mouse plasma was evaluated by the following method.

[0815] Mouse plasma was collected from the blood of C57BF/6J (Charles River) by a method known in the art. 200 pF of 200 mM PBS (Sigma, P4417) was added to 800 pF of mouse plasma to give 1 mF. Furthermore, sodium azide was added at a final concentration of 0.1% as an antiseptic. Then, each antibody (Hr9, H89/F118, and H553/F118) was added to the above-mentioned mouse plasma to a final concentration of 0.2 mg/mF. At this point, a portion of the sample was collected as the initial sample. The remaining sample was stored at 40°C. One week and two weeks after storage, a portion of each sample was collected, and they were used as the one-week-stored sample and the two-week-stored sample. All samples were frozen at -80°C and stored until each analysis was performed.

[0816] Next, anti-IF-8 antibodies contained in mouse plasma were evaluated for their human IF-8-neutralizing activity as follows: CXCR1 and CXCR2 are known receptors for human IF-8. The PathHunter(registered trademark) CH0-K1 CXCR2 β-Arrestin cell line (DiscoveRx Co., Cat.# 93-0202C2) expresses human CXCR2, and is a cell line artificially produced so as to emit chemiluminescence when human IF-8-mediated signals are transmitted. While it is not particularly limited, the human IF-8-neutralizing

224

WO 2017/046994

PCT/JP2016/003616 activity possessed by an anti-human IL-8 antibody can be evaluated using this cell. When human IL-8 is added to the culture solution of the cells, a certain amount of chemiluminescence is exhibited in a manner dependent on the concentration of the added human IL-8. When human IL-8 and an anti-human IL-8 antibody are added together to the culture solution, human IL-8 signal transduction may be blocked upon binding of the anti-human IL-8 antibody to human IL-8. As a result, chemiluminescence caused by addition of human IL-8 will be inhibited by the anti-human IL-8 antibody, and the chemiluminescence will be weaker than when the antibody is not added, or there will be no chemiluminescence at all. Therefore, as the human IL-8 neutralizing activity possessed by the antibody becomes stronger, the degree of chemiluminescence becomes weaker; and as the human IL-8 neutralizing activity possessed by the antibody becomes weaker, the degree of chemiluminescence becomes stronger.

[0817] This is the same for an antibody that has been added to mouse plasma and stored for a certain period of time. If the neutralizing activity of the antibody does not change due to storage in mouse plasma, the degree of the above-mentioned chemiluminescence before and after storage should not change. On the other hand, in the case of an antibody whose neutralizing activity decreases due to storage in mouse plasma, the degree of chemiluminescence by use of a stored antibody will increase as compared to that before storage.

[0818] Then, the above-mentioned cell line was used to examine whether the neutralizing activity of an antibody stored in mouse plasma was maintained. First, the cell line was suspended in the AssayComplete(tm) Cell Plating 0 Reagent, and then seeded into a 384-well plate at 5000 cells/well. One day after starting of the cell culture, an experiment was performed below for determining the concentration of human IL-8 to be added. Serially diluted human IL-8 solutions, which contain final human IL-8 concentrations from 45 nM (400 ng/mL) to 0.098 nM (0.1 ng/mL), were added to the cell culture solution. Next, a detection reagent was added according to the protocol of the product, and the relative chemiluminescence level was detected using a chemiluminescence detector. From this result, reactivity of the cells towards human IL-8 was confirmed, and the human IL-8 concentration suitable for confirming the neutralizing activity of anti-human IL-8 antibodies was determined. Here, the human IL-8 concentration was set to 2 nM.

[0819] Next, the aforementioned anti-human IL-8 antibody-added mouse plasma was used to evaluate the neutralizing activities of the antibodies contained therein. Human IL-8 at the concentration determined above and the aforementioned anti-human IL-8 antibody-containing mouse plasma were added to the cell culture. The amount of mouse plasma to be added was determined so as to contain stepwise concentrations of the anti-human IL-8 antibody in the range of 2 pg/mL (13.3 nM) to 0.016 pg/mL (0.1

225

WO 2017/046994

PCT/JP2016/003616 nM). Next, detection reagents were added according to the product protocol, and the relative chemiluminescence levels were detected using a chemiluminescence detector.

[0820] Here, relative values for the relative chemiluminescence levels at each antibody concentration were calculated by defining the average relative chemiluminescence level in wells without addition of human IL-8 and antibody as 0%, and by defining the average relative chemiluminescence level in wells that have been added with only human IL-8 but no antibody as 100%.

[0821] The results of human IL-8 inhibition assay using human CXCR2-expressing cells are shown in Fig. 25A, which shows results from the initial sample (without preservative treatment in mouse plasma), Fig. 25B, which shows results for the samples stored at 40°C for one week, and Fig. 25C, which shows results for the samples stored at 40°C for two weeks.

[0822] As a result, differences in the human IL-8-neutralizing activity before and after storage in mouse plasma were not observed for Hr9 and H89/L118. On the other hand, H553/L118 showed decrease in the human IL-8-neutralizing activity after two-week storage. Therefore, the human IL-8-neutralizing activity of H553/L118 readily decreases in mouse plasma as compared to that of Hr9 and H89/L118, and H553/L118 was shown to be an antibody having unstable properties in terms of the IL-8 neutralizing activity.

Example 13 [0823] Production of antibodies with reduced predicted immunogenicity score using an in silico system (13-1) Predicted immunogenicity score of various IL-8-binding antibodies

Generation of anti-drug antibodies (ADA) influences the efficacy and pharmacokinetics of therapeutic antibodies, and brings about serious side effects in some cases; and therefore, clinical utility and drug efficacy of therapeutic antibodies may be limited by the generation of ADA. The immunogenicity of therapeutic antibodies is known to be affected by many factors, and, there are many reports describing the importance of effector T cell epitopes in the therapeutic antibodies.

[0824] In silico tools for predicting T cell epitopes such as Epibase (Lonza), iTope/TCED (Antitope), and EpiMatrix (EpiVax) have been developed. Using these in silico tools,

T cell epitopes in each of the amino acid sequences can be predicted (Walle et al., Expert Opin. Biol. Ther. 7(3):405-418 (2007)), and the potential immunogenicity of therapeutic antibodies can be evaluated.

[0825] Here, EpiMatrix was used to calculate the immunogenicity scores of each of the anti IL-8 antibodies. EpiMatrix is a system for predicting the immunogenicity of a protein of interest by automatically designing sequences of peptide fragments by sectioning

226

WO 2017/046994

PCT/JP2016/003616 the amino acid sequence of the protein to be predicted for its immunogenicity by nine amino acids, and then calculating their ability to bind eight major MHC Class II alleles (DRBl*0101, DRBl*0301, DRBl*0401, DRBl*0701, DRBl*0801, DRBl*1101, DRB 1*1301, and DRB 1*1501) (De Groot et al., Clin. Immunol. 131(2):189-201 (2009)).

[0826] The immunogenicity scores of the heavy chains and light chains of each anti-IL-8 antibody, which were calculated as described above, are shown in the EpiMatrix Score column of Table 19. Furthermore, regarding the EpiMatrix Scores, immunogenicity scores corrected for the Tregitope content are shown in the tReg Adjusted Epx Score column. Tregitope is a peptide fragment sequence present in large amounts mainly in native antibody sequences, and is a sequence considered to inhibit immunogenicity by activating regulatory T cells (Tregs).

[0827] Furthermore, regarding these scores, the sum of the scores for the heavy and light chains is shown in the Total column.

[0828] [Table 19]

	Heavy Chain	Lig	it Chain	Total
Antibody Name	EpiMatrix 7 Score	tReg Adjusted Epx Score	EpiMatrix Score	tReg Adjusted Epx Score	EpiMatrix Score	tReg Adjusted Epx Score
hWS-4	62.44	12.18	22.64	-23,89	85,08	-11.71
Hr9	56.52	6.27	22.64	-23.89	79.16	-17.62
H89/L118	57.99	7.74	7.16	-39.36	65.15	-31.62
H496/L118	54.13	3,87	7,16	-39.36	61.29	-35.49
H553/L118	47,88	-2.37	7.16	-39.36	55.04	-41.73

[0829] According to these results, both the EpiMatrix Score and the tReg Adjusted Epx Score showed that the immunogenicity scores of H89/L118, H496/L118, and H553/L118 were decreased as compared to that of hWS-4, which is a known humanized anti-human IL-8 antibody.

[0830] Furthermore, with EpiMatrix, it is feasible to compare the frequency of ADA development predicted for the antibody molecule as a whole by considering the heavychain and light-chain scores with the actual frequency of ADA development caused by various commercially available antibodies. Results of performing such analysis are shown in Fig. 26. Due to system limitations, the notations used in Fig. 26 are WS4 for hWS-4, HR9 forHr9, H89L118 forH89/L118, H496L118 for H496/L118, and H553L118 for H553/L118.

[0831] As shown in Fig. 26, the frequency of ADA development in humans caused by various commercially available antibodies is known to be 45% for Campath (Alemtuzumab), 27% for Rituxan (Rituximab), and 14% for Zenapax (Daclizumab).

On the other hand, while the frequency of ADA development predicted from the amino acid sequence was 10.42% for hWS-4 which is a known humanized anti-human IL-8

227

WO 2017/046994

PCT/JP2016/003616 antibody, the frequency of H89/L118 (5.52%), H496/L118 (4.67%), or H553/L118 (3.45%) newly identified herein were significantly lower in comparison to that of hWS-4.

[0832] (13-2) Production of modified antibodies with lowered predicted immunogenicity scores

As described above, the immunogenicity scores of H89/L118, H496/L118, and H553/L118 were lower in comparison to that of hWS-4; however, as is apparent from Table 19, the immunogenicity scores for the heavy chain are higher than those for the light chains, which suggests that there is still room for improvement in the amino acid sequences of the heavy chain in particular from the viewpoint of immunogenicity. Then, a search was conducted in the heavy chain variable region of H496 for amino acid modifications that can decrease the immunogenicity score. As a result of diligent search, three variants, H496vl in which alanine at position 52c according to Rabat numbering was substituted with aspartic acid, H496v2 in which glutamine at position 81 was substituted with threonine, and H496v3 in which serine at position 82b was substituted with aspartic acid were found. Furthermore, H1004 that contains all three of these modifications was produced.

[0833] The results of immunogenicity scores calculated by a method similar to that of Example 13-1 are shown in Table 20.

[0834] [Table 20]

	Heavy Chain	Light Chain	Total
Antibody Name	EpiMatrix Score	tReg Adjusted Epx Score	EpiMatrix Score	tReg Adjusted Epx Score	EpiMatrix Score	tReg Adjusted Epx Score
H496/L118	54.13	3.87	7.16	-39.36	61.29	-35.49
H496V1/L118	32.17	-18.08	7.16	-39.36	39.33	-57.44
H496v2/L118	45.26	-5.00	7.16	-39.36	52.42	-44.36
H496V3/L118	38,27	-11.98	7.16	-39.36	45.43	-51.34
H1004/L118	10.79	-39.47	7.16	-39.36	17.95	-78.83
H1004/L395	10.79	-39.47	7.79	-38.74	18.58	-78.21

[0835] The three heavy chains, H496vl, H496v2, and H496v3, all of which contain a single modification, showed decreased immunogenicity scores in comparison to that of H496. Furthermore, H1004, that contains a combination of three modifications, achieved a remarkable improvement of the immunogenicity score.

[0836] Here, in addition to LI 18, L395 was identified as the light chain appropriate for combination with Hl004. Therefore, in the calculation of immunogenicity scores, both the LI 18 combination and the L395 combination were used. As indicated in Table 20, H1004/L118 and H1004/L395, which are combinations of heavy and light chains, also showed very low immunogenicity scores.

[0837] Next, the frequency of ADA development for these combinations was predicted in a manner similar to Example 13-1. The results are shown in Fig. 27. The notations used

228

WO 2017/046994 PCT/JP2016/003616 in Fig. 27 are VI for H496vl/L118, V2 for H496v2/L118, V3 for H496v3/L118, H1004L118 for H1004/L118, and H1004L395 for H1004/L395.

[0838] Surprisingly, H1004/L118 and H1004/L395, which have remarkably lowered immunogenicity scores, also showed improvement in the predicted values for the frequency of ADA development, and showed a predicted value of 0%.

[0839] (13-3) Measurement of the IL-8-binding affinity of H1004/L395

H1004/L395 which is an antibody comprising H1004-IgGlm (SEQ ID NO:91) as the heavy chain and L395-k0MT (SEQ ID NO:82) as the light chain was produced. The binding affinity of H1004/L395 for human IL-8 was measured as described below using BIACORE T200 (GE Healthcare).

[0840] The following two running buffers were used, and measurements were carried out at the respective temperatures: (1) 0.05% tween20, 40 mM ACES, 150 mM NaCl, pH 7.4, 40°C; and (2) 0.05% tween20, 40 mM ACES, 150 mM NaCl, pH 5.8, 37°C.

[0841] An appropriate amount of Protein A/G (PIERCE) was immobilized onto the Sensor chip CM4 (GE Healthcare) by the amine coupling method and the antibodies of interest were captured. Next, a diluted human IL-8 solution or a running buffer (used as a reference solution) was injected to allow interaction of the antibodies captured onto the sensor chip with human IL-8. For the running buffer, either one of the abovementioned solutions was used, and the respective buffers were also used to dilute human IL-8. To regenerate the sensor chip, 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) were used. KD (M) of each antibody for human IL-8 was calculated based on the association rate constant kon (1/Ms) and dissociation rate constant koff (1/s) which are kinetic parameters calculated from sensorgrams obtained by the measurements.

The BIACORE T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

[0842] The measurement results are shown in Table 21. In comparison to H89/L118,

H1004/L395, with lowered immunogenicity score, had an equivalent KD for human IL-8 at neutral pH, but increased KD and koff at acidic pH; and it was shown to have the property of dissociating readily from IL-8 in the endosome.

[0843] [Table 21-1]

Antibody Name	pH	kon (1/Ms)	koff (1/s)	KD (M)	kon Ratio (pH7.4/pH5.8)	koff Ratio (pH5.8/pH7.4)	KD Ratio (pH5.8/pH7.4)
H89/L118	pH 7.4 pH 5.8	7.51 £+05 1.29E+05	1.29E-04 528E-03	1.72E-10 4.88E-08	5.8	48.7	283.7
H1004/L395	pH 7.4 pH 5.8	1.02E+06 3.06E+05	1.55E-04 3.38E-02	1.51E-10 1.10E-07	3.3	218.1	728.5

Example 14 [0844] Production and evaluation of the pH-dependent IL-8-binding antibody H1009/L395 (14-1) Production of various pH-dependent IL-8-binding antibodies

229

WO 2017/046994

PCT/JP2016/003616

H1004/L395, which has pH-dependent IL-8 binding ability and also a lowered immunogenicity score was obtained by the evaluation shown in Example 13. Subsequently, a dedicated investigation was carried out to produce variants that have these favorable properties as well as stability in mouse plasma.

[0845] The following modified antibodies were produced based on H1004/L395 by introducing various modifications.

[0846] [Table 21-2]

Heavy Chain

Hl 00 4	A52cD/R57P/Q8lT/S82bD/Y97H
H0932	A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H
Hl 000	D31E/A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H
H13Q9	A52cD/G54Y/Y55H/R57P/Q8lT/S82bD/Y97H
Hl 02 2	A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/Y97H
Hl 02 3	A52cD/T56H/R57P/Q81T/S82bD/Y97H
H1028	A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/Y97H
H1029	S30D/D31K/A52cD/G54H/Y55H/R57P/Q81T/S82bD/Y97H
H1031	S30D/D31K/A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/Y97H
H1032	530D/D31K/A52cD/T56H/R57P/Q81T/S82bD/Y97H
H1037	S30D/D31K/A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/Y97H
H104 0	D31E/A52cD/G54H/Y55H/T56H/R57P/Q81T/S82bD/Y97H
H1041	D31E/A52cD/T56H/R57P/Q81T/S82bD/Y97H
H104 6	D31E/A52cD/G54Y/Y55H/T56H/R57P/Q81T/S82bD/Y97H
Hl 04 7	S 3 0 D/D31K/A5 2cD/R57P/Q81T/S82bD/Y97H
H1O4 8	D31E/A52eD/R57P/Q81T/582bD/Y97B
H104 9	S30D/D31K/A52cD/G54Y/Y55H/R57P/Q81T/S82bD/Y97H
H1050	D31E/A52cD/G54Y/Y55H/R57P/Q81T/S82bD/Y97H

[0847] [Table 21-3]

L395	N50K/L54H/Q89K
L442	S31E/N50K/LS4H/Q89K

[0848] A total of 36 types of antibodies were produced by combining the 18 types of heavy chains and two types of light chains described above. Various evaluations were performed on these antibodies as indicated below.

[0849] The human IL-8-binding affinities under neutral and acidic pH conditions were measured in a manner similar to the method of Example 13-3. Among the obtained results, KD at pH 7.4, and KD and koff at pH 5.8 are shown in Table 22.

[0850] Next, stability in terms of IL-8 binding upon storage of the antibodies in PBS was evaluated by the method indicated below.

[0851] The respective antibodies were dialyzed overnight against DPBS (Sigma-Aldrich), and then the concentration of each of the antibodies was adjusted to 0.1 mg/mL. At this point, some of the antibody samples were collected as initial samples. The remaining samples were stored at 50°C for one week, and then collected as samples for the thermal acceleration test.

230

WO 2017/046994 PCT/JP2016/003616 [0852] Next, BIACORE measurement of the IL-8-binding affinity was carried out as follows using the initial samples and samples for the thermal acceleration test.

[0853] The levels of human IL-8 binding to the modified antibodies were analyzed using BIACORE T200 (GE Healthcare). Measurements were carried out at 40°C by using 0.05% tween20, 40 mM ACES, and 150 mM NaCl at pH 7.4 as the running buffer.

[0854] An appropriate amount of Protein A/G (PIERCE) was immobilized onto the Sensor chip CM4 (GE Healthcare) by the amine coupling method and the antibodies of interest was captured. Next, a diluted human IL-8 solution or a running buffer (used as a reference solution) was injected to allow interaction of the antibodies captured onto the sensor chip with human IL-8. The running buffer was also used to dilute human IL 8. To regenerate the sensor chip, 25 mM NaOH and 10 mM glycine-HCl (pH 1.5) were used. The measured binding level of human IL-8 and the amount of antibodies captured at that binding level were extracted using the BIACORE T200 Evaluation Software (GE Healthcare).

[0855] The amount of human IL-8-binding per 1000 RU of the amount of antibody captured was calculated for the initial samples and the samples for the thermal acceleration test. Furthermore, the ratio of the human IL-8-binding level for the initial samples to that for samples of the thermal acceleration test was calculated.

[0856] The resulting ratios of IL-8-binding level of the initial samples to that for samples of the thermal acceleration test are shown in Table 22 as well.

[0857]

231

WO 2017/046994

PCT/JP2016/003616 [Table 22]

Antibody	pH7.4 KD	pHS.8 KD	pH5.8 koff	Ratio of IL-8 Binding Amount (Thermal Acceleration/lnitial)
H0089/L0118	1.7E-10	4.9E-08	6.3E-03	0,61
H0932/L0395	1.6E-10	1.1E-07	5.7E-02	0.56
H0932/L0442	2.1E-10	7.9E-08	2.2E-02	0.56
H1000/L0395	1.4E-10	8.9E-08	2.0E-02	0.57
H1000/L0442	2.0E-10	7.1E-08	1.7E-02	0.57
H1004/L0395	1.5E-10	1.1E-07	3.4E-02	0.58
H1004/L0442	2.2E-10	7.7E-08	2.0E-02	0,59
H1009/L0395	7.1E-11	8.7E-08	1.0E-02	0.64
H1009/L0442	1.1E-10	6.3E-08	6.0E-03	0,64
H1022/L0395	2.7E-10	2.9E-07	1.2E+01	0.47
H1022/L0442	3.6E-10	1.8E-07	2.0E-02	0.46
H1023/L0395	7.6E-11	9.2E-08	1.8E-02	0,54
H1023/L0442	1.2E-10	7.1E-08	1.7E-02	0.55
H1028/L0395	1.8E-10	2.1E-07	1.0E+01	0.55
H1028/L0442	2.4E-10	1.4E-07	1.3E-01	0.56
H1029/L0395	8.6E-11	5.5E-08	8.0E-03	0.59
H1029/L0442	1.4E-10	4.8E-08	8.5E-03	0.58
H1031/L0395	1.5E-10	9.9E-08	4.6E-02	0.48
H1031/L0442	2.1E-10	8.9E-08	3.9E-02	0.47
H1032/L0395	4.2E-11	5.0E-08	4. IE-03	0.61
H1032/L0442	7.8E-11	4.3E-08	5.9E-03	0.61
H1037/L0395	9.4E-11	7.0E-08	1.5E-02	0.55
H1037/10442	1.3E-1Q	6.1E-08	1.5E-02	0.57
H1040/L0395	2.6E-10	2.4E-07	4.6E-02	0.44
H1040/L0442	3.4E-10	1.4E-07	2.1E+01	0.49
H1041/L0395	8.0E-11	7.1E-08	1.3E-02	0.55
H1041/L0442	1.2E-10	6.1E-08	1.5E-02	0.56
H1046/L0395	1.8E-10	1.6E-07	1.2E-02	0.56
H1046/L0442	2.3E-10	1.1E-07	1.2E-02	0.55
H1047/L0395	9.5E-11	4.7E-08	6.0E-03	0.65
H1047/L0442	1.5E-10	4.7E-08	4.6E-03	0.64
H1048/L0395	1.5E-10	9.0E-08	6.4E-03	0.59
H1048/L0442	2.1E-10	6.7E-08	1.5E-02	0.59
H1049/L0395	2.5E-11	3.8E-08	4.0E-03	0.65
H1049/L0442	5.3E-11	3.3E-08	4.5E-03	0.65
H1050/L0395	6.6E-11	7.7E-08	5.0E-03	0.64
H105G/L0442	9.9E-11	5.4E-08	7.6E-03	0.64

[0858] By the above-mentioned examination, H1009/L395 which is an antibody comprising H1009-IgGlm (SEQ ID NO:92) as the heavy chain and L395-k0MT as the light chain was obtained.

[0859] As shown in Table 22, in comparison to H89/L118, H1009/L395 had a slightly enhanced human IL-8-binding affinity at neutral pH, but on the other hand, a decreased binding affinity at acidic pH, that is, pH-dependence had been further strengthened. Furthermore, when exposed to severe conditions such as at 50°C in PBS, H1009/L395 had a slightly enhanced stability in IL-8 binding when compared to that of H89/L118.

[0860] Accordingly, H1009/L395 was selected as an antibody whose neutralizing activity in mouse plasma may be stably maintained, while keeping its pH-dependent IL-8 binding

232

WO 2017/046994

PCT/JP2016/003616 ability.

[0861] (14-2) Stability evaluation of H1009/L395

Next, in a manner similar to the method of Example 12-3, it was evaluated whether the IL-8 neutralizing activity of H1009/L395 is stably maintained in mouse plasma. Here, H1009/L395-F1886s which will be described in detail later in Example 19 was used. This antibody has the same variable region as that of H1009/L395, and a constant region having modifications that enhance FcRn binding under acidic pH conditions and modifications for reducing its binding towards FcyR(s) in comparison to those of the native human IgGl. The variable region of H1009/L395, especially the region around HVR, is responsible for human IL-8-binding and IL-8-neutralizing activity of this antibody, and modifications introduced into the constant region are considered not to affect these properties.

[0862] Evaluation of the stability in mouse plasma was performed as follows. 150 pL of 200 mM phosphate buffer (pH 6.7) was added to 585 pL of mouse plasma. Then, sodium azide was added as an antiseptic at a final concentration of 0.1%. Each antibody (Hr9, H89/L118, or H1009/L395-F1886s) was added to the above-mentioned mouse plasma at a final concentration of 0.4 mg/mL. At this point, a portion of the sample was collected as the initial sample. The remaining sample was stored at 40°C. One week and two weeks after the start of storage, a portion of each sample was collected, and they were used as the sample stored for one week and the sample stored for two weeks. All samples were frozen at -80°C and stored until each analysis was performed.

[0863] Measurement of the human IL-8-neutralizing activity was carried out using human CXCR2-expressing cells by a method similar to that of Example 12-3. However, the concentration of human IL-8 used to confirm the neutralizing activity of an anti-human IL-8 antibody this time was 1.2 nM.

[0864] The results of human IL-8 inhibition assay obtained using the above-mentioned antibodies with human CXCR2-expressing cells are shown in Fig. 28A, which shows results for the initial sample (without storage treatment in mouse plasma), Fig. 28B, which shows results for the samples stored at 40°C for one week, and Fig. 28C, which shows results for the samples stored at 40°C for two weeks.

[0865] As a result, surprisingly, the human IL-8-neutralizing activity was maintained in

H1009/L395-F1886s even after it was stored in mouse plasma at 40°C for two weeks, and the IL-8-neutralizing activity was more stably maintained than in the case of H553/L118.

[0866] 114-31 Mouse PK assay using H1009/L395

The rate of human IL-8 elimination by H1009/H395 in mice was evaluated by the following method. H1009/L395, H553/L118, and H998/L63 were used as the antibodies. Administration to mice and blood collection, and measurement of the human

233

WO 2017/046994

PCT/JP2016/003616

IL-8 concentration in mouse plasma were carried out by the method shown in Example 11.

[0867] The resulting data on the concentration of human IL-8 in plasma are shown in Fig. 29, and the values of human IL-8 clearance (CL) from mouse plasma are shown in Table 23.

[0868] [Table 23]

	Human IL-8 CL (mL/d/kg)
Antibody Name	H998/L63	H553/L118	H1009/L395
#1	21.4	773.2	705.0
#2	27.5	497.6	777.3
#3	24.7	879.8	737.7
Average (N = 3)	24.5	716.9	740.0
Standard Deviation	3.0	197.2	36.2

[0869] As a result, the rate of human IL-8 elimination in mice when H1009/L395 was administered at 2 mg/kg was equivalent to that of H553/L118, and it was shown that H1009/L395 achieves nearly 100% free IL-8 in the endosome. The value of clearance (CL) which quantitatively represents the rate of human IL-8 elimination from mouse plasma was shown to be approximately 30-fold higher than that of H998/L63.

[0870] Without being particularly limited, the effect of increasing the rate of human IL-8 elimination can be understood as follows. Generally, in a living body where antigens are maintained at nearly constant concentrations, production rates and elimination rates of antigens will also be maintained at nearly constant values. When antibodies are administered under such conditions, even in cases where the antigen production rates are not affected, the rates of antigen elimination may change due to the complex formation of antigen with antibodies. Generally, since the antigen-elimination rate is greater than the antibody-elimination rate, in such cases, the elimination rate of antigens that have formed complexes with antibodies decreases. When the antigen elimination rate decreases, the antigen concentration in plasma increases, but the degree of increase in this case may also be defined by the ratio of the elimination rate when the antigen is present alone to the elimination rate when the antigen forms a complex. That is, in comparison to the elimination rate when the antigen is present alone, if the elimination rate when a complex is formed is decreased to one tenth, the antigen concentration in the plasma of the antibody-administered organism may increase up to approximately ten times that before antibody administration. Here, clearance (CL) may be used as the elimination rate. More specifically, increase of the antigen concentration (antigen accumulation) that takes place after antibody administration to an organism may be defined by the antigen CL under each of the conditions before antibody administration and after antibody administration.

[0871] Here, the presence of an approximately 30-fold difference in CL of human IL-8 when

234

WO 2017/046994

PCT/JP2016/003616

H998/L63 and H1009/L395 were administered suggests that there may be an approximately 30-fold difference between the levels of increase in the human IL-8 concentration in plasma when these antibodies are administered to humans. Furthermore, generation of a 30-fold difference in the human IL-8 concentration in plasma indicates that there will also be approximately a 30-fold difference in the amount of antibodies necessary for completely blocking the biological activity of human IL-8 under the respective conditions. That is, in comparison to H998/L63, H1009/L395 can block the biological activity of IL-8 in plasma at approximately 1/30 of the amount, which is a very small amount of antibody. Furthermore, when H1009/L395 and H998/L63 are individually administered to humans at the same dose, H1009/L395 will be able to block the biological activity of IL-8 for a longer period of time with greater strength. To block the biological activity of IL-8 for a long period of time, it is necessary that the IL-8-neutralizing activity is stably maintained. As shown in Example 14, experiments using mouse plasma have elucidated that H1009/L395 can maintain its human IL8-neutralizing activity for a long period of time. H1009/L395 which has these noteworthy properties was also shown to be an antibody that has superior effects from the viewpoint of the efficacy in neutralizing IL-8 in vivo.

Example 15 [0872] Evaluation of extracellular matrix-binding using the pH-dependent IL-8-binding antibody H1009/L395

The excellent 30-fold greater effect of H1009/L395 in eliminating human IL-8 as shown in Example 14 was a surprising effect. It is known that the rate of antigen elimination when a pH-dependent antigen-binding antibody is administered depends on the rate of uptake of the antibody-antigen complex into cells. That is, if the rate of the pH-dependent antigen-binding antibody uptake into cells increases when an antigen-antibody complex is formed in comparison to when the complex is not formed, the antigen-eliminating effect of the pH-dependent antibody can be increased. Known methods for increasing the rate of uptake of an antibody into cells include the method of conferring the FcRn-binding ability under neutral pH conditions to an antibody (WO 2011/122011), the method for enhancing the binding ability of an antibody towards FcyR(s) (WO 2013/047752), and the method that uses promotion of the formation of complexes containing a polyvalent antibody and a polyvalent antigen (WO 2013/081143).

[0873] However, the above-mentioned technique is not used in the constant regions of H1009/L395. Furthermore, while IL-8 is known to form a homodimer, human IL-8 bound by H1009/L395 has been found to exist in the form of a monomer because H1009/L395 recognizes the homodimer-forming surface of human IL-8. Therefore,

235

WO 2017/046994

PCT/JP2016/003616 this antibody will not form polyvalent complexes.

[0874] More specifically, while the above-mentioned technique is not used for H1009/L395,

H1009/L395 showed a 30-fold greater human IL-8-eliminating effect.

[0875] Then, the inventors carried out the following discussion as a possible factor that may bring about the aforementioned properties of pH-dependent IL-8-binding antibodies represented by H1009/L395. However, the following is only a possibility surmised by the inventors based on the technical background, and the content of Disclosure C is not limited to the content of the following discussion.

[0876] Human IL-8 is a protein that has a high isoelectric point (pi), and the theoretical isoelectric point calculated by a known method is approximately 10. That is, under neutral pH conditions, human IL-8 is a protein whose charge is shifted towards the positive side. pH-dependent IL-8-binding antibodies represented by H1009/L395 are also proteins whose charge is shifted towards the positive side, and the theoretical isoelectric point of H1009/L395 is approximately 9. That is, the isoelectric point of a complex produced by binding of H1009/L395, a protein that has a high isoelectric point and is originally rich in positive charges, to human IL-8 which has a high isoelectric point will be higher than that of H1009/L395 alone.

[0877] As shown in Example 3, increasing the isoelectric point of an antibody, which includes increasing the number of positive charges and/or decreasing the number of negative charges on the antibody, can be considered to increase non-specific uptake of the antibody-antigen complex into cells. The isoelectric point of complex formed between an anti-IL-8 antibody and human IL-8 which has a high isoelectric point is higher compared to that of the anti-IL-8 antibody alone, and the complex may be taken up more readily into cells.

[0878] As described earlier, affinity for the extracellular matrix is also a factor that may influence uptake into cells. Then, it was examined whether there is a difference in extracellular matrix binding between an antibody alone and a complex with a human IL8-antibody.

[0879] Evaluation of the amount of antibody binding to the extracellular matrix by the ECL (electrochemiluminescence) method

Extracellular matrix (the BD Matrigel Basement Membrane Matrix / manufactured by BD) was diluted to 2 mg/mL using TBS (Takara, T903). The diluted extracellular matrix was dispensed into the MULTI-ARRAY 96well Plate, High bind, Bare (manufactured by Meso Scale Discovery: MSD) at 5 pL per well, and immobilized overnight at 4°C. Then, blocking was performed using 20 mM ACES buffer (pH 7.4) containing 150 mM NaCl, 0.05% Tween20, 0.5% BSA, and 0.01% NaN₃.

[0880] The antibodies to be evaluated were prepared as follows. The antibody samples to be added alone were prepared by diluting each antibody to 9 ug/mL using Buffer 1 (20

236

WO 2017/046994

PCT/JP2016/003616 mM ACES buffer containing 150 mM NaCl, 0.05% Tween20, and 0.01% NaN₃, at pH 7.4), and then further diluting them using Buffer2 (20 mM ACES buffer containing 150 mM NaCl, 0.05% Tween20, 0.1% BSA, and 0.01% NaN₃, at pH 7.4) to a final concentration of 3 pg/mL.

[0881] On the other hand, the antibody samples to be added as a complex with human IL-8 were prepared by adding human IL-8 at ten times the molar concentration of the antibody to an antibody sample, then diluting each antibody using Buffer-1 so that the antibody concentration became 9 pg/mL, respectively, and then further diluting each of them using Buffer-2 to a final antibody concentration of 3 pg/mL. At this point, the human IL-8 concentration was approximately 0.6 pg/mL. This was shaken at room temperature for one hour for complex formation.

[0882] Next, solutions of the antibody alone or the antibody as a complex were added to the plate from which the blocking solution had been removed, and this was shaken at room temperature for one hour. Then, after removal of the antibody-alone solution or the complex solution, Buffer-1 containing 0.25% Glutaraldehyde was added. Then, after the plate was allowed to stand for 10 minutes, it was washed with DPBS (manufactured by Wako Pure Chemical Industries) containing 0.05% Tween20. An antibody for ECL detection was prepared by sulfo-tagging the goat anti-human IgG (gamma) (manufactured by Zymed Laboratories) using the Sulfo-Tag NHS Ester (manufactured by MSD). The antibody for ECL detection was diluted with Buffer-2 to be 1 pg/mL, added to the plate, and then shaken in the dark at room temperature for one hour. The antibody for ECL detection was removed, a solution produced by 2-fold dilution of the MSD Read Buffer T (4x) (manufactured by MSD) using ultrapure water was added, and then the amount of luminescence was measured by SECTOR Imager 2400 (manufactured by MSD).

[0883] The results are shown in Fig. 30. Interestingly, all of the anti-IL-8 antibodies such as H1009/L395 hardly showed any binding to the extracellular matrix as the antibody alone (-IL8), but bound to the extracellular matrix upon complex formation with human IL-8 (+hIL8).

[0884] As described above, the property of anti-IL-8 antibodies to acquire affinity for the extracellular matrix by binding to human IL-8 has not been elucidated. Furthermore, without being limited, combining such properties with pH-dependent IL-8-binding antibodies can increase the rate of IL-8 elimination more efficiently.

Example 16 [0885] Mouse PK assay using non-FcRn-binding antibodies

The following method was used to confirm whether a complex between human IL-8 and a pH-dependent IL-8-binding antibody is formed and uptake of that complex into

237

WO 2017/046994 PCT/JP2016/003616 cells increases in mice.

[0886] First, an antibody variant comprising the variable region of H1009/L395 and an Fc region deficient in binding affinity to various Fc receptors was produced. Specifically, as modifications for deleting the binding ability towards human FcRn under acidic pH conditions, the heavy chain H1009-IgGl was subjected to substitution of alanine for isoleucine at position 253 and aspartic acid for serine at position 254, according to EU numbering. Furthermore, as modifications for deleting the binding to mouse FcyR(s), leucine at position 235 was substituted with arginine, glycine at position 236 was substituted with arginine, and serine at position 239 was substituted with lysine. H1009-F1942m (SEQ ID NO:93) was produced as a heavy chain containing four of these modifications. Furthermore, H1009/L395-F1942m comprising H1009-F1942m as the heavy chain and L395-k0MT as the light chain was produced.

[0887] Since antibody that has this Fc region is deficient in the FcRn binding affinity under acidic pH conditions, it is not transferred from the endosome into plasma. Therefore, such antibody is quickly eliminated from plasma in a living body as compared to antibody that comprises native Fc region. In this case, after the antibody that comprises native Fc region is taken up into cells, only a portion of them that is not salvaged by FcRn is degraded after being transferred to the lysosome, but in the case of antibody comprising Fc region that does not comprise FcRn-binding affinity, all of the antibody taken up into the cells are degraded in lysosomes. More specifically, in the case of antibody that comprises such modified Fc region, the rate of elimination of the administered antibody from plasma may be equivalent to the rate of incorporation into cells. That is, the rate of intracellular uptake of the antibody whose FcRn-binding affinity has been deleted can also be confirmed by measuring the rate of elimination of these antibodies from plasma.

[0888] Then, whether intracellular uptake of the complex formed between

H1009/L395-F 1942m and human IL-8 increases as compared to the uptake of H1009/L395-F 1942m alone was tested. Specifically, whether the rate of elimination of the antibody from plasma will change when the antibody is administered alone and when the antibody is administered upon formation of a complex with human IL-8 was tested.

[0889] The respective biokinetics of the anti-human IL-8 antibody was evaluated in cases when the anti-human IL-8 antibody was administered alone to human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse; Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)) and when human IL-8 and the anti-human IL-8 antibody were administered simultaneously to the human FcRn transgenic mice. The anti-human IL-8 antibody solution (200 pg/mL), and a mixed solution of human IL-8 (10 pg/mL) and the anti-human IL-8 antibody (200 pg/mL) were individually administered once at

238

WO 2017/046994

PCT/JP2016/003616 mL/kg to the tail vein. In this case, since the anti-human IL-8 antibody was present in sufficient excess over human IL-8, almost all of human IL-8 was considered to be bound to the antibody. Blood was collected five minutes, two hours, seven hours, one day, and two days after the administration. The collected blood was immediately centrifuged at 4°C and 15,000 rpm for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set to -20°C or below until measurements were taken.

[0890] The anti-human IL-8 antibody concentration in mouse plasma was measured by an electrochemiluminescence method. First, to the Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery) which had been blocked overnight at room temperature using a PBS-Tween solution containing 5% BSA (w/v), an Anti-Human Kappa Light Chain Goat IgG Biotin (IBL) was allowed to react at room temperature for one hour to produce an anti-human antibody-immobilized plate. Samples for calibration curve containing the anti-human IL-8 antibody at concentrations of 3.20, 1.60, 0.800, 0.400, 0.200, 0.100, and 0.0500 pg/mL in plasma and samples for mouse plasma measurement diluted 100-fold or higher were prepared. Each sample was mixed with human IL-8, and then dispensed at 50 pL per well into the anti-human antibodyimmobilized plate, and then stirred at room temperature for one hour. Human IL-8 was adjusted to a final concentration of 333 ng/mL.

[0891] Then, an anti-human IL-8 antibody (prepared in-house) comprising a mouse IgG constant region was added to the plate, and was allowed to react at room temperature for one hour. Furthermore, the Anti-Mouse IgG (BECKMAN COULTER) rutheniumlabeled with the SULFO-TAG NHS Ester (Meso Scale Discovery) was added to the plate, and this was allowed to react for one hour. Then, immediately after the Read Buffer T(xl) (Meso Scale Discovery) was dispensed into the plate, measurement was carried out using SECTOR Imager 2400 (Meso Scale Discovery). The anti-human IL-8 antibody concentration was calculated based on the response in the calibration curve using the analytical software, the SOFTmax PRO (Molecular Devices).

[0892] Antibody concentrations in mouse plasma obtained as a result are shown in Fig. 31, and the antibody clearance under the respective conditions are shown in Table 24.

[0893] [Table 24]

Antibody Name	IL8	CL
Mg/kg	mL/d/kg
H1009 1.395-F1942m		134
H1009/L395-F1942m	100	291

[0894] The rate of intracellular uptake of the complex of H1009/L395-F1942m and human IL-8 was shown to be increased by at least 2.2 fold compared to the uptake rate of H1009/L395-F 1942m. Here, it is noted as at least 2.2-fold because of the following reason which is included as one of the possibilities that the value may actually be

239

WO 2017/046994

PCT/JP2016/003616

5-fold, 10-fold, or 30-fold. As the rate of elimination of human IL-8 from mouse plasma is very rapid compared to the rate of elimination of H1009/L395-L 1942m, the proportion of H1009/L395-L 1942m bound by human IL-8 in plasma quickly decreases after administration. More specifically, even when administered simultaneously with human IL-8, not all H1009/L395-L 1942m present in the plasma are in the human IL8-bound form, and in fact, at approximately seven hours after administration, most of them already exist in the free form. Since the uptake rate is evaluated under such conditions, even if the rate of intracellular uptake of the complex of

H1009/L395-L 1942m and human IL-8 has been actually increased five-fold, ten-fold, or 30-fold in comparison to the uptake rate of H1009/L395-L1942m, the results in this experiment system are reflected only partially; therefore, the effect may possibly be presented as an increase of 2.2-fold or so. Accordingly, from these obtained results, whereas the intracellular uptake rate of the complex of H1009/L395 and IL-8 was shown to be increased compared to the actual intracellular uptake rate of H1009/L395 in vivo, this effect is not limited to the obtained value of 2.2-fold increase.

[0895] Without being particularly limited, the following interpretation may be made from the findings obtained so far. When H1009/L395, which is a pH-dependent IL8-binding antibody, forms a complex with human IL-8, that complex has a higher isoelectric point and is shifted more towards a positive charge than when the antibody alone exists. At the same time, the affinity of the complex towards the extracellular matrix is more increased than the affinity of the antibody alone. Properties such as elevation of isoelectric point and enhancement of the extracellular matrix binding can be considered as factors that promote uptake of an antibody into cells in vivo. Furthermore, from mouse experiments, the rate of intracellular uptake of the complex of H1009/L395 and human IL-8 was shown to be increased 2.2-fold or greater compared to the uptake rate of H1009/L395. From the above, the theoretical explanation as well as the in vitro properties and in vivo phenomena consistently support the hypothesis that H1009/L395 and human IL-8 form a complex to promote uptake of the complex into cells, and leads to a remarkable increase in the elimination of human IL-8.

[0896] Several antibodies against IL-8 have been reported to date, but there has been no report so far on the increase of binding affinity to the extracellular matrix upon complex formation with IL-8 and the increase in uptake of the complexes into cells.

