KR20180125036A - Il-8-binding antibodies and uses thereof - Google Patents

Abstract

In a non-exclusive embodiment, it provides novel IL-8 antibodies that are excellent as pharmaceuticals.

Description

Antibodies that bind to IL-8 and their use {IL-8-BINDING ANTIBODIES AND USES THEREOF}

[Cross reference of related applications]

This application relates to Japanese Priority Patent Application No. 2015-185254 for which it applied to Japan on September 18, 2015, and claims priority to this application. The contents of this application are incorporated by reference in their entirety.

In one non-exclusive aspect, the present disclosure provides an anti-IL-8 antibody, a pharmaceutical composition comprising the antibody, a nucleic acid encoding the antibody, and a host cell containing the nucleic acid. For example, a method for producing and using an IL-8 antibody and pharmaceutical composition in the treatment of a disease related to IL-8 is also provided.

Antibodies are attracting attention as pharmaceuticals because they have high stability in plasma and have few side effects. There are a number of IgG-type therapeutic antibodies, and many therapeutic antibodies are still being developed (Reichert et al., Nat. Biotechnol. 23:1073-1078 (2005) (Non-Patent Document 1); Pavlou et al. ., Eur. J. Pharm. Biopharm. 59(3):389-396(2005) (Non-Patent Document 2)). On the other hand, various technologies have been developed for the second generation of therapeutic antibodies, and these include technologies for improving the function of the sound processing device, antigen binding ability, pharmacokinetics, or stability, and reducing the risk of immunogenicity ( Kim et al., Mol. Cells. 20 (1):17-29 (2005) (Non-Patent Document 3)). Since therapeutic antibodies generally have a very high dosage, the development of therapeutic antibodies faces problems such as difficulty in preparing subcutaneous administration formulations and high manufacturing costs. A method for improving the pharmacokinetics, pharmacokinetics, and antigen binding properties of a therapeutic antibody provides a means for reducing the dosage and manufacturing cost associated with the therapeutic antibody.

Substitution of amino acid residues in the constant region provides one method of improving the pharmacokinetics of an antibody (Hinton et al., J. Immunol. 176 (1):346-356 (2006) (Non-Patent Document) 4); Ghetie et al., Nat. Biotechnol. 15(7):637-640(1997)) (Non-Patent Document 5)). The affinity maturation technique provides a method of increasing the antigen-neutralizing ability of an antibody (Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471(2005) (Non-Patent Document 6)) ; Wu et al., J. Mol. Biol. 368:652 (2007) (Non-Patent Document 7)), antigen-binding activity by introducing mutations into the amino acid residues of the CDRs and/or framework regions of the antibody variable domain It is possible to increase By improving the antigen binding properties of the antibody, it is possible to improve the biological activity of the antibody in vitro, or to reduce the dosage, and it is also possible to improve the drug efficacy in vivo (in vivo) (Wu et al., J. Mol. Biol. 368:652-665 (2007) (Non-Patent Document 8)).

Since the amount of antigen that can be neutralized per antibody molecule depends on the affinity of the antibody for the antigen, it is possible to neutralize the antigen with a small amount of antibody by increasing the affinity. It is conventionally possible to enhance the affinity of an antibody for an antigen using a variety of known methods (e.g., Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471( 2005) (Refer to Non-Patent Document 6). Moreover, it is theoretically possible to neutralize one molecule of antigen (two antigens if the antibody is bivalent) with one molecule of antibody if covalent binding to the antigen can be achieved and the affinity can be infinite. However, one of the limitations of the development of therapeutic antibodies so far is that one molecule of antibodies typically binds to only one molecule of antigen (a binary antigen in the case of a bivalent antibody) and neutralizes it. Recently, by using an antibody that binds to an antigen in a pH-dependent manner (hereinafter, also referred to as a "pH-dependent antibody" or a "pH-dependent binding antibody"), it is possible to bind and neutralize one molecule of an antigen. (See, for example, WO2009/125825 (Patent Document 1); Igawa et al., Nat. Biotechnol. 28:1203-1207 (2010) (Non-Patent Document 9)). The pH-dependent antibody binds strongly to the antigen under neutral pH conditions in plasma, and dissociates the antigen under acidic pH conditions in the endosomes of cells. When the antibody is recycled into plasma by FcRn after dissociation of the antigen, it freely binds to and neutralizes other antigenic molecules. Therefore, it becomes possible to repeatedly bind and neutralize a plurality of antigenic molecules with one pH-dependent antibody.

Antibody recycling characteristics by paying attention to the difference in the concentration of calcium (Ca) ions in plasma and in the endosome, and using an antibody having an antigen-antibody interaction exhibiting calcium dependence (hereinafter, also referred to as "calcium ion concentration-dependent antibody") It has been recently reported that can be achieved (WO2012/073992 (Patent Document 2)). (Hereinafter, the pH-dependent antibody and the calcium ion concentration-dependent antibody are collectively referred to as "pH/Ca concentration-dependent antibody".)

IgG antibodies have long plasma retention by binding to FcRn. The binding of the IgG antibody to FcRn is strong under acidic pH conditions (eg, pH 5.8), but hardly binds under neutral pH conditions (eg, pH 7.4). The IgG antibody is non-specifically absorbed by cells, but returns to the cell surface by binding to FcRn in the endosome under acidic pH conditions in the endosome. IgG then dissociates from FcRn under neutral pH conditions in plasma.

Since pH-dependent antibodies modified to increase FcRn binding under neutral pH conditions have the ability to repeatedly bind to antigen molecules and eliminate them from plasma, it is possible to eliminate antigens from plasma by administration of such antibodies. It is reported that it is (WO2011/122011 (Patent Document 3)). According to this report, the pH-dependent antibody modified to increase FcRn binding under neutral pH conditions (e.g., pH 7.4) is compared with a pH-dependent antibody containing the Fc region of a native IgG antibody. It is possible to further accelerate antigen disappearance (WO2011/122011 (Patent Document 3)).

On the other hand, when a mutation is introduced into the Fc region of an IgG antibody and the binding to FcRn is lost under acidic pH conditions, it cannot be recycled from the endosome into the plasma, so the plasma retention of the antibody is significantly impaired. . Therefore, as a method of improving the plasma retention of an IgG antibody, a method of improving the binding to FcRn under acidic pH conditions has been reported. By introducing amino acid alteration in the Fc region of an IgG antibody to improve binding to FcRn under acidic pH conditions, the recycling efficiency from the endosome to the plasma is increased, and as a result, the retention in plasma can be improved. have. For example, modified M252Y/S254T/T256E (YTE; Dall'Acqua et al., J. Biol. Chem. 281:23514-235249 (2006) (Non-Patent Document 10)), M428L/N434S (LS; Zalevsky et al. al., Nat. Biotechnol. 28:157-159 (2010)) (Non-Patent Document 11), and N434H (Zheng et al., Clin. Pharm. & Ther. 89(2):283-290(2011) ( It is reported that non-patent document 12)) brings about an increased antibody half-life compared to native IgG1.

However, the antibody containing such an Fc region variant, which has increased binding to FcRn under neutral pH conditions or acidic pH conditions, may be concerned that immunogenicity or the rate at which aggregates occur will deteriorate. In addition, it has been reported that the binding of anti-drug antibodies (hereinafter also referred to as “conventional ADA”) (eg, rheumatoid factor) present in patients before administration of therapeutic antibodies is increased (WO2013/ 046722 (Patent Document 4), WO2013/046704 (Patent Document 5)). WO2013/046704 (Patent Document 5) shows that the Fc region modified variant containing a specific mutation (as a representative, two residues of Q438R/S440E according to EU numbering), increases binding to FcRn under acidic pH conditions. It is reported that it is made, and a significant decrease in binding to the rheumatoid factor was also shown compared to the unmodified native Fc. However, in WO2013/046704 (Patent Document 5), it is not particularly demonstrated that this Fc region modified variant has superior plasma retention compared to an antibody having a native Fc region.

Therefore, there is a demand for a safe and more advantageous Fc region modified variant that does not show binding to the existing ADA and further improves the retention in plasma.

As an effector function of IgG antibody, antibody-dependent cytotoxic activity (hereinafter referred to as "ADCC"), complement dependent cytotoxic activity (hereinafter referred to as "CDC"), and phagocytosis of target cells via IgG antibody Phosphorus antibody dependent cell mediated phagocytosis (ADCP) has been reported. In order for IgG antibodies to mediate ADCC activity or ADCP activity, antibody receptors present on the Fc region of IgG antibodies and effector cells such as killer cells, natural killer cells, or activated macrophages (Initiation A in this specification is described. In the present range, binding with "Fcγ receptor", "FcgR", "Fc gamma receptor" or "FcγR") is required. In humans, as proteins of the FcγR family, isoforms of FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb have been reported, and each allotype has also been reported (Jefferis et al., Immunol. Lett. 82:57-65 ( 2002) (Non-Patent Document 13)). The balance of the affinity of the antibody for each of the activated receptors including FcγRIa, FcγRIIa, FcγRIIIa, or FcγRIIIb and inhibitory receptors including FcγRIIb is an important factor in optimizing the effector function of the antibody.

So far, various techniques for increasing or improving the activity of therapeutic antibodies against antigens have been reported. For example, since the binding activity of an antibody to active FcγR plays an important role in the cytotoxic activity of the antibody, it targets a membrane-type antigen and also causes cytotoxicity due to increased binding activity to active FcγR. Antibodies with increased activity are being developed. For example, WO2000/042072 (Patent Document 6); WO2006/019447 (Patent Document 7); Lazar et al., Proc. Nat. Acad. Sci. USA. 103:4005-4010 (2006) (Non-Patent Document 14); Shinkawa et al., J. Biol. Chem. 278, 3466-3473 (2003) (Non-Patent Document 15); Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998) (Non-Patent Document 16); Clynes et al., Nat. Med. See 6:443-446 (2000) (Non-Patent Document 17). Similarly, since the binding activity to inhibitory FcγR (FcyRIIb in humans) plays an important role in immunosuppressive activity and agonist activity, antibodies targeting membrane antigens with increased binding activity to inhibitory FcγR (Li et al., Proc. Nat. Acad. Sci. USA. 109 (27): 10966-10971 (2012) (Non-Patent Document 18)). In addition, the influence of the binding of an antibody binding to a soluble antigen to FcγR has been studied mainly in terms of its side effects (Scappaticci et al., J. Natl. Cancer Inst. 99 (16):1232-1239 (2007). ) (Non-Patent Document 19)). For example, when an antibody with increased binding to FcγRIIb is used as a pharmaceutical product, it is expected to reduce the risk that the anti-drug antibody is produced (Desai et al., J. Immunol. 178(10):6217-6226( 2007) (Non-Patent Document 20)).

In recent years, amino acid modifications have been introduced into the Fc region of an IgG antibody to increase the binding activity of an antibody targeting a soluble antigen to an active and/or inhibitory FcγR, thereby reducing the loss of the corresponding antigen from the serum. It has been reported that further acceleration was possible (WO2012/115241 (Patent Document 8), WO2013/047752 (Patent Document 9), WO2013/125667 (Patent Document 10), and WO2014/030728 (Patent Document 11)). In addition, the binding activity to FcγRIIb is almost unchanged from that of the native IgG antibody, but a modified Fc region has been found that lowered the activity against other active FcγR (WO2014/163101 (Patent Document 12)).

Since the retention of the soluble antigen in plasma is very short compared to the antibody having a recycling mechanism via FcRn, the soluble antigen is an antibody having the recycling mechanism (e.g., it does not have the characteristics of a pH/Ca concentration-dependent antibody. Antibody), it is possible to exhibit elevated plasma retention and plasma concentration. Therefore, for example, when a soluble antigen in plasma has multiple types of physiological functions, even if one type of physiological function is blocked by binding of the antibody, the retention of the antigen in plasma due to the binding of the antibody. And/or as a result of the increase in the plasma concentration, it is also conceivable that the concentration of the antigen in the plasma further exacerbates the pathogenic symptoms in which other physiological functions become pathogens. In such a case, for the purpose of accelerating the disappearance of the antigen, in addition to the method of applying the above-described modifications to the antibody, for example, a plurality of pH/Ca concentration-dependent antibodies and a plurality of antigens are multivalent immune complexes. A method of increasing the binding to FcRn, FcγR, and complement receptors by using the formation of is reported (WO2013/081143 (Patent Document 13)).

Even when the Fc region is not altered, the isoelectric point (pI) of the antibody is raised or lowered by altering the amino acid residue so that the charge of the amino acid residue that may be exposed on the surface of the variable region of the antibody is changed. Regardless of the type, it has been reported that it is possible to control the blood half-life of the antibody without significantly reducing the antigen-binding activity of the antibody (WO2007/114319 (Patent Document 14): Amino acid substitution technology mainly in FR) ; WO2009/041643 (Patent Document 15): amino acid substitution technology mainly in CDR). In these documents, it is shown that the plasma half-life of the antibody can be extended by lowering the pi of the antibody, and conversely, the half-life in the plasma of the antibody can be shortened by increasing the pi of the antibody.

Regarding the alteration of the charge of the amino acid residue in the constant region of the antibody, in particular, by altering the charge of a specific amino acid residue in the CH3 domain and raising the pi of the antibody, it is possible to promote the uptake of the antigen into the cell. It has been reported, and it is also described that the modification does not preferably interfere with the binding to FcRn (WO2014/145159 (Patent Document 16)). In addition, by altering the charge of amino acid residues in the constant region of the antibody (mainly the CH1 domain) to decrease pI, the half-life in the plasma of the antibody can be extended, and by combining with mutations in amino acid residues that increase binding to FcRn, While increasing the binding to FcRn, it has been reported that the half-life of the antibody in plasma can be extended (WO2012/016227 (Patent Document 17)).

On the other hand, when such a modification technique intended to increase or decrease the pi of an antibody is combined with a technique other than a modification technique that increases or decreases binding to FcRn or FcγR, the plasma retention of the antibody or antigen from the plasma About the presence or absence of promotion of disappearance, the effect is not clear.

The extracellular matrix (ECM) is a structure that covers cells in vivo, and is mainly composed of glycoproteins such as collagen, proteoglycan, fibronectin, and laminin. The role of ECM in vivo is to arrange a microenvironment for cells to survive, and ECM is important in various activities performed by cells, such as cell proliferation and cell adhesion.

It has been reported that ECM is involved in the in vivo dynamics of proteins administered to a living body. A review was conducted on the blood concentration when the VEGF-Trap molecule, which is a fusion protein of VEGF receptor and Fc, was administered subcutaneously (Holash et al., Proc. Natl. Acad. Sci., 99(17):11393-11398( 2002) (Non-Patent Document 21)). The VEGF-Trap molecule with high pI has low bioavailability due to its low concentration in plasma when administered subcutaneously. The modified VEGF-Trap molecule whose pI is lowered by amino acid substitution increases the concentration in plasma, thereby improving the bioavailability. In addition, since the change in the bioavailability and the binding strength to ECM are correlated, it has become clear that the bioavailability of the VEGF-Trap molecule when administered subcutaneously depends on the binding strength of the subcutaneous site to the ECM.

In WO2012/093704 (Patent Document 18), it is reported that there is an inverse correlation between the binding of an antibody to ECM and retention in plasma, so that antibody molecules that do not bind to ECM are compared to antibodies that bind to ECM. , Has better plasma retention.

As described above, with the aim of improving the in vivo bioavailability of proteins and their retention in plasma, techniques for lowering the binding properties to the extracellular matrix have been reported. On the other hand, the advantage by increasing the binding of the antibody to the ECM was not known until now.

Human IL-8 (Interleukin 8) is a member of the chemokine family, which is 72 or 77 amino acid residues in length. The term "chemokine" is a generic term for a family of proteins having a molecular weight of 8 to 12 kDa and containing four cysteine residues forming an intermolecular disulfide bond. Chemokines are classified into CC chemokines, CXC chemokines, C chemokines, and CA3C chemokines according to the characteristics of the cysteine alignment method. IL-8 is classified as a CXC chemokine and is also called CXCL8.

IL-8 exists in the form of monomers and homodimers in solution. The IL-8 monomer contains an antiparallel β sheet, and has a structure in which the α helix at the C-terminus crosses the β sheet and covers it. The IL-8 monomer contains two disulfide bridges between cysteine 7 and cysteine 34 and between cysteine 9 and cysteine 50 for the 72 amino acid form of IL-8. The IL-8 homodimer is stabilized by a non-covalent interaction between the β-sheets of the two monomers because there is no covalent bond between the molecules of the homodimer.

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) (Non-Patent Document 22)).

In general, chemokines are generally only detectable or weakly detectable in normal tissues, but are strongly detected in the inflamed area, promote infiltration of leukocytes into the tissue inflamed area, and are involved in causing inflammation. IL-8 is a known inflammation-inducing cytokine that activates neutrophils and enhances the expression of cell adhesion molecules, thereby enhancing adhesion of neutrophils to vascular endothelial cells. In addition, IL-8 also has chemotactic capacity, and IL-8 produced in damaged tissue promotes chemotaxis of neutrophils adhered to vascular endothelial cells into tissues, causing inflammation accompanying neutrophil infiltration. do. It is also known that IL-8 is a potent angiogenesis factor for endothelial cells and is involved in the promotion of tumor angiogenesis.

Examples of inflammatory diseases associated with elevated (eg, excess) IL-8 levels include inflammatory skin diseases such as inflammatory keratosis (eg dry ringworm), atopic dermatitis, and contact dermatitis; Chronic inflammatory diseases which are autoimmune diseases, such as joint rheumatism, systemic lupus erythematosus (SLE), and Bechosis; Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; Inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, and drug-induced allergic hepatitis; Inflammatory kidney diseases such as glomerulonephritis; Inflammatory respiratory diseases such as bronchitis and asthma; Inflammatory chronic vascular diseases such as atherosclerosis; Multiple sclerosis, stomatitis, vocalitis, and inflammation associated with the use of artificial organs and/or artificial blood vessels, and the like. Elevated (eg, excess) IL-8 levels may also include malignant tumors such as ovarian cancer, lung cancer, prostate cancer, gastric cancer, breast cancer, melanoma, head and neck cancer, and kidney cancer; Sepsis due to infection; Cystic fibrosis; And pulmonary fibrosis (e.g., Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619(2014), which is incorporated by reference in its entirety in this specification) (ratio Refer to Patent Document 22).

For some of these diseases, human anti-IL-8 antibodies having high affinity have been developed as pharmaceutical compositions (Desai et al., J. Immunol. 178(10):6217-6226(2007) (non-patent literature) 23)), they are not on the market yet. Until now, only one type is sold as a pharmaceutical composition containing an anti-IL-8 antibody, and this is a mouse anti-IL-8 antibody against dry ringworm as an external drug. There is a need for novel anti-IL-8 antibodies for the treatment of diseases.

WO2009/125825 WO2012/073992 WO2011/122011 WO2013/046722 WO2013/046704 WO2000/042072 WO2006/019447 WO2012/115241 WO2013/047752 WO2013/125667 WO2014/030728 WO2014/163101 WO2013/081143 WO2007/114319 WO2009/041643 WO2014/145159 WO2012/016227 WO2012/093704

Reichert et al., Nat. Biotechnol. 23:1073-1078(2005) Pavlou et al., Eur. J. Pharm. Biopharm. 59(3):389-396(2005) Kim et al., Mol. Cells. 20(1):17-29(2005) Hinton et al., J. Immunol. 176(1):346-356(2006) Ghetie et al., Nat. Biotechnol. 15(7):637-640(1997)) Rajpal et al., Proc. Natl. Acad. Sci. USA 102(24):8466-8471(2005) Wu et al., J. Mol. Biol. 368:652 (2007) Wu et al., J. Mol. Biol. 368:652-665 (2007) Igawa et al., Nat. Biotechnol. 28:1203-1207(2010) Dall'Acqua et al., J. Biol. Chem. 281:23514-235249 (2006) Zalevsky et al., Nat. Biotechnol. 28:157-159 (2010)) Zheng et al., Clin. Pharm. & Ther. 89(2):283-290(2011) Jefferis et al., Immunol. Lett. 82:57-65(2002) Lazar et al., Proc. Nat. Acad. Sci. USA. 103: 4005-4010 (2006) Shinkawa et al., J. Biol. Chem. 278, 3466-3473(2003) Clynes et al., Proc. Natl. Acad. Sci. U SA 95:652-656 (1998) Clynes et al., Nat. Med. 6:443-446(2000) Li et al., Proc. Nat. Acad. Sci. USA. 109 (27):10966-10971(2012) Scappaticci et al., J. Natl. Cancer Inst. 99 (16):1232-1239 (2007) Desai et al., J. Immunol. 178(10):6217-6226(2007) Holash et al., Proc. Natl. Acad. Sci., 99(17):11393-11398(2002) Russo et al., Exp. Rev. Clin. Immunol. 10(5):593-619(2014) Desai et al., J. Immunol. 178(10):6217-6226(2007)

In one non-exclusive aspect, the non-limiting purpose of embodiments of Initiation A is to improve pharmacokinetic properties for antibodies, such as ion concentration dependent antigen binding to improve antibody half-life and/or antigen clearance from plasma. It is to provide a molecule with

In one non-exclusive aspect, the non-limiting object of embodiments of Initiation B is to provide safe and more advantageous Fc region variants with increased half-life and reduced binding to existing anti-drug antibodies (ADA). will be.

In one non-exclusive aspect, a non-limiting object of the embodiment of Initiation C is to provide an anti-IL-8 antibody having a pH dependent binding affinity for IL-8. A further embodiment relates to an anti-IL-8 antibody that, when administered to a subject, has the effect of rapidly losing IL-8 compared to a reference antibody. In another embodiment, Initiation C relates to an anti-IL-8 antibody capable of stably maintaining the activity of neutralizing IL-8 when administered to an individual. In some embodiments, the anti-IL-8 antibody exhibits reduced immunogenicity. In a further embodiment, Initiation C relates to a method of making and using the anti-IL-8 antibody. Another alternative, non-limiting object of Initiation C is to provide a novel IL-8 antibody that can be included in a pharmaceutical composition.

In one non-exclusive aspect within the range of disclosure A in this specification, the inventors surprisingly found that an ion concentration-dependent antibody (ion concentration-dependent antigen-binding domain ("binding to an antigen depending on the condition of ion concentration The ability of an antibody) containing an antigen-binding domain whose activity changes") to disappear from plasma) can be promoted by increasing the isoelectric point (pI) by altering at least one of the amino acid residues exposed on the surface of the antibody. Found that there is. In another non-exclusive aspect, the present inventors have found that ion concentration dependent antibodies with elevated pi can further enhance the binding of the antibody to the extracellular matrix. The inventors of the present invention have found that this can increase the disappearance of antigens from plasma by increasing the binding property of the antibody to the extracellular matrix, although not bound to a specific theory.

In one non-exclusive aspect within the range of disclosure B in this specification, the present inventors do not show binding to an anti-drug antibody (conventional ADA), and can further improve plasma retention. Extensive studies were conducted on the safer and more advantageous Fc region variants. As a result, the present inventors surprisingly, in order to achieve the plasma retention of the antibody and maintain a significant decrease in binding to the rheumatoid factor, the amino acid at position 434 according to EU numbering was substituted with Ala (A). In addition, it was found that an Fc region variant containing a specific two-residue mutation (typically, Q438R/S440E according to EU numbering) as a combination of mutations of amino acid residues is preferable.

In one non-exclusive aspect within the range of the disclosure C in this specification, the present inventors have prepared a pH-dependent anti-IL-8 antibody (an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner). I made a number of them. And, as a result of various tests, the present inventors discovered a pH-dependent anti-IL-8 antibody having an effect of rapidly eliminating IL-8 compared to a reference antibody when administered to an individual. In some embodiments, Initiation C relates to a pH dependent anti-IL-8 antibody capable of stably maintaining IL-8 neutralizing activity. In a further non-limiting embodiment, the pH dependent anti-IL-8 antibody has reduced immunogenicity and good expression levels.

In addition, in the range of initiation C, the present inventors have found that anti-IL- containing an Fc region in which the binding affinity to FcRn at acidic pH is increased than that of the native Fc region to FcRn. 8 Succeeded in obtaining antibodies. In an alternative embodiment, the present inventors obtain an anti-IL-8 antibody containing an Fc region in which the binding affinity for the existing ADA is lower than the binding affinity for the existing ADA of the native Fc region. I succeeded in doing it. In an alternative embodiment, the present inventors have succeeded in obtaining an anti-IL-8 antibody containing an Fc region whose half-life in plasma is greater than that of the native Fc region. In an alternative embodiment, the present inventors obtain a pH-dependent anti-IL-8 antibody containing an Fc region in which the binding affinity to the effector receptor is lower than that of the native Fc region to the effector receptor. I succeeded in doing it. In a separate aspect, the present inventors have discovered a nucleic acid encoding the anti-IL-8 antibody. In another aspect, the inventors have also discovered a host comprising the nucleic acid. In another aspect, the present inventors have found a method for producing the anti-IL-8 antibody comprising culturing the host. In another aspect, the present inventors have discovered a method for promoting the loss of IL-8 from the individual than a reference antibody, comprising administering the anti-IL-8 antibody to the individual.

In one embodiment, the disclosure A relates to the following, without limitation.

[1] An antibody containing an antigen-binding domain whose antigen-binding activity changes depending on the condition of the ion concentration, wherein at least one amino acid residue that can be exposed on the surface of the antibody is modified, so that the isoelectric point (pI) is Rising antibodies.

[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 having a higher antigen-binding activity under a high ionic concentration condition than under a low ionic 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 KD (acidic pH range)/KD (neutral pH range), which is the ratio of KD in the acidic pH range to the antigen and KD in the neutral pH range, is 2 or more.

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

[7] The antibody of any one of [1] to [6], which is capable of promoting the disappearance of the antigen from plasma as compared with the antibody before the modification.

[8] The antibody of any one of [1] to [7], wherein the binding activity to the extracellular matrix is increased as compared with the antibody before the modification.

[9] The antibody according to any one of [1] to [8], wherein the alteration of the amino acid residue is a substitution of an amino acid residue.

[10] alteration of the amino acid residue,

(a) substitution from a negatively charged amino acid residue to a non-charged amino acid residue;

(b) substitution from a negatively charged amino acid residue to a positively charged amino acid residue; And

(c) substitution of amino acid residues having positive charges from amino acid residues having no charge

The antibody of any one of [1] to [9] selected from the group consisting of.

[11] Among [1] to [10], wherein the antibody contains a variable region and/or a constant region, and the amino acid residue modification is a modification of the amino acid residue in the variable region and/or the constant region. Either antibody.

[12] The antibody of [11], wherein the variable region contains a complementarity determining region (CDR) and/or a framework region (FR).

[13] The variable region includes a heavy chain variable region and/or a light chain variable region, and at least one amino acid residue is according to Kabat numbering,

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

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

(c) 1st, 3rd, 7th, 8th, 9th, 11th, 12th, 16th, 17th, 18th, 20th, 22nd, 37th, 38th in the light chain variable region FR Ranked, 39th, 41st, 42nd, 43rd, 45th, 46th, 49th, 57th, 60th, 63rd, 65th, 66th, 68th, 69th, 70th, 74th, 76th, 77th, 79th, 80th, 81st, 85th, 100th, 103rd, 105th, 106th, 107th, and 108th; And

(d) 24th, 25th, 26th, 27th, 52nd, 53rd, 54th, 55th, and 56th in the CDR of the light chain variable region

The antibody of [12], which is selected from the group consisting of, and is modified at a position in the CDR or FR.

[14] at least one amino acid residue,

(a) In the FR of the heavy chain variable region, 8th, 10th, 12th, 13th, 15th, 16th, 18th, 23rd, 39th, 41st, 43rd, 44th, 77th, 82nd Place, 82a place, 82b place, 83 place, 84 place, 85 place, and 105 place;

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

(c) 16th, 18th, 37th, 41st, 42nd, 45th, 65th, 69th, 74th, 76th, 77th, 79th, and 107th in the FR of the light chain variable region; And

(d) 24th, 25th, 26th, 27th, 52nd, 53rd, 54th, 55th, and 56th in the CDR of the light chain variable region

The antibody of [13], which is selected from the group consisting of, and is modified at a position in the CDR or FR.

[15] At least one amino acid residue, according to EU numbering,

196th, 253th, 254th, 256th, 258th, 278th, 280th, 281th, 282th, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th , 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400 Ranked, 401th, 402th, 413th, 415th, 418th, 419th, 421th, 424th, 430th, 433th, 434th, and 443th

The antibody of any one of [11] to [14], which is selected from the group consisting of, and is modified at a position in a constant region.

[16] at least one amino acid residue,

254th, 258th, 281th, 282th, 285th, 309th, 311th, 315th, 327th, 330th, 342th, 343th, 345th, 356th, 358th, 359th, 361th , 362, 384, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434, and , 443th

The antibody of [15], which is selected from the group consisting of, and is modified at a position in a normal region.

[17] At least one amino acid residue, according to EU numbering,

282, 309, 311, 315, 342, 343, 384, 399, 401, 402, and, 413

The antibody of [16], which is selected from the group consisting of, and is modified at a position in a constant region.

[18] The above constant region has a binding activity to Fc gamma receptor (FcγR), compared with a reference antibody containing the constant region of native IgG, the binding activity to FcγR is increased under neutral pH conditions, The antibody of any one of [1] to [17].

[19] The antibody of [18], wherein the FcγR is FcγRIIb.

[20] The constant region has a binding activity to one or more active FcγRs selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa, and has a binding activity to FcγRIIb, and a native IgG Compared with a reference antibody that differs only in that it has a constant region, which is a normal region of, [1] to [ 17].

[21] Compared with the reference antibody, which differs only in that the constant region has a binding activity to FcRn and a constant region that is a natural IgG constant region, FcRn under neutral pH conditions (for example, pH 7.4) The antibody of any one of [1] to [20], wherein the binding activity to is increased.

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

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

[24] A pharmaceutical composition containing the antibody of any one of [1] to [23].

[25] The pharmaceutical composition of [24], for promoting the disappearance of antigens from plasma.

[26] The pharmaceutical composition of [24] or [25], for enhancing 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 containing an antigen-binding domain whose antigen-binding activity varies depending on conditions of ion concentration, comprising the step of culturing the host cell of [29] and recovering the antibody from the culture of the cell. Containing, manufacturing method.

[30A] A method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity varies depending on the condition of ion concentration, wherein at least one amino acid residue that can be exposed on the surface of the antibody so as to increase the isoelectric point (pI) A manufacturing method including a step of changing.

[30B] At least one amino acid residue,

(I) According to Kabat numbering, (a) 1st, 3rd, 5th, 8th, 10th, 12th, 13th, 15th, 16th, 18th, 19th in the FR of the heavy chain variable region , 23rd, 25th, 26th, 39th, 41st, 42nd, 43rd, 44th, 46th, 68th, 71st, 72nd, 73rd, 75th, 76th, 77th, 81 Place, 82nd place, 82a place, 82b place, 83 place, 84th place, 85th place, 86th place, 105th place, 108th place, 110th place, and 112th place; (b) 31st, 61st, 62nd, 63rd, 64th, 65th, and 97th position in the CDR of the heavy chain variable region; (c) 1st, 3rd, 7th, 8th, 9th, 11th, 12th, 16th, 17th, 18th, 20th, 22nd, 37th, 38th in the light chain variable region FR Ranked, 39th, 41st, 42nd, 43rd, 45th, 46th, 49th, 57th, 60th, 63rd, 65th, 66th, 68th, 69th, 70th, 74th, 76th, 77th, 79th, 80th, 81st, 85th, 100th, 103rd, 105th, 106th, 107th, and 108th; And (d) in the CDR of the light chain variable region, selected from the group consisting of 24th, 25th, 26th, 27th, 52nd, 53rd, 54th, 55th, and 56th position in the CDR or FR location; or

(II) According to EU numbering, 196th, 253th, 254th, 256th, 258th, 278th, 280th, 281th, 282th, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th, 342th, 343th, 345th, 356th, 358th, 359th, 361th, 362th, 373th, 382th, 384th, 385th, 386th, 387th , 389th, 399th, 400th, 401st, 402th, 413th, 415th, 418th, 419th, 421st, 424th, 430th, 433th, 434th, and 443th The method of [30A], which is changed at the position in the normal region.

[31] alteration of the amino acid residue,

(a) substitution from a negatively charged amino acid residue to a non-charged amino acid residue;

(b) substitution from a negatively charged amino acid residue to a positively charged amino acid residue;

(c) substitution from a non-charged amino acid residue to a positively charged amino acid residue; And

(d) substitution with histidine or insertion of histidine in CDR or FR

The method of [30A] or [30B], including modifications selected from the group consisting of.

[32] Compared to the reference antibody,

(a) selecting an antibody capable of promoting the disappearance of antigen from plasma;

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

(c) selecting an antibody with increased binding activity to FcγR under neutral pH conditions (eg, pH 7.4);

(d) selecting an antibody with increased binding activity to FcγRIIb under neutral pH conditions (eg, pH 7.4);

(e) While the binding activity to FcγRIIb is maintained or increased, the binding activity to one or more active FcγRs preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa decreases. Selecting an antibody that has been prepared;

(f) selecting an antibody with increased FcRn-binding activity under neutral pH conditions (for example, pH 7.4);

(g) selecting an antibody with an elevated isoelectric point (pI);

(h) confirming the isoelectric point (pI) of the recovered antibody, and then selecting an antibody with an elevated isoelectric point (pI); And

(i) The process of selecting an antibody whose antigen-binding activity is changed or increased depending on the condition of the ion concentration

The method of any one of [30] or [30A] to [30C], optionally further comprising any one or more of.

Indication A, in an alternative embodiment, relates, but is not limited to, the following.

[A1] An antibody having a constant region, wherein at least one amino acid residue selected from the group of the same change site as the change site in the group defined in [15] or [16] is modified in the normal region. , Antibodies.

[A2] further has a heavy chain variable region and/or a light chain variable region, the variable region has a CDR and/or FR, and the same change site as in the group specified in [13] or [14] The antibody of [A1], wherein at least one amino acid residue selected from the group has been modified in the CDRs and/or FRs.

[A3] An antibody having a constant region, wherein at least one amino acid residue selected from the group of the same change site as the change site in the group specified in [15] or [16] in the normal region is pI Antibody, which has been altered so that it is elevated.

[A4] further has a heavy chain variable region and/or a light chain variable region, the variable region has a CDR and/or FR, and is the same as the change site in the group specified in [13] or [14]. The antibody of [A3], wherein at least one amino acid residue selected from the group has been modified in the CDRs and/or FRs.

[A5] An antibody containing an antigen-binding domain whose antigen-binding activity changes depending on the condition of ion concentration, wherein the antibody has a constant region, and a modified site in the group specified in [15] or [16] The antibody, wherein at least one amino acid residue selected from the group of the same modification sites as has been altered in the constant region.

[A6] further has a heavy chain variable region and/or a light chain variable region, the variable region has a CDR and/or FR, and is the same as the change site in the group specified in [13] or [14]. The antibody of [A5], wherein at least one amino acid residue selected from the group has been modified in the CDRs and/or FRs.

[A7] Use of the antibody of any one of [1] to [23] and [A1] to [A6] in the manufacture of a medicament for promoting the disappearance of antigen from plasma.

[A8] Use of the antibody of any one of [1] to [23] and [A1] to [A6] in the manufacture of a medicament for enhancing the binding to the extracellular matrix.

[A9] Use of the antibody of any one of [1] to [23] and [A1] to [A6] to eliminate an antigen from plasma.

[A10] Use of the antibody of any one of [1] to [23] and [A1] to [A6] for enhancing binding to an extracellular matrix.

[A11] An antibody obtained by the method of any one of [30], [30A], [30B], [31], and [32].

According to various embodiments, one or a plurality of elements described in any one of the above [1] to [30], [30A], [30B], [31], [32], and [A1] to [A11] Combinations of some or all of are included in Disclosure A unless such a combination is technically contradictory based on common technical knowledge in the art. For example, in some embodiments, initiation A includes a method for producing a modified antibody comprising an antigen binding domain that promotes the loss of antigen from plasma compared to before antibody modification, including:

(a)

(I) According to Kabat numbering, (a) 1st, 3rd, 5th, 8th, 10th, 12th, 13th, 15th, 16th, 18th, 19th in the FR of the heavy chain variable region , 23rd, 25th, 26th, 39th, 41st, 42nd, 43rd, 44th, 46th, 68th, 71st, 72nd, 73rd, 75th, 76th, 77th, 81 Place, 82nd place, 82a place, 82b place, 83 place, 84th place, 85th place, 86th place, 105th place, 108th place, 110th place, and 112th place; (b) 31st, 61st, 62nd, 63rd, 64th, 65th, and 97th position in the CDR of the heavy chain variable region; (c) 1st, 3rd, 7th, 8th, 9th, 11th, 12th, 16th, 17th, 18th, 20th, 22nd, 37th, 38th in the light chain variable region FR Ranked, 39th, 41st, 42nd, 43rd, 45th, 46th, 49th, 57th, 60th, 63rd, 65th, 66th, 68th, 69th, 70th, 74th, 76th, 77th, 79th, 80th, 81st, 85th, 100th, 103rd, 105th, 106th, 107th, and 108th; And (d) a position in the CDR or FR selected from the group consisting of position 24, 25, 26, 27, 52, 53, 54, 55, 56 in the CDR of the light chain variable region. in; or

(II) According to EU numbering, 196th, 253th, 254th, 256th, 258th, 278th, 280th, 281th, 282th, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th, 342th, 343th, 345th, 356th, 358th, 359th, 361th, 362th, 373th, 382th, 384th, 385th, 386th, 387th , 389th, 399th, 400th, 401st, 402th, 413th, 415th, 418th, 419th, 421th, 424th, 430th, 433th, 434th, and 443th

At a position in a normal region, selected from the group consisting of,

Modifying at least one amino acid residue that may be exposed on the surface of the antibody;

(b) a step in which the antigen-binding domain is modified in a manner in which the resulting antigen-binding activity changes according to ion concentration conditions, wherein (a) and (b) can be performed simultaneously or sequentially;

(c) culturing the host cell so that the nucleic acid encoding the modified antibody is expressed; And

(d) The step of recovering the modified antibody from the host cell culture.

In a further embodiment, the method optionally further comprises any one or a plurality of the following:

Compared with the antibody before modification,

(e) selecting an antibody capable of promoting the disappearance of antigen from plasma;

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

(g) selecting an antibody with increased binding activity to FcγR under neutral pH conditions (eg, pH 7.4);

(h) selecting an antibody with increased binding activity to FcγRIIb under neutral pH conditions (eg, pH 7.4);

(i) While the binding activity to FcγRIIb is maintained or increased, the binding activity to one or more active FcγRs preferably selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa decreases. Selecting an antibody that has been prepared;

(j) selecting an antibody with increased FcRn-binding activity under neutral pH conditions (for example, pH 7.4);

(k) selecting an antibody with an elevated isoelectric point (pI);

(l) checking the isoelectric point (pI) of the recovered antibody, and then selecting an antibody with an elevated isoelectric point (pI); And

(m) A step of selecting an antibody whose antigen-binding activity is changed or increased depending on the condition of the ion concentration.

Other embodiments of Disclosure A relate to the following inventions, for example and without limitation.

[D1]

(a) according to EU numbering,

196th, 253th, 254th, 256th, 258th, 278th, 280th, 281th, 282th, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th , 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400 Ranked, 401th, 402th, 413th, 415th, 418th, 419th, 421th, 424th, 430th, 433th, 434th, and 443th

A step of modifying the nucleic acid encoding the antibody before modification so that the charge of at least one amino acid residue changes at a position selected from the group consisting of;

(b) culturing the host cell so that the nucleic acid is expressed; And

(c) the step of recovering the antibody from the host cell culture

A method for producing a modified antibody in which the half-life in plasma is prolonged or shortened as compared to the antibody before modification, including; or,

[D2]

According to EU numbering,

196th, 253th, 254th, 256th, 258th, 278th, 280th, 281th, 282th, 285th, 286th, 307th, 309th, 311th, 315th, 327th, 330th , 342, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 389, 399, 400 Ranked, 401th, 402th, 413th, 415th, 418th, 419th, 421th, 424th, 430th, 433th, 434th, and 443th

Step of modifying at least one amino acid residue at a position selected from the group consisting of

A method for extending or shortening the half-life in plasma of the antibody comprising a.

Disclosure B relates, in one embodiment, to, for example, but not limited to, the following.

[33] An Fc region variant comprising an FcRn-binding domain, wherein the FcRn-binding domain is Ala at position 434 according to EU numbering; Glu, Arg, Ser or Lys at position 438; And Glu, Asp or Gln at position 440, Fc region modified.

[34] The FcRn binding domain, according to EU numbering, Ala at position 434; Arg or Lys at position 438; And the Fc region variant of [33], including Glu or Asp at position 440.

[35] The FcRn binding domain is Ile or Leu at position 428 according to EU numbering; And/or the Fc region variant of [33] or [34], further comprising Ile, Leu, Val, Thr or Phe at position 436.

[36] The FcRn binding domain, according to EU numbering, Leu at position 428; And/or the Fc region variant of [35], comprising Val or Thr at position 436.

[37] The FcRn binding domain, according to EU numbering, 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; M428L/N434A/Q438R/S440E; M428L/N434A/Q438R/S440D; M428L/N434A/Q438K/S440E; M428L/N434A/Q438K/S440D; M428L/N434A/Y436T/Q438R/S440E; M428L/N434A/Y436T/Q438R/S440D; M428L/N434A/Y436T/Q438K/S440E; M428L/N434A/Y436T/Q438K/S440D; M428L/N434A/Y436V/Q438R/S440E; M428L/N434A/Y436V/Q438R/S440D; M428L/N434A/Y436V/Q438K/S440E; M428L/N434A/Y436V/Q438K/S440D; L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; And a combination of substituted amino acids selected from the group consisting of L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E .

[38] The FcRn binding domain,

According to EU numbering, the following:

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 a combination of substituted amino acids selected from the group consisting of L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E.

[39] The Fc region variant of any one of [33] to [38], wherein the binding activity to FcRn is increased under acidic pH conditions (for example, pH 5.8) compared to the Fc region of a native IgG. .

[40] The Fc region variant of any one of [33] to [39], wherein the binding activity to an anti-drug antibody (ADA) is not significantly increased under neutral pH conditions compared to the 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] Compared with the Fc region of a native IgG, the clearance (CL) in plasma is reduced, the residence time in plasma is increased, or the half-life (t1/2) in plasma is increased, [33] The Fc region modified variant of any one of to [41].

[43] According to EU numbering, compared with the reference Fc region variant comprising a combination of substituted amino acids of N434Y/Y436V/Q438R/S440E, the retention in plasma is increased, among [33] to [42] Any one Fc region variant.

[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], for increasing the plasma retention of the antibody.

[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] An Fc region variant containing an FcRn-binding domain, or a method for producing an antibody containing the variant, wherein the host cell of [50] is cultured, and an Fc region variant or a variant thereof is obtained from the cell culture. A manufacturing method comprising the step of recovering the antibody comprising a.

[52]

(a) a step of selecting an Fc region variant having increased FcRn-binding activity under acidic pH conditions compared to the Fc region of a native IgG;

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

(c) a step of selecting an Fc region variant with increased retention in plasma compared to the Fc region of a native IgG; And

(d) selecting an antibody containing an Fc region variant capable of promoting the disappearance of antigen from plasma compared to a reference antibody containing the Fc region of a native IgG;

The method of [51], optionally further comprising any one or more steps selected from the group consisting of.

[53] The obtained Fc region variant or the antibody containing the variant is Ala at position 434 according to EU numbering; Glu, Arg, Ser, or Lys at position 438; And a step of substituting an amino acid in a form containing Glu, Asp, or Gln at position 440, an Fc region variant comprising an FcRn binding domain or a method for producing an antibody comprising the variant.

Disclosure B relates, in one embodiment, to, for example, but not limited to, the following.

[B1] The use of the Fc region modified variant of any one of [33] to [43] or the antibody of [44] or [45] in the manufacture of a medicament for enhancing plasma retention.

[B2] Any of [33] to [43] in the manufacture of a medicament for not significantly increasing the binding activity to an anti-drug antibody (ADA) under neutral pH conditions compared to the Fc region of a native IgG. Use of one Fc region variant, or the antibody of [44] or [45].

[B3] Use of the Fc region modified variant of any one of [33] to [43] or the antibody of [44] or [45] to increase retention in plasma.

[B4] In comparison with the Fc region of a native IgG of the Fc region modified variant of any one of [33] to [43], or of the antibody of [44] or [45], an anti-drug antibody (ADA ) To not significantly increase the binding activity.

[B5] An Fc region variant obtained by the method of any one of [51], [52], and [53], or an antibody containing the variant.

According to various embodiments, the combination of some or all of one or a plurality of elements described in any one of [33] to [53] and [B1] to [B5] above, such a combination is applicable in the art. It is included in Disclosure B unless it is technically contradictory based on the technical common sense in For example, in some embodiments, Fc region variants comprising an FcRn binding domain, which may include:

(a) Ala at 434, according to EU numbering; Glu, Arg, Ser, or Lys at position 438; And Glu, Asp, or Gln at position 440;

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

(c) Ile or Leu at 428, according to EU numbering; Ala on 434; Ile, Leu, Val, Thr, or Phe at 436; Glu, Arg, Ser, or Lys at position 438; And Glu, Asp, or Gln at position 440;

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

(e) Leu at 428, according to EU numbering; Ala on 434; Val or Thr at 436; Glu, Arg, Ser, or Lys at position 438; And Glu, Asp, or Gln at position 440; or

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

In addition, in one embodiment, the disclosure C relates to the following, for example, without limitation.

[54] An isolated anti-IL-8 antibody that binds to human IL-8, comprising at least one amino acid substitution according to at least one of the following (a) to (f), which binds to IL-8 in a pH-dependent manner. Anti-IL-8 antibody:

(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.

[55] comprising an amino acid substitution of tyrosine at position 9 in the amino acid sequence of SEQ ID NO: 68, arginine at position 11 in the amino acid sequence of SEQ ID NO: 68, and tyrosine at position 3 in the amino acid sequence of SEQ ID NO: 69, [ 54] anti-IL-8 antibody.

[56] The anti-IL-8 antibody of [54] or [55], further comprising an amino acid substitution 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] comprising an amino acid substitution of asparagine at position 1 in the amino acid sequence of SEQ ID NO: 71, leucine at position 5 in the amino acid sequence of SEQ ID NO: 71, and glutamine at position 1 in the amino acid sequence of SEQ ID NO: 72 54] to [56] of any one of the anti-IL-8 antibody.

[58] (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) the amino acid sequence of SEQ ID NO: 74 The anti-IL-8 antibody of any one of [54] to [57], comprising HVR-H3.

[59] (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) the amino acid sequence of SEQ ID NO: 76 The anti-IL-8 antibody of any one of [54] to [58], comprising HVR-L3.

[60] The anti-IL-8 antibody of any one of [54] to [59], comprising 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], comprising an Fc region having at least one property selected from the following properties (a) to (f):

(a) the binding affinity of the Fc region to FcRn at acidic pH is greater than that of the native Fc region to FcRn;

(b) the binding affinity of the Fc region to the existing ADA is lower than the binding affinity of the native Fc region to the existing ADA;

(c) the plasma half-life of the Fc region is greater than the plasma half-life of the native Fc region;

(d) the clearance in the plasma of the Fc region is less than the clearance in the plasma of the native Fc region; And

(e) the binding affinity of the Fc region to the effector receptor is lower than that of the native Fc region to the effector receptor; And

(f) The binding to the extracellular matrix is increased.

[62] 1 selected from the group consisting of the Fc region, 235th, 236th, 239th, 327th, 330th, 331st, 428th, 434th, 436th, 438th, and 440th according to EU numbering The anti-IL-8 antibody of [61], comprising an amino acid substitution at the above position.

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

[64] The anti-IL-8 antibody of [63], wherein the Fc region contains 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 contains amino acid substitutions of L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

[66] An anti-IL-8 antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 81 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 82.

[67] An anti-IL-8 antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 80 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 82.

[68] The 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, comprising the step of culturing the host of [70].

[72] The method for producing an anti-IL-8 antibody of [71], comprising the step of isolating the antibody from the 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 according to any one of [54] to [67], for use in a pharmaceutical composition.

[75] The anti-IL-8 antibody according to any one of [54] to [67], for use in the treatment of a disease in which IL-8 is excessively present.

[76] The use of the anti-IL-8 antibody according to any one of [54] to [67] in the manufacture of a pharmaceutical composition for a disease in which IL-8 is excessively present.

[77] A method of treating a patient having a disease in which IL-8 is excessive, comprising the step of administering to an individual the anti-IL-8 antibody of any one of [54] to [67].

[78] A method for promoting the loss of IL-8 from the subject, comprising the step of administering to the subject the anti-IL-8 antibody of any one of [54] to [67].

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

[80] As a method for producing an anti-IL-8 antibody containing a variable region whose binding activity to IL-8 is dependent on pH,

(a) the step of evaluating the binding of the anti-IL-8 antibody and the extracellular matrix,

(b) selecting an anti-IL-8 antibody with high binding to the extracellular matrix,

(c) culturing a host containing a vector containing a nucleic acid encoding the antibody, and

(d) the step of isolating the antibody from the culture medium

Containing, the method for producing an anti-IL-8 antibody.

Initiation C relates to the following in an alternative embodiment.

[C1] 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 the accumulation of IL-8 having biological activity .

[C2] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] in inhibiting the accumulation of IL-8 having biological activity.

[C3] Use of the anti-IL-8 antibody according to 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 according to any one of [54] to [67] and [C26] to [C31] in inhibiting angiogenesis.

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

[C6] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] in inhibiting neutrophil migration promotion.

[C7] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for use in inhibiting accumulation of IL-8 having biological activity.

[C8] A method for inhibiting the accumulation of biologically active IL-8, comprising administering to an individual the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] .

[C9] A pharmaceutical composition for inhibiting accumulation of IL-8 having biological activity, comprising the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31].

[C10] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for use in inhibiting angiogenesis.

[C11] A method for inhibiting angiogenesis in an individual, comprising the step of administering to the individual the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31].

[C12] A pharmaceutical composition for inhibiting angiogenesis, comprising the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31].

[C13] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for use in inhibiting neutrophil migration promotion.

[C14] A method of inhibiting the promotion of neutrophil migration in an individual, comprising the step of administering to an individual the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31].

[C15] A pharmaceutical composition for inhibiting neutrophil migration promotion, comprising the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31].

[C16] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for use in the treatment of a disease in which IL-8 is excessively present.

[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 disease in which IL-8 is excessively present .

[C18] The use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] in the treatment of a disease in which IL-8 is excessively present.

[C19] A disease in which IL-8 is excessively present in an individual, comprising the step of administering to the individual the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] Method of treatment.

[C20] A pharmaceutical composition for treating a disease in which IL-8 is excessive, comprising the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31].

[C21] The anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31], for use in promoting the disappearance of IL-8.

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

[C23] Use of the anti-IL-8 antibody according to any one of [54] to [67] and [C26] to [C31] in promoting the disappearance of IL-8.

[C24] A method for promoting loss of IL-8 in an individual, comprising the step of administering to the individual the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31] .

[C25] A pharmaceutical composition for promoting disappearance of IL-8, comprising the anti-IL-8 antibody of any one of [54] to [67] and [C26] to [C31].

[C26] Amino acid at one or more positions selected from the group consisting of 235th, 236th, 239th, 327th, 330th, 331st, 428th, 434th, 436th, 438th, and 440th according to EU numbering An anti-IL-8 antibody comprising an Fc region comprising a substitution.

[C27] The anti-IL-8 antibody according to [C26], comprising an Fc region having at least one property selected from the following properties (a) to (f):

(a) the binding affinity of the Fc region to FcRn at acidic pH is greater than that of the native Fc region to FcRn;

(b) the binding affinity of the Fc region to the existing ADA is lower than the binding affinity of the native Fc region to the existing ADA;

(c) the plasma half-life of the Fc region is greater than the plasma half-life of the native Fc region;

(d) the clearance in the plasma of the Fc region is less than the clearance in the plasma of the native Fc region;

(e) the binding affinity of the Fc region to the effector receptor is lower than that of the native Fc region to the effector receptor; And

(f) The binding to the extracellular matrix is increased.

[C28] comprising 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 according to EU numbering, [ C26] or an anti-IL-8 antibody of [C27].

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

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

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

[C32] The 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, comprising the step of culturing the host cell of [C34].

[C36] A method for producing the antibody, further comprising a step of isolating the anti-IL-8 antibody of any one of [C26] to [C31] 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 having a disease in which IL-8 is excessive, comprising the step of administering to an individual the anti-IL-8 antibody of any one of [C26] to [C31].

[C39] A method for promoting the loss of IL-8 from the individual, comprising administering to the individual the anti-IL-8 antibody of any one of [C26] to [C31].

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

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

According to various embodiments, the combination of some or all of one or more elements described in any one of [54] to [80] and [C1] to [C41] above, such a combination is applicable to the art. It is included in Disclosure C unless it is technically contradictory based on the technical common sense.

Fig. 1 is a human IL-6 receptor-binding antibody (Low_pI-IgG1) in which the constant region is a natural IgG1, which binds to a human IL-6 receptor in a pH-dependent manner, and an antibody in which the pI of the variable region in the antibody is increased. When (High_pI-IgG1) is administered to a human FcRn transgenic mouse, the change in the concentration of human IL-6 receptor in the plasma of the mouse is shown.
Fig. 2 is a human IL-6 receptor-binding antibody (Low_pI-F939) that binds to human IL-6 receptor in a pH-dependent manner and confers binding to FcRn under neutral pH conditions, and the variable in the antibody. The change in the concentration of human IL-6 receptor in the plasma of the mouse is shown when antibodies (Middle_pI-F939, High_pI-F939) with elevated pI of the region were respectively administered to human FcRn transgenic mice.
Fig. 3 is a human IL-6 receptor-binding antibody (Low_pI-F1180) that binds to human IL-6 receptor in a pH-dependent manner and increases binding to FcγR under neutral pH conditions, and the variable in the antibody. The change in the concentration of human IL-6 receptor in the plasma of the mouse is shown when antibodies (Middle_pI-F1180, High_pI-F1180) with elevated pI of the region were respectively administered to human FcRn transgenic mice.
Fig. 4 is a human IL-6 receptor-binding antibody (Low_pI-IgG1) in which the antibody binds to human IL-6 receptor in a pH-dependent manner, and the constant region is a natural IgG1, and the Fc region in the antibody is under neutral pH conditions. Antibodies (Low_pI-F11) containing Fc region modifiers with increased binding to FcRn, and antibodies (High_pI-IgG1, High_pI-F11) which raised the pI of the variable regions of these antibodies, soluble in plasma The transition of the human IL-6 receptor concentration in the plasma of the mouse when administered to human FcRn transgenic mice in which the human IL-6 receptor concentration is maintained in a normal state is shown.
Figure 5 shows the pH-dependent binding to the human IL-6 receptor, 3 kinds of antibodies with different pi (Low_pI-IgG1, Middle_pI-IgG1, and High_pI-IgG1), and pH-dependent for human IL-6 receptor. It shows the degree of each binding to the extracellular matrix of two kinds of antibodies (Low_pI(NPH)-IgG1, and High_pI(NPH)-IgG1) that do not show binding and have different pIs. In addition, in the range in which the indication A in this specification is described, "NPH" means that it is pH independent.
Fig. 6 is a soluble human FcγRIIb of an antibody containing an Fc region variant in which each pI is elevated by altering 1 amino acid residue in the constant region of the antibody Ab1H-P600 that binds to IgE in a pH-dependent manner. The degree of binding to (measured by BIACORE (registered trademark)) is expressed as a relative value with the value of Ab1H-P600 as 1.00.
Fig. 7 shows the rate of intracellular uptake of antibodies containing Fc region variants in which each pI is elevated by altering one amino acid residue in the constant region of Ab1H-P600 to hFcγRIIb-expressing cell lines, The value in Ab1H-P600 was evaluated as 1.00 and expressed as a relative value.
Fig. 8 shows the degree of binding of Fv4-IgG1, which has the Fc region of native human IgG1, to rheumatoid factors in individual serum of RA patients.
Fig. 9 shows the degree of binding of Fv4-YTE with an Fc region variant with increased binding to FcRn to rheumatoid factors in individual serum of RA patients.
Fig. 10 shows the degree of binding of Fv4-LS with an Fc region variant with increased binding to FcRn to rheumatoid factors in individual serum of RA patients.
Fig. 11 shows the degree of binding of Fv4-N434H, which has an Fc region variant with increased binding to FcRn, to rheumatoid factors in individual serum of RA patients.
Fig. 12 shows the degree of binding of Fv4-F1847m to the rheumatoid factor in individual serum of RA patients, having an Fc region variant with increased binding to FcRn.
Fig. 13 shows the degree of binding of Fv4-F1848m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients.
Fig. 14 shows the degree of binding of Fv4-F1886m to rheumatoid factor in individual serum of RA patients, having an Fc region variant with increased binding to FcRn.
Fig. 15 shows the degree of binding of Fv4-F1889m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients.
Fig. 16 shows the degree of binding of Fv4-F1927m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients.
Fig. 17 shows the degree of binding of Fv4-F1168m, which has an Fc region variant with increased binding to FcRn, to rheumatoid factor in individual serum of RA patients.
Fig. 18 is for each antibody containing Fv4-IgG1 having an Fc region of native human IgG1 and a novel Fc region variant having an Fc region variant with increased binding to various FcRn, in serum of RA patients. The average value of the binding property to the rheumatoid factor is shown.
FIG. 19 is an anti-human IgE antibody, OHB-IgG1 having an Fc region of native human IgG1, and each antibody comprising a novel Fc region variant having an Fc region variant with increased binding to FcRn (OHB -LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m) were administered to a Philippine monkey, the concentration of various anti-human IgE antibodies in the plasma of the Philippine monkey Shows the trend.
Fig. 20 is an anti-human IL-6 receptor antibody, Fv4-IgG1 having an Fc region of natural human IgG1, or Fv4-F1718 having increased binding to FcRn under acidic pH conditions in the antibody is administered. In one case, the change in plasma concentration of the anti-human IL-6 receptor antibody in human FcRn transgenic mice is shown.
Fig. 21 shows a sensorgram obtained when the binding of H998/L63 and Hr9 to IL-8 at pH 7.4 and pH 5.8 is measured by Biacore.
22 shows the transition of human IL-8 concentration in plasma of the mouse when H998/L63 or H89/L118 mixed with human IL-8 was administered to a mouse at 2 mg/kg.
Fig. 23 shows the transition of human IL-8 concentration in plasma of the mouse when H89/L118 mixed with human IL-8 is administered to a mouse at 2 mg/kg or 8 mg/kg.
Fig. 24 shows the transition of human IL-8 concentration in plasma of the mouse when H89/L118 or H553/L118 mixed with human IL-8 is administered to mice at 2 mg/kg or 8 mg/kg, respectively.
Fig. 25A shows the change in the relative value of the amount of chemiluminescence by the antibody of Hr9, H89/L118, or H553/L118 before storage in plasma, depending on the antibody concentration.
Fig. 25B shows the relative value of the amount of chemiluminescence by the antibody of Hr9, H89/L118, or H553/L118 after storage in plasma for one week, depending on the antibody concentration.
Fig. 25C shows the transition of Hr9, H89/L118, or H553/L118 relative to the amount of chemiluminescence by the antibody after storage in plasma for 2 weeks, depending on the antibody concentration.
26 shows the predicted frequency of ADA occurrence of each anti-IL-8 antibody (hWS4, Hr9, H89/L118, H496/L118, or H553/L118) predicted by EpiMatrix and the predicted frequency of ADA occurrence of other existing therapeutic antibodies. Show.
Figure 27 shows the predicted frequency of ADA occurrence of each anti-IL-8 antibody (H496/L118, H496v1/L118, H496v2/L118, H496v3/L118, H1004/L118, or H1004/L395) predicted by EpiMatrix and other conventional treatments. It shows the predicted frequency of the occurrence of ADA of the soluble antibody.
Fig. 28A shows the relative value of the chemiluminescence amount of Hr9, H89/L118, or H1009/L395-F1886s by the antibody before storage in plasma, depending on the antibody concentration.
Fig. 28B shows the relative value of the amount of chemiluminescence by the antibody of Hr9, H89/L118, or H1009/L395-F1886s after storage in plasma for 1 week, depending on the antibody concentration.
Fig. 28C shows the relative value of the amount of chemiluminescence by the antibody of Hr9, H89/L118, or H1009/L395-F1886s after storage in plasma for 2 weeks, depending on the antibody concentration.
Fig. 29 shows the transition of human IL-8 concentration in plasma of the mouse when H1009/L395, H553/L118, and H998/L63 mixed with human IL-8 were administered to mice, respectively.
Fig. 30 shows the degree of binding to the extracellular matrix when Hr9, H89/L118 or H1009/L395 is added to the extracellular matrix alone and when added in combination with human IL-8.
Fig. 31 shows plasma of the mouse when an antibody having the variable region of H1009/L395 and an Fc region that does not bind to FcRn (F1942m) is administered alone or in combination with human IL-8 to human FcRn transgenic mice. Shows the change in the concentration of the antibody in.
32 shows the predicted frequency of ADA occurrence of H1009/L395 and H1004/L395 predicted by EpiMatrix and the predicted frequency of ADA occurrence of other existing therapeutic antibodies.
Figure 33 is an antibody (H89/L118-F1168m, H89/L118) having an H89/L118-IgG1 variable region of H89/L118 and an Fc region of native human IgG1, and an Fc region variant with increased binding to FcRn. -F1847m, H89/L118-F1848m, H89/L118-F1886m, H89/L118-F1889m, and H89/L118-F1927m) were administered to Philippine monkeys, respectively, in plasma of the Philippine monkeys. Shows the change in antibody concentration.
Fig. 34 shows the binding properties to various FcγRs of an antibody having a variable region of H1009/L395, whose Fc region is a variant (F1886m, F1886s, or F1974m).
Fig. 35 shows the change in the concentration of human IL-8 in plasma of the mouse when an anti-IL-8 antibody mixed with human IL-8 is administered to a human FcRn transgenic mouse. The anti-IL-8 antibody at this time is H1009/L395-IgG1 (2 mg/kg) containing the variable region of H1009/L395 and the Fc region of native human IgG1, or the variable region of H1009/L395 and the modified Fc region. It was H1009/L395-F1886s (2, 5 or 10 mg/kg) containing.
Figure 36 is a philippine monkey administration of Hr9-IgG1 or H89/L118-IgG1 containing all of the Fc regions of native human IgG1, or H1009/L395-F1886s or H1009/L395-F1974m containing all of the modified Fc regions. In the case, the change in the concentration of each antibody in the plasma of the Philippine monkey is shown.
Fig. 37 shows the temporal profile of plasma IgE concentrations of several anti-IgE antibodies in C57BL6J mice from the viewpoint of antibody variable region modification.
38A shows sensorgrams of octets of 25 selected, pH dependent and/or calcium dependent antigen binding clones.
FIG. 38B is a continuing view of FIG. 38A.
FIG. 38C is a continuation of FIG. 38B.
FIG. 38D is a continuation of FIG. 38C.
39 shows the temporal profile of plasma C5 concentration of several anti-C5 bispecific antibodies in C57BL6J mice from the viewpoint of antibody variable region modification.
Fig. 40 shows the temporal profile of plasma IgE concentrations of several anti-IgE antibodies in C57BL6J mice from the viewpoint of alteration of the antibody constant region.