[0897] Furthermore, based on the finding that an increase in the intracellular uptake of the anti-IL-8 antibodies is observed when the antibodies form complexes with IL-8, one may consider that the anti-IL-8 antibodies that have formed complexes with IL-8 in plasma are quickly taken up into cells, while the free antibodies which have not formed complexes with IL-8 tend to be retained in plasma without being taken up into cells. In this case, when the anti-IL-8 antibody is pH-dependent, the anti-IL-8 antibody which

240

WO 2017/046994

PCT/JP2016/003616 has been taken up into the cells releases the IL-8 molecule in the cells and then returns to the outside of the cells, and then it can bind to another IL-8 molecule; and therefore, increase in the intracellular uptake upon complex formation may have a further effect of eliminating IL-8 more strongly. That is, selecting anti-IL-8 antibodies with increased binding to the extracellular matrix or anti-IL-8 antibodies with increased uptake into cells may also be another embodiment of Disclosure C.

Example 17 [0898] Immunogenicity prediction of the pH-dependent IL-8-binding antibody H1009/L395 using an in silico system

Next, the immunogenicity score and frequency of ADA development were predicted for H1009/L395 by a method similar to that of Example 13-1. The results are shown in Table 25 and Fig. 32. In Fig. 32, H1009/L395 is noted as H1009L395.

[0899] [Table 25]

	Heavy Chain	Light Chain	Total
Antibody Name	EpiMatrix Score	tReg Adjusted Epx Score	EpiMatrix Score	tReg Adjusted Epx Score	EpiMatrix Score	tReg Adjusted Epx Score
hWS-4	62.44	12.18	22.64	-23.89	85.08	-11.71
H1004/L395	10.79	-39.47	7.79	-38.74	18.58	-78.21
H1009/L395	9.62	-40.64	7.79	-38.74	17.41	-79.38

[0900] The results in Table 25 show that H1009/L395 has the same level of low immunogenicity scores as H1004/L395. Furthermore, the frequency of ADA development predicted for H1009/L395 from the results in Fig. 32 was 0%, and this was also similar to that of H1004/L395.

[0901] Accordingly, the predicted immunogenicity was greatly decreased for H1009/L395 in comparison to the known anti-human IL-8 antibody hWS-4. Therefore, H1009/L395 is considered to have very low immunogenicity in humans, and to be able to stably maintain the anti-IL-8-neutralizing activity for a long period of time.

Example 18 [0902] Cynomolgus monkey PK assay using an H89/L118 variant with enhanced FcRnbinding ability under acidic pH conditions

As described in the Examples above, among the cases where the antibodies have native IgGl as their constant region, the pH-dependent IL-8-binding antibody H1009/L395 is an antibody that has superior properties. However, such antibodies can also be used as antibodies containing amino acid substitutions in the constant region, for example, those containing an Fc region with enhanced FcRn binding at acidic pH, as exemplified in Example 5. Therefore, H89/L118 was used to confirm that the Fc region with enhanced FcRn binding at acidic pH can also function in a pH-dependent IL-8-binding antibody.

241

WO 2017/046994 PCT/JP2016/003616 [0903] (18-1) Production of an H89/L118 Fc region-modified antibody with enhanced FcRn binding at acidic pH

Various modifications for enhancing FcRn binding as described in Example 5-1 were introduced into the Fc region of H89/L118. Specifically, the following variants were produced by introducing the modifications used in F1847m, F1848m, F1886m, F1889m, F1927m, and FI 168m into the Fc region of H89-IgGl: (a) H89/L118-IgGl comprising H89-IgGlm (SEQ ID NO:94) as the heavy chain and L118-K0MT as the light chain; (b) H89/L118-F1168m comprising H89-F1168m (SEQ ID NO:95) as the heavy chain and L118-K0MT as the light chain; (c) H89/L118-F1847m comprising H89-F1847m (SEQ ID NO:96) as the heavy chain and LI 18-K0MT as the light chain; (d) H89/L118-F 1848m comprising H89-F1848m (SEQ ID NO:97) as the heavy chain and L118-K0MT as the light chain; (e) H89/L118-F1886m comprising H89-F1886m (SEQ ID NO:98) as the heavy chain and LI 18-K0MT as the light chain; (f) H89/L118-F1889m comprising H89-F1889m (SEQ ID NO:99) as the heavy chain and L118-K0MT as the light chain; and (g) H89/L118-F1927m comprising H89-F1927m (SEQ ID NO: 100) as the heavy chain and L118-K0MT as the light chain. Cynomolgus monkey PK assays using these antibodies were carried out by the method shown below.

[0904] 118-21 Cynomolgus monkey PK assay of novel Fc region variant-containing antibodies

After administration of anti-human IL-8 antibodies to cynomolgus monkeys, biokinetics of the anti-human IL-8 antibodies was evaluated. An anti-human IL-8 antibody solution was intravenously administered once at 2 mg/kg. Blood was collected five minutes, four hours, one day, two days, three days, seven days, ten days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately centrifuged at 4°C and 15,000 rpm for ten minutes to obtain plasma. The separated plasma was stored in a freezer set to -60°C or below until measurements were taken.

[0905] The anti-human IL-8 antibody concentration in cynomolgus monkey plasma was measured by an electrochemiluminescence method. First, the Anti-hKappa Capture Ab (Antibody Solutions) was dispensed into a MULTI-ARRAY 96-well Plate (Meso Scale Discovery), and was stirred at room temperature for one hour. Then, a PBS-Tween solution containing 5% BSA (w/v) was used for blocking at room temperature for two hours to prepare an anti-human antibody-immobilized plate. Samples for calibration curve containing an anti-human IL-8 antibody at concentrations of 40.0, 13.3, 4.44, 1.48, 0.494, 0.165, and 0.0549 pg/mL in plasma and samples for cynomolgus monkey plasma measurement diluted 500-fold or more were prepared, 50 pL of the solutions were dispensed into each well of the anti-human antibody-immobilized plate, and the

242

WO 2017/046994

PCT/JP2016/003616 [0906] [0907] [0908] [0909] [0910] solutions were stirred at room temperature for one hour. Then, the Anti-hKappa Reporter Ab, Biotin conjugate (Antibody Solutions) was added to the aforementioned plate, and allowed to react at room temperature for one hour. After further adding the SULFO-TAG Labeled Streptavidin (Meso Scale Discovery) and allowing to react at room temperature for one hour, the Read Buffer T(xl) (Meso Scale Discovery) was dispensed into the plate, and measurements were taken immediately using SECTOR Imager 2400 (Meso Scale Discovery). The anti-human IL-8 antibody concentration was calculated based on the response in the calibration curve using the analytical software, the SOFTmax PRO (Molecular Devices).

The results obtained for the half-life (t 1/2) and clearance (CL) of each of the antibodies are shown in Table 26, and changes in the antibody concentration in cynomolgus monkey plasma are shown in Fig. 33.

[Table 26]

Antibody Name	tt/2 day	CL mL/d/kg
H89/L118-IgG1	11.9	2.95
H89/L118-F1168m	24.1	3.21
H89/L118-F1847m	27.9	2.09
H89/L118-F1848m	25.3	1.74
H89/L118-F1886m	45.1	1.34
H89/L118~F1889m	39.5	1.75
H89/L118-F1927m	30.3	2.13

The above results confirmed that all of the Fc region variants show prolonged retention in plasma in comparison to the antibody that has a native IgGl Fc region. In particular, H89/L118-F1886m showed the most desirable blood kinetics.

Example 19

Fc region with lowered binding ability towards FcyRs

The Fc region of a native human IgGl is known to bind to Fey receptor(s) (hereinafter, referred to as FcyR(s)) on various cells of the immune system, and exhibit effector functions such as ADCC and ADCP on target cells.

On the other hand, IL-8 is a soluble cytokine, and anti-IL-8 antibodies used as pharmaceuticals are mainly expected to show pharmacological actions by neutralizing the functions of IL-8 at sites where IL-8 is present in excess. Such sites where IL-8 is present in excess are not particularly limited, and for example, may be inflamed sites.

It is known that generally at such inflamed sites, various immune cells gather and are activated. Transmitting unintended activation signals to these cells via Fc receptors and inducing activities such as ADCC and ADCP in unintended cells are not always favorable. Therefore, without being particularly limited, from a safety point of view, it may be preferable that anti-IL-8 antibodies administered in vivo have low affinity

243

WO 2017/046994

PCT/JP2016/003616 towards FcyR(s).

[0911 ] (19-1) Production of modified antibodies with lowered binding towards FcvRs

Amino acid modifications were further introduced into the Fc region of

H1009/F395-F1886m with the objective of reducing the binding ability towards various human and cynomolgus monkey FcyRs. Specifically, H1009-F1886s (SEQ ID NO:81) was produced by subjecting the H1009-F1886m heavy chain to each of the following substitutions: R for L at position 235, R for G at position 236, and K for S at position 239, according to EU numbering. Similarly, H1009-F1974m (SEQ ID NO:80) was produced by subjecting H1009-F1886m to substitution of R for L at position 235 and R for G at position 236, according to EU numbering, and substituting the region from position 327 to position 331 according to EU numbering with that of the native human IgG4 sequence. H1009/L395-F1886s and H1009/L395-F 1974m were produced as antibodies having these heavy chains, and L395-k0MT as the light chain.

[0912] (19-2) Confirmation of the affinity towards various human FcyRs

Next, the affinities of the H1009/L395-F1886s or H1009/L395-F1974m towards the soluble forms of FcyRIa or FcyRIIIa in human or cynomolgus monkey were confirmed by the following method.

[0913] Assays were performed for the binding of the H1009/L395-F1886s or the

H1009/L395-F 1974m to the soluble forms of FcyRIa or FcyRIIIa in human or cynomolgus monkey using BIACORE T200 (GE Healthcare). Soluble FcyRIa and FcyRIIIa in both human and cynomolgus monkey were produced in the form of Histagged molecules by methods known to those of ordinary skill in the art. An appropriate amount of rProtein L (BioVision) was immobilized onto the Sensor chip CM4 (GE Healthcare) by the amine coupling method and antibody of interest was captured. Next, soluble FcyRIa or FcyRIIIa was injected with a running buffer (used as a reference solution), and was made to interact with the antibodies captured onto the sensor chip. HBS-EP+ (GE Healthcare) was used as the running buffer, and HBS-EP+ was also used to dilute the soluble FcyRIa or FcyRIIIa. To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements were carried out at 20°C.

[0914] The results are shown in Fig. 34. Here, the notations used for human FcyRIa, human FcyRIIIa, cynomolgus monkey FcyRIa, and cynomolgus monkey FcyRIIIa are in the same order: hFcyRIa, hFcyRIIIa, cynoFcyRIa, and cynoFcyRIIIa, respectively. H1009/L395-F1886m was shown to bind to all FcyRs, but on the other hand, the H1009/L395-F1886s and H1009/L395-F1974m were confirmed not to bind to any of the FcyRs.

[0915] 119-31 Mouse IL-8 elimination assay of Fc variants

Next, for the H1009/L395-F1886s and H1009/L395-F 1974m, the rate of human IL-8 elimination and the retention in plasma of the antibodies in mice were confirmed by

244

WO 2017/046994

PCT/JP2016/003616 the following experiment. Here, three doses of H1009/L395-F1886s, 2 mg/kg, 5 mg/ kg, and 10 mg/kg, were used for the evaluation so that the effects of increasing the antibody dosage can also be evaluated for H1009/L395-F1886s.

[0916] After simultaneous administration of human IL-8 and an anti-human IL-8 antibody to human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse; Jackson Laboratories; Methods Mol. Biol. 602:93-104 (2010)), the biokinetics of human IL-8 was evaluated. A mixed solution of human IL-8 (10 pg/mL) and an anti-human IL-8 antibody (200 pg/mL, 500 pg/mL, or 1000 pg/mL) was administered once at 10 mL/kg through the tail vein. In this case, since the anti-human IL-8 antibody was present in sufficient excess over human IL-8, almost all of human IL-8 was considered to be bound to the antibody. Blood was collected five minutes, two hours, four hours, seven hours, one day, two days, three days, seven days, 14 days, 21 days, and 28 days after the administration. The collected blood was immediately centrifuged at 4°C and 15,000 rpm for 15 minutes to obtain plasma. The separated plasma was stored in a freezer set to -20°C or below until measurements were taken.

[0917] The human IL-8 concentration in mouse plasma was measured by a method similar to that of Example 11. The resulting data on the human IL-8 concentration in plasma is shown in Fig. 35, and the values of human IL-8 clearance (CL) from mouse plasma are shown in Table 27.

[0918] First, H1009/L395 comprising the Fc region of a native IgGl and

H1009/L395-F1886s comprising the modified Fc region were shown to have equivalent human IL-8-eliminating effects when the 2 mg/kg-administered groups were compared.

[0919] Next, when the dosage of the H1009/L395-F1886s antibody was changed, significant difference in the human IL-8 clearance values was not observed between the 2 mg/kg and 10 mg/kg doses while there was a slight difference in the plasma IL-8 concentration one day after administration. This strongly suggests that antibodies comprising the variable region of H1009/L395 showed sufficient IL-8-eliminating effects even when the antibodies were administered at high doses.

[0920] [Table 27]

Antibody Name	Dose	Human IL-8 CL (mL/d/kg)
H1009/L395	2 mg/kg	740
H1009/L395-F1886S	2 mg/kg	628
H1009/L395-F1886s	5 mg/kg	458
H1009/L395-F1886s	10 mg/kg	560

[0921] (19-4) Cynomolgus monkey PK assay of Fc variants

Next, plasma retention of antibodies in cynomolgus monkeys was verified by the following method using H1009/L395-F1886s or H1009/L395-F 1974m.

245

WO 2017/046994 PCT/JP2016/003616 [0922] Biokinetics of an anti-human IL-8 antibody were evaluated in case that the antihuman IL-8 antibody was administered alone or in case that human IL-8 and the antihuman IL-8 antibody were simultaneously administered to cynomolgus monkeys. An anti-human IL-8 antibody solution (2 mg/mL) or a mixed solution of human IL-8 (100 pg/kg) and an anti-human IL-8 antibody (2 mg/kg) was intravenously administered once at 1 mL/kg. Blood was collected five minutes, four hours, one day, two days, three days, seven days, ten days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration. The collected blood was immediately centrifuged at 4°C and 15,000 rpm for ten minutes to obtain plasma. The separated plasma was stored in a freezer set to -60°C or below until measurements were taken.

[0923] The anti-human IL-8 antibody concentration in cynomolgus monkey plasma was measured by the method of Example 18. The resulting data on the anti-human IL-8 antibody concentration in plasma is shown in Fig. 36, and the values for the half-life (t i/₂) and clearance (CL) of the anti-human IL-8 antibody from cynomolgus monkey plasma are shown in Table 28.

[0924] First, in comparison to Hr9 and H89/L118 which have the Fc region of a native human IgGl, H1009/L395-F1886s which has an Fc region with improved functions was shown to have significantly prolonged plasma retention.

[0925] Furthermore, when H1009/L395-F1886s was administered simultaneously with human IL-8, the change in plasma concentration was equivalent to that when the antibody was administered alone. Without being particularly limited, the following discussion is possible from this finding. As described above, intracellular uptake of the complex of H1009/L395 and human IL-8 has been shown to be increased compared to the uptake of H1009/L395 alone. Generally, high-molecular-weight proteins are thought to be incorporated non-specifically or in a receptor-dependent manner into cells, then transferred to the lysosome and degraded by various degrading enzymes present in the lysosome. Therefore, if the rate of uptake of the protein into cells increases, the plasma retention of that protein is likely to worsen as well. However, in the case of an antibody, it has the property of being returned to the plasma by FcRn in the endosome; and therefore, as long as the salvaging by FcRn functions sufficiently, plasma retention may not be affected even if the rate of intracellular uptake is accelerated. Here, even when H1009/L395-F1886s was administered simultaneously with human IL-8 to cynomolgus monkeys, plasma retention was not affected. This indicates the possibility that while the rate of antibody uptake into cells is increased for H1009/L395-F1886s, the antibody is sufficiently salvaged by FcRn such that it can return to the plasma.

[0926] Furthermore, another Fc variant H1009/L395-F 1974m also showed equivalent plasma retention to that of H1009/L395-F1886s. While these Fc variants have been in246

WO 2017/046994

PCT/JP2016/003616 traduced with different modifications that decrease the binding ability to various FcyRs as describe above, they have been shown not to affect the plasma retention of the antibodies themselves. From the above, plasma retention of both H1009/L395-F1886s and H1009/L395-F 1974m in cynomolgus monkeys was shown to be remarkably prolonged and extremely satisfactory in comparison to that of antibodies that have the native IgGl Fc region.

[0927] [Table 28]

	tl/2	Gt
	day	mL/d/kg
HrS	20.26	3. 72
H89/L118	11.88	2.95
H1OO9/L395-F1886S	35. 75	1.64
H1QQ9/L395—FI 886 s +hIL-8	72.24	1.11
H10G9/L395-F1974m +hIL-8	43. 78	1.60

[0928] As demonstrated in the above-mentioned Examples, by comprising pH-dependent IL-8 binding ability with a feature of being quickly taken up into cells as a complex with IL-8, H1009/L395 achieved for the first time as an antibody that increases significantly the rate of human IL-8 elimination in vivo. Furthermore, the IL-8-binding affinity of this antibody under neutral pH conditions is also increased compared to the known hWS-4 antibody, and the antibody can neutralize human IL-8 more strongly under neutral pH conditions such as in plasma. In addition, it is an antibody that has excellent stability under plasma conditions, and whose IL-8 neutralizing activity does not decrease after it is administered in vivo. Furthermore, H1009/L395, constructed based on Hr9 which has a greatly improved production level as compared to the hWS4, is an antibody suitable for manufacturing from the viewpoint of production level. Moreover, in in silico immunogenicity prediction, the antibody showed a very low score for its immunogenicity, and this score was significantly lower in comparison to those of the known hWS-4 antibody and several other known commercially available antibodies. That is, it is expected that H1009/L395 would hardly generate ADA in humans, and would be able to be used safely for a long period of time. Accordingly, in comparison to known anti-human IL-8 antibodies, H1009/L395 shows improvement in various aspects, and is very useful as a pharmaceutical.

[0929] H1009/L395 which has the native IgG Fc region is sufficiently useful as described above; however, variants of H1009/L395 comprising the functionally-improved Fc region can also be used appropriately as antibodies with enhanced utility. Specifically, it is possible to increase the FcRn binding under acidic pH conditions to prolong plasma retention and to maintain effects for a longer period of time. Furthermore, variants comprising the Fc region introduced with modification(s) that decrease the binding ability to FcyR(s) can be used as high-safety therapeutic antibodies to avoid

247

WO 2017/046994

PCT/JP2016/003616 unintended activation of immune cells and generation of cytotoxic activity in the administered organism. As such Fc variants, the use of F1886s or F1974m exploited herein is particularly favorable, but it is not limited to these Fc variants; and as long as the Fc variant has similar functions, therapeutic antibodies comprising other modified Fc regions are used as an embodiment of Disclosure C.

[0930] As a result, the antibodies of Disclosure C including H1009/L395-F1886s and H1009/L395-F 1974m generated by the inventors through dedicated research can maintain a condition where the biological activity of human IL-8 is strongly inhibited both safely and for a long period of time. Here, levels that could not be achieved by known anti-IL-8 antibodies have been realized, and these antibodies of Disclosure C are expected to be used as high-quality finished anti-IL-8 antibody pharmaceuticals. Example 20 [0931] Anti-factor IXa/factor X bispecific antibodies

The humanized anti-factor IXa/factor X bispecific antibodies disclosed in

WO2012/067176 bind to human factor IXa and factor X and induce co-aggregation activity of blood. A humanized anti-factor IXa/factor X bispecific antibody F8M(Q499-zl21/J327-zll9/L404-k: H chain (SEQ ID NO:330)/H chain (SEQ ID NO:331)/common L chain (SEQ ID NO:332)) described in WO2012/067176 was utilized in this example and F8M comprises two different H chains and two same common L chains. F8M was produced by the method described in Examples of WO2012/067176.

[0932] (20-1) Production of anti-factor IXa/factor X bispecific antibodies

The following three antibodies were produced by the method of Reference Example as anti-factor IXa/factor X bispecific antibodies based on F8M: (a) F8M-F1847mv, which is a conventional antibody comprising F8M-F1847mvl (SEQ ID NO:323) and F8M-F1847mv2 (SEQ ID NO:324) as the heavy chains and F8ML (SEQ ID NO:325) as the light chain; (b) F8M-F1868mv, which is a conventional antibody comprising F8M-F1868mvl (SEQ ID NO:326) and F8M-F1868mv2 (SEQ ID NO:327) as the heavy chains and F8ML (SEQ ID NO:325) as the light chain; and (c) F8M-F1927mv, which is a conventional antibody comprising F8M-F1927mvl (SEQ ID NO:328) and F8M-F1927mv2 (SEQ ID NO:329) as the heavy chains and F8ML (SEQ ID NO:325) as the light chain.

[0933] The heavy chain sequences include the same Fc variant sequences regarding the enhancement of FcRn binding and the reduction of the rheumatoid factor binding mentioned in Example 5 as follows:

[0934]

248

WO 2017/046994

PCT/JP2016/003616 [Table 29]

Sequence Name	Name in Example 5
F8M-F1847mvl (SEQ ID NO:323)	F1847m
F8M-F1847mv2 (SEQ ID Ν(Τ324)	F1847m
F8M-F1868mvl (SEQ ID NCK326)	F1868m
F8M-F1868mv2 (SEQ ID NO:327)	F1868m
F8M-Fl927mvl (SEQ ID NO:328)	F1927m
F8M-F1927mv2 (SEQ ID NCK329)	F1927m

[0935] (20-2) Pharmacokinetic study of monoclonal antibodies. F8M-F1847mv.

F8M-F1868mv. and F8M-F1927mv. in cvnomolgus monkey

Pharmacokinetics of monoclonal antibodies, F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv, after single bolus intravenous administration at the dose of 0.6 mg/kg to male cynomolgus monkey were each evaluated. The plasma concentrations of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were determined by a sandwich ELISA. The pharmacokinetic parameters were calculated using WinNonlin ver 6.4 software. As shown in Table 30, the half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were 29.3 day, 54.5 day, and 35.0 day, respectively. The PK study of F8M using cynomolgus monkey was conducted in a different day at the dose of 6 mg/ kg, and the half-life was revealed to be 19.4 day. It was clarified that the half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were longer than F8M. This suggests that the half-life of an anti-factor IXa/X bispecific antibody could be prolonged by the same modification on the Fc region sequence with that mentioned in Example 5 above.

[0936] [Table 30]

Half-lives of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv and F8M after intravenous administration to male cynomolgus monkey

	F8M-F1847mv	F8M-F1868mv	F8M-F1927mv	F8M
Half-life (day)	29.3	54.5	35.0	19.4

Example 21 [0937] Evaluation of clearance of IgE from plasma using pi-increased Fab variants

To enhance the clearance of human IgE, pi increased substitutions in the Fab portion of antibodies were evaluated in this example using pH-dependent antigen-binding antibodies. The method of adding amino acid substitutions to the antibody variable region to increase pi is not particularly limited, but for example, it can be performed by the method described in W02007/114319 or W02009/041643. Amino acid substitutions introduced into the variable region are preferably those that decrease the number of negatively charged amino acids (such as aspartic acid and glutamic acid) while increasing the positively charged amino acids (such as arginine and lysine). Fur249

WO 2017/046994

PCT/JP2016/003616 thermore, amino acid substitutions may be introduced at any position in the antibody variable region. Without particular limitation, the sites for introducing amino acid substitutions are preferably positions where amino acid side chains may be exposed on the antibody molecule surface.

[0938] (21-1) Production of antibodies with increased pi bv modification of amino acids in the variable region

The tested antibodies are summarized in Table 32 and Table 33.

[0939] The heavy chain, AblH003 (also called H003, SEQ ID NO: 144) was prepared by introducing pi-increasing substitution H32R into AblH (SEQ ID NO:38). Other heavy chain variants were also prepared by introducing respective substitutions represented in Table 32 into AblH according to the method shown in Reference Example 1. All the heavy chain variants were expressed with AblL (SEQ ID NO:39) as light chain. The pH-dependent binding profile of this antibody is summarized in Table 5 (Abl).

[0940] Similarly, we also evaluated the pi-increasing substitution in light chain.

[0941] The light chain, AblLOOlT (also called L001, SEQ ID NO: 164) was prepared by introducing pi-increasing substitution G16K into AblL. Other light chain variants were also prepared by introducing respective substitutions represented in Table 33 into AblL according to the method shown in Reference Example 1. All the light chain variants were expressed with AblH as heavy chain.

[0942]

250

WO 2017/046994

PCT/JP2016/003616 [Table 32]

Heavy Chain Variants of AblH evaluated in this Example

			Imaging	BIACORE
Sample Name (H Chain 1 L Chain)	Variant		fold	fold
AblH/AblL	original Abl		1.00	1.00
AblH003/AblL	H003	H32R	no data	no data
AblH005m/AblL	H005	P41R/G44R	1.73	0.92
AblHOlO/AblL	H010	T77R	1.68	1.05
AblH012/AblL	H012	D82aN/S82bR	2.71	0.96
AblH013/AblL	H013	D82aG/S82bR	2.95	1.02
Ab1H014/AblL	H014	D82aS/S82bR	2.36	0.91
AblH016/AblL	HO 16	E85G	1.51	1.04
AblH018/AblL	HO 18	A93K	0.00	-0.01
AblH026m/AblL	H026	P41R/G44R/T77R	0.76	no data
AblH027/AblL	H027	T77R/D82aN/S82bR	2.78	1.21
AblH028/AblL	H028	T77R/D82aG/S82bR	3.04	1.37
AblH029/AblL	H029	T77R/D82aS/S82bR	1.80	1.33
AblH030/AblL	H030	T77R/E85G	1.50	1.27
AblH031m/AblL	H031	T77R/A93K	0.03	0.06
AblH032/AblL	H032	D82aG/S82bR/E85G	0.52	no data
AblH034/AblL	H034	Q13K	1.12	1.21
AblH035/AblL	H035	G15R	0.06	1.28
AblH039/AblL	11039	S64K	0.77	1.57
AblH041m/AblL	H041	Q105R	1.03	1.52
AblH045/AblL	H045	S82bR	2.00	0.76

[0943]

251

WO 2017/046994

PCT/JP2016/003616 [Table 33]

Light Chain Variants of AblL evaluated in this Example

			Imaging	BIACORE
Sample Name (H Chain / L Chain)	Variant		fold	fold
AblH/AblL	original Ab 1		1.00	1.00
AblH/AblLOOl	L001	G16K	2.11	1.03
AblH/AblL002	L002	Q24R/E27Q	1.43	1.03
AblH/AblL003	L003	Q24R/E27R	3.14	1.09
AblH/AblL004	L004	Q24K/E27K	1.42	1.01
AblH/AblL005	L005	A25K7S26K	1.82	0.84
AblH/AblL006	L006	A25R/S26R	6.82	1.18
AblH/AblL007	L007	Q37R	1.82	1.06
AblH/AblL008	L008	G41R/Q42K	1.70	1.07
AblH/AblL009	L009	L46R/Y49K	0.02	-0.02
AblH/AblLOlO	L010	S52R/S56R	2.72	0.98
AblH/AblLOll	L011	S52K/S56K	1.21	1.02
AblH/AblL012	L012	S65R/T69R	1.28	0.97
AblH/AblL013	L013	T74K/S77R	3.31	1.70
AblH/AblL014	L014	S76R/Q79K	4.47	1.08
AblH/AblL015	L015	G16K/Q24R/E27R	2.11	1.25
AblH/AblL016	L016	Q24R/E27R/Q37R	3.33	1.36
AblH/AblL017	L017	Q24R/E27R/G41R/Q42K	2.90	1.27
AblH/AblL018	L018	Q24R/E27R/L46R/Y49K	0.01	0.07
AblH/AblL019	L019	Q24R/E27R/S52R/S56R	3.88	1.17
Ab1H/AblL020	L020	Q24R/E27R/S52K/S56K	4.61	1.22
AblH/ Ab1L021	L021	Q24R/E27R/S65R/T69R	11.43	1.36
AblH/AblL022	L022	Q24R/E27R/T74K/S77R	19.05	1.45
AblLl/AblL023	L023	Q24R/E27R/S76R/Q79K.	13.15	1.39
AblH/AblL024	L024	G16K/A25R/S26R	0.73	No data
AblH/AblL025	L025	A25R/S26R/Q37R	2.03	1.39
AblH/AblL026	L026	A25R7S26R/G41R/Q42K	1.28	No data
AblH/AblL028	L028	A25R/S26R/S52R/S56R	6.33	1.46
AblH/AblL029	L029	A25R/S26R/S52K/S56K	9.84	1.23
AblH/AblL030	L030	A25R/S26R/S65R/T69R	7.19	1.16
AblH/AblL032	L032	A25R/S26R/S76R/Q79K	2.67	No data
AblH/AblL033	L033	Q24R/E27R/G41R/Q42K/S65R/T69R	6.68	1.26
Ab1H/AblL034	L034	Q24R/E27R/S52R/S56R/S65R/T69R	9.81	1.71
Ab1H/AblL035	L035	Q24R/E27R/S65R/T69R/T74K/S77R	19.56	1.49
AblH/Ab1L036	L036	Q24R/E27R/S65R/T69R/S76R/Q79K	17.04	1.48
AblH/AblL037	L037	Q24R/E27R/G41R/Q42K/T74K/S77R	8.62	1.38
AblH/AblL038	L038	Q24R7E27R/S52R/S56R/T74K/S77R	15.13	1.47
AblH/AblL039	L039	Q24R/E27R/T74K/S76R/S77R/Q79K	26.95	0.99
AblH/Ab1L040	L040	Q24R/E27R/G41R/Q42K/S76R/Q79K	5.29	1.23
AblH/AblL041	L041	Q24R/E27R/S52R/S56R/S76R/Q79K	11.86	1.35
AblH/AblL061	L061	Q42K/S76R	4.86	1.02
AblH/AblL062	L062	S65R/Q79K	2.93	0.98

[0944] (21-2) Human FcyRIIb-binding assay by BIACORE using pi-increased variants

Regarding the produced Fc region variant-containing antibodies, binding assays between soluble human FcyRIIb and antigen-antibody complexes were performed using BIACORE T200 (GE Healthcare). Soluble human FcyRIIb (NCBI accession

252

WO 2017/046994

PCT/JP2016/003616

NM_004001.3) was produced in the form of a His-tagged molecule by a method known in the art. An appropriate amount of an anti-His antibody was fixed onto Sensor chip CM5 (GE Healthcare) by the amine coupling method using a His capture kit (GE Healthcare) to capture human FcyRIIb. Next, an antibody-antigen complex and a running buffer (as a reference solution) were injected, and interaction was allowed to take place with the human FcyRIIb captured onto the sensor chip. 20 mM N(2-Acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl₂, and 0.05% (w/v) Tween 20 at pH 7.4 was used as the running buffer, and the respective buffer was also used to dilute the soluble human FcyRIIb. To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements were carried out at 25°C. Analyses were performed based on binding (RU) calculated from sensorgrams obtained by the measurements, and relative values when the binding amount of AblH/AblL (original Abl) was defined as 1.00 are shown. To calculate the parameters, the BIACORE T100 Evaluation Software (GE Healthcare) was used.

[0945] The SPR analysis results are summarized in Tables 32 and 33. A few variants were shown to have enhanced binding toward human FcyRIIb fixed on the BIACORE sensor chip.

[0946] The antibodies produced by introducing the pi-increasing modification(s) into the variable region are antibodies in which the charge of the variable region is more positively charged when compared with those before introduction of the modification(s). Therefore, the Coulombic interaction between the variable region (positive charge) and the sensor chip surface (negative charge) can be considered to have been strengthened by the pi-increasing amino acid modifications. Furthermore, such effects are expected to take place similarly on the same negatively charged cell membrane surface; therefore, they are also expected to show an effect of accelerating the speed of uptake into cells in vivo.

[0947] Here, about 1.2 fold or more of the binding to hFcyRIIb of the variants compared to the binding to hFcyRIIb of original Abl was considered to have strong charge effect on binding of an antibody to hFcyRIIb on the sensor chip.

[0948] Among the pi-increased heavy chain variants, the antibody with Q13K, G15R, S64K, T77R, D82aN, D82aG, D82aS, S82bR, E85G or Q105R substitution(s) (according to Kabat numbering), alone or in combination, showed higher binding to hFcyRIIb. The single amino acid substitution or combination of these substitutions in heavy chain is supposed to have strong charge effect on binding to hFcyRIIb on the sensor chip. Thus, one or more of positions that are expected to show an effect of accelerating the speed or rate of uptake into cells in vivo by introducing the pi-increasing modification into the heavy chain variable region of an antibody can include, for example, positions 13, 15, 64, 77, 82a, 82b, 85 and 105 according to Kabat numbering. An amino acid sub253

WO 2017/046994

PCT/JP2016/003616 stitution introduced at such position(s) can be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

[0949] In pi-increased light chain variants, the antibody with G16K, Q24R, A25R, S26R, E27R, Q37R, G41R, Q42K, S52K, S52R, S56K, S56R, S65R, T69R, T74K, S76R, S77R, Q79K substitution(s) (according to Kabat numbering), alone or in combination, shows higher binding to human FcYRIIb. The single amino acid substitution or combination of these substitutions in light chain is supposed to have strong charge effect on binding to human FcyRIIb on the sensor chip. Thus, one or more of positions that are expected to show an effect of accelerating the speed or rate of uptake into cells in vivo by introducing the pi-increasing modification into the light chain variable region of an antibody can include, for example, positions 16, 24, 25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77, and 79 according to Kabat numbering. An amino acid substitution introduced at such position(s) can be arginine or lysine.

[0950] (21-3) Cellular uptake of pi-increased Fab region variant-containing antibodies

To evaluate the rate of intracellular uptake into an hFcYRIIb-expressing cell line using the produced Fab region variant-containing antibodies, the assay similar to (4-5) above was performed, provided that the amount of antigen taken up was presented as relative values to the AblH/AblL (original Abl) value which is taken as 1.00.

[0951] The quantification results of cellular uptake were summarized in Tables 32 and 33. Strong fluorescence derived from the antigen in the cells was observed in several Fc variants. Here, about 1.5 fold or more of the fluorescence intensity of the antigen taken up into the cells of the variants compared to the fluorescence intensity of original Abl was considered to have strong charge effect on an antigen taken up into the cells.

[0952] Among the pi-increased heavy chain variants, the antibody with P41R, G44R, T77R, D82aN, D82aG, D82aS, S82bR or E85G substitution(s) (according to Kabat numbering), alone or in combination, showed stronger antigen uptake into the cells.

The single amino acid substitution or combination of these substitutions in heavy chain is supposed to have strong charge effect on antigen antibody complex uptake into the cells. Thus, one or more of positions that are expected to cause uptake of an antigenantibody complex into cells more quickly or more frequently by introducing the plincreasing modification into the heavy chain variable region of an antibody can include, for example, positions 41, 44, 77, 82a, 82b or 85, according to Kabat numbering. An amino acid substitution introduced at such position(s) can be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

[0953] In pi-increased light chain variants, the antibody with G16K, Q24R, A25R, A25K, S26R, S26K, E27R, E27Q, E27K, Q37R, G41R, Q42K, S52K, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitution(s) (according to Kabat numbering), alone or in combination, showed stronger antigen uptake into the cells. The single

254

WO 2017/046994

PCT/JP2016/003616 amino acid substitution or a combination of these substitutions in light chain is supposed to have strong charge effect on antigen antibody complex uptake into the cells. The variants with four or more amino acid substitutions tended to show stronger charge effect than those variants with lesser amino acid substitutions. One or more of positions that are expected to cause uptake of an antigen-antibody complex into cells more quickly or more frequently by introducing the pi-increasing modification into the light chain variable region of an antibody can include, for example, positions 16, 24,

25, 26, 27, 37, 41, 42, 52, 56, 65, 69, 74, 76, 77 or 79, according to Kabat numbering. An amino acid substitution introduced at such position(s) can be glutamine, arginine or lysine, and preferably arginine or lysine.

[0954] While not being restricted to a particular theory, this result can be explained as follows: the antigen and antibodies added to the cell culture solution form antigenantibody complexes in the culture solution. The antigen-antibody complexes bind to human FcyRIIb expressed on the cell membrane via the antibody Fc region, and are taken up into the cells in a receptor-dependent manner. Antibodies used in this experiment binds to antigen in a pH-dependent manner; therefore, the antibody can dissociate from the antigen in the endosomes (acidic pH conditions) inside the cells. Since the dissociated antigen is transported to lysosome and accumulate, it fluoresces inside the cells. Thus, a strong fluorescence intensity inside the cell is thought to indicate that the uptake of the antigen-antibody complexes into the cells is taking place more quickly or more frequently.

[0955] (21-4) Evaluation of clearance of human IgE in mouse co-injection model

Some anti-IgE antibodies with pH-dependent antigen-binding (original Abl,

AblH/AblL013, AblH/AblL014, AblH/AblL007) were tested in mice co-injection model to evaluate their ability to accelerate the clearance of IgE from plasma. In coinjection model, C57BL6J mice (Jackson Laboratories) were administered by single i.v. injection with IgE pre-mixed with the anti-IgE antibody, respectively. All groups received 0.2 mg/kg IgE with 1.0 mg/kg of anti-IgE antibodies. Total IgE plasma concentration was determined by anti-IgE ELISA. First, anti-human IgE (clone 107, MABTECH) was dispensed into a microWell plate (Nalge nunc International), and left for two hours at room temperature or overnight at 4°C to prepare an anti-human IgE antibody-immobilized plate. Samples for standard curve and samples were mixed with excess amount of the anti-IgE antibody (prepared in house) to form a uniform structure of immune complex. These samples were added into the anti-human IgE antibodyimmobilized plate, and left for overnight at 4°C. Then, these samples were reacted with human GPC3 core protein (prepared in house), biotinylated anti-GPC3 antibody (prepared in house), Streptavidin Poly HRP80 Conjugate (Stereospecific Detection Technologies) for one hour in order. After that, SuperSignal(registered trademark)

255

WO 2017/046994

PCT/JP2016/003616

ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific) were added. Chemical luminescence was read with SpectraMax M2 (Molecular Devices). The concentration of human IgE was calculated using SOFTmax PRO (Molecular Devices). Fig. 37 describes the IgE plasma concentration time profile in C57BL6J mice.

[0956] After administration of pi-increased Fab variants with pH-dependent antigenbinding, plasma total IgE concentration was lower than that of original Abl. These results indicate that the antigen-antibody immune complex of high pi variants with pH dependent antigen-binding could bind more strongly to plasma membrane receptor such as FcyRs, which increase the cellular uptake of antigen-antibody immune complex. The antigen uptaken into the cells could release from antibody inside the endosome effectively, resulted in accelerated elimination of IgE. IgE concentration of mice treated with AblH/AblL007, which showed weak efficacy in vitro study, was higher than that of other pi-increase Fab variant-containing antibodies. These results also suggest that for speculating an evaluation of clearance of an antigen from plasma in vivo, the sensitivity of the in vitro system using the fluorescence intensity by InCell Analyzer 6000 described above may be higher than that of the in vitro BIACORE system described above.

Example 22 [0957] Evaluation of clearance of C5 from plasma using pi-increased Fab variants

To enhance the clearance of human IgE, pi-increased substitutions in the Fab portion of an antibody were evaluated in this Example using pH-dependent antigen-binding antibodies.

[0958] (22-1) Preparation of C5 [Expression and purification of recombinant human C51

Recombinant human C5 (NCBI GenBank accession number: NP_001726.2, SEQ ID

NO:207) was expressed transiently using FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Conditioned media expressing human C5 was diluted with equal volume of milliQ water, then applied to a Q-sepharose FF or Q-sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden), followed by elution with NaCl gradient. Fractions containing human C5 were pooled, then salt concentration and pH was adjusted to 80mM NaCl and pH6.4, respectively. The resulting sample was applied to a SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden) and eluted with a NaCl gradient. Fractions containing human C5 were pooled and subjected to CHT ceramic Hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA, USA). Human C5 eluate was then applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing human C5 was pooled and stored at -150°C. Either in-house prepared recombinant human C5 or plasma derived human C5 (CALBIOCHEM, Cat#204888) was used for the study.

256

WO 2017/046994 PCT/JP2016/003616 [0959] Expression and purification of recombinant cynomolgus monkey C5 (NCBI

GenBank accession number: XP_005580972, SEQ ID NO:208) was done exactly the same way as the human counterpart.

[0960] (22-2) Preparation of synthetic calcium library

A gene library of antibody heavy chain variable regions which were used as synthetic human heavy chain libraries consist of 10 heavy chain libraries. Germ-line frameworks VH1-2, VH1-69, VH3-23, VH3-66, VH3-72, VH4-59, VH4-61, VH4-b, VH5-51, and VH6-1 were selected for this library based on germ-line frequency in human B-cell repertoires, and biophysical properties of V-gene families. The synthetic human heavy chain library was diversified at the antibody-binding site mimicking human B cell antibody repertoires.

[0961] A gene library of antibody light chain variable regions were designed to have calcium binding motif and were diversified at the positions which would contribute to antigen recognition, referring to human B cell antibody repertoires. The design of a gene library of antibody light chain variable regions which exert characteristics for calcium-dependent binding to antigens is described in WO 2012/073992.

[0962] The combination of a heavy chain variable region library and a light chain variable region library is inserted in a phagemid vector, and a phage library was constructed, referring to (de Heard et al., Meth. Mol. Biol. 178:87-100 (2002)). A trypsin-cleavage site was introduced into the phagemid vector at a linker region between Fab and pill protein. Modified M13KO7 helper phage which has a trypsin-cleavage site between N2 and CT domains at genelll was used for Fab displayed phage preparation.

[0963] (22-3) Isolation of calcium dependent anti-C5 antibodies

The phage display library was diluted with TBS supplemented with BSA and CaCl₂ at the final concentration of 4% and 1.2 mM, respectively. As a panning method, conventional magnetic beads selection was applied referring to general protocols (Junutula et al., J. Immunol. Methods 332(1-2):41-52 (2008), D'Mello et al., J. Immunol. Methods 247 (1-2):191-203 (2001), Yeung et al., Biotechnol. Prog. 18(2):212-220 (2002), Jensen et al., Mol. Cell Proteomics 2(2):61-69 (2003). As magnetic beads, NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin) were applied. Human C5 (CALBIOCHEM, Cat#204888) was labelled with EZ-Link NHS-PEG4-Biotin (PIERCE, Cat No. 21329).

[0964] The initial round of phage selection, the phage display library was incubated with biotinylated human C5 (312.5 nM) for 60 minutes at room temperature. Phages that displayed binding Fab variants were then captured using magnetic beads.

[0965] After incubation with beads for 15 minutes at room temperature, the beads were washed three times with 1 mL of TBS containing 1.2 mM CaCl₂ and 0.1% Tween20,

257

WO 2017/046994

PCT/JP2016/003616 and the beads were washed twice with 1 mL of TBS containing 1.2 mM CaCl₂. Phages were eluted by re-suspending the beads with TBS containing 1 mg/mL trypsin for 15 minutes. The eluted phages were infected with ER2738 and rescued by the helper phage. The rescued phages were precipitated with polyethylene glycol, re-suspended with TBS supplemented with BSA and CaCl₂ at the final concentration of 4% and 1.2 mM, respectively and used in the next round of panning.

[0966] After 1st round of panning, the phages were selected for its calcium dependency, in which the antibody binds to C5 stronger in the presence of calcium ion. In the second and third round, the panning was performed in the same manner as the first round except by using 50 nM (second round) or 12.5 nM (third round) of biotinylated antigen and finally eluted with 0.1 mL of elution buffer (50mM MES, 2mM EDTA, 150mM NaCl, pH5.5) and contacted with 1 pL of 100 mg/mL trypsin to select for its calcium dependency. After selection, selected phage clones were converted to IgG format.

[0967] Binding ability of converted IgG antibodies against human C5 were assessed under two different conditions: association and dissociation at 1.2 mM CaCl₂-pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl₂) and association at 1.2 mM CaCl₂-pH 7.4 (20 mM MES, 150 mM NaCl, 1.2 mM CaCl₂) and dissociation at 3pM CaCl₂-pH 5.8 (20 mM MES, 150 mM NaCl, 3 pM CaCl₂), at 30°C using Octet RED384 system (Pall Life Sciences). 25 clones of pH-Calcium dependent antigen binding clones were isolated. The sensorgrams of these antibodies are shown in Lig. 38.

[0968] (22-4) Identification of anti-C5 bispecific antibody

Lrom the clones isolated in Example B-3, nine pH or calcium dependent anti-C5 antibody clones were selected for further analysis (CLP0008, 0011, 0015, 0016, 0017, 0018, 0019, 0020, 0021). Some amino acid substitutions were introduced to the CFP0016 heavy chain variable region by a method generally known to those of ordinary skill in the art to improve properties of the antibodies like physicochemical properties. This CFP0016 variant, CFP0016H019, was used for further analysis instead of CFP0016. The amino acid sequences of VH and VL regions of these nine antibodies are described in Table 34. In this table, names described in brackets represent the abbreviated names.

[0969]

258

WO 2017/046994

PCT/JP2016/003616 [Table 34]

Clone Name and Amino Acid Sequence of Selected Antibodies

Clone Name	VH Name	VH SEQ ID	VL Name	VL SEQ ID
CFP0008 (08)	CFP0008H (08H)	NO: 209	CFP0008L (08L)	NO: 210
CFP0011 (11)	CFP0011H (11H)	NO: 211	CFP0011L (11L)	NO: 212
CFP0015 (15)	CFP0015H (15H)	NO: 213	CFP0015L (15L)	NO: 214
CFP0016H019 (16H019)	CFP0016H019 (16H019)	NO: 215	CFP0016L (16L)	NO: 216
CFP0017 (17)	CFP0017H (17H)	NO: 217	CFP0017L (17L)	NO: 218
CFPOO18 08)	CFP0018H (18H)	NO: 219	CFP0018L (18L)	NO: 220
CFP0019(19)	CFP0019H (19H)	NO: 221	CFP0019L (19L)	NO: 222
CFP0020 (20)	CFP0020H (20H)	NO: 223	CFP0020L (20L)	NO: 224
CFP0021 (21)	CFP0021H(21H)	NO: 225	CFP0021L (21L)	NO: 226

[0970] The full-length genes having nucleotide sequences encoding antibody heavy chain and light chain were synthesized and prepared by a method generally known to those of ordinary skill in the art. Heavy chain and light chain expression vectors were prepared by inserting the obtained plasmid fragments into vectors for expression in mammalian cells. The obtained expression vectors were sequenced by a method generally known to those of ordinary skill in the art. For expression of antibodies, the prepared plasmids were transiently transfected to FreeStyle293-F cell line (Thermo Fisher Scientific). Purification from the conditioned media expressing antibodies was conducted by a method generally known to those of ordinary skill in the art using rProtein A Sepharose Fast Flow (GE Healthcare).

[0971 ] 122-51 Generation and characterization of pH dependent anti-C5 bispecific antibody

Bispecific antibodies, which recognize two different epitopes of C5, were generated by combination of CFP0020 and CFP0018. Bispecific antibody was prepared as IgG format having two different clones of Fab in each binding site of the antibody and was prepared using a method generally known to those of ordinary skill in the art. In this bispecific IgG antibody, two heavy chains comprise distinct heavy chain constant regions (GldPl, SEQ ID NO:227 and GldNl, SEQ ID NO:228) from each other so as to efficiently form a heterodimer of the two heavy chains. The anti-C5 bispecific antibody comprising the binding sites of anti-C5 MAb X and anti-C5 MAb Y is represented as X//Y.

[0972] By introducing some amino acid substitutions into heavy chain and light chain CDR by a method generally known to those of ordinary skill in the art, we obtained light chain communization variant of 20//18, which we named 'optimized 20//18' (consisted by two heavy chains: CFP0020H0261-GldPl, SEQ ID NO:229 and

CFP0018H0012-GldNl, SEQ ID NO:230 and common light chain: CFP0020L233-k0, SEQ ID NO:231).

[0973] The kinetics parameters of optimized 20//18 against recombinant human C5 were

259

WO 2017/046994

PCT/JP2016/003616 assessed under two different conditions (e.g. (A) association and dissociation at pH 7.4 and (B) association at pH 7.4 and dissociation at pH 5.8), at 37°C using BIACORE T200 instrument (GE Healthcare). Protein A/G (Pierce, Cat No. #21186) or anti-human IgG (Fc) antibody (within Human Antibody Capture Kit; GE Healthcare, Cat No. BR1008-39) was immobilized onto a Series S CM4 (GE Healthcare, Cat No. BR-1005-34) by amine coupling method. Anti-C5 antibodies were captured on an immobilized molecule, and then human C5 was injected. The running buffers used were ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl₂, 0.05% Tween 20). Kinetics parameters at both pH conditions were determined by fitting the sensorgrams with 1:1 binding -RI (without bulk effect adjustment) model using BIACORE T200 Evaluation software, version 2.0 (GE Healthcare). Kinetic parameters, association rate (ka), dissociation rate (kd), and binding affinity (KD) at pH 7.4, and dissociation rate (kd) determined by only calculating the dissociation phase at each pH conditions, are described in Table 35. Optimized 20//18 showed faster dissociation at pH 5.8 against human C5 compared with dissociation rate at pH 7.4.

[0974] [Table 35]

Kinetic Parameters of 20//18 Variants against Human C5 under two Different Conditions

	pH 7.4	pH 7.4	pH 5.8
ka	kd	KD	kd (only dissociation)
optimized 20//18	3.17E+05	1.87E-04	5.89E-10	1.36E-04	4.84E-02

[0975] (22-6) Production of antibodies with increased pi by modification of amino acids in the variable region

The tested antibodies are summarized in Tables 36 and 37.

[0976] The heavy chain, CFP0020H0261-001-GldPl (also called 20H001, SEQ ID

NO:232) was prepared by introducing pi-increasing substitution P41R/G44R into CFP0020H0261-GldPl (SEQ ID NO:229). Similarly, the heavy chain, CFP0018H0012-002-GldNl (also called 18H002, SEQ ID NO:251) was prepared by introducing pi-increasing substitution T77R/E85R into CFP0018H0012-GldNl (SEQ ID NO:230). Other heavy chain variants were also prepared by introducing respective substitutions represented in Table 36 into CFP0020H0261-GldPl and CFP0018H0012-GldNl respectively, according to the method shown in Reference Example 1. The heavy chain variants of both CFP0020H0261-GldPl variants and CFP0018H0012-GldNl variants were expressed with CFP0020L233-k0 (SEQ ID NO:231) as light chain to obtain bi-specific antibody.

[0977] Similarly, we also evaluated the pi-increasing substitution in light chain. The light chain, CFP0020L233-001-k0 (also called 20L233-001, SEQ ID NO:271) was prepared by introducing pi-increasing substitution G16K into CFP0020L233-k0. Other light chain variants were also prepared by introducing respective substitutions represented in

260

WO 2017/046994

PCT/JP2016/003616

Heavy Chain Variants of CFP0020H0261-001-GldPl and CFP0018H0012-001-GldNl evaluated in this Example

Table 37 into CFP0020L233-k0 according to the method shown in Reference Example 1. All the light chain variants were expressed with CFP0020H0261-GldPl and CFP0018H0012-GldNl as heavy chain to obtain bi-specific antibody.

[0978] [Table 36]

Sample Name (Heavy Chain 1/ Heavy Chain 2 / Light Chain)	Variant	Mutation (Heavy Chain 1)	Mutation (Heavy Chain 2)	Imaging fold	BIACORE fold
CFP0020H0261-GldPl /CFPOO 18H0012-GldNl /CFP0020L233-k0	original Ab2			1.00	1.00
CFP0020H0261 -001 -G1 dP 1 /CFPOO18H0012-002-G1 dN 1 /CFP0020L233-k0	20H001/ 18H002	P41R/G44R	T77R/E85G	2.27	0.91
CFP0020H0261-002-G1 dP 1 /CFP0018H0012-002-G 1 dN 1 /CFP0020L233-k0	20H002/ 18H002	Q77R/A85R	T77R/E85G	2.46	1.09
CFP0020H0261-003-G1 dP 1 /CFPOO18H0012-003-G1 dN 1 /CFP0020L233-k0	20H003/ 18H003	L18R	G8R	1.69	0.97
CFP0020H0261 -005-G1 dP 1 /CFPOO18HOO12-005-G1 dN 1 /CFP0020L233-k0	20H005/ 18H005	S15R	G15R	1.36	0.96
CFP0020H0261 -008-G1 dP 1 /CFPOO1 SHOO 12-008-G1 dN 1 /CFP0020L233-k0	20H008/ 18H008	G32R	Y32R	0.00	0.20
CFP0020H0261 -009-G1 dP 1 /CFP0018H0012-009-G1 dN 1 /CFP0020L233-k0	20H009/ 18H009	Q39K	Q39K	1.45	1.01
CFP0020H0261 -013-G1 dP 1 /CFP00l8H0012-0l3-GldNl /CFP0020L233-k0	20H013/ 18H013	L63R	F63R	2.64	1.52
CFP0020H0261-GldPl /CFPOO 18H0012-014-G 1 dN 1 /CFP0020L233-k0	20H/ 18H014	-	Q64K	0.86	0.80
CFP0020H0261-GldPl /CFPOO 18H0012-016-G1 dN 1 /CFP0020L233-k0	20H/ 18H016	-	F63R/Q64K	1.90	1.08
CFP0020H0261 -018-G1 dP 1 /CFPOO 18H0012-018-G 1 dN 1 /CFP0020L233-k0	20H018/ 18H018	Q77R	T77R	2.80	0.96

261

WO 2017/046994

PCT/JP2016/003616

CFP0020H0261 -019-G1 dP 1 /CFP0018H0012-019-G 1 dN 1 /CFP0020L233-k0	20H019/ 18H019	L82K	L82K	2.47	1.61
CFP0020H0261 -020-G1 dP 1 /CFPOO18HO012-020-G1 dN 1 /CFP0020L233-k0	20H020/ 18H020	S82aN/S82bR	S82aN/S82bR	1.45	0.93
CFP0020H0261 -021 -G1 dP 1 /CFPOO18H0012-021 -G1 dN 1 /CFP0020L233-k0	20H021/ 18H021	S82aG/S82bR	S82aG/S82bR	0.74	0.85
CFP0020H0261-022-G 1 dP 1 /CFPOO 18H0012-022-G1 dN 1 /CFP0020L233-k0	20H022/ 18H022	S82bR	S82bR	1.25	0.86
CFP0020H0261 -023-G1 dP 1 /CFP0018H0012-023-GldNl /CFP0020L233-k0	20H023/ 18H023	V82cR	L82cR	0.58	0.52
CFP0020H026l-GldPl /CFPOO 1 SHOO 12-024-G 1 dN 1 /CFP0020L233-k0	20H/ 18H024	-	E85G	0.99	0.72
CFP0020H0261 -025-G1 dP 1 /CFP0018H0012-025-GldNl /CFP0020L233-k0	20H025/ 18H025	D86G	D86G	0.68	0.76
CFP0020H0261-026-G1 dP 1 /CFP0018H0012-026-GldNl /CFP0020L233-k0	20H026/ 18H026	A93K	A93K	0.01	0.23
CFP0020H0261-GldPl /CFPOO 18H0012-027-G1 dN 1 /CFP0020L233-k0	20H/ 18H027	-	Q105R	1.22	0.79
CFP0020H0261 -032-G1 dP 1 /CFPOO 18H0012-032-G1 dN 1 /CFP0020L233-k0	20H032/ 18H032	L82K/S82bR	L82K/S82bR	1.47	2.25
CFP0020H0261 -035-G1 dP 1 /CFPOO 18H0012-GldNl /CFP0020L233-k0	20H035/ 18H	S82bR/T83R	-	1.03	1.15
CFP0020H0261-036-G1 dP 1 /CFPOO 1 SHOO 12-GldNl /CFP0020L233-k0	20H036/ 18H	T83R	-	2.02	1.05
CFP0020F10261 -037-G1 dP 1 /CFPOO 18HOO12-037-G1 dN 1 /CFP0020L233-k0	20H037/ 18H037	V71R/A85G	A71R/E85G	0.34	0.49

[0979] [Table 37]

Light Chain Variants of CFP0020L233 evaluated in this Example

Sample Name	Variant	Mutation	Imaging fold	BIACORE fold
CFP0020H0261-GldPl /CFPOO 18H0012-GldNl /CFP0020L233-k0	original Ab2		1.00	1.00
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-001-k0	20L233- 001	G16K	2.20	1.59
CFP0020H0261-GldPl /CFPOO 18H0012-GldNl	20L233- 002	Q27R	1.08	0.92

262

WO 2017/046994

PCT/JP2016/003616

/CFP0020L233-002-kO
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-003-k0	20L233- 003	A25R/S26R	0.38	0.32
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-004-k0	20L233- 004	S52K/S56K	1.24	0.75
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-005-k0	20L233- 005	T74K/S77R	3.32	1.83
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-006-kO	20L233- 006	S76R/Q79K	4.85	1.89
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-007-kO	20L233- 007	Q27K	1.18	0.95
CFP0020H026l-GldP1 /CFP0018H0012-GldNl /CFP0020L233-008-kO	20L233- 008	A25K/S26K	0.29	0.29
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-009-kO	20L233- 009	Q37R	0.99	0.78
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-010-k0	20L233- 010	G41R	1.77	1.01
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-011-k0	20L233- 011	L46R/Y49K	0.00	0.14
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-012-k0	20L233- 012	S52R/S56R	1.02	0.68
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-013-k0	20L233- 013	S65R/T69R	1.24	No data
CFP0020H0261-GldP1 /CFP0018H0012-GldNl /CFP0020L233-016-k0	20L233- 016	G41R/T74K/S77R	6.58	1.96
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-017-k0	20L233- 017	L46R/Y49K/T74K/S77R	0.00	0.18
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-018-k0	20L233- 018	S52R/S56R/T74K/S77R	46.97	1.43
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-019-k0	20L233- 019	S65R/T69R/T74K/S77R	53.25	7.72
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-021-k0	20L233- 021	Q27R/S76R/Q79K	20.90	1.77
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-022-k0	20L233- 022	G41R/S76R/Q79K	27.3	2.04

263

WO 2017/046994

PCT/JP2016/003616

CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-023-k0	20L233- 023	L46R/Y49K/S76R/Q79K	0.2	0.18
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-024-k0	20L233- 024	S52R/S56R/S76R/Q79K	114.7	2.16
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-025-kO	20L233- 025	S65R/T69R/S76R/Q79K	75.6	3.18
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-027-kO	20L233- 027	Q27R/G41R/T74K/S77R	4.1	2.24
CFP0020H0261-GldP1 /CFP0018H0012-GldNl /CFP0020L233-028-k0	20L233- 028	G41R/S52R/S56R/T74K/S77R	18.1	1.94
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-029-k0	20L233- 029	G41R/S65R/T69R/T74K/S77R	28.8	9.63
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-031-k0	20L233- 031	Q27R/S52R/S56R/T74K/S77R	21.0	1.78
CFP0020H0261-GldPl /CFP0018H00l2-GldNl /CFP0020L233-032-k0	20L233- 032	S52R/S56R/S65R/T69R/T74K/S77 R	64.1	11.09
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-034-k0	20L233- 034	Q27R/S65R/T69R/T74K/S77R	30.5	8.61
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-036-k0	20L233- 036	Q27R/G41R/S76R/Q79K	14.5	2.45
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-037-k0	20L233- 037	G41R/S52R/S56R/S76R/Q79K	53.0	2.43
CFP0020H026l-GldP1 /CFP0018H0012-GldNl /CFP0020L233-038-kO	20L233- 038	G41R/S65R/T69R/S76R/Q79K	45.9	4.24
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-040-k0	20L233- 040	Q27R/S52R/S56R/S76R/Q79K	61.6	2.32
CFP0020H0261-GldPl /CFPOOl8HO012-G1dNl /CFP0020L233-041-k0	20L233- 041	S52R/S56R/S65R/T69R/S76R7Q79 K	96.2	5.28
CFP0020H0261-GldPl /CFP0018H00l2-G1dNl /CFP0020L233-043-k0	20L233- 043	Q27R/S65R/T69R/S76R/Q79K	49.3	3.51
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-044-kO	20L233- 044	S76R	1.61	1.25
CFP0020H0261-GldPl /CFP0018H0012-GldNl /CFP0020L233-045-k0	20L233- 045	S65R/Q79K	3.66	1.46

[0980] (22-7) Human FcyRIIb-binding assay by BIACORE using pi-increased variants

Regarding the produced Fc region variant-containing antibodies, binding assays between soluble hFcyRIIb and antigen-antibody complexes were performed using

BIACORE T200 (GE Healthcare). Soluble hFcyRIIb was produced in the form of a

264

WO 2017/046994

PCT/JP2016/003616

His-tagged molecule by a method known in the art. An appropriate amount of an antiHis antibody was fixed onto Sensor chip CM5 (GE Healthcare) by the amine coupling method using a His capture kit (GE Healthcare) to capture hFcYRIIb. Next, an antibody-antigen complex and a running buffer (as a reference solution) were injected, and interaction was allowed to take place with the hFcYRIIb captured onto the sensor chip. 20 mM N-(2-Acetamido)-2-aminoethanesulfonic acid, 150 mM NaCl, 1.2 mM CaCl₂, and 0.05% (w/v) Tween 20 at pH 7.4 was used as the running buffer, and the re spective buffer was also used to dilute the soluble hFcYRIIb. To regenerate the sensor chip, 10 mM glycine-HCl at pH 1.5 was used. All measurements were carried out at 25°C. Analyses were performed based on binding (RU) calculated from sensorgrams obtained by the measurements, and relative values when the binding amount of CFP0020H0261-GldPl/CFP0018H0012-GldNl/CFP0020F233-k0 (original Ab2) was defined as 1.00 are shown. To calculate the parameters, the BIACORE T100 Evaluation Software (GE Healthcare) was used.

[0981] The SPR analysis results are summarized in Tables 36 and 37. A few variants were shown to have enhanced binding toward hFcYRIIb fixed on the BIACORE sensor chip. Here, about 1.2 fold or more of the binding to hFcYRIIb of the variants compared to the binding to hFcYRIIb of original Ab2 was considered to have strong charge effect on binding of an antibody to hFcYRIIb on the sensor chip.

[0982] Among the pi-increased heavy chain variants, the antibody with F63R, F63R, F82K or S82bR substitutions (according to Kabat numbering) showed higher binding to hFcYRIIb. The single amino acid substitution or a combination of these substitutions in heavy chain is supposed to have strong charge effect on binding to hFcYRIIb on the sensor chip. Thus, one or more of positions that are expected to show an effect of accelerating the speed or rate of uptake into cells in vivo by introducing the pi-increasing modification into the heavy chain variable region(s) of an antibody can include, for example, position 63, 82 or 82b according to Kabat numbering. An amino acid substitution introduced at such position(s) can be arginine or lysine.

[0983] In the pi-increased light chain variants, the antibody with G16K, Q27R, G41R,

S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions (according to Kabat numbering) showed higher binding to hFcYRIIb. The single amino acid substitution or a combination of these substitutions in light chain is supposed to have strong charge effect on binding to human FcYRIIb on the sensor chip. Thus, one or more of positions that are expected to show an effect of accelerating the speed or rate of uptake into cells in vivo by introducing the pi-increasing modification into the light chain variable region of an antibody can include, for example, positions 16, 27, 41, 52, 56, 65, 69, 74, 76, 77 or 79, according to Kabat numbering. An amino acid substitution introduced at such position(s) can be arginine or lysine. The variants with four or more

265

WO 2017/046994

PCT/JP2016/003616 amino acid substitutions tended to show stronger charge effect than those variants with lesser amino acid substitutions.

[0984] (22-8) Cellular uptake of pi-increased Fab region variant-containing antibodies

To evaluate the rate of intracellular uptake into an hFcYRIIb-expressing cell line using the produced Fab region variant-containing antibodies, the following assay was performed.

[0985] An MDCK (Madin-Darby canine kidney) cell line that constitutively expresses hFcYRIIb was produced by known methods. Using these cells, intracellular uptake of antigen-antibody complexes was evaluated. Specifically, Alexa555 (Life Technlogies) was used to label human C5 according to an established protocol, and antigen-antibody complexes were formed in a culture solution with the antibody concentration being 10 mg/mL and the antigen concentration being 10 mg/mL. The culture solution containing the antigen-antibody complexes was added to culture plates of the above-mentioned MDCK cells which constitutively express hFcYRIIb and incubated for one hour, and then the fluorescence intensity of the antigen taken up into the cells was quantified using InCell Analyzer 6000 (GE healthcare). The amount of antigen taken up was presented as relative values to the original Ab2 value which is taken as 1.00.

[0986] The quantification results of cellular uptake were summarized in Tables 36 and 37. Strong fluorescence derived from the antigen in the cells was observed in several heavy chain and light chain variants. Here, about 1.5 fold or more of the fluorescence intensity of the antigen taken up into the cells of the variants compared to the fluorescence intensity of original Ab2 was considered to have strong charge effect on an antigen taken up into the cells.

[0987] Among the pi-increased heavy chain variants, the antibody with G8R, L18R, Q39K,

P41R, G44R, L63R, F63R, Q64K, Q77R, T77R, L82K, S82aN, S82bR, T83R, A85R or E85G substitution(s) (according to Kabat numbering) showed stronger antigen uptake into the cells. The single amino acid substitution or a combination of these substitutions in heavy chain is supposed to have strong charge effect on antigen antibody complex uptake into the cells. Thus, one or more of positions that are expected to cause uptake of an antigen-antibody complex into cells more quickly or more frequently by introducing the pi-increasing modification into the heavy chain variable region(s) of an antibody can include, for example, positions 8, 18, 39, 41, 44, 63, 64, 77, 82, 82a, 82b, 83, or 85, according to Kabat numbering. An amino acid substitution introduced at such position(s) can be asparagine, glycine, serine, arginine or lysine, and preferably arginine or lysine.

[0988] In the pi-increased light chain variants, the antibody with G16K, Q27R, S27R,

G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R or Q79K substitutions (according to Kabat numbering) showed stronger antigen uptake into the cells. The

266

WO 2017/046994

PCT/JP2016/003616 single amino acid substitution or a combination of these substitutions in light chain is supposed to have strong charge effect on antigen antibody complex uptake into the cells. The variants with four or more amino acid substitutions tended to show stronger charge effect than those variants with lesser amino acid substitutions. As shown in Example 21, (21-3), the combination of 42K and 76R substitution is effective in IgE antibody. In the case of C5 antibody, however, the amino acid of Rabat numbering 42 is already lysine, so we can observe the charge effect of 42K/76R by the single substitution of 76R. The fact that the variant with 76R substitution had strong charge effect also in C5 antibody shows the combination of 42K/76R has strong charge effect regardless of antigen. Thus, one or more of positions that are expected to cause uptake of an antigen-antibody complex into cells more quickly or more frequently by introducing the pi-increasing modification into the light chain variable region(s) of an antibody can include, for example, positions 16, 27, 41, 52, 56, 65, 69, 74, 76, 77 or 79, according to Rabat numbering. An amino acid substitution introduced at such position(s) can be arginine or lysine.

[0989] 122-91 Evaluation of clearance of C5 in mouse co-iniection model

Some anti-C5 bispecific antibodies (original Ab2, 20L233-005, 20L233-006 and

20L233-009) were tested in mice co-injection model to evaluate their ability to accelerate the clearance of C5 from plasma. In co-injection model, C57BL6J mice (Jackson Laboratories) were administered by single i.v. injection with C5 pre-mixed with the anti-C5 bispecific antibody, respectively. All groups received 0.1 mg/kg C5 with 1.0 mg/kg of anti-C5 bispecific antibodies. Total C5 plasma concentration was determined by anti-C5 ECLIA. First, anti-human C5 mouse IgG was dispensed into an ECL plate, and left for overnight at 5 °C to prepare an anti-human C5 mouse IgGimmobilized plate. Samples for standard curve and samples were mixed with an antihuman C5 rabbit IgG. These samples were added into the anti-human C5 mouse IgGimmobilized plate, and left for one hour at room temperature. Then, these samples were reacted with HRP-conjugated anti-rabbit IgG (Jackson Immuno Research). After the plate was incubated for one hour at room temperature, a sulfo-tag conjugated antiHRP antibody was added. ECL signal was read with Sector Imager 2400 (Meso Scale discovery). The concentration of human C5 was calculated from the ECL signal in the standard curve using SOFTmax PRO (Molecular Devices). Fig. 39 describes the C5 plasma concentration time profile in C57BL6J mice.

[0990] Compared to original Ab2, all of the bispecific antibodies with pi-increased substitution(s) tested in this study demonstrated rapid C5 clearance from plasma. Therefore, amino acid substitution(s) on T74K/S77R, S76R/Q79K and Q37R in light chain are suggested to accelerate elimination of C5-antibody immune complex also in vivo. Furthermore, C5 elimination of 20L233-005 and 20L233-006 was faster than that

267

WO 2017/046994

PCT/JP2016/003616 of 20L233-009, that was consistent with in vitro imaging and BIACORE analysis. These results suggest that even the position(s) which seems unlikely to contribute for clearance of an antigen from plasma in vivo, examined under either the in vitro system using the fluorescence intensity by InCell Analyzer 6000 or the in vitro BIACORE system described above, can be found out to contribute for that by using the more sensitive in vivo system. These results also suggest that for speculating an evaluation of clearance of an antigen from plasma in vivo, the sensitivity of the in vitro system using the fluorescence intensity by InCell Analyzer 6000 described above may be higher than that of the in vitro BIACORE system described above.

Example 23 [0991] Evaluation of clearance of IgE from plasma using pi-increased Fc variants

To enhance the clearance of human IgE or human C5, pi-increased substitutions in the Fc portion of antibodies were evaluated using pH dependent antibodies. The method of adding amino acid substitutions to the antibody constant region to increase pi is not particularly limited, but for example, it can be performed by the method described in WO2014/145159.

[0992] (23-1) Production of antibodies with increased-pl by a single amino acid modification in the constant region

The tested antibodies are summarized in Table 38. The heavy chain, Ab 1H-P 1394m (SEQ ID NO:307) was prepared by introducing a pi-increasing substitution Q31 IK into AblH. Other heavy chain variants were also prepared by introducing respective substitutions represented in Table 38 into AblH according to the method shown in Reference Example 1. All the heavy chain variants were expressed with AblL as light chain.

[0993]

268

WO 2017/046994

PCT/JP2016/003616 [Table 38]

			Imaging	BIACORE
Antibody Name (Heavy Chain / Light Chain)	Variant	Mutation	fold	fold
AblH/AblL	original Abl	—	1.00	1.00
AblH-P1394m/AblL	Pl 394m	Q311K	1.31	1.18
AblH-P1398m/AblL	P1398m	D413K	3.45	1.23
AblH-P1466m/AblL	P1466m	Q311R	1.90	1.22
AblH-P1468m/AblL	P1468m	N315R	1.42	1.13
AblH-P1469m/AblL	Pl 469m	N315K	1.93	1.13
Ab 1H-P 1470m/AblL	Pl470m	N384R	1.50	1.19
AblH-P1471m/Ab1L	Pl 471m	N384K	0.71	1.19
Abl H-P1480m/Ab1 L	Pl 480m	Q342R	1.08	1.03
AblH-P1481m/AblL	P1481m	Q342K	1.83	1.08
Ab 1H-P 1482m/AblL	Pl 482m	P343R	4.90	1.46
Ab 1H-P 1483m/AblL	P1483m	P343K	1.99	1.02
AblH-P1512m/AblL	P1512m	D401R	2.98	1.24
AblH-P1513m/AblL	P1513m	D401K	2.57	1.21
AblH-P1514m/AblL	Pl 514m	G402R	1.22	1.20
AblH-P15l5m/AblL	Pl 515m	G402K	0.93	1.19
AblH-P1653m/AblL	P1653m	D413R	3.96	0.79

[0994] (23-2) Human FcyRIIb-binding assay by BIACORE using pi-increased Fc region variant-containing antibodies

To evaluate the charge effect on FcRYRIIb-binding of antigen-antibody complex formed by using the antibodies described in Table 38, FcRYRIIb-binding assay was performed in a similar manner with those described in Example 21, (21-2). Assay results are shown in Table 38. Here, about 1.2 fold or more of the binding to hFcYRIIb of the variants compared to the binding to hFcYRIIb of original Abl was considered to have strong charge effect on binding of an antibody to hFcYRIIb on the sensor chip.

[0995] Among the pi-increased variants with a single amino acid substitution from original Abl, the antigen-antibody complex made by several variants such as Pl398m,

Pl466m, Pl482m, Pl512m, P1513m, andP1514m showed highest binding to hFcYRIIb. The single amino acid substitution on D413K, Q311R, P343R, D401R, D401K, G402R, Q311K, N384R, N384K, or G402K is supposed to have strong charge effect on binding to hFcYRIIb on the sensor chip. Thus, a single position that is expected to show an effect of accelerating the speed or rate of uptake into cells in vivo by introducing the pi-increasing modification into the constant or Fc region of an antibody can include, for example, positions 311, 343, 384, 401, 402, or 413, according to EU numbering. An amino acid substitution introduced at such position can be arginine or lysine.

[0996] (23-3) Cellular uptake of pi-increased Fc region variant-containing antibodies

To evaluate the intracellular uptake of antigen-antibody complex formed by the an269

WO 2017/046994

PCT/JP2016/003616 tibodies described in Table 38, cell imaging assay was performed in a similar manner with those described in Example 21, (21-3). Assay results are shown in Table 38. Here, about 1.5 fold or more of the fluorescence intensity of the antigen taken up into the cells of the variants compared to the fluorescence intensity of original Abl was considered to have strong charge effect on an antigen taken up into the cells.

[0997] Among the pi-increased variants with a single amino acid substitution from original Abl, the antigen-antibody complex made by several variants such as Pl398m,

P1466m, P1469m, P1470m, P1481m, P1482m, P1483m, P1512m, P1513m and P1653m showed stronger antigen uptake into the cells. The single amino acid substitution on D413K, Q311R, N315K, N384R, Q342K, P343R, P343K, D401R, D401K or D413R is supposed to have strong charge effect on antigen antibody complex uptake into the cells. Thus, a single position that is expected to cause uptake of an antigen-antibody complex into cells more quickly or more frequently by introducing the pi-increasing modification into the constant or Fc region of an antibody can include, for example, positions 311, 315, 342, 343, 384, 401, or 413, according to EU numbering. An amino acid substitution introduced at such position can be arginine or lysine.

[0998] (23-4) Evaluation of clearance of human IgE in mouse co-injection model

Some anti-IgE antibodies with pH-dependent antigen-binding (original Abl,

P1466m, P1469m, P1470m, P1480m, P1482m, P1512m, P1653m) were tested in mice co-injection model to evaluate their ability to accelerate the clearance of IgE from plasma. The assays were performed in a similar way with Example 21, (21-4). Fig. 40 describes the plasma concentration time profile in C57BL6J mice.

[0999] After administration of high pi variants (only a single amino acid substitution) with pH-dependent antigen-binding, the plasma total IgE concentration was lower than that of original Abl except for Pl480m. Pl480m, which showed weak efficacy the both in vitro studies, did not accelerate elimination of IgE. Furthermore, the plasma total IgE concentration in mice treated with high pi variant without pH-dependent antigenbinding was significantly higher than that of high pi variant with pH-dependent antigen-binding (data not shown). These results indicate that the cellular uptake of antigen-antibody immune complex increase by introducing the pi-increasing modification. The antigen uptake into the cells in complex with a pH-dependent antigenbinding antibody could release from antibody inside endosome effectively, resulted in accelerated elimination of IgE. These results suggest that even the substituted position which seems unlikely to contribute for clearance of an antigen from plasma in vivo, examined under the in vitro BIACORE system described above, can be found out to contribute for that by using the more sensitive in vivo system. These results also suggest that for speculating an evaluation of clearance of an antigen from plasma in

270

WO 2017/046994

PCT/JP2016/003616 vivo, the sensitivity of the in vitro system using the fluorescence intensity by InCell Analyzer 6000 described above may be higher than that of the in vitro BIACORE system described above.

[1000] REFERENCE EXAMPLE 1

Construction of expression vectors of amino acid-substituted IgG antibodies

Mutants were prepared using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) by the method described in the appended instruction manual. Plasmid fragments containing the mutants were inserted into animal cell expression vectors to construct desired H-chain and L-chain expression vectors. The nucleotide sequences of the obtained expression vectors were determined by methods known in the art.

[1001] REFERENCE EXAMPLE 2

Expression and purification of IgG antibodies

Antibodies were expressed using the following method. The human embryonic kidney cancer cell-derived HEK293H cell line (Invitrogen) was suspended in DMEM medium (Invitrogen) supplemented with 10% Fetal Bovine Serum (Invitrogen). The cells were plated at 10 mL per dish in dishes for adherent cells (10 cm in diameter; CORNING) at a cell density of 5 to 6 x 10⁵ cells/mL and cultured in a CO₂ incubator (37°C, 5% CO₂) for one day. Then, the medium was removed by aspiration, and 6.9 mL of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plasmid was introduced into the cells by the lipofection method. The resulting culture supernatants were collected, centrifuged (approximately 2,000 g, 5 minutes, room temperature) to remove cells, and sterilized by filtering through the 0.22-pm filter MILLEX (registered trademark)-GV (Millipore) to obtain supernatants. Antibodies were purified from the obtained culture supernatants by methods known in the art using the rProtein A Sepharose™ Fast Flow (Amersham Biosciences). To determine the concentration of the purified antibody, absorbance was measured at 280 nm using a spectrophotometer. Antibody concentrations were calculated from the determined values using an absorbance coefficient calculated by the method described in Pace et al., Protein Science 4:2411-2423(1995).

[1002] REFERENCE EXAMPLE 3

Preparation of a soluble human IL-6 receptor

A recombinant soluble human IL-6 receptor, which is an antigen, was prepared in the manner described below. A CHO cell line that constitutively expresses soluble human IL-6 receptor composed of an amino acid sequence of the 1st to 357th amino acid from the N terminus as reported in Mullberg et al., J. Immunol. 152:4958-4968 (1994) was constructed using a method known in the art. Soluble human IL-6 receptor was expressed by culturing this CHO line. Soluble human IL-6 receptor was purified from the culture supernatant of the obtained CHO line in two steps: Blue Sepharose 6 FF

271

WO 2017/046994

PCT/JP2016/003616 column chromatography and a gel filtration column chromatography. The fraction that was eluted as the main peak in the final step was used as the final purified product.

2Ί2

WO 2017/046994

PCT/JP2016/003616 [Claim 1] [Claim 2]

Claims (11)

1. Claims

   An isolated anti-IL-8 antibody that binds to human IL-8, which comprises at least one amino acid substitution(s) in at least one of (a) to (f) below, and binds to IL-8 in a pH-dependent manner:

   (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:67;

   (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:68;

   (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:69;

   (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:70;

   (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO :71; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:72. The anti-IL-8 antibody of claim 1, which comprises a member selected from the group consisting of :

   (a) amino acid substitutions of tyrosine at position 9 of the amino acid sequence of SEQ ID NO:68, arginine at position 11 of the amino acid sequence of SEQ ID NO:68, and tyrosine at position 3 of the amino acid sequence of SEQ ID NO:69;

   (b) amino acid substitutions of tyrosine at position 9 of the amino acid sequence of SEQ ID NO:68, alanine at position 6 of the amino acid sequence of SEQ ID NO:68, glycine at position 8 of the amino acid sequence of SEQ ID NO:68, arginine at position 11 of the amino acid sequence of SEQ ID NO:68, and tyrosine at position 3 of the amino acid sequence of SEQ ID NO:69; and (c) amino acid substitutions of asparagine at position 1 of the amino acid sequence of SEQ ID NO:71, leucine at position 5 of the amino acid sequence of SEQ ID NO:71, and glutamine at position 1 of the amino acid sequence of SEQ ID NO:72;

   (d) amino acid substitutions of tyrosine at position 9 of the amino acid sequence of SEQ ID NO:68, arginine at position 11 of the amino acid sequence of SEQ ID NO:68, and tyrosine at position 3 of the amino acid sequence of SEQ ID NO:69, asparagine at position 1 of the amino acid sequence of SEQ ID NO:71, leucine at position 5 of the amino acid sequence of SEQ ID NO:71, and glutamine at position 1 of the amino acid sequence of SEQ ID NO:72; and (e) amino acid substitutions of tyrosine at position 9 of the amino acid sequence of SEQ ID NO:68, alanine at position 6 of the amino acid sequence of SEQ ID NO:68, glycine at position 8 of the amino acid

   273

   WO 2017/046994

   PCT/JP2016/003616 [Claim 3] [Claim 4] [Claim 5] [Claim 6] [Claim 7] [Claim 8] sequence of SEQ ID NO:68, arginine at position 11 of the amino acid sequence of SEQ ID NO:68, tyrosine at position 3 of the amino acid sequence of SEQ ID NO:69, asparagine at position 1 of the amino acid sequence of SEQ ID NO:71, leucine at position 5 of the amino acid sequence of SEQ ID NO:71, and glutamine at position 1 of the amino acid sequence of SEQ ID NO:72.

   The anti-IL-8 antibody of claim 1 or 2, which comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:67, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:73, and (c) HVRH3 comprising the amino acid sequence of SEQ ID NO:74.

   The anti-IL-8 antibody of any one of claims 1 to 3, which comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:70, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:75, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. The anti-IL-8 antibody of any one of claims 1 to 4, which comprises the heavy chain variable region of SEQ ID NO:78 and the light chain variable region of SEQ ID NO:79.

   An anti-IL-8 antibody, which comprises an Fc region comprising amino acid substitution(s) at one or more positions selected from the group consisting of positions 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440, according to EU numbering.

   The anti-IL-8 antibody of claim 6, which comprises an Fc region having at least one property selected from the properties of (a) to (f) below:

   (a) increased binding affinity for FcRn of the Fc region relative to the binding affinity for FcRn of a native Fc region at acidic pH;

   (b) reduced binding affinity of the Fc region for pre-existing ADA relative to the binding affinity of a native Fc region for the pre-existing ADA;

   (c) increased plasma half-life of the Fc region relative to the plasma half-life of a native Fc region;

   (d) reduced plasma clearance of the Fc region relative to the plasma clearance of a native Fc region;

   (e) reduced binding affinity of the Fc region for an effector receptor relative to the binding affinity of a native Fc region for the effector receptor; and (f) increased binding to extracellular matrix.

   The anti-IL-8 antibody of claim 6 or 7, which comprises an Fc region

   274

   PCT/JP2016/003616

   WO 2017/046994 [Claim 9] [Claim 10] [Claim 11] [Claim 12] [Claim 13] [Claim 14] [Claim 15] [Claim 16] [Claim 17] [Claim 18] [Claim 19] [Claim 20] [Claim 21] [Claim 22] comprising one or more amino acid substitutions selected from the group consisting of L235R, G236R, S239K, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E, according to EU numbering.

   The anti-IL-8 antibody of claim 8, which comprises an Fc region comprising amino acid substitutions of (a) L235R, G236R, S239K, M428L, N434A, Y436T, Q438R and S440E; or (b) L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R and S440E, according to EU numbering.

   The anti-IL-8 antibody of claim 6 that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:80 or SEQ ID NO:81 and a light chain comprising the amino acid sequence of SEQ ID NO:82.

   An isolated nucleic acid encoding the anti-IL-8 antibody of any one of claims 1 to 10.

   A vector comprising the nucleic acid of claim 11.

   A host cell comprising the vector of claim 12.

   A method for producing an anti-IL-8 antibody, which comprises culturing the host cell of claim 13.

   The method of claim 14, which further comprises isolating the antibody from the host cell culture.

   A pharmaceutical composition comprising the anti-IL-8 antibody of any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

   A method for treating a patient that has a disorder with the presence of excess IL-8, which comprises administering the anti-IL-8 antibody of any one of claims 1 to 10 to the individual.