In the following, non-limiting embodiments of the disclosures A, B, or C will be described. All of the embodiments described in the present examples described below include any patent practices, customs, laws, etc. that may limit the interpretation of the contents described in this example in the country where the patent rights of the present patent application are intended. It is described with the intention that it is not bound by limitation, and it is intended that it will be understood that it is described also in the term of "specific content for carrying out the invention".

Initiation A or Initiation B

In some embodiments, initiation A is an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to conditions of ion concentration, wherein at least one amino acid residue that can be exposed on the surface of the antibody is altered. An antibody with an elevated isoelectric point (pI) by its presence (in the range of initiation A, it is also referred to as "an ion concentration dependent antibody with an elevated pi"", and the antigen-binding domain in the antibody is referred to as "an ion concentration with an elevated pi Also referred to as "dependent antigen binding domain".). In this invention, in an ion concentration-dependent antibody, if at least one amino acid residue that can be exposed on the surface of the antibody is altered and the isoelectric point (pI) is elevated (for example, when the antibody is administered in vivo ,) can promote the loss of antigen from plasma; Moreover, it is based in part on the surprising findings of the present inventors that an ion concentration dependent antibody with elevated pi can enhance the binding of the antibody to the extracellular matrix. This invention relates to the concept of an ion concentration-dependent antigen-binding domain or an ion-concentration-dependent antibody, and an antibody whose pi is elevated by alteration of at least one amino acid residue that can be exposed to the surface (in the range of initiation A, " Also referred to as "antibodies with elevated pi"; On the other hand, in the range of initiation A, on the contrary, antibodies with lower pi by altered at least one amino acid residue that can be exposed to the surface are "antibodies with lower pi" It is also partly based on the surprising findings of the present inventors that the combination of two concepts completely different from the concept of (referred to as "referred to as") brings about this advantageous effect. Accordingly, this invention is classified as a kind of pioneer invention, which can bring about remarkable technological innovation in the field to which the disclosure A belongs (for example, in the medical field).

For example, as an antibody comprising an antigen-binding domain in which at least one amino acid residue that may be exposed on the surface of the antibody has been altered, the pI is elevated, and the antigen-binding activity is changed according to the condition of the ion concentration. Antibodies in which the antigen-binding domain has been further modified are naturally also included in the range described in the disclosure A in the present specification (such antibodies are also in the range where disclosure A in this specification is described, similarly, "pI is increased. Is also referred to as an "ion concentration dependent antibody").

For example, for at least one amino acid residue that may be exposed on the surface of the antibody, the antibody (natural type antibody (e.g., a native type Ig antibody, preferably a native type IgG antibody) or a reference antibody or parent antibody ( For example, the antibody has a charge different from that of at least one amino acid residue at the corresponding position in the antibody before modification, or the antibody is in the process of libraryization or libraryization, etc.)), and the antibody as a whole Antibodies containing an ion concentration-dependent antigen-binding domain with an elevated pI are naturally also included in the disclosure A described in the present specification (such antibodies are also included in the disclosure A in this specification, similarly, "pI Is also referred to as an "elevated ion concentration dependent antibody").

For example, an antibody (natural type antibody (e.g., a native type Ig antibody, preferably a native type IgG antibody) or a reference antibody or parent antibody (e.g., modified Antibodies with elevated pi by altering at least one amino acid residue that can be exposed on the surface of the antibody in the antibody before adding or in the process of libraryization or before)) , Of course, is included in the disclosure A described in the present specification (such an antibody is also referred to as "an ion concentration-dependent antibody with elevated pi" within the range in which the disclosure A in this specification is described.).

For example, for the purpose of raising the pi of the antibody, an antibody containing an ion concentration-dependent antigen-binding domain in which at least one amino acid residue that can be exposed on the surface of the antibody has been altered is, of course, the disclosure A described herein. (Such antibodies are also referred to as "ion concentration-dependent antibodies with elevated pi" within the range in which the disclosure A in this specification is described.)

In the range describing the disclosures A and B in this specification, "amino acid" includes not only natural amino acids but also non-natural amino acids. In the range describing the disclosures A and B in this specification, the amino acid or amino acid residue is a one-letter code (e.g., A) or a three-letter code (e.g., Ala), or in combination with them (e.g. For example, Ala(A)) may be described.

In the context of the disclosures A and B, when "modification of amino acids", "modification of amino acid residues", or equivalent words are used, they are, but are not limited to, a specific one or more of the amino acid sequence of the antibody. Molecularly modified or one or more (e.g., more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10) amino acids (residues) of For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) may be understood as adding, deletion, substitution, or insertion of amino acids. The addition, deletion, substitution, or insertion of an amino acid can be performed on a nucleic acid encoding an amino acid sequence, for example, by a site-specific mutagenesis method (Kunkel et al., Proc. Natl. Acad. Sci. USA 82, 488-492). (1985)) or overlap extension PCR; Or using affinity maturation of the antibody or chain shuffling of the heavy or light chain of the antibody; Alternatively, it can be carried out by selection by antigen panning using a phage display library (Smith et al., Methods Enzymol. 217, 228-257 (1993)). These may be appropriately performed alone or in combination. Such amino acid modifications are not limited, but one or more amino acid residues in the amino acid sequence of the antibody are preferably substituted with other amino acids (respectively). The addition, deletion, substitution, or insertion of amino acids, and alteration of the amino acid sequence by humanization or chimerization can be performed by methods known in the art. Similarly, even if amino acids (residues) such as addition, deletion, substitution, or insertion of amino acids are changed or altered in the variable region of the antibody or the constant region of the antibody used when producing the antibody of initiation A or B as a recombinant antibody, do.

In one embodiment of the range of disclosures A and B in this specification, the substitution of an amino acid (residue) means substitution with another amino acid (residue), for example, the following (a) to (c) and You may design to change about the same point.

(a) a backbone structure of a polypeptide in a region of a sheet structure or a screw structure;

(b) charge or hydrophobicity at the target site, or

(c) the size of the side chain.

Amino acid residues are classified into, for example, the following groups based on the characteristics of the side chains included in their structure: (1) hydrophobicity: norleucine, Met, Ala, Val, Leu, and Ile; (2) neutral, hydrophilic: Cys, Ser, Thr, Asn, and Gin; (3) acidic: Asp and Glu; (4) basic: His, Lys, and Arg; (5) residues that influence the orientation of the chain: Gly and Pro; And (6) aromaticity: Trp, Tyr, and Phe.

Substitution of amino acid residues within each of these groups is referred to as conservative substitution, while substitution of amino acid residues between other groups is referred to as non-conservative substitution. Substitution of amino acid residues may be conservative substitution, non-conservative substitution, or a combination thereof. In order to substitute amino acids other than natural amino acids, a plurality of known methods may be appropriately employed (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)). For example, you may use a cell-free translation system (Clover Direct (Protein Express)), which contains a tRNA in which a non-natural amino acid is bound to an amber suppressor tRNA complementary to one of the stop codons, the UAG codon (amber codon). .

It is understood that within the range of disclosures A and B in this specification, the structure of "antigen" is not limited to a specific structure as long as the antigen contains an epitope that binds to an antibody. The antigen may be an inorganic substance or an organic substance. As the antigen, for example, any ligand containing various cytokines such as interleukin, chemokine, and cell proliferation factor may be used, or, for example, exist in a soluble form in a biological fluid such as plasma or modified to a soluble form. The resulting receptor can, of course, be used as an antigen. Non-limiting examples of such soluble receptors include, for example, Mullberg et al., J. Immunol. The soluble IL-6 receptor described in 152 (10), 4958-4968 (1994) can be exemplified. In addition, the antigen may be monovalent (eg, soluble IL-6 receptor) or polyvalent (eg, IgE).

In one embodiment, the antigen to which the antibodies of initiation A and B can bind is a subject (in the range in which the disclosures A and B in this specification are described, the administration (application) of the antibody is intended, and in fact , Any animal, for example, may be human, mouse, etc.) in the biological fluid (for example, the biological fluid discussed in WO2013/125667, preferably, plasma, intertissue fluid, lymph fluid, ascites, or pleural fluid, etc.) It is preferably a soluble antigen present, but may be a membrane antigen.

Within the range of disclosures A and B in this specification, "prolongation of half-life in plasma" or "shortening of half-life in plasma" of the target molecule (either antigen or antibody), or words that are synonymous with them, are in plasma. In addition to the parameter called half-life (t1/2), it can be expressed more specifically by any other parameters such as average plasma residence time, plasma clearance (CL), and area under the concentration curve (AUC) ("Pharmaco Kinetics: Understanding by Practice" (Nanjando)). For example, these parameters can be specifically evaluated by performing a noncompartmental analysis in accordance with a procedure attached to the in vivo dynamic analysis software WinNonlin (Pharsight). It is known to those skilled in the art that these parameters are usually correlated with each other.

In the range of disclosures A and B in this specification, "epitope" refers to an antigenic determinant present in an antigen, and refers to a site on an antigen to which an antigen-binding domain in an antibody binds. Thus, for example, an epitope can be defined based on its structure. In addition, the epitope can be defined also by the antigen-binding activity of the antibody that recognizes the epitope. When the antigen is a peptide or a polypeptide, it is also possible to specify the epitope by amino acid residues constituting the epitope. In addition, when the epitope is a sugar chain, it is also possible to specify the epitope based on a specific sugar chain structure. The antigen-binding domains in initiation A and B may bind to the same or different epitopes in the antigen.

The linear epitope may be an amino acid primary sequence. Such linear epitopes typically comprise at least 3, and usually at least 5, such as 8 to 10, or 6 to 20 amino acids, as native sequences.

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

In the range of disclosures A and B in this specification, "antibody" is not particularly limited as long as it can bind to a target antigen, and is used in the broadest sense. As a non-limiting example of the antibody, a commonly known antibody (for example, a natural immunoglobulin (abbreviated as "Ig")) or a molecule or variant derived therefrom, such as Fab, Fab', F (ab') ₂ , Diabodies, ScFv (Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP404,097; WO93/11161; Peer et al., Nature Nanotechnology 2 : 751-760 (2007)), a low molecular weight antibody (minibody) (Orita et al., Blood 105: 562-566 (2005)), a framework protein, a flaky antibody (a flaky antibody described in WO2005/063816) Including all embodiments of), multispecific antibodies (e.g., bispecific antibodies: antibodies having specificity for two different epitopes, antibodies that recognize different antigens and antibodies that recognize different epitopes on the same antigen. Including) is also included. In the range describing the disclosures A and B in this specification, the "bispecific antibody" is not limited, but may be produced as an antibody molecule having a common L chain described in, for example, WO2005/035756, Or, as described in WO2008/119353, a method of mixing two types of conventional antibodies having an IgG4-like constant region and causing an exchange reaction between the two types of such antibodies (for those skilled in the art, as "Fab arm exchange" method It is also known). As an alternative embodiment, an antibody having a structure in which the heavy chain variable region and the light chain variable region are linked as a single chain (eg, sc(Fv) ₂ ) may be used. Or an antibody-like molecule (e.g., scFv) in which the heavy chain variable region (VH) and the light chain variable region (VL) are linked to the scFv (or sc(Fv) ₂ ) are linked to the Fc region (the normal region from which the CH1 domain is deleted). -Fc) may be used. _{The multispecific antibody consisting of scFv-Fc has a (scFv) 2-} Fc type structure in which the first polypeptide is VH1-linker-VL1-Fc and the second polypeptide is VH2-linker-VL2-Fc. Alternatively, an antibody-like molecule in which a single domain antibody is linked to an Fc region (Marvin et al., Curr. Opin. Drug Discov. Devel., 9(2): 184-193(2006)), an Fc fusion protein (e.g. For example, immunoadhesin) (US2013/0171138), functional fragments thereof, functional equivalents thereof, and modified sugar chain modifications thereof may be used. In this specification, the natural type IgG (e.g., natural type IgG1) refers to an antibody that contains the same amino acid sequence as that of an IgG (e.g., natural type IgG1) found in nature, and is substantially encoded by an immunoglobulin gamma gene. It means a polypeptide belonging to the class. The natural IgG may be a mutant or the like naturally occurring therefrom.

Typically, when an antibody has a structure substantially the same as or similar to a native IgG, a Y-shaped four-stranded structure (two heavy chain polypeptides and two light chain polypeptides) may be used as the basic structure. Typically, the heavy and light chains can be bonded together via a disulfide bond (SS bond) to form a heterodimer. Such heterodimers may be bonded together via a disulfide bond to form a Y-shaped heterotetramer. On the other hand, two heavy chains or light chains may be the same or different.

For example, by papain digestion of an IgG antibody, the hinge region in which the Fab region of the heavy chain is linked to the Fc region (in the range describing the disclosures A and B in this specification, it is also referred to as "hinge") By cutting, two Fabs (regions) and one Fc (region) can be obtained. Typically, the Fab region has an antigen binding domain. Phagocytes such as leukocytes and macrophages have a receptor capable of binding to this Fc region (Fc receptor), and through this Fc receptor, the antibody bound to the antigen can be recognized and the antigen can be phagocytosed (opsonin action). Further, the Fc region has an effector function that is involved in mediating an immune response such as ADCC or CDC, and causes a reaction after the antibody binds to an antigen. It is known that the effector function of an antibody differs depending on the type (isotype) of the immunoglobulin. The Fc region of the IgG class may, for example, represent a region from cysteine at position 226 (according to EU numbering) or from proline at position 230 to C-terminus, but is not limited thereto. In addition, the Fc region is suitably obtained by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, and then eluting the fraction adsorbed on a protein A or protein G column. Can be.

In the range described in the disclosures A and B in this specification, the positions of amino acid residues present in the variable region (CDR and/or FR) of the antibody are indicated by Kabat, and the amino acid residues present in the constant region or the Fc region. Positions are indicated by EU numbering based on the amino acid positions of Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991).

As described in detail in WO2013/125667 (for example, paragraphs 0121 to 0125), in the range describing the disclosures A and B in this specification, the term "library" may be the same or different from each other. , A plurality of antibodies having sequence diversity, a plurality of fusion polypeptides containing an antibody, or a molecule (group), such as a nucleic acid or polynucleotide encoding these amino acid sequences, may be referred to. For example, the library may contain at least 10 ⁴ molecules, more preferably at least 10 ⁵ molecules, more preferably at least 10 ⁶ molecules, particularly preferably at least 10 ⁷ molecules or more of the antibody. The library may be a phage library. The term “consisting primarily of” means that a certain percentage of a number of independent clones having different sequences in the library are occupied by antibodies that may have different antigen-binding activities. In one embodiment, an immune library constructed based on an antibody gene derived from lymphocytes of an animal immunized with a specific antigen, a patient with an infectious disease, a human with an elevated blood antibody level after vaccination, or a patient with cancer or autoimmune disease, It can be suitably used as a randomized variable region library. In an alternative embodiment, a naive library constructed from an antibody gene derived from a lymphocyte of a normal person and containing a naive sequence, which is an antibody sequence that does not contain a bias in its repertoire, can also be suitably used as a randomized variable region library ( Gejima et al., Human Antibodies 11: 121-129 (2002); Cardoso et al., Scand. J. Immunol. 51: 337-344 (2000)). The amino acid sequence containing a naive sequence can be said to be obtained from such a naive library. In an alternative embodiment, a synthetic library in which the CDR sequences of the V gene or the reconstructed functional V gene in genomic DNA are replaced with a synthetic oligonucleotide set containing a sequence encoding a codon set of appropriate length is also randomized. It can be suitably used as a variable region library. In this case, since sequence diversity of the CDR3 gene is observed, it is also possible to substitute only the heavy chain CDR3 sequence. A standard method for calculating amino acid diversity in the variable region of an antibody may be to increase the variation of amino acid residues at positions that can be exposed on the surface of the antibody.

In one embodiment, when the antibody of initiation A or B has a structure substantially identical or similar to, for example, a native Ig antibody, the antibody is typically a variable region ("V region") [ Heavy chain variable region ("VH region") and light chain variable region ("VL region")] and normal region ("C region") [heavy chain constant region ("CH region") and light chain constant region ("CL region")] Has. The CH region can be further divided into three types: CH1 to CH3. Typically, the Fab region of the heavy chain contains the VH region and CH1, and typically, the Fc region of the heavy chain contains CH2 and CH3. Typically, the hinge region is located between CH1 and CH2. Moreover, typically, the variable region has a complementarity determining region (“CDR”) and a framework region (“FR”). Typically, in the VH region and the VL region, there are three CDRs (CDR1, CDR2, and CDR3) and four FRs (FR1, FR2, FR3, and FR4), respectively. Typically, all six CDRs present in the variable regions of the heavy and light chains interact to form an antigen-binding domain of an antibody. On the other hand, when there is only one CDR, it is known that the binding affinity for an antigen is lower than when there are 6 CDRs, but it still has the ability to recognize and bind to an antigen.

Ig antibodies can be divided into several classes (isotypes) based on differences in the structure of the normal region. In many mammals, they are classified into five classes of immunoglobulins: IgG, IgA, IgM, IgD, and IgE based on differences in the structure of the normal region. Moreover, in humans, IgG has four subclasses of IgG1, IgG2, IgG3, and IgG4, and IgA has two subclasses of IgA1 and IgA2. Heavy chains can be divided into γ chains, μ chains, α chains, δ chains, and ε chains according to the difference in the normal region, and based on this difference, five classes of IgG, IgM, IgA, IgD, and IgE ( Isotype) of immunoglobulins. On the other hand, there are two types of light chains, λ chain and κ chain, and all immunoglobulins have any one of them.

In one embodiment, when the antibody of initiation A or B has a heavy chain, the heavy chain is, for example, any one of γ chain, μ chain, α chain, δ chain, and ε chain, or any one of them. May be derived from, or when the antibody of initiation A or B has a light chain, the light chain may be, for example, either a κ chain or a λ chain, or may be derived from either. In addition, in the range described in the disclosure A and B in this specification, the antibody is not limited, but any isotype (e.g., IgG, IgM, IgA, IgD, or IgE), and any subclass (e.g. For example, human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, mouse IgG1, IgG2a, IgG2b, and IgG3) may be used, and may be derived from any of them.

In the range of disclosures A and B in this specification, the "antigen-binding domain" may have any structure as long as it binds to the target antigen. Examples of such domains include, for example, the variable regions of the heavy and light chains of an antibody (eg, 1 to 6 CDRs); Modules of about 35 amino acids called A domain, which are included in Avimer, which is a cell membrane protein present in the body (WO2004/044011 and WO2005/040229); Adnectin (WO2002/032925) comprising a 10Fn3 domain that binds to a protein in fibronectin, a glycoprotein expressed on cell membranes; Affibody having an IgG binding domain constituting a bundle of three helixes consisting of 58 amino acids of protein A as a skeleton (WO1995/001937); A region exposed on the molecular surface of an ankyrin repeat (AR) having a structure in which one turn containing 33 amino acid residues, two antiparallel helixes, and subunits having one loop are repeatedly stacked. DARPin (Designed Ankyrin Repeat Protein) (WO2002/020565); In lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL), 8 highly conserved antiparallel strands support one side of the barrel structure twisted in the center direction, anti- Kalin et al. (WO2003/029462); And a leucine-rich-repeat (LRR) module that does not have an immunoglobulin structure and is rich in leucine residues of variable lymphocyte receptors (VLR), which are used as an immune system for acquiring non-malignant species such as eel and eel. A concave area formed by a parallel sheet structure inside the horseshoe-shaped structure stacked repeatedly is mentioned (WO2008/016854). Preferred antigen-binding domains for initiation A or B include antigen-binding domains containing variable regions of heavy and light chains of an IgG antibody, and more specifically, ScFv, single-chain antibody, Fv, and scFv ₂ (single-chain Fv ₂ ), Fab, and F(ab') ₂ and the like.

In one embodiment of Initiation A, the "ion concentration" means, although not particularly limited, a hydrogen ion concentration (pH) or a metal ion concentration. Herein, "metal ion" refers to Group I elements such as alkali metals and copper group elements excluding hydrogen, Group II elements such as alkaline earth metals and zinc group elements, Group III elements excluding boron, carbon and silicon Any one of ions of metal elements such as group IV elements, elements belonging to each A subgroup of groups V, VI and VII, and metal elements such as antimony, bismuth, and polonium, except for Group IV elements, elements of group VIII such as iron group and platinum group elements I can. A metal atom has the property of becoming a cation by releasing valence electrons. This is called the ionization tendency. Metals with a high tendency to ionize are considered to be chemically active.

In one embodiment of Disclosure A, a suitable metal ion may be a calcium ion, as described in detail in WO2012/073992 or WO2013/125667.

In one embodiment of Initiation A, the "ion concentration condition" may be a condition in which attention is paid to the difference in the biological behavior of the ion concentration-dependent antibody at a low ion concentration and a high ion concentration. In addition, "the antigen-binding activity changes depending on the ion concentration condition" means that the antigen-binding activity of the ion concentration-dependent antigen-binding domain or the ion concentration-dependent antibody at initiation A or B is under low ion concentration. It can mean that it changes under high ionic concentration. For example, a case in which the antigen-binding activity is higher (strong) or lower (weak) under the condition of high ionic concentration than the antigen-binding activity under the condition of low ionic concentration is not limited thereto.

In one embodiment of Initiation A, the ion concentration may be a hydrogen ion concentration (pH) or a calcium ion concentration. When the ion concentration is a hydrogen ion concentration (pH), the ion concentration-dependent antigen-binding domain can also be referred to as a "pH-dependent antigen-binding domain". When the ion concentration is a calcium ion concentration, the "calcium ion concentration-dependent antigen It can also be referred to as "binding domain".

In one embodiment in the context of initiation A, the ion concentration-dependent antigen-binding domain, the ion concentration-dependent antibody, the ion concentration-dependent antigen-binding domain with elevated pi, and the ion concentration-dependent antibody with elevated pi, Depending on the conditions of, the antigen-binding domain or the antigen-binding domain may contain at least one amino acid residue that changes the binding activity of the antibody, and may be obtained from a library mainly composed of antibodies having different sequences (having diversity). As the antigen-binding domain, it may be preferable to exist in the light chain variable region (which may be altered) and/or the heavy chain variable region (which may be altered). Further, a library may be prepared by combining such a light chain variable region or heavy chain variable region with a heavy chain variable region or light chain variable region produced as a randomized variable region sequence library. As a non-limiting example of the library, when the ion concentration is a hydrogen ion concentration or a calcium ion concentration, for example, as the light chain variable region, SEQ ID NO: 1 (Vk1), SEQ ID NO: 2 (Vk2), SEQ ID NO: 3 ( Vk3), or a light chain variable region sequence in which amino acid residues of a germline sequence such as SEQ ID NO: 4 (Vk4) are substituted with at least one amino acid residue capable of changing antigen-binding activity depending on ion concentration, A library in which a heavy chain variable region prepared as a randomized variable region sequence library is combined is exemplified. Moreover, when the ion concentration is calcium ion concentration, for example, the heavy chain variable region sequence of SEQ ID NO: 5 (6RL#9-IgG1) or SEQ ID NO: 6 (6KC4-1#85-IgG1), and randomization variable Libraries in which a light chain variable region prepared as a region sequence library or a light chain variable region having a germ-line sequence is combined are mentioned.

In one embodiment, when the ion concentration is a calcium ion concentration, the high calcium ion concentration is not particularly limited to a specific value, but between 100 μM and 10 mM, between 200 μM and 5 mM, between 400 μM and 3 mM, between 200 μM and 2 mM , Or may be a concentration selected between 400 μM and 1 mM. A concentration selected between 500 µM and 2.5 mM close to the calcium ion concentration in plasma (in blood) of in vivo is also preferably mentioned. The low calcium ion concentration is not particularly limited to a specific value, but a concentration selected between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, or between 1 μM and 5 μM, or between 2 μM and 4 μM May be. A concentration selected between 1 µM and 5 µM close to the calcium ion concentration in the early endosome of in vivo is also preferably mentioned.

Whether the antigen-binding domain to the antigen or the binding activity of the antibody containing the domain changes depending on the condition of the metal ion concentration (e.g., calcium ion concentration), for example, in the context of the disclosure A in this specification. It can be easily determined using a known method such as the method described in or described in WO2012/073992. For example, the binding activity of an antigen-binding domain to an antigen or an antibody containing the domain can be measured and compared under a low calcium ion concentration and a high calcium ion concentration. At this time, it is preferable to make the conditions other than the calcium ion concentration the same. In addition, conditions other than the calcium ion concentration when measuring the antigen-binding activity can be appropriately selected by a person skilled in the art. The antigen-binding activity may be measured under conditions of, for example, HEPES buffer, 37°C, or may be measured using BIACORE (GE Healthcare) or the like.

In one embodiment in the context of initiation A, an ion concentration-dependent antigen-binding domain, an ion concentration-dependent antibody, an ion concentration-dependent antigen-binding domain with an elevated pi, or an ion concentration-dependent antibody with an elevated pI, against an antigen. , It is preferable that the binding activity is higher under the condition of a high calcium ion concentration than under the condition of a low calcium ion concentration. In this case, the ratio of the antigen-binding activity under the low calcium ion concentration condition to the antigen-binding activity under the high calcium ion concentration condition is not limited, but KD ( The value of the ratio of KD under conditions of dissociation constant) and high calcium ion concentration, that is, KD (3 μM of Ca)/KD (2 mM of Ca) is preferably 2 or more, more preferably 10 or more, and more preferably It may be 40 or more. The upper limit of the value of KD (3 μM Ca)/KD (2 mM Ca) is not limited, and may be any value such as 400, 1000, and 10000.

As a value of the antigen-binding activity, when the antigen is a soluble antigen, a dissociation constant (KD) can be used, but when the antigen is a membrane antigen, an apparent dissociation constant (KD) can be used. KD and apparent KD can be measured by a known method, for example, BIACORE (GE healthcare), a Scatchard plot, a flow cytometer, or the like.

Alternatively, as another index indicating the ratio of the binding activity, for example, a dissociation rate constant (kd) may be used. When using the dissociation rate constant (kd) instead of the dissociation constant (KD) as an index indicating the ratio of the antigen-binding activity, the dissociation rate constant (kd) and high calcium ion concentration conditions under the condition of low calcium ion concentration to the antigen The ratio of the dissociation rate constant (kd) under, that is, the value of kd (the condition of the low calcium ion concentration)/kd (the condition of the high calcium ion concentration) is preferably 2 or more, more preferably 5 or more, and more preferably It is preferably 10 or more, and even more preferably 30 or more. The upper limit of the value of kd (condition for low calcium ion concentration)/kd (condition for high calcium ion concentration) is not limited, and may be any value such as 50, 100, 200.

When the antigen is a soluble antigen, it is possible to use the dissociation rate constant (kd) as a value of the antigen-binding activity. When the antigen is a membrane antigen, it is possible to use the apparent dissociation rate constant (kd). The dissociation rate constant (kd) and the apparent dissociation rate constant (kd) can be measured by a known method, for example, BIACORE (GE healthcare) or a flow cytometer.

In one embodiment, a calcium ion concentration-dependent antigen-binding domain or calcium ion concentration-dependent antibody, or a method for producing a library thereof, having a higher antigen-binding activity under a condition of a high calcium ion concentration than under a condition of a low calcium ion concentration, or Although the screening method is not limited, for example, the method described in WO2012/073992 (for example, paragraphs 0200 to 0213) can be exemplified.

As such, for example,

(a) determining the antigen-binding activity of the antigen-binding domain or antibody under conditions of low calcium ion concentration;

(b) determining the antigen-binding activity of the antigen-binding domain or antibody under conditions of high calcium ion concentration; And

(c) Step of selecting an antigen-binding domain or antibody whose antigen-binding activity under conditions of low calcium ion concentration is lower than that under conditions of high calcium ion concentration

It may be a method including a.

Or, for example,

(a) a step of contacting an antigen-binding domain or antibody, or a library thereof with an antigen under conditions of high calcium ion concentration;

(b) incubating the antigen-binding domain or antibody bound to the antigen in step (a) under conditions of low calcium ion concentration; And

(c) the step of isolating the antigen-binding domain or antibody dissociated in step (b)

It may be a method including a.

Or, for example,

(a) a step of contacting an antigen-binding domain or an antibody or a library thereof with an antigen under conditions of low calcium ion concentration;

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

(c) a step of binding the antigen-binding domain or antibody selected in step (b) to the antigen under conditions of high calcium ion concentration; And

(d) the step of isolating the antigen-binding domain or antibody bound to the antigen in step (c)

It may be a method including a.

Or, for example,

(a) a step of contacting an antigen-binding domain, an antibody, or a library thereof on a column on which the antigen is immobilized under conditions of high calcium ion concentration;

(b) a step of eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under conditions of low calcium ion concentration; And

(c) the step of isolating the antigen-binding domain or antibody eluted in step (b)

It may be a method including a.

Or, for example,

(a) passing the antigen-binding domain or antibody or their library through a column on which the antigen is immobilized under conditions of low calcium ion concentration, and recovering the antigen-binding domain or antibody eluted without binding to the column;

(b) a step of binding the antigen-binding domain or antibody recovered in step (a) to an antigen under conditions of high calcium ion concentration; And

(c) the step of isolating the antigen-binding domain or antibody bound to the antigen in step (b)

It may be a method including a.

Or, for example,

(a) a step of contacting an antigen-binding domain or an antibody, or a library thereof with an antigen under conditions of high calcium ion concentration,

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

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

(d) the step of isolating an antigen-binding domain or antibody whose antigen-binding activity in step (c) is weaker than the criteria selected in step (b)

It may be a method including a.

Each step in these various screening methods may be repeated a plurality of times, or may be appropriately combined to obtain an optimal molecule. As the conditions for the low calcium ion concentration and the high calcium ion concentration, the above-described conditions may be appropriately selected, respectively. Thereby, a desired calcium ion concentration-dependent antigen-binding domain or a calcium ion concentration-dependent antibody can be obtained.

In the context of Initiation A, in one embodiment, the antigen-binding domain or antibody as a starting material has an elevated pi, e.g., by altering the charge of at least one amino acid residue that may be exposed on its surface. It may have a modified antigen-binding domain or an antibody. In an alternative embodiment, at least one amino acid that can be exposed to the surface of an antigen-binding domain or antibody that raises pi when an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain is introduced into the sequence. In addition to the alteration of the charge of the residue, alteration may be introduced.

Alternatively, in the context of the present disclosure A, for example, an existing antigen-binding domain or antibody, an existing library (phage library, etc.); Hybridomas obtained by immunizing animals, antibodies produced from B cells derived from immunized animals, or libraries thereof may be used; Alternatively, antigen-binding domains, antibodies, or libraries obtained by introducing natural or non-natural amino acid mutations capable of chelating calcium (described later) into them (e.g., libraries in which the content of amino acids capable of chelating calcium is increased, Alternatively, a library in which an amino acid capable of chelating calcium is introduced into a specific site may be used.

In one embodiment in the context of initiation A, when the ion concentration is a calcium ion concentration, the ion concentration-dependent antigen-binding domain, or an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain with elevated pi, is, The kind is not limited as long as it is an amino acid capable of forming a calcium-binding motif. For example, calcium binding motifs are well known to those skilled in the art (e.g. 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: 9: 349-354 (1990)); Wurzburg et al. (Structure. 14(6): 1049-1058 (2006)).Therefore, any of type C lectins, for example ASGPR, CD23, MBR, or DC-SIGN When the antigen-binding domain has a calcium-binding motif, the antigen-binding activity of the domain may change depending on the condition of the calcium ion concentration In addition to the above, examples of such a calcium-binding motif are included in the antigen-binding domain. The calcium-binding motif described in SEQ ID NO: 7 (corresponding to "Vk5-2") can be mentioned.

In one embodiment in the context of initiation A, when the ion concentration is a calcium ion concentration, the ion concentration-dependent antigen-binding domain, or a metal as an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain with elevated pi Amino acids having chelating activity can be used. As an example of an amino acid having metal chelating activity, any amino acid can be suitably used as long as it forms a calcium-binding motif, but specifically, an amino acid having electron donating is exemplified. Although not limited, Ser(S), Thr(T), Asn(N), Gln(Q), Asp(D), Glu(E), etc. are suitably mentioned as the amino acid.

The position of the amino acid having such metal chelating activity in the antigen-binding domain is not limited to a specific position. In one embodiment, the amino acid may be at any position in the heavy chain variable region and/or the light chain variable region which may form an antigen-binding domain. For example, at least one amino acid residue that changes the binding activity of an antibody to an antigen in a calcium ion concentration-dependent manner is the heavy and/or light chain CDRs (one or more of CDR1, CDR2 and CDR3) and/or FR (FR1). , At least one of FR2, FR3, and FR4). The amino acid residue may be located at one or more of positions 95, 96, 100a, and 101 according to Kabat numbering in CDR3 of the heavy chain, for example, and Kabat numbering in CDR1 of the light chain. It may be located at one or more of 30th, 31st, and 32nd positions according to, and may be located at position 50 according to Kabat numbering in CDR2 of the light chain, and/or Kabat numbering in CDR3 of the light chain. You can also be placed in 92nd place according to. These amino acid residues may be arranged alone or in combination.

It is known that troponin C, calmodulin, parvalbumin, myosin light chain and the like have a plurality of calcium binding sites, and are thought to originate from a common origin in molecular evolution. It is possible to design one or more of the light chain CDR1, CDR2, and CDR3 such that the binding motif is included. For example, for the above purposes, a cadherin domain; EF hand contained in calmodulin; The C2 domain included in protein kinase C; Gla domain contained in blood coagulation protein factor IX; C-type lectins contained in asialoglycoprotein receptors or mannose-coupled receptors; A domain contained in the LDL receptor; Annexin; Thrombospondin type 3 domain; And EGF-like domains can be used as appropriate.

In one embodiment, when the ion concentration is the hydrogen ion concentration (pH), the condition of the concentration of the proton, that is, the atomic nucleus of the hydrogen atom is treated as synonymous with the condition of the hydrogen index (pH). When the active amount of hydrogen ions in the aqueous solution ^{is expressed as aH +} , the pH is defined as ^{-log10aH +.} When the ionic strength in the aqueous solution is lower (for example, less than ^{10 -3 ), aH +} is approximately equal to the hydrogen ionic strength. For example, since the 25 ℃, 1 atm ion product of ^{Kw = aH + * aOH = 10} -14 in the water, the pure aH ⁺ = aOH = 10 ^{- 7.} In this case, pH=7 is neutral, an aqueous solution with a pH less than 7 is acidic, and an aqueous solution with a pH greater than 7 is alkaline. Therefore, the hydrogen ion concentration condition refers to the difference in the biological behavior of the pH-dependent antibody in the high hydrogen ion concentration (pH acidic region) and the low hydrogen ion concentration (pH neutral region) as a condition of hydrogen ion concentration or a condition of pH. It may be one condition. For example, in the context of Initiation A, "the antigen-binding activity under conditions of high hydrogen ion concentration (pH acidic region) is lower than the antigen-binding activity under conditions of low hydrogen ion concentration (pH neutral region)" Is, 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 an elevated pI, or an ion concentration-dependent antibody with an elevated pI, is pH 6.7 to pH 10.0, preferably Is pH 6.7 to pH 9.5, more preferably pH 7.0 to pH 9.0, more preferably pH 7.0 to pH 8.0 than at a pH selected from pH 4.0 to pH 6.5, preferably pH 4.5 to pH 6.5, Preferably it may mean that it is weak in a pH selected from pH 5.0 to pH 6.5, more preferably pH 5.5 to pH 6.5. Preferably, the expression may mean that the antigen-binding activity at pH in the early endosome of in vivo is weaker than the antigen-binding activity at pH in plasma of in vivo. For example, it may mean that the antibody's antigen-binding activity at pH 5.8 is weaker than the antigen-binding activity at pH 7.4, for example.

Whether the antigen-binding domain to the antigen or the binding activity of the antibody containing the domain is changed depending on the condition of the hydrogen ion concentration, for example, in the context of initiation A, a word described in this specification or described in WO2009/125825 It can be easily determined using a known method such as the Say method. For example, an antigen-binding domain for a target antigen or an antigen-binding activity of an antibody containing the domain may be measured and compared under a low hydrogen ion concentration and a high hydrogen ion concentration. At this time, it is preferable to make the conditions other than the hydrogen ion concentration the same. In addition, when measuring the antigen-binding activity, conditions other than the hydrogen ion concentration can be appropriately selected by a person skilled in the art, and for example, HEPES buffer may be measured under conditions of 37°C, or BIACORE (GE Healthcare) You may measure using etc.

In the range describing the disclosure A in this specification, unless otherwise specified from the context, "neutral pH" ("low hydrogen ion concentration", "high pH", "neutral pH condition" or "neutral pH" Is not particularly limited to a specific value, but may be preferably selected from pH 6.7 to pH 10.0, pH 6.7 to pH 9.5, pH 7.0 to pH 9.0, or pH 7.0 to pH 8.0. Preferably, the neutral pH range may be a pH of 7.4, which is close to the pH of the plasma (blood) of in vivo, but for convenience of measurement, for example, a pH of 7.0 may be used.

In the range describing the disclosure A in this specification, unless otherwise specified from the context, "pH acidic range" ("high hydrogen ion concentration", "low pH", "acidic pH condition" or "acidic pH") Is not particularly limited to a specific value, but may be preferably selected from pH 4.0 to pH 6.5, pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. . Preferably, the acidic pH range may be a pH of 5.8 close to the hydrogen ion concentration in the early endosome of in vivo, but for convenience of measurement, for example, pH 6.0 may be used.

In one embodiment in the context of initiation A, when the ion concentration is a hydrogen ion concentration, the ion concentration-dependent antigen-binding domain, the ion concentration-dependent antibody, the ion concentration-dependent antigen-binding domain with elevated pi, or the pi is elevated. It is preferable that the antigen-binding activity of the ionic concentration-dependent antibody is higher under neutral pH conditions than under acidic pH conditions. In this case, the ratio of the antigen-binding activity under neutral pH conditions and the antigen-binding activity under acidic pH conditions is not limited, but preferably, the dissociation constant ( The ratio of KD) and KD in a neutral pH condition, that is, the value of KD (pH acidic region)/KD (pH neutral region) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more may be sufficient. The upper limit of the value of KD (pH acidic range)/KD (pH neutral range) is not limited, and may be any value such as 400, 1000, and 10000.

In an alternative embodiment, as another index indicating the ratio of the binding activity, for example, a dissociation rate constant (kd) may be used. When using the dissociation rate constant (kd) instead of the dissociation constant (KD) as an index indicating the ratio of the binding activity, the dissociation rate constant (kd) and the low hydrogen ion concentration are The ratio of the dissociation rate constant (kd) under conditions, that is, the value of kd (pH acidic region)/kd (pH neutral region) may be 2 or more, 5 or more, 10 or more, or 30 or more. The upper limit of the value of kd (pH acidic range)/kd (pH neutral range) is not limited, and may be any value such as 50, 100, 200, or the like.

The value of the antigen-binding activity can be expressed as a dissociation rate constant (kd) when the antigen is a soluble antigen, while such a value is an apparent dissociation rate constant when the antigen is a membrane antigen. It is possible to express by (apparent kd). The dissociation rate constant (kd) and the apparent dissociation rate constant (apparent kd) can be measured by using a known method, for example, BIACORE (GE healthcare) or a flow cytometer.

In one embodiment, a pH-dependent antigen-binding domain or pH-dependent antibody having a higher antigen-binding activity under a neutral pH condition than under an acidic pH condition, or a method for preparing or screening a library thereof, is not limited, but examples For example, the method described in WO2009/125825 (for example, paragraphs 0158 to 0190) may be included.

This method, for example,

(a) determining the antigen-binding activity of the antigen-binding domain or antibody under acidic pH conditions;

(b) determining the antigen-binding activity of the antigen-binding domain or antibody under neutral pH conditions; And

(c) Step of selecting an antigen-binding domain or antibody whose antigen-binding activity under acidic pH conditions is lower than that under neutral pH conditions.

It may be a method including a.

Or, for example,

(a) a step of contacting an antigen-binding domain or antibody, or a library thereof with an antigen under neutral pH conditions;

(b) incubating the antigen-binding domain or antibody bound to the antigen in step (a) under acidic pH conditions; And

(c) the step of isolating the antigen-binding domain or antibody dissociated in step (b)

It may be a method including a.

Or, for example,

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

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

(c) a step of binding the antigen-binding domain or antibody selected in step (b) to an antigen under neutral pH conditions; And

(d) the step of isolating the antigen-binding domain or antibody bound to the antigen in step (c)

It may be a method including a.

Or, for example,

(a) a step of contacting an antigen-binding domain or antibody, or a library thereof, in a neutral pH condition to a column on which an antigen is immobilized,

(b) the step of eluting the antigen-binding domain or antibody bound to the column in step (a) from the column under acidic pH conditions, and

(c) the step of isolating the antigen-binding domain or antibody eluted in step (b)

It may be a method including a.

Or, for example,

(a) passing an antigen-binding domain or antibody, or a library thereof under acidic pH conditions through a column to which the antigen is immobilized, and recovering the antigen-binding domain or antibody eluted without binding to the column;

(b) a step of binding the antigen-binding domain or antibody recovered in step (a) to an antigen under neutral pH conditions; And

(c) the step of isolating the antigen-binding domain or antibody bound to the antigen in step (b)

It may be a method including a.

Or, for example,

(a) a step of contacting an antigen-binding domain or antibody, or a library thereof with an antigen under neutral pH conditions;

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

(c) incubating the antigen-binding domain or antibody obtained in step (b) under acidic pH conditions; And

(d) the step of isolating an antigen-binding domain or antibody whose antigen-binding activity in step (c) is weaker than the criteria selected in step (b)

It may be a method including a.

Each step in these various screening methods may be repeated a plurality of times or may be combined. As the acidic pH condition and the neutral pH condition, the above-described conditions may be appropriately selected. Thereby, a desired pH-dependent antigen-binding domain or a pH-dependent antibody can be obtained.

In the context of Initiation A, in one embodiment, the antigen-binding domain or antibody as a starting material has an elevated pi, e.g., by altering the charge of at least one amino acid residue that may be exposed on its surface. It may have a modified antigen-binding domain or an antibody. In an alternative embodiment, at least one amino acid that can be exposed to the surface of an antigen-binding domain or antibody that raises pi when an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain is introduced into the sequence. In addition to the alteration of the charge of the residue, alteration may be introduced.

Alternatively, in the context of the present disclosure A, for example, from an existing antigen-binding domain or antibody, an existing library (phage library, etc.), a hybridoma obtained by immunizing an animal, or B cells derived from an immunized animal. The produced antibody or its library may be used. Alternatively, for example, antigen-binding domains, antibodies, or libraries obtained by introducing mutations (described later) of natural or non-natural amino acids having a side chain pKa of 4.0 to 8.0 (e.g., a side chain pKa of 4.0 to 8.0). A library in which the content of mutations in natural or non-natural amino acids of 8.0 is increased, or a library in which mutations of natural or non-natural amino acids having a side chain pKa of 4.0 to 8.0 are introduced into a specific site) may be used. Such a preferred antigen-binding domain is, for example, as described in WO2009/125825, wherein at least one amino acid residue involves substitution and/or insertion with an amino acid having a side chain pKa of 4.0 to 8.0. It may have an amino acid sequence in the domain.

In one embodiment in the context of the present disclosure A, the site where the mutation of the amino acid whose side chain has a pKa of 4.0 to 8.0 is introduced is not limited, and the mutation is more acidic than the neutral pH range compared to before substitution or insertion. In the reverse, the value of the antigen-binding activity weakens (KD (pH acidic range)/KD (pH neutral range)), or the value of kd (pH acidic range)/kd (pH neutral range) increases. ) As long as, it may be introduced in any part. When the antibody has a variable region or CDR, the site may be a site within the variable region or CDR. The number of amino acids to be substituted or inserted can be appropriately determined by a person skilled in the art and may be one or more. Further, in addition to the above substitutions or insertions, deletions, additions, insertions and/or substitutions, or alterations of other amino acids may be performed. The substitution or insertion of the side chain with an amino acid having a pKa of 4.0 to 8.0 may be performed randomly by a scanning method such as histidine scanning method using histidine instead of alanine in an alanine scanning method known to those skilled in the art, and/or this The value of KD (pH acidic region)/KD (pH neutral region) or kd (pH acidic region)/kd compared to before mutation among antigen-binding domains or antibodies obtained by random substitution or insertion mutation by amino acids, or their libraries. An antibody with an increased value of (neutral pH) may be selected.

In addition, antigen-binding domains or antibodies that are before or after these mutations, or in which the binding activity in the neutral pH region to the antigen is not significantly reduced, is not substantially reduced, is substantially equivalent, or is increased. In other words, when an antigen-binding domain or antibody is preferred, wherein the activity is maintained at least 10% or more, preferably 50% or more, more preferably 80% or more, even more preferably 90% or more, or higher. have. When the antigen-binding domain or antibody binding activity is reduced by substitution or insertion of an amino acid having a pKa of 4.0 to 8.0, for example, substitution or deletion of one or more amino acids at sites other than the substitution or insertion site, The binding activity may be restored or increased by addition or insertion.

In an alternative embodiment, the amino acid whose side chain has a pKa of 4.0 to 8.0 may be at any position in the heavy chain variable region and/or the light chain variable region which may form an antigen-binding domain. For example, at least one amino acid residue having a pKa of 4.0 to 8.0 of the side chain is a heavy chain and/or a light chain CDR (one or more of CDR1, CDR2 and CDR3) and/or FR (of FR1, FR2, FR3 and FR4). One or more). Such amino acid residues are not limited, but amino acid residues at one or more of positions 24, 27, 28, 31, 32, and 34 according to Kabat numbering in CDR1 of the light chain variable region; Amino acid residues at one or more of positions 50, 51, 52, 53, 54, 55, and 56 according to Kabat numbering in CDR2 of the light chain variable region; And/or amino acid residues at one or more of positions 89, 90, 91, 92, 93, 94, and 95a according to Kabat numbering in the CDR3 of the light chain variable region. These amino acid residues may be included alone or in combination as long as the binding activity of the antibody to the antigen is changed depending on the conditions of the hydrogen ion concentration.

In one embodiment within the scope of the present disclosure A, any amino acid residue can be suitably used as the amino acid residue that changes the antigen-binding domain or antibody-binding activity depending on the hydrogen ion concentration condition. Specific examples of such amino acid residues include amino acids having a side chain pKa of 4.0 to 8.0. As amino acids having such electron donating, in addition to natural amino acids such as His (H) and Glu (E), histidine analog (US2009/0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr ( pKa 7.21), and non-natural amino acids such as 3,5-I2-Tyr (pKa 7.38) (Heyl et al., Bioorg. Med. Chem. 11 (17): 3761-3768 (2003)) are exemplified. Further, suitable examples of the amino acid residue include amino acids having a side chain pKa of 6.0 to 7.0, particularly His(H).

In the range where the disclosure A in this specification is described, the isoelectric point (pI) refers to either the theoretical isoelectric point or the experimentally measured isoelectric point, unless explicitly stated and unless it is contrary to the context. It is understood that it may be, and is also referred to as "pI".

For example, the pI value can be experimentally measured by isoelectric point electrophoresis. On the other hand, the theoretical pI value can be calculated using gene and amino acid sequence analysis software (Genetyx, etc.).

In one embodiment, the pI of the antibody to which the pI is raised or the antibody of initiation A is an antibody (natural type antibody (e.g., a natural type Ig antibody, preferably a natural type IgG antibody) or a reference antibody (e.g., For example, whether or not the antibody is elevated compared to the antibody before modification, or the antibody before libraryization or in the process of libraryization)), in addition to or instead of the above method, for example, mouse, rat , Combination of antibody pharmacokinetic tests using plasma of rabbits, dogs, monkeys, or humans with methods such as BIACORE, cell proliferation assay, ELISA, enzyme immunoassay (EIA), radioimmunoassay (RIA), or fluorescence immunization It is possible to determine by implementing it.

In the range described in the disclosure A in this specification, "amino acid residues that can be exposed on the surface" can refer to amino acid residues usually located on the surface of the polypeptide constituting the antibody. The "amino acid residue located on the surface of a polypeptide" refers to an amino acid residue whose side chain can contact a solvent molecule (usually a water molecule). However, it is not necessary that all of the side chains contact the solvent molecule, and when even a part of the side chain contacts the solvent molecule, the amino acid residue may be defined as "an amino acid located on the surface." In addition, amino acid residues located on the surface of the polypeptide may also contain amino acid residues that are located close to the surface of the antibody, so that even if the side chain is part of it, other amino acid residues that are in contact with the solvent molecule can be charged with each other. For example, by homology modeling using commercially available software, a person skilled in the art can create a homology model of a polypeptide or an antibody. Alternatively, a method such as X-ray crystal structure analysis may be used. For example, amino acid residues that may be exposed to the surface can be determined using coordinates from a three-dimensional model of the antibody, using a computer program such as the InsightII program (Accelrys). The area exposed to the surface is an algorithm known in the art (for example, Lee and Richards (J. Mol. Biol. 55: 379-400 (1971)); Connolly (J. Appl. Cryst. 16: 548). -558 (1983)) The determination of the site that can be exposed on the surface can be done using software suitable for protein modeling and three-dimensional structure information obtained from the antibody. Examples of available software include SYBYL Biopolymer Module Software (Tripos Associates) If the algorithm requires input of size parameters by the user, the "size" of the probe used in the calculation is, for example, about a radius. It may be set to 1.4 Å or less In addition, the method of determining the area and area exposed on the surface using software for personal computers is Pacios (Pacios, Comput. Chem. 18 (4): 377-386 (1994) and J. Mol. Model. 1: 46-53 (1995)) Based on the above information, an appropriate amino acid residue located on the surface of the polypeptide constituting the antibody can be selected.

As a method of raising the pI of a protein, for example, reducing the number of amino acids having a negative charge on the side chain (e.g., aspartic acid and glutamic acid) under conditions of neutral pH, and/or reducing the number of amino acids having a positive charge on the side chain (e.g. For example, increasing the number of arginine, lysine, and histidine) is mentioned. Although the theory is well known to those skilled in the art, an amino acid residue having a negative charge on the side chain has a negative charge expressed as -1 under a pH condition sufficiently higher than the pKa of the side chain. For example, the theoretical pKa of the aspartic acid side chain is 3.9, and under conditions of neutral pH (for example, in a solution at pH 7.0), the side chain has a negative charge expressed as -1. Conversely, an amino acid residue having a positive charge on the side chain has a positive charge expressed as +1 under a pH condition that is sufficiently lower than the pKa of the side chain. For example, the theoretical pKa of the arginine side chain is 12.5, and under conditions of neutral pH (for example, in a solution at pH 7.0), the side chain has a positive charge expressed as +1. Under the conditions of neutral pH (for example, in a solution of pH 7.0), the amino acid residues that do not have a charge on the side chain are 15 kinds of natural amino acids, namely, alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, It is known to include asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. It is naturally understood that non-natural amino acids may be used as amino acids that change pI.

From the above, as a method of raising the pI of a protein under conditions of neutral pH (for example, in a solution of pH 7.0), for example, aspartic acid (residue) or glutamic acid ( Residue) (which has a negative charge of -1 on the side chain) is substituted with an amino acid (residue) that does not have a charge on the side chain, so that a change in charge of +1 can be imparted to the protein. Further, for example, by substituting an amino acid (residue) that does not have a charge on the side chain with arginine or lysine (which has a positive charge of +1 on the side chain), a change in charge of +1 can be imparted to the protein. In addition, by substituting aspartic acid or glutamic acid (having a negative charge of -1 on the side chain) with arginine or lysine (having a positive charge of +1 on the side chain), a charge change of +2 can be given at a time. Alternatively, an amino acid having no charge on the side chain and/or an amino acid having a positive charge on the side chain is added or inserted into the amino acid sequence of a protein, or an amino acid that is present in the amino acid sequence of a protein and does not have a charge on the side chain and/or By deleting an amino acid having a negative charge on the side chain, it is possible to raise the pI of the protein. For example, amino acid residues located at the N-terminus or C-terminus of a protein have a charge derived from the main chain (NH ₃ ^{+ of the amino group at the N-terminus, COO-} of the carbonyl group at the C-terminus) in addition to the charge derived from the side chain. It makes sense. Therefore, the pI of the protein can be increased by any addition, deletion, substitution or insertion of the functional groups derived from these main chains.

The effect of changing the net charge or pI of a protein obtained by changing one or more amino acids (residues) in the amino acid sequence by paying attention to the presence or extent of the charge possessed by the amino acid (residue) is an antibody It will be understood by those skilled in the art that it does not depend only on (or substantially) the type of the amino acid sequence itself or the type of target antigen, but depends on the type or number of amino acid residues added, deleted, substituted or inserted.

Antibodies that have been modified to have a pI elevated by alteration of at least one amino acid residue that can be exposed on the surface of the antibody ("antibody that raises pi" or "antibody that raises pi"), for example, WO2007/ As described or suggested in 114319, WO2009/041643, WO2014/145159 or WO2012/016227, it can be absorbed into cells more rapidly, or it can promote the loss of antigen from plasma.

Of the many isotypes of antibodies, for example, IgG antibodies have a sufficiently large molecular weight, so their main metabolic pathway is not due to renal excretion. It is known that IgG antibodies having the Fc region as part of the molecule are recycled by the salvage pathway via FcRn, and thus have a long in vivo half-life. It is thought that IgG antibodies are mainly metabolized by metabolic pathways in endothelial cells (He et al., J. Immunol. 160(2): 1029-1035(1998)). In other words, it is considered that the IgG antibody nonspecifically absorbed into the endothelial cells is recycled by binding to FcRn, while the IgG antibody that could not be bound is metabolized. When the Fc region is altered so that the binding activity to FcRn is reduced, the half-life in plasma of the IgG antibody may be shortened. On the other hand, it has become clear that the plasma half-life of the pi-raising antibody has a high correlation and is dependent on pi, as described, for example, in WO2007/114319 and WO2009/041643. That is, from the fact that the plasma half-life of the antibody which raised the pI described in the above document was shortened without altering the amino acid sequence constituting the Fc that may bring about the acquisition of immunogenicity, scFv, Fab, or Fc fusion protein and Likewise, it has been suggested that the technique for increasing the pi can be widely applied even to any type of antibody molecule, which is a major metabolic pathway in which kidney excretion is a major metabolic pathway.

The pH concentration in the biological fluid (eg, plasma) is in the neutral pH range. Although not bound by a specific theory, the pI-raising antibody in the biological fluid has a higher net positive charge due to the increase in pI, and as a result, as a result, compared to the antibody in which the pI is not rising, it physically reaches the surface of the endothelial cell carrying a net negative charge. Can be attracted more strongly by chemical coulomb interactions; As a result of the antibody being absorbed into cells through non-specific binding, it is considered that the half-life of the antibody in the plasma is shortened, or the loss of the antigen from the plasma is increased. Moreover, by raising the pI of the antibody, the absorption of the antibody (or the complex of the antigen and the antibody) into the cells and/or the intracellular permeability is increased, which in turn lowers the concentration of the antibody in the plasma, resulting in the bioavailability of the antibody. It is thought to lower the blood pressure and/or shorten the plasma half-life of the antibody; It is thought that these phenomena can occur widely in vivo regardless of cell type, tissue type, organ type, and the like. In addition, when an antibody forms a complex with an antigen and is absorbed into a cell, the pI of the antigen in addition to the pI of the antibody may affect the increase or decrease of absorption into the cell.

In one embodiment, as a method for producing or screening an antibody with elevated pi, for example, WO2007/114319 (for example, paragraphs 0060 to 0087), WO2009/041643 (for example, paragraphs 0115-), and WO2014/ 145159, and the method described in WO2012/016227 can be illustrated. As the method, for example,

(a) a step of modifying a nucleic acid encoding an antibody containing the amino acid residue so that the pI of the antibody is increased and the charge of at least one amino acid residue that may be exposed on the surface of the antibody is changed;

(b) culturing the host cell so that the nucleic acid is expressed; And

(c) the step of recovering the antibody from the host cell culture

It may be a method including a.

Or, for example,

(a') a step of modifying a nucleic acid encoding an antibody containing the amino acid residue so that the charge of at least one amino acid residue that may be exposed on the surface of the antibody is altered;

(b') culturing the host cell so that the nucleic acid is expressed;

(c') recovering the antibody from the host cell culture, and

(d') Step of selecting (arbitrarily, by confirming or measuring) an antibody with an elevated pI compared to the antibody before modification

It may be a method including a. Here, the antibody as a starting material, the antibody before modification, or the reference antibody may be, for example, an ion concentration dependent antibody. Alternatively, an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain may be included in the sequence upon alteration of the amino acid residue.

Alternatively, the method may simply include a step of culturing the host cell obtained in step (b) or (b') and a step of recovering the antibody from the cell culture.

In an alternative embodiment, for example,

A method for producing a multispecific antibody comprising a first polypeptide and a second polypeptide, and, optionally, a third polypeptide and a fourth polypeptide,

(A) a step of modifying a nucleic acid encoding a first polypeptide and/or a second polypeptide, and, optionally, a third polypeptide and/or a fourth polypeptide, wherein any one or a plurality of the polypeptides increases the pi of the antibody. So that the charge of at least one amino acid residue that may be exposed on the surface of the polypeptide is altered, comprising the amino acid residue;

(B) a step of culturing the host cell so that the nucleic acid is expressed; And

(C) Step of recovering multispecific antibodies from host cell culture

It may be a method including a.

Or, the method, for example,

(A') a step of modifying a nucleic acid encoding a first polypeptide and/or a second polypeptide, and optionally a third polypeptide and/or a fourth polypeptide, wherein any one or a plurality of the polypeptides is exposed to the surface of the polypeptide. Comprising the amino acid residue such that the charge of at least one amino acid residue that may be changed is altered;

(B') a step of culturing the host cell so that the nucleic acid is expressed;

(C') recovering the multispecific antibody from the host cell culture; And

(D') Step of selecting (arbitrarily, confirming) an antibody with an elevated pI compared to the antibody before modification

You may include.

Here, for example, an antibody as a starting material, an antibody before modification, or a reference antibody may be an ion concentration dependent antibody. Alternatively, an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain may be contained in the sequence upon alteration of the amino acid residue.

Alternatively, the method may simply include a step of culturing the host cell obtained in step (B) or (B') and a step of recovering the antibody from the cell culture. In this case, the polypeptide to which the nucleic acid is modified is a homomultimer of the first polypeptide, a homomultimer of the second polypeptide, or a heteromultimer of the first and second polypeptide (and, optionally, a homomultimer of a third polypeptide). , A homomultimer of the fourth polypeptide, or a heteromultimer of the third and fourth polypeptides) in some cases.

In an alternative embodiment, for example, as a method for producing a humanized antibody or human antibody having a short half-life in plasma, a CDR selected from the group consisting of a human-derived CDR, a non-human animal-derived CDR, and a synthetic CDR. ; Human-derived FR; And in the antibody comprising a human constant region, (I) at least one that can be exposed on the surface of at least one region selected from the group consisting of the CDR, the FR, and the constant region so that the pI of the antibody is raised. A method including a step of modifying an amino acid residue into an amino acid residue having a charge different from that of the amino acid residue present at the corresponding position before the modification may be employed.

Alternatively, for example, a CDR selected from the group consisting of a human-derived CDR, a non-human animal-derived CDR, and a synthetic CDR; Human-derived FR; And in an antibody comprising a human normal region,

(I') 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, the FR, and the constant region is different from the amino acid residue present at the corresponding position before modification. A step of changing into an amino acid residue having a charge; And

(II') Step of selecting (arbitrarily, confirming) an antibody with an elevated pI compared to the antibody before modification

It may be a method including a.

Here, for example, an antibody as a starting material, an antibody before modification, or a reference antibody may be an ion concentration dependent antibody. Alternatively, an amino acid that changes the binding activity of the ion concentration-dependent antigen-binding domain may be contained in the sequence upon alteration of the amino acid residue.

Or, for example, an existing antigen-binding domain or antibody, an existing library (phage library, etc.); Hybridomas obtained by immunizing animals or antibodies prepared from B cells derived from immunized animals, or libraries thereof; Or in the above antigen-binding domain, antibody, or library thereof, for example, based on any one of the above-described embodiments, prepared by modifying at least one amino acid residue that may be exposed to the surface, the pI is elevated. An antigen-binding domain or antibody, or a library thereof may be used.

In one embodiment of the antibody of initiation A, the antibody before or before modification (natural antibody (e.g., native Ig antibody, preferably native IgG antibody), or reference antibody or parent antibody (e.g., modified The value of pI is preferably, for example, 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 may be increased, and in order to significantly shorten the half-life in plasma of the antibody, the value of pI is, for example, 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 may rise. Those skilled in the art routinely determine the optimal pI value of the antibody of initiation A, as appropriate, taking into account the balance of pharmacological effect and toxicity, and, e.g., the number of antigen-binding domains of the antibody or the pI of the antigen, depending on the purpose of the sacrificial application. I can. Although not bound by a specific theory, in one embodiment, the antibody of initiation A has an ion concentration-dependent antigen-binding domain, thereby allowing a single antibody molecule to react against a plurality of antigens while traveling between plasma and endosomes of cells. In addition to the property of repeating the binding, the net positive charge of the antibody increases as a result of the increase in pi, which is advantageous because the antibody is absorbed more rapidly in the cell. These properties may shorten the plasma half-life of the antibody, increase the binding activity of the antibody to the extracellular matrix, or promote the loss of antigen from the plasma. The optimum pI value may be determined to utilize such characteristics.

In one embodiment in the context of initiation A, an ion concentration-dependent antibody with an elevated pi of initiation A is an antibody (natural antibody (e.g., a natural antibody (e.g. For example, a native Ig antibody, preferably a native IgG antibody), or a reference antibody or a parent antibody (e.g., an antibody before modification, or an antibody before or in the process of libraryization, etc.), these are ionic (May be a concentration dependent antibody), preferably, the loss of antigen from plasma (when the antibody is administered in vivo), for example at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 Times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25 times, 4.5 times, 4.75 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times or 10 times or more may be promoted, or the binding activity to the extracellular matrix is preferably, for example, at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25 times, 4.5 times, 4.75 times, 5 times or more may be increased.

In one embodiment in the context of initiation A, the ion concentration-dependent antibody with an elevated pI of initiation A is an antibody before introducing the ion concentration-dependent antigen-binding domain (natural antibody (e.g., a native Ig antibody, Preferably, a native IgG antibody), or a reference antibody or a parent antibody (e.g., an antibody prior to modification, or an antibody before or in the course of libraryization, etc.), these may be antibodies with an elevated pI. Yes), preferably, (when the antibody is administered in vivo), the loss of antigen from plasma is, for example, at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times , 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25 times, 4.5 times, 4.75 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 It may promote at least twice, 8.5 times, 9 times, 9.5 times, or 10 times or more, or the binding activity to the extracellular matrix is preferably, for example, at least 1.1 times, 1.25 times, 1.5 times, 1.75 times, 2 It may increase by 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4.25 times, 4.5 times, 4.75 times, 5 times or more.

In one embodiment, an antibody before or before modification (a native antibody (e.g., a native Ig antibody, which may be a native IgG antibody), or a reference antibody or parent antibody (e.g., an antibody before modification) , Or, prior to libraryization or in the process of libraryization, etc.), these may be ion concentration-dependent antibodies or antibodies with elevated pi), compared with the binding activity of the antibody of initiation A to the extracellular matrix. The assay method for determining whether or not it is increased is not limited. For example, an assay can be performed using an ELISA system that detects binding of an antibody to an extracellular matrix, where an antibody is added to a plate on which the extracellular matrix is solidified, and a labeled antibody against the antibody is prepared. Add. Alternatively, as described in Examples 1 to 4 of this specification, WO2012/093704, etc., an electrochemiluminescence method (ECL) capable of detecting the extracellular matrix binding ability with a higher sensitivity may be used. In this method, for example, a mixture of an antibody and a ruthenium antibody is added to a plate on which the extracellular matrix is solidified, and the binding of the antibody and the extracellular matrix is measured using an ECL system, which is measured based on electrochemiluminescence of ruthenium. , Can be carried out. The concentration of the antibody to be added can be arbitrarily set, and the concentration to be added can be increased in order to increase the detection sensitivity of binding to the extracellular matrix. Such an extracellular matrix, as long as it contains glycoproteins such as collagen, proteoglycan, fibronectin, laminin, entaxin, fibrin, and percan, may be derived from animals or plants, and may be derived from animals in some cases. For example, an extracellular matrix derived from an animal such as human, mouse, rat, monkey, rabbit, or dog can be used. For example, a human-derived natural extracellular matrix may be used as an indicator of the plasma pharmacokinetics of antibodies in humans. As a condition for evaluating the binding of the antibody to the extracellular matrix, a neutral pH range near pH 7.4 under physiological conditions is preferable, but it does not have to be a neutral region, and the binding is evaluated in an acidic pH range (for example, around pH 6.0) You can do it. Alternatively, when evaluating the binding of the antibody to the extracellular matrix, the assay may be performed by coexisting with the antigen molecule to which the antibody binds, and evaluating the binding activity of the antigen-antibody complex to the extracellular matrix.

In one embodiment, the antibody of initiation A is an antibody before at least one amino acid residue is altered or altered in order to raise pi (natural type antibody (e.g., a natural type Ig antibody, preferably a natural type IgG antibody)). Alternatively, compared to a reference antibody (for example, an antibody before antibody modification, or an antibody before or during library construction)), the antigen-binding activity can be maintained (substantially). In this case, "maintaining (substantially) the binding activity to the antigen" means at least 50% or more, preferably 60% or more, more preferably 70%, compared to the binding activity of the antibody before or before modification. Or it may mean that it has an activity of 75% or more, even more preferably 80%, 85%, 90%, or 95% or more. Alternatively, the antibody of Initiation A only needs to maintain the binding activity enough to maintain its function when binding to the antigen, and therefore, for example, the affinity measured at 37°C under physiological conditions is 100 nM or less, Preferably 50 nM or less, more preferably 10 nM or less, and still more preferably 1 nM or less may be used.

In one embodiment of Initiation A, “modification of at least one amino acid residue that may be exposed on the surface of the antibody” or an expression of its synonym is added to at least one amino acid residue that may be exposed on the surface of the antibody. , Deletion, substitution, and insertion. The modification may preferably include substitution of at least one amino acid residue.

For example, as the substitution of the amino acid residue, in the amino acid sequence of the antibody of interest, substitution of an amino acid residue having a negative charge on the side chain to an amino acid residue having no charge on the side chain, or an amino acid having no charge on the side chain Substitution from a residue to an amino acid residue having a positive charge on the side chain, and substitution from an amino acid residue having a negative charge on the side chain to an amino acid residue having a positive charge on the side chain, these may be used alone or in appropriate combinations. You can do it. For example, as the insertion or addition of the amino acid residue, in the amino acid sequence of the antibody of interest, the insertion or addition of an amino acid having no charge on the side chain, and/or the insertion or addition of an amino acid having a positive charge on the side chain. These can be mentioned, and these can be implemented individually or in combination suitably. For example, as the deletion of the amino acid residue, in the amino acid sequence of the antibody of interest, deletion of an amino acid residue having no charge on the side chain, and/or deletion of an amino acid residue having a negative charge on the side chain. There exist, and these can be implemented individually or in combination suitably.

Those skilled in the art can appropriately combine one or more of these additions, deletions, substitutions, and insertions in the amino acid sequence of the antibody of interest. Since only the net pI of the antibody of initiation A must be raised, modifications may be performed that lead to a decrease in local charge of the amino acid residue. For example, if desired, modifications may be performed to lower (slightly) the pI of an antibody whose pI is rising (excessively). For other purposes (for example, to increase the stability of the antibody or decrease the immunogenicity), the local charge of the amino acid residue may be lowered as a result of alteration of at least one amino acid residue at the same time or at different times. As such an antibody, an antibody of a library prepared for a specific purpose may be used.

In one embodiment, among amino acids (residues) used for alteration of at least one amino acid residue that may be exposed on the surface of an antibody, the natural amino acid is as follows: An amino acid having a negative charge on the side chain is Glu(E) Or may be Asp(D); Amino acids that do not have a charge on the side chain are Ala(A), Asn(N), Cys(C), Gln(Q), Gly(G), His(H), Ile(I), Leu(L), Met (M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), or Val(V); An amino acid having a positive charge on the side chain may be His(H), Lys(K), or Arg(R).

As detailed in Examples 1 to 4 of this specification, in a solution at a neutral pH (for example, pH 7.0), lysine and arginine when present as residues in the antibody have a positive charge, while almost 100% , It is considered that only about 9% of histidine when present as a residue in the antibody has a positive charge, and most of the rest have no charge. Therefore, it is preferable to select Lys(K) or Arg(R) as an amino acid having a positive charge on the side chain.

In one embodiment, it is preferable that the antibody of initiation A has 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, and/or CDR (e.g., one or more of CDR1, CDR2 and CDR3) and/or FR (e.g. For example, there are cases where it is desirable to have 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 the sequence and type thereof include, for example, an IgG type constant region (preferably, human IgG1, human IgG2, human IgG3. Alternatively, a human IgG4 type constant region, a human κ chain constant region, and a human λ chain constant region) may be used. You may use the modified body in which these normal regions were altered.

In one embodiment, the modification of at least one amino acid residue that can be exposed on the surface of the antibody may be any of a single amino acid modification or a combination of modifications of a plurality of amino acids. Preferably, a method in which a plurality of amino acid substitutions are combined and introduced into a site where an amino acid can be exposed on the antibody surface is exemplified. Moreover, although not limited, it is preferable that such a plurality of amino acid substitutions are introduced at positions that are three-dimensionally close to each other. For example, amino acids that may be exposed on the surface of the antibody molecule (but are not limited, preferably, amino acids having a negative charge on the side chain (e.g., Glu(E) or Asp(D))) are positively charged on the side chain. In the case of substitution with an amino acid (e.g., Lys(K) or Arg(R)); or in the case of using an amino acid with an existing positive charge (e.g., Lys(K), or Arg(R)) , One or more amino acids that are three-dimensionally close to the amino acid (in some cases, an amino acid embedded in the antibody molecule may be included) is substituted with an amino acid having a positive charge, and as a result, localized to a position that is three-dimensionally close to the amino acid. Here, the definition of "a three-dimensionally close position" is not particularly limited, but, for example, within 20 Å, preferably within 15 Å, more preferably within 10 Å, 1 It may mean a state in which several or a plurality of amino acid substitutions have been introduced, whether the desired amino acid substitution site is exposed on the surface of the antibody molecule, or which amino acid substitution site is close to another amino acid substitution site or the existing amino acid. Whether or not it can be determined by a known method such as X-ray crystal structure analysis.

As a method of having a plurality of positive charges at sites that are three-dimensionally adjacent to each other, in addition to the above method, a method using an amino acid originally having a positive charge in a native IgG constant region may be used. Examples of such amino acids include arginine at positions 255, 292, 301, 344, 355, and 416 according to EU numbering; And 121, 133, 147, 205, 210, 213, 214, 218, 222, 246, 248, 274, 288, 290, 317, 320, 322, 326, 334, 338, 340, 360, 370 according to EU numbering. , 392, 409, 414, and 439 lysine. It is possible to have a plurality of positive charges in the three-dimensionally adjacent sites by performing substitutions using amino acids having a positive charge at positions that are three-dimensionally close to these positively charged amino acids.

In addition, when the antibody of initiation A has a variable region (which may be altered), amino acid residues that are not masked by antigen binding (i.e., which can still be exposed to the surface) may be altered, and/or antigen binding. At the site masked with, amino acid alteration may not be introduced, or amino acid alteration may be performed that does not (substantially) inhibit antigen binding. In addition, in the case of altering the amino acid residues present in the ion concentration-dependent binding domain that can be exposed on the surface of the antibody molecule, it is necessary to change the binding activity of the antibody to the antigen according to the condition of the ion concentration with respect to the antigen-binding domain. The amino acid of the antigen-binding domain may be altered so that the binding activity of possible amino acid residues (e.g., those in the calcium-binding motif, or histidine insertion site and/or histidine substitution site) is not (substantially) attenuated by alteration. Alternatively, the amino acid residue may be modified at a site other than the amino acid residue capable of changing the antigen-binding activity of the antibody depending on the ion concentration conditions. On the other hand, if the amino acid residues present in the ion concentration binding domain that can be exposed on the surface of the antibody molecule have already been modified, the pI of the antibody is not lowered below the acceptable level, and according to the conditions of the ion concentration, the antigen The position and type of amino acid residues capable of changing the binding activity of the antibody against the antibody may be selected. When the pI of the antibody is lower than the allowable level, the pI of the entire antibody may be increased by altering at least one amino acid residue that may be exposed on the surface of the antibody molecule.

Although not limited, Preferably, a FR sequence having a high pi may be selected from a human germline FR sequence or a sequence of a region equivalent thereto, and the amino acid may be modified in some cases.

When the antibody of initiation A has an FcγR-binding domain (which may be a binding domain for any isoform or allotype of FcγR as described later) and/or a constant region (may be altered) having an FcRn-binding domain, desired Accordingly, the modified site of at least one amino acid residue that can be exposed on the surface of the normal region may be an amino acid residue other than the amino acid residue in the FcγR-binding domain and/or the FcRn-binding domain. Alternatively, when the amino acid residue in the FcγR-binding domain and/or the FcRn-binding domain is selected as a modification site, a site that does not (substantially) affect the binding activity or binding affinity to FcγR and/or FcRn, or In some cases, it is desirable to select a biologically or pharmacologically acceptable site, even if it has an effect.

In one embodiment, at least one amino acid residue that can be exposed on the surface of the variable region (which may be altered) is altered to produce an antibody of initiation A whose pI is elevated, at least one amino acid residue alteration site Is, but is not limited to, according to Kabat numbering, (a) 1st, 3rd, 5th, 8th, 10th, 12th, 13th, 15th, 16th, 18th in the FR of the heavy chain variable region Ranked, 19th, 23rd, 25th, 26th, 39th, 41st, 42nd, 43rd, 44th, 46th, 68th, 71st, 72nd, 73rd, 75th, 76th, 77th, 81st, 82nd, 82a, 82b, 83rd, 84th, 85th, 86th, 105th, 108th, 110th, and 112th; (b) 31st, 61st, 62nd, 63rd, 64th, 65th, and 97th position in the CDR of the heavy chain variable region; (c) 1st, 3rd, 7th, 8th, 9th, 11th, 12th, 16th, 17th, 18th, 20th, 22nd, 37th, 38th in the light chain variable region FR Ranked, 39th, 41st, 42nd, 43rd, 45th, 46th, 49th, 57th, 60th, 63rd, 65th, 66th, 68th, 69th, 70th, 74th, 76th, 77th, 79th, 80th, 81st, 85th, 100th, 103rd, 105th, 106th, 107th, and 108th; And (d) in the CDR of the light chain variable region, it may be selected from the group consisting of 24th, 25th, 26th, 27th, 52nd, 53rd, 54th, 55th, and 56th position, and each position after modification. The amino acid in is not limited, such as Lys (K), Arg (R), Gln (Q), Gly (G), Ser (S), or Asn (N). It can be selected from any of amino acids. In some embodiments, of the aforementioned amino acid positions, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 are modified. In some embodiments, of the amino acid positions described above, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 are altered.

In one embodiment, the following positions among the positions to be modified are used to assist in raising the pI of the antibody of initiation A by combining them with other positions that may themselves have a sufficient effect to increase the pI of the antibody. I can. Such a position to assist in raising the pi can be selected from the group consisting of, for example, position 27, 52, 56, 65, and 69 according to Kabat numbering with respect to the light chain variable region. .

Moreover, the modification site of at least one amino acid residue in the CDR and/or FR is not limited, but (a) the 8th, 10th, 12th, 13th, 15th position in the FR of the heavy chain variable region , 16th, 18th, 23rd, 39th, 41st, 43rd, 44th, 77th, 82nd, 82a, 82b, 83rd, 84th, 85th, and 105th; (b) 31st, 61st, 62nd, 63rd, 64th, 65th, and 97th position in the CDR of the heavy chain variable region; (c) 16th, 18th, 37th, 41st, 42nd, 45th, 65th, 69th, 74th, 76th, 77th, 79th, and 107th in the FR of the light chain variable region; And (d) in the CDR of the light chain variable region, it may be selected from the group consisting of 24th, 25th, 26th, 27th, 52nd, 53rd, 54th, 55th, and 56th position. In some embodiments, of the aforementioned amino acid positions, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 are modified. In some embodiments, of the amino acid positions described above, 1 to 20, 1 to 15, 1 to 10, and 1 to 5 are modified.

For example, when the modified site of at least one amino acid residue is selected from the group including the group described above, the kind of amino acid after modification in the heavy chain variable region is, for example,

(a) in 8th place, 8K, 8R, 8Q, 8G, 8S, or 8N; (b) in 13th place, 13K, 13R, 13Q, 13G, 13S, or 13N; (c) in 15th place, 15K, 15R, 15Q, 15G, 15S, or 15N; (d) in 16th place, 16K, 16R, 16Q, 16G, 16S, or 16N; (e) in the 18th place, 18K, 18R, 18Q, 18G, 18S, or 18N; (f) in 39th place, 39K, 39R, 39Q, 39G, 39S, or 39N; (g) in the 41st place, 41K, 41R, 41Q, 41G, 41S, or 41N; (h) at position 43, 43K, 43R, 43Q, 43G, 43S, or 43N; (i) at 44th place, 44K, 44R, 44Q, 44G, 44S, or 44N; (j) at 63rd place, 63K, 63R, 63Q, 63G, 63S, or 63N; (k) at 64th place, 64K, 64R, 64Q, 64G, 64S, or 64N; (l) at 77th place, 77K, 77R, 77Q, 77G, 77S, or 77N; (m) at 82nd place, 82K, 82R, 82Q, 82G, 82S, or 82N; (n) 82aK, 82aR, 82aQ, 82aG, 82aS, or 82aN at position 82a; (o) 82bK, 82bR, 82bQ, 82bG, 82bS, or 82bN on 82b; (p) 83K, 83R, 83Q, 83G, 83S, or 83N at 83rd place; (q) 84K, 84R, 84Q, 84G, 84S, or 84N at 84th place; (r) at 85th place, 85K, 85R, 85Q, 85G, 85S, or 85N; Or (s) in the 105th place, it is 105K, 105R, 105Q, 105G, 105S, or 105N. In some embodiments, any combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the aforementioned amino acid positions is altered. In some embodiments, any combination of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 of the aforementioned amino acid positions is modified.

Non-limiting examples of combinations of modified amino acid positions in the heavy chain variable region are, for example, according to Kabat numbering,

Any two or more selected from the group consisting of 16th, 43rd, 64th, and 105th place; Any two or more selected from the group consisting of 77th, 82a, and 82b; 77th and 85th; 41st and 44th; 82a and 82b; 82nd and 82b places; 82b and 83; Or at positions 63 and 64, and the amino acid at each position after the modification is not limited, but from the viewpoint of the charge of the side chain, any of the aforementioned amino acids, such as Lys(K), Arg(R), It can be selected from Gln(Q), Gly(G), Ser(S), or Asn(N).

Specific combinations are, 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.

Similarly, the kind of amino acid after modification in the light chain variable region is, for example, (a) at position 16, 16K, 16R, 16Q, 16G, 16S, or 16N; (b) in the 18th place, 18K, 18R, 18Q, 18G, 18S, or 18N; (c) at position 24, 24K, 24R, 24Q, 24G, 24S, or 24N; (d) in 25th place, 25K, 25R, 25Q, 25G, 25S, or 25N; (e) in 26th place, 26K, 26R, 26Q, 26G, 26S, or 26N; (f) at 27th place, 27K, 27R, 27Q, 27G, 27S, or 27N; (g) in 37th place, 37K, 37R, 37Q, 37G, 37S, or 37N; (h) at 41st place, 41K, 41R, 41Q, 41G, 41S, or 41N; (i) in 42nd place, 42K, 42R, 42Q, 42G, 42S, or 42N; (j) at 45th place, 45K, 45R, 45Q, 45G, 45S, or 45N; (k) 52K, 52R, 52Q, 52G, 52S, or 52N at 52nd place; (l) in the 53rd position, 53K, 53R, 53Q, 53G, 53S, or 53N; (m) at 54th place, 54K, 54R, 54Q, 54G, 54S, or 54N; (n) in 55th place, 55K, 55R, 55Q, 55G, 55S, or 55N; (o) at 56th place, 56K, 56R, 56Q, 56G, 56S, or 56N; (p) at 65th place, 65K, 65R, 65Q, 65G, 65S, or 65N; (q) 69K, 69R, 69Q, 69G, 69S, or 69N in 69th position; (r) in the 74th place, 74K, 74R, 74Q, 74G, 74S, or 74N; (s) at 76th place, 76K, 76R, 76Q, 76G, 76S, or 76N; (t) at 77th place, 77K, 77R, 77Q, 77G, 77S, or 77N; (u) 79K, 79R, 79Q, 79G, 79S, or 79N at 79th place; And (v) 107K, 107R, 107Q, 107G, 107S, or 107N in the 107th position. In some embodiments, any combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the aforementioned amino acid positions is modified. In some embodiments, any combination of 1 to 20, 1 to 15, 1 to 10, or 1 to 5 of the combinations of amino acid positions described above is modified.

Non-limiting examples of combinations of modified amino acid positions in the light chain variable region include, for example, positions 24 and 27 according to Kabat numbering; 25th and 26th; 41st and 42nd; 42nd and 76th; 52nd and 56th; 65th and 79th; 74th and 77th; 76th and 79th; Any two or more selected from the group consisting of 16th, 24th, and 27th; Any two or more selected from the group consisting of 24th, 27th, and 37th; Any two or more selected from the group consisting of 25th, 26th, and 37th; Any two or more selected from the group consisting of 27th, 76th, and 79th place; Any two or more selected from the group consisting of 41st, 74th, and 77th; Any two or more selected from the group consisting of 41st, 76th, and 79th; Any two or more selected from the group consisting of 24th, 27th, 41st, and 42nd place; Any two or more selected from the group consisting of 24th, 27th, 52nd, and 56th; Any two or more selected from the group consisting of 24th, 27th, 65th, and 69th; Any two or more selected from the group consisting of 24th, 27th, 74th, and 77th; Any two or more selected from the group consisting of 24th, 27th, 76th, and 79th place; Any two or more selected from the group consisting of 25th, 26th, 52nd, and 56th; Any two or more selected from the group consisting of 25th, 26th, 65th, and 69th; Any two or more selected from the group consisting of 25th, 26th, 76th, and 79th; Any two or more selected from the group consisting of 27th, 41st, 74th, and 77th; Any two or more selected from the group consisting of 27th, 41st, 76th, and 79th; Any two or more selected from the group consisting of 52nd, 56th, 74th, and 77th; Any two or more selected from the group consisting of 52nd, 56th, 76th, and 79th; Any two or more selected from the group consisting of the 65th, 69th, 76th, and 79th place; Any two or more selected from the group consisting of 65th, 69th, 74th, and 77th; Any two or more selected from the group consisting of 18th, 24th, 45th, 79th, and 107th; Any two or more selected from the group consisting of 27th, 52nd, 56th, 74th, and 77th; At least two arbitrary places selected from the group consisting of 27th, 52nd, 56th, 76th, and 79th place; Any two or more selected from the group consisting of 27th, 65th, 69th, 74th, and 77th; Any two or more selected from the group consisting of 27th, 65th, 69th, 76th, and 79th place; At least two arbitrary places selected from the group consisting of 41st, 52nd, 56th, 74th, and 77th; At least two arbitrary places selected from the group consisting of 41st, 52nd, 56th, 76th, and 79th; At least two arbitrary places selected from the group consisting of 41st, 65th, 69th, 74th, and 77th; Any two or more selected from the group consisting of 41st, 65th, 69th, 76th, and 79th place; Any two or more selected from the group consisting of 24th, 27th, 41st, 42nd, 65th, and 69th; Any two or more selected from the group consisting of 24th, 27th, 52nd, 56th, 65th, and 69th; Any two or more selected from the group consisting of 24th, 27th, 65th, 69th, 74th, and 77th; Any two or more selected from the group consisting of 24th, 27th, 65th, 69th, 76th, and 79th place; Any two or more selected from the group consisting of 24th, 27th, 41st, 42nd, 74th, and 77th; Any two or more selected from the group consisting of 24th, 27th, 52nd, 56th, 74th, and 77th; Any two or more selected from the group consisting of 24th, 27th, 41st, 42nd, 76th, and 79th; Any two or more selected from the group consisting of 24th, 27th, 52nd, 56th, 76th, and 79th place; Any two or more selected from the group consisting of 24th, 27th, 74th, 76th, 77th, and 79th place; Any two or more selected from the group consisting of 52nd, 56th, 65th, 69th, 74th, and 77th; Or any two or more selected from the group consisting of the 52nd, 56th, 65th, 69th, 76th, and 79th position, and the amino acid at each position after modification is not limited, but the viewpoint of the charge of the side chain In, it can be selected from any of the aforementioned amino acids, for example Lys(K), Arg(R), Gln(Q), Gly(G), Ser(S), or Asn(N).

Specific combinations include, 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.

The effect of increasing pI by alteration of several amino acid residues in the variable region does not depend only (or substantially) on the amino acid sequence constituting the antibody itself or the type of target antigen, but the type of amino acid residue to be substituted or The dependence on the number has already been explained or demonstrated in WO2007/114319 or WO2009/041643 on the basis of rationale, homology modeling, or experimental techniques. In addition, it has been demonstrated that even after some amino acids have been modified, it can be expected by those skilled in the art that the antigen-binding activity to a plurality of antigens is maintained (substantially), or is maintained with at least a high probability.

For example, in WO2009/041643, specifically, in the heavy chain FR of the humanized glypican 3 antibody represented by SEQ ID NO: 8, as a modified site of an amino acid residue that can be exposed on the surface, according to Kabat numbering, 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, It has been shown that positions 87, 89, 90, 107, 110, 112, and 114 are preferred. In addition, it is reported that the amino acid residue at position 97 according to Kabat numbering is preferable because almost all antibodies are exposed to the surface. Further, in WO2009/041643, it is also shown that amino acid residues at positions 52, 54, 62, 63, 65, and 66 are preferable in the heavy chain CDRs of the antibody. In addition, in the light chain FR of the humanized glypican 3 antibody represented by SEQ ID NO: 9, 1, 3, 7, 8, 9, 11, 12, 16, 17, 18, 20, 22, 43, according to Kabat numbering, 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, It has also been shown that amino acid residues at positions 111 and 112 are preferred. In addition, it has also been shown that amino acid residues at positions 24, 27, 33, 55, and 59 are preferable in the light chain CDRs of the antibody. Moreover, in WO2009/041643, as a site capable of altering amino acid residues that may be exposed on the surface while maintaining antigen-binding activity, specifically, the heavy chain CDR of an anti-human IL-6 receptor antibody represented by SEQ ID NO: 10. It is also shown that amino acid residues at positions 31, 64, and 65 according to Kabat numbering are preferable. In addition, it is also shown that in the light chain CDRs of the anti-human IL-6 receptor antibody represented by SEQ ID NO: 11, amino acid residues at positions 24, 27, 53, and 55 according to Kabat numbering are preferable. In addition, as a site capable of altering amino acid residues that may be exposed on the surface while maintaining antigen-binding activity, specifically, in the heavy chain CDR of the anti-human IL-6 receptor antibody represented by SEQ ID NO: 12, Kabat It has also been shown that the amino acid residue at position 31 is preferable according to the numbering. In addition, it is also shown that in the light chain CDRs of the anti-human IL-6 receptor antibody represented by SEQ ID NO: 13, amino acid residues at positions 24, 53, 54, and 55 according to Kabat numbering are preferable. In addition, in WO2009/041643, Kabat numbering in the heavy chain CDR of the anti-human glypican 3 antibody represented by SEQ ID NO: 14 as a site capable of altering amino acid residues exposed to the surface while maintaining antigen-binding activity. According to, it has also been shown that amino acid residues at positions 61, 62, 64, and 65 are preferable. In addition, it is also shown that in the light chain CDRs of the anti-human glypican 3 antibody represented by SEQ ID NO: 15, amino acid residues at positions 24 and 27 according to Kabat numbering are preferable. In addition, as a site capable of modifying amino acid residues that may be exposed on the surface while maintaining antigen-binding activity, in the heavy chain CDR of the anti-human IL-31 receptor antibody represented by SEQ ID NO: 16, according to Kabat numbering It has also been shown that amino acid residues at positions 61, 62, 64, and 65 are preferable. Further, in WO2009/041643, it is also shown that amino acid residues at positions 24 and 54 according to Kabat numbering are preferable in the light chain CDRs of the anti-human IL-31 receptor antibody represented by SEQ ID NO: 17. Similarly, in WO2007/114319, antibodies hA69-PF, hA69-p18, hA69-N97R, hB26-F123e4, hB26-p15, and For hB26-PF, it was reported that the change in pI was demonstrated by isoelectric point electrophoresis, and that the binding activity to their antigen, factor IXa or factor X, was comparable to that of the antibody before or before the change. have. It was also reported that by administering these antibodies to mice, a high correlation was shown between the pI of each antibody and the plasma clearance (CL), the retention time in plasma, and the plasma half-life (T1/2) of each antibody. In addition, WO2007/114319 demonstrates that amino acid residues at positions 10, 12, 23, 39, 43, 97, and 105 of the variable region are preferable as a modified site for amino acid residues that may be exposed on the surface.

In an alternative or additional embodiment, for example, X-ray crystal structure analysis, or antibody constant region (but not limited, preferably it is a human normal region, more preferably a human Ig-type normal region, , By using a known method such as a homology model constructed by homology modeling from a human IgG-type normal region), by identifying amino acid residues that may be exposed on the surface of the antibody constant region, You may determine the modified site of at least one amino acid residue for producing an antibody of initiation A whose pI is rising. Although not limited, the modified site of at least one amino acid residue that can be exposed on the surface of the normal region is preferably 196th, 253th, 254th, 256th, 257th, 258th according to EU numbering. , 278th, 280th, 281th, 282th, 285th, 286th, 306th, 307th, 308th, 309th, 311th, 315th, 327th, 330th, 342th, 343th, 345 Above, 356th, 358th, 359th, 361th, 362th, 373th, 382th, 384th, 385th, 386th, 387th, 388th, 389th, 399th, 400th, 401th, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 can be selected from the group consisting of, preferably, 254, 258 , 281th, 282th, 285th, 309th, 311th, 315th, 327th, 330th, 342th, 343th, 345th, 356th, 358th, 359th, 361th, 362th, 384 Ranked, 385, 386, 387, 389, 399, 400, 401, 402, 413, 418, 419, 421, 433, 434, and 443 May be selected from, more preferably, it may be selected from the group consisting of 282th, 309th, 311th, 315th, 342th, 343th, 384th, 399th, 401st, 402th, and 413th, The amino acid at each site after modification is not limited, but can be selected from the aforementioned amino acids such as Lys(K), Arg(R), Gln(Q), or Asn(N) from the viewpoint of the charge of the side chain. Thus, for example, when the modified site of at least one amino acid residue is selected from the group including the group described above, the type of amino acid after the modification at each site is, for example, hereinafter: 254K at position 254, 254R, 254Q, or 254N; At 258, 258K, 258R, 258Q, or 258N; At position 281, 281K, 281R, 281Q, or 281N; At position 282, 282K, 282R, 282Q, or 282N; At 285, 285K, 285R, 285Q, or 285N; In 309th place, 309K, 309R, 309Q, or 309N; At 311, 311K, 311R, 311Q, or 311N; At 315, 315K, 315R, 315Q, or 315N; At 327 position, 327K, 327R, 327Q, or 327N; In 330th place, 330K, 330R, 330Q, or 330N; At position 342, 342K, 342R, 342Q, or 342N; At 311, 343K, 343R, 343Q, or 343N; In 345, 345K, 345R, 345Q, or 345N; At 356, 356K, 356R, 356Q, or 356N; At 358 place, 358K, 358R, 358Q, or 358N; At 359, 359K, 359R, 359Q, or 359N; At position 361, 361K, 361R, 361Q, or 361N; At position 362, 362K, 362R, 362Q, or 362N; At 384, 384K, 384R, 384Q, or 384N; At 385, 385K, 385R, 385Q, or 385N; At 386, 386K, 386R, 386Q, or 386N; At position 387, 387K, 387R, 387Q, or 387N; At position 389, 389K, 389R, 389Q, or 389N; At 399, 399K, 399R, 399Q, or 399N; In the 400th place, 400K, 400R, 400Q, or 400N; At 401, 401K, 401R, 401Q, or 401N; At 402, 402K, 402R, 402Q, or 402N; 413K, 413R, 413Q, or 413N; At position 418, 418K, 418R, 418Q, or 418N; At 419, 419K, 419R, 419Q, or 419N; 421K, 421R, 421Q, or 421N; At 433 place, 433K, 433R, 433Q, or 433N; At position 434, 434K, 434R, 434Q, or 434N; And 443 above, it may be 443K, 443R, 443Q, or 443N.

Further, in an alternative embodiment, as the modified site of at least one amino acid residue and the type of amino acid after the modification, 345R or 345K, and/or 430R, 430K, 430G or 435T according to EU numbering may be included. .

In one embodiment of the antibody of Initiation A, at least one amino acid residue that can be exposed on the surface of the variable region (which may be altered) is altered as described above, and the constant region (may be altered). At least one amino acid residue that can be exposed on the surface may be altered as described above, so that the net pI of the antibody may increase.

In the range described in the disclosures A and B in this specification, in the case where the initiation A or B antibody is an IgG-type antibody or a molecule derived therefrom, the heavy chain constant region of the antibody is IgG1, IgG2, IgG3, or IgG4. The normal region of the type may be included. In the initiation A or B, although it is not limited, the heavy chain constant region may be a human heavy chain constant region. It is known that several allotypes exist in human IgG. That is, it has been reported that some differences exist between individuals in the amino acid sequence of the human IgG normal region (Methods Mol. Biol. 882:635-80 (2012); Sequences of proteins of immunological interest, NIH Publication No. 91-3242). As an example, human IgG1 normal region (SEQ ID NO: 18), human IgG2 normal region (SEQ ID NO: 19), human IgG3 normal region (SEQ ID NO: 20), and human IgG4 normal region (SEQ ID NO: 21), respectively. Can be lifted.

Among these, for example, as human IgG1, an allotype called G1m1,17 or G1m3 is known. These allotypes have differences in amino acid sequence. That is, G1m1,17 has aspartic acid at position 356 and leucine at position 358 according to EU numbering, while G1m3 has glutamic acid at position 356 and methionine at position 358. However, for the reported allotypes, no reports were made suggesting that there is a significant difference in the function or properties of the intrinsic antibody. Therefore, various studies were conducted using specific allotypes, and the results are not limited to allotypes used to obtain these examples, and the same effect is expected for any allotype. It will be easily guessed by those skilled in the art. From the above, in the range where the disclosures A and B in this specification are described, when it is expressed as "human IgG1", "human IgG2", "human IgG3", or "human IgG4", the allotype is a specific It is not limited to allotypes, and it is assumed that all reported allotypes can be included.

In an alternative or additional embodiment of initiation A or B, the light chain constant region of the antibody will comprise any normal region of the κ chain (IgK) or λ chain (IgL1, IgL2, IgL3, IgL6, or IgL7) type. I can. Although not limited, it is preferable that the light chain constant region is a human light chain constant region. As for the human κ chain constant region and the human λ chain constant region, a plurality of allotype sequences by genopolymorphism are described in Sequences of proteins of immunological interest, NIH Publication No. It has been reported in 91-3242. Examples of such allotypes include a human κ chain constant region (SEQ ID NO: 22) and a human λ chain constant region (SEQ ID NO: 23). However, for the reported allotypes, no reports were made suggesting that there is a significant difference in the function or properties of the intrinsic antibody. Therefore, when referring to a specific allotype in the range of disclosure A and B in this specification, all allotypes (hereinafter, these are collectively referred to as a natural (human) IgG (type) normal region. ), that the same effect is expected can be easily inferred by those skilled in the art.

In addition, since the Fc region of a native IgG antibody constitutes a part of the constant region of a native IgG antibody, when the antibody of initiation A or B is, for example, an IgG type antibody or a molecule derived therefrom, the antibody May have an Fc region contained in the normal region of a native IgG (IgG1, IgG2, IgG3, or IgG4 type) (hereinafter, these are collectively referred to as a native (human) IgG (type) Fc region.) . The Fc region of a native IgG can mean an Fc region comprising the same amino acid sequence as an Fc region originating from an IgG found in nature. Specific examples of the Fc region of native human IgG include, as described above, a human IgG1 normal region (SEQ ID NO: 18), a human IgG2 normal region (SEQ ID NO: 19), a human IgG3 normal region (SEQ ID NO: 20), or a human IgG4 The Fc region contained in the normal region (SEQ ID NO: 21) (the Fc region of the IgG class is, for example, from cysteine at position 226 according to EU numbering to the C terminus, or from proline at position 230 according to EU numbering to the C terminus May mean).

In one embodiment, the antibody of initiation A and B is a natural (preferably, human) constant region (heavy chain constant region and/or light chain constant region) of a native (preferably human) IgG, or native (preferably, human) IgG. In the Fc region of, amino acid substitutions, additions, deletions, or modifications may be included in one or more modifications selected from insertion.

In the range described in the disclosure A in the present specification, WO2013/081143 is an ion concentration-dependent antibody capable of forming, for example, a multimeric antigen and a multivalent immune complex (a multivalent complex of an antigen and an antibody), and, A multispecific ion concentration dependent antibody or a multiparatopic ion concentration dependent antibody capable of forming a multivalent immune complex (a multivalent complex of an antigen and an antibody) by recognizing two or more epitopes present on a monomeric antigen , Avidity through at least two or more polyvalent constant regions (may be altered) or Fc regions (may be altered) contained in these antibody molecules; the strength of binding between a plurality of epitopes and a plurality of paratopes. The sum of), it is possible to bind more strongly to FcγR, FcRn, and complement receptors; And as a result, it is reported that the antibody is absorbed more rapidly in the cell. Therefore, when modified to have an elevated pI by altering at least one amino acid residue that can be exposed on the surface of the antibody, the above ion concentration-dependent antibody capable of forming a multimeric immune complex with a multimeric antigen or a monomeric antigen In addition, it can be used as an antibody of initiation A (an ion concentration-dependent antibody with an elevated pi). An ion concentration-dependent antibody with an elevated pI capable of forming a multivalent immune complex with a multimeric antigen or a monomeric antigen is compared with an ion concentration-dependent antibody with an elevated pI that cannot form a multivalent immune complex. One of skill in the art will recognize that it can be absorbed into. In addition, in one embodiment, the antibody of Initiation A may have increased binding activity to FcRn and/or FcγR under conditions of neutral pH, and in this case, a multimeric antigen or a monomeric antigen and a multivalent immune complex It can also be understood by those skilled in the art that an ion concentration-dependent antibody with an elevated pI that can be formed can be absorbed into cells more rapidly.

In one embodiment, the antibody of Initiation A may be a flat-armed antibody (including all embodiments of the flat-armed antibody described in WO2005/063816). Typically, a brachial antibody is an antibody that lacks one of the two Fab regions of a common IgG antibody, and is not limited, but can be produced, for example, by the method described in WO2005/063816. Although not limited, for example, in an IgG-type antibody in which the heavy chain has a structure of VH-CH1-Hinge-CH2-CH3, the Fab region at a site on the N-terminal side than Hinge (for example, VH or CH1) When one is cleaved, the antibody is expressed in a form containing an extra sequence, and if one of the Fab regions is cleaved at a site on the C-terminal side (for example, CH2) than Hinge, the Fc region becomes incomplete. . Therefore, although not limited, from the viewpoint of the stability of the antibody molecule, it is preferable to produce a brachial antibody by cutting at the hinge region (Hinge) of one of the two Fab regions of the IgG-type antibody. It is more preferable that the heavy chain after cleavage is connected via an uncleaved heavy chain and an intramolecular disulfide bond. It has been reported in WO2005/063816 that such a flaky antibody has increased stability compared to that of a Fab molecule. By preparing such a flaky antibody, an antibody with an elevated or lowered pI can also be produced. Moreover, when the ion concentration-dependent antigen-binding domain is introduced into an antibody with elevated pI, which is a flat-armed antibody, the half-life in plasma of the antibody is further shortened compared to an antibody with an elevated pI that does not have an ion concentration-dependent antigen-binding domain It may or may further promote the uptake of the antibody into the cells, or may further promote the loss of the antigen from the plasma, or may further increase the affinity of the antibody to the extracellular matrix.

Although not bound by a specific theory, although it is not particularly limited, as an embodiment in which the effect of accelerating intracellular uptake by a brachial antibody is expected, a case in which the pI of the soluble antigen is lower than that of the antibody may be assumed. The net pI of a complex consisting of an antibody and an antigen can be calculated by a known method by viewing the complex as one molecule. In this case, the lower the pI of the soluble antigen, the lower the net pI of the complex, and the higher the pI of the soluble antigen, the higher the net pI of the complex. In addition, with respect to a normal IgG antibody molecule (having two Fabs), when one soluble antigen with a low pI is bound and two soluble antigens with a low pI are bound, the latter is the complex. The net pI is lowered. When such a conventional antibody is converted into a single-armed antibody, only one antigen can be bound to one antibody molecule, but it becomes possible to suppress a decrease in the pi of the complex due to the binding of the second antigen. In other words, when the pI of the soluble antigen is lower than the pI of the antibody, it is considered that the pI of the complex is increased as compared with the normal antibody by conversion to a brachial antibody, thereby accelerating absorption into the cell.

In addition, although not limited, in the case of a normal IgG-type antibody molecule (having two Fabs), when the pI of the Fab is low compared to the pI of the Fc, it is converted into a brachial antibody and consists of a brachial antibody and an antigen The net pi of the complex is high. Moreover, when performing such conversion into a brachial antibody, from the viewpoint of stability of the brachial antibody, it is preferable to cleave one Fab in the hinge region located at the boundary between the Fab and the Fc. At this time, by selecting a site where the pI of the brachial antibody rises to a desired degree, it is expected that the pI will rise effectively.

Therefore, by calculating the theoretical pI of the antibody (theoretical pI of Fc, the theoretical pI of Fab), and the theoretical pI of soluble antigens, and predicting the relationship to the difference between their theoretical pI values, it is possible to convert the antibody into a brachial antibody. Thus, those skilled in the art that it is possible to increase the pi of the antibody and to accelerate the uptake of the antigen into the cell accordingly, regardless of (or substantially) only (or substantially) based on the amino acid sequence of the antibody itself and the type of soluble antigen. I understand.

In one embodiment, the antibody of initiation A or B may be a multispecific antibody, but is not limited, but the multispecific antibody may be a bispecific antibody. The multispecific antibody is a multispecific antibody comprising a first polypeptide and a second polypeptide. Herein, "multispecific antibody comprising a first polypeptide and a second polypeptide" means an antibody that binds to at least two or more different antigens or at least two or more different epitopes within the same antigen. It is preferable that the 1st polypeptide and the 2nd polypeptide can contain a heavy chain variable region, and it is more preferable that the CDR and/or FR are contained in the said variable region. In another embodiment, it is preferable that the first polypeptide and the second polypeptide can each contain a heavy chain constant region. Further, in another embodiment, the multispecific antibody may include a third polypeptide and a fourth polypeptide each comprising a light chain variable region and preferably a light chain constant region, respectively. In this case, the first to fourth polypeptides may associate together to form a multispecific antibody.

In one embodiment, when the antibody of initiation A is a multispecific antibody, and the multispecific antibody contains a heavy chain constant region, in order to lower the pI, for example, the sequence of IgG2 or IgG4 at position 137; The sequence of IgG1, IgG2 or IgG4 at position 196; The sequence of IgG2 or IgG4 at position 203; The sequence of IgG2 at position 214; The sequence of IgG1, IgG3 or IgG4 at position 217; The sequence of IgG1, IgG3 or IgG4 at position 233; The sequence of IgG4 at position 268; The sequence of IgG2, IgG3 or IgG4 at position 274; The sequence of IgG1, IgG2 or IgG4 at position 276; The sequence of IgG4 at position 355; The sequence of IgG3 at position 392; The sequence of IgG4 at position 419; Alternatively, an IgG1, IgG2, or IgG4 sequence at position 435 may be used. On the other hand, in order to raise pI, for example, the sequence of IgG1 or IgG3 at position 137; The sequence of IgG3 at position 196; The sequence of IgG1 or IgG3 at position 203; The sequence of IgG1, IgG3 or IgG4 at position 214; The sequence of IgG2 at position 217; The sequence of IgG2 at position 233; The sequence of IgG1, IgG2 or IgG3 at position 268; The sequence of IgG1 at position 274; The sequence of IgG3 at position 276; The sequence of IgG1, IgG2 or IgG3 at position 355; The sequence of IgG1, IgG2 or IgG4 at position 392; The sequence of IgG1, IgG2 or IgG3 at position 419; Alternatively, the sequence of IgG3 at position 435 may be used.

In one embodiment, when the antibody of initiation A has two heavy chain constant regions, the pIs of the two heavy chain constant regions may be the same or different from each other. As such a heavy chain constant region, a heavy chain constant region of IgG1, IgG2, IgG3, and IgG4 whose original pIs are different may be used. Alternatively, a pI difference may be introduced between the two heavy chain constant regions. The modified site of at least one amino acid residue for introducing a pI difference into the normal region may be the above-described position, or, for example, at position 137, position 196 according to EU numbering of the heavy chain constant region described in WO2009/041643, It may be a site selected from the group consisting of 203, 214, 217, 233, 268, 274, 276, 297, 355, 392, 419, and 435. Alternatively, since pI difference occurs by removing the sugar chain from the heavy chain constant region, the sugar chain may be removed by altering the amino acid residue at position 297, which is the sugar chain addition position.

In one embodiment, the antibody of initiation A or B may be a polyclonal antibody or a monoclonal antibody, and a monoclonal antibody derived from a mammal is preferable. Monoclonal antibodies include those produced by hybridomas or those produced by host cells transformed with an expression vector containing an antibody gene by a genetic engineering technique. The antibody of initiation A or B may be, for example, an antibody such as a chimeric antibody, a humanized antibody, or an antibody produced by affinity maturation, or a molecule derived therefrom.

In one embodiment, the antibody of the initiation A or B is not limited, but any animal species (e.g., human; or mouse, rat, hamster, rabbit, monkey, Philippine monkey, Himalayan monkey, mantle baboon, chimpanzee, Non-human animals such as goats, sheep, dogs, cattle, or camels) or any bird may be derived, and the antibody is preferably derived from humans, monkeys or mice.

In one embodiment, the antibody of initiation A or B may be an Ig-type antibody, preferably an IgG-type antibody.

In the range described in the disclosures A and B in this specification, the Fc receptor (also referred to as "FcR") is capable of binding to an Fc region or an Fc region variant possessed by an immunoglobulin (antibody) or a molecule derived therefrom. Indicates a receptor protein. In the range described in the disclosure A in this specification, for example, Fc receptors for IgG, IgA, IgE, and IgM are known as FcγR, FcαR, FcεR, and FcμR, respectively. In the range of disclosures A and B in this specification, the Fc receptor may be, for example, FcRn (also referred to as "neoplastic Fc receptor").

In the range described in the disclosure A in this specification, "FcγR" refers to a receptor protein capable of binding to an IgG1, IgG2, IgG3, or IgG4 antibody, or the Fc region or Fc region variant of a molecule derived therefrom. And substantially includes any one or more, or all members of the family of proteins encoded in the FcγR gene. In humans, this family includes FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including isoform FcγRIIa (including allotypes H131 (H-type) and R131 (R-type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; And FcγRIII (CD16) comprising isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and all undiscovered human FcγRs and FcγR isoforms And allotypes are included, but are not limited thereto. Furthermore, FcγRIIb1 and FcγRIIb2 have been reported as splicing variants of human FcγRIIb (hFcγRIIb). In addition, a splicing variant called FcγRIIb3 has also been reported (Brooks et al., J. Exp. Med, 170: 1369-1385 (1989)). In addition to these, hFcγRIIb includes all splicing variants registered in NCBI as NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. In addition, hFcγRIIb includes all previously reported polymorphisms, for example, FcγRIIb (Li 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 genotypes reported later.

FcγR may be derived from any organism, and may include those derived from humans, mice, rats, rabbits, or monkeys, but is not limited thereto. Mouse FcγRs include FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), and all undiscovered mouse FcγRs and FcγR isoforms and allotypes Not limited. Suitable examples of such FcγR include human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), or FcγRIIIB (CD16). On the other hand, since FcγR exists as an in vivo film type, it may be appropriately used in an experimental system after artificially converting it into a soluble type.

As discussed in WO2014/163101 as an example, the polynucleotide sequence and amino acid sequence of FcγRI may be sequences described in NM_000566.3 and NP_000557.1, respectively; The polynucleotide sequence and amino acid sequence of FcγRIIA may be sequences described in BC020823.1 and AAH20823.1, respectively; The polynucleotide sequence and amino acid sequence of FcγRIIB may be sequences described in BC146678.1 and AAI46679.1, respectively; The polynucleotide sequence and amino acid sequence of FcγRIIIA may be sequences described in BC033678.1 and AAH33678.1, respectively; The polynucleotide sequence and amino acid sequence of FcγRIIIB may be sequences described in BC128562.1 and AAI28563.1, respectively (RefSeq accession numbers are shown.).

In FcγRIIa, there are two types of polymorphisms in which the amino acid at position 131 of FcγRIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med. 172: 19-25, 1990).

In FcγRI (CD64) including FcγRIa, FcγRIb and FcγRIc, and FcγRIII (CD16) including FcγRIIIa (including allotypes V158 and F158), the α chain binding to the Fc region of IgG produces an activation signal in cells. It is linked to a common γ chain with the ITAM that carries it. FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2) is a GPI anchor protein. On the other hand, the cytoplasmic domain of FcγRII (CD32) including isoform FcγRIIa (including allotypes H131 and R131) and FcγRIIc contains ITAM. These receptors are expressed in many immune cells such as macrophages, mast cells, and antigen-presenting cells. Activation signals transmitted by binding of these receptors to the Fc region of IgG promote phagocytosis of macrophages, production of inflammatory cytokines, degranulation of mast cells, and hyperactivity of antigen presenting cells. The FcγR having the ability to transmit an activation signal in this way is also referred to as an active FcγR within the range of disclosures A and B in this specification.

On the other hand, the intracytoplasmic domain of FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) contains ITIM that transmits an inhibitory signal. In B cells, the activation signal from BCR is suppressed by crosslinking of FcγRIIb and B cell receptor (BCR), resulting in suppression of BCR antibody production. In macrophages, phagocytosis and the ability to produce inflammatory cytokines are suppressed by crosslinking of FcγRIII and FcγRIIb. The FcγR having the ability to transmit an inhibitory signal in this way is also referred to as an inhibitory Fcγ receptor in the range where the disclosures A and B in this specification are described.

In the range described in the disclosure A in this specification, whether or not the binding activity of the antibody or Fc region (variant) to various FcγRs was increased compared to the antibody or Fc region (variant) before modification, or (substantially) maintained Whether or not it is being reduced or not, can be evaluated according to a method known to those skilled in the art. Such a method is not particularly limited, and the one described in this example may be used, for example, BIACORE (Proc. Natl. Acad. Sci. USA (2006) 103 (11) using surface plasmon resonance (SPR) phenomenon. ), 4005-4010) may be used. Alternatively, for example, an ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay) other than ELISA or fluorescence activated cell sorting (FACS) may be used. In these assays, the extracellular domain of human FcγR may be used as a soluble antigen (for example, WO2013/047752).

As the pH condition for measuring the binding activity of the FcγR binding domain and FcγR contained in the antibody or the Fc region (variant), acidic or neutral pH conditions can be suitably used. As the temperature used in the measurement conditions, the binding activity (binding affinity) of the FcγR-binding domain and FcγR can be evaluated, for example, at an arbitrary temperature of 10°C to 50°C. A preferred temperature for determining the binding activity (binding affinity) of the human FcγR binding domain and FcγR is, for example, 15°C to 40°C. More preferably, for example, at 20° C., such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, 35 Any temperature up to °C is likewise used to determine the binding activity (binding affinity) of the FcγR-binding domain and FcγR. The temperature of 25°C is a non-limiting example.

In one embodiment, when the antibody of initiation A or B has a constant region (may be altered), the constant region is an Fc region or a modified Fc region (preferably, a human Fc region or a human Fc region). Variant), and preferably has an FcγR-binding domain in the range of initiation A, and an FcRn-binding domain in the range described initiation A and B in this specification.

In one embodiment, when the antibody of initiation A has an FcγR-binding activity, it may have an FcγR-binding domain, preferably a human FcγR-binding domain. The FcγR binding domain is not particularly limited as long as the antibody has binding activity or affinity to FcγR at acidic pH and/or neutral pH, or may be a domain having activity to bind FcγR directly or indirectly.

In one embodiment, when the antibody of initiation A has binding activity to FcγR, the antibody has a binding activity to FcγR under conditions of neutral pH as compared to a reference antibody containing a constant region of a native IgG. It is desirable that it is increased. Here, although not limited, the reference antibody including the constant region of the initiating A antibody and the native IgG is preferably a region other than the constant region of the initiating A antibody in which one or more amino acid residues have been altered. It is preferable from the viewpoint of comparing the binding activity of both to FcγR to have the same amino acid sequence (for example, in the variable region).

In one embodiment, when the antibody of initiation A has binding activity to FcγR, or if the binding activity to FcγR is increased under neutral pH conditions (e.g., pH 7.4), it is not bound by theory. , The antibody has an ion concentration-dependent antigen-binding domain, thereby repeatedly binding to a plurality of antigens with a single antibody molecule through endosomes in plasma and cells; The property of being rapidly absorbed into cells by increasing the pi and also increasing the positive charge throughout the antibody; And it is considered to have a property of being rapidly absorbed into cells by increasing the binding activity to FcγR under conditions of neutral pH. As a result, it is possible to further shorten the half-life of the antibody in plasma, or to further increase the binding activity of the antibody to the extracellular matrix, or to further promote the disappearance of antigen from the plasma. Antibodies are thought to be advantageous. Those skilled in the art can routinely determine the optimal antibody pI value in order to utilize these properties.

In one embodiment, the FcγR binding domain having a higher binding activity to FcγR than the binding activity to FcγR of a natural human IgG Fc region or a constant region of a natural human IgG where the sugar chain bound at position 297 according to EU numbering is a fucose-containing sugar chain, It can be produced by altering the amino acid residues of the Fc region or the normal region of type human IgG (refer to WO2013/047752). In addition, as the FcγR binding domain, domains of all structures that bind to FcγR can be used. In this case, the FcγR-binding domain may be prepared without requiring the introduction of amino acid modifications, or may increase the affinity for FcγR by introducing additional modifications. As such an FcγR binding domain, 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)), a Fab fragment antibody that binds to FcγRIIIa, a camel-derived single domain antibody and a single chain Fv antibody, and The FcγRI-binding cyclic peptides described in Bonetto et al., FASEB J. 23(2):575-585 (2009), etc. are mentioned. Whether the binding activity of the FcγR binding domain to FcγR is higher than that of the Fc region of a natural human IgG, which is a fucose-containing sugar chain, or a normal region of the sugar chain bound at position 297 according to EU numbering, according to the above-described method. Can be appropriately evaluated using.