   Use of the anti-IL-8 antibody of any one of claims 1 to 10 in the manufacture of a pharmaceutical composition for treating a disorder with the presence of excess IL-8.

   A method for inhibiting angiogenesis in an individual, wherein the method comprises administering the anti-IL-8 antibody of any one of claims 1 to 10 to the individual.

   Use of the anti-IL-8 antibody of any one of claims 1 to 10 in the manufacture of a pharmaceutical composition for inhibiting angiogenesis.

   A method for inhibiting facilitation of neutrophil migration in an individual, wherein the method comprises administering the anti-IL-8 antibody of any one of claims 1 to 10 to the individual.

   Use of the anti-IL-8 antibody of any one of claims 1 to 10 in the man275

   WO 2017/046994

   PCT/JP2016/003616 [Claim 23] ufacture of a pharmaceutical composition for inhibiting facilitation of neutrophil migration.

   A method for producing an anti-IL-8 antibody comprising a variable region with a pH-dependent IL-8-binding activity, wherein the method comprises:

   (a) evaluating binding of an anti-IL-8 antibody with extracellular matrix, (b) selecting an anti-IL-8 antibody with strong binding to the extracellular matrix, (c) culturing a host that comprises a vector comprising a nucleic acid encoding the antibody, and (d) isolating the antibody from the culture solution.

   1/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 1]

   10000 o

   —·- Low_pl-lgG1

   High_pl-lgG1

   0.1

   0 1 2 3 4 5 6 7

   Time (Days) [Fig. 2]

   0.1

   0 12 3

   -^b-Low_pl-F939 ·Ά·< Middle_pl-F939

   High_pl-F939

   Time (Days)
2. 2/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 3]

   Time (Days) [Fig. 4] ο E cu σ>

   8s <O Vi _j 35 — CL _ω c 3 = o “ ω ro c *;

   ΕΦ o

   -> c I o o

   Time (Days)
3. 3/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 5] oo h- © in < η cm T- o

   ECL Signal Intensity (X10E4)
4. 4/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 6]

   Q

   O

   LO >

   Ί3

   C in

   E o

   u .2

   Sen a cz> ο ο o <z> ez> o ca>

   io o m ο «χ» o in θ «·>

   CO co CM CM

   -sr~- θ θ θ
5. 5/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 7]

   Uptake into FcgRIlb-expressing Cells
6. 6/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 8]
7. 7/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 10]

   N434H
8. 8/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 12]

   F1847m
9. 9/29

   WO 2017/046994

   PCT/JP2016/003616 [Fig. 14]

   FlSSim

   2000

   1800

   1600

   1400

   Ί¹ *

   FJSSSro

   2000 «2515
10. 10/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 16] fllSSm

    2000 — 1800 · 1800 ‘ Ml. Bi IB (fc JK , BB ·β I H 01, IB H B. BB BB B IB IB. IB BB IB BB .BB 01 BB H BB ° § g § g g s S is § 3 S g 8 S 3 § s 8 §°gggggg§sggsggSH8 isssSSSS gogggss m eh oft <h fift <?% 3% So
11. 11/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 18]

    250

    Average of 30 donors [Fig. 19]

    C

    100.00

    10.00

    1.00

    0.10

    0.01

    0,00 ♦OHB-lgGl ♦0H8~F1847m *GHB-Fl848m

    -OHB-F1886m

    OHB-F1889m ♦O«B-F1927m ♦OHB-M434A

    0 20 40 60

    Time (Days)

    WO 2017/046994

    PCT/JP2016/003616 nnv

    7 14 21

    Time (Days) •O»* Fv4-lgGl

    -Fv4-fl718

    13/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 21]

    o o O O O O IO © O CM CM T“ V i

    in

    Q.

    14/29

    WO 2017/046994

    PCT/JP2016/003616

    Ό™· H998/L6S β—H88/LT18 [Fig. 23]

    Time (Days) —β—H89/L118 2mg/kg

    8mg/kg

    15/29

    WO 2017/046994

    PCT/JP2016/003616

    Antibody Concentration [nM]

    16/29

    WO 2017/046994 PCT/JP2016/003616 [Fig. 25B]

    Antibody Concentration [nM] [Fig. 25C]

    Antibody Concentration [nM]

    17/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 26]

    Campath (45%)·

    Rituxan (27%)-*

    Zenapax (14%)Humicade (7%)50%

    4<’>

    30%

    20%

    10%

    -WS4 (10.42%), HR9 (8.62%)

    -H89L118 (5.52%), H496L118 (4.67%), H553L118 (3.45%)

    20%

    Zenapax (14%)—*J

    Humicade (7%)

    Mylotarg (2.9%) Avastin (0%)

    H496L118 (4.67%), V2 (2.99%)

    V3 (1.95%), V1 (1.23%), H1004L395 (0%), H1004L118 (0%)

    18/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 28A]

    100.00

    Antibody Concentraion [nM] [Fig. 28B]

    Antibody Concentraion [nM]

    19/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 28C]

    Antibody Concentraion [nM] [Fig. 29]

    -Ο— H998/L63 -·— H553/L118 ❖ - H1009/L395

    20/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 30]

    450000

    400000

    350000

    300000

    250000

    200000

    150000

    100000

    50000 co c

    <

    o

    Z en oo x

    in

    Ch

    H1009/1395

    -118 J +ML8 [Fig. 31]

    0 0.5 1 1.5 2 2.5

    Time (Days)

    21/29

    WO 2017/046994

    PCT/JP2016/003616

    22/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 34]

    23/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 35]

    -O-H1009/L395

    2mg/kg ••♦“H1009/L395-F1886s

    2mg/kg

    - <-H10Q9O95~F1886s 5mg/kg

    -♦—HI 009/L395-F1886s lOmg/kg

    0 1 2

    Time (Days) «♦•HSS/llM-tgGl —·—H1G09/I335F1886S *·Ο·· H1009/L39Sf1886s +MD8 ••□••HMO/L395FlSTWm +hIL-8

    24/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 37]

    10000

    Plasma IgE concentration (ng/mL)

    25/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 38A]

    Tins (tec) ^Tlme i»·’]

    ~ association: 1.2mM CaCI₂-pH7.4, dissociation: 1.2mM CaCI₂-pH7.4 —« association: 1.2mM CaCI₂-pH7.4, dissociation: 3μΜ CaCI₂-pH5.8

    26/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 38B]

    Tine |seej association: 1.2mM CaCI₂-pH7.4, dissociation: 1.2mM CaCI₂-pH7.4 association: 1.2mM CaCI₂-pH7.4, dissociation: 3μΜ CaCI₂-pH5.8

    27/29

    WO 2017/046994 [Fig. 38C]

    PCT/JP2016/003616 loading Sjnffe ID tSUEl 3JQ55

    Time Uec{

    Time iWCZfl LU-41 « 80 1 20 WO 200 240

    Tire («J

    Loading Sample ID C5C2f 1-3,1131

    0 « 80 120 160 » 240

    Time(tecJ

    Loading Sampl* ID C5€2Eb3_W05 am

    0 40 80 120 160 2C0 240

    T«eiseci — association: 1.2mM CaCI₂-pH7.4, dissociation: 1.2mM CaCI₂-pH7.4 — association: 1.2mM CaCI₂-pH7.4, dissociation: 3μΜ CaCI₂-pH5.8

    28/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 38D]

    Loading Sample (D C5C2£1-3J?8 a*Q a»

    0.00

    0 2S

    0.21

    014

    0.07 /

    0.00 or « 80 120 1M - 200 2M7

    Time (see)

    0 40 60 120 160 200 240

    Tnejsee)

    Loarfnq Sampte IDLoading Sample <'i

    018

    012

    016 <00

    0.040,00

    0- « -80 120 160 20' Tine bed <' W ^:(l UH GD J» 240 lime t*ec«

    Loading Sample ID C5C2E1-3_1O36 „oading SamptelD’ C5C2EU_1371

    0 30 g ⁰²⁰ 0.107

    0,00

    0.20 0/16012 0.08 0 04 Over

    0 «7 StT 12D 1ST 200 2«

    Time (seel

    40 SO 120 160 200 240

    Τ» [see) toacM Sample IE). C5C2E1-3J1O?

    030 ' £ 0 20 ϋ· (GO / /

    association: 1.2mM CaCI₂-pH7.4, dissociation: 1,2mM CaCI₂-pH7.4 association: 1.2mM CaCI₂-pH7.4, dissociation: 3μΜ CaCI₂-pH5.8

    0,00

    0 «1 W: 120 «7 200 - 240

    TimefecJ

    29/29

    WO 2017/046994

    PCT/JP2016/003616 [Fig. 39]

    E δ» c

    o *5 ns

    4-» c

    Φ o

    c o

    o m

    O ns

    E (Λ ro ql

    0.5

    0.01

    -❖-original Ab2 «20L233-005

    O20L233-006

    A20L233-009

    0.001

    Time (day) [Fig. 40] ns

    E (Λ ns

    0.1

    0.5

    1 1.5

    Time (day)

    2.5

    -$-Ab1 H/Ab1 L «Ab1H-P1466m/Ab1L δAb1H-P1469m/Ab1L • o Ab1H-P1470m/Ab1L

    X Ab1H-P1480m/Ab1L o Ab1H-P1482m/Ab1L + Ab1H-P1512m/Ab1L — Ab1H-P1653m/Ab1L

    SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA

    <120> IL-8-BINDING ANTIBODIES Ϊ AND USES THEREOF <130> C1-A1502Y1P2 <150> JP 2015-185254 <151> 2015-09-18 <160> 332 <170> PatentIn version 3.5 <210> 1 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Vk1 <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

    100

    105 <210> 2 <211> 112 <212> PRT <213> Artificial Sequence <220>

    <223> Vk2 <400> 2

    Asp Gly 1 Ile Val Met Thr 5 Gln Ser Pro Leu Ser 10 Leu Pro Val Thr Pro 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asp Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Val 85 90 95 Leu Arg Asn Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Gln 100 105 110

    <210> 3 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Vk3

    <400> 3

    Glu Gly 1 Ile Val Met Thr 5 Gln Ser Pro Ala Thr 10 Leu Ser Leu Ser Pro 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

    <210> 4 <211> 112 <212> PRT <213> Artificial Sequence <220>

    <223> Vk4 <400> 4

    Asp Ile Val Met Thr Gln Ser Pro Glu Ser Leu Val Leu Ser Leu Gly 1 5 10 15

    Gly Thr Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30

    Ser Asn Asn Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Val Thr Leu Leu Phe Ser Trp Ala Ser Ile Arg Asp Ser Gly 50 55 60 Pro Asp Thr Arg Phe Ser Ala Ser Gly Ser Gly Thr Glu Phe Thr Leu 65 70 75 Ile Ser Asp Leu Gln Ala Glu Asp Ala Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Lys Arg Ala Pro Ser Phe Gly Gln Gly Thr Lys Leu Gln Ile 100 105 110 <210> 5 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 6RL#9 >-IgG1 <400> 5 Gln Val Ala Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Phe Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val

    Tyr

    Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Asp Pro Gly Gly Gly Glu Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 6 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> 6KC4- -1#85 -IgG1 <400> 6 Glu Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Val Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser 50 55 60 Lys Gly Tyr Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

    Cys

    85 90 95

    Ala Arg Asp Ala Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Thr Asp Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Calcium-] Binding ] Domain <400> 7 Glu Thr Thr Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Thr Pro Gly 1 5 10 15 Asp Lys Val Asn Ile Ser Cys Lys Ala Ser Gln Asp Ile Asp Asp Asp 20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Ala Ala Ile Phe Ile Ile 35 40 45 Gln Glu Ala Thr Thr Leu Val Pro Gly Ile Ser Pro Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu Ser 65 70 75 Glu Asp Ala Ala Tyr Tyr Phe Cys Leu Gln His Asp Asn Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

    100

    105 <210> 8 <211> 115 <212> PRT <213> Homo Sapian <400> 8

    Gln Ala 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115

    <210> 9 <211> 112 <212> PRT <213> Homo Sapian <400> 9 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15

    Glu Ser Pro Ala Ser 20 Ile Ser Cys Arg Ser 25 Ser Gln Ser Leu Val 30 His Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 10 <211> 449 <212> PRT <213> Homo Sapian <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser

    Leu

    50 55 60

    Lys Ser 65 Ser Arg Val Thr Met 70 Leu Arg Asp Thr Ser 75 Lys Asn Gln Phe Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Leu Ala Arg Ile Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

    Pro

    225 230 235

    240

    Ser Ser Val Phe Leu Phe 245 Pro Pro Lys Pro Lys 250 Asp Thr Leu Met Ile 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

    Lys

    405

    410

    415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445

    Lys <210> 11 <211> 214 <212> PRT <213> Homo Sapian <400> 11

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Ile Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

    105

    Ala

    100 Pro Ser Val Phe Ile Phe Pro Gly 115 Thr Ala Ser Val Val Cys Leu Ala 130 135 Lys Gln Val Gln Trp Lys Val Asp 145 150 160 Glu Ser Val Thr Glu Gln Asp Ser 165 Ser Thr Leu Thr Leu Ser Lys Tyr 180 Ala Cys Glu Val Thr His Gln Ser 195 Phe Asn 210 Arg Gly Glu Cys <210> 12 <211> 433 <212> PRT <213> Homo Sapian <400> 12 Gln Val Gln Leu Gln Glu Ser Gln 1 5 Thr Leu Ser Leu Thr Cys Thr Asp 20

    110

    Pro Ser Asp Glu Gln Leu Lys

    120 125

    Leu Asn Asn Phe Tyr Pro Arg

    140

    Asn Ala Leu Gln Ser Gly Asn

    155

    Ser Lys Asp Ser Thr Tyr Ser

    170

    Ala Asp Tyr Glu Lys His Lys

    185 190

    Gly Leu Ser Ser Pro Val Thr

    200 205

    Gly Pro Gly Leu Val Arg Pro

    Val Ser Gly Tyr Ser Ile Thr

    25 30

    Ser

    Glu

    Ser

    Leu

    175

    Val

    Lys

    Ser

    Ser

    His Ala Trp Ser Trp Val Arg Gln Trp 35 40

    Ile Gly Tyr Ile Ser Tyr Ser Gly Leu 50 55

    Lys Ser Arg Val Thr Met Leu Arg Ser

    65 70

    Pro Pro Gly Arg Gly 45 Leu Glu Ile Thr Thr Tyr Asn Pro Ser 60 Asp Thr Ser Lys Asn Gln Phe 75 80

    Leu Arg Leu Ser Cys

    Ser Val

    Thr Ala Ala Asp

    Thr Ala Val

    Tyr Tyr

    Ala Arg Gly

    Ser Leu Ala Arg Thr

    Thr Ala Met Asp

    Tyr

    Trp

    Gly Gln

    100

    105

    110

    Pro Leu Ala Pro Ser Ser Lys Leu

    115

    Ser Thr

    Ser

    Gly Gly Thr Ala

    Ala

    120

    125

    Gly Cys Trp

    130

    Leu Val

    Lys Asp Tyr

    Phe

    Pro Glu

    Pro Val

    Thr Val

    Ser

    135

    140

    Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Leu 145 150 155 160

    Phe

    Pro Ala Val

    Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

    165 170 175

    Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

    180 185 190

    Ser Asn Thr Lys Val Asp Lys Lys

    195

    Lys Val Glu Pro

    200

    Lys

    Ser Cys Asp

    205

    Thr Pro His 210 Thr Cys Pro Pro Cys 215 Ser Val Phe Leu Phe Pro Pro Ser 225 230 240 Arg Thr Pro Glu Val Thr Cys Asp 245 Pro Glu Val Lys Phe Asn Trp Asn 260 Ala Lys Thr Lys Pro Arg Glu Val 275 Val Ser Val Leu Thr Val Leu Glu 290 295 Tyr Lys Cys Lys Val Ser Asn Lys 305 310 320 Thr Ile Ser Lys Ala Lys Gly Thr 325 Leu Pro Pro Ser Arg Asp Glu Thr 340 Cys Leu Val Lys Gly Phe Tyr Glu 355 Ser Asn Gly Gln Pro Glu Asn Leu 370 375

    Ala Pro Glu Leu 220 Leu Gly Gly Pro Lys Asp Thr Leu Met Ile 235 Val Val Asp Val Ser His Glu 250 255 Val Asp Gly Val Glu Val His 265 270 Gln Tyr Asn Ser Thr Tyr Arg 285 Gln Asp Trp Leu Asn Gly Lys 300 Ala Leu Pro Ala Pro Ile Glu 315 Pro Arg Glu Pro Gln Val Tyr 330 335 Thr Lys Asn Gln Val Ser Leu 345 350 Ser Asp Ile Ala Val Glu Trp 365 Tyr Lys Thr Thr Pro Pro Val 380

    Asp Ser Lys Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 385 390 395 400 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 405 410 415 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 420 425 430 Lys <210> 13 <211> 214 <212> PRT <213> Homo Sapian <400> 13 Asp Ile Gly Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Gly Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Ser Gly Pro Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln 65 70 75 Glu Asp Tyr Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 85 90 95

    Thr Ala Phe Gly Gln 100 Gly Thr Lys Val Glu 105 Ile Lys Arg Thr Val 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 14 <211> 445 <212> PRT <213> Homo Sapian <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp

    Tyr

    Glu Ile Met His 35 Trp Ile Arg Gln Pro 40 Pro Gly Lys Gly Leu 45 Glu Trp Gly Ala Ile Asn Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Arg Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

    195 200 205

    Val Cys Asp 210 Lys Lys Val Glu Pro 215 Lys Ser Cys Asp Lys 220 Thr His Thr Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

    Gln

    370 375 380 Pro Glu Gly Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 15 <211> 219 <212> PRT <213> Homo Sapian <400> 15 Asp Ile Gly Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Arg Ser Leu Val His Ser 20 25 30 Asn Arg Ala Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Arg Pro Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val 50 55 60 Asp Arg Ile Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75

    Ser Asn Arg Val Glu Ala 85 Glu Asp Val Gly Val 90 Tyr Tyr Cys Ser Gln 95 Thr His Val Pro Pro Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 16 <211> 447 <212> PRT <213> Homo Sapian <400> 16

    Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

    1 5 10 15

    Ser Tyr Val Lys Val 20 Ser Cys Lys Ala Ser 25 Gly Tyr Thr Phe Thr 30 Gly Ile Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Tyr Asp Asp Gly Pro Tyr Thr Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190

    Pro His Ser Ser 195 Asn Phe Gly Thr Lys Pro Ser Asn Thr Lys Val Cys 210 215 Val Glu Cys Pro Pro Cys Pro Val 225 230 240 Phe Leu Phe Pro Pro Lys Pro Thr 245 Pro Glu Val Thr Cys Val Val Glu 260 Val Gln Phe Asn Trp Tyr Val Lys 275 Thr Lys Pro Arg Glu Glu Gln Ser 290 295 Val Leu Thr Val Val His Gln Lys 305 310 320 Cys Lys Val Ser Asn Lys Gly Ile 325 Ser Lys Thr Lys Gly Gln Pro Pro 340 Pro Ser Arg Glu Glu Met Thr Leu 355

    Thr Tyr Thr Cys Asn 205 Val Asp Lys Thr Val Glu Arg Lys Ser 220 Pro Pro Val Ala Gly Pro Ser 235 Asp Thr Leu Met Ile Ser Arg 250 255 Asp Val Ser His Glu Asp Pro 265 270 Gly Val Glu Val His Asn Ala 285 Asn Ser Thr Phe Arg Val Val 300 Trp Leu Asn Gly Lys Glu Tyr 315 Pro Ala Pro Ile Glu Lys Thr 330 335 Glu Pro Gln Val Tyr Thr Leu 345 350 Asn Gln Val Ser Leu Thr Cys 365

    Val Lys Asn Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Gly Gln Ser Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp 385 390 395 400 Asp Gly Arg Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 17 <211> 214 <212> PRT <213> Homo Sapian <400> 17 Asp Ile Gly Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Phe 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Gly Ala Lys Thr Leu Ala Lys Gly Val Pro Ser Arg Phe Ser 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

    Pro

    Glu Leu Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 His Tyr Glu Ser Pro 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

    <210> 18 <211> 330 <212> PRT <213> Homo Sapian <400> 18

    Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

    Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

    Leu

    180

    185

    190

    His Asn Gln Asp 195 Trp Leu Lys Ala Leu Pro Ala Gly 210 Gln Pro Arg Glu Pro Glu 225 240 Leu Thr Lys Asn Gln Tyr 245 Pro Ser Asp Ile Ala Asn 260 Asn Tyr Lys Thr Thr Phe 275 Leu Tyr Ser Lys Leu Asn 290 Val Phe Ser Cys Ser Thr 305 320 Gln Lys Ser Leu Ser 325

    Asn Gly Lys 200 Glu Tyr Pro Ile 215 Glu Lys Thr Gln 230 Val Tyr Thr Leu Val Ser Leu Thr Cys 250 Val Glu Trp Glu 265 Ser Pro Pro Val 280 Leu Asp Thr Val 295 Asp Lys Ser Val 310 Met His Glu Ala Leu Ser Pro Gly Lys 330

    Lys Cys Lys 205 Val Ser Ile Ser 220 Lys Ala Lys Pro 235 Pro Ser Arg Asp Leu Val Lys Gly Phe 255 Asn Gly Gln Pro 270 Glu Ser Asp Gly 285 Ser Phe Arg Trp 300 Gln Gln Gly Leu His Asn His Tyr

    315 <210> 19 <211> 326 <212> PRT <213> Homo Sapian <400> 19

    Ala Arg 1 Ser Thr Lys Gly 5 Pro Ser Val Phe Pro 10 Leu Ala Pro Cys Ser 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

    Asn

    165 170 175

    Ser Trp Thr Phe Arg 180 Val Val Ser Val Leu 185 Thr Val Val His Gln 190 Asp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

    Leu

    305 310 315

    320

    Ser Leu Ser Pro Gly Lys 325 <210> 20 <211> 377 <212> PRT <213> Homo Sapian <400> 20

    Ala Arg 1 Ser Thr Lys Gly 5 Pro Ser Val Phe Pro 10 Leu Ala Pro Cys Ser 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

    165

    170

    175

    Pro Val Lys Asp Thr 180 Leu Met Ile Ser Arg 185 Thr Pro Glu Val Thr 190 Cys Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

    Ile

    340

    345

    350

    Phe Gln Ser Cys 355 Ser Val Met His Glu 360 Ala Leu His Asn Arg 365 Phe Thr Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 21 <211> 327 <212> PRT <213> Homo Sapian <400> 21 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

    Lys

    115 120 125

    Asp Val Thr 130 Leu Met Ile Ser Arg 135 Thr Pro Glu Val Thr 140 Cys Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285

    Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 22 <211> 107 <212> PRT <213> Homo Sapian <400> 22 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

    100

    105 <210> 23 <211> 105 <212> PRT <213> Homo Sapian <400> 23

    Gln Glu 1 Pro Lys Ala Ala 5 Pro Ser Val Thr Leu 10 Phe Pro Pro Ser Ser 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90 95 Lys Thr Val Ala Pro Thr Glu Cys Ser

    100 105 <210> 24 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-IgG1 <400> 24 Gln Val Gln Leu Glu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15

    Thr Asp Leu Ser Leu 20 Thr Cys Ala Val Ser 25 Gly His Ser Ile Ser 30 His His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190

    Ser Pro Ser Leu 195 Gly Thr Gln Thr Tyr 200 Ile Cys Asn Val Asn 205 His Lys Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

    Thr

    355 360 365

    Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380

    Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    Ala Leu His 435

    Asn His Tyr Thr

    Gln Lys 440

    Ser Leu

    Ser Leu 445

    Ser

    Pro <210> 25 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3(High pI)-IgG1 <400> 25 Pro Gly 10 Leu Val Lys Pro Ser 15 Gln Gln 1 Val Gln Leu Gln 5 Glu Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45

    Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60

    Lys Tyr 65 Gly Arg Val Thr Leu Gln Met Asn Ser Cys 85 Ala Arg Ser Leu Ala Gly 100 Thr Leu Val Thr Val Phe 115 Pro Leu Ala Pro Ser Leu 130 Gly Cys Leu Val Lys Trp 145 160 Asn Ser Gly Ala Leu Leu 165 Gln Ser Ser Gly Leu Ser 180 Ser Ser Leu Gly Thr Pro 195 Ser Asn Thr Lys Val Lys 210 Thr His Thr Cys Pro Pro 225 240

    Ile 70 Ser Arg Asp Asn Leu Arg Ala Glu Asp 90 Arg Thr Thr Ala 105 Met Ser Ser Ala 120 Ser Thr Ser Lys 135 Ser Thr Ser Asp 150 Tyr Phe Pro Glu Thr Ser Gly Val His 170 Tyr Ser Leu Ser 185 Ser Gln Thr Tyr 200 Ile Cys Asp Lys 215 Lys Val Glu Pro 230 Cys Pro Ala Pro

    Ser 75 Lys Asn Thr Leu Thr Ala Val Tyr Tyr 95 Asp Tyr Trp Gly 110 Gln Lys Gly Pro 125 Ser Val Gly Gly 140 Thr Ala Ala Pro 155 Val Thr Val Ser Thr Phe Pro Ala Val 175 Val Val Thr Val 190 Pro Asn Val Asn 205 His Lys Pro Lys 220 Ser Cys Asp Glu Leu Leu Gly Gly

    235

    Ser Ser Val Phe Leu Phe 245 Pro Pro Arg Thr Pro Glu Val Thr Cys Asp 260 Pro Glu Val Lys Phe Asn Trp Asn 275 Ala Lys Thr Lys Pro Arg Glu Val 290 295 Val Ser Val Leu Thr Val Leu Glu 305 310 320 Tyr Lys Cys Lys Val Ser Asn Lys 325 Thr Ile Ser Lys Ala Lys Gly Thr 340 Leu Pro Pro Ser Arg Asp Glu Thr 355 Cys Leu Val Lys Gly Phe Tyr Glu 370 375 Ser Asn Gly Gln Pro Glu Asn Leu 385 390 400 Asp Ser Asp Gly Ser Phe Phe Lys 405

    Pro Lys 250 Asp Thr Leu Met Ile 255 Val Val Asp Val Ser His Glu 265 270 Val Asp Gly Val Glu Val His 285 Gln Tyr Asn Ser Thr Tyr Arg 300 Gln Asp Trp Leu Asn Gly Lys 315 Ala Leu Pro Ala Pro Ile Glu 330 335 Pro Arg Glu Pro Gln Val Tyr 345 350 Thr Lys Asn Gln Val Ser Leu 365 Ser Asp Ile Ala Val Glu Trp 380 Tyr Lys Thr Thr Pro Pro Val 395 Tyr Ser Lys Leu Thr Val Asp 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 26 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-IgG1-F939 <400> 26

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110

    Thr Phe Leu Val 115 Thr Val Ser Ser Ala 120 Ser Thr Lys Gly Pro 125 Ser Val Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro 225 230 235 240 Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285

    Ala Val Lys 290 Thr Lys Pro Arg Glu 295 Glu Gln Tyr Asn Ser 300 Thr Tyr Arg Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Tyr His Val Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 27 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3(High_pI)-F939

    <400> 27 Gln Gln 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155

    160

    Asn Leu Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Ala Val 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro 225 230 235 240 Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

    Lys

    325 330 335

    Thr Thr Ile Ser Lys 340 Ala Lys Gly Gln Pro 345 Arg Glu Pro Gln Val 350 Tyr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Tyr His Val Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 28 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-IgG1-F1180 <400> 28

    Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

    Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30

    His Trp Ala Trp 35 Ser Trp Val Arg Gln 40 Pro Pro Gly Glu Gly 45 Leu Glu Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205

    Cys Leu Val Lys Glu

    370

    Ser Asn Thr Lys Val Lys

    210

    Thr His Thr Cys Pro

    Pro

    225

    240

    Ser Val Phe Leu Phe Ser

    245

    Arg Thr Pro Glu Val Asp

    260

    Pro Glu Val Lys Phe Asn

    275

    Ala Lys Thr Lys Pro Val

    290

    Val Ser Val Leu Thr

    Glu

    305

    320

    Tyr Lys Cys Lys Val Lys

    325

    Thr Ile Ser Lys Ala Thr

    340

    Leu Pro Pro Ser Arg Thr

    355

    Asp Lys 215 Lys Val Glu Pro 230 Cys Pro Ala Pro Pro Pro Lys Pro Lys 250 Thr Cys Val Val 265 Val Asn Trp Tyr 280 Val Asp Arg Glu 295 Glu Gln Tyr Val 310 Leu His Gln Asp Ser Asn Lys Ala Tyr 330 Lys Gly Gln Pro 345 Arg Asp Glu Leu 360 Thr Lys

    Gly Phe Tyr

    375

    Pro Ser Asp

    Pro Lys 220 Ser Cys Asp Glu 235 Leu Leu Gly Gly Asp Thr Leu Met Ile 255 Asp Val Ser His 270 Glu Gly Val Glu 285 Val His Asn Ser 300 Thr Tyr Arg Trp 315 Leu Asn Gly Lys Pro Ala Pro Ile Glu 335 Glu Pro Gln Val 350 Tyr Asn Gln Val 365 Ser Leu

    Ile Ala Val

    Glu Trp

    380

    Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Leu 430 His Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 29 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3(High pI)-F1180 <400> 29 Gly 10 Leu Val Lys Pro Ser 15 Gln Gln 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45

    Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60

    Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75

    Leu Cys Gln Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Val Tyr Tyr 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

    Ser

    245 250 255

    Arg Asp Thr Pro Glu 260 Val Thr Cys Val Val 265 Val Asp Val Ser His 270 Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Tyr Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu

    420

    425

    430

    Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 30 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-IgG1-F11 <400> 30

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125

    Pro Leu Leu 130 Ala Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly 140 Thr Ala Ala Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> <211> <212> <213> 31 447 PRT Artificial Sequence <220> <223> VH3(High_pI )-F11

    <400> 31

    Gln Gln 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

    Leu

    165 170 175

    Gln Ser Ser Ser Gly 180 Leu Tyr Ser Leu Ser 185 Ser Val Val Thr Val 190 Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

    340

    345

    350

    Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365

    Cys Glu Leu 370 Val Lys Gly Phe Tyr 375 Pro Ser Asp Ile Ala 380 Val Glu Trp Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 32 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> VL3-CK <400> 32

    Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Gly Ser Ala Ser Val 1 5 10 15

    Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Thr Asp Ile Ser Ser His 20 25 30

    Leu Asn Trp Tyr Gln Gln Lys Ile

    Pro Gly Lys Ala

    Pro

    Glu Leu Leu

    Tyr Gly Tyr 50 Gly Ser His Leu Leu 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

    <210> 33 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> VL3(High_pI)-CK <400> 33

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Thr Asp Ile Ser Ser His 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Ile Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

    Ala

    130 135 140

    Lys Gln 145 160 Val Gln Trp Lys Val 150 Asp Asn Glu Ser Val Thr Glu Gln Asp Ser Ser 165 Ser Thr Leu Thr Leu Ser Lys Ala Tyr 180 Ala Cys Glu Val Thr His Gln Gly Ser 195 200 Phe Asn 210 Arg Gly Glu Cys

    Ala Leu Gln Ser Gly Asn Ser

    155

    Lys Asp Ser Thr Tyr Ser Leu

    170 175

    Asp Tyr Glu Lys His Lys Val

    185 190

    Leu Ser Ser Pro Val Thr Lys

    205 <210> 34 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H54 <400> 34

    Gln Val Glu Gln Leu Gln Glu Ser Gly 1 5

    Thr Asp Leu Ser Leu 20 Thr Cys Ala Val Gln Ala Trp Ser Trp Val Arg Gln Trp 35 40 Ile Gly Tyr Ile Ser Tyr Ser Gly Leu 50 55 Lys Gly Arg Val Thr Ile Ser Arg

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Asp

    25 30

    Pro Pro Gly Glu Gly Leu Glu

    Ile Thr Asn Tyr Asn Pro Ser

    Asp Thr Ser Lys Asn Gln Phe

    Ser

    Leu Cys Lys Leu Ser Ser 85 Val Thr Ala Ala Asp 90 Thr Ala Ala Tyr Tyr 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

    Pro

    225 230 235

    240

    Ser Ser Val Phe Leu Phe 245 Pro Pro Arg Thr Pro Glu Val Thr Cys Asp 260 Pro Glu Val Lys Phe Asn Trp Asn 275 Ala Lys Thr Lys Pro Arg Glu Val 290 295 Val Ser Val Leu Thr Val Leu Glu 305 310 320 Tyr Lys Cys Lys Val Ser Asn Lys 325 Thr Ile Ser Lys Ala Lys Gly Thr 340 Leu Pro Pro Ser Arg Asp Glu Thr 355 Cys Leu Val Lys Gly Phe Tyr Glu 370 375 Ser Asn Gly Gln Pro Glu Asn Leu 385 390 400 Asp Ser Asp Gly Ser Phe Phe Lys 405

    Pro Lys 250 Asp Thr Leu Met Ile 255 Val Val Asp Val Ser His Glu 265 270 Val Asp Gly Val Glu Val His 285 Gln Tyr Asn Ser Thr Tyr Arg 300 Gln Asp Trp Leu Asn Gly Lys 315 Ala Leu Pro Ala Pro Ile Glu 330 335 Pro Arg Glu Pro Gln Val Tyr 345 350 Thr Lys Asn Gln Val Ser Leu 365 Ser Asp Ile Ala Val Glu Trp 380 Tyr Lys Thr Thr Pro Pro Val 395 Tyr Ser Lys Leu Thr Val Asp 410 415

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

    420 425 430

    Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 35 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> L28 <400> 35 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly Ser Glu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70 75 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

    Gly

    115

    120

    125

    Thr Ala Ala 130 Ser Val Val Cys Leu 135 Leu Asn Asn Phe Tyr 140 Pro Arg Glu Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 36 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H(WT) <400> 36

    Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15

    Thr Asp Leu Ser Leu 20 Thr Cys Thr Val Ser 25 Gly Tyr Ser Ile Thr 30 Ser His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu

    Trp

    Ile Leu Gly 50 Tyr Ile Ser Tyr Ser 55 Gly Ile Thr Thr Tyr 60 Asn Pro Ser Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

    Lys

    210

    215

    220

    Thr Pro 225 240 His Thr Cys Pro Pro 230 Cys Pro Ala Pro Glu 235 Leu Leu Gly Gly Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

    Leu

    385

    400

    390

    395

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 37 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> L(WT) <400> 37

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro

    Tyr

    Thr Ala Phe Gly Gln 100 Gly Thr Lys Val Glu 105 Ile Lys Arg Thr Val 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 38 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H <400> 38

    Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser His Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Leu Ser 30 Asn Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

    180 185

    Ser Val Val

    Thr Val

    190

    Pro

    Ser Pro Ser Leu 195 Gly Thr Gln Thr Tyr 200 Ile Cys Asn Val Asn 205 His Lys Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

    355

    360

    365

    Cys Glu Leu 370 Val Lys Gly Phe Tyr 375 Pro Ser Asp Ile Ala 380 Val Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Lys 405 410 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Glu 420 425 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 <210> 39 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Ab1L <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Gly

    Glu Trp

    Pro Val

    Val Asp

    415

    Met His

    430

    Ser Pro

    Ser Val

    Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly 20 25 30

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Gln Pro Pro Lys 45 Leu Leu Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser

    Gly

    50 55 60

    Ser Pro 65 Gly Ser Gly Thr Glu 70 Tyr Ala Leu Thr Ile 75 Ser Ser Leu Gln Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 40 <211> 444 <212> PRT <213> Artificial Sequence <220>

    <223> Ab2H <400> 40

    Gln Pro 1 Ser Val Glu Glu 5 Ser Gly Gly Arg Leu 10 Val Thr Pro Gly Thr 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Phe Asn 20 25 30 Met Asp Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Ile Gly 35 40 45 Ala Val Ser Thr Gly Gly Ser Ala Tyr Tyr Ala Lys Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Ala Val Asp Leu Lys Ile Thr 65 70 75 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Val Asp 85 90 95 Ser Ser Gly Trp Gly Tyr Phe Asp Leu Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

    Gly

    145 150 155

    160

    Ala Ser Leu Thr Ser Gly 165 Val His Thr Phe Pro 170 Ala Val Leu Gln Ser 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

    Ser

    325 330 335

    Lys Pro Ala Lys Gly 340 Gln Pro Arg Glu Pro 345 Gln Val Tyr Thr Leu 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440

    <210> 41 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Ab2L <400> 41 Ala Ile Glu Met Thr Gln Thr Pro Phe Ser Val Gly 1 5 10

    Ser Ala Ala

    Val

    Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Asn Ile Tyr Ser Ser 20 25 30

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Gln Pro Pro Lys 45 Leu Leu Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Phe Ile Ser Ser 85 90 95 Thr Asp Phe Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205

    IS

    Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 42 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab3H <400> 42

    Gln Ser 1 Ser Leu Glu Glu 5 Ser Gly Gly Asp Leu 10 Val Lys Pro Gly Ala 15 Leu Thr Leu Thr Cys Thr Thr Ser Val Phe Asp Leu Ser Ser Tyr Tyr 20 25 30 Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ala Cys Val Tyr Ala Thr Ser Gly Ser Thr Asp Tyr Ala Thr Trp Ala 50 55 60 Lys Gly Arg Leu Thr Ile Ser Lys Ala Ser Ser Thr Thr Val Thr Leu 65 70 75 Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Gly Gly Ile Tyr Gly Gly Asp Gly Phe Asn Leu Trp Gly Pro Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

    Leu

    130 135 140

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 43 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> Ab3L <400> 43

    Ala Gly 1 Phe Glu Leu Thr 5 Gln Thr Pro Ala Ser 10 Val Glu Ala Ala Val 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Ser Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Thr Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Asp Pro Ser Ile Ile 85 90 95 Asp Gly Phe Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

    Tyr

    165 170 175

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu His

    180 185 190

    Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Val

    195 200 205

    Lys

    Pro

    Thr Lys Ser 210

    Phe Asn Arg Gly Glu 215

    Cys

    <210> 44 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> hGPC3-derived peptide <400> 44 Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro Asn 1 5 10

    Lys

    Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser Lys 20 25 <210> 45 <400> 000 45 <210> 46 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> VH3-IgG1m <400> 46 Gln Val Glu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 1 5 10

    Pro

    Pro

    Asp

    Leu

    Thr Leu Ser Leu Thr Cys Ala Val Ser Asp

    Gly His Ser Ile Ser

    Ser

    His

    His Trp Ala Trp 35 Ser Trp Val Arg Gln 40 Pro Pro Gly Glu Gly 45 Leu Glu Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

    195 200 205

    Ser Lys Asn 210 Thr Lys Val Asp Lys 215 Lys Val Glu Pro Lys 220 Ser Cys Asp Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

    Glu

    370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 47 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> VH3-YTE <400> 47 Gln Val Glu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser

    1 5 10 15

    Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp

    20 25 30

    His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp

    35 40 45

    Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

    50 55 60

    Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

    Leu Cys Gln Met Asn Ser 85 Leu Arg Ala Glu Asp 90 Thr Ala Val Tyr Tyr 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile

    Thr

    245 250 255 Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Ala Leu His 435 Asn His Tyr Thr

    Phe Ser Cys Ser Val Met His

    425 430

    Lys Ser Leu Ser Leu Ser Pro 445 <210> 48 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-LS <400> 48

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Thr Leu Ser Leu Thr Cys Ala Asp 20 His Ala Trp Ser Trp Val Arg Trp 35 Ile Gly Phe Ile Ser Tyr Ser Leu 50 55 Gln Gly Arg Val Thr Ile Ser Tyr 65 70 Leu Gln Met Asn Ser Leu Arg Cys 85 Ala Arg Ser Leu Ala Arg Thr Gly 100 Thr Leu Val Thr Val Ser Ser Phe

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly His Ser Ile Ser His

    25 30

    Pro Pro Gly Glu Gly Leu Glu

    Ile Thr Asn Tyr Asn Pro Ser

    Asp Asn Ser Lys Asn Thr Leu

    75 80

    Glu Asp Thr Ala Val Tyr Tyr

    90 95

    Ala Met Asp Tyr Trp Gly Glu

    105 110

    Ser Thr Lys Gly Pro Ser Val

    115 120 125

    Pro Leu Leu 130 Ala Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly 140 Thr Ala Ala Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425 430 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 49 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-N434H <400> 49

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

    Leu

    165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335

    Thr Thr Ile Ser Lys 340 Ala Lys Gly Gln Pro 345 Arg Glu Pro Gln Val 350 Tyr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His His His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 50 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3- F1847m <400> 50 Gln Val Gln Glu Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu

    Trp

    Ile Leu Gly 50 Phe Ile Ser Tyr Ser 55 Gly Ile Thr Asn Tyr 60 Asn Pro Ser Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

    Lys

    210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380

    Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro

    435 440 445 <210> 51 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-F1848m <400> 51 Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

    Cys

    Ala Gly Arg Ser Leu 100 Ala Arg Thr Thr Ala 105 Met Asp Tyr Trp Gly 110 Glu Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255

    Arg Asp Thr Pro Glu 260 Val Thr Cys Val Val 265 Val Asp Val Ser His 270 Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    Ala Leu His Ala His Val Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445 <210> 52 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-F1886m <400> 52

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

    Leu

    130 135 140

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425 430 Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445

    <210> 53 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> VH3-F1889m <400> 53

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    100

    Gln Ser Ser Ser Gly 180 Leu Tyr Ser Leu Ser 185 Ser Val Val Thr Val 190 Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350

    101

    Leu Thr Pro Pro 355 Ser Arg Glu Glu Met 360 Thr Lys Asn Gln Val 365 Ser Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425 430 Ala Leu His Ala His Val Thr Arg Lys Glu Leu Ser Leu Ser Pro

    435 440 445 <210> 54 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-F1927m Gly Pro Gly 10 Leu Val Lys Pro Ser 15 <400> 54 Leu Gln 5 Glu Ser Gln Glu 1 Val Gln Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45