In one embodiment of initiation A, examples of the starting FcγR binding domain include an Fc region of (human) IgG or a constant region of (human) IgG as appropriate. Any Fc region or constant region may be used as a starting Fc region or a starting normal region, as long as the starting Fc region or the variant of the starting normal region can bind to human FcγR in the neutral pH region. In addition, the Fc region or the constant region obtained by adding additional modifications to the starting Fc region or the starting constant region to which the amino acid residue modification has already been applied to the Fc region or the constant region is also suitable as an Fc region or a constant region in start A. Can be used. The starting Fc region or starting constant region may mean a polypeptide itself, a composition comprising a starting Fc region or a starting constant region, or an amino acid sequence encoding a starting Fc region or a starting constant region. The starting Fc region or the starting normal region may contain a known Fc region or a known normal region produced by recombinant technology. The origin of the starting Fc region or the starting normal region is not limited, but can be obtained from any organism that is a non-human animal, or a human. In addition, the starting FcγR binding domain can be obtained from Philippine monkey, marmoset, rhesus monkey, chimpanzee, or human. Preferably, the starting Fc region or the starting constant region may be obtained from human IgG1, but is not limited to a specific class of IgG. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be suitably used as the starting FcγR binding domain. Similarly, in the range described in the disclosure A in this specification, it is possible to use an Fc region or a constant region such as an IgG class or subclass from any organism, preferably as a starting Fc region or a starting constant region. it means. Examples of native IgG variants or modifications can be found in known literature (e.g. 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), WO2009/086320, WO2008/092117, WO2007/041635, and WO2006/105338) But is not limited to them.

In one embodiment, as an example of the starting FcγR binding domain, or the amino acid residues of the starting Fc region or the starting normal region, one or more mutations, for example, of amino acid residues different from the amino acid residues of the starting Fc region or starting normal region substitution; Insertion of one or more amino acid residues relative to the amino acid residues of the starting Fc region or the starting normal region; Alternatively, deletion of one or more amino acid residues from the amino acid residues of the starting Fc region or the starting constant region may be included. Preferably, the amino acid sequence of the Fc region or the normal region after the modification may be an amino acid sequence including at least a portion of the Fc region or the normal region that cannot occur naturally. Such variants inevitably have less than 100% sequence identity or similarity to the starting Fc region or starting normal region. For example, the modifications may be from about 75% to less than 100%, more preferably from about 80% to less than 100%, even more preferably from about 85% to the amino acid sequence of the starting Fc region or starting normal region. It has an amino acid sequence identity or similarity of less than 100%, even more preferably about 90% to less than 100%, and most preferably about 95% to less than 100%. In a non-limiting example, at least one amino acid differs between the starting Fc region or the starting normal region and the Fc region or normal region in which the starting A is modified.

In one embodiment, an Fc region or a constant region having binding activity to FcγR in the acidic pH range and/or neutral pH range, which may be included in the antibody of Initiation A, can be obtained by any method. Specifically, by modifying the amino acid of a human IgG antibody that can be used as a starting Fc region or a starting constant region, an Fc region or a modified variant of the constant region having binding activity to FcγR in the neutral pH region can be obtained. have. As the Fc region of an IgG antibody suitable for modification or the constant region of an IgG-type antibody, for example, the Fc region or constant region of human IgG (IgG1, IgG2, IgG3 or IgG4, or their variants) and variants naturally occurring therefrom are exemplified. I can. Regarding the Fc region or constant region of human IgG1, human IgG2, human IgG3, or human IgG4 antibodies, several allotype sequences due to polymorphism are described in "Sequences of proteins of immunological interest", NIH Publication No. Although described in 91-3242, in the indication A, any of them may be used. In particular, regarding the sequence of human IgG1, the amino acid sequence at positions 356 to 358 according to EU numbering may be DEL or EEM.

In a further embodiment in the range of Initiation A, the modification to another amino acid is not limited as long as the variant has a binding activity to FcγR in the neutral pH range. Such amino acid alteration positions are, for example, WO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072, WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635, WO2008/092117, WO2005/ Reported in 070963, WO2006/020114, WO2006/116260, WO2006/023403, WO2013/047752, WO2006/019447, WO2012/115241, WO2013/125667, WO2014/030728, WO2014/163101, WO2013/118858, and WO2014/030750 have.

As an amino acid modification site in a constant region or an Fc region for increasing the binding activity to FcγR in the neutral pH region, for example, the Fc region or the constant region of a human IgG antibody described in WO2013/047752 , According to EU numbering, 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237 , 238th, 239th, 240th, 241st, 243th, 244th, 245th, 246th, 247th, 249th, 250th, 251st, 254th, 255th, 256th, 258th, 260 Rank, 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 Rank, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, One or more positions selected from the group consisting of 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436 and 440 Can be mentioned. By such alteration of amino acid residues, binding to FcγR in the Fc region or constant region of the IgG antibody under conditions of neutral pH can be increased. In WO2013/047752, the amino acid at position 221 according to EU numbering, for example, according to EU numbering, as a preferred modification in the IgG-type normal region or the Fc region is either Lys or Tyr; The amino acid at position 222 is any one of Phe, Trp, Glu, and Tyr; The amino acid at position 223 is any one of Phe, Trp, Glu, and Lys; 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, Lys, and Trp; The amino acid at position 227 is one of Glu, Gly, Lys, and Tyr; The amino acid at position 228 is one of Glu, Gly, Lys, and Tyr; The amino acid at position 230 is any one of Ala, Glu, Gly, and Tyr; The amino acid at position 231 is one of Glu, Gly, Lys, Pro, and Tyr; The amino acid at position 232 to any one of Glu, Gly, Lys, and Tyr; Amino acid at position 233 to any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; Amino acid at position 234 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 235 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 236 to any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 237 as any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 238 as any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 239 as any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; The amino acid at position 240 is any one of Ala, Ile, Met, and Thr; The amino acid at position 241 to any one of Asp, Glu, Leu, Arg, Trp, and Tyr; The amino acid at position 243 to any one of Glu, Leu, Gln, Arg, Trp, and Tyr; The amino acid at position 244 as His; Amino acid at position 245 as Ala; The amino acid at position 246 to any one of Asp, Glu, His, and Tyr; Amino acid at position 247 to any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr; The amino acid at position 249 is one of Glu, His, Gln, and Tyr; The amino acid at position 250 as either Glu or Gln; The amino acid at position 251 as Phe; The amino acid at position 254 is 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 is any one of Ala, Met, and Pro; The amino acid at position 258 as 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, Ile, and Thr; Amino acid at position 263 to any one of Ala, Ile, Met, and Thr; The amino acid at position 264 as any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; The amino acid at position 265 as any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 266 is any one of Ala, Ile, Met, and Thr; The amino acid at position 267 as any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, and Tyr; The amino acid at position 268 to any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp; The amino acid at position 269 to any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 270 as any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr; The amino acid at position 271 as any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 272 as any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 273 is either Phe or Ile; The amino acid at position 274 as any one of Asp, Glu, Phe, Gly, His, Ile, 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 as any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; Amino acid at position 278 as any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; Amino acid at position 279 as Ala; The amino acid at position 280 to any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr; 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 as any one of Ala, Gly, His, Ile, 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, Gln, Trp, and Tyr; The amino acid at position 286 is one of Glu, Gly, Pro, and Tyr; Amino acid at position 288 to any one of Asn, Asp, Glu, and Tyr; The amino acid at position 290 as any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr; The amino acid at position 291 to any one of Asp, Glu, Gly, His, Ile, Gln, and Thr; The amino acid at position 292 is any one of Ala, Asp, Glu, Pro, Thr, and Tyr; The amino acid at position 293 to any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 294 as any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 295 as any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 296 as any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val; The amino acid at position 297 as any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 298 as any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr; The amino acid at position 299 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; The amino acid at position 300 as any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, and Trp; The amino acid at position 301 to any one of Asp, Glu, His, and Tyr; Amino acid at position 302 as Ile; The amino acid at position 303 is any one of Asp, Gly, and Tyr; The amino acid at position 304 as any one of Asp, His, Leu, Asn, and Thr; The amino acid at position 305 is one of Glu, Ile, Thr, and Tyr; The amino acid at position 311 is any one of Ala, Asp, Asn, Thr, Val, and Tyr; The amino acid at position 313 as Phe; The amino acid at position 315 as Leu; The amino acid at position 317 is either Glu or Gln; The amino acid at position 318 as any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr; The amino acid at position 320 as any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 322 as any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr; Amino acid at position 323 as Ile; Amino acid at position 324 to any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr; Amino acid at position 325 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 326 to any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr; Amino acid at position 327 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, and Tyr; The amino acid at position 328 as any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 329 as any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; The amino acid at position 330 as one of Cys, Glu, Phe, Gly, His, Ile, 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, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr; The amino acid at position 332 as any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; Amino acid at position 333 to any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr; The amino acid at position 334 to any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr; The amino acid at position 335 as any one of Asp, Phe, Gly, His, Ile, 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 is any one of Glu, His, and Asn; The amino acid at position 339 to any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr; The amino acid at position 376 is either Ala or Val; The amino acid at position 377 is either Gly or Lys; Amino acid at position 378 as Asp; The amino acid at position 379 as Asn; The amino acid at position 380 is any one of Ala, Asn, and Ser; The amino acid at position 382 is either Ala or Ile; The amino acid at position 385 as Glu; Amino acid at position 392 as Thr; The amino acid at position 396 as Leu; The amino acid at position 421 as Lys; Amino acid at position 427 as Asn; The amino acid at position 428 is either Phe or Leu; The amino acid at position 429 as Met; Amino acid at position 434 as Trp; Amino acid at position 436 as Ile; And modification of one or more amino acid residues selected from the group consisting of the amino acid at position 440 as any one of Gly, His, Ile, Leu, and Tyr. In addition, the number of amino acids to be modified is not particularly limited, and only one amino acid may be modified, and two or more amino acids may be modified. As a combination of modifications of two or more amino acids, it is described in Table 5 of WO2013/047752. Also in the antibody of initiation A, modifications of these amino acid residues may be appropriately introduced.

In one embodiment, (human) FcγR, for example, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and the binding activity of the antibody of initiation A (FcyR binding domain) against any one or more of FcγRIIIb is a native IgG (Fc Region or constant region), or a reference antibody comprising the starting Fc region or the starting constant region, in some cases higher than the binding activity to FcγR. For example, the binding activity to FcγR of the antibody of Initiation A (FcyR binding domain in) is 55% or more, 60% or more, 65% or more, 70% or more, compared with the binding activity to FcγR of the reference antibody. % 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, or compared with the binding activity of the reference antibody to FcγR, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more , 5 times or more, 7.5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, or 100 times or more have.

In a further embodiment, the degree of enhancement of the binding activity to inhibitory FcγR (FcγRIIb-1 and/or FcγRIIb-2) (in the neutral pH region) is the active FcγR (FcγRIa; FcγRIb; FcγRIc; allo FcγRIIIa including type V158; FcγRIIIa including allotype F158; FcγRIIIb including allotype FcγRIIIb-NA1; FcγRIIIb including allotype FcγRIIIb-NA2; FcγRIIa including allotype H131; or including allotype R131 It may be higher than the degree of increase in the binding activity to FcγRIIa).

In one embodiment, the antibody of initiation A may have binding activity to FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

In one embodiment, as an example of a suitable FcγR binding domain in initiation A, an FcγR binding domain having a higher binding activity to specific FcγR than other FcγR binding activities (FcyR binding domain having selective FcγR binding activity ). When an antibody (or an Fc region as an FcγR binding domain) is used, one antibody molecule can bind only to one FcγR molecule. Therefore, one antibody molecule in a state bound to an inhibitory FcγR cannot bind to another active FcγR, and one antibody molecule in a state bound to an active FcγR cannot bind to another active FcγR or an inhibitory FcγR. .

As described above, examples of the active FcγR include FcγRI (CD64), such as FcγRIa, FcγRIb, or FcγRIc; And FcγRIII (CD16), such as FcγRIIIa (eg, allotype V158 or F158), or FcγRIIIb (eg, allotype FcγRIIIb-NA1 or FcγRIIIb-NA2). On the other hand, FcγRIIb (for example, FcγRIIb-1 or FcγRIIb-2) is exemplified as a suitable example of the inhibitory FcγR.

In one embodiment, as the selective FcγR binding domain contained in the antibody of initiation A, an FcγR binding domain having higher binding activity to inhibitory FcγR than active FcγR may be used. For example, as the selective FcγR binding domain, FcγRI (CD64) such as FcγRIa, FcγRIb, or FcγRIc; FcγRIII (CD16) such as FcγRIIIa (eg, allotype V158 or F158) or FcγRIIIb (eg, FcγRIIIb-NA1 or FcγRIIIb-NA2); And FcγRIIa (including allotype H131 or R131); And FcγR binding domains having higher binding activity to FcγRIIb (such as FcγRIIb-1 and/or FcγRIIb-2) than any one or more of the active FcγRs selected from the group consisting of FcγRII (CD32) such as FcγRIIc. .

Moreover, whether or not the FcγR binding domain has a selective binding activity is determined by comparing the binding activity to each FcγR determined according to the method described above, for example, by comparing the KD value for the active FcγR to the inhibitory FcγR. It is possible to judge by comparing the value (ratio) divided by the KD value for each, that is, by comparing the FcγR selectivity index represented by the following formula (1).

[Formula 1] FcγR selectivity index=KD value for active FcγR/KD value for inhibitory FcγR

In Formula 1, the KD value for active FcγR means FcγRIa; FcγRIb; FcγRIc; FcγRIIIa comprising allotype V158 and/or F158; FcγRIIIb including FcγRIIIb-NA1 and/or FcγRIIIb-NA2; FcγRIIa comprising allotype H131 and/or R131; And a KD value for at least one of FcγRIIc, and the KD value for inhibitory FcγR refers to a KD value for FcγRIIb-1 and/or FcγRIIb-2. The active FcγR and inhibitory FcγR used in the measurement of the KD value may be selected from any combination. For example, a value (ratio) obtained by dividing the KD value for FcγRIIa including allotype H131 by the KD value for FcγRIIb-1 and/or FcγRIIb-2 may be used, but is not limited thereto.

FcγR selectivity index is, for example, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 3 or more, 5 or more, 6 or more, 7 or more, 8 or more , 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 More than, 90 or more, 95 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more , 410 or higher, 420 or higher, 430 or higher, 440 or higher, 450 or higher, 460 or higher, 470 or higher, 480 or higher, 490 or higher, 500 or higher, 520 or higher, 540 or higher, 560 or higher, 580 or higher, 600 or higher, 620 or higher, 640 More than, 660 or more, 680 or more, 700 or more, 720 or more, 740 or more, 760 or more, 780 or more, 800 or more, 820 or more, 840 or more, 860 or more, 880 or more, 900 or more, 920 or more, 940 or more, 960 or more, 980 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, 4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 or more, 7500 or more, 8000 or more, 8500 or more , 9000 or more, 9500 or more, 10000 or more, or 100000 or more, but is not limited thereto.

In one embodiment, according to EU numbering of human IgG (IgG1, IgG2, IgG3, or IgG4), the amino acid at position 238 or 328 is Asp or Glu, respectively. ), as described in particular in WO2013/125667, WO2012/115241, and WO2013/047752, FcγRIa, FcγRIb, FcγRIc, FcγRIIIa including allotype V158, FcγRIIIa including allotype F158, allotype FcγRIIIb-NA1 FcγRIIIb including, FcγRIIIb including allotype FcγRIIIb-NA2, FcγRIIa including allotype H131, FcγRIIa including allotype R131, and/or FcγRIIc, the binding activity to FcγRIIb-1 and/or FcγRIIb-2 Since it is high, it can be suitably used as an antibody of the initiation A containing an Fc region variant or a constant region variant. The antibody of Initiation A in this embodiment has binding activity to all active FcγR and FcγRIIb (in this specification, selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa), and also natural Compared with the reference antibody containing the constant region of type IgG or the Fc region of native IgG, its binding activity to the FcγRIIb is maintained or increased, and/or the binding activity to all active FcγRs is decreased.

In one embodiment of the antibody of Initiation A comprising an Fc region variant or a constant region variant, their binding activity to FcγRIIb is maintained as compared to the activity of a reference antibody having a constant region or an Fc region of a native IgG. Alternatively, they may be increased, and their binding activity to FcγRIIa (H type) and FcγRIIa (R type) may decrease. Such an antibody can have increased selectivity for FcγRIIb than for FcγRIIa.

In the range described in the disclosure A in this specification, the degree to which "the binding activity to all active FcγRs is reduced" is not limited, but 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, It may be 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.

In the range described in the disclosure A in the present specification, the degree to which "the binding activity to FcγRIIb is maintained or increased" or "retained or increased binding activity to FcγRIIb" is not limited, but 55% More than, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 100% or more, 101% or more, 102% or more, 103% or more, 104% More than, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 112% or more, 114% or more, 116% or more, 118% or more, 120% or more, 122% or more, 124% or more, 126% or more, 128% or more, 130% or more, 132% or more, 134% or more, 136% or more, 138% or more, 140% or more, 142% or more, 144% or more, 146% or more, 148% More than, 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, 195% or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times More than, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, It may be 10000 times or more, or 100000 times or more.

In the range described in the disclosure A in this specification, the degree to which "the binding activity to FcγRIIa (H type) and FcγRIIa (R type) is reduced", or "FcγRIIa (H type) and FcγRIIa (R type) The term "reduced binding activity to" means, 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% It may be less than or equal to 0.1%, less than or equal to 0.05%, less than or equal to 0.01%, or less than or equal to 0.005%.

Within the range described in the disclosure A in this specification, modifications that increase binding selectivity to FcγRIIb than FcγRIIa (R type) are likely to be preferable, and modifications that increase binding selectivity to FcγRIIb than FcγRIIa (H type) This may be more preferable, and preferable amino acid substitutions for such modifications are, as reported in WO2013/047752, for example, according to EU numbering (a) a modification of substituting Gly at position 237 with Trp; (b) a modification of substituting Phe for Gly at position 237; (c) a change in which Pro at position 238 is substituted with Phe; (d) a change in which Asn at position 325 is substituted with Met; (e) a change in which Ser at position 267 is substituted with Ile; (f) a change in which Leu at position 328 is substituted with Asp; (g) a modification of substituting Val for Ser at position 267; (h) a modification in which Leu at position 328 is substituted with Trp; (i) a modification in which Ser at position 267 is substituted with Gln; (j) a change in which Ser at position 267 is substituted with Met; (k) a modification in which Gly at position 236 is substituted with Asp; (l) a modification in which Ala at position 327 is substituted with Asn; (m) a modification to replace Asn at position 325 with Ser; (n) a change in which Leu at position 235 is substituted with Tyr; (o) a change in which Val at position 266 is substituted with Met; (p) a change in which Leu at position 328 is substituted with Tyr; (q) a change in which Leu at position 235 is substituted with Trp; (r) a change in which Leu at position 235 is substituted with Phe; (s) a change in which Ser at position 239 is substituted with Gly; (t) a modification of substituting Glu for Ala at position 327; (u) a modification in which Ala at position 327 is substituted with Gly; (v) a change in which Pro at position 238 is substituted with Leu; (w) a change in which Ser at position 239 is substituted with Leu; (x) a modification in which Leu at position 328 is substituted with Thr; (y) a change in which Leu at position 328 is substituted with Ser; (z) a change in which Leu at position 328 is substituted with Met; (aa) a change in which Pro at position 331 is substituted with Trp; (ab) a modification in which Pro at position 331 is substituted with Tyr; (ac) a change in which Pro at position 331 is substituted with Phe; (ad) a change in which Ala at position 327 is substituted with Asp; (ae) a modification in which Leu at position 328 is substituted with Phe; (af) a change in which Pro at position 271 is substituted with Leu; (ag) a modification in which Ser at position 267 is substituted with Glu; (ah) a change in which Leu at position 328 is substituted with Ala; (ai) a change in which Leu at position 328 is substituted with Ile; (aj) a change in which Leu at position 328 is substituted with Gln; (ak) a change in which Leu at position 328 is substituted with Val; (al) a change in which Lys at position 326 is substituted with Trp; (am) a modification in which Lys at position 334 is substituted with Arg; (an) a modification in which His at position 268 is substituted with Gly; (ao) a change in which His at position 268 is substituted with Asn; (ap) a modification of substituting Val for Ser at position 324; (aq) a change in which Val at position 266 is substituted with Leu; (ar) a change in which Pro at position 271 is substituted with Gly; (as) a modification in which Ile at position 332 is substituted with Phe; (at) a change in which Ser at position 324 is substituted with Ile; (au) a change in which Glu at position 333 is substituted with Pro; (av) a modification to replace Tyr at position 300 with Asp; (aw) a change in which Ser at position 337 is substituted with Asp; (ax) a modification to replace Tyr at position 300 with Gln; (ay) a modification to replace Thr at position 335 with Asp; (az) a modification in which Ser at position 239 is substituted with Asn; (ba) a change in which Lys at position 326 is substituted with Leu; (bb) a change in which Lys at position 326 is substituted with Ile; (bc) a modification of substituting Glu for Ser at position 239; (bd) a change in which Lys at position 326 is substituted with Phe; (be) a modification of substituting Val for Lys at position 326; (bf) a modification in which Lys at position 326 is substituted with Tyr; (bg) a change in which Ser at position 267 is substituted with Asp; (bh) a change in which Lys at position 326 is substituted with Pro; (bi) a change in which Lys at position 326 is substituted with His; (bj) a change in which Lys at position 334 is substituted with Ala; (bk) a change in which Lys at position 334 is substituted with Trp; (bl) a modification in which His at position 268 is substituted with Gln; (bm) a modification in which Lys at position 326 is substituted with Gln; (bn) a modification of substituting Glu for Lys at position 326; (bo) a change in which Lys at position 326 is substituted with Met; (bp) a change in which Val at position 266 is substituted with Ile; (bq) a modification of substituting Glu for Lys at position 334; (br) a modification to replace Tyr at position 300 with Glu; (bs) a change in which Lys at position 334 is substituted with Met; (bt) a modification of substituting Val for Lys at position 334; (bu) a modification in which Lys at position 334 is substituted with Thr; (bv) a modification of substituting Ser for Lys at position 334; (bw) a modification in which Lys at position 334 is substituted with His; (bx) a change in which Lys at position 334 is substituted with Phe; (by) a modification in which Lys at position 334 is substituted with Gln; (bz) a modification of substituting Pro for Lys at position 334; (ca) a change in which Lys at position 334 is substituted with Tyr; (cb) a change in which Lys at position 334 is substituted with Ile; (cc) a change in which Gln at position 295 is substituted with Leu; (cd) a change in which Lys at position 334 is substituted with Leu; (ce) a modification in which Lys at position 334 is substituted with Asn; (cf) a modification in which His at position 268 is substituted with Ala; (cg) a modification in which Ser at position 239 is substituted with Asp; (ch) a change in which Ser at position 267 is substituted with Ala; (ci) a change in which Leu at position 234 is substituted with Trp; (cj) a modification in which Leu at position 234 is substituted with Tyr; (ck) a modification in which Gly at position 237 is substituted with Ala; (cl) a modification in which Gly at position 237 is substituted with Asp; (cm) a change in which Gly at position 237 is substituted with Glu; (cn) a change in which Gly at position 237 is substituted with Leu; (co) a modification in which Gly at position 237 is substituted with Met; (cp) a modification in which Gly at position 237 is substituted with Tyr; (cq) a modification in which Ala at position 330 is substituted with Lys; (cr) a modification in which Ala at position 330 is substituted with Arg; (cs) a change in which Glu at position 233 is substituted with Asp; (ct) a modification in which His at position 268 is substituted with Asp; (cu) a modification of substituting Glu for His at position 268; (cv) a change in which Lys at position 326 is substituted with Asp; (cw) a modification of substituting Ser for Lys at position 326; (cx) a modification in which Lys at position 326 is substituted with Thr; (cy) a change in which Val at position 323 is substituted with Ile; (cz) a change in which Val at position 323 is substituted with Leu; (da) a modification in which Val at position 323 is substituted with Met; (db) a modification in which Tyr at position 296 is substituted with Asp; (dc) a modification of substituting Ala for Lys at position 326; (dd) a change in which Lys at position 326 is substituted with Asn; (de) It may be a modification in which Ala at position 330 is substituted with Met.

The above modification may be performed at only one place, or may be a combination of two or more places. Alternatively, as a preferred example of such a modification, those described in Tables 14 to 15, 17 to 24, and 26 to 28 of WO2013/047752, for example, human IgG (IgG1, IgG2, IgG3, or IgG4) 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, a human normal region or a variant of the human Fc region. 234, 237, 264, 265, 266, 267, 268, 269, 272, 296, 326, 327, 330, 331, 332, 333, and 396 One or more of the above may be substituted. In this case, as a variant, any one of Asp at 233, Tyr at 234, Asp at 237, Ile at 264, Glu at 265, Phe, Met, and Leu at 266 according to EU numbering, Any one of Ala, Glu, Gly, and Gln at position 267, any of Asp and Glu at position 268, Asp at position 269, Asp at position 272, Phe, Ile, Met, Asn, and any one of Gln, 296 Asp above, any one of Ala and Asp at 326, Gly at 327, Lys and Arg at 330, Ser at 331, Thr at 332, Thr, Lys, and Arg at 333. One, and Asp, Glu, Phe, Ile, Lys, Leu, Met, Gln, Arg, and a variant of the human Fc region containing one or more of any one or more of the 396 position. It is not limited to these.

In an alternative embodiment, the antibody of Initiation A comprising an Fc region variant or a constant region variant maintains the binding activity to FcγRIIb as compared to a reference antibody comprising a native IgG constant region or an Fc region. Alternatively, it may be increased, and the binding activity to FcγRIIa (H type) and FcγRIIa (R type) may be decreased. Preferred amino acid substitution sites for these variants are, for example, amino acids at position 238 according to EU numbering, and 233 positions, 234 positions, 235 positions, 237 positions according to EU numbering, as reported in WO2014/030728, 264th, 265th, 266th, 267th, 268th, 269th, 271th, 272th, 274th, 296th, 326th, 327th, 330th, 331th, 332th, 333th, 334th , At least one amino acid position selected from the group consisting of 355th, 356th, 358th, 396th, 409th and 419th position may be sufficient.

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

In an alternative embodiment, the antibody of Initiation A comprising an Fc region variant or a constant region variant maintains the binding activity to FcγRIIb as compared to a reference antibody comprising a native IgG constant region or an Fc region. In addition, binding activity to all active FcγR, among which FcγRIIa (R type) may be reduced. Preferred amino acid substitution sites for these variants are as reported in WO2014/163101, for example, in addition to amino acids at position 238 according to EU numbering, position 235, position 237, position 241, 268 according to EU numbering, It may be at least one amino acid position selected from position 295, 296, 298, 323, 324, and 330. More preferably, the variant is Asp at position 238, Phe at position 235, Gln or Asp at position 237, Met or Leu at position 241, Pro at position 268, Met or Val at position 295 according to EU numbering, Even if it has at least one amino acid selected from the amino acid group of Glu, His, Asn or Asp at position 296, Ala or Met at position 298, Ile at position 323, Asn or His at position 324, and His or Tyr at position 330 do.

In the range described in the disclosure A in this specification, the degree to which the "FcγRIIb-binding activity is maintained" is not limited, but is not limited to 55% or more, 60% or more, 65% or more, 70% or more, 75 % Or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more , 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 100% or more, 101% or more, 102% or more, 103 % Or more, 104% or more, 105% or more, 106% or more, 107% or more, 108% or more, 109% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 175% or more , Or more than twice.

In the range described in the disclosure A in this specification, the degree to which "all active FcγR, especially FcγRIIa (R-type) binding activity is reduced" is not limited, but 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.

In addition, in WO2014/030750, there are also reported modifications of the constant region and the Fc region in mice. In one embodiment, the antibody of initiation A or B may contain the said variant.

In the range of disclosures A and B in this specification, unlike FcγR belonging to the immunoglobulin superfamily, "FcRn", especially human FcRn, is structurally and structurally with the polypeptide of the major histocompatibility complex (MHC) class I. It is similar and exhibits 22% to 29% sequence identity to MHC class I molecules (Ghetie et al., Immunol. Today 18 (12), 592-598 (1997)). FcRn is expressed as a heterodimer composed of a transmembrane α or heavy chain complexed with a soluble β or light chain (β2 microglobulin). Like MHC, the α chain of FcRn contains three extracellular domains (α1, α2, and α3), and the short cytoplasmic domain allows the protein to bind to the cell surface. The α1 and α2 domains interact with the FcRn binding domain of the antibody Fc region (Raghavan et al., (Immunity 1: 303-315 (1994)).

FcRn is expressed in the maternal placenta and yolk sac of mammals, and is involved in the transfer of IgG from mother to fetus. In addition, in the intestine of newborn rodents in which FcRn is expressed, FcRn is involved in the migration of maternal IgG from ingested colostrum or milk across the dentate epithelium. FcRn is expressed in a variety of different tissues and endothelial cell lines of various species. FcRn is also expressed in human adult vascular endothelium, musculovascular system, and hepatic oriental capillaries. It is thought that FcRn plays a role in maintaining the plasma concentration of IgG by binding to IgG and recycling the IgG to serum. Typically, binding of FcRn to an IgG molecule is strictly pH dependent. Optimal binding is recognized in the acidic pH range below 7.0.

As an example, the polynucleotide sequence and amino acid sequence of human FcRn may be derived from the precursors described in NM_004107.4 and NP_004098.1 (including the signal sequence), respectively (in parentheses the RefSeq registration number is indicated.).

This precursor forms a complex with human β2-microglobulin in vivo. Therefore, for appropriate use in various experimental systems, a soluble human FcRn capable of forming a complex with human β2-microglobulin may be prepared using a known recombinant expression method. Using such a soluble human FcRn, an antibody or an Fc region modified variant may be evaluated for the binding activity to FcRn. In Initiation A or B, FcRn is not particularly limited as long as it is a form capable of binding to an FcRn-binding domain, but human FcRn is a preferred FcRn.

In the ranges described in the disclosures A and B in this specification, when the antibody or Fc region variant has a binding activity to FcRn, they may have a "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 or affinity for FcRn at acidic pH and/or neutral pH, or may be a domain having activity to bind FcRn directly or indirectly. As such a domain, the Fc region of an IgG-type immunoglobulin having an activity that directly binds to FcRn, albumin, albumin domain 3, anti-FcRn antibody, anti-FcRn peptide, and anti-FcRn framework molecule, and indirectly binds to FcRn. Molecules that bind to IgG and albumin having activity may be mentioned, but are not limited thereto. In the initiation A or B, a domain having FcRn-binding activity in an acidic pH range and/or a neutral pH range may be used. This domain can also be used without further alteration when it originally has FcRn-binding activity in an acidic pH range and/or a neutral pH range. When there is no or weak FcRn-binding activity in the acidic pH range and/or neutral pH range of the domain, the amino acid residue in the FcRn-binding domain in the antibody or Fc region modified body is altered, and the pH acidic range and/or pH neutral Inverse FcRn binding activity may be obtained. Alternatively, the amino acid of the domain having FcRn-binding activity in the original pH acidic region and/or the neutral pH region may be altered to further increase the FcRn-binding activity. By comparing the FcRn-binding activity in the acidic pH range and/or the neutral pH range before and after amino acid modification, the desired amino acid modification of the FcRn-binding domain can be found.

In some cases, the FcRn-binding domain is preferably a region that directly binds to FcRn. Preferred examples of the FcRn-binding domain include the antibody constant region and the Fc region. However, a region capable of binding to a polypeptide having FcRn-binding activity, such as albumin or IgG, can indirectly bind to FcRn via albumin or IgG. Therefore, the FcRn-binding region may be a region that binds to a polypeptide having binding activity to albumin or IgG. Although not limited, the FcRn-binding domain preferably has high FcRn-binding activity at neutral pH in order to promote the disappearance of antigen from plasma, and in order to improve the retention of the antibody in plasma, FcRn It is preferable that the binding domain has a high FcRn binding activity at an acidic pH. For example, an FcRn-binding domain having originally high FcRn-binding activity at neutral pH or acidic pH can be selected. Alternatively, an antibody or an amino acid in the Fc region modified body may be modified to impart FcRn binding activity at neutral pH or acidic pH. Alternatively, you may increase the existing FcRn-binding activity at neutral pH or acidic pH.

In the range described in the disclosures A and B in this specification, the binding activity of the antibody or Fc region (variant) to FcRn is increased compared to the antibody or Fc region (variant) before modification, or (substantially ) Whether being maintained or reduced, known methods, e.g., the methods described in the examples in this specification, and e.g. BIACORE, Scatchard plot, and flow cytometer (see WO2013/046722) You may evaluate using. In these assays, the extracellular domain of human FcRn may be used as a soluble antigen. Conditions other than pH when measuring the binding activity of the antibody or the Fc region (modified) to FcRn can be appropriately selected by those skilled in the art. For example, as described in WO2009/125825, it is possible to carry out the assay under the conditions of MES buffer and 37°C. Measurement of the binding activity to FcRn of the antibody or the Fc region (modified) may be evaluated by, for example, flowing FcRn as an analyte on a chip to which the antibody is immobilized.

The binding activity of the antibody or the Fc region (variant) to FcRn can be evaluated based on the dissociation constant (KD), the apparent dissociation constant (apparent KD), the dissociation rate (kd), and the apparent dissociation rate (apparent kd).

As the pH condition for measuring the binding activity of the FcRn binding domain and FcRn contained in the antibody or the Fc region (variant), an acidic pH condition or a neutral pH condition can be appropriately used. As the temperature conditions used for the measurement, the binding activity (binding affinity) of the FcRn-binding domain and FcRn can be evaluated at any temperature of 10°C to 50°C. Preferably, in order to determine the binding activity (binding affinity) of the human FcRn binding domain and FcRn, a temperature of 15°C to 40°C is used. More preferably, any of 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 The temperature of may be used. 25°C is a non-limiting example of such a temperature.

In one embodiment, when the initiating A or B antibodies 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 antibody has a binding activity or affinity for FcRn at acidic pH and/or neutral pH, and may be a domain having an activity that directly or indirectly binds to FcRn. In a specific embodiment, it is preferable that the antibody of initiation A or B has increased binding activity to FcRn under conditions of neutral pH as compared to a reference antibody containing, for example, a constant region of a native IgG. Do (see WO2013/046722). Although not limited, the reference antibody including the antibody of the initiation A or B and the constant region of a native IgG is preferably other than the normal region of the antibody of the initiation A or B in which one or more amino acid residues have been modified. In some cases, it is preferable to say that the amino acid sequence of the region (for example, the variable region) is the same from the viewpoint of comparing the binding activity to FcRn of both.

In one embodiment within the range described in the disclosure A in this specification, when the antibody of Initiation A has increased binding activity to FcRn under conditions of neutral pH, it is not limited to a specific theory, but disclosure The antibody of A has an ion concentration-dependent antigen-binding domain, so that it repeatedly binds to a plurality of antigens as a single antibody molecule through endosomes in plasma and cells; a property of being rapidly absorbed into cells by increasing the pi and increasing the positive charge as a whole of the antibody; And any two or more of the characteristics of being rapidly absorbed into cells by increasing the binding activity to FcRn under conditions of neutral pH. As a result, the half-life of the antibody in plasma can be further shortened, or the binding property of the antibody to the extracellular matrix can be further increased, or the disappearance of the antigen from the plasma can be further promoted. In order to take advantage of these characteristics, those skilled in the art may determine the optimum pI value for the initiating A antibody.

In the range describing the disclosures A and B in this specification, according to Yeung et al. (J. Immunol. 182:7663-7671 (2009)), the human FcRn binding activity of natural human IgG1 is in the acidic pH range ( pH 6.0), the KD is 1.7 μM, but the activity is hardly detectable in the neutral pH range. Therefore, in order to increase the FcRn binding activity in the neutral pH region, for example, the human FcRn binding activity in the acidic pH region is KD 20 μM or stronger, and the human FcRn binding activity in the neutral pH region is natural human Antibody or constant region variant or Fc region variant equivalent to or stronger than IgG; Preferably, the human FcRn-binding activity in the acidic pH region is KD 2.0 μM or stronger, and the human FcRn-binding activity in the neutral pH region KD 40 μM or higher, antibody or constant region modified or Fc region modified ; More preferably, the human FcRn-binding activity in the acidic pH range is KD 0.5 μM or stronger, and the human FcRn-binding activity in the neutral pH range KD 15 μM or more, antibody or constant region modified or Fc region dog It is preferable to use the variant as an antibody of initiation A or B. The KD value described above is determined by the method described in Yeung et al. (J. Immunol. 182: 7663-7671 (2009)) (by immobilizing an antibody on a chip and flowing human FcRn as an analyte). .

In the range described in the disclosures A and B in this specification, a domain of any structure that binds to FcRn can be used as the FcRn-binding domain. In that case, the FcRn binding domain can be produced without requiring the introduction of amino acid modifications, and furthermore, the affinity for FcRn may be enhanced by introducing modifications.

In the range described in the disclosures A and B in this specification, examples of the starting FcRn-binding domain include the Fc region or the constant region of (human) IgG. Any Fc region or normal region can be used as a starting Fc region or a starting normal region, as long as the starting Fc region or the variant of the starting normal region can bind to FcRn in the acidic pH region and/or in the neutral pH region. Alternatively, an Fc region or a constant region obtained by adding additional modifications to a starting Fc region or a starting normal region to which amino acid residue modifications have already been added to the Fc region or constant region can also be suitably used as the Fc region or the normal region. The starting Fc region or the starting normal region may contain a known Fc region produced by recombination. Depending on the context, the starting Fc region or starting constant region may mean a polypeptide itself, a starting Fc region or a composition comprising a starting constant region, or an amino acid sequence encoding a starting Fc region or a starting constant region. The origin of the starting Fc region or the starting normal region is not limited, but can be obtained from any organism or human of a non-human animal. In addition, the starting FcRn binding domain can be obtained from Philippine monkeys, marmosets, rhesus monkeys, chimpanzees, and humans. The starting Fc region or starting constant region may be obtained from human IgG1, but is not limited to any specific class of IgG. This means that the Fc region of human IgG1, IgG2, IgG3, or IgG4 can be appropriately used as the starting FcRn binding domain, and the Fc region or normal region of the class or subclass of IgG from any organism is used as the starting Fc. It means that it can be used as an area or a starting normal area. Examples of native IgG variants or modifications are, 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)); WO2009/086320; WO2008/092117; WO2007/041635; And WO2006/105338.

In the range described in the disclosure A and B in this specification, examples of the amino acid residues of the starting FcRn binding domain or the starting Fc region or the starting constant region include one or more mutations, for example, Substitutional mutations with amino acid residues different from the amino acid residues; Or insertion of one or more amino acid residues into the amino acid residues of the starting Fc region or starting normal region; Alternatively, deletion of one or more amino acid residues from the amino acid residues of the starting Fc region or the starting constant region may be included. Preferably, the amino acid sequence of the Fc region or the constant region after the modification is an amino acid sequence containing at least a portion of the Fc region or the constant region that cannot occur naturally. Such variants inevitably have less than 100% sequence identity or similarity to the starting Fc region or starting normal region. For example, the modifications may be from about 75% to less than 100%, more preferably from about 80% to less than 100%, even more preferably from about 85% to the amino acid sequence of the starting Fc region or starting normal region. It has an amino acid sequence identity or similarity of less than 100%, even more preferably about 90% to less than 100%, even more preferably about 95% to less than 100%. In a non-limiting example, at least one amino acid is different between the starting Fc region or the starting normal region and the Fc region or normal region in which the starting A or B is modified.

In the ranges described in the disclosures A and B in this specification, the Fc region or constant region having FcRn-binding activity in the acidic pH range and/or the neutral pH range may be obtained by any method. Specifically, by alteration of the amino acid of a human IgG-type antibody that can be used as a starting Fc region or a starting constant region, the Fc region or normal having binding activity to FcRn in the acidic pH range and/or the neutral pH range. A variant of the domain can be acquired. As the Fc region or constant region of an IgG-type antibody suitable for modification, for example, an Fc region or a constant region of human IgG (IgG1, IgG2, IgG3, and IgG4, and their variants) can be exemplified, and variants naturally occurring therefrom Also, it is included in the Fc region or the normal region of IgG. As the Fc region or constant region of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, plural allotype sequences by genopolymorphism are described in "Sequences of proteins of immunological interest", NIH Publication No. Although described in 91-3242, any of them may be used at the start A or B. In particular, as the sequence of human IgG1, the amino acid sequence at positions 356 to 358 according to EU numbering may be DEL or EEM.

In one embodiment of the initiation A or B, the modification to another amino acid is as long as the obtained modified product has FcRn-binding activity in the acidic pH range and/or the neutral pH range, preferably in the neutral pH range. , It is not particularly limited. As an amino acid modification site for enhancing the binding activity to FcRn under conditions of neutral pH, it is described, for example, in WO2013/046722 and the like. As such a modified site, for example, as described in WO2013/046722, in the Fc region or the normal region of a human IgG-type antibody, according to EU numbering, from 221st to 225th, 227th, 228th, 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, 325th, 327th to 339th, 341th, 343th, 345th, 360th, 362th, 370th, 375th to 378th, 380th, 382th, 385th to 387th, 389th, 396th, 414th , One or more positions selected from the group consisting of 416th, 423th, 424th, 426 to 438th, 440th and 442th place may be mentioned. In addition, in WO2013/046722, as part of a preferred modification in the normal region or the Fc region, for example, according to EU numbering, the amino acid at position 256 is Pro, the amino acid at position 280 is Lys, and the amino acid at position 339 is. 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, and the modification of one or more amino acids selected from the group consisting of Has been. In addition, the number of amino acids to be modified is not particularly limited, and the amino acid position of only one place may be changed, and two or more positions may be changed. By alteration of these amino acid residues, binding to FcRn in the Fc region or constant region of the IgG-type antibody under conditions of neutral pH can be increased. Initiation A or B antibody may be appropriately introduced with modifications of these amino acid residues.

In a further embodiment or an alternative embodiment, an amino acid modification site for enhancing the binding activity to FcRn under acidic pH conditions may also be appropriately used. Among such modified sites, one or more modified sites capable of increasing binding to FcRn even in the neutral pH range can be suitably used for initiation A or B. Such modified sites are reported in, for example, WO2011/122011, WO2013/046722, WO2013/046704, and WO2013/046722. The amino acid sites that allow such modifications in the constant region or the Fc region of a human IgG-type antibody and the types of amino acids after the modification are reported in Table 1 of WO2013/046722. In addition, in WO2013/046722, as a particularly preferred modified site in the normal region or the Fc region, for example, according to EU numbering, 237th, 238th, 239th, 248th, 250th, 252nd, 254th, 255th, 256th, 257th, 258th, 265th, 270th, 286th, 289th, 297th, 298th, 303th, 305th, 307th, 308th, 309th, 311th, 312th , 314th, 315th, 317th, 325th, 332th, 334th, 360th, 376th, 380th, 382th, 384th, 385th, 386th, 387th, 389th, 424th, 428 One or more amino acid positions selected from the group consisting of the above, 433 position, 434 position and 436 position may be mentioned. Also by alteration at these amino acid residue positions, the binding to human FcRn in the neutral pH range of the FcRn binding domain can be increased. In WO2013/046722, as part of a preferred modification in the IgG-type normal region or the Fc region, for example, according to EU numbering, (a) amino acid at position 237 as Met; (b) Ala for the amino acid at position 238; (c) Lys for the amino acid at position 239; (d) amino acid at position 248 to Ile; (e) one of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, and Tyr for the amino acid at position 250; (f) the amino acid at position 252 is one of Phe, Trp, and Tyr; (g) the amino acid at position 254 as Thr; (h) Glu for the amino acid at position 255; (i) the amino acid at position 256 is any one of Asp, Glu, and Gln; (j) the amino acid at position 257 is any one of Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, and Val; (k) the amino acid at position 258 as His; (l) the amino acid at position 265 as Ala; (m) the amino acid at position 270 as Phe; (n) the amino acid at position 286 is either Ala or Glu; (o) the amino acid at position 289 as His; (p) Ala for the amino acid at position 297; (q) the amino acid at position 298 as Gly; (r) the amino acid at position 303 as Ala; (s) the amino acid at position 305 as Ala; (t) any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr for the amino acid at position 307; (u) any one of Ala, Phe, Ile, Leu, Met, Pro, Gln, and Thr for the amino acid at position 308; (v) any one of Ala, Asp, Glu, Pro, and Arg for the amino acid at position 309; (w) the amino acid at position 311 is any one of Ala, His, and Ile; (x) the amino acid at position 312 is either Ala or His; (y) the amino acid at position 314 is either Lys or Arg; (z) the amino acid at position 315 is either Ala or His; (aa) the amino acid at position 317 as Ala; (ab) the amino acid at position 325 as Gly; (ac) the amino acid at position 332 as Val; (ad) Leu for amino acid at position 334; (ae) the amino acid at position 360 as His; (af) amino acid at position 376 as Ala; (ag) the amino acid at position 380 as Ala; (ah) Ala for the amino acid at position 382; (ai) Ala for the amino acid at position 384; (aj) the amino acid at position 385 is either Asp or His; (ak) Pro for amino acid at position 386; (al) Glu for amino acid at position 387; (am) the amino acid at position 389 is either Ala or Ser; (an) Ala for the amino acid at position 424; (ao) any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr for the amino acid at position 428; (ap) Lys for amino acid at position 433; (aq) amino acid at position 434 as Ala, Phe, His, Ser, Trp, and Tyr; And (ar) modification of at least one amino acid residue selected from the group consisting of the amino acid at position 436 as His. In addition, the number of amino acids to be modified is not particularly limited, and only one position may be altered, and two or more positions may be altered. As a combination of modifications of two or more amino acids, it is described in Table 2 of WO2013/046722, for example. In the antibodies of initiation A and B, modifications of these amino acid residues may be appropriately introduced.

In one embodiment, the FcRn-binding activity of the FcRn-binding domain of the initiating A or B antibody is in the FcRn of a native IgG Fc region or constant region, or a reference antibody comprising a starting Fc region or a starting constant region. Compared with the binding activity to, it is increased. That is, the binding activity to FcRn of the Fc region variant or constant region variant of initiation A or B, or an antibody containing such a variant is higher than that of the reference antibody to FcRn. This indicates that the binding activity to FcRn of the antibody of initiation A or B is compared with the binding activity to FcRn of the reference antibody, for example, 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, more 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% It means that there is a possibility that it is more than 195%, more than 2 times, more than 2.5 times, more than 3 times, more than 3.5 times, more than 4 times, more than 4.5 times, or more than 5 times.

In one embodiment, in order not to increase the immunogenicity of the antibody when the antibody is administered in vivo (preferably in the human body), the amino acid sequence modified in the initiating A or B antibody is a human sequence (human It is preferred that the sequence found in the derived natural antibody) can be included. Alternatively, after the modification, a mutation may be introduced at a site other than the amino acid modification position in which one or more FRs (FR1, FR2, FR3, and FR4) are substituted with a human sequence. A method of replacing FR with a human sequence is known in the art, and Ono et al., Mol. Immunol. 36(6): Includes, but is not limited to, those reported in 387-395(1999). In addition, humanization methods are known in the art, and include, but are not limited to, those reported in Methods 36(1): 43-60(2005).

In one embodiment, the sequence of the framework region of the heavy and/or light chain variable region of the initiating A or B antibody (also referred to as “FR sequence”) may include a human germline framework sequence. If the framework sequence is a sequence of a completely human germline, then when administered to a human (for example, for the purpose of treating or preventing a disease), the antibody is expected to cause little or no immunogenic response. I can.

As the FR sequence, it is preferable that a completely human FR sequence, such as those shown in V-Base (vbase.mrc-cpe.cam.ac.uk/), can be included. These FR sequences can be suitably used for initiation A or B. Sequences of germline lines can be classified based on their similarity (Tomlinson et al. (J. Mol. Biol. 227: 776-798 (1992)); Williams et al. (Eur. J. Immunol. 23: 1456-1461 (1993)); And Cox et al. (Nat. Genetics 7: 162-168 (1994))). Moreover, a sequence of a suitable germline line can be appropriately selected from Vκ classified into 7 subgroups, Vλ classified into 10 subgroups, and VH classified into 7 subgroups.

As a fully human VH sequence, for example, a VH1 subgroup (e.g., VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45, VH1-46, VH1-58, And VH1-69); VH2 subgroups (eg, VH2-5, VH2-26, and VH2-70); VH3 subgroups (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); VH4 subgroups (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, and VH4-61); VH5 subgroup (VH5-51); VH6 subgroup (VH6-1); Alternatively, the VH sequence of the VH7 subgroup (VH7-4, and VH7-81) is preferably mentioned. These are also described, for example, in Matsuda et al. (J. Exp. Med. 188: 1973-1975 (1998)), and those skilled in the art can appropriately design antibodies based on their sequence information. Other completely human FR sequences or sequences of regions equivalent thereto may also be suitably used.

As a completely human Vκ sequence, for example, A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23, L24, O2 classified into the Vk1 subgroup, O4, O8, O12, O14, or O18; A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11 classified in the Vk2 subgroup; A11, A27, L2, L6, L10, L16, L20, and L25 classified in the Vk3 subgroup; B3 classified in the Vk4 subgroup; B2 (also referred to as "Vk5-2") classified in the Vk5 subgroup; Or A10, A14, and A26 classified as Vk6 subgroups (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)) are preferably mentioned.

As a completely human Vλ sequence, for example, V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1- classified into the VL1 subgroup 16, V1-17, V1-18, V1-19, V1-20, and V1-22; V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19 classified in the VL2 subgroup; V3-2, V3-3, and V3-4 classified in the VL3 subgroup; V4-1, V4-2, V4-3, V4-4, and V4-6 classified in the VL4 subgroup; Alternatively, V5-1, V5-2, V5-4, and V5-6, etc. classified into the VL5 subgroup (Kawasaki et al. (Genome Res. 7:250-261 (1997))) are preferably mentioned.

Usually, these FR sequences differ from each other in one or more amino acid residues. These FR sequences can be used for alteration of amino acid residues of an antibody. Moreover, examples of fully human FR sequences that can be used for modification include KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (e.g., Kabat et al. (1991) described above). ; See Wu et al. (J. Exp. Med. 132, 211-250 (1970)).

In the range describing the disclosures A and B in this specification, "flexible residue" means a light chain having several different amino acids when comparing the amino acid sequences of known and/or natural antibodies or antigen-binding domains. Variation of amino acid residues at a position in which the variable region or the heavy chain variable region exhibits high amino acid diversity can be said. Positions exhibiting high amino acid diversity are generally present within the CDRs. In determining such a wide variety of positions in known and/or native antibodies, the data provided by Kabat, Sequences of Proteins of Immunological Interest (National Institute of Health Bethesda Md.) (1987 and 1991) are available. It can be available. In addition, in a plurality of databases on the Internet (vbase.mrc-cpe.cam.ac.uk/, bioinf.org.uk/abs/index.html), the sequences and arrangements of a number of human light and heavy chains collected It is being provided. Information on these sequences and arrangements is useful for determining the position of flexible residues. Although not limited, for example, the amino acid residue is at any specific position, preferably 2 to 20, 3 to 19, 4 to 18, 5 to 17, 6 to 16, 7 to 15, 8 to 14, When there is a variation of 9 to 13, or 10 to 12 amino acid residues, the position can be judged to represent (high) diversity.

In one embodiment, when the antibody of initiation A or B contains all or part of the light chain variable region and/or the heavy chain variable region, the antibody may appropriately contain one or more flexible residues, as desired. I can understand. For example, in the sequence of the heavy chain and/or light chain variable region selected as the FR sequence originally containing amino acid residues that change the binding activity of the antibody to the antigen according to the condition of the ion concentration (hydrogen ion concentration or calcium ion concentration). , In addition to the amino acid residue, other amino acid residues may be designed to be included. In this case, as long as the binding activity of the antibody of initiation A or B to the antigen changes according to the condition of the ion concentration, for example, the number and position of the flexible residues can be determined without being limited to a specific embodiment. That is, at least one flexible residue may be included in the CDR sequence and/or the FR sequence of the heavy and/or light chain. For example, when the ion concentration is calcium ion concentration, as a non-limiting example of flexible residues that can be introduced into the light chain variable region sequence (Vk5-2 described above), the position of one or more amino acid residues listed in Table 1 or Table 2 Can be mentioned. Likewise, for example, in an ion concentration-dependent antibody or an antibody not having the ion concentration dependence, including all or part of the light chain variable region and/or the heavy chain variable region, at least one that can be exposed to the surface of the antibody. Also in the case where the amino acid residues of dogs are modified so that the pI rises, flexible residues can be appropriately introduced together.

(Location is indicated according to Kabat numbering.)

(Location is indicated by Kabat numbering.)

In one embodiment, when a chimeric antibody is humanized, the pI of the chimeric antibody is increased by modifying one or more amino acid residues that may be exposed on the surface of the chimeric antibody, compared with a chimeric antibody without such modification. Thus, a humanized antibody of initiation A or B with a shortened half-life in plasma can be produced. The alteration of amino acid residues that can be exposed on the surface of the humanized antibody may be performed before or simultaneously with humanization of the antibody. Alternatively, the pi of the humanized antibody may be further modified by using a humanized antibody as a starting material and modifying the amino acid residues that may be exposed on the surface thereof.

Adams et al. (Cancer Immunol. Immunother. 55(6): 717-727 (2006)), humanized antibodies trastuzumab (antigen: HER2), bevacizumab humanized using the FR sequence of the same human antibody. (Antigen: VEGF), and pertuzumab (antigen: HER2) in plasma have been reported to have almost the same pharmacokinetics. In particular, when humanization is performed using the same FR sequence, it can be understood that the pharmacokinetics in plasma are almost equivalent. In one embodiment of Initiation A, in addition to the humanization process, the plasma antigen concentration is lowered by raising the pi of the antibody by alteration of amino acid residues that may be exposed on the surface of the antibody. In an alternative embodiment of initiation A or B, human antibodies can be used. From the plasma of the first human antibody produced by modifying the amino acid residues that can be exposed on the surface of human antibodies produced from human antibody libraries, human antibody-producing mice, recombinant cells, etc. to increase the pi of the human antibody It is possible to increase the ability to lose antigen of

In one embodiment, the antibody of the present disclosure A may contain a modified sugar chain. Examples of antibodies with altered sugar chains include, for example, antibodies with altered glycosylation (WO99/54342), antibodies with a deletion of fucose (WO00/61739, WO02/31140, WO2006/067847, WO2006/067913, etc.), and And antibodies (such as WO02/79255) having a sugar chain having a bisecting GlcNAc.

In one embodiment, the antibody of initiation A or B may be used, for example, in a technique that exhibits increased anti-tumor activity against cancer cells, or in a technique that promotes the loss of an antigen harmful to a living body from plasma. do.

In an alternative embodiment, initiation A or B relates to a library of ion concentration dependent antigen binding domains with elevated pI or ion concentration dependent antibodies with elevated pI as described above.

In an alternative embodiment, initiation A or B relates to a nucleic acid (polynucleotide) encoding an ion concentration dependent antigen binding domain with elevated pI or an ion concentration dependent antibody with elevated pI described above. In a specific embodiment, a nucleic acid can be obtained by appropriately using a known method. As a specific embodiment, for example, WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012/115241, WO2013/047752, WO2013/125667, WO2014 /030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, and WO2012/093704, each of which may be incorporated herein by reference. have.

In one embodiment, the nucleic acid of initiation A or B may be an isolated or purified nucleic acid. The nucleic acid encoding the antibody of initiation A or B may be any gene, DNA or RNA, or other nucleic acid analogues.

When the amino acid in the antibody is modified within the range described in the disclosures A and B in this specification, the amino acid sequence of the antibody before the modification may be a known sequence, or may be the amino acid sequence of a newly obtained antibody. For example, the antibody can be obtained from an antibody library, or a nucleic acid encoding the antibody can be cloned and obtained from a hybridoma or B cell that produces a monoclonal antibody. As a method of obtaining a nucleic acid encoding an antibody from a hybridoma, the following techniques can be used: Immunization is performed according to a conventional immunization method using a target antigen or a cell expressing the target antigen as a sensitizing antigen. Technology; A technique of fusing the resulting immune cells with known parental cells according to a conventional cell fusion method; A technique of screening monoclonal antibody-producing cells (hybridoma) by a conventional screening method; A technique of synthesizing the cDNA of the variable region (V region) of the antibody using reverse transcriptase from the obtained hybridoma mRNA; And a technology for linking the cDNA with DNA encoding the antibody constant region (region C) of interest.

Although not limited to the following examples, the sensitizing antigens used to obtain nucleic acids encoding the heavy and light chains described above include both a complete antigen having immunogenicity and an incomplete antigen containing a hapten that does not exhibit immunogenicity. For example, a target full-length protein or a partial peptide of the protein can be used. In addition, it is known that substances composed of polysaccharides, nucleic acids, lipids, and other compositions can be antigens, and thus, in some embodiments, the antigen of the initiating A or B antibody is not particularly limited. The antigen can be prepared, for example, by a method using a baculovirus (see, for example, WO98/46777) or the like. The production of hybridomas is described, for example, in G. Kohler and C. Milstein, Methods Enzymol. 73: It can be done according to the method of 3-46 (1981). When the immunogenicity of the antigen is low, immunization may be performed by linking the antigen with a macromolecule having immunogenicity such as albumin. Alternatively, if necessary, a soluble antigen may be prepared by linking the antigen with another molecule. When a transmembrane molecule such as a membrane antigen (e.g., a receptor) is used as an antigen, it is possible to use a portion of the extracellular region of the membrane antigen as a fragment, and cells expressing the transmembrane molecule on the surface are used as an immunogen. Can also be done.

In some embodiments, antibody-producing cells can be obtained by immunizing animals with the appropriate sensitizing antigens described above. Alternatively, lymphocytes capable of producing antibodies can be immunized in vitro to prepare antibody-producing cells. A variety of mammals can be used for immunization and other conventional antibody production techniques. Animals of rodent, rabbit, and primate orders can be used in general. Rodents such as mice, rats, and hamsters, rabbits such as rabbits, and primates including monkeys such as Philippine monkeys, rhesus monkeys, mantle baboons, and chimpanzees may be exemplified. In addition, transgenic animals having a repertoire of human antibody genes are also known, and human antibodies can also be obtained by using such animals (eg WO96/34096; Mendez et al., Nat. Genet. 15: 146). -156 (1997); see WO93/12227, WO92/03918, WO94/02602, WO96/34096, and WO96/33735). Instead of the use of such transgenic animals, for example, sensitizing human lymphocytes in vitro with a cell expressing a desired antigen or a desired antigen, and then fusing the sensitized lymphocytes with human myeloma cells, such as U266. Thus, a desired human antibody having antigen-binding activity can also be obtained (Japanese Patent Publication No. Hei 1-59878).

For immunization of animals, for example, sensitizing antigens are appropriately diluted and suspended with phosphate buffered saline (PBS) or physiological saline, and adjuvants are mixed and emulsified as necessary, and then injected intraperitoneally or subcutaneously of the animal. Done by Thereafter, preferably, the sensitizing antigen mixed with Freund's incomplete adjuvant can be administered several times every 4 to 21 days. Confirmation of the production of the antibody can be performed, for example, by measuring the titer of a target antibody in the serum of an animal.

Hybridomas can be produced by fusing antibody-producing cells obtained from animals or lymphocytes immunized with a desired antigen with myeloma cells using a commonly used fusion agent (e.g., polyethylene glycol) (Goding, Monoclonal Antibodies: Principles. and Practice, Academic Press, 1986, 59-103). If necessary, hybridoma cells are cultured and proliferated, and the binding specificity of the antibody produced from the hybridoma is determined by, for example, immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA). Measure. Thereafter, if necessary, a hybridoma producing an antibody whose target specificity, affinity, or activity has been measured may be subcloned by a technique such as a limiting dilution method.

A probe capable of specifically binding to an antibody (e.g., an oligonucleotide complementary to a sequence encoding an antibody constant region) from a hybridoma or an antibody-producing cell (such as sensitized lymphocytes) with a nucleic acid encoding the selected antibody It can be cloned using. Alternatively, the nucleic acid can be cloned from mRNA by RT-PCR. The heavy and light chains used in the production of initiating A or B production antibodies may be derived from antibodies belonging to any class and subclass of, for example, Ig-type antibodies, and IgG may be preferred.

In one embodiment, for example, the nucleic acid encoding the amino acid sequence constituting the heavy chain (all or part of) of the antibody of initiation A or B and/or the light chain (all or part of) of the antibody is a genetic engineering technique. It is changed by For example, by altering the nucleic acid residue encoding the amino acid sequence related to the constituent components of the antibody, such as a mouse antibody, rat antibody, rabbit antibody, hamster antibody, sheep antibody, or camel antibody, the heterologous antigenicity to humans Genetically recombined antibodies, such as chimeric antibodies or humanized antibodies, which are accompanied by artificial sequence alteration for the purpose of reducing or the like may be appropriately prepared. For example, a DNA encoding the variable region of an antibody derived from a mouse is ligated to a DNA encoding a constant region of a human antibody, and a sequence encoding the ligated DNA is introduced into an expression vector, and then the obtained recombinant vector Is introduced into a host to express a gene to obtain a chimeric antibody. Humanized antibodies are also referred to as reshaped human antibodies, and CDRs of antibodies isolated from non-human mammals, such as mice, and FRs of human antibodies are linked in-frame to form coding sequences. It is an antibody. The DNA sequence encoding such a humanized antibody can be synthesized by an overlap extension PCR reaction using a plurality of oligonucleotides as a template. Materials and experimental methods for overlap extension PCR are described in WO98/13388 and the like. For example, DNA encoding the amino acid sequence of the variable region of the antibody of initiation A or B can be obtained by overlap extension PCR using a plurality of oligonucleotides designed to have overlapping nucleotide sequences. The overlapping DNA is then ligated in frame to form a coding sequence with a DNA encoding a normal region. The DNA thus linked may then be inserted into an expression vector so that the DNA is expressed, and the obtained vector may be introduced into a host or host cell. The antibody encoded by the DNA can be expressed by producing the host or culturing the host cell. The expressed antibody can be appropriately purified from the host culture medium or the like (EP239400; WO96/02576). Further, for example, the FR of a humanized antibody linked via a CDR may be selected so as to form an antigen-binding site suitable for the antigen in the CDR. If necessary, for example, amino acid residues constituting the FR of the variable region of the selected antibody may be modified by appropriate substitution.

In one embodiment, for expression of an antibody of initiation A or B or a fragment thereof, a nucleic acid cassette can be cloned into an appropriate vector. For this purpose, several types of vectors, for example phagemid vectors, can be used. Phagemid vectors may contain various constituents, including control sequences such as promoters or signal sequences, phenotypic selection genes, origins of replication, and other necessary components.

Methods for introducing desired amino acid modifications into antibodies have been established in the art. For example, at least one modified amino acid residue that may be exposed on the surface of the antibody of initiation A or B, and/or at least one capable of changing the binding activity of the antibody to the antigen depending on the condition of the ion concentration. Libraries can be constructed by introducing amino acids. In addition, if necessary, a flexible residue can be added using the method Kunkel et al. (Methods Enzymol. 154: 367-382 (1987)).

In an alternative embodiment, Initiation A relates to a vector comprising a nucleic acid encoding an ion concentration dependent antigen binding domain with an elevated pI described above or an ion concentration dependent antibody with an elevated pI described above. As a specific embodiment, for example, WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012/115241, WO2013/047752, WO2013/125667, WO2014 By the vectors described in /030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, and WO2012/093704, vectors can be obtained, all of them by reference It can be used in this specification.

In one embodiment, the nucleic acid encoding the embodiment of initiation A or B may be operably cloned (inserted) into a suitable vector and introduced into a host cell. For example, when E. coli (E. Coli) is used as a host, examples of the vector include a cloning vector, a pBluescript vector (made by Stratagene), or any of various commercially available vectors.

In one embodiment, as a vector containing the nucleic acid of initiation A or B, an expression vector is useful. An expression vector capable of expressing the polypeptide in vitro, in E. coli, in cultured cells, or in vivo can be used. For example, pBEST vector (manufactured by Promega) for expression in vitro, pET vector (manufactured by Invitrogen) for expression in E. coli, pME18S-FL3 vector for expression in cultured cells (GenBank Accession No. AB009864), and pME18S for expression in vivo. A vector (Takebe et al., Mol. Cell Biol. 8:466-472 (1988)) or the like may be used. Insertion of DNA into the vector can be performed by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons.See Section 11.4-11.11).

In an alternative embodiment, initiation A is a host or host cell comprising a vector comprising a nucleic acid encoding an ion concentration dependent antigen-binding domain with an elevated pi as described above or an ion concentration dependent antibody with an elevated pi above. It is about. In a specific embodiment, the host or host cell is, for example, WO2009/125825, WO2012/073992, WO2011/122011, WO2013/046722, WO2013/046704, WO2000/042072, WO2006/019447, WO2012/115241, WO2013 /047752, WO2013/125667, WO2014/030728, WO2014/163101, WO2013/081143, WO2007/114319, WO2009/041643, WO2014/145159, WO2012/016227, or WO2012/093704, and they All of each may be incorporated herein by reference.

The type of host cell of the initiation A or B is not particularly limited, and examples thereof include bacterial cells such as E. coli and various animal cells as host cells. The host cell can be suitably used as a production system for the production or expression of antibodies. Both eukaryotic cells and prokaryotic cells can be used.

Examples of eukaryotic cells used as host cells include animal cells, plant cells, and fungal cells. As animal cells, mammalian cells such as CHO (Puck et al., J. Exp. Med. 108: 945-956 (1995)), COS, HEK293, 3T3, myeloma, baby hamster kidney (BHK), HeLa , And Vero; Amphibian cells, for example African claw frog (Xenopus) oocytes (Valle et al., Nature 291:338-340 (1981)); And insect cells such as Sf9, Sf21, and Tn5. For example, introduction of recombinant vectors into host cells by using methods such as calcium phosphate method, DEAE dextran method, cationic liposome DOTAP (Boehringer Mannheim), electroporation method, and lipofection. You can do it.

Plant cells known to be useful as a protein production system include, for example, cells derived from Nicotiana tabacum and cells derived from duckweed (Lemna minor). The antibody of initiation A or B can be produced by culturing the union tissue derived from these cells. The protein production system derived from fungal cells includes yeast cells, for example, cells of the genus Saccharomyces such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; And filamentous fungi, for example, using cells of the genus Aspergillus such as Aspergillus niger. When prokaryotic cells are used, bacterial cell-based production systems can be used. The bacterial cell-based production system includes, for example, using Bacillus subtilis in addition to Escherichia coli.

In order to produce the initiating A or B antibody using the host cell, the host cell is transformed and cultured with an expression vector containing a nucleic acid encoding the initiating A or B antibody to express the nucleic acid. For example, when animal cells are used as a host, for example, DMEM, MEM, RPMI1640, and IMDM may be used as the culture medium, and these are suitable for serum stock solutions such as FBS or fetal fetal calf serum (FCS). Can be used together. In addition, cells can be cultured by serum-free culture.

On the other hand, as an in vivo production system for producing an antibody of initiation A or B, animals or plants can be used. For example, a nucleic acid encoding an antibody of initiation A or B is introduced into these animals or plants, and the antibody is produced in vivo in the animal or plant, and then recovered.

When an animal is used as a host, a living system using mammalian animals or insects can be used. As mammalian animals, goats, pigs, sheep, mice, and cattle are suitably used, but are not limited thereto (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). In addition, transgenic animals can also be used.

As an example, a nucleic acid encoding an antibody of initiation A or B is prepared as a fusion gene with a gene encoding a polypeptide specifically contained in milk, such as goat β casein. Next, a polynucleotide fragment containing this fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. An antibody of interest can be obtained from the milk produced by a transgenic goat born from a goat transplanted with an embryo or from the milk produced by its offspring. In order to increase the amount of milk containing the antibody produced from the transgenic goat, a hormone is appropriately administered to the transgenic goat (Ebert et al., Bio/Technology 12:699-702 (1994)).

As an insect producing an antibody of initiation A or B, for example, a silkworm can be used. When a silkworm is used, a baculovirus in which a polynucleotide encoding an antibody of interest is inserted into the viral genome is used in infection to the silkworm. The desired antibody can be obtained from the body fluid of the infected silkworm (Susumu et al., Nature 315: 592-594 (1985)).

When the plant is used for the production of antibodies of initiation A or B, tobacco can be used. When tobacco is used, a recombinant vector obtained as a result of inserting a polynucleotide encoding an antibody of interest into a plant expression vector, e.g., pMON530, is introduced into bacteria such as Agrobacterium tumefaciens. Can be. The obtained bacteria can be used for infection with tobacco, for example Nicotiana tabacum (Ma et al., Eur. J. Immunol. 24: 131-8 (1994)), and of the infected tobacco. The desired antibody can be obtained from the leaves. In addition, such modified bacteria can also be used for infection against duckweed (Lemna minor), and desired antibodies can be obtained from cloned infected duckweed cells (Cox et al., Nat. Biotechnol. 24). (12): 1591-1597 (2006)).

In order to secrete the antibody expressed in the host cell into the lumen of the endoplasmic reticulum, into the pericellular cavity, or into the extracellular environment, an appropriate secretion signal can be introduced into the target polypeptide. These signals may be endogenous to the antibody of interest or may be heterologous signals known in the art.

The antibody of initiation A or B thus obtained can be isolated from the host cell or inside or outside of the host (medium and milk, etc.) and purified as a substantially pure and homogeneous antibody. For example, chromatography column, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization are appropriately selected and combined. Antibodies can be appropriately isolated and purified. Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. These chromatography can be performed using liquid chromatography, such as HPLC and FPLC, for example. As a column used for affinity chromatography, a protein A column or a protein G column is mentioned. Examples of the protein A column include Hyper D, POROS, and Sepharose F.F. (manufactured by Pharmacia).

It is also possible to modify the antibody or partially remove the peptide by treating the antibody with an appropriate protein-modifying enzyme before or after purification of the antibody. As the protein-modifying enzyme, trypsin, chymotrypsin, lysylendopeptidase, protein kinase, glucosidase, and the like can be used, for example.

In an alternative embodiment, initiation A is a method for producing an antibody containing an antigen-binding domain whose antigen-binding activity changes depending on the ion concentration condition, and culturing a host cell or producing the above-described host ( raise), it may include a step of recovering the antibody from the culture of the cell or the secretion of the host, or the like, or by another method known in the art.

In one embodiment, initiation A comprises the steps of: (a) selecting an antibody capable of promoting the loss of an antigen from plasma, compared to a reference antibody; (b) selecting an antibody with increased binding activity to the extracellular matrix; (c) selecting an antibody with increased FcγR binding activity under conditions of neutral pH; (d) selecting an antibody with increased FcγRIIb binding activity under conditions of neutral pH; (e) selecting an antibody with reduced binding activity to at least one active FcγR selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb and FcγRIIa while maintaining or increasing FcγRIIb binding activity fair; (f) selecting an antibody with increased FcRn binding activity under conditions of neutral pH; (g) selecting an antibody with elevated pi; (h) confirming the pI of the recovered antibody, and then selecting an antibody with an elevated pI; And (i) selecting an antibody whose antigen-binding activity is changed or increased depending on the ion concentration condition.

Here, the reference antibody is not limited, but a natural type antibody (e.g., a natural type Ig antibody, preferably a natural type IgG antibody) and an antibody before modification (before libraryization or in the process of libraryization) Antibodies, for example, an ion concentration-dependent antibody before pi is elevated, or an antibody on which pi is elevated before an ion concentration-dependent antigen-binding domain is imparted).

After preparing the antibody of Initiation A, by performing a pharmacokinetic assay of the antibody using plasma of mice, rats, rabbits, dogs, monkeys, humans, etc., the antibody and the reference antibody were compared, and the antigen from plasma You may select an antibody whose disappearance is promoted.

Alternatively, after preparing the antibody of Initiation A, an antibody with increased binding to the extracellular matrix may be selected by comparing the extracellular matrix-binding ability of the antibody and the reference antibody using an electrochemiluminescence method or the like.

Alternatively, after preparing the antibody of Initiation A, using BIACORE (registered trademark) or the like, by comparing the binding activity of the antibody and the reference antibody to various FcγRs under neutral pH conditions, these various An antibody with increased binding activity to FcγR may be selected. In this case, the various FcγRs may be of the desired type, or may be, for example, FcγRIIb. Likewise, for example, while maintaining or increasing the binding activity to FcγRIIb (under conditions of neutral pH), 1 of an active FcγR selected from the group consisting of FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb, and FcγRIIa, etc. Antibodies with reduced binding activity to more than one dog may be selected. In this case, FcγR may be human FcγR.

Alternatively, after preparing the antibody of Initiation A, by comparing the binding activity of the antibody and the reference antibody to FcRn under conditions of neutral pH using BIACORE or other known techniques, the FcRn under neutral pH conditions. You may select an antibody with increased binding activity to. In this case, FcRn may be human FcRn.

Alternatively, after preparing the antibody of Initiation A, after evaluating the pI of the antibody using isoelectric point electrophoresis or the like, an antibody with an elevated pI may be selected as compared with a reference antibody. At this time, for example, even if the value of the pI, for example, 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 to select an antibody that rises Or 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 rising antibody may be selected do.

Alternatively, after preparing the antibody of Initiation A, by comparing the binding activity to the desired antigen of the antibody and the reference antibody under conditions of low ion concentration and under conditions of high ion concentration using BIACORE or the like, ions You may select an antibody whose binding activity to the antigen is changed or increased depending on the concentration conditions. The ion concentration may be, for example, a hydrogen ion concentration or a metal ion concentration, and in the case of a metal ion concentration, it may be, for example, a calcium ion concentration. Whether the binding activity is changed or increased, for example, (a) changes or promotes the uptake of antigen into cells, (b) the ability to bind to other antigen molecules multiple times is changed or increased, ( It can be evaluated on the basis of whether c) a decrease in the antigen concentration in plasma is being changed or promoted, or (d) the plasma retention of the antibody is changing. Alternatively, any two or more of these selection methods may be appropriately combined as desired.

In an alternative embodiment, initiation A is an antibody comprising an antigen-binding domain whose antigen-binding activity changes depending on the ion concentration condition, wherein at least one amino acid residue that can be exposed on the surface of the antibody is modified. It relates to a method for producing or screening an antibody ("ion concentration-dependent antibody with elevated pI") in which the pI is elevated by being produced. The production method is, for example, a related embodiment described in the range described in the disclosure A in this specification, for example, an embodiment of a production method or screening method for an antibody with an elevated pI, and the In an embodiment of a method for preparing or screening a calcium ion concentration-dependent antigen-binding domain or a calcium ion concentration-dependent antibody or their library having a higher antigen-binding activity under conditions of a higher calcium ion concentration than under conditions of a low calcium ion concentration , And/or the above, pH-dependent antigen-binding domain or pH-dependent antibody having higher antigen-binding activity under neutral pH conditions than under acidic pH conditions, or a method for preparing or screening a library thereof. , It can be implemented by appropriately combining as desired.

In an alternative embodiment, initiation A is an antibody comprising an antigen-binding domain whose antigen-binding activity changes depending on the ion concentration condition, wherein the binding activity to the extracellular matrix is increased, and the surface of the antibody There is provided a method for producing or screening an antibody in which at least one amino acid residue that can be exposed to is altered and its pi is elevated ("Ion concentration-dependent antibody with elevated pi"). This method may be carried out with the intention of raising the pI of the ion concentration-dependent antibody. Such a method includes, for example, a related embodiment described in the range describing the disclosure A in this specification, for example, an embodiment of a method for producing or screening an antibody with an elevated pI, and, Calcium ion concentration-dependent antigen-binding domain or calcium ion concentration-dependent antibody having a higher antigen-binding activity under conditions of higher calcium ion concentration than under the above-described conditions of low calcium ion concentration, or production methods or screening methods for their libraries An embodiment of a method for preparing or screening a pH-dependent antigen-binding domain or a pH-dependent antibody, or a library thereof, having a higher antigen-binding activity under conditions of neutral pH than the embodiment and/or acidic pH conditions, is desired. According to, it can be implemented by combining suitably. For example, by comparing the extracellular matrix-binding ability of the obtained antibody and the reference antibody using an electrochemiluminescence method or another known technique, an antibody with increased binding to the extracellular matrix may be selected.

Here, the reference antibody is not limited, but a natural type antibody (e.g., a natural type Ig antibody, preferably a natural type IgG antibody) and an antibody before modification (before libraryization or in the process of libraryization) An antibody, for example, an ion concentration-dependent antibody before pi is elevated, or an antibody on which pI is elevated before an ion concentration-dependent antigen-binding domain is imparted) may be used.

In an alternative embodiment, initiation A relates to a method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, the method comprising: raising the isoelectric point (pI). To do this, it includes a step of modifying at least one amino acid residue that may be exposed on the surface of the antibody. In some embodiments, the alteration of the amino acid residue comprises the following: (a) substitution of an amino acid residue having a negative charge with an amino acid residue having no charge; (b) replacing an amino acid residue having a negative charge with an amino acid residue having a positive charge; And (c) a modification selected from the group consisting of substituting an amino acid residue having no charge with an amino acid residue having a positive charge. In some embodiments, at least one modified amino acid residue is substituted with histidine. In a further embodiment, the antibody comprises a variable region and/or a constant region, and amino acid residues are modified in this variable region and/or a constant region. In a further embodiment, the at least one amino acid residue modified according to the method is, according to Kabat numbering, hereinafter: (a) 1st, 3rd, 5th, 8th position in the FR of the heavy chain variable region, 10th, 12th, 13th, 15th, 16th, 18th, 19th, 23rd, 25th, 26th, 39th, 41st, 42nd, 43rd, 44th, 46th, 68th , 71st, 72nd, 73rd, 75th, 76th, 77th, 81st, 82nd, 82a, 82b, 83rd, 84th, 85th, 86th, 105th, 108th, 110th Above, and 112th; (b) 31st, 61st, 62nd, 63rd, 64th, 65th, and 97th positions in the CDR of the heavy chain variable region; (c) 1st, 3rd, 7th, 8th, 9th, 11th, 12th, 16th, 17th, 18th, 20th, 22nd, 37th in the light chain variable region FR , 38th, 39th, 41st, 42nd, 43rd, 45th, 46th, 49th, 57th, 60th, 63rd, 65th, 66th, 68th, 69th, 70th, 74th Ranked, 76th, 77th, 79th, 80th, 81st, 85th, 100th, 103rd, 105th, 106th, 107th, and 108th; And (d) in the CDR of the light chain variable region, in a CDR or FR selected from the group consisting of 24th, 25th, 26th, 27th, 52nd, 53rd, 54th, 55th, and 56th position. In position. Meanwhile, in a further embodiment, at least one amino acid residue modified according to the method is, according to Kabat numbering, hereinafter: (a) 8th, 10th, 12th, 13th position in the FR of the heavy chain variable region , 15th, 16th, 18th, 23rd, 39th, 41st, 43rd, 44th, 77th, 82nd, 82a, 82b, 83, 84, 85, and 105th; (b) 31st, 61st, 62nd, 63rd, 64th, 65th, and 97th positions in the CDR of the heavy chain variable region; (c) In the FR of the light chain variable region, 16th, 18th, 37th, 41st, 42nd, 45th, 65th, 69th, 74th, 76th, 77th, 79th, and 107th top; And (d) in the CDR of the light chain variable region, in a CDR or FR selected from the group consisting of 24th, 25th, 26th, 27th, 52nd, 53rd, 54th, 55th, and 56th position. In position. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method, at acidic pH (e.g. pH 5.8) and neutral pH (e.g. pH 7.4), comparing the KD of the antibody produced according to the method with the corresponding antigen. It further includes a process. In a further embodiment, the method comprises a step of selecting an antibody with an antigen-related KD (pH acidic region (e.g. pH 5.8))/KD (pH neutral region (e.g. pH 7.4)) of 2 or higher. Includes. In some embodiments, the method further comprises a step of comparing the antigen-binding activity of the antibody produced according to the method under conditions of high ion concentration (eg, hydrogen ion or calcium ion concentration) and under low ion concentration conditions. Includes. In a further embodiment, the method further comprises a step of selecting an antibody having a higher (eg, twice) antigen binding activity under a high ionic concentration than under a low ionic concentration. In some embodiments, the ion concentration is a calcium ion concentration, and the high calcium ion concentration is selected between 100 μM to 10 mM, 200 μM to 5 mM, 400 μM to 3 mM, 200 μM to 2 mM, or 400 μM to 1 mM. Can be. In some cases, a concentration selected between 500 μM and 2.5 mM, which is close to the concentration of calcium ions in plasma (blood) in vivo, is also preferable. In some embodiments, the low calcium ion concentration may be selected between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, or between 1 μM and 5 μM, or between 2 μM and 4 μM. have. In some cases, a concentration selected between 1 μM and 5 μM, which is close to the initial endosome calcium ion concentration in vivo, is also preferred. In some embodiments, the lower limit of the value of KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (e.g., KD (3 μM Ca)/KD (2 mM Ca)) value is 2 or more, 10 or more. , Or 40 or more, and its upper limit is 400 or less, 1000 or less, or 10000 or less. In some alternative embodiments, the lower limit of the kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) (e.g., kd (3 μM 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, the ion concentration is the hydrogen ion concentration, and the low hydrogen ion concentration (pH neutral range) is from pH 6.7 to pH 10.0, pH 6.7 to pH 9.5, pH 7.0 to pH 9.0, or pH 7.0 to pH 8.0. Can be chosen. As for the low hydrogen ion concentration, pH 7.4, which is close to the pH in plasma (blood) in vivo, is preferable. For simplicity of measurement, for example, pH 7.0 may be used. In some embodiments, the high hydrogen ion concentration (pH acidic range) may be selected from pH 4.0 to pH 6.5, pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. In the acidic pH range, pH 5.8, which is close to the hydrogen ion concentration of the initial endosome in vivo, is preferable. For simplicity of measurement, for example, pH 6.0 may be used. In some embodiments, the lower limit of KD (pH acidic range)/KD (pH neutral range) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more. , Its upper limit is 400 or less, 1000 or less, or 10000 or less. In some embodiments, the method compares the loss of antigen from plasma after administration of the antibody produced by the method when compared with a reference antibody that differs only in that it does not include the modification introduced by the method. It further includes the process of doing. In a further embodiment, the method is an antibody that promotes (e.g., doubles) the loss of antigen from plasma produced by the method when compared with an antibody that does not contain the modification introduced by the method. It further includes a step of selecting. In some embodiments, the method further comprises a step of comparing the binding of the antibody produced by the method to an extracellular matrix when compared with an antibody different only in that it does not include the modification introduced by the method. do. In a further embodiment, the method has increased extracellular matrix binding (e.g., doubled when bound to an antigen) when compared with an antibody that differs only in that it does not include the modification introduced by the method. It further comprises a step of selecting the antibody produced by the above method having,. In a further embodiment, an antibody (natural type antibody (e.g., a native type Ig antibody, preferably a native type IgG antibody)), or a reference antibody prior to modification or alteration of at least one amino acid residue to raise pi When compared with (for example, an antibody before antibody modification, or an antibody before or during library construction), the antibody produced by the above method maintains (substantially) the antigen-binding activity. In this case, "the antigen-binding activity is (substantially) maintained" means 50% or more, more preferably 60% or more, more preferably 70%, compared to the binding activity of the antibody before modification or modification. Or it may mean having an activity of 75% or more, even more preferably 80%, 85%, 90%, or 95% or more.

In a further embodiment, initiation A relates to a method for producing an antibody comprising an antigen-binding domain whose antigen-binding activity changes according to ion concentration conditions, the method comprising: raising the isoelectric point (pI). For this purpose, it includes a step of modifying at least one amino acid residue that may be exposed on the surface of the normal region of the antibody. In some embodiments, the alteration of the amino acid residue comprises the following: (a) substitution of an amino acid residue having a negative charge with an amino acid residue having no charge; (b) replacing an amino acid residue having a negative charge with an amino acid residue having a positive charge; And (c) a modification selected from the group consisting of substituting an amino acid residue having no charge with an amino acid residue having a positive charge. In some embodiments, at least one modified amino acid residue is substituted with histidine. In a further embodiment, the antibody comprises a variable region and/or a constant region, and amino acid residues are modified in this variable region and/or a constant region. In a further embodiment, the at least one amino acid residue modified according to the method is, according to EU numbering, hereinafter: 196th, 253th, 254th, 256th, 258th, 278th, 280th, 281th, 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 It is in a position among the normal regions selected from the group consisting of the above, 430th, 433th, 434th, and 443th. In a further embodiment, the at least one amino acid residue modified according to the method is, according to EU numbering, hereinafter: 254th, 258th, 281th, 282th, 285th, 309th, 311th, 315th, 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. In a further embodiment, the at least one amino acid residue modified according to the method is, according to EU numbering, hereinafter: 282th, 309th, 311th, 315th, 342th, 343th, 384th, 399th, It is in a position among the normal regions selected from the group consisting of 401st, 402th, and 413th. In some embodiments, the method further comprises a step of comparing the antigen-binding activity of the antibody produced according to the method under conditions of high ion concentration (eg, hydrogen ion or calcium ion concentration) and under low ion concentration conditions. Includes. In a further embodiment, the method further comprises a step of selecting an antibody having a higher antigen-binding activity under a high ionic concentration than under a low ionic concentration. In some embodiments, the method compares the loss of antigen from plasma after administration of the antibody produced by the method when compared with a reference antibody that differs only in that it does not include the modification introduced by the method. It further includes the process of doing. In a further embodiment, the method is an antibody that promotes (e.g., doubles) the loss of antigen from plasma produced by the method when compared with an antibody that does not contain the modification introduced by the method. It further includes a step of selecting. In some embodiments, the method includes a step of comparing the binding of an antibody produced by the method to an extracellular matrix when compared with an antibody that differs only in that it does not include the modification introduced by the method. In a further embodiment, the method has increased extracellular matrix binding (e.g., when it is bound to an antigen, doubled when compared to a reference antibody that differs only in that it does not include the modification introduced by the method). ), further comprising a step of selecting an antibody produced by the above method. In some embodiments, the method comprises the Fcγ receptor (FcγR) binding activity of the antibody produced by the method under neutral pH (e.g., pH 7.4) of a reference antibody comprising a normal region of a native IgG. It includes the process of comparing with the binding activity. In a further embodiment, the method is an antibody produced by the method having increased FcγR binding activity under neutral pH (e.g., pH 7.4) when compared to a reference antibody comprising a normal region of a native IgG. Including the process of selecting. In some embodiments, the antibody selected, produced by the above method, has increased FcγRIIb binding activity under neutral pH. In some embodiments, the selected antibody produced by the above method is preferably FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb when compared with a reference antibody that differs only in that the normal region is of a native IgG. And FcγRIIa has a binding activity to at least one active FcγR and to FcγRIIb selected from the group consisting of, optionally, FcγRIIb binding activity is maintained or increased, and the binding activity to active FcγR is decreased. . In some embodiments, the method is a step of comparing the FcRn binding activity of the antibody produced by the method under neutral pH conditions when compared with a reference antibody that differs only in that the normal region is of a native IgG. It additionally includes. In a further embodiment, the method, when compared with a reference antibody that differs only in that the normal region is of a native IgG, increased FcRn binding activity under neutral pH conditions produced by the method (e.g. , 2 times). In some embodiments, an antibody (natural antibody (e.g., a native Ig antibody, preferably a native IgG antibody)), or a reference antibody prior to modification or alteration of at least one amino acid residue to raise pi When compared with (for example, an antibody before antibody modification, or an antibody before or during library construction), the antibody produced by the above method maintains (substantially) the antigen-binding activity. In this case, "the antigen-binding activity is (substantially) maintained" means at least 50% or more, preferably 60% or more, more preferably 70%, compared to the binding activity of the antibody before modification or modification. Or it may mean having an activity of 75% or more, even more preferably 80%, 85%, 90%, or 95% or more.

In a further embodiment, the method comprises a step of altering at least one amino acid residue that may be exposed on the surface of the variable and constant regions of the antibody to raise the isoelectric point (pI). In a further embodiment, at least one amino acid residue modified according to the method is at a position in the above-described normal region. In a further embodiment, at least one amino acid residue modified according to the above method is at a position in the aforementioned variable region. In a further embodiment, the at least one amino acid residue modified according to the method is at a position in the above-described constant region, and the at least one amino acid residue modified according to the method is at a position in the aforementioned variable region. have. In some embodiments, the antigen is a soluble antigen. In some embodiments, the method, at acidic pH (e.g. pH 5.8) and neutral pH (e.g. pH 7.4), comparing the KD of the antibody produced according to the method with the corresponding antigen. It further includes a process. In a further embodiment, the method comprises selecting an antibody having a KD (pH acidic region)/KD (pH neutral region) for the antigen of 2 or higher. In some embodiments, the method further comprises a step of comparing the antigen-binding activity of the antibody produced according to the method under conditions of high ion concentration (eg, hydrogen ion or calcium ion concentration) and under low ion concentration conditions. Includes. In a further embodiment, the method further comprises a step of selecting an antibody having a higher antigen-binding activity under a high ionic concentration than under a low ionic concentration. In some embodiments, the ion concentration is a calcium ion concentration, and the high calcium ion concentration is selected between 100 μM to 10 mM, 200 μM to 5 mM, 400 μM to 3 mM, 200 μM to 2 mM, or 400 μM to 1 mM. Can be. In some cases, a concentration selected between 500 μM and 2.5 mM is also preferred. In some embodiments, the low calcium ion concentration may be selected between 0.1 μM and 30 μM, between 0.2 μM and 20 μM, between 0.5 μM and 10 μM, or between 1 μM and 5 μM, or between 2 μM and 4 μM. have. In some cases, a concentration selected between 1 μM and 5 μM is also preferred. In some embodiments, the lower limit of the value of KD (low calcium ion concentration condition)/KD (high calcium ion concentration condition) (e.g., KD (3 μM Ca)/KD (2 mM Ca)) value is 2 or more, 10 or more. , Or 40 or more, and its upper limit is 400 or less, 1000 or less, or 10000 or less. In some alternative embodiments, the lower limit of the kd (low calcium ion concentration condition)/kd (high calcium ion concentration condition) (e.g., kd (3 μM 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, the ion concentration is the hydrogen ion concentration, and the low hydrogen ion concentration (pH neutral range) is from pH 6.7 to pH 10.0, pH 6.7 to pH 9.5, pH 7.0 to pH 9.0, or pH 7.0 to pH 8.0. Can be chosen. As for the low hydrogen ion concentration, pH 7.4, which is close to the pH of plasma (blood) in vivo, is preferable. For simplicity of measurement, for example, pH 7.0 may be used. In some embodiments, the high hydrogen ion concentration (pH acidic range) may be selected from pH 4.0 to pH 6.5, pH 4.5 to pH 6.5, pH 5.0 to pH 6.5, or pH 5.5 to pH 6.5. The pH acidic range may be, for example, a pH of 5.8 or a pH of 6.0. In some embodiments, the lower limit of KD (pH acidic range)/KD (pH neutral range) (e.g., KD (pH 5.8)/KD (pH 7.4)) is 2 or more, 10 or more, or 40 or more. , Its upper limit is 400 or less, 1000 or less, or 10000 or less.

In some embodiments, the method compares the loss of antigen from plasma after administration of the antibody produced by the method when compared with a reference antibody that differs only in that it does not include the modification introduced by the method. It further includes the process of doing. In a further embodiment, the method is an antibody that promotes (e.g., doubles) the loss of antigen from plasma produced by the method when compared with an antibody that does not contain the modification introduced by the method. It further includes a step of selecting. In some embodiments, the method further comprises a step of comparing the binding of the antibody produced by the method to an extracellular matrix when compared with an antibody different only in that it does not include the modification introduced by the method. do. In a further embodiment, the method has increased extracellular matrix binding (e.g., binding to an antigen) produced by the method when compared with an antibody different only in that it does not include the modification introduced by the method. If so, a step of selecting an antibody having 5 times) is further included. In some embodiments, the method comprises the Fcγ receptor (FcγR) binding activity of the antibody produced by the method under neutral pH (e.g., pH 7.4) of a reference antibody comprising a normal region of a native IgG. It further comprises a step of comparing with the binding activity. In a further embodiment, the method has an increased FcγR binding activity under neutral pH (e.g., pH 7.4) when compared to the Fcγ-binding activity of a reference antibody comprising a normal region of a native IgG. It includes a step of selecting an antibody produced by the method. In some embodiments, the antibody selected, produced by the above method, has increased FcγRIIb binding activity under neutral pH. In some embodiments, the selected antibody produced by the above method is preferably FcγRIa, FcγRIb, FcγRIc, FcγRIIIa, FcγRIIIb when compared with a reference antibody that differs only in that the normal region is of a native IgG. And FcγRIIa has a binding activity to at least one active FcγR and to FcγRIIb selected from the group consisting of, optionally, FcγRIIb binding activity is maintained or increased, and the binding activity to active FcγR is decreased. . In some embodiments, the method is a step of comparing the FcRn binding activity of the antibody produced by the method under neutral pH conditions when compared with a reference antibody that differs only in that the normal region is of a native IgG. It additionally includes. In a further embodiment, the method, when compared with a reference antibody that differs only in that the normal region is of a native IgG, increased FcRn binding activity under neutral pH conditions produced by the method (e.g. , 2 times). In a further embodiment, an antibody (natural type antibody (e.g., a native type Ig antibody, preferably a native type IgG antibody)), or a reference antibody prior to modification or alteration of at least one amino acid residue to raise pi When compared with (for example, an antibody before antibody modification, or an antibody before or during library construction), the antibody produced by the above method maintains (substantially) the antigen-binding activity. In this case, "the antigen-binding activity is (substantially) maintained" means at least 50% or more, preferably 60% or more, more preferably 70%, compared to the binding activity of the antibody before modification or modification. Or it may mean having an activity of 75% or more, even more preferably 80%, 85%, 90%, or 95% or more.

In an alternative embodiment, initiation A relates to an antibody obtained by the above-described method for producing an antibody of initiation A or a screening method.

In an alternative embodiment, Initiation A relates to a composition or a pharmaceutical composition comprising the antibody of Initiation A described above. In one embodiment, the pharmaceutical composition of Initiation A is a biological fluid (preferably, plasma) in the subject (when the antibody of Initiation A is administered (applied) to a subject (preferably in vivo)). Etc.), may be a pharmaceutical composition for promoting the disappearance of the antigen and/or for enhancing the binding to the extracellular matrix. The pharmaceutical composition of Initiation A may optionally contain a pharmaceutically acceptable carrier. Here, the pharmaceutical composition may generally refer to a drug for treatment, prevention, diagnosis, or examination of a disease.

The composition or pharmaceutical composition of Initiation A can be suitably formulated. In some embodiments, they can be used parenterally in the form of injections, for example in sterile solutions or suspensions with water or any other pharmaceutically acceptable liquid. The composition is suitably combined with a pharmaceutically acceptable carrier or medium, and may be suitably formulated in a unit dose required for generally accepted pharmaceutical practice. Such pharmaceutically acceptable carriers or media are, but are not limited to, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, and binders. Includes. The amount of the active ingredient in the composition can be adjusted so as to obtain an appropriate dose within a specified range.

In some embodiments, the composition of Initiation A or the pharmaceutical composition may be administered by parenteral administration. The composition or pharmaceutical composition may be suitably formulated as, for example, an injectable, nasal administration, transpulmonary administration, or transdermal administration composition. The composition or pharmaceutical composition can be appropriately administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

In some embodiments, the sugar initiation is an antibody in which at least one amino acid residue that may be exposed to the surface is altered, thereby raising the pi (antibodies with elevated pi); A method for producing the antibody; Or (when the antibody is administered in vivo to a subject), the use of the antibody in promoting the disappearance of the antigen from the plasma is provided. It can be understood that the range in which the disclosure A in the present specification is described and the corresponding description in the present embodiment are appropriately applied mutatis mutandis to these embodiments. In another embodiment, the sugar disclosure may include an antibody in which at least one amino acid residue that may be exposed to the surface has been altered, thereby lowering the pi ("antibody that lowers the pi"); A method for producing the antibody; Alternatively, the use of the antibody in improving plasma retention (when the antibody is administered in vivo to a subject) is provided. The inventors have found that by introducing a specific amino acid mutation into a specific site of the amino acid sequence of the constant region, the pi can be raised and the uptake of the antibody into the cell can be promoted. Those skilled in the art can understand that by introducing amino acids with different side chain charge characteristics into the site, conversely, by reducing the pI and inhibiting the absorption of the antibody into cells, the retention of the antibody in the plasma can be prolonged. have. It can be understood that the range in which the disclosure A in this specification is described and the corresponding description in the present embodiment are appropriately applied mutatis mutandis to these embodiments.

In one embodiment, the sugar disclosure provides a method for producing a modified antibody having an extended or decreased half-life in plasma compared to the half-life before the modification of the antibody, and the method includes the following steps. : (a) According to EU numbering, the following positions: 196th, 253th, 254th, 256th, 257th, 258th, 278th, 280th, 281th, 282th, 285th, 286th, 306th Above, 307th, 308th, 309th, 311th, 315th, 327th, 330th, 342th, 343th, 345th, 356th, 358th, 359th, 361th, 362th, 373th, 382, 384, 385, 386, 387, 388, 389, 399, 400, 401, 402, 413, 415, 418, 419, 421, 424 , A step of modifying a nucleic acid encoding an antibody prior to alteration to change the charge of at least one amino acid residue positioned at a position selected from the group consisting of 430, 433, 434, and 443; (b) culturing a host cell in order to produce an antibody by expressing the modified nucleic acid; And (c) recovering the produced antibody from the host cell culture.

A further embodiment provides a method for extending or reducing the half-life of an antibody in plasma, the method comprising the following positions: 196th, 253rd, 254th, 256th, 257, according to EU numbering. Above, 258th, 278th, 280th, 281th, 282th, 285th, 286th, 306th, 307th, 308th, 309th, 311th, 315th, 327th, 330th, 342th, 343, 345, 356, 358, 359, 361, 362, 373, 382, 384, 385, 386, 387, 388, 389, 399, 400 , At least one amino acid positioned at a position selected from the group consisting of 401, 402, 413, 415, 418, 419, 421, 424, 430, 433, 434, and 443 It includes the process of modifying the residue.

These methods may further include a step of determining whether the half-life in the plasma of the recovered and/or modified antibody is prolonged or decreased compared to that of the antibody prior to modification.

Charge change can be achieved by amino acid substitution. In some embodiments, the substituted amino acid residues are amino acid residues of the following groups (a) and (b): (a) Glu(E) and Asp(D); And (b) Lys(K), Arg(R), and His(H).

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, a humanized antibody, or a human antibody. In some embodiments, the antibody is a multispecific antibody, eg, a bispecific antibody.

Initiation B

In a non-limiting embodiment, Initiation B relates to Fc region modified variants, their use and production methods, and the like.

In the range of disclosures A and B in this specification, "Fc region modified" means, for example, an Fc region in which at least one amino acid of the Fc region of a native IgG antibody has been modified with another amino acid. Alternatively, it may mean an Fc region in which at least one amino acid of such an Fc region modified variant is further modified with another amino acid. Here, such an Fc region modified body includes not only the Fc region into which the amino acid modification has been introduced, but also an Fc region having the same amino acid sequence as the Fc region.

In an alternative embodiment, initiation B is an Fc region variant comprising an FcRn-binding domain, wherein the FcRn-binding domain is Ala at position 434; Glu, Arg, Ser, and Lys at position 438; And it relates to an Fc region variant containing any one of Glu, Asp, and Gln at position 440 (in the range where disclosure B in this specification is described, the Fc region variant is, for convenience, "new Fc region dogs. It is also called "variant").

In practice, the Fc region variant of initiation B can be introduced into virtually any antibody (e.g., a multispecific antibody, such as a bispecific antibody), regardless of the type of target antigen. For example, the anti-agent IXa factor/factor X bispecific antibody is such an Fc region variant as shown in Example 20 (e.g., F8M-F1847mv [F8M-F1847mv1 as heavy chain) (SEQ ID NO: 323 ) And F8M-F1847mv2 (SEQ ID NO: 324), and F8ML as light chain (SEQ ID NO: 325)]; F8M-F1868mv [F8M-F1868mv1 as heavy chain (SEQ ID NO: 326) and F8M-F1868mv2 (SEQ ID NO: 327), And F8ML as light chain (SEQ ID NO: 325)]; And F8M-F1927mv [F8M-F1927mv1 as heavy chain (SEQ ID NO: 328) and F8M-F1927mv2 (SEQ ID NO: 329) and F8ML as light chain (SEQ ID NO: 325)]) It can be produced by using.

As described above, WO2013/046704 introduces a mutation that increases binding to FcRn under acidic conditions, as well as a specific mutation (as a representative, two residue mutations called Q438R/S440E according to EU numbering). It has been reported that one Fc region variant exhibited a significant decrease in binding to rheumatoid factors. However, in WO2013/046704, it did not appear that the Fc region modified variant whose rheumatoid factor binding was lowered by the modification of Q438R/S440E was superior in plasma retention as compared to an antibody having a native Fc region. Therefore, there has been a demand for a safe and more advantageous Fc region modified variant that does not show binding to existing ADA and further improves plasma retention. The present inventors disclose herein a safe and more advantageous Fc region variant that does not show binding to an anti-drug antibody (such as conventional ADA) and further improves plasma retention. In particular, in order to increase the retention of the antibody in plasma and to maintain a significant decrease in binding to the rheumatoid factor, surprisingly, the substitution of amino acid at position 434 to Ala (A) according to EU numbering and a specific two residues It is disclosed for the first time herein that an Fc region variant comprising a mutation (as a representative, Q438R/S440E) as a combination of mutations of amino acid residues is preferred.

Therefore, the novel Fc region variant of disclosure B described in the present specification provides an advantageous and surprising improvement over the Fc region variant described in WO2013/046704, and all aspects described in WO2013/046704 are described in this specification. It is used by reference in.

In one embodiment, initiation B provides a combination of novel amino acid substitutions in the FcRn-binding domain that enhances the FcRn-binding activity of the antibody in the acidic pH range and the neutral pH range, particularly in the acidic pH range.

In one embodiment, the Fc region variant of initiation B is Ala at position 434, according to EU numbering; Glu, Arg, Ser, and any one of Lys at position 438; And Glu, Asp, and Gln at position 440; Also, Ala at 434; Arg or Lys at position 438; And it is more preferable to include Glu or Asp at position 440. Preferably, the Fc region variant of initiation B is Ile or Leu at position 428, according to EU numbering; And/or, at position 436, any one of Ile, Leu, Val, Thr, and Phe is further included. Fc region variant, according to EU numbering, Leu at position 428; And/or, it is more preferable to include Val or Thr at position 436.

In one embodiment, the Fc region variant of initiation B may be an Fc region variant of a native Ig antibody, and is an Fc region variant of a native IgG (IgG1, IgG2, IgG3, or IgG4 type) antibody. It is more preferable. About the native Fc region, it is included in part in the range which describes the indication A and B in this specification. In Initiation B, more specifically, the native Fc region may mean an unmodified Fc region or a naturally occurring Fc region, and the amino acid residue in the Fc region is unmodified natural Ig antibody. It is more preferable that it is an Fc region which is not formed or a naturally occurring Fc region. The antibody from which the Fc region is derived may be Ig, such as IgM or IgG, and may be, for example, human IgG1, IgG2, IgG3, or IgG4. In one embodiment, human IgG1 may be used. In addition, the (reference) antibody containing a native Fc region may be an antibody containing an unmodified Fc region or a naturally occurring Fc region.

The 428th, 434th, 438th, and 440th positions are common in all Fc regions of human natural IgG1, IgG2, IgG3, and IgG4 antibodies. However, at position 436, all of the Fc regions of human native IgG1, IgG2, and IgG4 type antibodies are Tyr(Y), while in the Fc region of human native IgG3 type antibodies, Phe(F). . On the other hand, Stapleton et al., (Nature Comm. 599 (2011)) reported that human IgG3 allotypes having an amino acid substitution of R435H according to EU numbering showed a half-life in plasma comparable to that of IgG1 in humans. Therefore, the present inventors also conceived that by introducing the amino acid substitution of R435H in addition to the amino acid substitution at position 436, it is possible to increase the binding to FcRn under acidic conditions and improve the retention in plasma.

In addition, in WO2013/046704, as a substitution of two residues of amino acids leading to a significant decrease in rheumatoid factor binding when combined with substitution of amino acids capable of increasing binding to FcRn under acidic conditions, specifically, According to EU numbering, the Q438R/S440E, Q438R/S440D, Q438K/S440E, and Q438K/S440D are also reported.

Thus, in a preferred embodiment, the Fc region variant of initiation B, the FcRn binding domain, according to EU numbering, (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; (l) 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) M428L/N434A/Q438R/S440E; (v) M428L/N434A/Q438R/S440D; (w) M428L/N434A/Q438K/S440E; (x) M428L/N434A/Q438K/S440D; (y) M428L/N434A/Y436T/Q438R/S440E; (z) M428L/N434A/Y436T/Q438R/S440D; (aa) M428L/N434A/Y436T/Q438K/S440E; (ab) M428L/N434A/Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad) M428L/N434A/Y436V/Q438R/S440D; (ae) M428L/N434A/Y436V/Q438K/S440E; (af) M428L/N434A/Y436V/Q438K/S440D; (ag) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; And (ah) a combination of substituted amino acid positions selected from the group consisting of L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E.

In addition, in a further preferred embodiment, the Fc region variant of initiation B, the FcRn binding domain, according to EU numbering, (a) N434A/Q438R/S440E; (b) N434A/Y436T/Q438R/S440E; (c) N434A/Y436V/Q438R/S440E; (d) M428L/N434A/Q438R/S440E; (e) M428L/N434A/Y436T/Q438R/S440E; (f) M428L/N434A/Y436V/Q438R/S440E; (g) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; And (h) a combination of substituted amino acids selected from the group consisting of L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E.

In one embodiment, it is preferable that the Fc region modified variant of initiation B has increased FcRn-binding activity under acidic pH conditions as compared to that of a native IgG.

On the other hand, the increase in the binding activity (binding affinity) of the FcRn binding domain to FcRn in a certain pH range was compared with the FcRn binding activity (binding affinity) measured against the native FcRn binding domain, and measured FcRn It may correspond to an increase in binding activity (binding affinity). In this case, KD (natural Fc region) /KD (initiating B Fc region variant) showing a difference in binding activity (binding affinity) is at least 1.5 times, 2 times, 3 times, 4 times, 5 times, It may be 10 times, 15 times, 20 times, 50 times, 70 times, 80 times, 100 times, 500 times, or 1000 times. Such an increase may occur in the acidic pH range and/or in the neutral pH range, but in light of the mechanism of action of the initiation B, there are cases where the increase in the acidic pH range is desirable.

In some embodiments, the Fc region variant of initiation B whose binding activity to FcRn is increased under the conditions of the acidic pH range is more active to FcRn than the Fc region of native IgG (e.g., pH 6.0 and At 25°C) is, for example, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 50 times, It is 75 times, 100 times, 200 times, 500 times, or 1000 times or more. In some embodiments, the increased FcRn-binding activity of the Fc region modified variant in the acidic pH range may be at least 5 times greater than the FcRn-binding activity of the native IgG Fc region, or may be at least 10 times greater.

By manipulating the FcRn-binding domain by introducing amino acid substitutions, the stability of the antibody may decrease (WO2007/092772). Since proteins with insufficient stability tend to aggregate easily during storage, the stability of drug proteins is extremely important in the manufacture of pharmaceuticals. Therefore, development of a stable antibody preparation may become difficult due to a decrease in stability caused by substitution in the Fc region (WO2007/092772).

In addition, the purity of drug proteins with respect to monomeric and high molecular weight species is also important in drug development. The wild-type IgG1 after protein A purification does not contain a significant amount of high molecular weight species, but if the FcRn binding domain is manipulated by introduction of a substitution, a large number of high molecular weight species may be generated. In this case, such high molecular weight species may need to be removed from the raw drug substance by a purification process.

In addition, substitution of amino acids in the antibody may have a negative result, for example, an increase in the immunogenicity of the therapeutic antibody, which in turn inhibits the production of cytokine storms and/or anti-drug antibodies (ADA). Can cause. Since ADA affects the effect and pharmacokinetics of therapeutic antibodies and sometimes causes serious side effects, the usefulness and efficacy of therapeutic antibodies in clinical practice may be limited. Many factors affect the immunogenicity of therapeutic antibodies, and the presence of effector T cell epitopes is one of them. Likewise, the presence of existing antibodies to therapeutic antibodies is also considered to be problematic. Examples of such conventional antibodies are rheumatoid factor (RF), an autoantibody against the Fc portion of an antibody (ie, IgG) (antibody against its own protein). Rheumatoid factor is particularly found in patients with systemic erythematosus (SLE) or in patients with joint rheumatism. In arthritis patients, RF and IgG are linked to form an immune complex, which contributes to the disease process. Recently, it has been reported that humanized anti-CD4IgG1 antibodies with Asn434His mutations induce significant rheumatoid factor binding (Zheng et al., Clin. Pharmacol. Ther. 89(2):283-290(2011)). By detailed study, it was confirmed that the Asn434His mutation in human IgG1 increases the binding of rheumatoid factor to the Fc region of the antibody as compared with the parent human IgG1.

RF is a polyclonal autoantibody against human IgG, and the epitope of RF in the sequence of human IgG differs depending on the clone, but the RF epitope is located in the CH2/CH3 interfacial region and in the CH3 domain that can overlap with the FcRn binding epitope. Seems to be. Therefore, mutations for increasing the binding activity to FcRn at neutral pH can further increase the binding activity to specific clones of RF.

The term "anti-drug antibody" or "ADA" in Initiation B may mean an endogenous antibody that has binding activity to an epitope located on a therapeutic antibody, and thus can bind to the therapeutic antibody. have. The term "conventional anti-drug antibody" or "conventional ADA" may refer to an anti-drug antibody that is present in the blood of a patient and can be detected prior to administration of the therapeutic antibody to the patient. In some embodiments, the existing ADA is a human antibody. In a further embodiment, the existing ADA is a rheumatoid factor.

The binding activity of the Fc region (variant) of an antibody against conventional ADA can be exhibited by, for example, electrochemiluminescence (ECL) reaction at acidic pH and/or neutral pH. ECL assays are described, for example, in Moxness et al. (Clin. Chem. 51:1983-1985 (2005)) and Example 6. The assay can be performed under conditions of, for example, MES buffer and 37°C. The antigen-binding activity of an antibody can be determined, for example, by BIACORE (registered trademark) analysis.

The binding activity to the existing ADA can be evaluated at any temperature of 10°C to 50°C. In some embodiments, to determine the binding activity (binding affinity) between a human Fc region and a human existing ADA, a temperature of 15° C. to 40° C., for example, between 20° C. and 25° C., or 25°C is used. In a further embodiment, the interaction between the human existing ADA and the human Fc region is measured at pH 7.4 (or pH 7.0) and 25°C.

In the range describing Initiation B in this specification, the (existing) ADA-binding activity is significantly increased, or the term synonymous with it is the (conventional) Fc region variant of Initiation B for ADA. Alternatively, the measured binding activity (binding affinity) (i.e., KD) of the antibody containing the variant is measured binding of the reference Fc region variant to the (conventional) ADA or a reference antibody containing the variant. Compared to the activity (binding affinity), for example, 0.55 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, It may mean that it is increased by 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, or 2.3 times or more. This increase in binding activity to existing ADA can be observed in individual patients or groups of patients.

In one embodiment, the terms "patients" and "patients" used in Initiation B are not limited, and this includes, a process of treatment in which a disease is affected and a therapeutic antibody is administered. All humans in the can be included. The patient may be a human suffering from an autoimmune disease such as arthritis disease or systemic erythematosus (SLE). Arthritis disease may include joint rheumatism.

In one embodiment of Initiation B, the fact that the binding activity to the existing ADA is significantly increased in individual patients is an antibody comprising an Fc region variant measured in a patient (e.g., a therapeutic antibody ), the binding activity to the existing ADA of the reference antibody is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or more, compared to the binding activity to the existing ADA of the reference antibody. It may mean becoming. Alternatively, it may be that the ECL response to the antibody is preferably greater than 250, or at least 500, or at least 1000, or meanwhile at least 2000. Preferably, the increase may be an increase compared to the ECL response of a reference antibody that is less than 500 or 250. Between the binding activity of the reference antibody to the existing ADA and such binding activity of the antibody having the Fc region variant, the ECL response is specifically less than 250 to at least 250, at least less than 250 to at least 500, less than 500 to 500 or more. , It is preferably in the range of less than 500 to 1000 or more, or less than 500 to at least 2000, but is not limited thereto.

In one embodiment, the increase in the binding activity to existing ADA is that in the patient population, (a) the binding activity to FcRn at acidic pH and (b) to the existing ADA at neutral pH. The measured proportion of patients with an ECL response of at least 500 (preferably at least 250) to an antibody comprising an Fc region variant having increased binding activity to the reference antibody is at least 500 (preferably at least 250 or higher), e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50 compared to the proportion of patients having an ECL response to the reference antibody. It can mean a percentage increase.

In one embodiment of initiation B, the reduction in the binding activity to existing ADA means that the measured binding activity (i.e., KD or ECL reaction) of the antibody containing the Fc region variant is relative to the reference antibody. It may mean that it is lowered compared to the measured binding activity. Such deterioration can be observed in individual patients or groups of patients. In individual patients, the affinity of the antibody containing the Fc region variant for the existing ADA at neutral pH is significantly lowered, the binding activity to the existing ADA at the neutral pH measured in the patient is significantly reduced. , It means that, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% lowered compared to the binding activity to the existing ADA at neutral pH measured for the reference antibody. can do.

Alternatively, the fact that the binding activity of the antibody containing the Fc region modified variant to the existing ADA in individual patients is significantly lowered is the ECL of the antibody used to be 500 or more (preferably 1000 or more, or 2000 or more). It can mean that the response is measured, for example, less than 500, preferably less than 250, compared to the ECL response of the reference antibody.

In a preferred embodiment, the Fc region variant of initiation B and the antibody containing the variant have low binding activity to existing ADA at neutral pH. Specifically, the binding activity of the antibody containing the Fc region variant of Initiation B with respect to the existing ADA at neutral pH is a native IgG against existing ADA at neutral pH (e.g., pH 7.4). Compared with the binding activity of a reference antibody containing the Fc region of, it is preferable that it is lower or not significantly increased. Low binding activity (binding affinity) for existing ADA or that the affinity is at baseline level may be, but is not limited to, an ECL response in an individual patient of less than 500, or less than 250. . In any patient population, the fact that the binding activity to the existing ADA is low is not limited thereto, but, for example, 90% of patients, preferably 95% of patients, more preferably in the patient population. Perhaps in 98% of patients, the ECL response may be less than 500.

The Fc region variant of initiation B or the antibody containing the variant does not significantly increase the binding activity to (existing) ADA in plasma at neutral pH, and FcRn at neutral pH and/or acidic pH. In some cases, it is desirable to select those with increased binding activity. It is preferable that the FcRn binding activity in acidic pH (for example, pH 5.8) is increased. In one embodiment, it is preferable that the Fc region variant does not significantly increase the binding activity to ADA under conditions of neutral pH (e.g., pH 7.4) compared to the Fc region of a native IgG. , The ADA is an existing ADA, and is preferably a rheumatoid factor (RF).

In one embodiment, the Fc region modified variant of initiation B has increased binding activity to FcRn under conditions of acidic pH as compared to the Fc region of native IgG, thereby reducing plasma clearance (CL). Or, there are cases where it is desirable that the residence time in plasma is prolonged, or the half-life (t1/2) in plasma is prolonged. It is well known in the art that they are correlated with each other.

In one embodiment, the Fc region modified of initiation B has increased binding activity to FcRn under conditions of acidic pH as compared to the Fc region of native IgG, but has binding activity to ADA under conditions of neutral pH. In some cases, it is preferable that the plasma clearance (CL) is not significantly increased, the plasma residence time is extended, or the plasma half-life (t1/2) is extended. The ADA may be an existing ADA, and it is preferable that it is a rheumatoid factor (RF).

In one embodiment, the Fc region variant of initiation B is compared with a reference Fc region variant comprising a combination of amino acid substitutions of N434Y/Y436V/Q438R/S440E according to EU numbering, and the retention in plasma is improved. It is advantageous because it is becoming.

In Examples 5 to 7, the Fc region modified F1718 described in WO2013/046704 (4 mutations N434Y/Y436V/Q438R/S440E are introduced into the Fc region) and a novel Fc region modified F1848m (N434A). Four mutations of /Y436V/Q438R/S440E are introduced.) Plasma retention of the two Fc region modified variants is compared. The difference between the amino acid mutations of both Fc region variants was that the amino acid mutation introduced at position 434 according to EU numbering, F1718 is Y (tyrosine), and F1848m is A (alanine). Nevertheless, F1848m showed improved plasma retention compared to that of native IgG1, whereas F1718 did not show improved plasma retention (see Example (7-2)). Therefore, it is preferable that the Fc region modified variant of initiation B has improved plasma retention as compared to a reference Fc region variant comprising a combination of substituted amino acids of N434Y/Y436V/Q438R/S440E. There are cases. On the other hand, from the experimental results of this Example (5-2) and this Example (7-3), among the various Fc region variants, F1847m, F1886m, F1889m, and F1927m have longer residence times in plasma than F1848m. It is recognized that it is becoming. Therefore, the Fc region variant of initiation B including F1848m as well as F1847m, F1886m, F1889m, or F1927m is in plasma compared to the reference Fc region variant including substitution of N434Y/Y436V/Q438R/S440E. Those skilled in the art can understand that the retention property is improving.

Binding to FcγR or complement proteins can also cause undesirable effects (eg, inappropriate platelet activation). Fc region modifiers that do not bind to effector receptors such as the FcγRIIa receptor may be safer and/or more effective. In some embodiments, the Fc region variant of initiating B has weak binding activity to the effector receptor or does not bind to the effector receptor. Examples of effector receptors include activating FcγR, particularly FcγRI, FcγRII, and FcγRIII. FcγRI includes FcγRIa, FcγRIb and FcγRIc, and subtypes thereof. FcγRII includes FcγRIIa (having two allotypes R131 and H131) and FcγRIIb. FcγRIII includes FcγRIIIa (having two allotypes V158 and F158) and FcγRIIIb (having two allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). Antibodies that have weak binding activity to effector receptors or that do not bind to them are, for example, antibodies containing a silent Fc region, and antibodies not containing an Fc region (e.g., Fab, F(ab)' ₂ , scFv, sc(Fv) ₂ , diabodies).