    102

    Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu

    50 55 60

    Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

    65 70 75 80

    Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

    85 90 95

    Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly

    100 105 110

    Thr Leu Val Thr Val Phe

    115

    Ser Ser Ala

    Ser Thr Lys

    Gly Pro

    Ser Val

    120

    125

    Pro Leu Ala Pro Leu

    130

    Ser Ser Lys

    135

    Ser Thr

    Ser

    Gly Gly Thr Ala

    Ala

    140

    Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Trp 145 150 155 160

    Ser

    Asn Ser Gly Ala Leu Thr Ser Gly Val Leu

    165

    His

    170

    Thr

    Phe

    Pro Ala Val

    175

    Gln Ser Ser Gly Leu Tyr Ser

    180

    Ser Leu Ser

    185

    Ser Val Val

    Thr Val

    Pro

    190

    Ser

    Pro

    Ser Leu Gly Thr

    Gln Thr

    Tyr

    195

    200

    Ile Cys Asn Val Asn His Lys

    205

    Ser Asn Thr Lys Lys

    210

    Val Asp Lys

    215

    Lys Val Glu Pro

    Lys

    220

    Ser Cys Asp

    103

    Thr Pro 225 240 His Thr Cys Pro Pro 230 Cys Pro Ala Pro Glu 235 Leu Leu Gly Gly Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

    Leu

    385 390 395

    400

    104

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Leu 430 His Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

    435 440 445 <210> 55 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-F1168m <400> 55 Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    105

    Ala Gly Arg Ser Leu 100 Ala Arg Thr Thr Ala 105 Met Asp Tyr Trp Gly 110 Glu Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

    Asp

    260 265 270

    106

    Pro Asn Glu Val 275 Lys Phe Asn Trp Tyr 280 Val Asp Gly Val Glu 285 Val His Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445

    107 <210> 56 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> OHBH-IgG1 <400> 56

    Gln Glu 1 Val Thr Leu Lys 5 Thr Leu Thr Leu Thr Phe 20 Asn Met Asp Trp Val Ile 35 Gly Ala Val Ser Thr Lys 50 Gly Arg Phe Thr Ile Leu 65 Thr Ile Thr Asn Met Ala 85 Arg Val Asp Ser Ser Gly 100 Thr Leu Val Thr Val Phe 115 Pro Leu Ala Pro Ser Leu 130

    Glu Ser Gly Gly Arg 10 Cys Thr Val Ser 25 Gly Arg Gln Pro 40 Pro Gly Gly Gly 55 Ser Ala Tyr Ser 70 Lys Asp Thr Ser Asp Pro Val Asp Thr 90 Gly Trp Gly Tyr 105 Phe Ser Ser Ala 120 Ser Thr Ser Lys 135 Ser Thr Ser

    Leu Val Lys Pro Thr 15 Phe Ser Leu Ser 30 Ser Lys Gly Leu 45 Glu Trp Tyr Ala 60 Lys Trp Ala Lys 75 Asn Gln Val Val Ala Thr Tyr Phe Cys 95 Asp Leu Trp Gly 110 Gln Lys Gly Pro 125 Ser Val Gly Gly 140 Thr Ala Ala

    108

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

    Glu

    305 310 315

    320

    109

    Tyr Lys Lys Cys Lys Val 325 Ser Asn Lys Ala Leu 330 Pro Ala Pro Ile Glu 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 57 <211> 218 <212> PRT <213> Artificial Sequence <220>

    <223> OHBL-CK <400> 57

    Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Gly Ser Ala Ser Val 1 5 10 15

    110

    Asp Ser Arg Val Thr 20 Ile Thr Cys Gln Ala 25 Ser Glu Asn Ile Tyr 30 Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Ser Tyr Tyr Phe Ile Ser Ser 85 90 95 Thr Asp Phe Asn Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

    Lys

    180 185 190

    111

    His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

    195 200 205

    Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 58 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> OHBH-LS <400> 58

    Gln Glu 1 Val Thr Leu Lys 5 Glu Ser Gly Gly Arg 10 Leu Val Lys Pro Thr 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Phe 20 25 30 Asn Met Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ala Val Ser Thr Gly Gly Ser Ala Tyr Tyr Ala Lys Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu 65 70 75 Thr Ile Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Val Asp Ser Ser Gly Trp Gly Tyr Phe Asp Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

    112

    Phe

    115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285

    113

    Ala Val Lys 290 Thr Lys Pro Arg Glu 295 Glu Gln Tyr Asn Ser 300 Thr Tyr Arg Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425 430 Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 59 <211> 447 <212> PRT <213> Artificial Sequence

    114 <220>

    <223> OHBH-N434A

    <400> 59 Gln Glu 1 Val Thr Leu Lys 5 Glu Ser Gly Gly Arg 10 Leu Val Lys Pro Thr 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Phe 20 25 30 Asn Met Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ala Val Ser Thr Gly Gly Ser Ala Tyr Tyr Ala Lys Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu 65 70 75 Thr Ile Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Val Asp Ser Ser Gly Trp Gly Tyr Phe Asp Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155

    160

    115

    Asn Leu Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Ala Val 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335

    116

    Thr Thr Ile Ser Lys 340 Ala Lys Gly Gln Pro 345 Arg Glu Pro Gln Val 350 Tyr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 60 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> OHBH-F1847m <400> 60 Gln Val Glu Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr 1 5 10 15

    Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Phe

    20 25 30

    117

    Asn

    Ile

    Gly

    Lys

    Gly

    Leu

    Met Asp Trp Val Arg Gln Pro

    35 40

    Ala Val Ser Thr Gly Gly Ser

    50 55

    Arg Phe Thr Ile Ser Lys Asp

    Pro Gly Lys Gly Leu 45 Glu Trp Ala Tyr Tyr Ala Lys Trp Ala 60 Thr Ser Lys Asn Gln Val Val 75 80

    Thr

    Ala

    Ile Thr Asn Met Asp

    Pro Val Asp

    Thr Ala Thr Tyr

    Phe Cys

    Arg

    Gly

    Val Asp Ser

    Ser Gly Trp Gly Tyr

    Phe Asp Leu Trp

    Gly Gln

    100

    105

    110

    Thr

    Phe

    Leu Val

    Thr Val

    Ser Ser Ala

    Ser Thr Lys

    Gly Pro

    Ser Val

    115

    120

    125

    Pro

    Leu

    Leu Ala Pro

    Ser Ser Lys

    Ser Thr

    Ser

    Gly Gly Thr Ala

    Ala

    130

    135

    140

    Gly

    Trp

    145

    160

    Cys

    Leu Val

    Lys Asp Tyr

    Phe

    Pro Glu

    Pro Val

    Thr Val

    Ser

    150

    155

    Asn

    Leu

    Gln

    Ser

    Ser

    Pro

    Ser Gly Ala Leu 165 Thr Ser Gly Ser Ser Gly Leu Tyr Ser Leu 180 Ser Leu Gly Thr Gln Thr Tyr 195 200

    Val His Thr Phe Pro Ala Val 170 175 Ser Ser Val Val Thr Val Pro 185 190 Ile Cys Asn Val Asn His Lys 205

    118

    Cys Leu Val Lys Glu

    370

    Ser Asn Thr Lys Val Lys

    210

    Thr His Thr Cys Pro

    Pro

    225

    240

    Ser Val Phe Leu Phe Ser

    245

    Arg Thr Pro Glu Val Asp

    260

    Pro Glu Val Lys Phe Asn

    275

    Ala Lys Thr Lys Pro Val

    290

    Val Ser Val Leu Thr

    Glu

    305

    320

    Tyr Lys Cys Lys Val Lys

    325

    Thr Ile Ser Lys Ala Thr

    340

    Leu Pro Pro Ser Arg Thr

    355

    Asp Lys 215 Lys Val Glu Pro 230 Cys Pro Ala Pro Pro Pro Lys Pro Lys 250 Thr Cys Val Val 265 Val Asn Trp Tyr 280 Val Asp Arg Glu 295 Glu Gln Tyr Val 310 Leu His Gln Asp Ser Asn Lys Ala Leu 330 Lys Gly Gln Pro 345 Arg Glu Glu Met 360 Thr Lys

    Gly Phe Tyr

    375

    Pro Ser Asp

    Pro Lys 220 Ser Cys Asp Glu 235 Leu Leu Gly Gly Asp Thr Leu Met Ile 255 Asp Val Ser His 270 Glu Gly Val Glu 285 Val His Asn Ser 300 Thr Tyr Arg Trp 315 Leu Asn Gly Lys Pro Ala Pro Ile Glu 335 Glu Pro Gln Val 350 Tyr Asn Gln Val 365 Ser Leu

    Ile Ala Val

    Glu Trp

    380

    119

    Ser Asn Gly Gln Leu Pro Glu Asn 385 390 400

    Asp Lys Ser Asp Gly Ser 405 Phe Phe Ser Glu Arg Trp Gln 420 Gln Gly Asn Ala Leu His 435 Ala His Thr Thr

    Tyr Lys Thr Thr Pro Pro Val

    395

    Tyr Ser Lys Leu Thr Val Asp

    410 415

    Phe Ser Cys Ser Val Met His

    425 430

    Lys Glu Leu Ser Leu Ser Pro 445 <210> 61 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> OHBH-F1848m <400> 61

    Gln Glu 1 Val Thr Leu Lys 5 Glu Ser Thr Leu Thr Leu Thr Cys Thr Phe 20 Asn Met Asp Trp Val Arg Gln Ile 35 Gly Ala Val Ser Thr Gly Gly Lys 50 55 Gly Arg Phe Thr Ile Ser Lys Leu 65 70

    Gly Arg Leu Val Lys Pro Thr

    10 15

    Ser Gly Phe Ser Leu Ser Ser

    25 30

    Pro Gly Lys Gly Leu Glu Trp

    Ala Tyr Tyr Ala Lys Trp Ala

    Thr Ser Lys Asn Gln Val Val

    75 80

    120

    Thr Ala Ile Thr Asn Met 85 Asp Pro Val Asp Thr 90 Ala Thr Tyr Phe Cys 95 Arg Val Asp Ser Ser Gly Trp Gly Tyr Phe Asp Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255

    121

    Arg Asp Thr Pro Glu 260 Val Thr Cys Val Val 265 Val Asp Val Ser His 270 Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    122

    Ala Leu His Ala His Val Thr 435

    Lys Glu Leu Ser Leu Ser Pro 445 <210> 62 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> OHBH-F1886m <400> 62

    Gln Glu 1 Val Thr Leu Lys 5 Glu Ser Thr Leu Thr Leu Thr Cys Thr Phe 20 Asn Met Asp Trp Val Arg Gln Ile 35 Gly Ala Val Ser Thr Gly Gly Lys 50 55 Gly Arg Phe Thr Ile Ser Lys Leu 65 70 Thr Ile Thr Asn Met Asp Pro Ala 85 Arg Val Asp Ser Ser Gly Trp Gly 100 Thr Leu Val Thr Val Ser Ser Phe 115

    Gly Arg Leu Val Lys Pro Thr

    10 15

    Ser Gly Phe Ser Leu Ser Ser

    25 30

    Pro Gly Lys Gly Leu Glu Trp

    Ala Tyr Tyr Ala Lys Trp Ala

    Thr Ser Lys Asn Gln Val Val

    75 80

    Asp Thr Ala Thr Tyr Phe Cys

    90 95

    Tyr Phe Asp Leu Trp Gly Gln

    105 110

    Ser Thr Lys Gly Pro Ser Val

    125

    123

    Pro Leu Leu 130 Ala Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly 140 Thr Ala Ala Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    124

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425 430 Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445

    <210> 63 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> OHBH-F1889m <400> 63

    125

    Gln Glu 1 Val Thr Leu Lys 5 Glu Ser Gly Gly Arg 10 Leu Val Lys Pro Thr 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Phe 20 25 30 Asn Met Asp Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ala Val Ser Thr Gly Gly Ser Ala Tyr Tyr Ala Lys Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu 65 70 75 Thr Ile Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Val Asp Ser Ser Gly Trp Gly Tyr Phe Asp Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    126

    Gln Ser Ser Ser Gly 180 Leu Tyr Ser Leu Ser 185 Ser Val Val Thr Val 190 Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350

    127

    Leu Thr Pro Pro 355 Ser Arg Glu Glu Met 360 Thr Lys Asn Gln Val 365 Ser Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425 430 Ala Leu His Ala His Val Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445

    <210> 64 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> OHBH-F1927m <400> 64 Gln Val Glu Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr 1 5 10 15

    Thr Leu Thr Leu Thr Cys Thr Val Phe

    Ser Gly Phe

    Ser Leu Ser

    Ser

    Asn Met Asp Trp Val Arg Gln Ile

    Pro Pro

    Gly Lys

    Gly Leu Glu Trp

    128

    Gly Lys Ala 50 Val Ser Thr Gly Gly 55 Ser Ala Tyr Tyr Ala 60 Lys Trp Ala Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu 65 70 75 Thr Ile Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Val Asp Ser Ser Gly Trp Gly Tyr Phe Asp Leu Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220

    129

    Thr Pro 225 240 His Thr Cys Pro Pro 230 Cys Pro Ala Pro Glu 235 Leu Leu Gly Gly Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

    Leu

    385 390 395

    130

    400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Leu 430 His Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro

    435 440 445 <210> 65 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> VH3-F1718 <400> 65

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

    131

    Ala Gly Arg Ser Leu 100 Ala Arg Thr Thr Ala 105 Met Asp Tyr Trp Gly 110 Glu Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

    Asp

    260 265 270

    132

    Pro Asn Glu Val 275 Lys Phe Asn Trp Tyr 280 Val Asp Gly Val Glu 285 Val His Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Tyr His Val Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445

    133

    <210> 66 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> IL8 molecule <400> 66 Ala Val Leu Pro Arg Ser Ala Lys Glu Leu Arg Cys Gln Cys Ile Lys 1 5 10 15

    Thr Tyr Ser Lys Pro Phe His Pro Lys Phe Ile Lys Glu Leu Arg Val

    20 25 30

    Ile Glu Ser Gly Pro His Cys Ala Asn Thr Glu Ile Ile Val Lys Leu

    35 40 45

    Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn Trp Val Gln

    50 55 60

    Arg Val Val Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser 75

    65 70 <210> 67 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HVR-H1of WS4 <400> 67 Asp Tyr Tyr Leu Ser 1 5 <210> 68 <211> 19 <212> PRT <213> Artificial Sequence <220>

    134 <223> HVR-H2 of WS4 <400> 68

    Leu Ser 1 Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala 15 5 10 Val Lys Gly

    <210> 69 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 of WS4 <400> 69 Glu Asn Tyr Arg Tyr Asp Val Glu Leu Ala Tyr 1 5 10 <210> 70 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HVR-L1 of WS4 <400> 70 Arg Ala Ser Glu Ile Ile Tyr Ser Tyr Leu Ala 1 5 10

    <210> 71 <211> 7 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L2 of WS4 <400> 71

    Asn Ala Lys Thr Leu Ala Asp 1 5 <210> 72

    135 <211> 9 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L3 of WS4 <400> 72

    Gln His His Phe Gly Phe Pro Arg Thr 1 5 <210> 73 <211> 19 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H2 of H1009 <400> 73

    Leu Ile Arg Asn Lys Asp Asn Tyr His Thr Pro Glu Tyr Ser Ala Ser

    1 5 10 15

    Val Lys Gly <210> 74 <211> 11 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H3 of H1009 <400> 74

    Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr

    1 5 <210> <211> <212> <213> 75 7 PRT Artificial Sequence <220> <223> HVR-L2 of L395 <400> 75

    136

    Lys Ala Lys Thr His Ala Asp 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L3 of L395 <400> 76

    Lys His His Phe Gly Phe Pro Arg Thr 1 5 <210> 77 <211> 122 <212> PRT <213> Artificial Sequence <220>

    <223> Heavy chain variable region of Hr9 <400> 77

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95

    137

    Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120

    <210> 78 <211> 122 <212> PRT <213> Artificial Sequence <220>

    <223> Heavy chain variable region of H1009 <400> 78

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Arg Asn Lys Asp Asn Tyr His Thr Pro Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Thr Met Ser Asp Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110

    138

    Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 79 <211> 107 <212> PRT <213> Artificial Sequence <220>

    <223> Light chain variable region of L395 <400> 79

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ile Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Lys Thr His Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Ile Ala Thr Tyr Tyr Cys Lys His His Phe Gly Phe Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

    <210> 80 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H1009-F1974m

    139 <400> 80

    Gln Val Gly 1 Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Arg Asn Lys Asp Asn Tyr His Thr Pro Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Thr Met Ser Asp Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175

    140

    Ala Thr Val Leu Gln 180 Ser Ser Gly Leu Tyr 185 Ser Leu Ser Ser Val 190 Val Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg 225 230 235 240 Arg Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

    141

    340

    345

    350

    Val Val Tyr Thr 355 Leu Pro Pro Ser Arg 360 Glu Glu Met Thr Lys 365 Asn Gln Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Leu His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu 435 440 445

    Ser Pro 450

    <210> 81 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H1009-F1886s <400> 81 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

    20 25 30

    142

    Tyr Val Leu Ser 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Gly Leu Ile Arg Asn Lys Asp Asn Tyr His Thr Pro Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Thr Met Ser Asp Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205

    143

    His Ser Lys 210 Pro Ser Asn Thr Lys 215 Val Asp Lys Lys Val 220 Glu Pro Lys Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg 225 230 235 240 Arg Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

    144

    370

    375

    380

    Glu Trp Pro Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Leu His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu 435 440 445 Ser Pro 450 <210> 82 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> L395 -k0MT <400> 82 Asp Ile Gly Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ile Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Lys Thr His Ala Asp Gly Val Pro Ser Arg Phe Ser

    Gly

    50 55 60

    145

    Ser Pro 65 Gly Ser Gly Thr Asp 70 Phe Thr Phe Thr Ile 75 Ser Ser Leu Gln Glu Asp Ile Ala Thr Tyr Tyr Cys Lys His His Phe Gly Phe Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 83 <211> 450 <212> PRT <213> Artificial Sequence

    146 <220>

    <223> hWS4H-IgG1

    <400> 83 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Glu Gly 1 Val Gln Leu Leu 5 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr 65 70 75 Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155

    160

    147

    Val Pro Ser Trp Asn Ser 165 Gly Ala Leu Thr Ser 170 Gly Val His Thr Phe 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335

    148

    Ile Gln Glu Lys Thr 340 Ile Ser Lys Ala Lys 345 Gly Gln Pro Arg Glu 350 Pro Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450 <210> 84 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> hWS4L-k0MT <400> 84

    Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

    1 5 10 15

    149

    Asp Tyr Arg Val Thr 20 Ile Thr Cys Arg Ala 25 Ser Glu Ile Ile Tyr 30 Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His His Phe Gly Phe Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

    Tyr

    180 185 190

    150

    Ala Ser Cys Glu 195 Val Thr His Gln Gly 200 Phe Asn 210 Arg Gly Glu Cys

    Leu Ser Ser Pro Val Thr Lys

    205

    <210> <211> <212> <213> 85 450 PRT Artificial Sequence <220> <223> Hr9-IgG1 <400> 85 Gln Val Gln Leu Val Glu Ser Gly Gly 1 5

    Ser Leu Arg Leu Ser Cys Ala Ala Tyr

    Tyr Leu Ser Trp Val Arg Gln Ala Val

    35 40

    Gly Leu Ile Arg Asn Lys Ala Asn Ala

    50 55

    Ser Val Lys Gly Arg Phe Thr Ile Ser

    65 70

    Leu Tyr Leu Gln Met Ser Ser Leu Tyr

    Tyr Cys Ala Arg Glu Asn Tyr Arg Trp

    100

    Gly Gly Leu Val Gln Pro Gly

    10 15

    Ser Gly Phe Thr Phe Ser Asp

    25 30

    Pro Gly Lys Gly Leu Glu Trp

    Gly Tyr Thr Arg Glu Tyr Ser

    Ser Arg Asp Asp Ser Lys Asn

    Lys Thr Glu Asp Thr Ala Val

    90 95

    Tyr Asp Val Glu Leu Ala Tyr

    105 110

    Gly Gln Gly Thr

    Leu Val

    Thr

    Val Ser

    Ser Ala

    Ser Thr Lys

    Gly

    151

    Pro

    115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285

    152

    Val Thr His 290 Asn Ala Lys Thr Lys 295 Pro Arg Glu Glu Gln 300 Tyr Asn Ser Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450

    153 <210> 86 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> H89-IgG1 <400> 86

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

    154

    Thr

    145 150 155

    160

    Val Pro Ser Trp Asn Ser 165 Gly Ala Leu Thr Ser 170 Gly Val His Thr Phe 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

    Asn

    305 310 315

    320

    155

    Gly Lys Glu Tyr Lys Cys Lys Val Pro

    325

    Ser Asn Lys Ala Leu

    330

    Pro Ala

    335

    Ile Glu Lys Thr Ile Gln 340

    Ser Lys Ala Lys

    Gly Gln Pro Arg Glu

    Pro

    345

    350

    Val Tyr Thr Leu Pro Pro Val

    355

    Ser Arg Asp

    Glu Leu Thr

    Lys

    Asn Gln

    360

    365

    Ser Leu Thr Cys Val

    370

    Leu Val Lys

    375

    Gly Phe Tyr

    Pro

    Ser Asp

    380

    Ile Ala

    Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Pro 385 390 395 400

    Thr Thr

    Pro Val Leu Asp Ser Asp Gly Ser Phe Thr

    405

    Phe Leu Tyr Ser Lys

    410

    Leu

    415

    Val Val Asp Lys Ser 420 Arg Met His Glu Ala Leu Leu 435

    Trp Gln Gln Gly 425 Asn His Asn His Tyr Thr 440

    Val Phe Ser Cys 430 Ser Gln Lys Ser Leu Ser 445

    Ser Pro 450

    <210> 87 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> L63-k0MT <400> 87

    156

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ile Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Ala Lys Thr His Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His His Phe Gly Phe Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    157

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

    180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

    195 200 205

    Phe Asn Arg Gly Glu Cys 210 <210> 88 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> L118-k0MT <400> 88

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ile Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Ala Lys Thr His Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Ile Ala Thr Tyr Tyr Cys Lys His His Phe Gly Phe Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

    158

    100

    105

    110

    Pro Gly Ser Val 115 Phe Ile Phe Pro Pro 120 Ser Asp Glu Gln Leu 125 Lys Ser Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210

    <210> <211> <212> <213> 89 450 PRT Artificial Sequence <220> <223> H998-IgG1 <400> 89 Gln Val Gln Leu Val Glu Ser Gly 1 5

    Gly Gly Gly

    Leu Val

    Gln

    Pro

    Gly

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30

    159

    Tyr Val Leu Ser 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Leu Arg Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Thr Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn Tyr Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205

    160

    His Ser Lys 210 Pro Ser Asn Thr Lys 215 Val Asp Lys Lys Val 220 Glu Pro Lys Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

    161

    370

    375

    380

    Glu Trp Pro Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro 450 <210> 90 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H553 IgG1 <400> 90 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Gly His Thr Pro Glu Tyr Ser

    Ala

    50 55 60

    162

    Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

    65 70 75 80

    Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

    85 90 95

    Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp

    100 105 110

    Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

    115 120 125

    Ser Val Phe Thr

    130

    Pro Leu Ala Pro

    135

    Ser Ser Lys

    Ser Thr

    140

    Ser Gly Gly

    Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Thr 145 150 155 160

    Pro Glu

    Pro Val

    Val

    Pro

    Ser Trp Asn

    Ser Gly Ala Leu Thr

    Ser Gly Val

    His

    Thr

    Phe

    165

    170

    175

    Ala Val Leu Gln Ser Thr

    180

    Ser Gly Leu Tyr

    185

    Ser Leu Ser

    Ser Val Val

    190

    Val Pro Asn

    Ser Ser

    Ser Leu Gly Thr

    Gln Thr

    Tyr

    195

    200

    Ile Cys Asn Val

    205

    His Lys Ser

    210

    Pro

    Ser Asn Thr Lys

    Val Asp Lys

    Lys Val Glu Pro

    Lys

    215

    220

    Cys Asp Lys Thr His Thr Cys Leu 225 230

    Pro Pro Cys

    Pro Ala

    Pro Glu Leu

    235

    163

    240

    Gly Leu Gly Pro Ser Val 245 Phe Leu Phe Pro Pro 250 Lys Pro Lys Asp Thr 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

    164

    Thr

    405 Val Asp Lys Ser Arg Trp Gln Val 420 Met His Glu Ala Leu His Asn Leu 435

    410 415

    Gly Asn Val Phe Ser Cys Ser

    425 430

    Tyr Thr Gln Lys Ser Leu Ser

    445

    Ser Pro 450 <210> 91 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> H1004-IgG1m <400> 91

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Ser Leu Arg Leu Ser Cys Ala Tyr 20 Tyr Leu Ser Trp Val Arg Gln Val 35 Gly Leu Ile Arg Asn Lys Asp Ala 50 55 Ser Val Lys Gly Arg Phe Thr Ser 65 70 Leu Tyr Leu Thr Met Ser Asp Tyr 85

    Gly Gly Leu Val Gln Pro Gly

    10 15

    Ser Gly Phe Thr Phe Ser Asp

    25 30

    Pro Gly Lys Gly Leu Glu Trp

    Gly Tyr Thr Pro Glu Tyr Ser

    Ser Arg Asp Asp Ser Lys Asn

    75 80

    Lys Thr Glu Asp Thr Ala Val

    90 95

    165

    Tyr Trp Cys Ala Arg 100 Glu Asn His Arg Tyr 105 Asp Val Glu Leu Ala 110 Tyr Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

    166

    260

    265

    270

    His Glu Glu Asp 275 Pro Glu Val Lys Phe 280 Asn Trp Tyr Val Asp 285 Gly Val Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

    167

    Leu

    435

    440

    445

    Ser Pro 450

    <210> <211> <212> <213> 92 450 PRT Artificial Sequence <220> <223> H1009-IgG1m <400> 92 Gln Val Gly . Gln Leu Val Glu Ser Gly 1 5

    Ser Leu Arg Leu Ser Cys Ala Ala Tyr

    Tyr Leu Ser Trp Val Arg Gln Ala Val

    35 40

    Gly Leu Ile Arg Asn Lys Asp Asn Ala

    50 55

    Ser Val Lys Gly Arg Phe Thr Ile Ser

    65 70

    Leu Tyr Leu Thr Met Ser Asp Leu Tyr

    Tyr Cys Ala Arg Glu Asn His Arg Trp

    100

    Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Gly Phe Thr Phe Ser Asp 25 30 Pro Gly Lys Gly Leu Glu Trp 45 Tyr His Thr Pro Glu Tyr Ser 60 Ser Arg Asp Asp Ser Lys Asn 75 Lys Thr Glu Asp Thr Ala Val 90 95 Tyr Asp Val Glu Leu Ala Tyr 105 110

    Gly Gln Gly Thr Leu Val Pro

    Thr Val Ser

    Ser Ala

    Ser Thr Lys

    Gly

    115

    120

    125

    168

    Ser Thr Val 130 Phe Pro Leu Ala Pro 135 Ser Ser Lys Ser Thr 140 Ser Gly Gly Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

    169

    290

    295

    300

    Tyr Asn 305 320 Arg Val Val Ser Val 310 Leu Thr Val Leu His 315 Gln Asp Trp Leu Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450 <210> 93 <211> 450

    170 <212> PRT <213> Artificial Sequence <220>

    <223> 1009-F1942m <400> 93

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly Leu 10 Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Arg Asn Lys Asp Asn Tyr His Thr Pro Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Thr Met Ser Asp Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155

    171

    160

    Val Pro Ser Trp Asn Ser 165 Gly Ala Leu Thr Ser 170 Gly Val His Thr Phe 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Arg 225 230 235 240 Arg Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ala Asp Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

    172

    Pro

    325

    330

    335

    Ile Gln Glu Lys Thr 340 Ile Ser Lys Ala Lys 345 Gly Gln Pro Arg Glu 350 Pro Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450 <210> 94 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> H89-IgG1m <400> 94

    Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

    173

    Ser Tyr Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Asp Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

    174

    180

    185

    190

    Val Asn Pro Ser 195 Ser Ser Leu Gly Thr 200 Gln Thr Tyr Ile Cys 205 Asn Val His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

    175

    Val

    355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450

    <210> 95 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H89-F1168m <400> 95 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Tyr

    Ser Gly Phe Thr

    Phe

    Ser Asp

    Tyr Leu Ser Trp Val Arg Gln Val

    Ala Pro

    Gly Lys

    Gly Leu Glu Trp

    176

    Gly Ala Leu 50 Ile Arg Asn Lys Ala 55 Asn Gly Tyr Thr Arg 60 Glu Tyr Ser Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

    Ser

    177

    210

    215

    220

    Cys Leu 225 240 Asp Lys Thr His Thr 230 Cys Pro Pro Cys Pro 235 Ala Pro Glu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

    178

    Pro

    385

    400

    390

    395

    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415

    Val Val Asp Lys Ser 420 Arg Trp Gln Gln Gly 425 Asn Val Phe Ser Cys 430 Ser Met His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser

    Leu

    435 440 445

    Ser Pro 450

    <210> 96 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H89-F1847m <400> 96 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

    20 25 30

    Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

    35 40 45

    Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala

    50 55 60

    Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

    179

    Leu Tyr Tyr Leu Gln Met 85 Ser Ser Leu Lys Thr 90 Glu Asp Thr Ala Val 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

    180

    Leu

    245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415

    181

    Val Asp Lys Ser Arg Trp Gln Val

    420

    Met His Glu Ala Leu His Ala Leu

    435

    Gly Asn Val Phe Ser Cys Ser

    425 430

    Thr Thr Arg Lys Glu Leu Ser

    445

    Ser Pro 450 <210> 97 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> H89-F1848m <400> 97

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Ser Leu Arg Leu Ser Cys Ala Tyr 20 Tyr Leu Ser Trp Val Arg Gln Val 35 Gly Leu Ile Arg Asn Lys Ala Ala 50 55 Ser Val Lys Gly Arg Phe Thr Ser 65 70 Leu Tyr Leu Gln Met Ser Ser Tyr 85 Tyr Cys Ala Arg Glu Asn His Trp

    Gly Gly Leu Val Gln Pro Gly

    10 15

    Ser Gly Phe Thr Phe Ser Asp

    25 30

    Pro Gly Lys Gly Leu Glu Trp

    Gly Tyr Thr Arg Glu Tyr Ser

    Ser Arg Asp Asp Ser Lys Asn

    75 80

    Lys Thr Glu Asp Thr Ala Val

    90 95

    Tyr Asp Val Glu Leu Ala Tyr

    182

    100

    105

    110

    Gly Pro Gln Gly 115 Thr Leu Val Thr Val 120 Ser Ser Ala Ser Thr 125 Lys Gly Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

    183

    Glu

    275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Ala His Val Thr Arg Lys Glu Leu Ser Leu 435 440 445

    184

    Ser Pro 450

    <210> 98 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H89- F1886m <400> 98 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Gly Leu Ala Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser 50 55 60 Ser Val Ser Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn 65 70 75 Leu Tyr Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val 85 90 95 Tyr Cys Trp Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

    Thr

    185

    130

    135

    140

    Ala Thr 145 160 Ala Leu Gly Cys Leu 150 Val Lys Asp Tyr Phe 155 Pro Glu Pro Val Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

    186

    Asn

    305

    320

    310

    315

    Gly Pro Lys Glu Tyr Lys 325 Cys Lys Val Ser Asn 330 Lys Ala Leu Pro Ala 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Leu His Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu 435 440 445

    Ser Pro 450

    <210> 99 <211> 450 <212> PRT <213> Artificial Sequence <220>

    187 <223> H89-F1889m <400> 99

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

    188

    Pro

    165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335

    189

    Ile Gln Glu Lys Thr 340 Ile Ser Lys Ala Lys 345 Gly Gln Pro Arg Glu 350 Pro Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Leu His Glu Ala Leu His Ala His Val Thr Arg Lys Glu Leu Ser

    Leu

    435 440 445

    Ser Pro 450

    <210> 100 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H89-F1927m <400> 100 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg

    Leu Ser Cys Ala

    Ala Ser

    Gly Phe Thr Phe

    Ser Asp

    190

    Tyr

    Tyr Val Leu Ser 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Gly Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

    191

    Asn

    195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365

    192

    Ser Val Leu 370 Thr Cys Leu Val Lys 375 Gly Phe Tyr Pro Ser 380 Asp Ile Ala Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400

    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415

    Val Val Asp Lys Ser 420 Arg Trp Gln Gln Gly 425 Asn Val Phe Ser Cys 430 Ser Leu His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser

    Leu

    435 440 445

    Ser Pro 450

    <210> <211> <212> <213> 101 450 PRT Artificial Sequence <220> <223> H496-IgG1 <400> 101 Gln Val Gln Leu Val Glu Ser Gly 1 5

    Gly

    Gly Gly Leu Val

    Gln

    Pro

    Gly

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30

    Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

    Gly Leu

    Ile Arg Asn Lys Ala

    Asn Gly

    Tyr Thr

    Pro Glu Tyr

    Ser

    193

    Ala

    Ser Ser 65 Val Lys Gly Arg Phe 70 Thr Ile Ser Arg Asp 75 Asp Ser Lys Asn Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Glu Asn His Arg Tyr Asp Val Glu Leu Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

    194

    Leu

    225 230 235

    240

    Gly Leu Gly Pro Ser Val 245 Phe Leu Phe Pro Pro 250 Lys Pro Lys Asp Thr 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

    Pro

    385 390 395

    400

    195

    Pro Thr Val Leu Asp Ser 405 Asp Gly Val Asp Lys Ser Arg Trp Gln Val 420 Met His Glu Ala Leu His Asn Leu 435 Ser Pro

    450

    Ser Phe Phe 410 Leu Tyr Ser Lys Gln Gly Asn Val Phe Ser Cys 425 430 His Tyr Thr Gln Lys Ser Leu 440 445

    Leu

    415

    Ser

    Ser <210> 102 <211> 5 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H1 <400> 102

    Ser Tyr Gly Met Leu 1 5 <210> 103 <211> 17 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H2 <400> 103

    Asp Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Lys

    1 5 10

    Val

    Gly <210> 104 <211> 8 <212> PRT

    196

    <213> Artificial Sequence

    <220> <223> HVR-H3 <400> 104

    Asp Arg Ile Ala Val Ala Asp Tyr 1 5

    <210> <211> <212> <213> 105 12 PRT Artificial Sequence

    <220> <223> HVR-L1 <400> 105

    Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

    1 5 10 <210> <211> <212> <213> 106 7 PRT Artificial Sequence

    <220> <223> HVR-L2 <400> 106

    Gly Ala Ser Ser Arg Ala Thr

    1 5 <210> <211> <212> <213> 107 8 PRT Artificial Sequence

    <220> <223> HVR-L3 <400> 107

    Gln Gln Tyr Gly Ser Ser Phe Thr

    1 5 <210> <211> <212> 108 5 PRT

    197 <213> Artificial Sequence <220>

    <223> HVR-H1 <400> 108

    Asn Tyr Gly Met His 1 5 <210> 109 <211> 17 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H2 <400> 109

    Val Ile Tyr Phe Glu Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val Lys

    1 5 10 15

    Gly <210> 110 <211> 9 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H3 <400> 110

    Ser Pro Tyr Gly Asp Tyr Leu Asp Tyr 1 5 <210> 111 <211> 12 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L1 <400> 111

    Arg Ala Ser Gln Thr Ile Asp Tyr Asn Tyr Leu His 1 5 10

    198

    <210> <211> <212> <213> 112 7 PRT Artificial Sequence

    <220> <223> HVR-L2 <400> 112

    Gly Thr Phe Ile Arg Ala Thr

    1 5 <210> <211> <212> <213> 113 9 PRT Artificial Sequence

    <220> <223> HVR-L3 <400> 113

    Gln Gln Phe Gly Arg Ser Pro Leu Thr

    1 5 <210> <211> <212> <213> 114 5 PRT Artificial Sequence

    <220> <223> HVR-H1 <400> 114

    Ser Tyr Gly Met Leu 1 5

    <210> <211> <212> <213> 115 17 PRT Artificial Sequence

    <220> <223> HVR-H2 <400> 115

    Asp Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

    199

    Gly <210> 116 <211> 8 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H3 <400> 116

    Asp Arg Ile Ala Val Ala Asp Tyr 1 5 <210> 117 <211> 12 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L1 <400> 117

    Arg Ala Ser Gln Ser Val Ser Ser Ser Phe Leu Ala 1 5 10 <210> 118 <211> 7 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L2 <400> 118

    Gly Ala Ser Ser Arg Ala Thr

    1 5 <210> 119 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3

    200 <400> 119

    Gln Gln Tyr Asp Ser Ser Phe Thr 1 5 <210> 120 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HVR-H1 <400> 120 Gly Tyr 1 Tyr Trp Thr 5 <210> 121 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HVR-H2 <400> 121 Glu Val Ser Ile His His Gly Ser Thr Asn Tyr Ser Pro Ser Leu Lys 1 5 10 15 <210> 122 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 <400> 122 Gly Gly 1 Ala Ala Ala Ala Leu 5 Asp Ser

    <210> 123 <211> 17 <212> PRT <213> Artificial Sequence <220>

    201 <223> HVR-L1 <400> 123

    Lys Ser Ser Gln Ser Val Leu Phe Ser Ser Asn Asn Arg Lys Tyr

    Leu 1 5 10 15

    Ala

    <210> <211> <212> <213> 124 7 PRT Artificial Sequence

    <220> <223> HVR-L2 <400> 124

    Trp Ala Ser Thr Arg Glu Ser

    1 5 <210> <211> <212> <213> 125 9 PRT Artificial Sequence

    <220> <223> HVR-L3 <400> 125

    Gln Gln Tyr Tyr Ser Thr Pro Phe Thr

    1 5 <210> <211> <212> <213> 126 5 PRT Artificial Sequence

    <220> <223> HVR-H1 <400> 126

    Ser Tyr Trp Met His 1 5 <210> 127

    202 <211> 17 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H2 <400> 127

    Glu Ile Asp Pro Ser Asp Ser Asn Thr Asn Tyr Asn Gln Lys Phe Lys

    1 5 10 15

    Gly <210> 128 <211> 7 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H3 <400> 128

    Glu Leu Leu His Ala Val Tyr 1 5 <210> 129 <211> 11 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L1 <400> 129

    Thr Ala Ser Gln Asp Ile His Lys Tyr Ile Ser 1 5 10

    <210> 130 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 <400> 130

    203

    Thr Ser Thr Leu Gln Pro 1 5

    <210> <211> <212> <213> 131 8 PRT Artificial Sequence

    <220> <223> HVR-L3 <400> 131

    Leu Gln Tyr Asp Asn Leu Trp Thr

    1 5 <210> <211> <212> <213> 132 5 PRT Artificial Sequence

    <220> <223> HVR-H1 <400> 132

    Asn Tyr Trp Ile Val 1 5

    <210> <211> <212> <213> 133 17 PRT Artificial Sequence

    <220> <223> HVR-H2 <400> 133

    Asp Leu Tyr Ser Gly Gly Gly Tyr Thr Phe Tyr Ser Glu Asn Phe

    Lys 1 5 10 15

    Gly

    <210> <211> <212> <213> 134 10 PRT Artificial Sequence

    204 <220>

    <223> HVR-H3 <400> 134

    Ser Gly Tyr Asp Arg Thr Trp Phe Ala His 1 5 10 <210> 135 <211> 11 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L1 <400> 135

    Gln Ala Ser Gln Asp Ile Glu Ser Tyr Leu 1 5 10

    Ser <210> 136 <211> 7 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L2 <400> 136

    Tyr Ala Thr Arg Leu Ala Asp <210> 137 <211> 9 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L3 <400> 137

    Leu Gln His Gly Glu Ser Pro Pro Thr <210> 138 <211> 5 <212> PRT <213> Artificial Sequence

    205 <220>

    <223> HVR-H1 <400> 138

    His Tyr Gly Met Tyr 1 5 <210> 139 <211> 17 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H2 <400> 139

    Val Ile Trp Tyr Asp Gly Ser Tyr Glu Tyr Asn Ala Asp Ser Val Lys

    1 5 10 15

    Gly <210> 140 <211> 8 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-H3 <400> 140

    Asp Arg Val Gly Leu Phe Asp Tyr 1 5 <210> 141 <211> 12 <212> PRT <213> Artificial Sequence <220>

    <223> HVR-L1 <400> 141

    Arg Ala Ser Gln Ser Ile Ser Ser Ser Tyr Leu Ala 1 5 10

    206

    <210> <211> <212> <213> 142 7 PRT Artificial Sequence <220> <223> HVR-L2 <400> 142 Gly Pro Ser Ser Arg Ala Thr 1 5 <210> 143 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 143 Gln Gln Tyr Ala Gly Ser Leu 1 5 <210> 144 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H003 <400> 144 Gln Val Gln Leu Val Glu Ser Gly 1 5

    Thr

    Ser Leu Arg Leu Ser Cys Ala Arg

    Asp Met Thr Trp Val Arg Gln Val

    Ser Ile Ile Asn Thr Tyr Gly Ser

    50 55

    Gly Gly Gly Leu Val Gln Pro 10 Ala Ser Gly Phe Thr Leu Ser 25 30 Ala Pro Gly Lys Gly Leu Glu 40 45 Asn His Trp Tyr Ala Asn Trp 60

    Gly

    Asn

    Tyr

    Ala

    207

    Gly Leu 65 Arg Phe Thr Ile Ser 70 Arg Asp Asn Ser Lys 75 Asn Thr Val Tyr Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

    Pro

    225 230 235

    208

    240

    Ser Ser Val Phe Leu Phe 245 Pro Pro Lys Pro Lys 250 Asp Thr Leu Met Ile 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

    209

    Lys

    405

    410

    415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 145 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H005 <400> 145

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Arg Gly Lys Arg Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110

    210

    Thr Phe Leu Val 115 Thr Val Ser Ser Ala 120 Ser Thr Lys Gly Pro 125 Ser Val Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

    211

    275

    280

    285

    Ala Val Lys 290 Thr Lys Pro Arg Glu 295 Glu Gln Tyr Asn Ser 300 Thr Tyr Arg Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 146 <211> 447

    212 <212> PRT <213> Artificial Sequence <220>

    <223> H010 <400> 146

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly Leu 10 Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155

    213

    160

    Asn Leu Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Ala Val 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

    214

    Lys

    325

    330

    335

    Thr Thr Ile Ser Lys 340 Ala Lys Gly Gln Pro 345 Arg Glu Pro Gln Val 350 Tyr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 147 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H012 <400> 147

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn

    His

    215

    Asp Val Met Thr 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Tyr Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asn Arg Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

    216

    195 200 205

    Ser Lys Asn 210 Thr Lys Val Asp Lys 215 Lys Val Glu Pro Lys 220 Ser Cys Asp Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