Examples of Fc regions that have weak binding activity to effector receptors or do not have corresponding binding activity are, for example, in Strohl et al. (Curr. Op. Biotech. 20(6):685-691(2009)). It is described. Specifically, for example, deglycosylated Fc regions (N297A and N297Q), and examples of silent Fc regions (IgG1-L234A/L235A, IgG1), which are Fc regions engineered to silence (or immunosuppress) effector functions. -H268Q/A330S/P331S, IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S, IgG4 -L235A/G237A/E318A, and IgG4-L236E) are described. WO2008/092117 discloses an antibody comprising a silent Fc region comprising the substitution G236R/L328R, L235G/G236R, N325A/L328R, or N325L/L328R (represented by the EU numbering system). WO2000/042072 discloses an antibody comprising a silent Fc region containing a substitution in one or more of EU233 position (the 233 position indicated by the EU numbering system), EU234 position, EU235 position, and EU237 position. In WO2009/011941, an antibody comprising a silent Fc region comprising deletion of residues from EU231 to EU238 is disclosed. Davis et al (J. Rheum. 34(11): 2204-2210 (2007)) discloses an antibody comprising a silent Fc region comprising the substitution C220S/C226S/C229S/P238S. Shields et al (J. Biol. Chem. 276 (9), 6591-6604 (2001)) discloses an antibody comprising a silent Fc region containing substitution D265A. Also in the Fc region modified variant of initiation B, modifications of these amino acid residues may be appropriately introduced.

The term "weak binding to an effector receptor" means that the binding activity to the effector receptor is, for example, 95% or less of the binding activity of an antibody comprising the Fc region of a native IgG, or a native IgG. Is 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 may mean binding activity.

The "silent Fc region" is a modified Fc region including one or more amino acid substitutions, insertions, additions, deletions, and the like that reduce binding to effector receptors compared to the native Fc region. Since the binding activity to the effector receptor can be greatly reduced, such silent Fc regions will no longer bind to the effector receptor. Examples of silent Fc regions include 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 may include an Fc region containing amino acid substitutions at one or a plurality of positions selected from the group consisting of, but is not limited to these. Also in the Fc region modified variant of initiation B, modifications of these amino acid sites may be appropriately introduced.

In a further embodiment, the silent Fc region is EU234, EU235, EU236, EU237, EU238, EU239, EU265, EU266, EU267, EU269, EU270, EU271, EU295, EU296, EU297, EU298, EU300, EU324, EU325, EU327 , EU328, EU329, EU331, and at one or a plurality of positions selected from the group consisting of EU332, preferably, EU235, EU237, EU238, EU239, EU270, EU298, EU325, and EU329 1 selected from the group consisting of At one or more positions, it has a substitution, and the substitution involves an amino acid residue selected from the following list:

The amino acid at position EU234 is preferably substituted with one of amino acids selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, and Thr.

The amino acid at position EU235 is preferably substituted with one of amino acids selected from the group consisting of Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, and Arg. do.

The amino acid at position EU236 is preferably substituted with one of amino acids selected from the group consisting of Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, and Tyr.

The amino acid at position EU 237 is preferably among amino acids selected from the group consisting of Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, and Arg. Is replaced by one.

The amino acid at position EU238 is preferably substituted with one of amino acids selected from the group consisting of Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, and Arg.

The amino acid at position EU239 is preferably substituted with one of amino acids selected from the group consisting of Gln, His, Lys, Phe, Pro, Trp, Tyr, and Arg.

The amino acid at position EU265 is preferably, among amino acids selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, and Val. Is replaced by one.

The amino acid at position EU266 is preferably substituted with one of amino acids selected from the group consisting of Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, and Tyr. do.

The amino acid at position EU267 is preferably substituted with one of amino acids selected from the group consisting of Arg, His, Lys, Phe, Pro, Trp, and Tyr.

The amino acid at position EU269 is preferably selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. It is substituted with one of the amino acids.

The amino acid at position EU270 is preferably selected from the group consisting of Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. It is substituted with one of the amino acids.

The amino acid at position EU271 is preferably substituted with one of amino acids selected from the group consisting of Arg, His, Phe, Ser, Thr, Trp, and Tyr.

The amino acid at position EU295 is preferably substituted with one of amino acids selected from the group consisting of Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, and Tyr.

The amino acid at position EU296 is preferably substituted with one of amino acids selected from the group consisting of Arg, Gly, Lys, and Pro.

The amino acid at position EU297 is preferably substituted with Ala.

The amino acid at position EU298 is preferably substituted with one of amino acids selected from the group consisting of Arg, Gly, Lys, Pro, Trp, and Tyr.

The amino acid at position EU 300 is preferably substituted with one of amino acids selected from the group consisting of Arg, Lys, and Pro.

The amino acid at position EU324 is preferably substituted with Lys or Pro.

The amino acid at position EU325 is preferably substituted with one of amino acids selected from the group consisting of Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, and Val.

The amino acid at position EU327 is preferably substituted with one of amino acids selected from the group consisting of Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The amino acid at position EU328 is preferably substituted with one of amino acids selected from the group consisting of Arg, Asn, Gly, His, Lys, and Pro.

The amino acid at position EU329 is preferably selected from the group consisting of Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, and Arg. It is substituted with one of the amino acids.

The amino acid at position EU330 is preferably substituted with Pro or Ser.

The amino acid at position EU331 is preferably substituted with one of amino acids selected from the group consisting of Arg, Gly, and Lys.

The amino acid at position EU332 is preferably substituted with one of amino acids selected from the group consisting of Arg, Lys, and Pro.

Preferably, the silent Fc region is substituted with Lys or Arg at position EU235, substituted with Lys or Arg at position EU237, substituted with Lys or Arg at position EU238, substituted with Lys or Arg at position EU239, EU270. Substitution with Phe in the above, Gly in EU298, Gly in EU325, or Lys or Arg in EU329 may be included. More preferably, the silent Fc region may contain a substitution with arginine at position EU235 or a substitution with lysine at position EU239. More preferably, the silent Fc region may comprise the substitution L235R/S239K. Also in the Fc region modified form of start B, you may introduce the modification of these amino acid residues as appropriate.

In one embodiment, the antibody containing the Fc region variant of initiation B has weak binding activity to a complement protein or does not bind to a complement protein. In some embodiments, the complement protein is C1q. In some embodiments, the weak binding activity to the complement protein is 1/10 or less, 1/50 or less, 1/10 or less, 1/50 or less, compared to the binding activity to the complement protein of an antibody comprising the Fc region of a native IgG or native IgG. It is a binding activity to a complement protein, which is reduced to /100 or less. The binding activity of the Fc region to a complement protein can be reduced by alteration of amino acids such as amino acid substitution, insertion, addition, or deletion.

In one embodiment, the evaluation of the binding activity to (human) FcRn in the neutral pH region and/or the acidic pH region of the Fc region variant of initiation B or the antibody containing the variant is performed in the same manner as described above. Can also be done.

In one embodiment, the method of modifying the antibody constant region for producing the Fc region variant of initiation B is, for example, by examining a plurality of constant region isotypes (IgG1, IgG2, IgG3, and IgG4), and It may be based on selecting an isotype in which the antigen-binding activity in the acidic region decreases and/or the dissociation rate in the acidic pH region is accelerated. An alternative method is to introduce an amino acid substitution into the amino acid sequence of a native IgG isotype to lower the antigen-binding activity in the acidic pH range (for example, pH 5.8), and/or the acidic pH range. It may be based on increasing the dissociation rate in. The sequence of the hinge region of the antibody constant region differs greatly depending on the isotype (IgG1, IgG2, IgG3, and IgG4), and the difference in the amino acid sequence of the hinge region can greatly affect the antigen-binding activity. By selecting an appropriate isotype according to the type of epitope, it is possible to select an isotype that lowers the antigen-binding activity in the acidic pH range and/or accelerates the dissociation rate in the acidic pH range. . In addition, since the difference in the amino acid sequence of the hinge region greatly affects the antigen-binding activity, the amino acid substitution site of the natural isotype amino acid sequence may be a hinge region.

In an alternative embodiment, initiation B is the release of the antibody containing the Fc region variant of initiation B described above, which has been absorbed into the cell in an antigen-bound state, to the outside of the cell in a state not bound to an antigen. The use of such antibodies to promote is provided. Here, "release of an antibody absorbed into a cell in a state bound to an antigen, in a state not bound to an antigen, to the outside of the cell" means that all antibodies absorbed into the cell in a state bound to the antigen are not bound to the antigen. It does not necessarily mean that it is released out of the cell in a state. Compared to before alteration of the FcRn-binding domain (e.g., before increasing the FcRn-binding activity in the acidic pH range of the antibody), the proportion of the antibody released outside the cell without binding to the antigen is It just needs to be high. It is preferable that the antibody released to the outside of the cell maintains antigen-binding activity.

The term "antigen elimination ability in plasma" or synonymous therewith refers to the ability to eliminate antigens from plasma when an antibody is administered in vivo or when an antibody is secreted in vivo. Therefore, "the ability of the antibody to lose antigen in plasma is increased" may mean that the rate at which antigen is lost from plasma is increased when the antibody is administered, for example, compared to before the FcRn-binding domain is altered. have. The increase in the activity of the antibody to eliminate antigens from plasma can be evaluated, for example, by administering a soluble antigen and an antibody in vivo, and measuring the plasma concentration of the soluble antigen after administration. The soluble antigen may be an antigen to which an antibody is bound, or an antigen to which an antibody is not bound, and the concentrations can be determined as "antibody-binding antigen concentration in plasma" and "antibody-free antigen concentration in plasma", respectively. . The latter is synonymous with "the concentration of free antigen in plasma". The "total antigen concentration in plasma" may mean a concentration obtained by adding an antibody-binding antigen concentration and an antibody-non-binding antigen concentration.

In an alternative embodiment, initiation B provides a method of extending the plasma retention time of an antibody comprising the Fc region variant of initiation B described above. Natural human IgG can bind to FcRn derived from non-human animals. For example, since native human IgG can bind to mouse FcRn more strongly than human FcRn (Ober et al., Intl. Immunol. 13(12): 1551-1559(2001)), the characteristics of the antibody were confirmed. In order to do this, the antibody can be administered to the mouse. As another example, a mouse (Roopenian et al., Meth. Mol. Biol. 602: 93-104 (2010)) also expresses a human FcRn gene as a transgene, although the original FcRn gene is destroyed. It is also preferred for evaluating antibodies.

Within the range of disclosures A and B in this specification, it is possible to determine the plasma concentration of the free antigen that does not bind to the antibody, or the ratio of the free antigen concentration to the total antigen concentration (for example, Ng et al., Pharm. Res. 23(1):95-103(2006)). Alternatively, when an antigen exhibits a certain function in vivo, whether the antigen binds to an antibody (antagonist molecule) that neutralizes the function of the antigen can be evaluated by testing whether the function of the antigen is neutralized. . Whether or not the function of the antigen is being neutralized can be evaluated by measuring any in vivo marker that reflects the function of the antigen. Whether or not an antigen binds to an antibody (agonist molecule) that activates the function of the antigen can be evaluated by measuring any in vivo marker that reflects the function of the antigen.

Measurement of the plasma concentration of free antigen, measurement of the ratio of the amount of free antigen in plasma to the total amount of antigen in plasma, and measurement of an in vivo marker are not particularly limited, but after a certain period of time has elapsed after the antibody is administered. There are cases where it is desirable to perform measurement. In the context of Initiation B, "after a certain period of time has elapsed since the antibody is administered" is not particularly limited, and a person skilled in the art can appropriately determine the period according to the nature of the administered antibody, for example, administration of the antibody. After 1 day, after 3 days, after 7 days, after 14 days, or after 28 days, etc. are mentioned. Herein, the term "antigen concentration in plasma" is either "total antigen concentration in plasma", which is the sum of the concentration of antibody-binding antigen and non-antibody antigen, or "free antigen concentration in plasma", which is the concentration of non-antibody-binding antigen. It can mean one.

The antigen/antibody molar ratio can be calculated by the formula: C=A/B, where the A value is the molar concentration of the antigen at each time point, and the B value is the molar concentration of the antibody at each time point, The C value is the molar concentration of the antigen per molar concentration of the antibody at each time point (antigen/antibody molar ratio).

A smaller C value indicates a higher antigen elimination efficiency near the antibody, and a larger C value indicates a lower antigen elimination efficiency near the antibody.

In some embodiments, when initiation B is administered, the antigen/antibody molar ratio is 2 times, 5 times, 10 times compared to the case of administering a reference antibody comprising the Fc region of a native human IgG as a human FcRn binding domain. It can be reduced by times, 20 times, 50 times, 100 times, 200 times, 500 times, 1,000 times or more.

The reduction of the total antigen concentration or the antigen/antibody molar ratio in plasma can be evaluated, for example, as described in Examples 6, 8 and 13 of WO2011/122011 using a method known in the art. More specifically, when the antibody of interest in Initiation B does not cross-react with the mouse counterpart antigen, human FcRn transgenic mouse line 32 or line 276 (Jackson Laboratories, Methods Mol. Biol. 602: 93-104) (2010)) can be evaluated based on either the antigen-antibody simultaneous injection model or the steady-state antigen injection model. When the antibody cross-reacts with the mouse counterpart, it can be evaluated by simply injecting the antibody into human FcRn transgenic mouse strain 32 or strain 276 (Jackson Laboratories). In the simultaneous injection model, a mixture of antibody and antigen is administered to mice. In the steady state antigen injection model, in order to achieve a constant plasma antigen concentration, a mouse is embedded with an infusion pump filled with an antigen solution, and then the antibody is injected into the mouse. All test antibodies are administered at the same dose. The total antigen concentration in plasma, the free antigen concentration in plasma, and the antibody concentration in plasma can be measured at an appropriate time point.

After 2 days, 4 days, 7 days, 14 days, 28 days, 56 days, or 84 days after administration, the total antigen concentration or free antigen concentration in plasma, or the antigen/antibody molar ratio is measured, and initiation B The long-term effect of the antibody of can be evaluated. In other words, in order to evaluate the properties of the antibody, the antigen concentration in plasma for a long period of time is 2 days, 4 days, 7 days, 14 days, 28 days, 56 days, or 84 days after administration of the antibody. It can be determined by measuring the total antigen concentration or free antigen concentration in plasma, or the antigen/antibody molar ratio. Whether a decrease in the plasma antigen concentration or the antigen/antibody molar ratio is achieved by the antibody can be determined by evaluating the decrease at one or a plurality of time points described above.

After 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration, the total antigen concentration or free antigen concentration in plasma or the antigen/antibody molar ratio is measured, and start B The short-term effect of the antibody of can be evaluated. In other words, in order to evaluate the properties of the antibody, the antigen concentration in the plasma for a short period of time is 15 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours after administration of the antibody. It can be determined by measuring the total antigen concentration or free antigen concentration in plasma, or the antigen/antibody molar ratio. When it is difficult to measure plasma retention in humans, mice (e.g., normal mice, human antigen-expressing transgenic mice, or human FcRn-expressing transgenic mice, etc.) or monkeys (e.g., Philippine monkeys, etc. ) On the basis of plasma retention in humans may be predicted.

In an alternative embodiment, initiation B relates to an antibody comprising the Fc region variant of initiation B described above. Various embodiments of the antibody are described within the range of disclosures A and B in the present specification, and can be applied mutatis mutandis as long as they do not contradict common knowledge in the art and do not contradict the context.

In one embodiment, the antibody containing the Fc region variant of initiation B is affected by autoimmune diseases, transplant rejection (graft versus host disease), other inflammatory diseases or allergic diseases, as described in WO2013/046704. It is advantageous as a therapeutic antibody for the treatment of human patients who are doing it.

In one embodiment, the antibody containing the Fc region modified variant of initiation B may have a modified sugar chain. Examples of antibodies with altered sugar chains include, for example, antibodies with altered glycosylation (WO99/54342), antibodies with a deletion of fucose (WO00/61739, WO02/31140, WO2006/067847, WO2006/067913), and bi And antibodies having a sugar chain having a sectioning GlcNAc (WO02/79255). In one embodiment, the antibody may be deglycosylated. In some embodiments, the antibody comprises a mutation in the heavy chain glycosylation site to prevent glycosylation at the site, for example as described in WO2005/03175. Such non-glycosylated antibodies can be prepared by altering the heavy chain glycosylation site, i.e., introducing substitution N297Q or N297A indicated by EU numbering, and expressing this protein in an appropriate host cell.

In an alternative embodiment, Initiation B relates to a composition or pharmaceutical composition comprising an antibody comprising such an Fc region variant. As long as the composition or various embodiments of the pharmaceutical composition within the range of disclosures A and B in this specification are not contradictory to the ordinary technical knowledge in the art and are not contradictory in the context, it is applied mutatis mutandis. May be. The composition can be used in order to improve the retention in plasma (in the subject when the antibody of initiation B is administered (applied) to the subject).

In an alternative embodiment, initiation B relates to a nucleic acid encoding an antibody comprising the aforementioned Fc region variant or the variant. Various embodiments described for nucleic acids within the range of disclosures A and B in the present specification may be applied mutatis mutandis as long as they do not contradict the technical common sense of those skilled in the art and do not contradict the context. Alternatively, initiation B relates to a vector containing the nucleic acid. The various embodiments within the range describing the disclosures A and B in the present specification may be applied mutatis mutandis as long as they do not contradict the technical common sense of those skilled in the art and do not contradict the context. Alternatively, initiation B relates to a host or host cell containing the vector. The various embodiments within the range describing the disclosures A and B in the present specification may be applied mutatis mutandis as long as they do not contradict the technical common sense of those skilled in the art and do not contradict the context.

In an alternative embodiment, initiation B is an Fc region variant containing an FcRn binding domain, or a method for producing an antibody containing the variant, by culturing the above-described host cell or by culturing the above-described host. , A production method comprising the step of recovering the Fc region variant or an antibody containing the variant from the culture of the cell or the secretion of the host. In this case, initiation B is a step of (a) selecting an Fc region variant having increased binding activity to FcRn under conditions of acidic pH compared to that of the native IgG Fc region; (b) a step of selecting an Fc region variant whose binding activity to (existing) ADA is not significantly increased under conditions of neutral pH as compared with the Fc region of a native IgG; (c) comparing the Fc region of native IgG, selecting an Fc region variant having improved plasma retention; And (d) compared to a reference antibody containing the Fc region of a native IgG, the step of selecting an antibody containing an Fc region variant capable of promoting the loss of antigen from plasma is optionally added. You may include a manufacturing method to include as.

Here, although not limited, the antibody containing the Fc region modified variant of the initiation B produced and the "reference antibody containing the Fc region of a native IgG" are said to be identical to each other except for the Fc region portion to be compared. In some cases, this is preferable in evaluating the plasma retention of the Fc region variant of initiation B. In addition, in some cases, it is preferable that FcRn is human FcRn.

For example, after preparing an antibody containing the Fc region variant of initiation B, using BIACORE (registered trademark) or other known technology, the acidity of the reference antibody containing the antibody and the Fc region of a native IgG By comparing the binding activity to FcRn under conditions of pH (e.g., pH 5.8), an Fc region variant or an antibody containing the variant, which has increased binding activity to the FcRn under acidic pH conditions, is selected. Can also be done.

Alternatively, for example, after preparing an antibody containing the Fc region variant of initiation B, electrochemiluminescence (ECL) or known techniques are used to obtain a reference antibody containing the antibody and the Fc region of a native IgG. , By comparing the binding activity to ADA under conditions of neutral pH, the Fc region variant or antibody containing the variant may be selected for which the binding activity to the ADA is not significantly increased under conditions of neutral pH. .

Or, for example, after preparing an antibody containing the Fc region variant of initiation B, for example, by carrying out a pharmacokinetic test of the antibody using plasma of mice, rats, rabbits, dogs, monkeys, or humans, The antibody and the reference antibody containing the Fc region of a native IgG were compared, and the Fc region variant or antibody containing the variant was selected for which it was confirmed that the retention in plasma is improved in the subject. Can also be done.

Alternatively, for example, after preparing an antibody containing the Fc region variant of initiation B, for example, by performing a pharmacokinetic test of the antibody using plasma of a mouse, rat, rabbit, dog, monkey, or human. , The antibody may be compared with a reference antibody containing the Fc region of a native IgG, and an Fc region variant or an antibody containing the variant may be selected, in which the disappearance of the antigen from plasma is promoted.

Alternatively, for example, these selection methods may be appropriately combined as desired.

In one embodiment, initiation B relates to a method for producing an Fc region variant containing an FcRn-binding domain, or an antibody containing the variant, wherein the method is the obtained Fc region variant or the The antibody containing the variant is Ala at position 434 according to EU numbering; Glu, Arg, Ser, or Lys at position 438; And a step of substituting an amino acid to include Glu, Asp, or Gln at position 440. In a further embodiment, such a method is that the obtained Fc region variant or antibody comprising the variant is, according to EU numbering, Ile or Leu at position 428, and/or Ile, Leu at position 436, It includes a step of substituting amino acids to further include Val, Thr, or Phe. In a further embodiment, the amino acid is the Fc region modified product produced and obtained according to the method, or the antibody containing the modified, according to EU numbering, Leu at position 428 and/or Val or Thr at position 436. It is substituted to further include.

In one embodiment, initiation B relates to a method for producing an Fc region variant containing an FcRn-binding domain, or an antibody containing the variant, wherein the method is the obtained Fc region variant or the The antibody containing the variant is Ala at position 434 according to EU numbering; Arg or Lys at position 438; And a step of substituting an amino acid to include Glu or Asp at position 440. In a further embodiment, such a method is that the obtained Fc region variant or antibody comprising the variant is, according to EU numbering, Ile or Leu at position 428, and/or Ile, Leu at position 436, It includes a step of substituting amino acids to further include Val, Thr, or Phe. In a further embodiment, the amino acid is the Fc region modified product produced and obtained according to the method, or the antibody containing the modified, according to EU numbering, Leu at position 428 and/or Val or Thr at position 436. It is substituted to further include.

In one embodiment, such a method includes all amino acids at positions 434, 438, and 440, respectively, Ala; Glu, Arg, Ser, or Lys; And a step of substituting with Glu, Asp, or Gln. In a further embodiment, such a method is that the obtained Fc region variant or antibody comprising the variant is, according to EU numbering, Ile or Leu at position 428, and/or Ile, Leu at position 436, It includes a step of substituting amino acids to further include Val, Thr, or Phe. In a further embodiment, the amino acid is the Fc region modified product obtained by producing according to the method or the antibody containing the modified product, according to EU numbering, Leu at position 428, and/or Val or Thr at position 436. It is substituted to further include.

In an alternative embodiment, Initiation B relates to an Fc region modified variant obtained by any of the above-described production methods of Initiation B or an antibody containing the Fc region variant.

In an alternative embodiment, initiation B is the (conventional) ADA-binding activity of an antibody comprising an Fc region variant having increased binding activity to FcRn at acidic pH, comprising the following steps: How to lower it; And the binding activity to FcRn at acidic pH (e.g., pH 5.8) is increased, and the binding activity to the existing ADA is reduced, providing a method for producing an Fc region variant: (a) Reference antibody Compared with, the step of providing an antibody containing an Fc region (variant) with increased binding activity to FcRn at acidic pH, and (b) in the Fc region, according to EU numbering, (i ) Amino acid substitution by Ala at position 434; (ii) amino acid substitution by Glu, Arg, Ser or Lys at position 438; And (iii) an amino acid substitution by Glu, Asp or Gln at position 440, (iv) optionally, an amino acid substitution by Ile or Leu at position 428; And/or (v) optionally, a step of introducing an amino acid substitution by Ile, Leu, Val, Thr or Phe at position 436.

In some embodiments, the preferred Fc domain (variant) in step (a) is the Fc domain (variant) of human IgG. In addition, in order to increase the binding activity to FcRn at acidic pH and to reduce the (conventional) binding activity to ADA in the neutral pH region (e.g., pH 7.4), the Fc region (variant) is , (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; (l) 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) M428L/N434A/Q438R/S440E; (v) M428L/N434A/Q438R/S440D; (w) M428L/N434A/Q438K/S440E; (x) M428L/N434A/Q438K/S440D; (y) M428L/N434A/Y436T/Q438R/S440E; (z) M428L/N434A/Y436T/Q438R/S440D; (aa) M428L/N434A/Y436T/Q438K/S440E; (ab) M428L/N434A/Y436T/Q438K/S440D; (ac) M428L/N434A/Y436V/Q438R/S440E; (ad) M428L/N434A/Y436V/Q438R/S440D; (ae) M428L/N434A/Y436V/Q438K/S440E; (af) M428L/N434A/Y436V/Q438K/S440D; (ag) L235R/G236R/S239K/M428L/N434A/Y436T/Q438R/S440E; And (ah) a combination of substituted amino acids selected from the group consisting of L235R/G236R/A327G/A330S/P331S/M428L/N434A/Y436T/Q438R/S440E.

Optionally, the method further comprises a step of (c) confirming that the (conventional) ADA-binding activity of the antibody containing the produced Fc region variant is lowered compared to the binding activity of the reference antibody. You may include it.

Alternatively, the method does not significantly increase the binding activity of the (conventional) antibody to ADA at neutral pH, and the antibody absorbed into the cell in a state bound to the antigen, in a state that is not bound to the antigen. It may be used as a method of promoting release to the outside of cells.

Initiation C

Initiation C is an anti-IL-8 antibody described below, a nucleic acid encoding the antibody, a pharmaceutical composition containing the antibody, a method for producing the antibody, and the antibody in the treatment of diseases related to IL-8. It relates to the use of. On the other hand, the meaning of the terms described below may be applied mutatis mutandis to all descriptions of the disclosure C in this specification in addition to known embodiments of the art, without contrary to the technical common sense of those skilled in the art.

I. Definitions in the scope of the disclosure C

In the range describing the disclosure C in this specification, "acidic pH" refers to a pH that can be selected from pH 4.0 to pH 6.5, for example. In one embodiment, the acidic pH is, 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 certain embodiments, the term acidic pH refers to pH 5.8.

In the range describing the disclosure C in this specification, "neutral pH" refers to a pH that can be selected from pH 6.7 to pH 10.0, for example. In one embodiment, the neutral pH is, 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, but are not limited thereto. In certain embodiments, the term neutral pH refers to pH 7.4.

The term "IL-8" in Disclosure C is all vertebrates, primates (e.g. humans, Filipino monkeys, rhesus monkeys), and other mammals (e.g., dogs or rabbits), unless otherwise indicated. ) Refers to any native IL-8 derived. The term "IL-8" also includes full-length IL-8, unprocessed IL-8, and all forms of IL-8 resulting from processing in cells. The term "IL-8" also includes derivatives of native IL-8, such as splice variants and Allel variants. The amino acid sequence of an exemplary human IL-8 is shown in SEQ ID NO: 66.

The terms “anti-IL-8 antibody” and “antibody that binds to IL-8” refer to IL-8 with sufficient affinity, which is useful as a diagnostic and/or therapeutic agent by the antibody targeting IL-8. It refers to an antibody capable of binding.

In one embodiment, the degree of binding of the anti-IL-8 antibody to an unrelated non-IL-8 protein may be, for example, less than about 10% of the binding of the antibody to IL-8.

In the range describing the disclosure C in this specification, "affinity" is generally the total of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Means the strength of the sum. Unless otherwise indicated, "binding affinity" in the range described in the disclosure C in this specification refers to intrinsic binding that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). Means affinity. The affinity of a molecule X for its partner Y can generally be expressed as the dissociation constant (KD). The binding affinity may be measured using a method known to those skilled in the art, including those described in the range in which the disclosure C in this specification is described.

In certain embodiments, the antibody that binds to IL-8 is, for example, ≤1000nM, ≤100nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM, or ≤0.001nM (e.g., 10 ^-8 M or less, 10 ^-8 M to 10 ^-13 M, 10 ^-9 M to 10 ^-13 M).

The term "antibody" in the range describing the disclosure C in this specification is used in the broadest sense, and is not limited thereto, but as long as it exhibits a desired antigen-binding activity, a monoclonal antibody, Polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) and antibody fragments.

The term "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks, for example, 50%, 60%, 70%, or 80% or more of the binding of the reference antibody to the antigen, and vice versa, the reference antibody Inhibits, for example, 50%, 60%, 70%, or 80% or more of the binding of the antibody to its antigen. Here, an exemplary contention assay may be used, but is not limited thereto.

A "chimeric antibody" refers to an antibody in which a part of the heavy and/or light chain is derived from a specific source or species, and the remaining part is derived from another source or species.

The "humanized" antibody refers to a chimeric antibody containing an amino acid residue derived from a non-human HVR and an amino acid residue derived from a human FR. In certain embodiments, the humanized antibody may contain substantially at least one, typically two variable regions, and in the variable region, all (or substantially all) HVRs (e.g. CDRs) are non-human antibodies. It corresponds to HVR of, and all (or substantially all) FRs correspond to FRs of human antibodies. The humanized antibody may contain at least a part of an antibody constant region derived from a human antibody, depending on the case.

The term "monoclonal antibody" used in the range describing the disclosure C in this specification means an antibody obtained from a population of substantially homogeneous antibodies, that is, includes, for example, a mutation occurring in nature, or Or, except for potential modified antibodies, such as those occurring in the production of monoclonal antibody preparations and generally present in small amounts, the individual antibodies constituting the population are the same, and/or on the same epitope. Combine. In contrast to polyclonal antibody preparations that generally contain other antibodies to other determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" denotes the characteristics of an antibody obtained from a population of substantially homogeneous antibodies, and should not be interpreted as requiring the production of the antibody by any specific method. For example, the monoclonal antibody used according to the initiation C is not limited, but the hybridoma method, the recombinant DNA method, the phage display method, and a transgenic animal containing all or part of the human immunoglobulin locus are used. Such methods and other exemplary methods for making monoclonal antibodies, made by a variety of techniques, including methods, are described herein.

In the scope of the disclosure C in this specification, the "natural antibody" refers to an immunoglobulin molecule that has various structures that occur naturally. For example, one embodiment of a native IgG antibody is a heterotetrameric glycoprotein of about 150,000 Daltons consisting of two identical light chains and two identical heavy chains with a disulfide bond, but is not limited thereto. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, and three normal domains (CH1, CH2, and CH3) continue. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, and a normal light chain (CL) domain continues. The light chain of an antibody can be assigned to either of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its normal domain. As the normal domain used as the initiation C, any reported allotype (Arel) or any subclass/isotype may be used. The heavy chain constant region is not limited, but a constant region of a native IgG antibody (IgG1, IgG2, IgG3, and IgG4) can be used. For example, allel of IgG1 ^{is known as IGHG1 *} 01, IGHG1 ^* 02, IGHG1 ^* 03, IGHG1 ^* 04, and IGHG1 ^* 05 (see imgt.org), but all of these are as human native IgG1 sequences. Can be used. In addition, the sequence of the normal domain may be derived from a single allel or subclass/isotype, or may be derived from a plurality of alleles or subclass/isotype. That is, such an antibody is not limited to this, for example, the antibody that CH1 is ^{derived from IGHG1 *} 01, CH2 is ^{derived from IGHG1 *} 02, and CH3 is ^{derived from IGHG1 *} 01 is also included. .

In the range described in the disclosure C in this specification, "effector function" refers to a biological activity attributable to the Fc region of an antibody, and may vary depending on the isotype of the antibody. Examples of effector functions of antibodies include C1q binding and complement dependent cellular disorders; Fc receptor binding; Antibody dependent cell mediated cell disorder (ADCC); Phagocytosis; Down regulation of cell surface receptors (eg, B cell receptors); And B cell activation.

The term "Fc region" in the range describing the disclosure C in this specification is used to define the C-terminal region of an immunoglobulin heavy chain including at least a part of the normal region. The term includes native Fc regions and modified Fc regions. The native Fc region refers to the Fc region of a native antibody.

In one embodiment, the human IgG heavy chain Fc region extends from the amino acid residue of Cys226 or Pro230 to the carboxyl terminal of the heavy chain. However, lysine (Lys447) at the C-terminus of the Fc region or glycine-lysine (residues 446-447) may be present or not. Unless otherwise specified, the numbering of amino acid residues in the Fc region or the constant region in the range where the disclosure C in this specification is described is described in the following sources, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. It is by the EU numbering system, also called the EU Index, as described by the Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

In the range describing the disclosure C in this specification, "framework" or "FR" is a variable domain residue other than a high frequency variable region (HVR) residue. Typically, the FR of a variable domain consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the sequence of HVR and FR is usually shown in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2) -FR3-H3(L3)-FR4.

The "human consensus framework" in the range describing the disclosure C in this specification is a framework representing the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Usually, the human immunoglobulin VL or VH sequence is selected from a subgroup of variable domain sequences. Usually, subgroups of sequences are described in Kabat et al., Sequences of proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al., above. In one embodiment, for VH, the subgroup is subgroup III by Kabat et al., above.

The "acceptor human framework" for the purpose within the range of the disclosure C in this specification is a framework comprising the amino acid sequence of a VL or VH framework obtained from a human immunoglobulin framework or a human consensus framework. to be. The acceptor human framework "obtained from" the human immunoglobulin framework or the human consensus framework may contain an amino acid sequence identical thereto, or may contain an existing amino acid sequence substitution. In some embodiments, the number of existing amino acid substitutions is 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 VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

The term "variable region" or "variable domain" in the range describing the disclosure C in this specification refers to the domain of the heavy or light chain of an antibody, which is involved in the binding of the antibody to an antigen. The variable regions of the heavy and light chains of a native antibody (VH and VL, respectively) usually have a similar structure, and each domain contains four conserved framework regions (FR) and three high frequency variable regions (HVR). (See Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). One VH or VL domain is sufficient to impart antigen binding specificity, but is not limited thereto. Moreover, antibodies that bind to a specific antigen may be isolated from antibodies that bind to the antigen using VH or VL domains in order to screen for a complementary library of VL or VH domains. For example, Portolano et al., J. Immunol. 150:880-887 (1993); See Clarkson et al., Nature 352:624-628 (1991).

The term "high frequency variable region" or "HVR" used in the range describing the disclosure C in this specification is highly variable in sequence ("complementarity determining region" or "CDR"), and/or It means each region of the variable domain of an antibody that forms a structurally defined loop (“highly variable loop”), and/or contains an antigen-contacting moiety (“antigen contact”). In general, antibodies contain 6 high frequency variable regions; There are 3 (H1, H2, H3) in VH and 3 (L1, L2, L3) in VL.

Exemplary HVRs include, but are not limited to, the following: (a) amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1) , 53-55 (H2), and 96-101 (H3), a high frequency variable loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs with amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 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 with amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) Contact (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); And (d) HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49 Combinations with (a), (b), and/or (c), including -65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVRs and other residues in the variable region (eg, FR residues, etc.) can be numbered as shown in Kabat et al. mentioned above.

In the scope of the disclosure C in this specification, "individual" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs and horses, etc.), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (mouse Or rats, etc.). In some embodiments, the “individual” is a human.

In the range described in the disclosure C in this specification, an "isolated" antibody is one isolated from a component of its natural environment. In some embodiments, the antibody is, for example, by chromatography (e.g., ion exchange or reverse phase HPLC), or by electrophoresis (e.g. SDS-PAGE, isoelectric point electrophoresis (IEF), capillary electrophoresis). ), purified to a measured purity of greater than 95% or greater than 99%. A review of methods for measuring the purity of antibodies can be found in, for example, Flatman et al., J. Chromatogr. See B 848:79-87 (2007).

In the scope of the disclosure C in this specification, the "isolated" nucleic acid refers to a nucleic acid molecule isolated from an element of the natural environment. The isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains a nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or at a position different from its natural chromosomal position.

The term "isolated nucleic acid encoding an anti-IL-8 antibody" in the scope of the disclosure C in this specification refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the anti-IL-8 antibody. And such nucleic acids are, for example, in one vector or in a separate vector, or at one or more locations in a host cell.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which foreign nucleic acids have been introduced within the scope of the disclosure C in this specification. Includes his descendants. Host cells include "transformants" and "transformed cells", which include both primary transformed cells and progeny derived therefrom regardless of the number of passages. The progeny may not be completely identical in the content of the parent cell and the nucleic acid, or may contain mutations. Variant progeny having the same functional or biological activity as initially selected or screened from the transformed cell are also included here.

The term "vector" used within the range of the disclosure C in this specification refers to a nucleic acid molecule capable of amplifying another nucleic acid linked thereto. The term includes a vector introduced into a host cell and introduced into its genome, and a vector as a self-replicating nucleic acid structure. Certain vectors are capable of directing the expression of nucleic acids to which they are functionally linked. Such a vector is referred to as "expression vector" in this specification.

Within the scope of disclosure C in this specification, the term "package insert" means efficacy, usage, dosage, administration, combination therapy, information on contraindications, and/or warnings regarding the use of such therapeutic products. Used to refer to instructions customarily included in commercial packages of therapeutic products, including.

In the range described in the disclosure C in this specification, "percent (%) amino acid sequence identity" with respect to the reference polypeptide sequence means introducing a gap if necessary to obtain the maximum percent sequence identity, and any conservative degree of substitution. It is defined as the percentage of the amino acid residues in the candidate sequence identical to the amino acid residues of the reference polypeptide sequence after sequence alignment, which is not considered to be part of sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished by various methods within the skill range of those skilled in the art, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Co., Ltd.) can be achieved by using publicly available computer software. Those of skill in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for the purpose here, the% amino acid sequence identity value can be obtained, for example, by using the sequence comparison computer program ALIGN-2. ALIGN-2 is produced by Genentech, and the source code of ALIGN-2 is submitted to the US Copyright Office, Washington, D.C., 20559, along with user documents, and is registered under US copyright registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, South San Francisco, California, or is compiled from the source code. The ALIGN-2 program is compiled for use in a UNIX (registered trademark) operating system, for example, digital UNIX (registered trademark) V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In situations in which ALIGN-2 is used for amino acid sequence comparison, the% amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (or a given amino acid sequence having or containing a specific% amino acid sequence identity to a given amino acid sequence B). The amino acid sequence A) is calculated as follows: 100 times fraction X/Y. Here, X is the number of amino acid residues scored as perfect matches by the program alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues of B. When the length of amino acid sequence A is different from the length of amino acid sequence B, it will be understood that the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A. Unless otherwise noted, all% amino acid sequence identity values herein can be obtained by using an ALIGN-2 computer program as shown in the range in which the disclosure C in this specification is described.

In the range describing the disclosure C in this specification, the "pharmaceutical composition" usually refers to a drug for treating, preventing, testing, or diagnosing a disease. The "pharmaceutically acceptable carrier" refers to constituent elements other than the active ingredient in the pharmaceutical composition, and is harmless to a subject. Such pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, and preservatives.

"Treatment" (and its grammatical variation, for example, "treat", or "treating") of the place used within the range of the disclosure C in this specification is treatment It refers to a clinical intervention to alter the natural course of the individual being subjected, which can be carried out either for prophylaxis or during the course of clinical pathology. Preferred effects of the treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, and disease state. Recovery or remission of, and a remission or improved prognosis. In one embodiment, the antibody of initiation C can be used to slow the progression of a disease or disease.

In the range described in the disclosure C in this specification, the term "effective amount" of an antibody or pharmaceutical composition means a period necessary to achieve a desired therapeutic or prophylactic result, and an effective amount when used in a required dose. do.

II. Composition and method

In one embodiment, initiation C is based on the availability of an anti-IL-8 antibody having a pH-dependent affinity for IL-8 as a pharmaceutical composition. The antibody of initiation C is useful, for example, for diagnosis or treatment of a disease in which IL-8 is excessively present.

A. Exemplary anti-IL-8 antibodies

In one embodiment, initiation C provides an anti-IL-8 antibody having a pH dependent affinity for IL-8.

In one embodiment, initiation C is at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in the amino acid sequence of (a) to (f) below. Anti-IL-8 antibodies comprising a sequence comprising a substitution and having a pH dependent affinity for IL-8 are provided: (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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72.

In another embodiment, initiation C includes substitution of at least one amino acid in at least any one of the following amino acid sequences (a) to (f), and pH-dependent affinity for IL-8. It provides an anti-IL-8 antibody having: (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.

Unless otherwise specified, amino acids may be substituted with any other amino acid. In one embodiment, the initiating C anti-IL-8 antibody comprises substitution of one or more amino acids at a position selected from the group consisting of: (a) at position 1 of the sequence of SEQ ID NO: 67. Aspartic acid; (b) the tyrosine at position 2 in the sequence of SEQ ID NO: 67; (c) the 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) an alanine at position 6 in the sequence of SEQ ID NO: 68; (l) 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) the tyrosine at position 9 of the sequence of SEQ ID NO: 68; (o) threonine at position 10 of 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) the tyrosine at position 13 in the sequence of SEQ ID NO: 68; (s) serine at position 14 of the sequence of SEQ ID NO: 68; (t) an alanine at position 15 of 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 of 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) the 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 of the sequence of SEQ ID NO: 69; (ah) an alanine at position 10 of the sequence of SEQ ID NO: 69; (ai) the tyrosine at position 11 of the sequence of SEQ ID NO: 69; (aj) arginine at position 1 of the sequence of SEQ ID NO: 70; (ak) an 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 of the sequence of SEQ ID NO: 70; (ao) isoleucine at position 6 of the sequence of SEQ ID NO: 70; (ap) the tyrosine at position 7 in the sequence of SEQ ID NO: 70; (aq) serine at position 8 of the sequence of SEQ ID NO: 70; (ar) the tyrosine at position 9 of the sequence of SEQ ID NO: 70; (as) leucine at position 10 of the sequence of SEQ ID NO: 70; (at) an alanine at position 11 of the sequence of SEQ ID NO: 70; (au) asparagine at position 1 of 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 of 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 of 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; (bc) 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 at 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 of the sequence of SEQ ID NO: 72.

In one embodiment, the initiating C anti-IL-8 antibody comprises substitution of one or more amino acids at a position selected from the group consisting of: (a) at position 6 of the sequence of SEQ ID NO: 68. Alanine; (b) glycine at position 8 in the sequence of SEQ ID NO: 68; (c) the tyrosine at position 9 of the sequence of SEQ ID NO: 68; (d) arginine at position 11 in the sequence of SEQ ID NO: 68; And (e) the tyrosine at position 3 in the sequence of SEQ ID NO: 69.

In one embodiment, the initiating C anti-IL-8 antibody comprises a combination of substitutions of amino acids 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) the tyrosine at position 9 of the sequence of SEQ ID NO: 68; (d) arginine at position 11 in the sequence of SEQ ID NO: 68; And (e) the tyrosine at position 3 in the sequence of SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody of initiation C comprises: (a) tyrosine at position 9 of the sequence of SEQ ID NO: 68; (b) arginine at position 11 in the sequence of SEQ ID NO: 68; And (c) substitution of an amino acid at the position of the tyrosine at position 3 in the sequence of SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody of initiation C comprises (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) the tyrosine at position 9 of the sequence of SEQ ID NO: 68; (d) arginine at position 11 in the sequence of SEQ ID NO: 68; And (e) substitution of an amino acid at the position of the tyrosine at position 3 in the sequence of SEQ ID NO: 69.

In one embodiment, the anti-IL-8 antibody of initiation C comprises: (a) substitution of alanine at position 6 in the sequence of SEQ ID NO: 68 with aspartic acid; (b) substitution of arginine at position 11 in the sequence of SEQ ID NO: 68 with proline; And (c) substitution of tyrosine at position 3 in the sequence of SEQ ID NO: 69 with histidine.

In one embodiment, the initiating C anti-IL-8 antibody comprises: (a) substitution of glycine at position 8 in the sequence of SEQ ID NO: 68 with tyrosine; (b) A substitution of tyrosine at position 9 in the sequence of SEQ ID NO: 68 with histidine is included.

In one embodiment, the anti-IL-8 antibody of initiation C comprises: (a) substitution of alanine at position 6 in the sequence of SEQ ID NO: 68 with aspartic acid; (b) substitution of glycine at position 8 in the sequence of SEQ ID NO: 68 with tyrosine; (c) substitution of the tyrosine at position 9 in the sequence of SEQ ID NO: 68 with histidine; (d) substitution of arginine at position 11 in the sequence of SEQ ID NO: 68 with proline; And (e) substitution of tyrosine at position 3 in the sequence of SEQ ID NO: 69 with histidine.

In one embodiment, the anti-IL-8 antibody of Initiation C comprises HVR-H2 comprising the amino acid sequence of SEQ ID NO: 73.

In one embodiment, the anti-IL-8 antibody of Initiation C comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 74.

In one embodiment, the anti-IL-8 antibody of initiation C is 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 the amino acid sequence of SEQ ID NO: 74. It includes HVR-H3 containing.

In one embodiment, the initiating C anti-IL-8 antibody comprises substitution of one or more amino acids at a position selected from the group consisting of: (a) at position 8 of the sequence of SEQ ID NO: 70. Serine; (b) asparagine at position 1 of 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 any two, three, or all four combinations of their substitutions.

In one embodiment, the initiating C anti-IL-8 antibody comprises a combination of substitutions of amino acids 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 of 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 any two, three, or all four combinations of their substitutions.

In one embodiment, the anti-IL-8 antibody of initiation C comprises: (a) asparagine at position 1 of the sequence of SEQ ID NO: 71; (b) leucine at position 5 in the sequence of SEQ ID NO: 71; And (c) substitution of an amino acid at the glutamine position at position 1 in the sequence of SEQ ID NO: 72.

In one embodiment, the anti-IL-8 antibody of initiation C comprises (a) serine at position 8 of the sequence of SEQ ID NO: 70; (b) asparagine at position 1 of the sequence of SEQ ID NO: 71; (c) leucine at position 5 in the sequence of SEQ ID NO: 71; And (d) substitution of an amino acid at the glutamine position at position 1 in the sequence of SEQ ID NO: 72.

In one embodiment, the anti-IL-8 antibody of initiation C comprises: (a) substitution of asparagine at position 1 in the sequence of SEQ ID NO: 71 with lysine; (b) substitution of leucine at position 5 in the sequence of SEQ ID NO: 71 with histidine; And (c) substitution of glutamine at position 1 in the sequence of SEQ ID NO: 72 with lysine.

In one embodiment, the anti-IL-8 antibody of initiation C comprises: (a) substitution of serine at position 8 in the sequence of SEQ ID NO: 70 with glutamic acid; (b) substitution of asparagine at position 1 in the sequence of SEQ ID NO: 71 with lysine; (c) substitution of leucine at position 5 in the sequence of SEQ ID NO: 71 with histidine; And (d) substitution of glutamine at position 1 in the sequence of SEQ ID NO: 72 with lysine.

In one embodiment, the anti-IL-8 antibody of initiation C comprises HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75.

In one embodiment, the anti-IL-8 antibody of Initiation C comprises HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76.

In one embodiment, the anti-IL-8 antibody of initiation C is 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 the amino acid sequence of SEQ ID NO: 76. It includes HVR-L3 comprising the sequence.

In one embodiment, the anti-IL-8 antibody of initiation C comprises: (a) an 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) the 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) substitution of an amino acid at the tyrosine position at position 103 of the sequence of SEQ ID NO: 77.

In one embodiment, the anti-IL-8 antibody of initiation C comprises: (a) substitution of alanine at position 55 in the sequence of SEQ ID NO: 77 with aspartic acid; (b) substitution of glycine at position 57 in the sequence of SEQ ID NO: 77 with tyrosine; (c) substitution of the tyrosine at position 58 in the sequence of SEQ ID NO: 77 with histidine; (d) substitution of arginine at position 60 in the sequence of SEQ ID NO: 77 with proline; (e) substitution of glutamine at position 84 in the sequence of SEQ ID NO: 77 with threonine; (f) substitution of serine at position 87 in the sequence of SEQ ID NO: 77 with aspartic acid; And (g) substitution of tyrosine at position 103 in the sequence of SEQ ID NO: 77 with histidine.

In one embodiment, the anti-IL-8 antibody of initiation C comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78.

In one embodiment, the anti-IL-8 antibody of initiation C comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79.

In one embodiment, the initiating C anti-IL-8 antibody 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 anti-IL-8 antibody comprising 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 is an anti-IL- 8 An antibody may be sufficient. The anti-IL-8 antibody comprising the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 79 is IL- 8 An anti-IL-8 antibody in which neutralizing activity is maintained stably may be used. The anti-IL-8 antibody containing the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 78 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 79 may be an antibody with low immunogenicity.

In addition, in an alternative embodiment, the anti-IL-8 antibody of initiation C contains substitution of at least one amino acid in at least any one of the following amino acid sequences (a) to (f), and IL- Also included are anti-IL-8 antibodies, which have a pH-dependent affinity for 8. (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.

In addition, in an alternative embodiment, the anti-IL-8 antibody of initiation C contains substitution of at least one amino acid in the following amino acid sequences (a) to (f), and is pH dependent on IL-8. Also included are anti-IL-8 antibodies with affinity: (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.

In addition, in an alternative embodiment, the anti-IL-8 antibody of initiation C contains substitution of at least one amino acid in at least any one of the following amino acid sequences (a) to (f), while IL- Also included are anti-IL-8 antibodies, which have a pH dependent affinity for 8. (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.

In addition, in an alternative embodiment, the anti-IL-8 antibody of initiation C contains substitution of at least one amino acid in at least any one of the following amino acid sequences (a) to (f), and IL- Also included are anti-IL-8 antibodies, which have a pH-dependent affinity for 8: (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) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

In addition, in an alternative embodiment, the anti-IL-8 antibody of initiation C contains substitution of at least one amino acid in at least any one of the following amino acid sequences (a) to (f), and IL- Also included are anti-IL-8 antibodies, which have a pH-dependent affinity for 8. (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.

In addition, in an alternative embodiment, the anti-IL-8 antibody of initiation C contains substitution of at least one amino acid in at least any one of the following amino acid sequences (a) to (f), and IL- Also included are anti-IL-8 antibodies, which have a pH dependent affinity for 8. (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.

In addition, in an alternative embodiment, the anti-IL-8 antibody of initiation C contains substitution of at least one amino acid in at least any one of the following amino acid sequences (a) to (f), and IL- Also included are anti-IL-8 antibodies, which have a pH dependent affinity for 8. (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.

In one embodiment, the anti-IL-8 antibody of initiation C has IL-8 neutralizing activity. The neutralizing activity of IL-8 may indicate an activity that inhibits the biological activity exhibited by IL-8, or may indicate an activity that inhibits IL-8 from binding to its receptor.

In an alternative embodiment, the anti-IL-8 antibody of initiation C is an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner. In the context of initiation C, an anti-IL-8 antibody that binds to IL-8 in a pH-dependent manner is the binding affinity to IL-8 at acidic pH compared to the binding affinity to IL-8 at neutral pH. This refers to the reduced antibody. For example, the pH-dependent anti-IL-8 antibody includes antibodies having higher affinity for IL-8 at neutral pH than at acidic pH. In one embodiment, the anti-IL-8 antibody of initiation C is at least 2 times, 3 times, 5 times, 10 times the affinity at neutral pH than the affinity at acidic pH for IL-8. , 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. In order to measure the binding affinity, it is not particularly limited, but a surface plasmon resonance method (such as BIACORE (registered trademark)) can be used. The binding rate constant (kon) and the dissociation rate constant (koff) are based on a simple one-to-one Langmuir binding model by simultaneously fitting sensorgrams of binding and dissociation. Trademark) It can be calculated using T200 Evaluation Software (GE Healthcare). The equilibrium dissociation constant (KD) is calculated as a koff/kon ratio. In order to screen for antibodies having different binding affinity depending on pH, it is not particularly limited, but in addition to the surface plasmon resonance method (BIACORE (registered trademark), etc.), the ELISA method or the binding equilibrium exclusion method (Kinetic Exclusion Assay; KinExA (trademark)) ), etc. can also be used. The pH-dependent IL-8 binding ability means a property of binding to IL-8 in a pH-dependent manner. In addition, whether or not the antibody can bind to IL-8 multiple times can be determined according to the method described in WO2009/125825.

In one embodiment, it is preferable that the anti-IL-8 antibody of initiation C has a small dissociation constant (KD) for IL-8 at neutral pH. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at neutral pH of, for example, 0.3 nM or less, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at neutral pH of, for example, 0.1 nM or less, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at neutral pH of, for example, 0.03 nM or less, but is not limited thereto.

In one embodiment, it is preferable that the anti-IL-8 antibody of initiation C has a small dissociation constant (KD) for IL-8 at pH 7.4. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at pH 7.4, for example, 0.3 nM or less, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at pH 7.4, for example 0.1 nM or less, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at pH 7.4, for example, 0.03 nM or less, but is not limited thereto.

In one embodiment, it is preferable that the anti-IL-8 antibody of initiation C has a large dissociation constant (KD) with respect to IL-8 at an acidic pH. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at an acidic pH of, for example, 3 nM or more, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at an acidic pH of, for example, 10 nM or more, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at an acidic pH of, for example, 30 nM or more, but is not limited thereto.

In one embodiment, it is preferable that the anti-IL-8 antibody of initiation C has a large dissociation constant (KD) for IL-8 at pH 5.8. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at pH 5.8 of, for example, 3 nM or more, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at pH 5.8 of, for example, 10 nM or more, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation constant for IL-8 at pH 5.8 of, for example, 30 nM or more, but is not limited thereto.

In one embodiment, it is preferable that the anti-IL-8 antibody of initiation C has higher binding affinity at neutral pH than at acidic pH for IL-8.

In one embodiment, the ratio of the dissociation constant in the acidic pH to the dissociation constant in the neutral pH [KD (acidic pH) /KD (neutral pH)] is, for example, the anti-IL-8 antibody of initiation C. 30 or more, but is not limited to this. In one embodiment, the ratio of the dissociation constant in the acidic pH to the dissociation constant in the neutral pH [KD (acidic pH) /KD (neutral pH)] is, for example, the anti-IL-8 antibody of initiation C. 100 or more, e.g. 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 not limited to this.

In one embodiment, the ratio of the dissociation constant at pH 5.8 to the dissociation constant at pH 7.4 [KD (pH 5.8) /KD (pH 7.4)] is 30 or more in the anti-IL-8 antibody of start C. It is not limited to this. In one embodiment, in the antibody of initiation C, the ratio of the dissociation constant at pH 5.8 to the dissociation constant at pH 7.4 [KD (pH 5.8) /KD (pH 7.4)] 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 not limited to this.

In one embodiment, it is preferable that the anti-IL-8 antibody of initiation C has a large dissociation rate constant (koff) at an acidic pH. In one embodiment, the antibody of initiation C has a dissociation rate constant at an acidic pH of, for example, 0.003 (1/s) or more, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation rate constant at an acidic pH of, for example, 0.005 (1/s) or more, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation rate constant at an acidic pH of, for example, 0.01 (1/s) or more, but is not limited thereto.

In one embodiment, it is preferable that the anti-IL-8 antibody of initiation C has a large dissociation rate constant (koff) at pH 5.8. In one embodiment, the antibody of initiation C has a dissociation rate constant at pH 5.8 of, for example, 0.003 (1/s) or more, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation rate constant at pH 5.8 of, for example, 0.005 (1/s) or more, but is not limited thereto. In one embodiment, the antibody of initiation C has a dissociation rate constant at pH 5.8 of, for example, 0.01 (1/s) or more, but is not limited thereto.

In one embodiment, it is preferable that the anti-IL-8 antibody of Initiation C maintains stable IL-8 neutralizing activity in a solution (eg, in PBS). Whether or not the activity is stably maintained in the solution can be confirmed by measuring whether the IL-8 neutralizing activity of the antibody of initiation C added to the solution changes before and after storage at which period and at which temperature. In one embodiment, the storage period is not limited to these, but is, for example, 1 week, 2 weeks, 3 weeks, or 4 weeks. In one embodiment, the storage temperature is, for example, but not limited to, 25°C, 30°C, 35°C, 40°C, or 50°C. In one embodiment, the storage temperature is not limited to this, but is, for example, 40° C., and the storage period is not limited thereto, but is, for example, 2 weeks. In one embodiment, the storage temperature is not limited to this, but is, for example, 50° C., and the storage period is not limited thereto, but is, for example, one week.

In one embodiment, it is preferable that the anti-IL-8 antibody of Initiation C maintains stable IL-8 neutralizing activity in vivo (eg, in plasma). Whether or not the activity is stably maintained in vivo, the IL-8 neutralizing activity of the antibody of initiation C added to the plasma of an animal (e.g., mouse) or human changes before and after storage at a certain period and at a certain temperature. It can be confirmed by measuring whether or not. In one embodiment, the storage period is not limited to these, but is, for example, 1 week, 2 weeks, 3 weeks, or 4 weeks. In one embodiment, the storage temperature is, for example, but not limited to, 25°C, 30°C, 35°C, or 40°C. In one embodiment, the storage temperature is not limited to this, but is, for example, 40° C., and the storage period is not limited thereto, but is, for example, 2 weeks.

In one embodiment, the rate at which the initiating C anti-IL-8 antibody is absorbed into the cell is higher when the antibody forms a complex with IL-8 than when the antibody is alone. Initiation C IL-8 antibody is more likely to be absorbed into cells when it is in a complex with IL-8 than when it is not in a complex with IL-8 in extracellular (for example, in plasma). .

In one embodiment, it is preferable that the anti-IL-8 antibody of Initiation C has a reduced predicted immunogenicity in a human host. "Immunogenicity is low" is not limited to this, for example, for a period sufficient to achieve a therapeutic effect, in at least half of the individuals administered with a sufficient amount of the antibody, the administered anti-IL-8 antibody is It may mean that it does not cause an immune response by. Causing an immune response may include the production of anti-drug antibodies. It is also possible to change "low immunogenicity" to "low production of anti-drug antibodies". The level of immunogenicity in humans can also be estimated by a T cell epitope prediction program, and this T cell epitope prediction program includes Epibase (Lonza), iTope/TCED (Antitope), EpiMatrix (EpiVax), and the like. EpiMatrix mechanically designs the sequence of a peptide fragment in which the amino acid sequence of a protein for which immunogenicity is to be measured is divided every 9 amino acids, and for them, eight kinds of major MHC class II alleles (DRB1 ^* 0101, DRB1 ^* 0301, DRB1 ^* 0401, DRB1 ^* 0701, DRB1 ^* 0801, DRB1 ^* 1101, DRB1 ^* 1301, and DRB1 ^* 1501) by predicting the binding capacity to predict the immunogenicity of the target protein (De Groot et al. , Clin. Immunol. May; 131(2):189-201(2009)). A sequence in which an amino acid in the amino acid sequence of an anti-IL-8 antibody has been modified can be analyzed using the T cell epitope prediction program described above, and a sequence with reduced immunogenicity can be designed. As a non-limiting example of a preferred amino acid modification site for reducing the immunogenicity of the anti-IL-8 antibody of initiation C, position 81 according to Kabat numbering in the heavy chain sequence of the anti-IL-8 antibody represented by SEQ ID NO: 78, and/or 82b The above amino acids are mentioned.

In one embodiment, initiation C provides a method for promoting the loss of IL-8 from the individual than when using a reference antibody, comprising the step of administering to the individual the anti-IL-8 antibody described in Initiation C. do. In one embodiment, initiation C relates to the use of the anti-IL-8 antibody described in Initiation C in promoting the loss of IL-8 from an individual than when using a reference antibody. In one embodiment, Initiation C relates to the anti-IL-8 antibody described in Initiation C for use in promoting the loss of IL-8 from an individual than when using a reference antibody. In one embodiment, initiation C is the use of the anti-IL-8 antibody described in Initiation C in the manufacture of a pharmaceutical composition for promoting the loss of IL-8 in vivo than when using a reference antibody. About. In one embodiment, initiation C relates to a pharmaceutical composition containing the anti-IL-8 antibody described in Initiation C, for promoting the loss of IL-8 than when using a reference antibody. In one embodiment, initiation C relates to a method of promoting the loss of IL-8 than when using a reference antibody, including the step of administering to a subject the anti-IL-8 antibody described in Initiation C. The reference antibody in the embodiment of initiation C is an anti-IL-8 antibody before modification to obtain an antibody of initiation C, or an antibody with strong IL-8 binding affinity at both acidic and neutral pH. Points to. The reference antibody may be an antibody containing the amino acid sequence of SEQ ID NO: 83 and SEQ ID NO: 84, or an antibody containing SEQ ID NO: 89 and SEQ ID NO: 87.

In one embodiment, initiation C includes the anti-IL-8 antibody described in Initiation C, characterized in that the anti-IL-8 antibody described in Initiation C binds to the extracellular matrix after binding to IL-8, It provides a pharmaceutical composition. In one embodiment, initiation C is the anti-IL described in Initiation C in the manufacture of a pharmaceutical composition, wherein the anti-IL-8 antibody described in Initiation C binds to the extracellular matrix after binding to IL-8. -8 relates to the use of antibodies.

In any of the above embodiments, the anti-IL-8 antibody may be humanized.

In one aspect, the antibody of initiation C comprises a heavy chain variable region in any one of the embodiments provided above, and a light chain variable region in any one of the embodiments provided above. In one embodiment, the antibody of initiation C includes the heavy chain variable region of SEQ ID NO: 78 and the light chain variable region of SEQ ID NO: 79, respectively, and post-translational modification of these sequences may also be included.

In a further aspect, the anti-IL-8 antibodies according to some of the above embodiments may introduce any of the features described in Sections 1 to 7 below, alone or in combination.

1. Chimeric and humanized antibodies

In some embodiments, the antibody provided at initiation C may be a chimeric antibody. Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567 or Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some instances, the chimeric antibody may include a non-human variable region (for example, a non-human primate such as a monkey, or a variable region derived from a mouse, rat, hamster, or rabbit) and a human constant region.

In some embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized in order to reduce immunogenicity in humans, while maintaining the specificity and affinity of the parent non-human antibody. In general, humanized antibodies comprise one or more variable regions, wherein HVRs, such as CDRs (or portions thereof) are derived from non-human antibodies, and FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody may optionally contain at least a portion of a human normal region. In some embodiments, several FR residues in a humanized antibody are directed to the corresponding residues of a non-human antibody (e.g., an antibody from which an HVR residue is derived), e.g., to maintain or improve the specificity or affinity of the antibody. It may be substituted.

Humanized antibodies and methods for their preparation are described, for example, in Almagro et al., Front. Biosci. 13:1619-1633 (2008), and furthermore, described, for example, below: Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (described for specificity determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (described for “resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (described for “FR shuffling”); And Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (described for "choice under guide" leading to FR shuffling).

The human framework regions used for humanization are, but are not limited to, framework regions selected using the "best-fit" method (see, for example, Sims et al. J. Immunol. 151:2296 (1993)). ), a framework region derived from the consensus sequence of a specific subgroup of human antibodies with the variable region of the heavy or light chain (eg 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 the screening of FR libraries (e.g. Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

2. Antibody fragment

In some embodiments, the antibody provided as initiation 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 reviews of certain antibody fragments, see Hudson et al., Nat. Med. See 9:129-134 (2003). For reviews of scFv fragments, see, eg, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. See 269-315 (1994) or WO 93/16185, U.S. Patent No. 5,571,894, U.S. Patent No. 5,587,458.

Diabodies are bivalent or bispecific and are antibody fragments having two antigen-binding sites. 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 90: 6444-6448 (1993) et al. Triabodina or tetrabodido Hudson et al., Nat. Med. 9:129-134 (2003).

A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain of the antibody, or all or part of the light chain variable domain. In some embodiments, the single domain antibody may be a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US Pat. No. 6,248,516 B1). Antibody fragments are not limited to this, but in addition to production by recombinant host cells (e.g., E. coli and phage) as described within the scope of the disclosure C in this specification, the proteolytic properties of the raw antibody It can be produced by various methods, including fire extinguishing.

3. Human Antibodies

In some embodiments, the antibody provided at initiation C may be a human antibody. Human antibodies can be produced by various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) or Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies can be prepared by administering an immunogen to a transgenic animal modified to produce a crude antibody having a human variable region or a crude human antibody in response to an antigen. Such animals typically contain all or part of the human immunoglobulin loci, which are substituted for the animal (non-human) immunoglobulin loci, exist outside the chromosome, or randomly introduced into the animal's chromosome. do. In such transgenic mice, the immunoglobulin locus of an animal (non-human) is usually inactivated. For a review of how to obtain human antibodies from transgenic animals, see: Lonberg, Nat. Biotech. 23:1117-1125 (2005). For XENOMOUSE ^™ technology, see US Patent Nos. 6,075,181 and 6,150,584. For HUMAB ^™ technology, see U.S. Patent No. 5,770,429. For KM MOUSE ^™ technology, see US Pat. No. 7,041,870. For VELOCIMOUSE ^TM technology, see US Patent Application Publication US 2007/0061900.

The human variable region derived from the unprocessed antibody produced from such an animal may be further modified by, for example, combination with other human constant regions. Human antibodies can also be produced by a hybridoma-based method. For the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heteromyeloma cell lines are described below (eg, 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 Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced by human B cell hybridomas are described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). As additional methods, for example, a method for producing a monoclonal human IgM antibody from a hybridoma cell line described in U.S. Patent No. 7,189,826, and, for example, Ni, Xiandai Mianyixue, 26(4) for human-human hybridomas. ): The method described in 265-268 (2006) is mentioned. Human hybridoma technology (trioma technology) is 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 can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such a variable domain sequence can be combined with a preferred human normal domain. Techniques for selecting human antibodies from antibody libraries are described below.

4. Library-derived antibodies

The antibody of initiation C can be isolated by screening a combinatorial library for antibodies having one or more desirable activities. Several methods are known, for example, for constructing phage display libraries, and for screening such libraries for antibodies possessing desirable binding properties. Such a method is described in Hoogenboom et al., Meth. Mol. Biol. 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), furthermore, for example 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 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); And Lee et al., J. Immunol. Meth. 284(1-2): 119-132(2004).

In any phage display method, a repertoire of sequences encoding VH and VL can be separately cloned by polymerase chain reaction (PCR), and randomly recombined into a phage library. In the obtained phage library, Winter et al., Ann. Rev. As described in Immunol., 12: 433-455 (1994), screening of antigen-binding phage is performed. Phage typically presents antibody fragments as single chain Fv (scFv) fragments or as Fab fragments.

Alternatively, as described in Griffiths et al., EMBO J, 12:725-734 (1993), the naive repertoire was cloned (for example from humans), and without any immunity, a wide range of nonmagnetic Antibodies of a single source can be provided against the antigen of the antagonist and also against the own antigen.

Finally, the naive library is synthesized by cloning an unreconstituted V-gene segment from stem cells and reconstructing in vitro using PCR primers containing a random sequence encoding a highly variable CDR3 region. It can also be constructed separately (hereinafter, see Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992); published publications for human antibody phage libraries are, for example, U.S. Patent No. 5,750,373 or , US published applications 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360, etc.). An antibody or antibody fragment isolated from a human antibody library is regarded as a human antibody or human antibody fragment.

5. Multispecific Antibodies

In certain embodiments, the antibody provided by initiation C may be a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies having binding specificities for at least two different sites.

In some embodiments, one of the binding specificities is for IL-8, and the other is for any other antigen.

In certain embodiments, the bispecific antibody is capable of binding to two different epitopes of IL-8. Bispecific antibodies can also be used to deploy cytotoxic agents to cells expressing IL-8. The bispecific antibody may be prepared as a full-length antibody or as an antibody fragment.

Techniques for producing multispecific antibodies are not limited thereto, but recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, And Traunecker et al., EMBO J. 10: 3655 (1991)) or the “knob-in-hole” method (see US Pat. No. 5,731,168). Multispecific antibodies are those that use electrostatic steering effect to produce Fc heterobimolecules (WO2009/089004A1), or those that cross-link two or more antibodies or fragments (US Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)) or producing a bispecific antibody using a leucine zipper (Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), or "dia. The production of bispecific antibody fragments using the "body" technique (Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)) or using a single chain Fv (scFv) dimer ( Gruber et al., J. Immunol. 152:5368 (1994)), or by other methods. The preparation of trispecific antibodies is described, for example, in Tutt et al., J. Immunol. 147: 60 (1991).

Antibodies engineered to have three or more functional antigen binding sites, including “Octopus antibodies”, are also included in this specification (see, eg, US 2006/0025576).

In the range described in the disclosure C in the present specification, the antibody or antibody fragment also includes "dual acting Fab" or "DAF" containing one antigen-binding site that also binds IL-8 and other different antigens. (See, for example, US 2008/0069820).

6. Antibody variants

The amino acid sequence modification of the antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by synthesizing a peptide. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be used to reach that final construct, as long as the final construct is an antibody with the desired characteristics described in the context of initiation C.

In one embodiment, initiation C provides an antibody variant having one or more amino acid substitutions. Such a substitution site may be any position of the antibody. Amino acids used for conservative substitutions appear in Table 10 under the heading of “conservative substitutions”. The amino acids used for typical substitutions that lead to more substantial changes appear under the heading of “Typical Substitutions” in Table 10, and are further described with regard to amino acid side chain classification.

Amino acids can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: 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.

Non-conservative substitution means exchanging a member in one of these classes for another class.

Insertion of amino acids means insertion of one or more amino acid residues into a sequence, and fusion of a polypeptide containing one, two, or three to 100 or more residues at the N-terminus and/or C-terminus. Includes that. Examples of such terminal insertion include antibodies having an N-terminal methionylated residue. Other insertional modifications of the antibody molecule include those resulting from fusion at the N-terminus or C-terminus of the antibody to an enzyme (eg, ADEPT) or a polypeptide that extends the plasma half-life of the antibody.

7. Glycosylated variants

In one embodiment, the antibody provided at initiation C may be glycosylated. The addition of the glycosylation site to the antibody or deletion from the antibody can be achieved by altering the amino acid sequence to prepare or eliminate the glycosylation site.

When the antibody contains an Fc region, the sugar chain added thereto may be altered. The naïve antibody produced by animal cells typically contains a branched or branched oligosaccharide, and the oligosaccharide is added to Asn297 of the CH2 domain of the Fc region by N linkage (Wright et al. TIBTECH 15 :26-32 (1997)). Oligosaccharides include fucose added to GlcNAc in the "stem" of the oligosaccharide structure of the second branch, and, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid. In one embodiment, the modification of the oligosaccharide of the initiating C antibody is performed in order to produce an antibody variant having certain improved properties.

8. Fc region variant

In one embodiment, one or more amino acid modifications are introduced into the Fc region of the antibody provided at initiation C, resulting in Fc region modifications. The Fc region modified is a naturally-occurring human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising a change (e.g., substitution) of one, two, or three or more amino acids. Includes those with ).

The anti-IL-8 antibody of Initiation C is not limited to these, but may contain an Fc region having at least one of the following five properties: (a) FcRn of a native Fc region at an acidic pH The binding affinity of the Fc region to FcRn, which is greater than the binding affinity to; (b) the binding affinity of the Fc region to the existing ADA, which is lower than the binding affinity of the native Fc region to the existing ADA; (c) the plasma half-life of the Fc region, which is longer than the plasma half-life of the native Fc region; (d) the plasma clearance of the Fc region, which is less than the plasma clearance of the native Fc region; And (e) the binding affinity of the Fc region to the effector receptor, which is lower than the binding affinity of the native Fc region to the effector receptor. In some embodiments, the Fc region has two, three or four of the aforementioned properties. In one embodiment, the Fc region variant includes an Fc region variant whose binding affinity to FcRn is increased at an acidic pH. An Fc region modified variant with increased binding affinity to FcRn has a binding affinity for FcRn maximally 2 times, 3 times, 4 times, 5 times compared to an antibody containing the Fc region of a native IgG. , 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, or 100-fold Fc region variants are included, but are not limited thereto.

In one embodiment, the Fc region variant contains a safe and advantageous Fc region variant that does not show binding to existing ADA and at the same time improves plasma retention. The term "ADA" in the context of initiation C means an endogenous antibody having binding affinity for an epitope located on a therapeutic antibody. The term "conventional ADA" in the context of Initiation C means an anti-drug antibody that can be detected by being present in the blood of a patient before administration of the therapeutic antibody to the patient. Existing ADA contains rheumatoid factor. An existing Fc region variant having a low binding affinity for ADA means that the binding affinity for ADA is 1/10 or less, 1/50 or less, or 1/ as compared to an antibody containing the Fc region of a native IgG. It includes, but is not limited to, the Fc region variant that has decreased to 100 or less.

In one embodiment, the Fc region variant includes an Fc region variant that has a low binding affinity for a complement protein or does not bind to a complement protein. Complement proteins include C1q. The Fc region variant having a low binding affinity to the complement protein is compared with an antibody containing the Fc region of a native IgG, and the binding affinity to the complement protein is 1/10 or less, 1/50 or less, or 1/ It includes, but is not limited to, the Fc region variant that has decreased to 100 or less.

In one embodiment, the Fc region variant includes an Fc region variant that has a low binding affinity for an effector receptor or no binding affinity for an effector receptor. Effector receptors include, but are not limited to, FcγRI, FcγRII, and FcγRIII. FcγRI is not limited, but includes FcγRIa, FcγRIb and FcγRIc, and subtypes thereof. FcγRII is not limited, but includes FcγRIIa (having two allotypes R131 and H131) and FcγRIIb. FcγRIII includes, but is not limited to, FcγRIIIa (having two allotypes V158 and F158) and FcγRIIIb (having two allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). With the Fc region modified variant having a low binding affinity to the effector receptor, the binding affinity to the effector receptor is at least 1/10 or less and 1/50 or less compared to the binding affinity of an antibody containing the Fc region of a native IgG. , Or Fc region modified by 1/100 or less, but is not limited to these.

In one embodiment, the Fc region variant is 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 according to EU numbering compared to the native Fc region. Includes an Fc region, including substitution of one or more amino acids at any one of the positions of the group consisting of the above and 440 positions.

In one embodiment, the Fc region modified is amino acid substitution at position 235, 236, 239, 428, 434, 436, 438, and 440 according to EU numbering compared to the native Fc region. Includes, including, an Fc region.

In one embodiment, the Fc region modified according to EU numbering compared to the native Fc region 235, 236, 327, 330, 331, 428, 434, 436, 438, and It includes an Fc region, including a substitution of an amino acid at position 440.

In one embodiment, the Fc region modified is 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. Includes.

In one embodiment, the Fc region variant comprises an Fc region comprising amino acid substitutions of M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the Fc region variant comprises an Fc region comprising amino acid substitutions of L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the Fc region variant comprises an Fc region comprising amino acid substitutions of L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E.

In one embodiment, the anti-IL-8 antibody of initiation 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 containing 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 IL-8 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 stably maintain IL-8 neutralizing activity in vivo (eg, in plasma). . 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 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 has a binding affinity for FcRn at acidic pH (e.g., pH 5.8), acidic pH It can contain an Fc region which is more enhanced than the binding affinity of the native Fc region to FcRn in. 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 has a binding affinity for the existing ADA and a binding parent for the existing ADA of the native Fc region. It may contain an Fc region that is lower than that of chemical conversion. 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 include an Fc region whose half-life in plasma is longer than that of the native Fc region. have. 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 has a binding affinity for the effector receptor than that of the native Fc region for the effector receptor. It may contain a reduced Fc region. In a further embodiment, the anti-IL-8 antibody comprises any 2, 3, 4, 5, 6, or all 7 combinations of the aforementioned features.

In one embodiment, the anti-IL-8 antibody of initiation 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 IL-8 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 stably maintain IL-8 neutralizing activity in vivo (eg, in plasma). . 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 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 has a binding affinity to FcRn at acidic pH, and binding to FcRn of the native Fc region. It may contain an Fc region that has increased more than affinity. 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 has a binding affinity for the existing ADA, and the binding affinity of the native Fc region to the existing ADA. It may contain an Fc region that is lower than that of chemical conversion. 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 include an Fc region whose half-life in plasma is longer than that of the native Fc region. have. 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 has a binding affinity to the effector receptor than that of the native Fc region to the effector receptor. It may contain a reduced Fc region. In a further embodiment, the anti-IL-8 antibody comprises any 2, 3, 4, 5, 6, or all 7 combinations of the aforementioned features.

In some embodiments, initiation C includes antibody variants with some, but not all, effector functions. Such antibody variants may be preferred candidates when no effector function is required or detrimental (such as complement or ADCC). In vitro and/or in vivo cytotoxicity assays known in the art are routinely done to ensure a reduction/complete deletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that an antibody lacks FcγR binding (ie, lacks ADCC activity) but maintains FcRn binding ability on the other hand.

Primary cultured cells and NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII on the other hand. Expression of FcR in hematopoietic cells is as described in Ravetch et al., Annu. Rev. Immunol. It is summarized in Table 3 on page 464 of 9:457-492 (1991). Examples of in vitro assays for evaluating ADCC activity of a target molecule are, but are not limited to, US Pat. No. 5,500,362, Hellstrom, et al., Proc. Natl. Acad. Sci. USA 83:7059-7063 (1986), Hellstrom et al., Proc. Natl. Acad. Sci. USA 82:1499-1502 (1985), U.S. Patent No. 5,821,337, and Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987). Alternatively, a non-radioactive isotope assay can be used for evaluation of effector cell function (e.g., for flow cytometry, ACTI (trademark) non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA; and CytoTox) 96 (trademark) non-radioactive cytotoxicity assay (Promega, Madison, WI)) Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.

Alternatively, or additionally, the ADCC activity of the antibody variant of interest can be determined in vivo, for example Clynes et al., Proc. Natl. Acad. Sci. In animal models, measurements can be made, as described in USA 95:652-656 (1998). The C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and that it lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO2006/029879 and WO2005/100402. In order to evaluate complement activation, a CDC assay may be performed (e.g., Gazzano-Santoro 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)). Determination of FcRn binding and clearance/half-life in vivo can be done by methods known in the art. (See for example Petkova et al., Intl. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those having a substitution at one or more of the residues at positions 238, 265, 269, 270, 297, 327, or 329 of the Fc region (US Pat. No. 6,737,056). Such an Fc region variant includes a so-called "DANA" Fc region variant (US Pat. No. 7,332,581) in which positions 265 and 297 are substituted with alanine, and among the residues at position 265, 269, 270, 297, or 327 It includes Fc region variants having substitutions in two or more.

Antibody variants with improved or reduced binding to the FcR group are described below (e.g., US Pat. No. 6,737,056; WO2004/056312, Shields et al., J. Biol. Chem. 9(2): 6591. See -6604 (2001)).

Antibodies with extended blood half-life and improved binding to FcRn at acidic pH are described in US2005/0014934. The antibodies of these descriptions contain an Fc region having one or more substitutions that increase the binding of the Fc region to FcRn. Such Fc region variants are 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413 of the Fc region. , 424, or a substitution at one or more selected from position 434, for example, a substitution at position 434 of the Fc region (US Pat. No. 7,371,826).

For other examples of Fc region variants, see Duncan et al., Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; And WO94/29351.

9. Antibody derivatives

In certain embodiments, the antibody provided in Initiation C may be further modified to further include a non-proteinaceous moiety known as prior art and readily available. The part suitable for producing an antibody derivative is not limited to this, but includes a water-soluble polymer. 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, polyvinylpyrrolidone, poly-1,3- Dioxolein, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer and random copolymer), poly(n-vinylpyrrolidone) polyethylene glycol, proppropylene glycol Homopolymers, prolylpropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohols, and mixtures thereof. Polyethylene glycol propion aldehyde is advantageous for industrialization because of its stability in water.

The polymer may have any molecular weight, and even if it is branched, it is not necessary to branch. The number of polymers added to the antibody may vary, and if two or more polymers are added, they may be the same or different. In general, the number and/or type of polymer used to make the derivative is not limited to this, but if the derivative of the antibody is used as a defined treatment, a review containing the specific property or function of the antibody to be improved. Can be determined based on

In another embodiment, a conjugate of an anti-IL-8 antibody of initiation C and a non-proteinaceous moiety that can be selectively heated to exposure to radiation may be provided. In one embodiment, the non-proteinaceous moiety is, for example, carbon nanotubes (eg, Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be any wavelength, and is not limited thereto, but includes a wavelength capable of heating the non-proteinaceous portion to a temperature at which cells near the antibody-non-proteinaceous portion die, although usually harmless to humans.

B. Recombinant method and composition

The anti-IL-8 antibody of Initiation C can be produced using, for example, a recombinant method or a composition described in US Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-IL-8 antibody set forth as Initiation C is provided. Such a nucleic acid may encode an amino acid sequence containing the VL of an antibody and/or an amino acid sequence containing the VH of an antibody (for example, a light chain and/or a heavy chain of an antibody). In a further embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In one embodiment, a host cell comprising such a nucleic acid is provided. In such an embodiment, the host cell comprises (1) a vector containing a nucleic acid encoding the VL of the antibody and the VH of the antibody, or (2) a first vector containing a nucleic acid encoding the VL of the antibody and the antibody. And a second vector containing a nucleic acid encoding VH (eg, being transformed with these vectors).

In one embodiment, the host is a eukaryote (eg, Chinese hamster ovary (CHO) cells or lymphocyte cells (eg Y0, NS0, SP20 cells)).

In one embodiment, a method for producing an anti-IL-8 antibody of initiation C is provided, and the method of producing the host cell comprising a nucleic acid encoding an anti-IL-8 antibody as set forth above is suitable for antibody expression. Culturing under conditions, and optionally (eg, from a host cell or a host cell culture medium) recovering the antibody.

For recombinant production of anti-IL-8 antibodies, for example, nucleic acids encoding antibodies as described above are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional techniques (for example, by using oligonucleotide probes that specifically bind to nucleic acids encoding heavy and light chains of an antibody).

Host cells suitable for expression or cloning of a vector encoding an antibody include prokaryotic cells or eukaryotic cells described in the range of the disclosure C in this specification. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (for expression of antibody fragments in E. coli, Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), also see pp. 245-254). After expression, the antibody can be isolated from the bacterial cell paste of the soluble fraction and further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeast are suitable as hosts for the expression or cloning of vectors encoding antibodies, and for them, the stock price of filamentous fungi or yeast whose glycosylation pathway is "humanized" It is included, allowing the production of antibodies with partial or complete human glycosylation patterns. Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. See 24:210-215 (2006).

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells are plants or insect cells. Although not particularly limited, baculovirus is used together with insect cells for transformation of cells of spodoptera frugiperda, and many baculovirus strains have been identified.

Plant cell cultures can also be used as hosts. See U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 ( ^{describes PLANTIBODIES TM} technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as a host. For example, mammalian cell lines suitable for growth in a suspended state are useful. Other examples of useful mammalian host cells include monkey kidney CV1 strain transformed with SV40 (COS-7); Human fetal kidney strain (293 cells, described in Graham et al., J. Gen Virol. 36:59 (1977)); Baby hamster kidney cells (BHK); Mouse sertoli cells (TM4 cells, described in Mather, Biol. Reprod. 23:243-251 (1980)); Monkey kidney cells (CV1); African savannah monkey kidney cells (VERO-76); Human cervical cancer (HELA); Canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); Human lung cells (W138); Human liver cells (Hep G2); Mouse mammary tumor (MMT060562); TRI cells (described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; And FS4 cells.

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)), Y0, NS0 And myeloma cell lines such as 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. See 255-268 (2003).

The antibody of initiation C produced by culturing a host cell containing a nucleic acid encoding the antibody as set forth above under conditions suitable for antibody expression is isolated from inside or outside the host cell (medium, milk, etc.) It can be purified as a pure and homogeneous antibody. Isolation and purification of the antibody may be suitably used for isolation/purification methods used in the purification of ordinary polypeptides, but is not limited thereto. For example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization are appropriately selected and combined. Antibodies can be appropriately isolated, and purified, but are not limited thereto. Examples of the chromatography include, but are not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography. These chromatography can be performed using liquid chromatography, for example, HPLC and FPLC. Examples of the columns used for affinity chromatography include, but are not limited to, a protein A column and a protein G column. Examples of the protein A column include, but are not limited to, Hyper D, POROS, Sepharose F.F. (manufactured by Pharmacia) and the like.

Focusing on the properties of the anti-IL-8 antibody, such as an increase in the binding activity to the extracellular matrix or an increase in the uptake of the complex into cells, initiation C is an antibody or intracellular with increased binding to the extracellular matrix. Methods of selecting antibodies with increased absorption are provided. In one embodiment, initiation C provides a method for producing an anti-IL-8 antibody comprising a variable region whose binding activity to IL-8 is pH dependent, comprising the following steps: (a) anti-IL -8 The process of evaluating the binding of the antibody to the extracellular matrix; (b) selecting an anti-IL-8 antibody with strong binding to the extracellular matrix; (c) culturing a host containing a vector containing a nucleic acid encoding the antibody; And (d) a step of isolating the antibody from the culture medium.

The evaluation of binding to the extracellular matrix is not particularly limited as long as it is a method known to those skilled in the art, and can be performed by any method. For example, an assay can be performed using an ELISA system that detects binding of an antibody to an extracellular matrix, where an antibody is added to a plate on which the extracellular matrix is solidified, and a labeled antibody against the antibody is prepared. Add. Alternatively, for example, an electrochemiluminescence method in which a mixture of an antibody and a ruthenium antibody is added to a plate on which the extracellular matrix is solidified, and the binding of the antibody and the extracellular matrix is measured based on electrochemiluminescence of ruthenium ( Such an assay can be performed using ECL (Electrochemiluminescence) method).

The anti-IL-8 antibody for which the binding to the extracellular matrix is evaluated in step (a) may be the antibody alone, or may be in contact with IL-8. In step (b), "selecting an anti-IL-8 antibody with strong binding to the extracellular matrix" indicates the binding of the anti-IL-8 antibody to the extracellular matrix when evaluating the binding to the extracellular matrix. A, higher than the value indicating the binding of the antibody used as a control and the extracellular matrix, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 Although it refers to selection of an anti-IL-8 antibody based on the criterion that it may be double, 50 times, 100 times, or more, the ratio is not particularly limited to this example. In the step of evaluating the binding of the anti-IL-8 antibody to the extracellular matrix, the conditions other than the presence or absence of IL-8 are preferably the same. The control anti-IL-8 antibody used for comparison with a plurality of anti-IL-8 antibodies to which the modification has been applied may be an anti-IL-8 antibody before modification, and in that case, it is preferable that conditions other than the presence or absence of IL-8 are the same. Specifically, one embodiment of Initiation C includes a step of selecting an antibody having a high level of binding to the extracellular matrix from among a plurality of anti-IL-8 antibodies that are not in contact with IL-8. In another embodiment, initiation C includes selecting an antibody having a high level of binding to the extracellular matrix from among a plurality of anti-IL-8 antibodies contacted with IL-8. In an alternative embodiment, in step (b), "selecting an anti-IL-8 antibody with strong binding to the extracellular matrix" means that the antibody is selected according to the presence or absence of IL-8 when evaluating binding to the extracellular matrix. It indicates that it is possible to select an antibody based on the criterion that the binding to the and extracellular matrix changes. For example, a value representing the binding of an anti-IL-8 antibody contacted with IL-8 to the extracellular matrix, and a value representing the binding of an anti-IL-8 antibody not contacted with IL-8 to the extracellular matrix. The ratio may be 2 to 1000. In addition, even if the ratio of the number is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 do.

C. Assay

Anti-IL-8 antibodies provided within the range of disclosure C in this specification are identified, screened, or characterized in terms of physical/chemical properties and/or biological activity according to various methods known in the art. I can.

1. Combined and other assays

In one embodiment, the antibody of the initiation C is, for example, ELISA, Western blot, a binding equilibrium exclusion method (Kinetic Exclusion Assay; KinExA (trademark)), and a surface plasmon resonance method using devices such as BIACORE (GE Healthcare). By known methods, antigen-binding activity can be investigated.

In one embodiment, binding affinity can be measured as follows using BIACORE T200 (GE Healthcare). An appropriate amount of a capture protein (eg, protein A/G (PIERCE)) is fixed on the sensor chip CM4 (GE Healthcare) by an amine coupling method, and the target antibody is captured therein. Next, the diluted antigen solution and running buffer (as a reference solution: for example, 0.05% tween20, 20mM ACES, 150mM NaCl, pH 7.4) are injected, and the antibody captured on the sensor chip and the antigen molecule are allowed to interact. A 10 mM glycine-HCl solution (pH 1.5) is used for regeneration of the sensor chip, and the measurement is carried out at a specified temperature (eg 37°C, 25°C, or 20°C). Based on the binding rate constant kon (1/Ms), which is a kinetic parameter calculated from the sensorgram obtained by measurement, and the dissociation rate constant koff (1/s), the KD (M) for the antigen of each antibody is Is calculated. BIACORE T200 Evaluation Software (GE Healthcare) is used to calculate each parameter.

In one embodiment, the quantification of IL-8 can be carried out as follows. A plate immobilized with an anti-human IL-8 antibody containing a mouse IgG constant region was prepared. A solution containing IL-8 bound to a humanized IL-8 antibody that does not compete with the anti-human IL-8 antibody is dispensed onto the immobilized plate and stirred. Thereafter, biotinylated anti-human Igκ light chain antibody was added to react for a certain period of time, and SULFO-Tag labeled streptavidin was further added to react for a certain period of time. Immediately after addition of the assay buffer, it is measured with SECTOR Imager 2400 (Meso Scale Discovery).

2. Active Assay

In one aspect, an assay is provided to identify anti-IL-8 antibodies having biological activity. The biological activity includes, for example, the activity of neutralizing IL-8, and the activity of blocking the signal of IL-8. Initiation C also provides antibodies with such biological activity in vivo and/or exovivo.

In one embodiment, the method of determining the degree of neutralization of IL-8 is not particularly limited, but it is also possible to measure by the following method. PathHunter ^TM CHO-K1 CXCR2 β-Arrestin Cell Line (DiscoveRx, catalog number 93-0202C2) expresses human CXCR2, known as one of the human IL-8 receptors, and chemilumines when a signal by human IL-8 is received. It is an artificially manufactured cell line to represent. When human IL-8 is added to the culture medium of the cells, the cells show chemiluminescence depending on the concentration of the added human IL-8. When human IL-8 and anti-human IL-8 antibody are added together in the culture medium, the anti-human IL-8 antibody can block signal transduction by IL-8, so that the chemiluminescence of the cell is performed by adding the antibody. It is weak or not detected at all compared to the case where it is not. That is, the stronger the human IL-8 neutralizing activity of the antibody, the weaker the degree of chemiluminescence, and the weaker the human IL-8 neutralizing activity of the antibody, the stronger the degree of chemiluminescence. Therefore, by confirming this difference, it is possible to evaluate the neutralizing activity of the anti-human IL-8 antibody against human IL-8.

D. Pharmaceutical formulation

The pharmaceutical formulation containing the anti-IL-8 antibody described in the range described in the disclosure C in this specification is an anti-IL-8 antibody having a desired degree of purity, and one or more arbitrary pharmaceutically acceptable It can be prepared in the form of a freeze-dried preparation or an aqueous solution preparation by mixing the carrier (see, for example, Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and are not limited to the following, but are not limited to buffer solutions such as phosphoric acid, citric acid, histidine and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives (e.g. octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclone Hexanol; 3-pentanol; and m-cresol); Low molecular 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 saccharides including glucose, mannose, or dextrin; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose, or sorbitol; Salt-forming counter ions such as sodium; Metal complexes (eg, Zn-protein complexes); And/or nonionic surfactants such as TWEEN (trademark), PLURONICS (trademark), or polyethylene glycol (PEG).

Examples of pharmaceutically acceptable carriers in this specification are, in addition, interstitial drug dispersants such as soluble hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, Examples include rHuPH20 (HYLENEX®, Baxter International, Inc.). Examples of sHASEGP comprising rHuPH20 and methods of use thereof are described in US Patent Application Publications 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more glycosaminoglycanases, such as chondroitinases.

Examples of lyophilized preparations of antibodies are described in US Pat. No. 6,267,958. Antibody aqueous solution formulations are described in U.S. Patent No. 6,171,586 and WO2006/044908, and the formulation of WO2006/044908 contains histidine-acetic acid buffer.

Within the scope of the disclosure C in this specification, the formulation may contain one or more active ingredients as necessary for the specific indication to be treated, preferably, they are complementary to each other, and side effects Does not affect Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

The active ingredient is, for example, in microcapsules prepared by coacervation technology or interfacial polymerization, for example in hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drugs. It may be captured in a delivery system (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in a macroemulsion. Such a technique is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained release formulations are also available. Suitable examples of sustained-release preparations include semipermeable matrices of hydrophobic solid polymers comprising the anti-IL-8 antibody of Initiation C, which matrices are in the form of moldings, for example films or microcapsules.

The formulations used for in vivo administration are generally sterile. Sterility is easily achieved, for example, by filtering through a sterile filtration membrane.

E. Treatment methods and therapeutic compositions

In some embodiments, the anti-IL-8 antibody provided in Initiation C is used in a method of treatment.

In one aspect, an anti-IL-8 antibody for use as a pharmaceutical composition is provided. In an alternative aspect, anti-IL-8 antibodies are provided for use in the treatment of diseases in which IL-8 is present in excess. In one embodiment, an anti-IL-8 antibody for use in a method of treating a disease in which IL-8 is present in excess is provided. In one embodiment, initiation C is a disease in which an excess of IL-8 is present (e.g., due to the excessive presence of IL-8, comprising the step of administering to the individual an effective amount of an anti-IL-8 antibody. Disease). In another embodiment, Initiation C provides an anti-IL-8 antibody for use in such a method. In one embodiment, initiation C relates to a pharmaceutical composition for the treatment of a disease in which IL-8 is excessively present, comprising an effective amount of an anti-IL-8 antibody. In one embodiment, initiation C relates to the use of an anti-IL-8 antibody in the manufacture of a pharmaceutical composition for a disease in which IL-8 is excessively present. In one embodiment, initiation C relates to the use of an effective amount of an IL-8 antibody in the treatment of a disease in which IL-8 is excessively present. As diseases in which IL-8 is present in excess, inflammatory skin diseases such as inflammatory keratosis (dry ringworm, etc.), atopic dermatitis, and contact dermatitis; Autoimmune diseases such as chronic joint rheumatism, systemic erythematosus (SLE), and chronic inflammatory diseases including Bechosis; Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; Inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, and drug-induced allergic hepatitis; Inflammatory kidney diseases such as glomerulonephritis; Inflammatory respiratory diseases such as bronchitis and asthma; Chronic inflammatory vascular diseases such as atherosclerosis; Multiple sclerosis; Stomatitis; Vocalitis; Inflammation that occurs when using artificial organs/artificial blood vessels; Malignant tumors such as ovarian cancer, lung cancer, prostate cancer, stomach cancer, breast cancer, melanoma, head and neck cancer, and kidney cancer; Sepsis due to infection; Cystic fibrosis; Pulmonary fibrosis; And acute lung injury, but are not limited thereto.

In an alternative embodiment, Initiation C provides an anti-IL-8 antibody for use in inhibiting the accumulation of IL-8 with biological activity. "Inhibiting the accumulation of IL-8" may be achieved by not increasing the amount of existing IL-8 in vivo, or by reducing the amount of existing IL-8 in vivo. In one embodiment, initiation C provides an anti-IL-8 antibody for inhibiting the accumulation of IL-8 in an individual in order to inhibit the accumulation of IL-8 having biological activity. Here, "IL-8 present in vivo" may be IL-8 forming a complex with an anti-IL-8 antibody, or IL-8 not forming a complex. Alternatively, it may be all IL-8 as their total. Here, "exists in vivo" may mean "is secreted in vivo out of the cell". In one embodiment, Initiation C provides a method of inhibiting the accumulation of biologically active IL-8 comprising administering to the individual an effective amount of an anti-IL-8 antibody. In one embodiment, initiation C relates to a pharmaceutical composition for inhibiting accumulation of IL-8 having biological activity, comprising an effective amount of an anti-IL-8 antibody. In one embodiment, initiation C relates to the use of an anti-IL-8 antibody in the manufacture of a pharmaceutical composition for inhibiting the accumulation of IL-8 having biological activity. In one embodiment, initiation C relates to the use of an effective amount of IL-8 antibody in inhibiting accumulation of IL-8 having biological activity. In one embodiment, the anti-IL-8 antibody of initiation C inhibits the accumulation of IL-8 as compared to an anti-IL-8 antibody that does not have a pH-dependent binding activity. In the above embodiment, the "individual" is preferably a human.

In an alternative embodiment, Initiation C provides an anti-IL-8 antibody for use in inhibiting angiogenesis (eg, angiogenesis). In one embodiment, initiation C is a method for inhibiting angiogenesis in an individual, comprising administering an effective amount of an anti-IL-8 antibody to the individual, and an anti-IL-8 antibody for use in the method. to provide. In one embodiment, initiation C relates to a pharmaceutical composition for inhibiting angiogenesis, comprising an effective amount of an anti-IL-8 antibody. In one embodiment, initiation C relates to the use of an anti-IL-8 antibody in the manufacture of a pharmaceutical composition for inhibiting angiogenesis. In one embodiment, initiation C relates to the use of an effective amount of an IL-8 antibody in inhibiting angiogenesis. The "individual" in the above embodiment is preferably a human.

In an alternative embodiment, initiation C provides an anti-IL-8 antibody for use in inhibiting neutrophil migration promotion. In one embodiment, initiation C is a method of inhibiting the promotion of neutrophil migration in an individual comprising the step of administering to the individual an effective amount of an anti-IL-8 antibody, and anti-IL-8 for use in the method. Antibodies are provided. In one embodiment, initiation C relates to a pharmaceutical composition for inhibiting the promotion of neutrophil migration in an individual containing an effective amount of an anti-IL-8 antibody. In one embodiment, Initiation C relates to the use of an anti-IL-8 antibody in the manufacture of a pharmaceutical composition for inhibiting the promotion of neutrophil migration in an individual. In one embodiment, initiation C relates to the use of an effective amount of an IL-8 antibody in inhibiting the promotion of neutrophil migration in an individual. The "individual" in the above embodiment is preferably a human.

In an alternative embodiment, Initiation C provides a pharmaceutical composition comprising an anti-IL-8 antibody provided herein, for example, for use in any one of the treatment methods described above. In one embodiment, the pharmaceutical composition contains the anti-IL-8 antibody provided in Initiation C, and a pharmaceutically acceptable carrier.

The antibody for initiation C can be used alone or in combination with other drugs in treatment. For example, the antibody of initiation C may be administered simultaneously with at least one additional therapeutic agent.

The antibody of Initiation C (and any additional therapeutic agent) can be administered by any suitable means, such as parenteral administration, intrapulmonary administration, and intranasal administration, and, if topical treatment is desired, intralesional administration. Examples of parenteral injection include intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, or subcutaneous administration. Although it partially depends on whether the administration is short-term or long-term, it can be administered by any suitable route, such as, for example, intravenous injection or injection such as subcutaneous injection. Although not limited to this, various dosing schedules, such as single or multiple administration over various time points, bolus administration, and pulse injection, can be performed.

Preferably, the antibody of Initiation C can be formulated, dosed, and administered in a format tailored to good medical practice. Factors to be considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the pharmaceutical composition, the method of administration, the schedule of administration, and known to the physician. Other factors. Although not required, in some cases, the antibody may be formulated with one or more agents currently used to prevent or treat the disease in question.

The effective amount of such other drugs depends on the amount of antibody in the formulation, the type of disease or treatment, and other factors described above. These are generally at the same dose and the same route of administration as described in the range describing the disclosure C in this specification, or 1 to 99% of the dose described in the range describing the disclosure C in this specification, or empirical /Can be used at any dose and any route determined clinically appropriately.

In the prophylaxis or treatment of a disease, the appropriate dosage of the antibody of initiation C (even when used alone or in combination with one or more additional therapeutic agents) is the type of disease to be treated, the type of antibody, and the type of disease. It depends on the severity and course, whether the administration of the antibody variant is for prophylactic or therapeutic purposes, the treatment so far, the clinical history of the patient and the response to the antibody, and the discretion of the attending physician. The antibody is appropriately administered to a patient by a single treatment or a plurality of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) of the antibody is administered, for example, as a single administration or multiple administrations, or continuous infusion. By the like, the initial candidate dosage for the patient can be obtained. A typical daily dosage may range from about 1 mg/kg to 100 mg/kg or more, depending on the above factors. In the case of repeated administration over several days or longer, depending on the situation, treatment may be maintained until a desired inhibitory effect is exhibited on disease symptoms. Certain typical dosages of the antibody will fall in the range of, for example, about 0.05 mg/kg to about 10 mg/kg. Thus, a dose of one or more, e.g. about 0.5 mg/kg, e.g. 2.0 mg/kg, e.g. 4.0 mg/kg or e.g. 10 mg/kg (or any combination thereof), is administered to the patient. It may be administered. Such administration may be performed intermittently, for example every week or every 3 weeks (for example, so that the patient receives from about 2 doses to about 20 doses, or about 6 doses of antibody). A single high dose administration may be performed first, followed by one or more low dose administration, but other administration regimens are also useful. The progress of this treatment can be easily traced by conventional techniques and assays.

F. Products

As yet another aspect of Initiation C, in this disclosure, there is provided a product comprising substances useful for the treatment, prevention, and/or diagnosis of the diseases described above. Such products include a container and a label or attached document accompanying the container. Suitable containers include, for example, bins, vials, and intravenous solution bags and the like. The container may be formed of various materials such as glass or plastic. Such a container contains a composition that is itself effective to diagnose, prevent, and/or treat a condition, or a composition in combination with other compositions effective to diagnose, prevent, and/or treat the condition, It may have an access port (for example, the container may be a solution bag for intravenous injection or a vial having a stopper pierced with a hypodermic needle). In the composition, at least one active ingredient is an antibody of initiation C. The label or accompanying document dictates that the composition is to be used to treat the selected condition.

Furthermore, the product may contain (a) a first container containing a composition containing the antibody of Initiation C, and (b) a second container containing a composition containing another cytotoxic agent or other therapeutic agent. The product of embodiment C may further contain an attached document indicating that the composition can be used to treat a particular condition. Instead, or in addition, the product may contain a second (or third) pharmaceutically acceptable buffer such as, for example, sterile water for injection (BWFI), phosphate buffered physiological saline, Ringer's solution, and dextrose solution. Additional containers may be included. The product may further contain other articles, such as other buffers, diluents, filters, needles, and syringes, as desired from a commercial point of view or from a user's point of view.

It is understood by those skilled in the art based on the technical common knowledge of those skilled in the art that any combination of all or all of the embodiments described in the present specification is included in the disclosure C as long as there is no technical contradiction.

Initiation A, B, or C

All technical background documents cited in this specification are incorporated in this specification by reference.

In this specification, the phrase “and/or” includes the meaning of the combination of terms before and after the scripture “and/or”, and it is understood that this includes all combinations of terms appropriately linked by the scripture.

In this specification, when used together with terms such as first, second, third, fourth, ..., it is understood that these elements do not have to be limited by those terms. These terms are used only to distinguish one element from other elements. For example, writing a first element as a second element, and similarly, writing a second element as a first element, starts A, B, It is understood that it is possible without departing from the range of and C.

Unless clearly stated otherwise, or unless contradicted by context, any term expressed in the singular form described in this specification also includes the plural form, and any term expressed in the plural form described in this specification is also the singular form. Also includes.

The terms used in this specification are used only to describe specific embodiments, and are not intended to limit the present disclosure. Unless other definitions are specified, terms (including technical terms and scientific terms) used herein are the same as those broadly understood by those skilled in the art to which disclosures A, B, and C belong. It is to be construed as having meaning, and should not be interpreted in an idealized or excessively formal sense.

The term "comprising" as used herein is intended to define the existence of the described matters (absence, process, element, number, etc.), except when a clearly different understanding is required from the context, and this The term does not exclude the presence of other matters (absence, process, element, number, etc.).

Embodiments of the disclosures A, B, and C are described with reference to schematic diagrams, and the diagrams may be exaggerated for the sake of explanation.

Numerical values described in this specification may be understood as values having a constant width, based on common technical knowledge of a person skilled in the art, as long as they do not contradict the context. For example, it is understood that the expression "1 mg" is described as "about 1 mg" accompanied by a constant fluctuation amount. For example, the expression "1 to 5" is "1, 2, 3, 4, 5," unless it is contrary to the context, and each value is specifically stated individually. It is understood that it will.

Example

Hereinafter, Initiation A is specifically described by Examples 1 to 4 and 21 to 23, Initiation B by Examples 5 to 7, 19 and 20, and Initiation C by Examples 8 to 19, respectively, but the present disclosure Is not limited to these examples. It is to be understood that several other embodiments may be envisaged in view of the above general description.

Example 1

Construction of pH-dependent human IL-6 receptor-binding human antibody with elevated pi

Fv4-IgG1 disclosed in WO2009/125825 is an antibody that binds to human IL-6 receptor in a pH-dependent manner, and uses VH3-IgG1 (SEQ ID NO: 24) as a heavy chain and VL3-CK (SEQ ID NO: 32) as a light chain. Includes. In order to increase the pi of Fv4-IgG1, for the variable region of Fv4-IgG1, the number of amino acids having a negative charge (e.g., aspartic acid, glutamic acid) is reduced, while amino acids having a positive charge (e.g., arginine, Lysine) was introduced. Specifically, in the heavy chain VH3-IgG1, according to Kabat numbering, glutamic acid at position 16 for glutamine, glutamic acid at position 43 for arginine, glutamine at position 64 for lysine, and glutamic acid at position 105 for glutamine As a result, VH3 (High_pI)-IgG1 (SEQ ID NO: 25) was prepared as a heavy chain with an elevated pI. Similarly, among the light chain VL3-CK, according to Kabat numbering, serine at position 18 is arginine, glutamine at position 24 is arginine, glutamic acid at position 45 is lysine, glutamic acid at position 79 is glutamine, and position 107 By substituting lysine for glutamic acid, VL3 (High_pI)-CK (SEQ ID NO: 33) was prepared as a light chain with an elevated pI. On the other hand, when introducing the substitution at position 79 of VL3-CK, although it is not intended to increase pI, a modification of substituting alanine at position 80 with proline and alanine at position 83 with isoleucine was simultaneously introduced.

An antibody shown below was produced by the method of Reference Example 2: (a) Low_pI-IgG1 containing VH3-IgG1 as the heavy chain and VL3-CK as the light chain; (b) Middle_pI-IgG1 comprising VH3-IgG1 as the heavy chain and VL3(High_pI)-CK as the light chain; And (c) VH3 (High_pI)-IgG1 as a heavy chain and VL3 (High_pI)-CK as a light chain, High_pI-IgG1.

Next, calculation of the theoretical pI of each produced antibody was performed using a method known in the art using GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION) (for example, Skoog et al. , Trends Analyt.Chem. 5(4): 82-83 (1986)). On the other hand, in the antibody molecule, all cysteine side chains were assumed to form disulfide bonds, and the contribution of the cysteine side chains to pKa was excluded.

Table 3 shows the calculated theoretical pI values. While the theoretical pI of Low_pI-IgG1 was 6.39, Middle_pI-IgG1 was 8.70 and High_pI-IgG1 was 9.30, and the theoretical pI value increased step by step.

Fv4-IgG1-F11 (hereinafter, Low_pI-F11), which imparts binding to FcRn under neutral pH conditions by introducing amino acid substitutions into the Fc region of Fv4-IgG1 and promotes uptake into cells via FcRn (hereinafter, Low_pI-F11 ) And Fv4-IgG1-F939 (hereinafter referred to as Low_pI-F939) are disclosed in WO2011/122011. Moreover, Fv4-IgG1-F1180 (hereinafter, Low_pI), which promotes uptake into cells via FcγR by increasing the binding properties to FcγR under neutral pH conditions by introducing amino acid substitutions into the Fc region of Fv4-IgG1 (hereinafter, Low_pI -Referred to as F1180) is disclosed in WO2013/125667. In Fv4-IgG1-F1180, amino acid modifications were introduced to increase the plasma retention of the antibody by simultaneously increasing FcRn binding under acidic pH conditions in the endosome. The pi of the antibody containing these novel Fc region variants was raised, and the antibody shown below was produced.

Specifically, VH3-IgG1-F11 (SEQ ID NO: 30) and VH3-IgG1-F939 (SEQ ID NO: 26) described in WO2011/122011, and VH3-IgG1-F1180 (SEQ ID NO: 28) described in WO2013/125667 For each, pI is increased by substituting glutamic acid at position 16 for glutamine, glutamic acid at position 43 for arginine, glutamine at position 64 for lysine, and glutamic acid at position 105 for glutamine according to Kabat numbering. VH3 (High_pI)-F11 (SEQ ID NO: 31), VH3 (High_pI)-F939 (SEQ ID NO: 27), and VH3 (High_pI)-F1180 (SEQ ID NO: 29) were prepared as heavy chains.

Using these heavy chains, the following antibodies were produced by the method of Reference Example 2: (1) Low_pI-F939 containing VH3-IgG1-F939 as heavy chain and VL3-CK as light chain; (2) Middle_pI-F939 comprising VH3(High_pI)-F939 as heavy chain and VL3-CK as light chain; (3) High_pI-F939 comprising VH3 (High_pI)-F939 as the heavy chain and VL3 (High_pI)-CK as the light chain; (4) Low_pI-F1180 containing VH3-IgG1-F1180 as a heavy chain and VL3-CK as a light chain; (5) Middle_pI-F1180 including VH3-IgG1-F1180 as a heavy chain and VL3(High_pI)-CK as a light chain; (6) High_pI-F1180 including VH3 (High_pI)-F1180 as a heavy chain and VL3 (High_pI)-CK as a light chain; (7) Low_pI-F11 containing VH3-IgG1-F11 as a heavy chain and VL3-CK as a light chain; And (8) High_pI-F11 comprising VH3 (High_pI)-F11 as a heavy chain and VL3 (High_pI)-CK as a light chain.

Next, calculation of the theoretical pI of each produced antibody was carried out in the same manner as the above-described method using GENETYX-SV/RC Ver 9.1.0 (GENETYX CORPORATION). Table 3 shows the calculated theoretical pI values. In the case of the antibody containing all the new Fc region variants, the theoretical pI value rose stepwise in the order of Low_pI, Middle_pI, and High_pI.

Example 2

Antigen loss effect of pH-dependent binding antibody with elevated pi

(2-1) In vivo assay of pH-dependent human IL-6 receptor-binding antibody modulating pI

Low_pI-IgG1, High_pI-IgG1, Low_pI-F939, Middle_pI-F939, High_pI-F939, Low_pI-F1180, Middle_pI-F1180 and High_pI-F1180, which are various pH-dependent human IL-6 receptor-binding antibodies produced in Example 1 The in vivo assay using was performed as follows.

In human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories, Methods Mol. Biol. 602:93-104 (2010)), prepared by the method of Reference Example 3 Soluble human IL-6 receptor (also referred to as "hsIL-6R"), anti-human IL-6 receptor antibody, and human immunoglobulin agent Sanglopor after simultaneous administration of soluble human IL-6 receptor In vivo dynamics were evaluated. A mixed solution of soluble human IL-6 receptor, anti-human IL-6 receptor antibody, and sanglophor (at concentrations of 5 μg/mL, 0.1 mg/mL, and 100 mg/mL, respectively) to the tail vein at a single dose of 10 mL/kg did. Since the anti-human IL-6 receptor antibody is present in a sufficient amount excessively with respect to the soluble human IL-6 receptor, it was considered that almost all of the soluble human IL-6 receptor is bound to the antibody. Blood was collected 15 minutes after administration, 7 hours, 1 day, 2 days, 3 days, and 7 days after 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 at -20°C or lower until measurement was performed.

(2-2) Measurement of soluble human IL-6 receptor concentration in plasma by electrochemiluminescence method

The concentration of soluble human IL-6 receptor in the plasma of mice was measured by electrochemiluminescence. Soluble human IL-6 receptor calibration samples prepared at a concentration of 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90 pg/mL and mouse plasma assay samples diluted 50 times or more, respectively, were SULFO-TAG NHS Ester. (Meso Scale Discovery) rutheniumized Monoclonal Anti-human IL-6R Antibody (R&D), Biotinylated Anti-human IL-6R Antibody (R&D), and Tocilizumab, a human IL-6 receptor binding antibody (CAS number: 375823-41 After mixing with -9), it was made to react at 37 degreeC overnight. The final concentration of Tocilizumab was prepared to be 333 μg/mL. Thereafter, the reaction solution was dispensed on a Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery). Further, after washing the reaction solution reacted at room temperature for 1 hour, Read Buffer T (x 4) (Meso Scale Discovery) was dispensed. Immediately thereafter, measurements were taken with SECTOR Imager 2400 (Meso Scale Discovery). The soluble human IL-6 receptor concentration was calculated using the analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve.

1, 2, and 3 show changes in plasma soluble human IL-6 receptor concentrations recognized in human FcRn transgenic mice after intravenous administration. Fig. 1 shows the effect of increasing antigen disappearance when the pI of the variable region is raised in the case where the normal region is a native IgG1. Fig. 2 shows the effect of increasing antigen disappearance when the pi of the variable region is raised in the antibody (F939) to which the binding property to FcRn is imparted under neutral pH conditions. Fig. 3 shows the effect of increasing antigen disappearance when the pI of the variable region is increased in the antibody (F1180) having increased binding properties to FcγR under neutral pH conditions.

In either case, it was shown that it is possible to accelerate the rate of antigen disappearance by the pH-dependent binding antibody by raising the pi of the antibody. In addition, it was shown that it was possible to further increase the rate of antigen disappearance compared to the case where only the pI of the pH-dependent binding antibody was raised by further imparting an increase in the binding property to FcRn or FcγR under neutral pH conditions. (Comparison between Fig. 1 and Figs. 2 and 3).

(2-3) pI Modulated pH-dependent human IL-6 receptor binding antibody In vivo Infusion Assay

In vivo assays using various pH-dependent human IL-6 receptor-binding antibodies, Low_pI-IgG1, High_pI-IgG1, Low_pI-F11, and High_pI-F11 prepared in Example 1 were carried out as follows.

Soluble human IL-6 receptor subcutaneously in the stomach of human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories, Methods Mol. Biol. 602: 93-104 (2010)) By embedding an infusion pump (MINI-OSMOTIC PUMP MODEL2004, alzet) filled with, a model animal in which the concentration of soluble human IL-6 receptor in plasma is maintained in a normal state was produced. In vivo kinetics after administration of the anti-human IL-6 receptor antibody administered to the model animal was evaluated.

Specifically, for the soluble human IL-6 receptor, in order to suppress the production of neutralizing antibodies that may be produced by the mouse itself, a monoclonal anti-mouse CD4 antibody obtained by a method known in the art is used. It was administered as a single dose to the tail vein at 20 mg/kg. Thereafter, an infusion pump filled with 92.8 μg/mL of a soluble human IL-6 receptor was embedded under the mouse's stomach. Three days after the infusion pump was implanted, the anti-human IL-6 receptor antibody was administered a single dose to the tail vein at 1 mg/kg. 15 minutes, 7 hours, 1 day, 2 days, 3 days or 4 days, 6 days or 7 days, 13 days or 14 days, 20 days or 21 days, and 27 days or after administration of anti-human IL-6 receptor antibody After 28 days had elapsed, blood was collected from the mice. Plasma was obtained by immediately centrifuging the collected blood at 4° C. and 15,000 rpm for 15 minutes. The separated plasma was stored in a freezer set at -20°C or lower until measurement was performed.

(2-4) Measurement of hsIL-6R concentration in plasma by electrochemiluminescence method

The concentration of hsIL-6R in the plasma of mice was measured by electrochemiluminescence. HsIL-6R calibration curve samples prepared at 250, 125, 62.5, 31.25, 15.61, 7.81, or 3.90 pg/mL and mouse plasma assay samples diluted 50 times or more were used as SULFO-TAG NHS Ester (Meso Scale Discovery). After mixing with the converted Monoclonal Anti-human IL-6R Antibody (R&D), Biotinylated Anti-human IL-6 R Antibody (R&D) and Tocilizumab, it was reacted overnight at 37°C. The final concentration of Tocilizumab was prepared to be 333 μg/mL. Thereafter, the reaction solution was dispensed on a Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery). After washing the reaction solution further reacted at room temperature for 1 hour, Read Buffer T (×4) (Meso Scale Discovery) was dispensed onto the plate. Immediately thereafter, measurements were taken with SECTOR Imager 2400 (Meso Scale Discovery). The hsIL-6R concentration was calculated using analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve.

Fig. 4 shows the measured human IL-6 receptor concentration trend. Regarding both an antibody (High_pI-IgG1) having an Fc region, which is an Fc region of native IgG1, and an antibody (High_pI-F11) containing a novel Fc region variant with increased binding to FcRn under neutral pH conditions, The concentration of soluble human IL-6 receptor in plasma was lowered when a high-pI antibody (also referred to as "High_pI") was administered compared to the case where a low-pI antibody (also referred to as "Low_pI") was administered.

Although not bound by a specific theory, the results obtained by these experiments can also be described as follows. When the Fc region of the administered antibody is that of a native IgG antibody, it is considered that absorption into cells is mainly caused by non-specific absorption (pinocytosis). Here, since the cell membrane has a negative charge, the administered antibody-antigen complex is thought to have a higher pI (i.e., the charge as a whole molecule is skewed toward the positive charge side), it is more likely to be closer to the cell membrane, and nonspecific absorption is more likely to occur. do. It is thought that even when an antibody with an elevated pI forms a complex with an antigen, the pI as the whole complex is increased compared to the original complex of the antibody and the antigen, thereby increasing absorption into cells. Therefore, for an antibody that binds to an antigen in a pH-dependent manner, by raising its pI, it is possible to further increase the rate of disappearance of the antigen from the plasma, and to keep the antigen concentration in the plasma lower. Do.

On the other hand, in these Examples, in the variable region of the antibody, the introduction of amino acid substitutions to reduce the number of negatively charged amino acids that can be exposed on the surface of the antibody molecule and/or increase the number of positively charged amino acids. By doing this, the pi of the antibody was raised. Those skilled in the art will understand that the effect obtained by such an increase in pI does not uniquely (or substantially) depend on the amino acid sequence constituting the antibody or the kind of target antigen, but rather, they can be expected to depend on pI. There will be. For example, in WO2007/114319 and WO2009/041643, schematically, the following is described.