    217

    Glu

    370 375 380

    Ser Leu 385 400 Asn Gly Gln Pro Glu 390 Asn Asn Tyr Lys Thr 395 Thr Pro Pro Val Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 148 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H013 <400> 148 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60

    Gly Arg Phe Thr Ile Ser Arg Leu

    Asp Asn

    Ser Lys Asn Thr Val Tyr

    218

    Gln Ala Ile Gly Arg Leu 85 Arg Ala Glu Asp Thr 90 Ala Thr Tyr Tyr Cys 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

    219

    Ser

    245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    220

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 149 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H014 <400> 149 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ser Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala 50 55 60 Gly Arg Leu Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 Gln Ile Ser Arg Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

    Phe

    221

    115 120 125

    Pro Leu Leu 130 Ala Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly 140 Thr Ala Ala Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    222

    Val

    290 295 300

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 150 <211> 447 <212> PRT <213> Artificial Sequence <220>

    223 <223> H016 <400> 150

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Gly Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

    224

    Leu

    165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335

    225

    Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Ser Asn Leu Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 151 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H018 <400> 151 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

    226

    Val

    Ser Ser Ile 50 Ile Asn Thr Tyr Gly 55 Asn His Trp Tyr Ala 60 Asn Trp Ala Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Lys 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

    227

    Lys

    210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380

    228

    Ser Asn Leu Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 152 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H026 <400> 152 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Arg Gly Lys Arg Leu Glu Tyr Val 35 40 45 Ser Ile Ser Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala 50 55 60 Gly Arg Leu Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Val Tyr 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

    229

    Ala

    Arg Gly Glu Thr His 100 His Gly Ser Ser Gly 105 Ile Asp His Trp Gly 110 Gln Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255

    230

    Arg Asp Thr Pro Glu 260 Val Thr Cys Val Val 265 Val Asp Val Ser His 270 Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    231

    Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 153 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H027 <400> 153

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Val Tyr Leu 65 70 75 Gln Ile Asn Arg Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

    232

    Leu

    130 135 140

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    233

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 154 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H028 <400> 154

    234

    Gln Val Gly 1 Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Val Tyr Leu 65 70 75 Gln Ile Gly Arg Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    235

    Gln Ser Ser Ser Gly 180 Leu Tyr Ser Leu Ser 185 Ser Val Val Thr Val 190 Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350

    236

    Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Ser Asn Leu Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 155 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H029 <400> 155 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45

    237

    Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser

    50 55 60

    Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Val Tyr Leu

    65 70 75 80

    Gln Ile Ser Arg Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

    85 90 95

    Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly

    100 105 110

    Thr Leu Val Thr Val Phe

    115

    Ser Ser Ala

    Ser Thr Lys

    Gly Pro

    Ser Val

    120

    125

    Pro Leu Ala Pro Leu

    130

    Ser Ser Lys

    135

    Ser Thr

    Ser

    Gly Gly Thr Ala

    Ala

    140

    Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Trp 145 150 155 160

    Ser

    Asn Ser Gly Ala Leu Thr Ser Gly Val Leu

    165

    His

    170

    Thr

    Phe

    Pro Ala Val

    175

    Gln Ser Ser Gly Leu Tyr Ser

    180

    Ser Leu Ser

    185

    Ser Val Val

    Thr Val

    Pro

    190

    Ser

    Pro

    Ser Leu Gly Thr

    Gln Thr

    Tyr

    195

    200

    Ile Cys Asn Val Asn His Lys

    205

    Ser Asn Thr Lys Lys

    210

    Val Asp Lys

    215

    Lys Val Glu Pro

    Lys

    220

    Ser Cys Asp

    238

    Thr Pro 225 240 His Thr Cys Pro Pro 230 Cys Pro Ala Pro Glu 235 Leu Leu Gly Gly Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

    Leu

    385 390 395

    400

    239

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 156 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H030 <400> 156

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Gly Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95

    240

    Arg Gly Glu Thr His 100 His Gly Ser Ser Gly 105 Ile Asp His Trp Gly 110 Gln Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270

    241

    Pro Asn Glu Val 275 Lys Phe Asn Trp Tyr 280 Val Asp Gly Val Glu 285 Val His Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    242 <210> 157 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H031 <400> 157

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Arg Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Lys 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140

    243

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

    Glu

    305 310 315

    320

    244

    Tyr Lys Lys Cys Lys Val 325 Ser Asn Thr Ile Ser Lys Ala Lys Gly Thr 340 Leu Pro Pro Ser Arg Glu Glu Thr 355 Cys Leu Val Lys Gly Phe Tyr Glu 370 375 Ser Asn Gly Gln Pro Glu Asn Leu 385 390 400 Asp Ser Asp Gly Ser Phe Phe Lys 405 Ser Arg Trp Gln Gln Gly Asn Glu 420 Ala Leu His Asn His Tyr Thr 435

    Ala Leu Pro Ala Pro Ile Glu

    330 335

    Pro Arg Glu Pro Gln Val Tyr

    345 350

    Thr Lys Asn Gln Val Ser Leu

    365

    Ser Asp Ile Ala Val Glu Trp

    380

    Tyr Lys Thr Thr Pro Pro Val

    395

    Tyr Ser Lys Leu Thr Val Asp

    410 415

    Phe Ser Cys Ser Val Met His

    425 430

    Lys Ser Leu Ser Leu Ser Pro 445 <210> 158 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H032 <400> 158

    Gln Val Gln Leu Val Glu Ser Gly

    1 5

    Gly Gly Leu Val Gln Pro Gly

    10 15

    245

    Ser His Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Leu Ser 30 Asn Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Gly Arg Leu Arg Ala Gly Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

    180 185

    Ser Val Val

    Thr Val

    190

    Pro

    246

    Ser Pro Ser Leu 195 Gly Thr Gln Thr Tyr 200 Ile Cys Asn Val Asn 205 His Lys Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365

    247

    Cys Leu Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Ser Asn Leu Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 159 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H034 <400> 159 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala

    Ser

    50 55 60

    248

    Gly Leu 65 Arg Phe Thr Ile Ser 70 Arg Asp Asn Ser Lys 75 Asn Thr Val Tyr Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

    Pro

    225 230 235

    240

    249

    Ser Ser Val Phe Leu Phe 245 Pro Pro Lys Pro Lys 250 Asp Thr Leu Met Ile 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

    250

    405

    410

    415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 160 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H035 <400> 160

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Arg 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110

    251

    Thr Phe Leu Val 115 Thr Val Ser Ser Ala 120 Ser Thr Lys Gly Pro 125 Ser Val Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285

    252

    Ala Val Lys 290 Thr Lys Pro Arg Glu 295 Glu Gln Tyr Asn Ser 300 Thr Tyr Arg Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 161 <211> 447 <212> PRT

    253

    <213> Artificial Sequence <220> <223> H039 <400> 161 Gln Val Gln Leu Val Glu Ser Gly 1 5

    Ser Leu Arg Leu Ser Cys Ala His

    Asp Met Thr Trp Val Arg Gln Val

    Ser Ile Ile Asn Thr Tyr Gly Lys

    50 55

    Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ala Ser Gly Phe Thr Leu Ser Asn 25 30 Ala Pro Gly Lys Gly Leu Glu Tyr 40 45 Asn His Trp Tyr Ala Asn Trp Ala 60

    Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

    65 70 75 80

    Gln Ile Asp Ala

    Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95

    Arg Glu Thr His Gly

    100

    His Gly Ser

    Ser Gly Ile Asp His

    105

    Trp Gly Gln

    110

    Thr Leu Val Thr Val Phe

    115

    Ser Ser Ala

    Ser Thr Lys

    Gly Pro

    Ser Val

    120

    125

    Pro Leu Ala Pro Leu

    130

    Ser Ser Lys

    135

    Ser Thr

    Ser

    Gly Gly Thr Ala

    Ala

    140

    Gly Cys Leu Val Lys Asp Tyr Trp 145 150 160

    Phe Pro Glu

    Pro Val

    Thr Val Ser

    155

    254

    Asn Leu Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Ala Val 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

    255

    325

    330

    335

    Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350

    Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365

    Cys Glu Leu 370 Val Lys Gly Phe Tyr 375 Pro Ser Asp Ile Ala 380 Val Glu Trp Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 162 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> H041 <400> 162 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30

    256

    Asp Val Met Thr 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Tyr Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205

    257

    Ser Lys Asn 210 Thr Lys Val Asp Lys 215 Lys Val Glu Pro Lys 220 Ser Cys Asp Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

    258

    370

    375

    380

    Ser Leu 385 400 Asn Gly Gln Pro Glu 390 Asn Asn Tyr Lys Thr 395 Thr Pro Pro Val Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 163 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H045 <400> 163 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75

    259

    Gln Ala Ile Asp Arg Leu 85 Arg Ala Glu Asp Thr 90 Ala Thr Tyr Tyr Cys 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

    260

    245

    250

    255

    Arg Asp Thr Pro Glu 260 Val Thr Cys Val Val 265 Val Asp Val Ser His 270 Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

    261

    Glu

    420 425 430

    Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 164 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L001 <400> 164

    Asp Lys 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125

    262

    Lys Pro Ser 130 Gly Thr Ala Ser Val 135 Val Cys Leu Leu Asn 140 Asn Phe Tyr Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 165 <211> 217 <212> PRT <213> Artificial Sequence

    <220> <223> L002 <400> 165 Asp Ile Gln Gly Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Ile Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45

    263

    Tyr Gly Asp 50 His Ser Thr Leu Ala 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    264 <210> 166 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L003 <400> 166

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

    265

    Gly

    145

    160

    150

    155

    Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190

    Lys Val Val Tyr 195 Ala Cys Glu Val Thr 200 His Gln Gly Leu Ser Ser Pro 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 167 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L004 Val 15 <400> 167 Pro Ser Ser 10 Val Ser Ala Ser Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Lys Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln

    Pro

    266

    Glu His Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Ala Asp Tyr Ala His 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 168 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L005

    267 <400> 168

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Lys Lys Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    268

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

    180 185 190

    Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 169 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L006 <400> 169

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Arg Arg Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95

    269

    Ser Thr Asp Asn Val 100 Phe Gly Gly Gly Thr 105 Lys Val Glu Ile Lys 110 Arg Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 170 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L007 <400> 170 Asp Ile Gly Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15

    Asp Arg Val Thr

    Ile Thr Cys

    Gln Ala

    Ser Glu Ser

    Ile Tyr Ser

    270

    Gly

    Leu Ile Ala Trp 35 Tyr Arg Gln Lys Pro 40 Gly Gln Pro Pro Lys 45 Leu Leu Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

    271

    Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 171 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L008 <400> 171

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125

    272

    Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

    130 135 140

    Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

    145 150 155

    160

    Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

    165 170 175

    Ser Leu Ser His

    Ser Thr

    Leu Thr

    Leu

    Ser Lys Ala Asp Tyr

    Glu Lys

    180

    185

    190

    Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Val 195 200 205

    Pro

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 172 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L009 <400> 172

    Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Gly 1 5 10

    Ser Ala

    Ser Val

    Asp Arg Val Thr Gly 20

    Ile Thr Cys Gln Ala

    Ser Glu Ser

    Ile Tyr Ser

    Leu Ala Trp Tyr Gln Gln Lys Ile

    Pro Gly Gln Pro

    Pro Lys Arg Leu

    273

    Lys Gly Asp 50 His Ser Thr Leu Ala 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    274 <210> 173 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L010 <400> 173

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Arg Thr Leu Ala Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

    275

    Gly

    145

    160

    150

    155

    Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190

    Lys Val Val Tyr 195 Ala Cys Glu Val Thr 200 His Gln Gly Leu Ser Ser Pro 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 174 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L011 <400> 174 Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Lys Thr Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro

    276

    Glu His Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Ala Asp Tyr Ala His 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 175 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L012

    277 <400> 175

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    278

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

    180 185 190

    Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 176 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L013 <400> 176 Asp Ile Gln Met Thr Gln Ser Gly

    Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly

    20 25 30

    Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

    35 40 45

    Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

    50 55 60

    Ser Gly Ser Gly Thr Glu Tyr Ala Leu Lys Ile Ser Arg Leu Gln Pro

    65 70 75

    Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His

    85 90 95

    279

    Ser Thr Asp Asn Val 100 Phe Gly Gly Gly Thr 105 Lys Val Glu Ile Lys 110 Arg Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 177 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L014 <400> 177 Asp Ile Gly Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15

    Asp Arg Val Thr

    Ile Thr Cys

    Gln Ala

    Ser Glu Ser

    Ile Tyr Ser

    280

    Gly

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Gln Pro Pro Lys 45 Leu Leu Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

    281

    Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 178 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L015 <400> 178

    Asp Lys 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125

    282

    Lys Pro Ser 130 Gly Thr Ala Ser Val 135 Val Cys Leu Leu Asn 140 Asn Phe Tyr Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 179 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L016 <400> 179

    Asp Ile Gln Met Gly Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30

    Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45

    283

    Tyr Gly Asp 50 His Ser Thr Leu Ala 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    284 <210> 180 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L017 <400> 180

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

    285

    Gly

    145

    160

    150

    155

    Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190

    Lys Val Val Tyr 195 Ala Cys Glu Val Thr 200 His Gln Gly Leu Ser Ser Pro 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 181 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L018 Val 15 <400> 181 Pro Ser Ser 10 Val Ser Ala Ser Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile 35 40 45 Lys Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln

    Pro

    286

    Glu His Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Ala Asp Tyr Ala His 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 182 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L019

    287 <400> 182

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Arg Thr Leu Ala Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    288

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

    180 185 190

    Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 183 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L020 <400> 183

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Lys Thr Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95

    289

    Ser Thr Asp Asn Val 100 Phe Gly Gly Gly Thr 105 Lys Val Glu Ile Lys 110 Arg Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 184 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L021 <400> 184 Asp Ile Gly Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15

    Asp Arg Val Thr

    Ile Thr Cys

    Arg Ala

    Ser Arg Ser

    Ile Tyr Ser

    290

    Gly

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Gln Pro Pro Lys 45 Leu Leu Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

    291

    Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 185 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L022 <400> 185

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125

    292

    Lys Pro Ser 130 Gly Thr Ala Ser Val 135 Val Cys Leu Leu Asn 140 Asn Phe Tyr Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 186 <211> 217 <212> PRT <213> Artificial Sequence

    <220> <223> L023 <400> 186 Asp Ile Gln Gly Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Ile Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45

    293

    Tyr Gly Asp 50 His Ser Thr Leu Ala 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    294 <210> 187 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L024 <400> 187

    Asp Lys 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Arg Arg Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

    295

    Gly

    145

    160

    150

    155

    Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190

    Lys Val Val Tyr 195 Ala Cys Glu Val Thr 200 His Gln Gly Leu Ser Ser Pro 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 188 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L025 Val 15 <400> 188 Pro Ser Ser 10 Val Ser Ala Ser Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Asp Arg Val Thr Ile Thr Cys Gln Arg Arg Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln

    Pro

    296

    Glu His Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Ala Asp Tyr Ala His 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 189 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L026

    297 <400> 189

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Arg Arg Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    298

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

    180 185 190

    Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 190 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L028 <400> 190

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Arg Arg Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Arg Thr Leu Ala Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95

    299

    Ser Thr Asp Asn Val 100 Phe Gly Gly Gly Thr 105 Lys Val Glu Ile Lys 110 Arg Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 191 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L029 <400> 191 Asp Ile Gly Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15

    Asp Arg Val Thr

    Ile Thr Cys

    Gln Arg

    Arg Glu Ser

    Ile Tyr Ser

    300

    Gly

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Gln Pro Pro Lys 45 Leu Leu Tyr Asp His Lys Thr Leu Ala Lys Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

    301

    Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 192 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L030 <400> 192

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Arg Arg Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125

    302

    Lys Pro Ser 130 Gly Thr Ala Ser Val 135 Val Cys Leu Leu Asn 140 Asn Phe Tyr Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 193 <211> 217 <212> PRT <213> Artificial Sequence

    <220> <223> L032 <400> 193 Asp Ile Gln Gly Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Arg Arg Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Ile Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45

    303

    Tyr Gly Asp 50 His Ser Thr Leu Ala 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    304 <210> 194 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L033 <400> 194

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

    305

    Gly

    145

    160

    150

    155

    Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190

    Lys Val Val Tyr 195 Ala Cys Glu Val Thr 200 His Gln Gly Leu Ser Ser Pro 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 195 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L034 <400> 195

    Asp Ile Gln Met Gly Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30

    Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45

    Tyr Asp His Arg Thr Leu Ala 55 Arg Gly Val Pro Ser 60 Arg Phe Ser Gly 50 Arg Gly Ser Gly Arg Glu Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro

    306

    Glu His Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Ala Asp Tyr Ala His 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 196 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L035

    307 <400> 196

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Glu Tyr Ala Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    308

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

    180 185 190

    Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 197 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L036 <400> 197

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Glu Tyr Ala Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95

    309

    Ser Thr Asp Asn Val 100 Phe Gly Gly Gly Thr 105 Lys Val Glu Ile Lys 110 Arg Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 198 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L037 <400> 198 Asp Ile Gly Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15

    Asp Arg Val Thr

    Ile Thr Cys

    Arg Ala

    Ser Arg Ser

    Ile Tyr Ser

    310

    Gly

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Arg Lys Pro Pro Lys 45 Leu Leu Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

    311

    Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 199 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L038 <400> 199

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Arg Thr Leu Ala Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125

    312

    Lys Pro Ser 130 Gly Thr Ala Ser Val 135 Val Cys Leu Leu Asn 140 Asn Phe Tyr Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 200 <211> 217 <212> PRT <213> Artificial Sequence

    <220> <223> L039 <400> 200 Asp Ile Gln Gly Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Ile Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45

    313

    Tyr Gly Asp 50 His Ser Thr Leu Ala 55 Ser Gly Val Pro Ser 60 Arg Phe Ser Ser Gly Ser Gly Thr Glu Tyr Ala Leu Lys Ile Arg Arg Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    314 <210> 201 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L040 <400> 201

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

    315

    Gly

    145

    160

    150

    155

    Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190

    Lys Val Val Tyr 195 Ala Cys Glu Val Thr 200 His Gln Gly Leu Ser Ser Pro 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 202 <211> 217 <212> PRT <213> Artificial Sequence <220>

    <223> L041 <400> 202 Ser Ser 10 Val Ser Ala Ser Val 15 Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Arg Thr Leu Ala Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Arg Ser Leu Lys

    Pro

    316

    Glu His Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Ala Asp Tyr Ala His 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 203 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L061

    317 <400> 203

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Val Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Arg Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His 85 90 95 Ser Asp Asn Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175

    318

    Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

    180 185 190

    Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

    195 200 205

    Thr Lys Ser Phe Asn Arg Gly Glu Cys

    210 215 <210> 204 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L062 <400> 204 Asp Ile Gln Met Thr Gln Ser Gly

    Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Ser Ile Tyr Ser Gly

    20 25 30

    Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

    35 40 45

    Tyr Asp His Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

    50 55 60

    Arg Gly Ser Gly Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Lys Pro

    65 70 75

    Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ala Asp Tyr Ala His His

    85 90 95

    319

    Ser Thr Asp Asn Val 100 Phe Gly Gly Gly Thr 105 Lys Val Glu Ile Lys 110 Arg Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

    <210> 205 <400> 000 205 <210> 206 <400> 000 206 <210> 207 <211> 1676 <212> PRT <213> Artificial <220>

    Sequence

    320 <223> Recombinant human C5 <400> 207

    Met Thr 1 Gly Leu Leu Gly 5 Ile Leu Cys Phe Leu 10 Ile Phe Leu Gly Lys 15 Trp Gly Gln Glu Gln Thr Tyr Val Ile Ser Ala Pro Lys Ile Phe Arg 20 25 30 Val Gly Ala Ser Glu Asn Ile Val Ile Gln Val Tyr Gly Tyr Thr Glu 35 40 45 Ala Phe Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe 50 55 60 Ser Tyr Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln 65 70 75 Asn Ser Ala Ile Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln 85 90 95 Asn Pro Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser 100 105 110 Lys Ser Lys Arg Met Pro Ile Thr Tyr Asp Asn Gly Phe Leu Phe Ile 115 120 125 His Thr Asp Lys Pro Val Tyr Thr Pro Asp Gln Ser Val Lys Val Arg 130 135 140 Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val 145 150 155 160 Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Val Asp Met Val Glu

    321

    Glu

    165 170 175 Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser 180 185 190 Asn Pro Arg Tyr Gly Met Trp Thr Ile Lys Ala Lys Tyr Lys Glu Asp 195 200 205 Phe Ser Thr Thr Gly Thr Ala Tyr Phe Glu Val Lys Glu Tyr Val Leu 210 215 220 Pro His Phe Ser Val Ser Ile Glu Pro Glu Tyr Asn Phe Ile Gly Tyr 225 230 235 240 Lys Asn Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg Tyr Phe Tyr 245 250 255 Asn Lys Val Val Thr Glu Ala Asp Val Tyr Ile Thr Phe Gly Ile Arg 260 265 270 Glu Asp Leu Lys Asp Asp Gln Lys Glu Met Met Gln Thr Ala Met Gln 275 280 285 Asn Thr Met Leu Ile Asn Gly Ile Ala Gln Val Thr Phe Asp Ser Glu 290 295 300 Thr Ala Val Lys Glu Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn 305 310 315 320 Lys Tyr Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe 325 330 335

    322

    Ser Tyr Glu Glu Ala 340 Glu Ile Pro Gly Ile 345 Lys Tyr Val Leu Ser 350 Pro Lys Leu Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile Pro 355 360 365 Tyr Pro Ile Lys Val Gln Val Lys Asp Ser Leu Asp Gln Leu Val Gly 370 375 380 Gly Val Pro Val Thr Leu Asn Ala Gln Thr Ile Asp Val Asn Gln Glu 385 390 395 400 Thr Ser Asp Leu Asp Pro Ser Lys Ser Val Thr Arg Val Asp Asp Gly 405 410 415 Val Ala Ser Phe Val Leu Asn Leu Pro Ser Gly Val Thr Val Leu Glu 420 425 430 Phe Asn Val Lys Thr Asp Ala Pro Asp Leu Pro Glu Glu Asn Gln Ala 435 440 445 Arg Glu Gly Tyr Arg Ala Ile Ala Tyr Ser Ser Leu Ser Gln Ser Tyr 450 455 460 Leu Tyr Ile Asp Trp Thr Asp Asn His Lys Ala Leu Leu Val Gly Glu 465 470 475 480 His Leu Asn Ile Ile Val Thr Pro Lys Ser Pro Tyr Ile Asp Lys Ile 485 490 495 Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile Ile His

    Phe

    500 505 510

    323

    Gly Ile Thr Arg 515 Glu Lys Phe Ser Pro Val Thr Gln Asn Met Val Tyr 530 535 Ile Val Thr Gly Glu Gln Thr Trp 545 550 560 Leu Asn Ile Glu Glu Lys Cys Ser 565 Pro Asp Ala Asp Ala Tyr Ser Met 580 Ala Thr Gly Met Asp Ser Trp Ala 595 Val Tyr Gly Val Gln Arg Gly Phe 610 615 Gln Phe Leu Glu Lys Ser Asp Leu 625 630 640 Asn Asn Ala Asn Val Phe His Asn 645 Ala Asn Ala Asp Asp Ser Gln Ile 660 Leu Arg Pro Arg Arg Thr Leu Ala 675

    Ala Ser Tyr Gln Ser 525 Ile Asn Ser Ser Arg Leu Leu Val Tyr 540 Glu Leu Val Ser Asp Ser Val 555 Asn Gln Leu Gln Val His Leu 570 575 Gly Gln Thr Val Ser Leu Asn 585 590 Ala Leu Ala Ala Val Asp Ser 605 Lys Lys Pro Leu Glu Arg Val 620 Gly Cys Gly Ala Gly Gly Gly 635 Ala Gly Leu Thr Phe Leu Thr 650 655 Asn Asp Glu Pro Cys Lys Glu 665 670 Lys Lys Ile Glu Glu Ile Ala 685

    324

    Lys Cys Tyr 690 Lys His Ser Val Val 695 Lys Lys Cys Cys Tyr 700 Asp Gly Ala Val Asn Asn Asp Glu Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu 705 710 715 720 Gly Pro Arg Cys Ile Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser 725 730 735 Gln Leu Arg Ala Asn Ile Ser His Lys Asp Met Gln Leu Gly Arg Leu 740 745 750 His Met Lys Thr Leu Leu Pro Val Ser Lys Pro Glu Ile Arg Ser Tyr 755 760 765 Phe Pro Glu Ser Trp Leu Trp Glu Val His Leu Val Pro Arg Arg Lys 770 775 780 Gln Leu Gln Phe Ala Leu Pro Asp Ser Leu Thr Thr Trp Glu Ile Gln 785 790 795 800 Gly Val Gly Ile Ser Asn Thr Gly Ile Cys Val Ala Asp Thr Val Lys 805 810 815 Ala Lys Val Phe Lys Asp Val Phe Leu Glu Met Asn Ile Pro Tyr Ser 820 825 830 Val Val Arg Gly Glu Gln Ile Gln Leu Lys Gly Thr Val Tyr Asn Tyr 835 840 845 Arg Thr Ser Gly Met Gln Phe Cys Val Lys Met Ser Ala Val Glu Gly

    325

    850

    855

    860

    Ile Ser 865 880 Cys Thr Ser Glu Ser 870 Pro Val Ile Asp His 875 Gln Gly Thr Lys Ser Lys Cys Val Arg Gln Lys Val Glu Gly Ser Ser Ser His Leu Val 885 890 895 Thr Phe Thr Val Leu Pro Leu Glu Ile Gly Leu His Asn Ile Asn Phe 900 905 910 Ser Leu Glu Thr Trp Phe Gly Lys Glu Ile Leu Val Lys Thr Leu Arg 915 920 925 Val Val Pro Glu Gly Val Lys Arg Glu Ser Tyr Ser Gly Val Thr Leu 930 935 940 Asp Pro Arg Gly Ile Tyr Gly Thr Ile Ser Arg Arg Lys Glu Phe Pro 945 950 955 960 Tyr Arg Ile Pro Leu Asp Leu Val Pro Lys Thr Glu Ile Lys Arg Ile 965 970 975 Leu Ser Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala Val Leu 980 985 990 Ser Gln Glu Gly Ile Asn Ile Leu Thr His Leu Pro Lys Gly Ser

    Ala

    995 1000 1005

    Glu Ala Glu Leu Met Ser Val Val Pro Val Phe Tyr Val Phe His 1010 1015 1020

    Tyr Leu Glu Thr Gly Asn His Trp Asn Ile Phe His Ser Asp Pro 1025 1030 1035

    326

    Leu Ile 1040 Glu Lys Gln Lys Leu 1045 Lys Lys Lys Leu Lys 1050 Glu Gly Met Leu Ser Ile Met Ser Tyr Arg Asn Ala Asp Tyr Ser Tyr Ser Val 1055 1060 1065 Trp Lys Gly Gly Ser Ala Ser Thr Trp Leu Thr Ala Phe Ala Leu 1070 1075 1080 Arg Val Leu Gly Gln Val Asn Lys Tyr Val Glu Gln Asn Gln Asn 1085 1090 1095 Ser Ile Cys Asn Ser Leu Leu Trp Leu Val Glu Asn Tyr Gln Leu 1100 1105 1110 Asp Asn Gly Ser Phe Lys Glu Asn Ser Gln Tyr Gln Pro Ile Lys 1115 1120 1125 Leu Gln Gly Thr Leu Pro Val Glu Ala Arg Glu Asn Ser Leu Tyr 1130 1135 1140 Leu Thr Ala Phe Thr Val Ile Gly Ile Arg Lys Ala Phe Asp Ile 1145 1150 1155 Cys Pro Leu Val Lys Ile Asp Thr Ala Leu Ile Lys Ala Asp Asn 1160 1165 1170 Phe Leu Leu Glu Asn Thr Leu Pro Ala Gln Ser Thr Phe Thr Leu 1175 1180 1185 Ala Ile Ser Ala Tyr Ala Leu Ser Leu Gly Asp Lys Thr His Pro 1190 1195 1200 Gln Phe Arg Ser Ile Val Ser Ala Leu Lys Arg Glu Ala Leu Val 1205 1210 1215 Lys Gly Asn Pro Pro Ile Tyr Arg Phe Trp Lys Asp Asn Leu Gln 1220 1225 1230 His Lys Asp Ser Ser Val Pro Asn Thr Gly Thr Ala Arg Met Val 1235 1240 1245

    327

    Glu Thr 1250 Thr Ala Tyr Ala Leu 1255 Leu Thr Ser Leu Asn 1260 Leu Lys Asp Ile Asn Tyr Val Asn Pro Val Ile Lys Trp Leu Ser Glu Glu Gln 1265 1270 1275 Arg Tyr Gly Gly Gly Phe Tyr Ser Thr Gln Asp Thr Ile Asn Ala 1280 1285 1290 Ile Glu Gly Leu Thr Glu Tyr Ser Leu Leu Val Lys Gln Leu Arg 1295 1300 1305 Leu Ser Met Asp Ile Asp Val Ser Tyr Lys His Lys Gly Ala Leu 1310 1315 1320 His Asn Tyr Lys Met Thr Asp Lys Asn Phe Leu Gly Arg Pro Val 1325 1330 1335 Glu Val Leu Leu Asn Asp Asp Leu Ile Val Ser Thr Gly Phe Gly 1340 1345 1350 Ser Gly Leu Ala Thr Val His Val Thr Thr Val Val His Lys Thr 1355 1360 1365 Ser Thr Ser Glu Glu Val Cys Ser Phe Tyr Leu Lys Ile Asp Thr 1370 1375 1380 Gln Asp Ile Glu Ala Ser His Tyr Arg Gly Tyr Gly Asn Ser Asp 1385 1390 1395 Tyr Lys Arg Ile Val Ala Cys Ala Ser Tyr Lys Pro Ser Arg Glu 1400 1405 1410 Glu Ser Ser Ser Gly Ser Ser His Ala Val Met Asp Ile Ser Leu 1415 1420 1425 Pro Thr Gly Ile Ser Ala Asn Glu Glu Asp Leu Lys Ala Leu Val 1430 1435 1440 Glu Gly Val Asp Gln Leu Phe Thr Asp Tyr Gln Ile Lys Asp Gly 1445 1450 1455

    328

    His Val 1460 Ile Leu Gln Leu Asn 1465 Ser Ile Pro Ser Ser 1470 Asp Phe Leu Cys Val Arg Phe Arg Ile Phe Glu Leu Phe Glu Val Gly Phe Leu 1475 1480 1485 Ser Pro Ala Thr Phe Thr Val Tyr Glu Tyr His Arg Pro Asp Lys 1490 1495 1500 Gln Cys Thr Met Phe Tyr Ser Thr Ser Asn Ile Lys Ile Gln Lys 1505 1510 1515 Val Cys Glu Gly Ala Ala Cys Lys Cys Val Glu Ala Asp Cys Gly 1520 1525 1530 Gln Met Gln Glu Glu Leu Asp Leu Thr Ile Ser Ala Glu Thr Arg 1535 1540 1545 Lys Gln Thr Ala Cys Lys Pro Glu Ile Ala Tyr Ala Tyr Lys Val 1550 1555 1560 Ser Ile Thr Ser Ile Thr Val Glu Asn Val Phe Val Lys Tyr Lys 1565 1570 1575 Ala Thr Leu Leu Asp Ile Tyr Lys Thr Gly Glu Ala Val Ala Glu 1580 1585 1590 Lys Asp Ser Glu Ile Thr Phe Ile Lys Lys Val Thr Cys Thr Asn 1595 1600 1605 Ala Glu Leu Val Lys Gly Arg Gln Tyr Leu Ile Met Gly Lys Glu 1610 1615 1620 Ala Leu Gln Ile Lys Tyr Asn Phe Ser Phe Arg Tyr Ile Tyr Pro 1625 1630 1635 Leu Asp Ser Leu Thr Trp Ile Glu Tyr Trp Pro Arg Asp Thr Thr 1640 1645 1650 Cys Ser Ser Cys Gln Ala Phe Leu Ala Asn Leu Asp Glu Phe Ala 1655 1660 1665

    329

    Glu Asp Ile Phe Leu Asn Gly Cys 1670 1675

    <210> <211> <212> <213> 208 1676 PRT Artificial Sequence <220> <223> cynomolgus monkey C5

    <400> 208

    Met Gly Leu Leu Gly Ile Leu Cys

    Thr 1 5

    Trp Gly Gln Glu Gln Thr Tyr Val Arg

    Val Gly Ala Ser Glu Asn Ile Val Glu

    35 40

    Phe Leu 10 Ile Phe Leu Gly Lys 15 Ile Ser Ala Pro Lys Ile Phe 25 30 Ile Gln Val Tyr Gly Tyr Thr 45

    Ala Phe Asp Ala Thr Ile Ser Ile Lys Ser Tyr Pro Asp Lys Lys Phe 50 55 60

    Ser Tyr Ser Ser Gly His Val His Leu Ser Ser Glu Asn Lys Phe Gln 65 70 75

    Asn Ser Ala Val Leu Thr Ile Gln Pro Lys Gln Leu Pro Gly Gly Gln 85 90 95

    Asn Gln Val Ser Tyr Val Tyr Leu Glu Val Val Ser Lys His Phe Ser 100 105 110

    Lys Ser Lys Lys Ile Pro Ile Ile

    115

    Thr Tyr Asp Asn Gly Phe Leu

    120

    125

    Phe

    330

    His Arg Thr 130 Asp Lys Pro Val Tyr 135 Thr Pro Asp Gln Ser 140 Val Lys Val Val Tyr Ser Leu Asn Asp Asp Leu Lys Pro Ala Lys Arg Glu Thr Val 145 150 155 160 Leu Thr Phe Ile Asp Pro Glu Gly Ser Glu Ile Asp Met Val Glu Glu 165 170 175 Ile Asp His Ile Gly Ile Ile Ser Phe Pro Asp Phe Lys Ile Pro Ser 180 185 190 Asn Pro Arg Tyr Gly Met Trp Thr Ile Gln Ala Lys Tyr Lys Glu Asp 195 200 205 Phe Ser Thr Thr Gly Thr Ala Phe Phe Glu Val Lys Glu Tyr Val Leu 210 215 220 Pro His Phe Ser Val Ser Val Glu Pro Glu Ser Asn Phe Ile Gly Tyr 225 230 235 240 Lys Asn Phe Lys Asn Phe Glu Ile Thr Ile Lys Ala Arg Tyr Phe Tyr 245 250 255 Asn Lys Val Val Thr Glu Ala Asp Val Tyr Ile Thr Phe Gly Ile Arg 260 265 270 Glu Asp Leu Lys Asp Asp Gln Lys Glu Met Met Gln Thr Ala Met Gln 275 280 285 Asn Thr Met Leu Ile Asn Gly Ile Ala Glu Val Thr Phe Asp Ser Glu 290 295 300

    331

    Thr Ala Val Lys Glu Leu Ser Tyr Tyr Ser Leu Glu Asp Leu Asn Asn

    305 310 315

    320

    Lys Tyr Leu Tyr Ile Ala Val Thr Val Ile Glu Ser Thr Gly Gly Phe

    325 330 335

    Ser Glu Glu Ala Glu Ile Pro Gly Ile Lys Tyr Val Leu Ser Pro Tyr

    340 345 350

    Lys Leu Asn Leu Val Ala Thr Pro Leu Phe Leu Lys Pro Gly Ile Pro

    355 360 365

    Tyr Ser Ile Lys Val Gln Val Lys Asp Ala Leu Asp Gln Leu Val Gly

    370 375 380

    Gly Val Pro Val Thr Leu Asn Ala Gln Thr Ile Asp Val Asn Gln Glu

    385 390 395

    400

    Thr Ser Asp Leu Glu Pro Arg Lys Ser Val Thr Arg Val Asp Asp Gly

    405 410 415

    Val Ala Ser Phe Val Val Asn Leu Pro Ser Gly Val Thr Val Leu Glu

    420 425 430

    Phe Asn Val Lys Thr Asp Ala Pro Asp Leu Pro Asp Glu Asn Gln Ala

    435 440 445

    Arg Glu Gly Tyr Arg Ala Ile Ala Tyr 450 455

    Leu Tyr Ile Asp Trp Thr Asp Asn Glu 465 470

    Tyr Ser Ser Leu 460 Ser Gln Ser His Lys Ala Leu Leu Val Gly

    475

    332

    480

    Tyr Ile Leu Asn Ile Ile 485 Val Thr Pro Lys Ser 490 Pro Tyr Ile Asp Lys 495 Thr His Tyr Asn Tyr Leu Ile Leu Ser Lys Gly Lys Ile Ile His Phe 500 505 510 Gly Thr Arg Glu Lys Leu Ser Asp Ala Ser Tyr Gln Ser Ile Asn Ile 515 520 525 Pro Val Thr Gln Asn Met Val Pro Ser Ser Arg Leu Leu Val Tyr Tyr 530 535 540 Ile Val Thr Gly Glu Gln Thr Ala Glu Leu Val Ser Asp Ser Val Trp 545 550 555 560 Leu Asn Ile Glu Glu Lys Cys Gly Asn Gln Leu Gln Val His Leu Ser 565 570 575 Pro Asp Ala Asp Thr Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met 580 585 590 Val Thr Gly Met Asp Ser Trp Val Ala Leu Thr Ala Val Asp Ser Ala 595 600 605 Val Tyr Gly Val Gln Arg Arg Ala Lys Lys Pro Leu Glu Arg Val Phe 610 615 620 Gln Phe Leu Glu Lys Ser Asp Leu Gly Cys Gly Ala Gly Gly Gly Leu 625 630 635 640 Asn Asn Ala Asn Val Phe His Leu Ala Gly Leu Thr Phe Leu Thr

    333

    Asn

    645 650 655 Ala Asn Ala Asp Asp Ser Gln Glu Asn Asp Glu Pro Cys Lys Glu Ile 660 665 670 Ile Arg Pro Arg Arg Met Leu Gln Glu Lys Ile Glu Glu Ile Ala Ala 675 680 685 Lys Tyr Lys His Leu Val Val Lys Lys Cys Cys Tyr Asp Gly Val Arg 690 695 700 Ile Asn His Asp Glu Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Val 705 710 715 720 Gly Pro Arg Cys Val Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser 725 730 735 Gln Leu Arg Ala Asn Asn Ser His Lys Asp Leu Gln Leu Gly Arg Leu 740 745 750 His Met Lys Thr Leu Leu Pro Val Ser Lys Pro Glu Ile Arg Ser Tyr 755 760 765 Phe Pro Glu Ser Trp Leu Trp Glu Val His Leu Val Pro Arg Arg Lys 770 775 780 Gln Leu Gln Phe Ala Leu Pro Asp Ser Val Thr Thr Trp Glu Ile Gln 785 790 795 800 Gly Val Gly Ile Ser Asn Ser Gly Ile Cys Val Ala Asp Thr Ile Lys 805 810 815

    334

    Ala Ser Lys Val Phe 820 Lys Asp Val Phe Leu 825 Glu Met Asn Ile Pro 830 Tyr Val Val Arg Gly Glu Gln Val Gln Leu Lys Gly Thr Val Tyr Asn Tyr 835 840 845 Arg Thr Ser Gly Met Gln Phe Cys Val Lys Met Ser Ala Val Glu Gly 850 855 860 Ile Cys Thr Ser Glu Ser Pro Val Ile Asp His Gln Gly Thr Lys Ser 865 870 875 880 Ser Lys Cys Val Arg Gln Lys Val Glu Gly Ser Ser Asn His Leu Val 885 890 895 Thr Phe Thr Val Leu Pro Leu Glu Ile Gly Leu Gln Asn Ile Asn Phe 900 905 910 Ser Leu Glu Thr Ser Phe Gly Lys Glu Ile Leu Val Lys Ser Leu Arg 915 920 925 Val Val Pro Glu Gly Val Lys Arg Glu Ser Tyr Ser Gly Ile Thr Leu 930 935 940 Asp Pro Arg Gly Ile Tyr Gly Thr Ile Ser Arg Arg Lys Glu Phe Pro 945 950 955 960 Tyr Arg Ile Pro Leu Asp Leu Val Pro Lys Thr Glu Ile Lys Arg Ile 965 970 975 Leu Ser Val Lys Gly Leu Leu Val Gly Glu Ile Leu Ser Ala Val Leu 980 985 990

    335

    Ser Arg Glu Gly Ile Asn Ile Leu Thr His Leu Pro Lys Gly Ser Ala

    995 1000 1005

    Glu Ala 1010 Glu Leu Met Ser Val 1015 Val Pro Val Phe Tyr 1020 Val Phe His Tyr Leu Glu Thr Gly Asn His Trp Asn Ile Phe His Ser Asp Pro 1025 1030 1035 Leu Ile Glu Lys Arg Asn Leu Glu Lys Lys Leu Lys Glu Gly Met 1040 1045 1050 Val Ser Ile Met Ser Tyr Arg Asn Ala Asp Tyr Ser Tyr Ser Val 1055 1060 1065 Trp Lys Gly Gly Ser Ala Ser Thr Trp Leu Thr Ala Phe Ala Leu 1070 1075 1080 Arg Val Leu Gly Gln Val His Lys Tyr Val Glu Gln Asn Gln Asn 1085 1090 1095 Ser Ile Cys Asn Ser Leu Leu Trp Leu Val Glu Asn Tyr Gln Leu 1100 1105 1110 Asp Asn Gly Ser Phe Lys Glu Asn Ser Gln Tyr Gln Pro Ile Lys 1115 1120 1125 Leu Gln Gly Thr Leu Pro Val Glu Ala Arg Glu Asn Ser Leu Tyr 1130 1135 1140 Leu Thr Ala Phe Thr Val Ile Gly Ile Arg Lys Ala Phe Asp Ile 1145 1150 1155 Cys Pro Leu Val Lys Ile Asn Thr Ala Leu Ile Lys Ala Asp Thr 1160 1165 1170 Phe Leu Leu Glu Asn Thr Leu Pro Ala Gln Ser Thr Phe Thr Leu 1175 1180 1185 Ala Ile Ser Ala Tyr Ala Leu Ser Leu Gly Asp Lys Thr His Pro 1190 1195 1200

    336

    Gln Phe 1205 Arg Ser Ile Val Ser 1210 Ala Leu Lys Arg Glu 1215 Ala Leu Val Lys Gly Asn Pro Pro Ile Tyr Arg Phe Trp Lys Asp Ser Leu Gln 1220 1225 1230 His Lys Asp Ser Ser Val Pro Asn Thr Gly Thr Ala Arg Met Val 1235 1240 1245 Glu Thr Thr Ala Tyr Ala Leu Leu Thr Ser Leu Asn Leu Lys Asp 1250 1255 1260 Ile Asn Tyr Val Asn Pro Ile Ile Lys Trp Leu Ser Glu Glu Gln 1265 1270 1275 Arg Tyr Gly Gly Gly Phe Tyr Ser Thr Gln Asp Thr Ile Asn Ala 1280 1285 1290 Ile Glu Gly Leu Thr Glu Tyr Ser Leu Leu Val Lys Gln Leu Arg 1295 1300 1305 Leu Asn Met Asp Ile Asp Val Ala Tyr Lys His Lys Gly Pro Leu 1310 1315 1320 His Asn Tyr Lys Met Thr Asp Lys Asn Phe Leu Gly Arg Pro Val 1325 1330 1335 Glu Val Leu Leu Asn Asp Asp Leu Val Val Ser Thr Gly Phe Gly 1340 1345 1350 Ser Gly Leu Ala Thr Val His Val Thr Thr Val Val His Lys Thr 1355 1360 1365 Ser Thr Ser Glu Glu Val Cys Ser Phe Tyr Leu Lys Ile Asp Thr 1370 1375 1380 Gln Asp Ile Glu Ala Ser His Tyr Arg Gly Tyr Gly Asn Ser Asp 1385 1390 1395 Tyr Lys Arg Ile Val Ala Cys Ala Ser Tyr Lys Pro Ser Lys Glu 1400 1405 1410

    337

    Glu Ser 1415 Ser Ser Gly Ser Ser 1420 His Ala Val Met Asp 1425 Ile Ser Leu Pro Thr Gly Ile Asn Ala Asn Glu Glu Asp Leu Lys Ala Leu Val 1430 1435 1440 Glu Gly Val Asp Gln Leu Phe Thr Asp Tyr Gln Ile Lys Asp Gly 1445 1450 1455 His Val Ile Leu Gln Leu Asn Ser Ile Pro Ser Ser Asp Phe Leu 1460 1465 1470 Cys Val Arg Phe Arg Ile Phe Glu Leu Phe Glu Val Gly Phe Leu 1475 1480 1485 Ser Pro Ala Thr Phe Thr Val Tyr Glu Tyr His Arg Pro Asp Lys 1490 1495 1500 Gln Cys Thr Met Phe Tyr Ser Thr Ser Asn Ile Lys Ile Gln Lys 1505 1510 1515 Val Cys Glu Gly Ala Thr Cys Lys Cys Ile Glu Ala Asp Cys Gly 1520 1525 1530 Gln Met Gln Lys Glu Leu Asp Leu Thr Ile Ser Ala Glu Thr Arg 1535 1540 1545 Lys Gln Thr Ala Cys Asn Pro Glu Ile Ala Tyr Ala Tyr Lys Val 1550 1555 1560 Ile Ile Thr Ser Ile Thr Thr Glu Asn Val Phe Val Lys Tyr Lys 1565 1570 1575 Ala Thr Leu Leu Asp Ile Tyr Lys Thr Gly Glu Ala Val Ala Glu 1580 1585 1590 Lys Asp Ser Glu Ile Thr Phe Ile Lys Lys Val Thr Cys Thr Asn 1595 1600 1605 Ala Glu Leu Val Lys Gly Arg Gln Tyr Leu Ile Met Gly Lys Glu 1610 1615 1620

    338

    Ala Leu Gln Ile Lys Tyr Asn Phe Thr Phe Arg Tyr Ile Tyr Pro 1625 1630 1635

    Leu Asp 1640 Ser Leu Thr Trp Ile 1645 Glu Tyr Trp Pro Arg 1650 Asp Thr Thr Cys Ser Ser Cys Gln Ala Phe Leu Ala Asn Leu Asp Glu Phe Ala 1655 1660 1665 Glu Asp Ile Phe Leu Asn Gly Cys 1670 1675

    <210> 209 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> CFP0008H <400> 209 Gln Val Ser Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

    20 25 30

    Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

    35 40 45

    Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

    50 55 60

    Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

    65 70 75 80

    Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

    85 90 95

    339

    Ala Arg Gly Val His Ile Lys Tyr Met Ile Gln Tyr Tyr Tyr Gly Ala

    100 105 110

    Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 210 <211> 107 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0008L <400> 210

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Glu Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asp Gly Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

    <210> 211

    340 <211> 127 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0011H <400> 211

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Met Ser Glu Phe Leu Gly Trp Ser Asn Tyr Tyr Ser Tyr 100 105 110 Pro Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125

    <210> 212 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CFP0011L

    341 <400> 212

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Glu Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Asn Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

    100 105

    <210> 213 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> CFP0015H <400> 213 Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Tyr

    Ala Ser

    Gly Phe Thr Phe

    Ser Ser

    342

    Ala Val Met Ser 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gln Ile Trp Tyr Asp Gln Trp Tyr Tyr Phe Asp Met 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

    115 120 <210> 214 <211> 107 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0015L <400> 214 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Glu Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

    Gly

    343

    Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75

    Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

    <210> 215 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> CFP0016H019 <400> 215 Gln Val Glu Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

    20 25 30

    Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

    35 40 45

    Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

    50 55 60

    Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

    65 70 75 80

    Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

    85 90 95

    344

    Ala Arg Asp Pro Tyr Tyr Ser Tyr Pro Trp Ser Thr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser

    115 120 <210> 216 <211> 107 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0016L <400> 216

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Glu Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

    <210> 217 <211> 119

    345 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0017H <400> 217

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Trp Gly Gly Ala Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser

    115 <210> <211> <212> <213> 218 107 PRT Artificial Sequence <220> <223> CFP0017L <400> 218

    346

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Asp Arg Val Thr Ile Thr Cys Arg Asp 20 Leu Ala Trp Tyr Gln Gln Lys Pro Ile 35 40 Tyr His Ala Ser Ser Leu Gln Ser Gly 50 55 Ser Gly Ser Gly Thr Asp Phe Thr Pro 65 70 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu 85 Thr Phe Gly Gln Gly Thr Lys Val 100

    Ser Ser Leu Ser Ala Ser Val

    10 15

    Ala Ser Gln Ser Ile Glu Asp

    25 30

    Gly Lys Ala Pro Lys Leu Leu

    Gly Val Pro Ser Arg Phe Ser

    Leu Thr Ile Ser Ser Leu Gln

    Gln Gln Ser Asp Ser Tyr Pro

    90 95

    Glu Ile Lys

    105

    <210> <211> <212> <213> 219 119 PRT Artificial Sequence <220> <223> CFP0018H <400> 219 Gln Val Gln Leu Val Gln Ser Gly Ser 1 5

    Ser Val Lys Val Ser Cys Lys Ala Tyr

    Ala Ile Ser Trp Val Arg Gln Ala

    Ala Glu Val Lys Lys Pro Gly

    10 15

    Ser Gly Gly Thr Phe Ser Ser

    25 30

    Pro Gly Gln Gly Leu Glu Trp

    347

    Met

    Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

    50 55 60

    Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

    65 70 75

    Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

    85 90 95

    Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly

    100 105 110

    Thr Leu Val Thr Val Ser Ser 115

    <210> 220 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CFP0018L <400> 220 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp Asp

    20 25 30

    Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

    35 40 45

    Tyr Glu Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

    50 55 60

    348

    Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75

    Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

    100 105 <210> 221 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> CFP0019H <400> 221 Gln Val Glu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser

    1 5 10 15

    Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly

    20 25 30

    Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

    35 40 45

    Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

    50 55 60

    Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

    65 70 75 80

    Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

    85 90 95

    349

    Ala Arg Tyr Tyr Val Trp Leu Gly Gly Pro Thr Tyr Met Asp Tyr Trp

    100 105 110

    Gly Gln Gly Thr Leu Val Thr Val Ser Ser

    115 120 <210> 222 <211> 107 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0019L <400> 222

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Glu Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Gly Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

    <210> 223 <211> 122 <212> PRT

    350 <213> Artificial Sequence <220>

    <223> CFP0020H <400> 223

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr Ser Thr Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120

    <210> 224 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L <400> 224

    351

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105

    <210> 225 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> CFP0021H <400> 225 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala His

    Ser Gly Phe Thr

    Phe

    Ser Asp

    Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

    352

    Gly Ala Arg 50 Thr Arg Asn Lys Ala 55 Asn Ser Tyr Thr Thr 60 Glu Tyr Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Thr Gly Met Met Tyr Trp Gly Ile Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser

    115 120 <210> 226 <211> 107 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0021L <400> 226

    Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Gly Ser Ala Ser Val 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Glu Asp Asp 20 25 30

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Lys Ala Pro Lys 45 Leu Leu Tyr His Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

    Gly

    50 55 60

    353

    Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75

    Glu Leu Asp Phe Ala Thr 85 Tyr Tyr Cys Gln Gln 90 His Asp Ser Tyr Pro 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

    100 105 <210> 227 <211> 328 <212> PRT <213> Artificial Sequence <220>

    <223> G1dP1 <400> 227

    Ala Lys 1 Ser Thr Lys Gly 5 Pro Ser Val Phe Pro 10 Leu Ala Pro Ser Ser 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

    354

    Cys

    100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270

    355

    Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285

    Leu Asn Tyr 290 Ser Lys Leu Thr Val 295 Asp Lys Ser Arg Trp 300 Gln Gln Gly Val Thr 305 320 Phe Ser Cys Ser Val 310 Met His Glu Ala Leu 315 His Asn His Tyr Gln Lys Ser Leu Ser 325 Leu Ser Pro

    <210> 228 <211> 328 <212> PRT <213> Artificial Sequence <220>

    <223> G1dN1 <400> 228

    Ala Lys 1 Ser Thr Lys Gly 5 Pro Ser Val Phe Pro 10 Leu Ala Pro Ser Ser 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

    356

    Lys

    Lys Cys Val Glu Pro 100 Lys Ser Cys Asp Lys 105 Thr His Thr Cys Pro 110 Pro Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255

    357

    Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270

    Asn Phe Tyr Lys 275 Thr Thr Pro Pro Val 280 Leu Asp Ser Asp Gly 285 Ser Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300

    Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315

    320

    Gln Glu Ser Leu Ser Leu Ser Pro 325 <210> 229 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020H0261-G1dP1_optimized 20 heavy chain <400> 229

    Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15

    Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30

    Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45

    Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60

    Lys Ser Arg Val Thr Ile Ser

    Val Asp

    Thr

    Ser Lys Asn Gln Phe

    358

    Ser

    Leu Cys Lys Leu Ser Ser 85 Val Thr Ala Ala Asp 90 Thr Ala Val Tyr Tyr 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

    Leu

    225 230 235

    240

    359

    Gly Leu Gly Pro Ser Val 245 Phe Leu Phe Pro Pro 250 Lys Pro Lys Asp Thr 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415

    360

    Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

    420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450 <210> 230 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0018H0012-G1dN1_optimized 18 heavy chain <400> 230

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln

    361

    Gly

    100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

    Asp

    260 265 270

    362

    Pro Asn Glu Val 275 Lys Phe Asn Trp Tyr 280 Val Asp Gly Val Glu 285 Val His Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445

    363 <210> 231 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-k0_optimized 20//18 light chain <400> 231

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

    364

    Gln

    145

    160

    150

    155

    Glu Ser Ser Val Thr Glu 165 Gln Asp Ser Thr Leu Thr Leu Ser Lys Tyr 180 Ala Cys Glu Val Thr His Gln Ser 195 Phe Asn 210 Arg Gly Glu Cys

    Ser Lys Asp Ser Thr Tyr Ser Leu

    170 175

    Ala Asp Tyr Glu Lys His Lys Val

    185 190

    Gly Leu Ser Ser Pro Val Thr Lys

    200 205

    <210> <211> <212> <213> 232 450 PRT Artificial Sequence <220> <223> CFP0020H0261-001-G1dP1 <400> 232 Gln Val Glu Gln Leu Gln Glu Ser Gly 1 5

    Thr Leu Ser Leu Thr Cys Ala Val Gly

    Tyr Tyr Trp Gly Trp Ile Arg Gln Trp

    35 40

    Ile Gly Ser Ile Tyr His Ser Gly Leu

    50 55

    Lys Ser Arg Val Thr Ile Ser Val Ser

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Ser

    25 30

    Pro Arg Gly Lys Arg Leu Glu

    Ser Thr Tyr Tyr Asn Lys Lys

    Asp Thr Ser Lys Asn Gln Phe

    365

    Leu Cys Lys Leu Ser Ser 85 Val Thr Ala Ala Asp 90 Thr Ala Val Tyr Tyr 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

    366

    Leu

    245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415

    367

    Val Asp Lys Ser Arg Trp Gln Gln Val

    420

    Met His Glu Ala Leu His Asn His Leu

    435 440

    Gly Asn Val Phe Ser Cys Ser

    425 430

    Tyr Thr Gln Lys Ser Leu Ser

    445

    Ser Pro 450

    <210> <211> <212> <213> 233 450 PRT Artificial Sequence <220> <223> CFP0020H0261-002-G1dP1 <400> 233 Gln Val Glu Gln Leu Gln Glu Ser Gly 1 5

    Thr Leu Ser Leu Thr Cys Ala Val Gly

    Tyr Tyr Trp Gly Trp Ile Arg Gln Trp

    35 40

    Ile Gly Ser Ile Tyr His Ser Gly Leu

    50 55

    Lys Ser Arg Val Thr Ile Ser Val Ser

    65 70

    Leu Lys Leu Ser Ser Val Thr Ala Cys

    Ala Arg His Asp Pro Thr Trp Tyr Trp

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Ser

    25 30

    Pro Pro Gly Lys Gly Leu Glu

    Ser Thr Tyr Tyr Asn Lys Lys

    Asp Thr Ser Lys Asn Arg Phe

    Arg Asp Thr Ala Val Tyr Tyr

    90 95

    His His Gly Tyr Phe Asp Tyr

    368

    100

    105

    110

    Gly Pro Gln Gly 115 Thr Leu Val Thr Val 120 Ser Ser Ala Ser Thr 125 Lys Gly Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

    369

    Glu

    275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    370

    Ser Pro 450 <210>

    <211>

    <212>

    <213>

    234

    450

    PRT

    Artificial Sequence <220>

    <223> CFP0020H0261-003-G1dP1 <400> 234

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Arg Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

    371

    130

    135

    140

    Ala Thr 145 160 Ala Leu Gly Cys Leu 150 Val Lys Asp Tyr Phe 155 Pro Glu Pro Val Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

    372

    Asn

    305

    320

    310

    315

    Gly Pro Lys Glu Tyr Lys 325 Cys Lys Val Ser Asn 330 Lys Ala Leu Pro Ala 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450

    <210> 235 <211> 450 <212> PRT <213> Artificial Sequence <220>

    373 <223> CFP0020H0261-005-G1dP1 <400> 235

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Arg 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

    374

    Pro

    165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335

    375

    Ile Gln Glu Lys Thr 340 Ile Ser Lys Ala Lys 345 Gly Gln Pro Arg Glu 350 Pro Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

    Leu

    435 440 445

    Ser Pro 450

    <210> 236 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-008-G1dP1 <400> 236 Gln Val Glu . Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15

    Thr Leu Ser

    Leu Thr Cys Ala Val

    Ser

    Gly Tyr Ser Ile

    Ser Ser

    376

    Arg

    Tyr Trp Tyr Trp 35 Gly Trp Ile Arg Gln 40 Pro Pro Gly Lys Gly 45 Leu Glu Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

    377

    Asn

    195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365

    378

    Ser Val Leu 370 Thr Cys Leu Val Lys 375 Gly Phe Tyr Pro Ser 380 Asp Ile Ala Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400

    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415

    Val Val Asp Lys Ser 420 Arg Trp Gln Gln Gly 425 Asn Val Phe Ser Cys 430 Ser Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

    Leu

    435 440 445

    Ser Pro 450

    <210> 237 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-009-G1dP1 <400> 237 Gln Val Glu . Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15

    Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly

    20 25 30

    Tyr Tyr Trp Gly Trp Trp

    Ile Arg Lys

    Pro

    Pro Gly Lys

    Gly Leu Glu

    Ile Gly Ser

    Ile Tyr His

    Ser

    Gly Ser

    Thr Tyr Tyr Asn Lys Lys

    379

    Leu

    Lys Ser 65 Ser Arg Val Thr Ile 70 Ser Val Asp Thr Ser 75 Lys Asn Gln Phe Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

    380

    Leu

    225 230 235

    240

    Gly Leu Gly Pro Ser Val 245 Phe Leu Phe Pro Pro 250 Lys Pro Lys Asp Thr 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

    Pro

    385 390 395

    400

    381

    Pro Val Leu Asp Ser Asp Gly Ser Thr

    405

    Val Asp Lys Ser Arg Trp Gln Gln Val

    420

    Met His Glu Ala Leu His Asn His Leu

    435 440

    Phe Phe Leu Tyr Ser Lys Leu

    410 415

    Gly Asn Val Phe Ser Cys Ser

    425 430

    Tyr Thr Gln Lys Ser Leu Ser

    445

    Ser Pro 450 <210> 238 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020H0261-013-G1dP1 <400> 238

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Thr Leu Ser Leu Thr Cys Ala Val Gly 20 Tyr Tyr Trp Gly Trp Ile Arg Gln Trp 35 40 Ile Gly Ser Ile Tyr His Ser Gly Arg 50 55 Lys Ser Arg Val Thr Ile Ser Val Ser 65 70 Leu Lys Leu Ser Ser Val Thr Ala

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Ser

    25 30

    Pro Pro Gly Lys Gly Leu Glu

    Ser Thr Tyr Tyr Asn Lys Lys

    Asp Thr Ser Lys Asn Gln Phe

    75 80

    Ala Asp Thr Ala Val Tyr Tyr

    382

    Cys

    Ala Trp Arg His Asp 100 Pro Thr Trp Tyr His 105 His Gly Tyr Phe Asp 110 Tyr Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255

    383

    Met Ser Ile Ser Arg 260 Thr Pro Glu Val Thr 265 Cys Val Val Val Asp 270 Val His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

    Val

    420 425 430

    384

    Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

    435 440 445

    Ser Pro 450 <210> 239 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020H0261-018-G1dP1 <400> 239

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Arg Phe Ser 65 70 75 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

    385

    Pro

    115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285

    386

    Val Thr His 290 Asn Ala Lys Thr Lys 295 Pro Arg Glu Glu Gln 300 Tyr Asn Ser Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450

    387 <210> 240 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020H0261-019-G1dP1 <400> 240

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Lys Lys Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

    388

    Thr

    145 150 155

    160

    Val Pro Ser Trp Asn Ser 165 Gly Ala Leu Thr Ser 170 Gly Val His Thr Phe 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

    Asn

    305 310 315

    320

    389

    Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Pro

    325 330

    Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Gln

    340 345 350

    Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Val

    355 360 365

    Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val

    370 375 380

    Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Pro

    385 390 395

    400

    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Thr

    405 410

    Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Val

    420 425 430

    Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Leu

    435 440 445

    Ala

    335

    Pro

    Gln

    Ala

    Thr

    Leu

    415

    Ser

    Ser

    Ser Pro 450 <210> 241 <211> 450 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020H0261-020-G1dP1 <400> 241

    390

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Lys Leu Asn Arg Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175

    391

    Ala Thr Val Leu Gln 180 Ser Val Pro Ser Ser Ser Asn 195 His Lys Pro Ser Asn Ser 210 Cys Asp Lys Thr His Leu 225 240 Gly Gly Pro Ser Val Leu 245 Met Ile Ser Arg Thr Ser 260 His Glu Asp Pro Glu Glu 275 Val His Asn Ala Lys Thr 290 Tyr Arg Val Val Ser Asn 305 320 Gly Lys Glu Tyr Lys Pro 325 Ile Glu Lys Thr Ile Gln 340

    Ser Gly Leu Tyr 185 Ser Leu Gly Thr 200 Gln Thr Thr Lys 215 Val Asp Lys Thr 230 Cys Pro Pro Cys Phe Leu Phe Pro Pro 250 Pro Glu Val Thr 265 Cys Val Lys Phe 280 Asn Trp Thr Lys 295 Pro Arg Glu Val 310 Leu Thr Val Leu Cys Lys Val Ser Asn 330 Ser Lys Ala Lys Gly

    345

    Leu Ser Ser Val 190 Val Tyr Ile Cys 205 Asn Val Lys Val 220 Glu Pro Lys Pro 235 Ala Pro Glu Leu Lys Pro Lys Asp Thr 255 Val Val Val Asp 270 Val Tyr Val Asp 285 Gly Val Glu Gln 300 Tyr Asn Ser His 315 Gln Asp Trp Leu Lys Ala Leu Pro Ala 335 Gln Pro Arg Glu 350 Pro

    392

    Val Val Tyr Thr 355 Leu Pro Pro Ser Ser Leu Thr Cys Leu Val Lys Val 370 375 Glu Trp Glu Ser Asn Gly Gln Pro 385 390 400 Pro Val Leu Asp Ser Asp Gly Thr 405 Val Asp Lys Ser Arg Trp Gln Val 420 Met His Glu Ala Leu His Asn Leu 435 Ser Pro

    450

    Arg Lys Glu Leu Thr Lys Asn Gln

    360 365

    Gly Phe Tyr Pro Ser Asp Ile Ala

    380

    Pro Glu Asn Asn Tyr Lys Thr Thr

    395

    Ser Phe Phe Leu Tyr Ser Lys Leu

    410 415

    Gln Gly Asn Val Phe Ser Cys Ser

    425 430

    His Tyr Thr Gln Lys Ser Leu Ser

    440 445

    <210> <211> <212> <213> 242 450 PRT Artificial Sequence <220> <223> CFP0020H0261-021-G1dP1 <400> 242 Gln Val Glu Gln Leu Gln Glu Ser Gly 1 5

    Thr Leu Ser Leu Thr Cys Ala Val Gly

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Ser

    25 30

    393

    Tyr Trp Tyr Trp 35 Gly Trp Ile Arg Ile Gly Ser Ile Tyr His Ser Leu 50 55 Lys Ser Arg Val Thr Ile Ser Ser 65 70 Leu Lys Leu Gly Arg Val Thr Cys 85 Ala Arg His Asp Pro Thr Trp Trp 100 Gly Gln Gly Thr Leu Val Thr Pro 115 Ser Val Phe Pro Leu Ala Pro Thr 130 135 Ala Ala Leu Gly Cys Leu Val Thr 145 150 160 Val Ser Trp Asn Ser Gly Ala Pro 165 Ala Val Leu Gln Ser Ser Gly Thr 180 Val Pro Ser Ser Ser Leu Gly Asn 195

    Pro Pro Gly Lys Gly 45 Leu Glu Ser Thr Tyr Tyr Asn Lys Lys 60 Asp Thr Ser Lys Asn Gln Phe 75 Ala Asp Thr Ala Val Tyr Tyr 90 95 His His Gly Tyr Phe Asp Tyr 105 110 Ser Ser Ala Ser Thr Lys Gly 125 Ser Lys Ser Thr Ser Gly Gly 140 Asp Tyr Phe Pro Glu Pro Val 155 Thr Ser Gly Val His Thr Phe 170 175 Tyr Ser Leu Ser Ser Val Val 185 190 Gln Thr Tyr Ile Cys Asn Val 205

    394

    His Ser Lys 210 Pro Ser Asn Thr Lys 215 Cys Asp Lys Thr His Thr Cys Leu 225 230 240 Gly Gly Pro Ser Val Phe Leu Leu 245 Met Ile Ser Arg Thr Pro Glu Ser 260 His Glu Asp Pro Glu Val Lys Glu 275 Val His Asn Ala Lys Thr Lys Thr 290 295 Tyr Arg Val Val Ser Val Leu Asn 305 310 320 Gly Lys Glu Tyr Lys Cys Lys Pro 325 Ile Glu Lys Thr Ile Ser Lys Gln 340 Val Tyr Thr Leu Pro Pro Ser Val 355 Ser Leu Thr Cys Leu Val Lys Val 370 375

    Asp Lys Lys Val 220 Glu Pro Lys Pro Cys Pro Ala Pro Glu Leu 235 Pro Pro Lys Pro Lys Asp Thr 250 255 Thr Cys Val Val Val Asp Val 265 270 Asn Trp Tyr Val Asp Gly Val 285 Arg Glu Glu Gln Tyr Asn Ser 300 Val Leu His Gln Asp Trp Leu 315 Ser Asn Lys Ala Leu Pro Ala 330 335 Lys Gly Gln Pro Arg Glu Pro 345 350 Lys Glu Leu Thr Lys Asn Gln 365 Phe Tyr Pro Ser Asp Ile Ala 380

    395

    Glu Pro 385 400 Trp Glu Ser Asn Gly 390 Gln Pro Thr Val Leu Asp Ser 405 Asp Gly Val Val Asp Lys Ser 420 Arg Trp Gln Met Leu His Glu 435 Ala Leu His Asn

    Pro Glu Asn Asn Tyr Lys Thr Thr

    395

    Ser Phe Phe Leu Tyr Ser Lys Leu

    410 415

    Gln Gly Asn Val Phe Ser Cys Ser

    425 430

    His Tyr Thr Gln Lys Ser Leu Ser

    440 445

    Ser Pro 450

    <210> <211> <212> <213> 243 450 PRT Artificial Sequence <220> <223> CFP0020H0261-022-G1dP1 <400> 243 Gln Val Glu . Gln Leu Gln Glu Ser Gly 1 5

    Thr Leu Ser Leu Thr Cys Ala Val Gly

    Tyr Tyr Trp Gly Trp Ile Arg Gln Trp

    35 40

    Ile Gly Ser Ile Tyr His Ser Gly Leu

    50 55

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Ser

    25 30

    Pro Pro Gly Lys Gly Leu Glu

    Ser Thr Tyr Tyr Asn Lys Lys

    396

    Lys Ser 65 Ser Arg Val Thr Ile 70 Ser Val Asp Thr Ser 75 Lys Asn Gln Phe Leu Lys Leu Ser Arg Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

    Leu

    225 230 235

    240

    397

    Gly Leu Gly Pro Ser Val 245 Phe Leu Phe Pro Pro 250 Lys Pro Lys Asp Thr 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415

    398

    Val Asp Lys Ser Arg Trp Gln Gln Val

    420

    Met His Glu Ala Leu His Asn His Leu

    435 440

    Gly Asn Val Phe Ser Cys Ser

    425 430

    Tyr Thr Gln Lys Ser Leu Ser

    445

    Ser Pro 450

    <210> <211> <212> <213> 244 450 PRT Artificial Sequence <220> <223> CFP0020H0261-023-G1dP1 <400> 244 Gln Val Glu Gln Leu Gln Glu Ser Gly 1 5

    Thr Leu Ser Leu Thr Cys Ala Val Gly

    Tyr Tyr Trp Gly Trp Ile Arg Gln Trp

    35 40

    Ile Gly Ser Ile Tyr His Ser Gly Leu

    50 55

    Lys Ser Arg Val Thr Ile Ser Val Ser

    65 70

    Leu Lys Leu Ser Ser Arg Thr Ala Cys

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Ser

    25 30

    Pro Pro Gly Lys Gly Leu Glu

    Ser Thr Tyr Tyr Asn Lys Lys

    Asp Thr Ser Lys Asn Gln Phe

    Ala Asp Thr Ala Val Tyr Tyr

    90 95

    399

    Ala Trp Arg His Asp 100 Pro Thr Trp Tyr His 105 His Gly Tyr Phe Asp 110 Tyr Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270

    400

    His Glu Glu Asp 275 Pro Glu Val Lys Phe 280 Asn Trp Tyr Val Asp 285 Gly Val Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    401

    Ser Pro 450 <210>

    <211>

    <212>

    <213>

    245

    450

    PRT

    Artificial Sequence <220>

    <223> CFP0020H0261-025-G1dP1 <400> 245

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Lys Leu Ser Ser Val Thr Ala Ala Gly Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125

    402

    Ser Thr Val 130 Phe Pro Leu Ala Pro 135 Ser Ser Lys Ser Thr 140 Ser Gly Gly Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

    Thr

    290 295 300

    403

    Tyr Asn 305 320 Arg Val Val Ser Val 310 Leu Thr Val Leu His 315 Gln Asp Trp Leu Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450 <210> 246 <211> 450 <212> PRT <213> Artificial Sequence

    404 <220>

    <223> CFP0020H0261-026-G1dP1 <400> 246

    Gln Glu 1 Val Gln Leu Gln 5 Glu Ser Gly Pro Gly 10 Leu Val Lys Pro Ser 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

    Thr

    145 150 155

    160

    405

    Val Pro Ser Trp Asn Ser 165 Gly Ala Leu Thr Ser 170 Gly Val His Thr Phe 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335

    406

    Ile Gln Glu Lys Thr 340 Ile Ser Lys Ala Lys 345 Gly Gln Pro Arg Glu 350 Pro Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

    Leu

    435 440 445

    Ser Pro 450

    <210> 247 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-032-G1dP1 <400> 247 Gln Val Glu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15

    407

    Thr

    Gly

    Tyr

    Trp

    Ile

    Leu

    Lys

    Ser

    Leu

    Cys

    Ala

    Trp

    Gly

    Pro

    Ser

    Thr

    Ala

    Thr

    145

    160

    Val

    Pro

    Ala

    Thr

    Leu Ser Leu 20 Thr Cys Ala Val Ser 25 Gly Tyr Ser Ile Ser 30 Ser Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Lys Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 70 75 Lys Lys Ser Arg Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 150 155 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190

    408

    Val Asn Pro Ser 195 Ser Ser Leu Gly Thr 200 Gln Thr Tyr Ile Cys 205 Asn Val His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365

    409

    Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380

    Glu Pro 385 400 Trp Glu Ser Asn Gly 390 Gln Pro Glu Asn Asn 395 Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

    Leu

    435 440 445

    Ser Pro 450

    <210> 248 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-035-G1dP1 <400> 248 Gln Val Glu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15

    Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly

    20 25 30

    Tyr Tyr Trp Gly Trp Ile Arg Trp

    Gln Pro 40

    Pro Gly Lys

    Gly Leu Glu

    410

    Ile Gly Ser Ile Tyr His Leu

    Ser Gly Ser Thr

    Tyr

    Tyr Asn Lys

    Lys

    Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

    65 70 75 80

    Leu Lys Cys

    Leu

    Ser Arg Val

    Arg Ala Ala Asp

    Thr Ala Val

    Tyr Tyr

    Ala Arg His Asp Trp

    100

    Pro Thr Trp Tyr His

    105

    His Gly Tyr

    Phe Asp Tyr

    110

    Gly Gln Gly Thr Leu Val Pro

    115

    Thr Val

    120

    Ser Ser Ala

    Ser Thr Lys

    Gly

    125

    Ser Val Phe Thr

    130

    Pro Leu Ala Pro

    135

    Ser Ser Lys

    Ser Thr

    Ser Gly Gly

    140

    Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Thr 145 150 155 160

    Pro Glu

    Pro Val

    Val

    Pro

    Ser Trp Asn

    Ser Gly Ala Leu Thr

    Ser Gly Val

    His

    Thr

    Phe

    165

    170

    175

    Ala Val Leu Gln Ser Thr

    180

    Ser Gly Leu Tyr

    185

    Ser Leu Ser

    Ser Val Val

    190

    Val Pro Ser Ser Ser Leu Gly Thr Asn 195 200

    His Lys Pro Ser Asn Thr Lys Val Ser 210 215

    Gln Thr Tyr Ile Cys 205 Asn Val Asp Lys Lys Val Glu Pro Lys 220

    411

    Asp Lys Thr His Thr 230 Cys Pro Pro Cys Pro 235 Ala Pro Glu Leu Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 310 315 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 390 395

    412

    400

    Pro Thr Val Leu Asp Ser 405 Asp Gly Ser Val Asp Lys Ser Arg Trp Gln Gln Val 420

    Met His Glu Ala Leu His Asn His Leu 435 440

    Phe Phe Leu Tyr Ser Lys Leu

    410 415

    Gly Asn Val Phe Ser Cys Ser

    425 430

    Tyr Thr Gln Lys Ser Leu Ser

    445

    Ser Pro 450

    <210> <211> <212> <213> 249 450 PRT Artificial Sequence <220> <223> CFP0020H0261-036-G1dP1 <400> 249 Gln Val Glu Gln Leu Gln Glu Ser Gly 1 5

    Thr Leu Ser Leu Thr Cys Ala Val Gly

    Tyr Tyr Trp Gly Trp Ile Arg Gln Trp

    35 40

    Ile Gly Ser Ile Tyr His Ser Gly Leu

    50 55

    Lys Ser Arg Val Thr Ile Ser Val Ser

    65 70

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Ser

    25 30

    Pro Pro Gly Lys Gly Leu Glu

    Ser Thr Tyr Tyr Asn Lys Lys

    Asp Thr Ser Lys Asn Gln Phe

    413

    Leu Cys Lys Leu Ser Ser 85 Val Arg Ala Ala Asp 90 Thr Ala Val Tyr Tyr 95 Ala Arg His Asp Pro Thr Trp Tyr His His Gly Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255

    414

    Met Ser Ile Ser Arg 260 Thr Pro Glu Val Thr 265 Cys Val Val Val Asp 270 Val His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

    415

    420

    425

    430

    Met His Glu Ala Leu His Asn His Leu

    435 440

    Tyr Thr Gln Lys Ser Leu Ser

    445

    Ser Pro 450

    <210> <211> <212> <213> 250 450 PRT Artificial Sequence <220> <223> CFP0020H0261-037-G1dP1 <400> 250 Gln Val Glu Gln Leu Gln Glu Ser Gly 1 5

    Thr Leu Ser Leu Thr Cys Ala Val Gly

    Tyr Tyr Trp Gly Trp Ile Arg Gln Trp

    35 40

    Ile Gly Ser Ile Tyr His Ser Gly Leu

    50 55

    Lys Ser Arg Val Thr Ile Ser Arg Ser

    65 70

    Leu Lys Leu Ser Ser Val Thr Ala Cys

    Ala Arg His Asp Pro Thr Trp Tyr Trp

    100

    Pro Gly Leu Val Lys Pro Ser

    10 15

    Ser Gly Tyr Ser Ile Ser Ser

    25 30

    Pro Pro Gly Lys Gly Leu Glu

    Ser Thr Tyr Tyr Asn Lys Lys

    Asp Thr Ser Lys Asn Gln Phe

    Gly Asp Thr Ala Val Tyr Tyr 90 95

    His His Gly Tyr Phe Asp Tyr

    105 110

    416

    Gly Pro Gln Gly 115 Thr Leu Val Thr Val 120 Ser Ser Ala Ser Thr 125 Lys Gly Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285

    417

    Val Thr His 290 Asn Ala Lys Thr Lys 295 Pro Arg Glu Glu Gln 300 Tyr Asn Ser Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445

    Ser Pro 450

    418 <210> 251 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0018H0012-002-G1dN1 <400> 251

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Arg Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140

    419

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

    Glu

    305 310 315

    420

    320

    Tyr Lys Lys Cys Lys Val 325 Ser Asn Lys Ala Leu 330 Pro Ala Pro Ile Glu 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445

    <210>

    <211>

    <212>

    <213>

    252

    447

    PRT

    Artificial Sequence <220>

    <223> CFP0018H0012-003-G1dN1

    <400> 252 Gln Val Gln Ser Leu Val Gln Ser Arg Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    421

    Ser Tyr Val Lys Val 20 Ser Cys Lys Ala Ser 25 Gly Gly Thr Phe Ser 30 Ser Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

    180 185

    Ser Val Val

    Thr Val

    190

    Pro

    422

    Ser Pro Ser Leu 195 Gly Thr Gln Thr Tyr 200 Ile Cys Asn Val Asn 205 His Lys Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

    423

    355

    360

    365

    Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380

    Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser 435 440 445

    Pro

    <210> 253 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-005-G1dN1 <400> 253 Gln Val Ser Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Arg 1 5 10 15

    Ser Val Lys Val Tyr

    Ser Cys Lys Ala

    Ser Gly Gly Thr

    Phe

    Ser Ser

    Ser Ile Met

    Ser Trp Val Arg

    Gln Ala

    Pro

    Gly Gln Gly Leu Glu

    Trp

    Gly Gly Ile Ile Pro Ile Phe Phe

    50 55

    Gly Thr Ala Asn

    Tyr Ala Gln Lys

    424

    Gln Tyr 65 Gly Arg Val Thr Ile 70 Thr Ala Asp Glu Ser 75 Thr Ser Thr Ala Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

    Pro

    225 230 235

    425

    240

    Ser Ser Val Phe Leu Phe 245 Pro Pro Lys Pro Lys 250 Asp Thr Leu Met Ile 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

    426

    Lys

    405

    410

    415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Ala Leu His 435 Asn His Tyr Thr Gln 440

    Phe Ser Cys Ser Val Met His

    425 430

    Glu Ser Leu Ser Leu Ser Pro 445

    <210> <211> <212> <213> 254 447 PRT Artificial Sequence <220> <223> CFP0018H0012-008-G1dN1 <400> 254 Gln Val Ser Gln Leu Val Gln Ser Gly 1 5

    Ser Val Lys Val Ser Cys Lys Ala Arg

    Ser Ile Ser Trp Val Arg Gln Ala Met

    35 40

    Gly Gly Ile Ile Pro Ile Phe Gly Phe

    50 55

    Gln Gly Arg Val Thr Ile Thr Ala Tyr

    65 70

    Met Glu Leu Ser Ser Leu Arg Ser Cys

    Ala Arg Gln Leu Tyr Gly Tyr Tyr Gly

    100

    Ala Glu Val Lys Lys Pro Gly

    10 15

    Ser Gly Gly Thr Phe Ser Ser

    25 30

    Pro Gly Gln Gly Leu Glu Trp

    Thr Ala Asn Tyr Ala Gln Lys

    Asp Glu Ser Thr Ser Thr Ala

    75 80

    Glu Asp Thr Ala Val Tyr Tyr

    90 95

    Glu Leu Asp Ile Trp Gly Gln

    105 110

    427

    Thr Phe Leu Val 115 Thr Val Ser Ser Ala 120 Ser Thr Lys Gly Pro 125 Ser Val Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

    428

    275

    280

    285

    Ala Val Lys 290 Thr Lys Pro Arg Glu 295 Glu Gln Tyr Asn Ser 300 Thr Tyr Arg Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 255 <211> 447

    429 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0018H0012-009-G1dN1 <400> 255

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ser Ile Ser Trp Val Arg Lys Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155

    430

    160

    Asn Leu Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Ala Val 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

    431

    Lys

    325

    330

    335

    Thr Thr Ile Ser Lys 340 Ala Lys Gly Gln Pro 345 Arg Glu Pro Gln Val 350 Tyr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro

    435 440 445

    <210> 256 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-013-G1dN1 <400> 256 Gln Val Ser Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    Ser Val Lys Val Ser Cys Lys Ala Ser Tyr

    Gly Gly Thr Phe

    Ser Ser

    432

    Ser Met Ile Ser 35 Trp Val Arg Gln Ala 40 Pro Gly Gln Gly Leu 45 Glu Trp Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Arg 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

    433

    195 200 205

    Ser Lys Asn 210 Thr Lys Val Asp Lys 215 Lys Val Glu Pro Lys 220 Ser Cys Asp Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

    434

    Glu

    370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro

    435 440 445

    <210> 257 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-014-G1dN1 <400> 257 Gln Val Ser Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

    20 25 30

    Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

    35 40 45

    Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

    50 55 60

    Lys Gly Arg Val Thr Ile Thr Tyr

    Ala Asp

    Glu Ser Thr

    Ser Thr Ala

    435

    Met Cys Glu Leu Ser Ser 85 Leu Arg Ser Glu Asp 90 Thr Ala Val Tyr Tyr 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

    436

    Ser

    245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    437

    Ser Arg Trp Gln Gln Gly Asn Val Glu

    420

    Ala Leu His Asn His Tyr Thr Gln 435 440

    Phe Ser Cys Ser Val Met His

    425 430

    Glu Ser Leu Ser Leu Ser Pro 445 <210> 258 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0018H0012-016-G1dN1 <400> 258

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ser Val Lys Val Ser Cys Lys Ala Tyr 20 Ser Ile Ser Trp Val Arg Gln Ala Met 35 40 Gly Gly Ile Ile Pro Ile Phe Gly Arg 50 55 Lys Gly Arg Val Thr Ile Thr Ala Tyr 65 70 Met Glu Leu Ser Ser Leu Arg Ser Cys 85 Ala Arg Gln Leu Tyr Gly Tyr Tyr Gly 100 Thr Leu Val Thr Val Ser Ser Ala Phe

    Ala Glu Val Lys Lys Pro Gly

    10 15

    Ser Gly Gly Thr Phe Ser Ser

    25 30

    Pro Gly Gln Gly Leu Glu Trp

    Thr Ala Asn Tyr Ala Gln Lys

    Asp Glu Ser Thr Ser Thr Ala

    75 80

    Glu Asp Thr Ala Val Tyr Tyr

    90 95

    Glu Leu Asp Ile Trp Gly Gln

    105 110

    Ser Thr Lys Gly Pro Ser Val

    438

    115 120 125

    Pro Leu Leu 130 Ala Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly 140 Thr Ala Ala Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    439

    Val

    290 295 300

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 259 <211> 447 <212> PRT <213> Artificial Sequence <220>

    440 <223> CFP0018H0012-018-G1dN1 <400> 259

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Arg Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

    441

    Leu

    165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335

    442

    Thr Thr Ile Ser Lys 340 Ala Lys Gly Gln Pro 345 Arg Glu Pro Gln Val 350 Tyr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro

    435 440 445

    <210> 260 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-019-G1dN1 <400> 260 Gln Val Ser Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    Ser Val Lys Val Tyr

    Ser Cys Lys Ala

    Ser Gly Gly Thr

    Phe

    Ser Ser

    Ser Ile

    Ser Trp Val Arg Gln

    Ala Pro

    Gly Gln Gly Leu Glu

    Trp

    443

    Met

    Gly Phe Gly 50 Ile Ile Pro Ile Phe 55 Gly Thr Ala Asn Tyr 60 Ala Gln Lys Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Lys Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

    444

    Lys

    210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380

    445

    Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 261 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0018H0012-020-G1dN1 <400> 261 Pro Gly 15 Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Asn Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

    446

    Cys

    Ala Gly Arg Gln Leu 100 Tyr Gly Tyr Tyr Glu 105 Leu Asp Ile Trp Gly 110 Gln Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255

    447

    Arg Asp Thr Pro Glu 260 Val Thr Cys Val Val 265 Val Asp Val Ser His 270 Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    448

    Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 262 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0018H0012-021-G1dN1 <400> 262

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Gly Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

    449

    Leu

    130 135 140

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    450

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 263 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0018H0012-022-G1dN1 <400> 263

    451

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Arg Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    452

    Gln Ser Ser Ser Gly 180 Leu Tyr Ser Leu Ser 185 Ser Val Val Thr Val 190 Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350

    453

    Leu Thr Pro Pro 355 Ser Arg Asp Glu Leu 360 Thr Lys Asn Gln Val 365 Ser Leu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro

    435 440 445

    <210> 264 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-023-G1dN1 <400> 264 Gln Val Ser . Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

    20 25 30

    Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

    35 40 45

    454

    Gly Phe Gly 50 Ile Ile Pro Ile Phe 55 Gly Thr Ala Asn Tyr 60 Ala Gln Lys Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Arg Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220

    455

    Thr Pro 225 240 His Thr Cys Pro Pro 230 Cys Pro Ala Pro Glu 235 Leu Leu Gly Gly Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

    Leu

    385 390 395

    400

    456

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser 435 440 445

    Pro

    <210> 265 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-024-G1dN1 <400> 265 Gln Val Ser . Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    Ser Val Lys Val Tyr

    Ser Cys Lys Ala

    Ser Gly Gly Thr

    Phe

    Ser Ser

    Ser Ile Met

    Ser Trp Val Arg

    Gln Ala

    Pro

    Gly Gln Gly Leu Glu

    Trp

    Gly Gly Ile Ile Pro Phe

    Ile

    Phe

    Gly Thr Ala Asn

    Tyr Ala

    Gln Lys

    Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

    65 70 75 80

    Met Glu Leu Ser Ser Leu Arg Cys

    Ser Gly Asp

    Thr Ala Val

    Tyr Tyr

    457

    Ala Gly Arg Gln Leu 100 Tyr Thr Leu Val Thr Val Phe 115 Pro Leu Ala Pro Ser Leu 130 Gly Cys Leu Val Lys Trp 145 160 Asn Ser Gly Ala Leu Leu 165 Gln Ser Ser Gly Leu Ser 180 Ser Ser Leu Gly Thr Pro 195 Ser Asn Thr Lys Val Lys 210 Thr His Thr Cys Pro Pro 225 240 Ser Val Phe Leu Phe Ser 245 Arg Thr Pro Glu Val Asp 260

    Gly Tyr Tyr Glu 105 Leu Ser Ser Ala 120 Ser Thr Ser Lys 135 Ser Thr Ser Asp 150 Tyr Phe Pro Glu Thr Ser Gly Val His 170 Tyr Ser Leu Ser 185 Ser Gln Thr Tyr 200 Ile Cys Asp Lys 215 Lys Val Glu Pro 230 Cys Pro Ala Pro Pro Pro Lys Pro Lys 250 Thr Cys Val Val Val

    265

    Asp Ile Trp Gly 110 Gln Lys Gly Pro 125 Ser Val Gly Gly 140 Thr Ala Ala Pro 155 Val Thr Val Ser Thr Phe Pro Ala Val 175 Val Val Thr Val 190 Pro Asn Val Asn 205 His Lys Pro Lys 220 Ser Cys Asp Glu 235 Leu Leu Gly Gly Asp Thr Leu Met Ile 255 Asp Val Ser His 270 Glu

    458

    Pro Asn Glu Val 275 Lys Phe Asn Trp Tyr 280 Val Asp Gly Val Glu 285 Val His Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445

    459 <210> 266 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0018H0012-025-G1dN1 <400> 266

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Gly Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140

    460

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

    Glu

    305 310 315

    320

    461

    Tyr Lys Lys Cys Lys Val 325 Ser Asn Lys Ala Leu 330 Pro Ala Pro Ile Glu 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro

    435 440 445 <210>

    <211>

    <212>

    <213>

    267

    447

    PRT

    Artificial Sequence <220>

    <223> CFP0018H0012-026-G1dN1

    <400> 267 Gln Val Gln Ser Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    462

    Ser Tyr Val Lys Val 20 Ser Cys Lys Ala Ser 25 Gly Gly Thr Phe Ser 30 Ser Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Lys Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

    180 185

    Ser Val Val

    Thr Val

    190

    Pro

    463

    Ser Pro Ser Leu 195 Gly Thr Gln Thr Tyr 200 Ile Cys Asn Val Asn 205 His Lys Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

    Thr

    355 360 365

    464

    Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380

    Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser 435 440 445

    Pro

    <210> 268 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-027-G1dN1 <400> 268 Gln Val Ser Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15

    Ser Val Lys Val Tyr

    Ser Cys Lys Ala

    Ser Gly Gly Thr

    Phe

    Ser Ser

    Ser Ile Met

    Ser Trp Val Arg

    Gln Ala

    Pro

    Gly Gln Gly Leu Glu

    Trp

    Gly Gly Ile Ile Pro Ile Phe Phe

    50 55

    Gly Thr Ala Asn

    Tyr Ala Gln Lys

    465

    Gln Tyr 65 Gly Arg Val Thr Ile 70 Thr Ala Asp Glu Ser 75 Thr Ser Thr Ala Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Leu Tyr Gly Tyr Tyr Glu Leu Asp Ile Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

    Pro

    225 230 235

    240

    466

    Ser Ser Val Phe Leu Phe 245 Pro Pro Lys Pro Lys 250 Asp Thr Leu Met Ile 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

    467

    405

    410

    415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Ala Leu His 435 Asn His Tyr Thr Gln 440

    Phe Ser Cys Ser Val Met His

    425 430

    Glu Ser Leu Ser Leu Ser Pro 445

    <210> <211> <212> <213> 269 447 PRT Artificial Sequence <220> <223> CFP0018H0012-032-G1dN1 <400> 269 Gln Val Ser Gln Leu Val Gln Ser Gly 1 5

    Ser Val Lys Val Ser Cys Lys Ala Tyr

    Ser Ile Ser Trp Val Arg Gln Ala Met

    35 40

    Gly Gly Ile Ile Pro Ile Phe Gly Phe

    50 55

    Gln Gly Arg Val Thr Ile Thr Ala Tyr

    65 70

    Met Glu Lys Ser Arg Leu Arg Ser Cys

    Ala Arg Gln Leu Tyr Gly Tyr Tyr Gly

    100

    Ala Glu Val Lys Lys Pro Gly

    10 15

    Ser Gly Gly Thr Phe Ser Ser

    25 30

    Pro Gly Gln Gly Leu Glu Trp

    Thr Ala Asn Tyr Ala Gln Lys

    Asp Glu Ser Thr Ser Thr Ala

    75 80

    Glu Asp Thr Ala Val Tyr Tyr

    90 95

    Glu Leu Asp Ile Trp Gly Gln

    105 110

    468

    Thr Phe Leu Val 115 Thr Val Ser Ser Ala 120 Ser Thr Lys Gly Pro 125 Ser Val Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285

    469

    Ala Val Lys 290 Thr Lys Pro Arg Glu 295 Glu Gln Tyr Asn Ser 300 Thr Tyr Arg Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 270 <211> 447 <212> PRT

    470 <213> Artificial Sequence <220>

    <223> CFP0018H0012-037-G1dN1 <400> 270

    Gln Ser 1 Val Gln Leu Val 5 Gln Ser Gly Ser Val Lys Val Ser Cys Lys Ala Tyr 20 Ser Ile Ser Trp Val Arg Gln Ala Met 35 40 Gly Gly Ile Ile Pro Ile Phe Gly Phe 50 55 Gln Gly Arg Val Thr Ile Thr Arg Tyr 65 70 Met Glu Leu Ser Ser Leu Arg Ser Cys 85 Ala Arg Gln Leu Tyr Gly Tyr Tyr Gly 100 Thr Leu Val Thr Val Ser Ser Ala Phe 115 120 Pro Leu Ala Pro Ser Ser Lys Ser Leu 130 135 Gly Cys Leu Val Lys Asp Tyr Phe Trp 145 150 160

    Ala Glu 10 Val Lys Lys Pro Gly 15 Ser Gly Gly Thr Phe Ser Ser 25 30 Pro Gly Gln Gly Leu Glu Trp 45 Thr Ala Asn Tyr Ala Gln Lys 60 Asp Glu Ser Thr Ser Thr Ala 75 Gly Asp Thr Ala Val Tyr Tyr 90 95 Glu Leu Asp Ile Trp Gly Gln 105 110 Ser Thr Lys Gly Pro Ser Val 125 Thr Ser Gly Gly Thr Ala Ala 140 Pro Glu Pro Val Thr Val Ser

    155

    471

    Asn Leu Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Ala Val 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

    472

    325

    330

    335

    Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350

    Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365

    Cys Glu Leu 370 Val Lys Gly Phe Tyr 375 Pro Ser Asp Ile Ala 380 Val Glu Trp Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    Ser Pro

    Ala Leu His Asn His Tyr Thr Gln Glu Ser Leu Ser Leu

    435 440 445 <210> 271 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-001-k0 <400> 271 Asp Ile ; Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Lys 1 5 10

    Ser Val

    Asp Arg Val Thr Ile Thr Cys Arg Ala Asp 20 25

    Ser Gln Ser

    Ile His Asn

    473

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Lys Ala Pro Lys 45 Leu Leu Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

    474

    Phe Asn Arg Gly Glu Cys 210 <210> 272 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-002-k0 <400> 272

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125

    475

    Thr Ala Ala 130 Ser Val Val Cys Leu 135 Leu Asn Asn Phe Tyr 140 Pro Arg Glu Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 273 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233- 003- k0 <400> 273 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Arg Arg Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

    476

    Gly

    Ser Pro 65 Gly Ser Gly Thr Asp 70 Phe Thr Leu Thr Ile 75 Ser Ser Leu Gln Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    <210> 274 <211> 214

    210

    477 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-004-k0 <400> 274

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Lys Glu Leu Gln Lys Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

    Gln

    145 150 155

    478

    160

    Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 275 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-005-k0 Pro Ser Ser 10 Leu Ser Ala Ser Val 15 <400> 275 Thr 5 Gln Ser Asp Gly 1 Ile Gln Met Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45

    Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

    Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75

    479

    Glu Tyr Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Tyr Ser Ser Tyr Pro 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

    <210> <211> <212> <213> 276 214 PRT Artificial Sequence <220> <223> CFP0020L233 -006-k0 <400> 276

    480

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    481

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

    180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

    195 200 205

    Phe Asn Arg Gly Glu Cys 210 <210> 277 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-007-k0 <400> 277

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

    482

    100

    105

    110

    Pro Gly Ser Val 115 Phe Ile Phe Pro Pro 120 Ser Asp Glu Gln Leu 125 Lys Ser Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 278 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-008-k0 <400> 278

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Lys Lys Gln Ser Ile His Asn

    Asp

    20 25 30

    483

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Lys Ala Pro Lys 45 Leu Leu Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

    484

    Phe Asn Arg Gly Glu Cys 210 <210> 279 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-009-k0 <400> 279

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125

    485

    Thr Ala Ala 130 Ser Val Val Cys Leu 135 Leu Asn Asn Phe Tyr 140 Pro Arg Glu Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 280 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233- 010- k0 <400> 280 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

    486

    Gly

    Ser Pro 65 Gly Ser Gly Thr Asp 70 Phe Thr Leu Thr Ile 75 Ser Ser Leu Gln Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    <210> 281 <211> 214

    210

    487 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-011-k0 <400> 281

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Lys Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

    Gln

    145 150 155

    488

    160

    Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 282 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-012-k0 Pro Ser Ser 10 Leu Ser Ala Ser Val 15 <400> 282 Thr 5 Gln Ser Asp Gly 1 Ile Gln Met Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45

    Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

    Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75

    489

    Glu Tyr Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Tyr Ser Ser Tyr Pro 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

    <210> <211> <212> <213> 283 214 PRT Artificial Sequence <220> <223> CFP0020L233 -013-k0 <400> 283

    490

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    491

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

    180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

    195 200 205

    Phe Asn Arg Gly Glu Cys 210 <210> 284 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-016-k0 <400> 284

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

    492

    100

    105

    110

    Pro Gly Ser Val 115 Phe Ile Phe Pro Pro 120 Ser Asp Glu Gln Leu 125 Lys Ser Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 285 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-017-k0 <400> 285

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn

    Asp

    20 25 30

    493

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Lys Ala Pro Lys 45 Arg Leu Lys Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

    494

    Phe Asn Arg Gly Glu Cys 210 <210> 286 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-018-k0 <400> 286

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125

    495

    Thr Ala Ala 130 Ser Val Val Cys Leu 135 Leu Asn Asn Phe Tyr 140 Pro Arg Glu Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 287 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233- 019- k0 <400> 287 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

    496

    Gly

    Arg Pro 65 Gly Ser Gly Arg Asp 70 Phe Thr Leu Lys Ile 75 Ser Arg Leu Gln Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    <210> 288 <211> 214

    210

    497 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-021-k0 <400> 288

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

    Gln

    145 150 155

    498

    160

    Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 289 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-022-k0 Pro Ser Ser 10 Leu Ser Ala Ser Val 15 <400> 289 Thr 5 Gln Ser Asp Gly 1 Ile Gln Met Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Ala Pro Lys Leu Leu Ile 35 40 45

    Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

    Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75

    499

    Glu Tyr Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Tyr Ser Ser Tyr Pro 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

    <210> <211> <212> <213> 290 214 PRT Artificial Sequence <220> <223> CFP0020L233 -023-k0 <400> 290

    500

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Lys Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    501

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

    180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

    195 200 205

    Phe Asn Arg Gly Glu Cys 210 <210> 291 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-024-k0 <400> 291

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

    502

    100

    105

    110

    Pro Gly Ser Val 115 Phe Ile Phe Pro Pro 120 Ser Asp Glu Gln Leu 125 Lys Ser Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 292 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-025-k0 <400> 292

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn

    Asp

    20 25 30

    503

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Lys Ala Pro Lys 45 Leu Leu Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

    504

    Phe Asn Arg Gly Glu Cys 210 <210> 293 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-027-k0 <400> 293

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125

    505

    Thr Ala Ala 130 Ser Val Val Cys Leu 135 Leu Asn Asn Phe Tyr 140 Pro Arg Glu Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 294 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233- 028- k0 <400> 294 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser

    506

    Gly

    Ser Pro 65 Gly Ser Gly Thr Asp 70 Phe Thr Leu Lys Ile 75 Ser Arg Leu Gln Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    <210> 295 <211> 214

    210

    507 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-029-k0 <400> 295

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

    Gln

    145 150 155

    508

    160

    Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 296 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-031-k0 Pro Ser Ser 10 Leu Ser Ala Ser Val 15 <400> 296 Thr 5 Gln Ser Asp Gly 1 Ile Gln Met Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45

    Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

    Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75

    509

    Glu Tyr Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Tyr Ser Ser Tyr Pro 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

    <210> <211> <212> <213> 297 214 PRT Artificial Sequence <220> <223> CFP0020L233 -032-k0 <400> 297

    510

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    511

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

    180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

    195 200 205

    Phe Asn Arg Gly Glu Cys 210 <210> 298 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-034-k0 <400> 298

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

    512

    100

    105

    110

    Pro Gly Ser Val 115 Phe Ile Phe Pro Pro 120 Ser Asp Glu Gln Leu 125 Lys Ser Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 299 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-036-k0 <400> 299

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile His Asn

    Asp

    20 25 30

    513

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Arg Lys Ala Pro Lys 45 Leu Leu Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

    514

    Phe Asn Arg Gly Glu Cys 210 <210> 300 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-037-k0 <400> 300

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125

    515

    Thr Ala Ala 130 Ser Val Val Cys Leu 135 Leu Asn Asn Phe Tyr 140 Pro Arg Glu Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 301 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> <400> CFP0020L233-038-k0 301 Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Gly 1 Ile Gln Met Thr 5 Gln Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Arg Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

    516

    Gly

    Arg Pro 65 Gly Ser Gly Arg Asp 70 Phe Thr Leu Thr Ile 75 Arg Ser Leu Lys Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    <210> 302 <211> 214

    210

    517 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-040-k0 <400> 302

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

    Gln

    145 150 155

    518

    160

    Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

    Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 303 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-041-k0 <400> 303 Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Gly 1 Ile Gln Met Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Arg Glu Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys

    Pro

    65 70 75 80

    519

    Glu Tyr Asp Phe Ala Thr 85 Tyr Tyr Cys Gln His 90 Tyr Ser Ser Tyr Pro 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210

    <210> <211> <212> <213> 304 214 PRT Artificial Sequence <220> <223> CFP0020L233 -043-k0 <400> 304

    520

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile His Asn Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175

    521

    Ser Thr Leu Thr Leu Ser Lys Tyr

    180

    Ala Cys Glu Val Thr His Gln Ser

    195

    Asp Tyr Glu Lys His Lys Val

    185 190

    Leu Ser Ser Pro Val Thr Lys

    205

    Phe Asn Arg Gly Glu Cys 210 <210> 305 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-044-k0 <400> 305

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Asp Arg Val Thr Ile Thr Cys Asp 20 Leu Ala Trp Tyr Gln Gln Lys Ile 35 Tyr Glu Ala Ser Glu Leu Gln Gly 50 55 Ser Gly Ser Gly Thr Asp Phe Pro 65 70 Glu Asp Phe Ala Thr Tyr Tyr Tyr 85 Thr Phe Gly Gln Gly Thr Lys Ala

    Ser Ser Leu Ser Ala Ser Val

    10 15

    Ala Ser Gln Ser Ile His Asn

    25 30

    Gly Lys Ala Pro Lys Leu Leu

    Gly Val Pro Ser Arg Phe Ser

    Leu Thr Ile Arg Ser Leu Gln

    75 80

    Gln His Tyr Ser Ser Tyr Pro

    90 95

    Glu Ile Lys Arg Thr Val Ala

    522

    100

    105

    110

    Pro Gly Ser Val 115 Phe Ile Phe Pro Pro 120 Ser Asp Glu Gln Leu 125 Lys Ser Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 306 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> CFP0020L233-045-k0

    <400> 306 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15

    Asp Arg Val Thr Ile Thr Cys Arg Ala Asp 20 25

    Ser Gln Ser

    Ile His Asn

    523

    Leu Ile Ala Trp 35 Tyr Gln Gln Lys Pro 40 Gly Lys Ala Pro Lys 45 Leu Leu Tyr Glu Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Lys Pro 65 70 75 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205

    524

    Phe Asn Arg Gly Glu Cys 210 <210> 307 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1394m <400> 307

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125

    525

    Pro Leu Leu 130 Ala Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly 140 Thr Ala Ala Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    526

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Lys Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 308 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1398m <400> 308

    527

    Gln Val Gly 1 Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    528

    Gln Ser Ser Ser Gly 180 Leu Tyr Ser Leu Ser 185 Ser Val Val Thr Val 190 Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350

    529

    Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Ser Asn Leu Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Lys Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 309 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H- -P1466m <400> 309 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45

    530

    Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser

    50 55 60

    Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

    65 70 75 80

    Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

    85 90 95

    Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly

    100 105 110

    Thr Leu Val Thr Val Phe

    115

    Ser Ser Ala

    Ser Thr Lys

    Gly Pro

    Ser Val

    120

    125

    Pro Leu Ala Pro Leu

    130

    Ser Ser Lys

    135

    Ser Thr

    Ser

    Gly Gly Thr Ala

    Ala

    140

    Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Trp 145 150 155 160

    Ser

    Asn Ser Gly Ala Leu Thr Ser Gly Val Leu

    165

    His

    170

    Thr

    Phe

    Pro Ala Val

    175

    Gln Ser Ser Gly Leu Tyr Ser

    180

    Ser Leu Ser

    185

    Ser Val Val

    Thr Val

    Pro

    190

    Ser

    Pro

    Ser Leu Gly Thr

    Gln Thr

    Tyr

    195

    200

    Ile Cys Asn Val Asn His Lys

    205

    Ser Asn Thr Lys Lys

    210

    Val Asp Lys

    215

    Lys Val Glu Pro

    Lys

    220

    Ser Cys Asp

    531

    Thr Pro 225 240 His Thr Cys Pro Pro 230 Cys Pro Ala Pro Glu 235 Leu Leu Gly Gly Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Arg Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

    Leu

    385 390 395

    532

    400

    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 310 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1468m <400> 310

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95

    533

    Arg Gly Glu Thr His 100 His Gly Ser Ser Gly 105 Ile Asp His Trp Gly 110 Gln Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270

    534

    Pro Asn Glu Val 275 Lys Phe Asn Trp Tyr 280 Val Asp Gly Val Glu 285 Val His Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Arg Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    535 <210> 311 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1469m <400> 311

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140

    536

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Lys Gly Lys

    Glu

    305 310 315

    537

    320

    Tyr Lys Lys Cys Lys Val 325 Ser Asn Lys Ala Leu 330 Pro Ala Pro Ile Glu 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 312 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1470m <400> 312 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15

    538

    Ser His Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Leu Ser 30 Asn Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

    Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

    180 185

    Ser Val Val

    Thr Val

    190

    Pro

    539

    Ser Pro Ser Leu 195 Gly Thr Gln Thr Tyr 200 Ile Cys Asn Val Asn 205 His Lys Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

    540

    355

    360

    365

    Cys Leu Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Ser Arg Leu Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 313 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H- P1471m <400> 313 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala

    Ser

    50 55 60

    541

    Gly Leu 65 Arg Phe Thr Ile Ser 70 Arg Asp Asn Ser Lys 75 Asn Thr Val Tyr Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

    Pro

    225 230 235

    542

    240

    Ser Ser Val Phe Leu Phe 245 Pro Pro Lys Pro Lys 250 Asp Thr Leu Met Ile 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Lys Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

    543

    Lys

    405

    410

    415

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 His Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 314 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1480m <400> 314

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110

    544

    Thr Phe Leu Val 115 Thr Val Ser Ser Ala 120 Ser Thr Lys Gly Pro 125 Ser Val Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

    545

    275

    280

    285

    Ala Val Lys 290 Thr Lys Pro Arg Glu 295 Glu Gln Tyr Asn Ser 300 Thr Tyr Arg Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Arg Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 315 <211> 447

    546 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1481m <400> 315

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly Leu 10 Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155

    547

    160

    Asn Leu Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Ala Val 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

    548

    Lys

    325

    330

    335

    Thr Thr Ile Ser Lys 340 Ala Lys Gly Lys Pro 345 Arg Glu Pro Gln Val 350 Tyr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

    435 440 445 <210> 316 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1482m

    <400> 316 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn

    His

    549

    Asp Val Met Thr 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Tyr Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

    550

    195 200 205

    Ser Lys Asn 210 Thr Lys Val Asp Lys 215 Lys Val Glu Pro Lys 220 Ser Cys Asp Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Arg Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

    551

    Glu

    370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Lys 405 410

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Phe 425 Ser Cys Ser Val Met 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445

    Val

    Asp

    415

    His

    Pro <210> 317 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1483m <400> 317

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly Leu Val 10 Gln Pro Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Leu

    Gly

    Asn

    Tyr

    Ala

    Tyr

    552

    Gln Ala Ile Asp Ser Leu 85 Arg Ala Glu Asp Thr 90 Ala Thr Tyr Tyr Cys 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

    553

    Ser

    245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Lys Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415

    554

    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 318 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H- -P1512m <400> 318 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ser Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala 50 55 60 Gly Arg Leu Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

    Phe

    555

    115 120 125

    Pro Leu Leu 130 Ala Pro Ser Ser Lys 135 Ser Thr Ser Gly Gly 140 Thr Ala Ala Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    556

    Val

    290 295 300

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Arg Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 319 <211> 447 <212> PRT <213> Artificial Sequence <220>

    557 <223> Ab1H-P1513m <400> 319

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ile Ile Asn Thr Tyr Gly Asn His Trp Tyr Ala Asn Trp Ala Ser 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

    558

    Leu

    165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335

    559

    Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Ser Asn Leu Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Asp Ser Lys Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 320 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H- P1514m <400> 320 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn His 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr

    560

    Val

    Ser Ser Ile 50 Ile Asn Thr Tyr Gly 55 Asn His Trp Tyr Ala 60 Asn Trp Ala Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 Gln Ile Asp Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Thr His His Gly Ser Ser Gly Ile Asp His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

    561

    Lys

    210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380

    562

    Ser Leu 385 400 Asn Gly Gln Pro Glu 390 Asn Asn Asp Ser Asp Arg Ser Phe Phe Leu Lys 405

    Ser Glu Arg Trp Gln 420 Gln Gly Asn Val Ala Leu His 435 Asn His Tyr Thr Gln 440

    <210> 321 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1515m <400> 321

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Ser Leu Arg Leu Ser Cys Ala Ala His 20 Asp Met Thr Trp Val Arg Gln Ala Val 35 40 Ser Ile Ile Asn Thr Tyr Gly Asn Ser 50 55 Gly Arg Phe Thr Ile Ser Arg Asp Leu 65 70 Gln Ile Asp Ser Leu Arg Ala Glu

    Tyr Lys Thr Thr Pro Pro Val 395

    Tyr Ser Lys Leu Thr Val Asp

    410 415

    Phe Ser Cys Ser Val Met His 425 430

    Lys Ser Leu Ser Leu Ser Pro 445

    Gly Gly Leu Val Gln Pro Gly 10 15

    Ser Gly Phe Thr Leu Ser Asn

    25 30

    Pro Gly Lys Gly Leu Glu Tyr 45

    His Trp Tyr Ala Asn Trp Ala 60

    Asn Ser Lys Asn Thr Val Tyr

    75 80

    Asp Thr Ala Thr Tyr Tyr Cys

    563

    Ala

    Arg Gly Glu Thr His 100 His Gly Ser Ser Gly 105 Ile Asp His Trp Gly 110 Gln Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255

    564

    Arg Asp Thr Pro Glu 260 Val Thr Cys Val Val 265 Val Asp Val Ser His 270 Glu Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Lys Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

    565

    Ala Leu His Asn His Tyr Thr Gln 435 440

    Lys Ser Leu Ser Leu Ser Pro 445 <210> 322 <211> 447 <212> PRT <213> Artificial Sequence <220>

    <223> Ab1H-P1653m <400> 322

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Gly Ser Leu Arg Leu Ser Cys Ala Ala His 20 Asp Met Thr Trp Val Arg Gln Ala Val 35 40 Ser Ile Ile Asn Thr Tyr Gly Asn Ser 50 55 Gly Arg Phe Thr Ile Ser Arg Asp Leu 65 70 Gln Ile Asp Ser Leu Arg Ala Glu Ala 85 Arg Glu Thr His His Gly Ser Ser Gly 100 Thr Leu Val Thr Val Ser Ser Ala Phe 115 120 Pro Leu Ala Pro Ser Ser Lys Ser

    Gly Gly Leu Val Gln Pro Gly

    10 15

    Ser Gly Phe Thr Leu Ser Asn

    25 30

    Pro Gly Lys Gly Leu Glu Tyr

    His Trp Tyr Ala Asn Trp Ala

    Asn Ser Lys Asn Thr Val Tyr

    75 80

    Asp Thr Ala Thr Tyr Tyr Cys

    90 95

    Gly Ile Asp His Trp Gly Gln

    105 110

    Ser Thr Lys Gly Pro Ser Val

    125

    Thr Ser Gly Gly Thr Ala Ala

    566

    Leu

    130 135 140

    Gly Trp 145 160 Cys Leu Val Lys Asp 150 Tyr Phe Pro Glu Pro 155 Val Thr Val Ser Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

    Val

    290 295 300

    567

    Val Glu 305 320 Ser Val Leu Thr Val 310 Leu His Gln Asp Trp 315 Leu Asn Gly Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Arg Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445

    <210> 323 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> F8M-F1847mv1 <400> 323

    568

    Gln Val Gly 1 Gln Leu Val 5 Glu Ser Gly Gly Gly 10 Leu Val Gln Pro Gly 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 Asp Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175

    569

    Pro Val Ala Val Leu 180 Gln Ser Ser Thr Val Pro Ser Ser Ser Leu Val 195 Asp His Lys Pro Ser Asn Thr Lys 210 215 Tyr Gly Pro Pro Cys Pro Pro Gly 225 230 240 Pro Ser Val Phe Leu Phe Pro Ile 245 Ser Arg Thr Pro Glu Val Thr Glu 260 Asp Pro Glu Val Gln Phe Asn His 275 Asn Ala Lys Thr Lys Pro Arg Arg 290 295 Val Val Ser Val Leu Thr Val Lys 305 310 320 Glu Tyr Lys Cys Lys Val Ser Glu 325 Lys Thr Ile Ser Lys Ala Lys Tyr 340

    Leu Tyr Ser Leu Ser Ser Val 185 190 Thr Gln Thr Tyr Thr Cys Asn 205 Val Asp Lys Arg Val Glu Ser 220 Pro Ala Pro Glu Phe Arg Arg 235 Lys Pro Lys Asp Thr Leu Met 250 255 Val Val Val Asp Val Ser Gln 265 270 Tyr Val Asp Gly Val Glu Val 285 Glu Gln Tyr Asn Ser Thr Tyr 300 His Gln Asp Trp Leu Asn Gly 315 Lys Gly Leu Pro Ser Ser Ile 330 335 Gln Pro Arg Glu Pro Gln Val 345 350

    570

    Thr Leu Leu Pro 355 Pro Ser Gln Lys Glu 360 Met Thr Lys Asn Gln 365 Val Ser Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser

    Pro

    435 440 445 <210> 324 <211> 444 <212> PRT <213> Artificial Sequence <220>

    <223> F8M- F1847mv2 <400> 324 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30 Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

    Met

    35 40 45

    571

    Gly Phe Asp 50 Ile Asn Thr Arg Ser 55 Gly Gly Ser Ile Tyr 60 Asn Glu Glu Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95 Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

    Pro

    210 215 220

    572

    Cys Phe 225 240 Pro Pro Cys Pro Ala 230 Pro Leu Phe Pro Pro Lys Pro Lys Pro 245 Glu Val Thr Cys Val Val Val Val 260 Gln Phe Asn Trp Tyr Val Asp Thr 275 Lys Pro Arg Glu Glu Gln Tyr Val 290 295 Leu Thr Val Leu His Gln Asp Cys 305 310 320 Lys Val Ser Asn Lys Gly Leu Ser 325 Lys Ala Lys Gly Gln Pro Arg Pro 340 Ser Gln Glu Glu Met Thr Lys Val 355 Lys Gly Phe Tyr Pro Ser Asp Gly 370 375 Gln Pro Glu Asn Asn Tyr Lys Asp 385 390

    Phe Arg Arg 235 Gly Pro Ser Val Thr Leu Met Ile Ser Arg Thr 250 255 Val Ser Gln Glu Asp Pro Glu 265 270 Val Glu Val His Asn Ala Lys 285 Ser Thr Tyr Arg Val Val Ser 300 Leu Asn Gly Lys Glu Tyr Lys 315 Ser Ser Ile Glu Lys Thr Ile 330 335 Pro Gln Val Tyr Thr Leu Pro 345 350 Gln Val Ser Leu Thr Cys Leu 365 Ala Val Glu Trp Glu Ser Asn 380 Thr Pro Pro Val Leu Asp Ser

    395

    573

    400

    Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415

    Gln His Glu Gly Asn 420 Val Phe Ser Cys Ser 425 Val Met His Glu Ala Leu 430 Ala His Thr Thr Arg Glu Glu Leu Ser Leu Ser Pro

    435 440 <210> 325 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> F8ML <400> 325

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Ser Ser 10 Leu Ser Ala Ser Val 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg Gln 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Gln Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 95

    574

    Thr Ala Phe Gly Gly 100 Gly Thr Lys Val Glu 105 Ile Lys Arg Thr Val 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys

    210 <210> 326 <211> 448 <212> PRT <213> Artificial Sequence <220>

    <223> F8M-F1868mv1 <400> 326 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15

    575

    Ser Tyr Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 Gly Phe Thr Phe Ser 30 Tyr Asp Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

    Val

    180 185 190

    576

    Thr Val Val Pro 195 Ser Ser Ser Leu Gly 200 Thr Gln Thr Tyr Thr 205 Cys Asn Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Arg Arg Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser

    Leu

    355 360 365

    577

    Thr Trp Cys 370 Leu Val Lys Gly Phe 375 Tyr Pro Ser Asp Ile 380 Ala Val Glu Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His 420 425 430 Glu Ala Leu His Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser

    Pro

    435 440 445 <210>

    <211>

    <212>

    <213>

    327

    444

    PRT

    Artificial Sequence <220>

    <223> F8M-F1868mv1 <400> 327

    Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15

    Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30

    Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45

    Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60

    578

    Gln Tyr 65 Asp Arg Val Ile Met 70 Thr Val Asp Lys Ser 75 Thr Asp Thr Ala Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95 Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Arg Arg Gly Pro Ser Val

    Phe

    225 230 235

    240

    579

    Leu Pro Phe Pro Pro Lys 245 Pro Lys Glu Val Thr Cys Val Val Val Val 260 Gln Phe Asn Trp Tyr Val Asp Thr 275 Lys Pro Arg Glu Glu Gln Tyr Val 290 295 Leu Thr Val Leu His Gln Asp Cys 305 310 320 Lys Val Ser Asn Lys Gly Leu Ser 325 Lys Ala Lys Gly Gln Pro Arg Pro 340 Ser Gln Glu Glu Met Thr Lys Val 355 Lys Gly Phe Tyr Pro Ser Asp Gly 370 375 Gln Pro Glu Asn Asn Tyr Lys Asp 385 390 400 Gly Ser Phe Phe Leu Tyr Ser Trp

    Thr Leu 250 Met Ile Ser Arg Thr 255 Val Ser Gln Glu Asp Pro Glu 265 270 Val Glu Val His Asn Ala Lys 285 Ser Thr Tyr Arg Val Val Ser 300 Leu Asn Gly Lys Glu Tyr Lys 315 Ser Ser Ile Glu Lys Thr Ile 330 335 Pro Gln Val Tyr Thr Leu Pro 345 350 Gln Val Ser Leu Thr Cys Leu 365 Ala Val Glu Trp Glu Ser Asn 380 Thr Pro Pro Val Leu Asp Ser 395 Leu Thr Val Asp Lys Ser Arg

    580

    405

    410

    415

    Gln His Glu Gly Asn 420 Val Phe Ser Ala His Thr 435 Thr Arg Glu Glu

    Ser Val Leu His Glu Ala Leu

    425 430

    Ser Leu Ser Pro <210> 328 <211> 448 <212> PRT <213> Artificial Sequence <220>

    <223> F8M-F1927mv1 <400> 328

    Gln Gly 1 Val Gln Leu Val 5 Glu Ser Ser Leu Arg Leu Ser Cys Ala Tyr 20 Asp Ile Gln Trp Val Arg Gln Val 35 Ser Ser Ile Ser Pro Ser Gly Val 50 55 Lys Gly Arg Phe Thr Ile Ser Tyr 65 70 Leu Gln Met Asn Ser Leu Arg Cys 85 Ala Arg Arg Thr Gly Arg Glu Tyr 100

    Gly Gly Leu Val Gln Pro Gly

    10 15

    Ser Gly Phe Thr Phe Ser Tyr

    25 30

    Pro Gly Lys Gly Leu Glu Trp

    Ser Thr Tyr Tyr Arg Arg Glu

    Asp Asn Ser Lys Asn Thr Leu

    75 80

    Glu Asp Thr Ala Val Tyr Tyr

    90 95

    Gly Gly Gly Trp Tyr Phe Asp

    105 110

    581

    Trp Gly Gly Gln 115 Gly Thr Leu Val Thr 120 Val Ser Ser Ala Ser 125 Thr Lys Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Arg Arg Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285

    582

    Asn Arg Ala 290 Lys Thr Lys Pro Arg 295 Glu Glu Gln Tyr Asn 300 Ser Thr Tyr Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His 420 425 430 Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445

    <210> 329 <211> 444

    583 <212> PRT <213> Artificial Sequence <220>

    <223> F8M-F1927mv2 <400> 329

    Gln Ala 1 Val Gln Leu Val 5 Gln Ser Gly Ser Glu 10 Leu Lys Lys Pro Gly 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn 20 25 30 Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe 50 55 60 Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr 65 70 75 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys 85 90 95 Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155

    584

    160

    Asn Leu Ser Gly Ala Leu 165 Thr Ser Gly Val His 170 Thr Phe Pro Ala Val 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Arg Arg Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

    585

    Ser

    325

    330

    335

    Lys Pro Ala Lys Gly 340 Gln Pro Arg Glu Pro 345 Gln Val Tyr Thr Leu 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His 420 425 430 Ala His Tyr Thr Arg Glu Glu Leu Ser Leu Ser Pro

    435 440 <210> 330 <211> 448 <212> PRT <213> Artificial Sequence <220>

    <223> Q499-z121 <400> 330 Gln Val Gly Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15

    Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr

    Gly Phe Thr Phe

    Ser Tyr

    586

    Asp Val Ile Gln 35 Trp Val Arg Gln Ala 40 Pro Gly Lys Gly Leu 45 Glu Trp Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Arg Arg Glu Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val

    587

    195

    200

    205

    Asp Lys His 210 Lys Pro Ser Asn Thr 215 Lys Val Asp Lys Arg 220 Val Glu Ser Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

    588

    Trp

    370

    375

    380

    Glu Ser Asn Gly Gln Pro Glu Val

    385 390

    400

    Leu Asp Ser Asp Gly Ser Phe Asp

    405

    Lys Ser Arg Trp Gln Glu Gly His

    420

    Glu Ala Leu His Asn Arg Tyr Pro

    435

    Asn Asn Tyr Lys Thr Thr Pro

    395

    Phe Leu Tyr Ser Lys Leu Thr

    410

    Asn Val Phe Ser Cys Ser Val

    425 430

    Thr Gln Lys Ser Leu Ser Leu

    440 445

    Pro

    Val

    415

    Met

    Ser

    <210> 331 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> J327-z119 <400> 331 Gln Val Gln Leu Val Gln Se Ala 1 5

    Ser Val Lys Val Ser Cys Lys Asn

    Asn Met Asp Trp Val Arg Gln Met

    Gly Asp Ile Asn Thr Arg Ser Phe

    50 55

    Gln Asp Arg Val Ile Met Thr

    Gly Ser Glu Leu Lys Lys Pro

    Ala Ser Gly Tyr Thr Phe Thr

    25 30

    Ala Pro Gly Gln Gly Leu Glu

    40 45

    Gly Gly Ser Ile Tyr Asn Glu

    Val Asp Lys Ser Thr Asp Thr

    Gly

    Asp

    Trp

    Glu

    Ala

    589

    Tyr

    Met Cys Glu Leu Ser Ser 85 Leu Arg Ser Glu Asp 90 Thr Ala Thr Tyr His 95 Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

    Phe

    225 230 235

    240

    590

    Leu Pro Phe Pro Pro Lys 245 Pro Lys Glu Val Thr Cys Val Val Val Val 260 Gln Phe Asn Trp Tyr Val Asp Thr 275 Lys Pro Arg Glu Glu Gln Tyr Val 290 295 Leu Thr Val Leu His Gln Asp Cys 305 310 320 Lys Val Ser Asn Lys Gly Leu Ser 325 Lys Ala Lys Gly Gln Pro Arg Pro 340 Ser Gln Glu Glu Met Thr Lys Val 355 Lys Gly Phe Tyr Pro Ser Asp Gly 370 375 Gln Pro Glu Asn Asn Tyr Lys Asp 385 390 400 Gly Ser Phe Phe Leu Tyr Ser Trp 405

    Thr Leu 250 Met Ile Ser Arg Thr 255 Val Ser Gln Glu Asp Pro Glu 265 270 Val Glu Val His Asn Ala Lys 285 Ser Thr Tyr Arg Val Val Ser 300 Leu Asn Gly Lys Glu Tyr Lys 315 Ser Ser Ile Glu Lys Thr Ile 330 335 Pro Gln Val Tyr Thr Leu Pro 345 350 Gln Val Ser Leu Thr Cys Leu 365 Ala Val Glu Trp Glu Ser Asn 380 Thr Pro Pro Val Leu Asp Ser 395 Leu Thr Val Asp Lys Ser Arg 410 415

    591

    Gln His Glu Gly Asn 420 Val Phe Ser Cys Asn His Tyr Thr Gln Glu Ser Leu 435 440

    Ser Val Met His Glu Ala Leu

    425 430

    Ser Leu Ser Pro <210> 332 <211> 214 <212> PRT <213> Artificial Sequence <220>

    <223> L404-k <400> 332

    Asp Gly 1 Ile Gln Met Thr 5 Gln Ser Pro Asp Arg Val Thr Ile Thr Cys Lys Gln 20 Leu Ala Trp Tyr Gln Gln Lys Pro Ile 35 40 Tyr Gln Ala Ser Arg Lys Glu Ser Gly 50 55 Ser Arg Tyr Gly Thr Asp Phe Thr Pro 65 70 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu 85 Thr Phe Gly Gly Gly Thr Lys Val Ala 100 Pro Ser Val Phe Ile Phe Pro Pro

    Ser Ser Leu Ser Ala Ser Val

    10 15

    Ala Ser Arg Asn Ile Glu Arg

    25 30

    Gly Gln Ala Pro Glu Leu Leu

    Gly Val Pro Asp Arg Phe Ser

    Leu Thr Ile Ser Ser Leu Gln

    75 80

    Gln Gln Tyr Ser Asp Pro Pro

    90 95

    Glu Ile Lys Arg Thr Val Ala

    105 110

    Ser Asp Glu Gln Leu Lys Ser

    592

    Gly

    115

    120

    125

    Thr Ala Ala 130 Ser Val Val Lys Gln Val Gln Trp Lys 145 160 Glu Ser Val Thr Glu Ser 165 Ser Thr Leu Thr Leu Tyr 180 Ala Cys Glu Val Thr Ser 195 Phe Asn 210 Arg Gly Glu

    Cys Leu 135 Leu Asn Asn Val 150 Asp Asn Ala Leu Gln Asp Ser Lys Asp 170 Ser Lys Ala Asp 185 Tyr His Gln Gly 200 Leu Ser Cys

    Phe Tyr 140 Pro Arg Glu Gln 155 Ser Gly Asn Ser Ser Thr Tyr Ser Leu 175 Glu Lys His Lys 190 Val Ser Pro Val 205 Thr Lys

    593