Since IgG antibodies have a sufficiently large molecular weight, their main metabolic pathway is not renal excretion. It is known that IgG antibodies having Fc have a long half-life by being recycled by the salvage pathway of FcRn expressed in cells including endothelial cells of blood vessels, and IgG is believed to be metabolized mainly in endothelial cells. have. In other words, it is thought that when IgG non-specifically taken up in endothelial cells binds to FcRn, IgG is recycled, and molecules that could not bind to FcRn are metabolized. The half-life in blood of IgG having lowered binding property to FcRn is shortened, and conversely, the half-life in blood can be lengthened by increasing the binding property to FcRn. As described above, the previous method of controlling the blood dynamics of IgG has been performed by changing the Fc to change the binding properties to FcRn, but WO2007/114319 (mainly amino acid substitution technology in the FR region) or WO2009/041643 (mainly CDR In the Examples of the amino acid substitution technique in the region), it was shown that the half-life in blood can be controlled without altering the Fc by changing the pi of the variable region of the antibody irrespective of the type of the target antigen. It is thought that the rate of absorption of non-specific IgG antibodies into endothelial cells depends on the physicochemical Coulomb interaction of the negatively charged cell surface and the IgG antibody. Therefore, by reducing (increasing) the pI of the IgG antibody, the coulomb interaction is reduced (increased), resulting in a decrease (increase) of nonspecific absorption into endothelial cells, resulting in a decrease (increase) of metabolism in endothelial cells. By doing so, it is thought that the pharmacokinetics in plasma could be controlled. Since the Coulomb interaction between endothelial cells and cell surface negative charges is a physicochemical interaction, it is thought that this interaction does not uniquely depend on the amino acid sequence constituting the antibody itself. Therefore, the plasma pharmacokinetics control method provided in this specification is not applied only to a specific antibody, but can be widely applied to any polypeptide including the variable region of the antibody. On the other hand, the reduction (increase) of the Coulomb interaction refers to a decrease (increase) in the Coulomb force expressed by the attractive force and/or an increase (decrease) in the Coulomb force expressed as a repulsive force.

As the amino acid substitution that realizes this, any of a single amino acid substitution or a combination of a plurality of amino acid substitutions may be used. In some embodiments, a method of introducing a single amino acid substitution or a combination of multiple amino acid substitutions at a position exposed on the surface of the antibody molecule is provided. Alternatively, a plurality of amino acid substitutions to be introduced may be arranged in three-dimensional proximity to each other. For example, when an amino acid that can be exposed on the surface of an antibody molecule is substituted with an amino acid having a positive charge (preferably arginine or lysine), or an amino acid having an existing positive charge (preferably arginine or lysine) is used. In the case of use, by further substituting one or a plurality of amino acids (in some cases, even one or a plurality of amino acids embedded in the antibody molecule) three-dimensionally close to these amino acids with amino acids having a positive charge, as a result, three-dimensional structural The inventors conceived that it may be desirable to produce a localized positive charge dense state at a location close to each other. Here, the definition of "stereo-structurally close position" is not particularly limited, for example, within 20 Å, preferably within 15 Å, or more preferably within 10 Å from each other, single amino acid substitution or multiple amino acid substitutions The state in which this is introduced can be meant. Whether the desired amino acid substitution is at any position exposed on the surface of the antibody molecule, or whether the amino acid substitution is at a close position can be determined by a known method such as X-ray crystal structure analysis.

As described above, the inventors of the present invention have noted that pI is an index indicating the state of charge of the entire molecule, and that neither the charge buried in the antibody molecule nor the charge on the surface of the antibody molecule are handled without distinction. By preparing an antibody molecule by comprehensively considering the charge effects such as surface charge of the antibody molecule or local charge concentration, it is possible to further accelerate the rate of antigen disappearance from plasma, and the concentration of antigen in plasma. It was also conceived that it was possible to keep it even lower.

Receptors such as FcRn and FcγR are expressed on the cell membrane, and antibodies with increased affinity for FcRn or FcγR under neutral pH conditions are thought to be mainly absorbed into cells through these Fc receptors. Since the cell membrane has a negative charge, the administered antibody-antigen complex has a higher pI (the charge as a whole molecule is skewed toward the positive charge side), the more it is closer to the cell membrane, and the absorption through the Fc receptor is more likely to occur. I think. Therefore, the affinity for FcRn or FcγR under neutral pH conditions is increased, and the antibody whose pI is further increased has increased absorption into cells via the Fc receptor even when a complex with an antigen is formed. I'm doing it. Therefore, even for an antibody that binds to an antigen in a pH-dependent manner, which has increased affinity for FcRn or FcγR under neutral pH conditions, the pI is increased to further increase the rate of antigen disappearance from plasma. It is possible to do so quickly, and it is possible to keep the antigen concentration in the plasma lower.

Example 3

Evaluation of the binding property of the pH-dependent binding antibody with elevated pi to the extracellular matrix

(3-1) Evaluation of binding ability to extracellular matrix

In order to evaluate how the antibody has a pH-dependent antigen-binding property and further altering the pI, the following experiment is performed to evaluate how it affects the binding ability to the extracellular matrix.

As in the method of Example 1, as antibodies showing pH-dependent binding to the IL-6 receptor, three antibodies with different pIs Low_pI-IgG1, Middle_pI-IgG1, and High_pI-IgG1 were prepared. Low_pI (NPH)-IgG1 containing H54 (SEQ ID NO: 34) and L28 (SEQ ID NO: 35) described in WO2009125825 as a conventional antibody that does not show pH-dependent binding to the IL-6 receptor, and H(WT ) (SEQ ID NO: 36) and L(WT) (SEQ ID NO: 37) containing High_pI(NPH)-IgG1, respectively, were prepared by the method of Reference Example 2.

For these antibodies, the theoretical pI was calculated in the same manner as in the method of Example 1. Table 4 shows the calculated theoretical pI. Antibodies that did not exhibit pH-dependent binding to the IL-6 receptor were also shown to have an elevated pI, similarly to antibodies exhibiting pH-dependent binding.

(3-2) by electrochemiluminescence (ECL) method Extracellular Evaluation of antibody binding to the matrix

Using TBS (Takara), the extracellular matrix (BD Matrigel basement membrane matrix/manufactured by BD) was diluted to 2 mg/mL. The diluted extracellular matrix was dispensed into MULTI-ARRAY 96well Plate, High bind, Bare (Meso Scale Discovery: manufactured by MSD), and 5 µL per well were dispensed, and solidified overnight at 4°C. Thereafter, a 20 mM ACES buffer containing 150 mM NaCl, 0.05% Tween20, 0.5% BSA, and 0.01% NaN ₃ , pH 7.4 was dispensed and blocked. Antibodies provided for evaluation were diluted to 30, 10, 3 μg/mL using a 20 mM ACES buffer, pH 7.4 (ACES-T buffer) containing 150 mM NaCl, 0.05% Tween20, and 0.01% NaN _{3, and then 150 mM} NaCl, 0.01% Tween20, 0.1% BSA, 0.01% NaN _{3 It} was further diluted using a 20mM ACES buffer, pH 7.4 (Dilution buffer) containing, and the final concentrations were 10, 3.3, and 1 μg/mL, respectively. To the plate from which the blocking solution was removed, the diluted antibody solution was added, followed by shaking at room temperature for 1 hour. The antibody solution was removed, ACES-T buffer containing 0.25% glutaraldehyde was added and allowed to stand for 10 minutes, and then the plate was washed with DPBS (manufactured by Wako Pure Chemical Industries, Ltd.) containing 0.05% Tween20. The antibody for ECL detection was prepared by converting Goat anti-human IgG (gamma) (manufactured by Zymed Laboratories) into Sulfo-Tag using Sulfo-Tag NHS Ester (manufactured by MSD). The detection antibody was diluted with a dilution buffer to 1 μg/mL, added to the plate, and shaken for 1 hour at room temperature under light shielding. The detection antibody was removed, and a solution in which MSD Read Buffer T (4x) (manufactured by MSD) was diluted twice with ultrapure water was added, and then the amount of light emission was measured with SECTOR Imager 2400 (manufactured by MSD).

The results are shown in FIG. 5. It was shown that both the antibody exhibiting pH-dependent binding property and the antibody not exhibiting pH-dependent binding property improved the binding property to the extracellular matrix by raising the pI. In addition, surprisingly, the effect of improving the binding property to the extracellular matrix by raising the pi was remarkable in the antibody binding to the antigen in a pH-dependent manner. In other words, the antibody (High_pI-IgG1), which binds to the antigen in a pH-dependent manner and has a high pI, showed the strongest affinity for the extracellular matrix.

Although not bound by a specific theory, the results obtained by these experiments can also be described as follows. As one method for imparting a pH-dependent antigen-binding property to an antibody, a method of introducing a histidine alteration into a variable region of an antibody is known (see, for example, WO2009/125825). It is known that histidine has an imidazoyl group in the side chain and does not have a charge under conditions of neutral to basic pH, but has a positive charge under acidic pH conditions. Using the property of histidine, by introducing histidine into the variable region of the antibody, in particular, the CDR located in the vicinity of the antigen-interacting site, the interaction site with the antigen between neutral and acidic pH conditions. It is possible to change the environment of the electric charge and the three-dimensional structure in. Such an antibody can be expected to change its affinity for an antigen in a pH-dependent manner. Such an effect obtained by the introduction of histidine is not uniquely (or substantially) dependent on the amino acid sequence constituting the antibody or the type of target antigen, but depends on the site where histidine is introduced or the number of histidine residues introduced. It will be understood by those skilled in the art.

In WO2009/041643, schematically, the following is described: protein-protein interactions consist of hydrophobic interactions, electrostatic interactions, and hydrogen bonds, and the strength of the bond is generally a binding constant, or apparent It can be expressed as an avidity. PH-dependent binding, in which the strength of the bond changes under neutral pH conditions (e.g., pH 7.4) and acidic pH conditions (e.g., pH 5.5 to pH 6.0), is a natural protein-protein interaction. Depends on For example, the binding of the above-described IgG molecule to FcRn, known as the salvage receptor of the IgG molecule, shows strong binding under acidic pH conditions and extremely weak binding under neutral pH conditions. Histidine residues are involved in most of these protein-protein interactions in which the binding changes in a pH-dependent manner. Since the pKa of the histidine residue is present in the vicinity of 6.0 to 6.5, the dissociation state of the proton of the histidine residue changes between the neutral pH condition and the acidic pH condition. That is, the histidine residue has no charge under neutral pH conditions and functions as a neutral hydrogen atom acceptor, and has a positive charge under acidic pH conditions and functions as a hydrogen atom donor. Also in the above-described IgG molecule-FcRn interaction, it has been reported that histidine residues present in the IgG molecule are involved in pH-dependent binding (Martin et al., Mol. Cell. 7(4):867-877 (2001) )).

Therefore, it becomes possible to impart pH dependence to the protein-protein interaction by substituting histidine residues for amino acid residues involved in the protein-protein interaction or introducing histidine at the interaction site. Such attempts have been made in protein-protein interactions between antibody-antigens, and antibody variants with reduced antigen-affinity under acidic pH conditions are obtained by introducing histidine into the CDR sequences of anti-oval lysozyme antibodies. It is successful in obtaining (Ito et al., FEBS Lett. 309(1): 85-88(1992)). Further, by introducing histidine into the CDR sequences, antibodies that specifically bind to the antigen at low pH in cancer tissues and weakly bind under neutral pH conditions have been reported (WO2003/105757).

On the other hand, the amino acid residue introduced to increase pi is preferably lysine, arginine, or histidine having a positively charged side chain. The standard pKa of 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 theory of acid-base equilibrium known in the art, these pKa values mean that the side chain of lysine has a positive charge of 50% in a solution of pH 10.5 and the remaining 50% has no charge. will be. In addition, as the pH of the solution increases, the ratio of the side chains of lysine to have a positive charge decreases, and in a solution of pH 11.5, the pH of which is 1 higher than the pKa of lysine, the ratio of positive charges is about 9%. Conversely, as the pH of the solution decreases, the proportion of having a positive charge increases, and in a solution having a pH of 9.5, which is 1 lower than the pKa of lysine, the proportion of having a positive charge becomes about 91%. This theory holds true for arginine and histidine as well. That is, almost 100% of lysine or arginine has a positive charge in a solution of neutral pH (for example, pH 7.0), while about 9% of histidine has a positive charge. Therefore, histidine has a positive charge under neutral pH conditions, but its degree is less than that of lysine or arginine, and as amino acids introduced to increase pI, lysine and arginine have been considered more preferable. Moreover, according to Holash et al. (Proc. Natl. Acad. Sci., 99(17): 11393-11398 (2002), as an alteration to increase binding to the extracellular matrix, an alteration that raises pI was introduced. Although it was thought that it was effective to do so, it was thought that the substitution with the introduction of lysine or arginine is more preferable amino acid modification than the substitution with the introduction of histidine for the reasons described above.

As disclosed for the first time in this specification, the affinity of the antibody to the extracellular matrix is surprisingly synergistic by combining the introduction of histidine for imparting pH-dependent antigen binding with the modification of the antibody that raises the pI. It is possible to ascend to. In fact, the pI of High_pI-IgG1 was 9.30, whereas the pI of High_pI(NPH)-IgG1 was 9.35, and the value of High_pI-IgG1 was slightly lower. Nevertheless, as for the actual affinity for the extracellular matrix, High_pI-IgG1 is clearly increasing, and this is not necessarily explained by the height of the pI alone, and alterations that increase pI and histidine alterations are not necessarily explained. It can be said that the synergistic effect of introducing in combination is shown. This result was a surprising and unexpected phenomenon.

However, since the pKa of the amino acid side chain in a protein is greatly influenced by the surrounding environment, it is necessary to be careful that the pKa theoretical value is not necessarily described above. That is, although the above technique is suggested in this specification based on a general scientific theory, it can be easily assumed that there may be many exceptions in the actual protein. For example, Hayes et al., J. Biol. Chem. In 250(18):7461-7472 (1975), when the pKa of histidine contained in myoglobin was experimentally determined, the value was centered around 6.0, but it was reported that there was a fluctuation from 5.37 to 8.05. Naturally, most of histidine having a high pKa has a positive charge under neutral pH conditions. Therefore, the above theory does not deny the effect of increasing pI by the introduction of histidine, and in the stereostructure of the actual protein, as seen in the case of lysine or arginine, also by amino acid alteration introducing histidine. It is also possible that the effect of increasing the pi can be exerted.

Example 4

Increase of pi by 1 amino acid substitution in the normal region

For an antibody that binds to an antigen in a pH-dependent manner, a method of increasing the pi of the antibody by introducing an amino acid substitution into the variable region of the antibody has been described. Moreover, the method of raising the pi of an antibody can also be implemented by making substitutions as little as about 1 amino acid in the antibody constant region.

Although it does not specifically limit as a method of raising pI by adding 1 amino acid substitution to an antibody constant region, For example, it can implement by the method described in WO2014/145159. As the amino acid substitution introduced into the normal region, as in the case of the variable region, while reducing the number of negatively charged amino acids (e.g., aspartic acid or glutamic acid), positively charged amino acids (e.g., arginine or lysine) It is desirable to introduce amino acid substitutions to increase.

Although not limited, the position where amino acid substitutions are introduced in the constant region is preferably a position at which the amino acid side chain can be exposed on the surface of the antibody molecule. A preferred example is a method of introducing a plurality of amino acid substitutions in combination at a location that may be exposed on the surface of such an antibody molecule. Alternatively, it is preferable that the plurality of amino acid substitutions introduced are positions that are three-dimensionally close to each other. Further, although not limited, the substitution of a plurality of amino acids to be introduced is preferably a substitution with an amino acid having a positive charge, resulting in a state having a plurality of positive charges at positions that are three-dimensionally close to each other in some cases. The definition of "stereo-structurally close position" is not particularly limited, but, for example, within 20 Å, preferably within 15 Å, or more preferably within 10 Å from each other, a single amino acid substitution or a plurality of amino acid substitutions is introduced. It can mean a state in which there is. Whether the desired amino acid substitution is at any position exposed on the surface of the antibody molecule, or whether the amino acid substitution at multiple positions is at a close position can be determined by a known method such as X-ray crystal structure analysis.

Further, as a method of giving a plurality of positive charges at positions that are three-dimensionally close to each other, in addition to the above method, there is also a method of using an amino acid originally having a positive charge in the IgG constant region. As an amino acid position having such a positive charge, for example, (a) arginine at position 255, 292, 301, 344, 355, or 416 according to EU numbering; And (b) 121th, 133th, 147th, 205th, 210th, 213th, 214th, 218th, 222th, 246th, 248th, 274th, 288th, 290th according to EU numbering. , 317, 320, 322, 326, 334, 338, 340, 360, 370, 392, 409, 414, or 439. By performing substitution with an amino acid having a positive charge at a position that is three-dimensionally close to these positively charged amino acids, it is possible to impart a plurality of positive charges to a position that is three-dimensionally close to each other.

(4-1) Preparation of pH-dependent anti-IgE-binding antibody

As the pH-dependent anti-human IgE antibody, the following three types of antibodies were prepared by the method of Reference Example 2: (1) Ab1H (SEQ ID NO: 38) as the heavy chain and Ab1L (SEQ ID NO: 39) as the light chain. Ab1, which is a conventional antibody; (2) Ab2, a conventional antibody comprising Ab2H (SEQ ID NO: 40) as a heavy chain and Ab2L (SEQ ID NO: 41) as a light chain; And (3) Ab3H (SEQ ID NO: 42) as a heavy chain and Ab3L (SEQ ID NO: 43) as a light chain.

(4-2) Evaluation of pH dependence on binding to human IgE

The affinity of Ab1 to human IgE at pH 7.4 and pH 5.8 was evaluated as follows. Using BIACORE T100 (GE Healthcare), kinetic analysis of human IgE and Ab1 was performed. Measurements were made using the following two types as running buffers: (1) 1.2mM CaCl ₂ /0.05% tween20, 20mM ACES, 150mM NaCl, pH 7.4; And (2) 1.2mM CaCl ₂ /0.05% tween20, 20mM ACES, 150mM NaCl, pH 5.8.

An appropriate amount of protein A/G (ACTIGEN) was immobilized on a sensor chip CM4 (GE Healthcare) by an amine coupling method, and a target antibody was captured therein. Next, an IgE dilution and a running buffer (as a reference solution) were injected, and human IgE was interacted with the antibody captured on the sensor chip. On the other hand, one of the above (1) and (2) was used as the running buffer, and each buffer was used for dilution of human IgE. For regeneration of the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 25°C. Based on the kinetic parameters, the binding rate constant ka (1/Ms) and the dissociation rate constant kd (1/s), calculated from the sensorgram obtained by the measurement, the KD (M) for human IgE of each antibody is calculated. Became. BIACORE T100 Evaluation Software (GE Healthcare) was used to calculate each parameter.

The affinity of Ab2 and Ab3 to human IgE at pH 7.4 and pH 5.8 was evaluated as follows. BIACORE T200 (GE Healthcare) was used to evaluate the binding activity of the anti-hIgE antibody to hIgE (dissociation constant KD (M)). Measurements were made using the following two types as running buffers: (1) 1.2mM CaCl ₂ /0.05% tween20, 20mM ACES, 150mM NaCl, pH 7.4; And (2) 1.2mM CaCl ₂ /0.05% tween20, 20mM ACES, 150mM NaCl, pH 5.8.

A peptide prepared by adding biotin to Lys present at the C-terminus of a chemically synthesized human Glypican 3 (aka GPC3) protein-derived peptide (having the amino acid sequence VDDAPGNSQQATPKDNEISTFHNLGNVHSPLK (SEQ ID NO: 44)) ") was added in an appropriate amount to the sensor chip SA (GE Healthcare), and fixed on the chip using the affinity of streptavidin and biotin. An appropriate concentration of hIgE was injected and immobilized on a chip by capturing the biotinylated GPC3 peptide. As an analyte, an appropriate concentration of anti-hIgE antibody was injected to interact with hIgE on the sensor chip. Thereafter, 10 mM Glycine-HCl, pH 1.5 was injected to reproduce the sensor chip. All measurements were performed at 37°C. The measurement results were analyzed by curve fitting using BIACORE T200 Evaluation Software (GE Healthcare), and the binding rate constant ka (1/Ms) and the dissociation rate constant kd (1/s) were calculated, based on the values. The dissociation constant KD(M) was calculated.

Table 5 shows the results. Both of the antibodies of Ab1, Ab2, and Ab3 have pH dependence on binding to human IgE, and their affinity under acidic pH conditions (pH 5.8) compared to their affinity under neutral pH conditions (pH 7.4), It turns out that it is weakening dramatically. From this, when these antibodies are administered to live animals, it is expected to exhibit an effect of accelerating the disappearance of human IgE, which is an antigen.

Table 6 shows the theoretical pI values (pI) calculated in the same manner as in the method of Example 1 for Ab1 to Ab3.

(4-3) normal In the realm Single amino acid To change due to pI Elevated Preparation of antibody

Ab1 produced in Example (4-1) is an antibody having a human natural IgG1 as a constant region. Here, in the Fc region of Ab1H, which is the heavy chain of Ab1, proline at position 238 according to EU numbering is substituted with aspartic acid, and serine at position 298 according to EU numbering is substituted with alanine to prepare Ab1H-P600. In addition, by introducing various single amino acid substitutions shown in Tables 7-1 and 7-2 into the Fc region of Ab1H-P600, various Fc variants were each produced by the method of Reference Example 2. On the other hand, in any Fc variant, Ab1L (SEQ ID NO: 39) was used as the light chain. The affinity of these antibodies for hFcγRII2b was equivalent to that of the P600 variant (data not shown).

[Table 7-1]

[Table 7-2]

(4-4) new Fc domain Variant Using an antibody containing, To BIACORE Human FcγRIIb binding assay

Using BIACORE (registered trademark) T200 (GE Healthcare), a binding assay of an antibody containing a modified Fc region between a soluble human FcγRIIb (aka "hFcγRIIb") and an antigen-antibody complex was performed. The soluble hFcγRIIb was prepared as a molecular type to which a His tag was attached using a method known to those skilled in the art. An appropriate amount of anti-His antibody was immobilized on the sensor chip CM5 (GE Healthcare) by the amine coupling method using His capture kit (GE Healthcare), and hFcγRIIb was captured therein. Next, an antibody-antigen complex and a running buffer (as a reference solution) were injected to interact with hFcγRIIb captured on a sensor chip. For the running buffer, 20mM N-(2-acetamide)-2-aminoethanesulfonic acid, 150mM NaCl, 1.2mM CaCl ₂ , 0.05% (w/v) Tween20, pH 7.4 were used, respectively, even for dilution of soluble hFcγRIIb. Was used. For regeneration of the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were performed at 25°C. Analysis is performed on the basis of the binding (RU) calculated from the sensorgram obtained by the measurement, and it is expressed as a relative value when the amount of binding in P600 is 1.00. BIACORE (registered trademark) T100 Evaluation Software (GE Healthcare) was used to calculate the parameters. The results are shown in Table 7-1 and Table 7-2 (refer to the column of "BIACORE" in the table) and Fig. 6. In some Fc variants, it has been shown that the affinity for hFcγRIIb immobilized on a sensor chip of BIACORE (registered trademark) is increased.

Although not bound by a specific theory, it is also possible to explain this result as follows. It is known that the sensor chip of BIACORE (registered trademark) has a negative charge, and the state of charge can be considered to be similar to the cell membrane surface. More specifically, the binding of the antigen-antibody complex to hFcγRIIb immobilized on a sensor chip of BIACORE having a negative charge is similar to that of the antigen-antibody complex binding to hFcγRIIb present on the surface of a cell membrane having a negative charge. Guess what to do.

An antibody produced by introducing a modification that increases pi with respect to the Fc region is an antibody in which the Fc region (normal region) has a greater positive charge compared to before introducing the modification. For this reason, it is possible to think that the Coulomb interaction between the Fc region (positive charge) and the sensor chip (negative charge) was strengthened by amino acid alteration that increases pI. Moreover, the effect is expected to occur similarly to the cell membrane surface having a negative charge, so it is expected to exhibit an effect of accelerating the rate of absorption into cells even in vivo.

From the above results, if the ratio of the binding of the variant to hFcγRIIb compared to the binding of Ab1H-P600 to hFcγRIIb is about 1.2 times or more, it seems that it will have a strong charging effect on the binding of the antibody to hFcγRIIb on the sensor chip. Therefore, as a change that is expected to bring about a charging effect, for example, 196th, 282th, 285th, 309th, 311th, 315th, 345th, 356th, 358th, 359th according to EU numbering, Changes to 361, 362, 382, 384, 385, 386, 387, 389, 399, 415, 418, 419, 421, 424, or 443 have. 282, 309, 311, 315, 345, 356, 359, 361, 362, 385, 386, 387, 389, 399, 418, 419, or 443 It is desirable to change the stomach. In addition, it is preferable that the amino acid substitution introduced at such a position is arginine or lysine. As another example of the amino acid mutation position from which such a charging effect can be expected, glutamic acid at position 430 according to EU numbering may be mentioned. As a preferable amino acid substitution introduced at position 430, it is preferable to substitute glycine or threonine among positively charged arginine or lysine, or non-charged residues.

(4-5) Uptake of antibodies containing Fc region variants by hFcγRIIb-expressing cells

The following assay was performed in order to evaluate the rate of intracellular uptake into hFcγRIIb-expressing cell lines using the produced antibody containing the novel Fc region variant.

Using a known method, a MDCK (Madin-Darby canine kidney) cell line that normally expresses hFcγRIIb was constructed. Using these cells, the intracellular uptake of the antigen-antibody complex was evaluated. Specifically, human IgE (antigen) was labeled according to a predetermined protocol using pHrodoRed (Life Technologies), and an antigen-antibody complex was prepared in the culture medium at an antibody concentration of 10.8 mg/mL and an antigen concentration of 12.5 mg/mL. Formed. To the culture plate of the hFcγRIIb normal expression MDCK cells, a culture medium containing an antigen-antibody complex was added and incubated for 1 hour, and then the fluorescence intensity of the antigen absorbed in the cells was measured using InCell Analyzer 6000 (GE healthcare). Quantified. The amount of antigen absorbed was expressed as a relative value when the value at P600 was 1.00.

The results are shown in Tables 7-1 and 7-2 (refer to the column of "Imaging" in the table) and Fig. 7. In some Fc variants, antigen-derived fluorescence in cells was strongly observed.

Although not bound by a specific theory, this result can be explained as follows. The antigen and antibody added to the cell culture solution form an antigen-antibody complex in the culture solution. The antigen-antibody complex binds to hFcγRIIb expressed on the cell membrane through the Fc region of the antibody, and is absorbed into cells in a receptor-dependent manner. Since Ab1 used in this experiment is an antibody that binds to an antigen in a pH-dependent manner, the antibody can dissociate from the antigen. The dissociated antigen is labeled with pHrodoRed as described above, and emits fluorescence in the endosome. That is, the fact that the fluorescence intensity in the cell is stronger compared to the control is considered to indicate that the intracellular absorption of the antigen-antibody complex occurs more rapidly or at a higher frequency.

Here, when the ratio of the fluorescence intensity of the antigen absorbed into the cells of the variant compared to the fluorescence intensity of Ab1H-P600 is about 1.05 times or more, it was shown that it has a charging effect on the antigen absorbed by the cell. When the ratio of the fluorescence intensity of the antigen absorbed by the cells of the variant compared to the fluorescence intensity of Ab1H-P600 was about 1.5 times or more, it was shown to have a strong charging effect on the antigen absorbed by the cell. Therefore, from the above results, it was shown that it is possible to accelerate intracellular uptake compared to before introducing the alteration by introducing an alteration that raises the pI at an appropriate position in the Fc region. Amino acid altered positions showing this action are, for example, 253th, 254th, 256th, 258th, 281th, 282th, 285th, 286th, 307th, 309th, 311th according to EU numbering, It is the 315th, 327th, 330th, 358th, 384th, 385th, 387th, 399th, 400th, 421th, 433th, or 434th. Preferably, 254th, 258th, 281th, 282th, 285th, 309th, 311th, 315th, 327th, 330th, 358th, 384th, 399th, 400th according to EU numbering, It is a change to the 421st, 433th, or 434th place. The amino acid substitution introduced at such a position is preferably arginine or lysine. Although not limited, the position of the amino acid substitution introduced into the constant region for the purpose of raising the pi of the antibody may be, for example, the amino acid residue at position 285 according to EU numbering. Alternatively, as another example, the amino acid substitution of the amino acid residue at position 399 according to EU numbering may be mentioned.

Example 5

Preparation of Fc variant with increased FcRn binding under acidic pH conditions to improve plasma retention

It is known that IgG antibodies absorbed into cells are returned to plasma by binding to FcRn under conditions of acidic pH in the endosome. Therefore, IgG antibodies generally have a longer half-life in plasma than proteins that do not bind to FcRn. A method of increasing the affinity for FcRn under acidic pH conditions by introducing amino acid alterations into the Fc region of an antibody using the property and increasing the plasma retention of the antibody is known. Specifically, M252Y/S254T/T256E(YTE) modification (Dall'Acqua et al., J. Biol. Chem. 281:23514-23524 (2006)) and M428L/N434S(LS) modification (Zalevsky et al. , Nat. Biotechnol. 28:157-159 (2010)), and N434H alteration (Zheng et al., Clinical Pharmacology & Therapeutics 89(2):283-290 (2011)) by amino acid alteration, and acidic pH conditions A method of improving the plasma retention of an antibody by increasing its affinity for FcRn is known.

On the other hand, as described above, it is also known that an Fc variant having increased FcRn affinity under acidic pH conditions exhibits an undesirable affinity for rheumatoid factor (RF) (WO2013/046704). Therefore, the following study was conducted for the purpose of producing an Fc variant capable of improving plasma retention, which has reduced or substantially no binding property to a rheumatoid factor.

(5-1) Preparation of an antibody containing a novel Fc region variant

Fc variants and several novel Fc variants (F1847m, F1848m, F1886m, F1889m, F1927m, which have increased affinity for FcRn under acidic pH conditions such as those containing known modifications YTE, LS, or N434H, etc.) F1168m) was produced as shown below.

In the Fc region of the heavy chain (VH3-IgG1m) of the anti-human IL-6 receptor antibody Fv4-IgG1, a sequence encoding a heavy chain in which amino acid modifications were introduced was prepared 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-IgG1 containing VH3-IgG1m (SEQ ID NO: 46) as the heavy chain and VL3-CK as the light chain; (b) Fv4-YTE comprising VH3-YTE as heavy chain (SEQ ID NO: 47) and VL3-CK as light chain; (c) Fv4-LS comprising VH3-LS as heavy chain (SEQ ID NO: 48) and VL3-CK as light chain; (d) Fv4-N434H comprising VH3-N434H as heavy chain (SEQ ID NO: 49) and VL3-CK as light chain; (e) Fv4-F1847m comprising VH3-F1847m as heavy chain (SEQ ID NO: 50) and VL3-CK as light chain; (f) Fv4-F1848m comprising VH3-F1848m as heavy chain (SEQ ID NO: 51) and VL3-CK as light chain; (g) Fv4-F1886m comprising VH3-F1886m as heavy chain (SEQ ID NO: 52) and VL3-CK as light chain; (h) Fv4-F1889m comprising VH3-F1889m as heavy chain (SEQ ID NO: 53) and VL3-CK as light chain; (i) Fv4-F1927m comprising VH3-F1927m as heavy chain (SEQ ID NO: 54) and VL3-CK as light chain; And (j) Fv4-F1168m comprising VH3-F1168m as a heavy chain (SEQ ID NO: 55) and VL3-CK as a light chain.

(5-2) Kinetic analysis of binding to human FcRn

An antibody containing VH3-IgG1m or the above variant as a heavy chain, and L(WT) (SEQ ID NO: 37) as a light chain was prepared by the method shown in Reference Example 2, and was directed against human FcRn as follows. The binding activity was evaluated.

Using BIACORE T100 (GE Healthcare), kinetic analysis of human FcRn and each antibody was performed. An appropriate amount of protein L (ACTIGEN) was fixed on a sensor chip CM4 (GE Healthcare) by an amine coupling method, and a target antibody was captured therein. Next, an FcRn dilution and a running buffer (as a reference solution) were injected, and human FcRn was allowed to interact with the antibody captured on the sensor chip. For the running buffer, 50mM sodium phosphate, 150mM NaCl, 0.05% (w/v) Tween20, pH 6.0 were used, and each buffer was used for dilution of FcRn. For regeneration of the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 25°C. Based on the binding rate constant ka (1/Ms), which is a kinetic parameter calculated from the sensorgram obtained by measurement, and the dissociation rate constant kd (1/s), the KD (M) for human FcRn of each antibody is Was calculated. BIACORE T100 Evaluation Software (GE Healthcare) was used to calculate each parameter.

The results are shown in Table 8.

Example 6

Evaluation of affinity for rheumatoid factors of antibodies containing Fc region modified variants with increased FcRn binding under acidic pH conditions

Anti-drug antibodies (ADA) affect the effectiveness and pharmacokinetics of therapeutic antibodies, and sometimes cause serious side effects.Therefore, the usefulness and efficacy of therapeutic antibodies in clinical settings will be limited by the production of ADA. I can. Many factors affect the immunogenicity of therapeutic antibodies, but the presence of an effector T cell epitope is one of the factors. In addition, the presence of ADA (also referred to as "existing ADA") that the patient has before administration of the therapeutic antibody is considered to be similarly problematic. In particular, in the case of an antibody for the treatment of patients with autoimmune diseases such as joint rheumatism (RA), rheumatoid factor (RF), an autoantibody against human IgG, may cause the problem of "conventional ADA". Recently, it has been reported that a humanized anti-CD4IgG1 antibody with an N434H (Asn434His) mutation induces remarkable rheumatoid factor binding (Zheng et al., Clinical Pharmacology & Therapeutics 89(2):283-290 (2011)). By detailed study, it was confirmed that the N434H mutation in human IgG1 increases the binding of rheumatoid factor to the Fc region of the antibody compared to that of the pro-human IgG1.

Rheumatoid factors are polyclonal autoantibodies to human IgG, and their epitopes in human IgG differ from clone to clone, but their epitopes are located in the CH2/CH3 interfacial region and in the CH3 domain, which may overlap with the FcRn binding epitope. Seems to be. Therefore, mutations that increase the binding activity (binding affinity) to FcRn may increase the binding activity (binding affinity) of the rheumatoid factor to a specific clone.

In fact, it is known that in Fc with increased affinity for FcRn at acidic or neutral pH, the binding of Fc to rheumatoid factor is similarly increased not only by N434H modification but also by many other amino acid modifications (WO2013 /046704).

On the other hand, in WO2013/046704, several amino acid modifications that selectively inhibit affinity for rheumatoid factors without affecting the affinity for FcRn are exemplified, among which Q438R/S440E, Q438R/S440D, which are a combination of two amino acid mutations. , Q438K/S440E and Q438K/S440D are shown. Therefore, it was verified whether it was possible to lower the binding property to rheumatoid factors by introducing Q438R/S440E for Fc, which was disclosed for the first time in this specification, and whose affinity under acidic pH conditions was newly increased.

(6-1) Fc domain Variant Of the containing antibody Rheumatoid Combination of factors Assay

The assay for binding to the rheumatoid factor was performed by electrochemiluminescence (ECL) at pH 7.4 using individual serum (Proteogenex) from 30 RA patients. A 50-fold diluted serum sample, biotin-labeled test antibody (1 μg/mL), and SULFO-TAG NHS Ester (Meso Scale Discovery)-labeled test antibody (1 μg/mL) were each mixed, and incubated at room temperature for 3 hours. . Thereafter, the mixture was added to a MULTI-ARRAY 96 well plate (Meso Scale Discovery) coated with Streptavidin, and the plate was incubated at room temperature for 2 hours, followed by washing. After adding Read Buffer T (×4) (Meso Scale Discovery) to each well, the plate was immediately set in SECTOR imager 2400 Reader (Meso Scale Discovery), and chemiluminescence was measured.

The results of this assay are shown in Figs. 8 to 17. Fv4-IgG1 with native human IgG1 (FIG. 8) showed only weak rheumatoid factor binding, whereas Fv4-YTE (FIG. 9) and Fv4-LS (FIG. 10), which are conventional Fc modifications with increased binding to FcRn. ), and Fv4-N434H (FIG. 11) significantly increased the rheumatoid factor binding in a plurality of donors. On the other hand, Fv4-F1847m (Figure 12), Fv4-F1848m (Figure 13), Fv4-F1886m (Figure 14), Fv4-F1889m (Figure 15), Fv4-F1927m, which are novel Fc region modifications with increased binding to FcRn. In (FIG. 16) and Fv4-F1168m (FIG. 17), both showed only weak rheumatoid factor binding, and it was shown that the binding to FcRn can remarkably inhibit the binding of rheumatoid factor due to increased alteration.

Fig. 18 shows the average value of binding affinity to rheumatoid factor in the serum of 30 RA patients for each variant. All of the six new variants showed lower affinity than the three existing variants (YTE, LS, N434H), and also showed lower affinity for rheumatoid factors even compared to native human IgG1. . From the above, when considering the clinical development of a therapeutic antibody with improved affinity for FcRn for autoimmune diseases such as articular rheumatism, the risk related to rheumatoid factors, which is a concern in the existing Fc variant, is: In the Fc variant disclosed for the first time in the specification, it is suppressed, and therefore, it is considered that it can be used more safely than the previously known Fc variant.

Example 7

PK evaluation in Philippine monkeys of the Fc variant with increased FcRn binding under acidic pH conditions

In Example 7, the effect of improving plasma retention in Philippine monkeys using an antibody containing a novel Fc region variant provided in this specification, which was confirmed that binding to a rheumatoid factor was inhibited, was as follows: It was evaluated by the method of.

(7-1) Preparation of an antibody containing a novel Fc region variant

The following anti-human IgE antibodies were produced: (a) OHBH-IgG1 containing OHBH-IgG1 (SEQ ID NO: 56) as a heavy chain and OHBL-CK (SEQ ID NO: 57) as a light chain; (b) OHB-LS comprising OHBH-LS (SEQ ID NO: 58) as heavy chain and OHBL-CK as light chain; (c) OHBH-N434A (SEQ ID NO: 59) as the heavy chain and OHB-N434A including OHBL-CK as the light chain; (d) OHBH-F1847m (SEQ ID NO: 60) as heavy chain and OHB-F1847m including OHBL-CK as light chain; (e) OHBH-F1848m (SEQ ID NO: 61) as heavy chain and OHB-F1848m comprising OHBL-CK as light chain; (f) OHBH-F1886m (SEQ ID NO: 62) as heavy chain and OHB-F1886m comprising OHBL-CK as light chain; (g) OHBH-F1889m (SEQ ID NO: 63) as heavy chain and OHB-F1889m including OHBL-CK as light chain; And (h) OHBH-F1927m (SEQ ID NO: 64) as heavy chain and OHB-F1927m comprising OHBL-CK as light chain.

(7-2) Monkey PK assay of antibodies containing novel Fc region variants

After administration of the anti-human IgE antibody to Philippine monkeys, the in vivo kinetics of the anti-human IgE antibody in plasma were evaluated. A solution of anti-human IgE antibody was administered intravenously at a single dose of 2 mg/kg. 5 minutes after administration, (2 hours), 7 hours, 1 day, 2 days, 3 days, (4 days), 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 Blood was collected at work. The collected blood was immediately centrifuged at 4° C. and 15,000 rpm for 5 minutes to obtain plasma. The separated plasma was stored in a freezer set at -80°C or lower until measurement was performed. As the anti-human IgE antibody, 8 types of OHB-IgG1, OHB-LS, OHB-N434A, OHB-F1847m, OHB-F1848m, OHB-F1886m, OHB-F1889m, and OHB-F1927m were used.

(7-3) Measurement of the concentration of anti-human IgE antibody in plasma by ELISA

The concentration of anti-human IgE antibody in the plasma of Philippine monkeys was measured by ELISA. First, an anti-human IgG κ chain antibody (Antibody Solution) was dispensed on Nunc-Immuno Plate, MaxiSorp (Nalge nunc International), and left to stand at 4° C. overnight to prepare an anti-human IgG κ chain antibody immobilized plate. As the concentration in plasma, a calibration curve sample of 640, 320, 160, 80, 40, 20, or 10 ng/mL, and a sample for measuring Philippine monkey plasma diluted 100 times or more were prepared. These calibration curve samples and plasma measurement samples were prepared so that Philippine monkey IgE (in-house preparation) was added at a concentration of 1 μg/ml. Thereafter, a sample was dispensed to an anti-human IgG κ chain antibody immobilized plate, and left standing at room temperature for 2 hours. Thereafter, an HRP-antihuman IgG γ-chain antibody (Southern Biotech) was dispensed and allowed to stand at room temperature for 1 hour. Thereafter, a color development reaction was performed using TMB Chromogen Solution (Life Technologies) as a substrate, and after stopping the reaction with 1N-Sulfuric acid (Wako), absorbance at 450 nm was measured with a microplate reader. The concentration of anti-human IgE antibody in monkey plasma was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices). Fig. 19 shows the change in the concentration of the anti-human IgE antibody in the measured monkey plasma. From the change in the concentration of anti-human IgE antibody in monkey plasma measured, Phoenix WinNonlin Ver. Using 6.2 (Pharsight Corporation), the disappearance clearance was calculated by moment analysis. Table 9 shows the calculated pharmacokinetic parameters. Samples from individuals with positive antibodies to the administration sample in plasma were excluded from the change in concentration of the anti-human IgE antibody in monkey plasma and the calculation of the clearance.

(7-4) Measurement of antibody against administration sample in plasma by electrochemiluminescence method

Antibodies in monkey plasma for the administration sample were measured by electrochemiluminescence. An equal amount of a ruthenium-labeled administration sample with SULFO-TAG NHS Ester (Meso Scale Discovery), an administration sample biotinylated with EZ-Link Micro Sulfo-NHS-Biotinylation Kit (Pierce), and a Philippine monkey plasma measurement sample were mixed in equal amounts, and then 4°C. In politics overnight. After the sample was added to the MULTI-ARRAY 96-well Streptavidin Gold Plate (Meso Scale Discovery), it was allowed to react at room temperature for 2 hours and washed. Thereafter, Read Buffer T (x4) (Meso Scale Discovery) was dispensed, and measurement was immediately performed with SECTOR Imager 2400 (Meso Scale Discovery).

As a result, it was confirmed that all of the novel Fc variants showed a significant improvement in plasma retention as compared to the Fc region of native IgG1.

(7-5) Mouse PK assay for Fc variant

In order to compare F1718, an Fc variant described in WO2013/046704 as an Fc variant for increasing FcRn binding at acidic pH, and F1848m, a newly discovered Fc variant, the following experiment was conducted.

A gene encoding a heavy chain in which amino acid modifications were introduced into the Fc region of the heavy chain (VH3-IgG1) of Fv4-IgG1 (anti-human IL-6 receptor antibody) was 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-IgG1 containing VH3-IgG1 as the heavy chain and VL3-CK as the light chain; And (b) Fv4-F1718 comprising VH3-F1718 as a heavy chain (SEQ ID NO: 65) and VL3-CK as a light chain.

In the tail vein of a human FcRn transgenic mouse (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories, Methods Mol. Biol. 602:93-104 (2010)), the anti-human IL-6 Receptor antibody was administered once at 1 mg/kg. Blood was collected at 15 minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after administration of the anti-human IL-6 receptor antibody. Plasma was obtained by immediately centrifuging the collected blood at 4° C. and 15,000 rpm for 15 minutes. The separated plasma was stored in a freezer at -20°C or lower until measurement was performed.

(7-6) Measurement of plasma concentration of anti-human IL-6 receptor antibody by ELISA

The concentration of anti-human IL-6 receptor antibody in mouse plasma was measured by ELISA. First, Anti-Human IgG (gamma-chain specific) F(ab')2 Fragment of Antibody (SIGMA) was dispensed onto Nunc-Immuno Plate, MaxiSorp (Nalge nunc International), and allowed to stand overnight at 4°C to prevent Anti-Human IgG (gamma-chain specific) F(ab')2 Fragment of Antibody (SIGMA). Human IgG immobilization plates were prepared. As a concentration in plasma, a calibration curve sample containing an anti-human IL-6 receptor antibody of 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, or 0.0125 μg/mL and a mouse plasma measurement sample diluted 100 times or more were prepared, respectively. A mixed solution to which 200 µL of 20 ng/mL soluble human IL-6 receptor was added to 100 µL of these calibration curve samples or plasma measurement samples was allowed to stand at room temperature for 1 hour. Then, the Anti-Human IgG immobilization plate in which the mixed solution was dispensed into each well was allowed to stand at room temperature for 1 hour. Thereafter, Biotinylated Anti-human IL-6R Antibody (R&D) was added and reacted at room temperature for 1 hour, and Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was added and the reaction solution reacted at room temperature for 1 hour. Component HRP Microwell Substrate (BioFX Laboratories) was used as a substrate. The absorbance at 450 nm of the reaction solution in each well in which the reaction was stopped by the addition of 1N-Sulfuric acid (Showa Chemical) was measured with a microplate reader. The antibody concentration in mouse plasma was calculated from the absorbance of the calibration curve using analysis software SOFTmax PRO (Molecular Devices).

The results are shown in FIG. 20. F1718, an Fc modification for enhancing FcRn binding at acidic pH, described in WO2013/046704, did not show the effect of prolonging antibody PK, and exhibited plasma retention equivalent to that of native IgG1.

In F1718, four mutations of N434Y/Y436V/Q438R/S440E are introduced into the Fc region. On the other hand, in F1848m disclosed for the first time in this specification, four mutations of N434A/Y436V/Q438R/S440E are introduced. The difference between the amino acid mutations introduced into these two types of Fc is that the amino acid mutation introduced at position 434 according to EU numbering is that F1718 is Y (tyrosine), and F1848m is A (alanine). In Example (7-2), F1848m showed improvement in plasma retention compared to native IgG1, but F1718 did not show improvement in plasma retention. From this, although not limited, it is suggested that A (alanine) is preferable as an amino acid mutation introduced at position 434 in order to improve plasma retention.

(7-7) Fv variant immunogenicity prediction score

The production of anti-drug antibodies (ADA) affects the effects and pharmacokinetics of therapeutic antibodies, and sometimes leads to serious side effects, so the usefulness and efficacy of therapeutic antibodies in clinical practice is the production of ADA. May be limited by Although it is known that the immunogenicity of a therapeutic antibody is affected by many factors, in particular, the importance of the effector T cell epitope possessed by a therapeutic antibody has been particularly reported in many cases.

As in silico tools for predicting T cell epitopes, Epibase (Lonza), iTope/TCED (Antitope), and EpiMatrix (EpiVax) have been developed. Using these in silico tools, it is possible to predict T cell epitopes in each amino acid sequence (Walle et al., Expert Opin. Biol. Ther. 7(3):405-418(2007)), the potential of therapeutic antibodies. Phosphorus immunogenicity evaluation becomes possible.

Using EpiMatrix, the immunogenicity score of the evaluated Fc variant was calculated. EpiMatrix mechanically designs the sequence of a peptide fragment in which the amino acid sequence of a protein for which immunogenicity is to be predicted is divided every 9 amino acids, and for them, 8 types of major MHC Class II alleles (DRB1 ^* 0101, DRB1 ^* 0301, DRB1 ^* 0401, DRB1 ^* 0701, DRB1 ^* 0801, DRB1 ^* 1101, DRB1 ^* 1301, and DRB1 ^* 1501) by calculating the binding capacity to predict the immunogenicity of the target protein (Clin. Immunol. 131 (2) ): 189-201 (2009)).

F1718 and F1756 (N434Y/Y436T/Q438R/S440E) described in WO2013/046704 contain the N434Y mutation. In contrast, the newly disclosed F1848m and F1847m contain the N434A mutation.

The immunogenicity scores of these four kinds of variants, that is, F1718, F1848m, F1756, and F1847m calculated as described above, are shown in the column of "EpiMatrix score" in Table 31. Moreover, for the EpiMatrix score, the immunogenicity score corrected in consideration of the inclusion of Tregitope is indicated in the column of "tReg Adjusted Epx score". Tregitope is a peptide fragment sequence mainly present in a large amount of natural antibody sequences, and is a sequence that is thought to suppress immunogenicity by activating a regulatory T cell (Treg).

From this result, even when seeing either "EpiMatrix score" and "tReg Adjusted Epx score", the immunogenicity scores of F1848m and F1847m, which are N434A variants, were lower than those of the N434Y variants. This suggests that A (alanine) is preferable as an amino acid mutation introduced at position 434 for a lower immunogenicity score.

Example 8

Construction of humanized anti-human IL-8 antibody

(8-1) Production of humanized anti-human IL-8 antibody hWS-4

The humanized anti-IL-8 antibody disclosed in U.S. Patent No. 6,245,894 blocks its physiological action by binding to human IL-8 (hIL-8). The modified humanized anti-IL-8 antibody can be produced by combining the variable region sequences of the heavy and light chains disclosed in US Pat. No. 6,245,894 and the constant region sequences of substantially any of a variety of known human antibodies. Therefore, the human antibody constant region sequence of these modified antibodies is not particularly limited, but a natural human IgG1 sequence or a natural human IgG4 sequence as the heavy chain constant region, and a natural human kappa sequence as the light chain constant region sequence can be used. have.

In the humanized IL-8 antibody disclosed in U.S. Patent No. 6,245,894, the coding sequence of hWS4H-IgG1 (SEQ ID NO: 83) in which the heavy chain variable region RVHg and the natural human anti-IgG1 sequence as the heavy chain constant region are combined is referred to. It was produced by the method of Example 1. Furthermore, the coding sequence of hWS4L-k0MT (SEQ ID NO: 84) in which the light chain variable region RVLa and the natural human κ sequence were combined as the light chain constant region was prepared by the method of Reference Example 1. An antibody combining the heavy and light chains described above was prepared to obtain a humanized WS-4 antibody (hereinafter, hWS-4).

(8-2) Preparation of humanized anti-human IL-8 antibody Hr9

A new humanized antibody was produced using a human consensus framework sequence different from the FR used in hWS-4.

Specifically, the hybrid sequence of VH3-23 and VH3-64 as the heavy chain FR1, the sequence shown in VH3-15 or VH3-49 as the FR2, and the sequence shown in VH3-72 as the FR3 (except 82a according to Kabat numbering) ), and as FR4, the sequence shown in JH1 was used. These sequences were ligated with the CDR sequences of the hWS-4 heavy chain to prepare Hr9-IgG1 (SEQ ID NO: 85), which is a heavy chain of a novel humanized antibody.

Next, two types of antibodies were prepared: hWS-4 having hWS4H-IgG1 as the heavy chain and hWS4L-k0MT as the light chain, and Hr9 having Hr9-IgG1 as the heavy chain and hWS4L-k0MT as the light chain. On the other hand, in the range in which the disclosure C in this specification is described, in the case where it is particularly desired to specify the light chain, Hr9 is also expressed as Hr9/hWS4L. Antibodies were expressed according to the protocol attached to the product using FreeStyle 293 F cells (Invitrogen). Purification of the antibody from the culture supernatant was performed by the method of Reference Example 2. As a result, antibodies in the amounts shown in Table 11 were obtained. Surprisingly, the expression level of Hr9 was about 8 times that of hWS-4.

(8-3) human IL-8 binding activity of hWS-4 and Hr9

The binding affinity of hWS-4 and Hr9 to human IL-8 was measured as follows using BIACORE T200 (GE Healthcare).

As a running buffer, a composition of 0.05% tween20, 20mM ACES, and 150mM NaCl (pH 7.4) was used. An appropriate amount of protein A/G (PEIRCE) was fixed on a sensor chip CM4 (GE Healthcare) by an amine coupling method, and the desired antibody was captured. Next, human IL-8 diluted solution and running buffer (as a reference solution) were injected, and human IL-8 was interacted with the antibody captured on the sensor chip. On the other hand, a solution of the above composition was used as a running buffer, and a sugar buffer was also used for dilution of human IL-8. For regeneration of the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 37°C. KD (M) for human IL-8 of each antibody based on the binding rate constant kon (1/Ms), which is the kinetic parameter calculated from the sensorgram obtained by the measurement, and the dissociation rate constant koff (1/s). Was calculated. BIACORE T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

Table 12 shows the results. It was confirmed that hWS-4 and Hr9 have equivalent binding affinity to human IL-8.

In the development of antibody pharmaceuticals, the production amount of the antibody molecule is an important factor, and a high production amount is generally preferred. According to the above examination, a sequence derived from a more appropriate human consensus framework to be combined with the HVR sequence of hWS-4 was selected to obtain Hr9 with improved production amount while maintaining the binding affinity for human IL-8. What was possible is a special point.

Example 9

Production of antibodies with pH-dependent IL-8 affinity

(9-1) Preparation of Hr9-modified antibody for imparting pH dependence

A test was conducted for the purpose of imparting pH-dependent IL-8 affinity to the Hr9 antibody obtained in Example 8.

While not wishing to be bound by any particular theory, antibodies with a pH dependent affinity for IL-8 may exhibit the following behavior in vivo. The antibody administered to an organism can bind strongly to IL-8 and block its function in an environment maintained at a neutral pH (for example, in plasma). A part of such a complex of IL-8 and antibody is absorbed into cells due to a non-specific interaction (pinocytosis) with a cell membrane (hereinafter referred to as non-specific absorption). Under conditions of acidic pH in the endosome, the antibody's binding affinity to IL-8 is weakened, so the antibody is dissociated from IL-8. Thereafter, the antibody dissociated from IL-8 can be returned to the outside of the cell via FcRn. The antibody thus returned to the extracellular (in plasma) can bind to other IL-8 again and block its function. It is considered that an antibody having a pH-dependent affinity for IL-8 can bind to IL-8 multiple times by the above mechanism.

On the other hand, in the case of an antibody that does not have the properties retained in the antibody, one molecule of the antibody can neutralize the antigen only once, and cannot neutralize the antigen multiple times. Usually, since an IgG antibody has two Fabs, one antibody molecule can neutralize two molecules of IL-8. On the other hand, it is thought that an antibody capable of binding to IL-8 multiple times can bind to IL-8 any number of times as long as it stays in the living body. For example, a pH-dependent IL-8-binding antibody absorbed 10 times into cells from administration to disappearance can neutralize up to 20 molecules of IL-8 even in one molecule. Therefore, an antibody capable of binding to IL-8 multiple times has the advantage of being able to neutralize many IL-8 molecules even with a smaller amount of antibody. In another aspect, the antibody capable of binding to IL-8 multiple times is said to be able to maintain a state capable of neutralizing IL-8 over a longer period of time than when the same amount of antibody that does not have the above-described properties is administered. Has the advantage of doing it. In addition, from another viewpoint, the antibody capable of binding to IL-8 multiple times is said to be able to block the biological activity of IL-8 more strongly than when the same amount of the antibody that does not have the above-described properties is administered. Has the advantage of doing it.

In order to realize these advantages, with the aim of creating an antibody capable of binding to IL-8 multiple times, amino acid modifications centered on histidine were introduced into the variable regions of Hr9-IgG1 and WS4L-k0MT. Specifically, the modified body shown in Table 13 was produced by the method of Reference Examples 1 and 2.

On the other hand, a notation such as "Y97H" shown in Table 13 indicates a mutation introduction position defined by Kabat numbering, an amino acid before mutation introduction, and an amino acid after mutation introduction. Specifically, the expression "Y97H" indicates that the amino acid residue at position 97 according to Kabat numbering was substituted from Y (tyrosine) to H (histidine). Moreover, when introducing a plurality of amino acid substitutions in combination, it is described as "N50H/L54H".

(9-2) pH-dependent IL-8 affinity

The human IL-8 binding affinity of the antibody produced in Example 9-1 was measured as follows using BIACORE T200 (GE Healthcare). The following two types of running buffers were used: (1) 0.05% tween20, 20mM ACES, 150mM NaCl, pH 7.4; And (2) 0.05% tween20, 20mM ACES, 150mM NaCl, pH 5.8.

An appropriate amount of protein A/G (PIERCE) was fixed on a sensor chip CM4 (GE Healthcare) by an amine coupling method, and the desired antibody was captured. Next, human IL-8 diluted solution and running buffer (as a reference solution) were injected, and human IL-8 was interacted with the antibody captured on the sensor chip. On the other hand, one of the above was used as the running buffer, and each buffer was used for dilution of human IL-8. For regeneration of the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 37°C. The KD (M) for human IL-8 of each antibody is based on the binding rate constant kon (1/Ms), which is the kinetic parameter calculated from the sensorgram obtained by the measurement, and the dissociation rate constant koff (1/s). Was calculated. BIACORE T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

The results are shown in Table 14-1. First, Hr9/L16 containing L54H alteration in the light chain has a slightly enhanced human IL-8 binding affinity at neutral pH (pH 7.4) than that of Hr9, while human at acidic pH (pH 5.8). The IL-8 binding affinity was lowered. On the other hand, anti-IL-8 antibodies (H89/WS4L, H89/L12, and H89/L16) produced by combining H89 containing Y97H modification in the heavy chain and various light chains all bind human IL-8 at acidic pH. While the affinity was lowered, the affinity for human IL-8 binding at neutral pH was also lowered.

[Table 14-1]

(9-3) Preparation and evaluation of modified antibodies for imparting pH dependence

The combination of the promising modifications found in 9-2 and the evaluation of new amino acid mutations were performed, and as a result, the following combinations were found.

[Table 14-2]

The variant was prepared by the method of Reference Examples 1 and 2, and the binding affinity to human IL-8 was evaluated in the same manner as in Example 9-2.

The result is combined with Table 14 and shown. H89/L63 having H89-IgG1 (SEQ ID NO: 86) as a heavy chain and L63-k0MT (SEQ ID NO: 87) as a light chain has a human IL-8 binding affinity at neutral pH (pH 7.4) equal to Hr9. As it remained, it showed the fall of the human IL-8 binding affinity in acidic pH (pH 5.8). Specifically, in H89/L63, both koff (dissociation rate constant) and KD (dissociation constant) at pH 5.8 were larger than Hr9. This means that under acidic pH conditions in the endosome, H89/L63 has a property that is easy to dissociate human IL-8.

Surprisingly, H89/L118, which has H89-IgG1 as a heavy chain and L118-k0MT (SEQ ID NO: 88) as a light chain, has a higher human IL-8 binding affinity (KD) under neutral pH conditions than Hr9, whereas, It was found that the human IL-8 binding affinity (KD) under acidic pH conditions was weaker than that of Hr9. Although not particularly limited, in general, in order to use an antibody capable of binding to an antigen multiple times as a pharmaceutical product, a pH-dependent antigen-binding antibody can strongly neutralize the antigen under neutral pH conditions (e.g., in plasma). So, it is desirable to have a strong binding affinity (small KD). On the other hand, under acidic pH conditions (e.g., in the endosome), the antibody has a large dissociation rate constant (koff) and/or a weak binding affinity ( It is preferable that the KD is large). H89/L118 obtained preferable properties compared with Hr9 in any of these neutral pH and acidic pH conditions.

Therefore, useful amino acid modifications such as Y97H for the heavy chain of Hr9 and N50H/L54H/Q89K for the light chain were found. Although it is not particularly limited, it has been shown that an excellent pH-dependent IL-8-binding antibody can be produced as a pharmaceutical, even by introducing amino acid modifications selected from these, singly or in combination of a plurality of them.

Although not bound by a specific theory, it is thought that an important factor when using a pH-dependent antigen-binding antibody as a pharmaceutical product is whether or not an antibody administered in vivo can dissociate an antigen in an endosome. For this, it is considered important that the binding is sufficiently weak (dissociation constant; KD is large) or sufficiently fast dissociation rate (dissociation rate constant; koff is large) under acidic pH conditions. Therefore, whether the KD or koff of H89/L118 obtained by BIACORE was sufficient to dissociate the antigen in the in vivo endosome was verified by the following experiment.

Example 10

Construction of high affinity antibody for mouse PK assay

There is no particular limitation as a method of confirming the effect of the antibody on the rate of disappearance of human IL-8 in mice. In one example, the method includes administering an antibody to a mouse in a mixed state with human IL-8, and then comparing the rate of disappearance of human IL-8 from the mouse plasma.

Here, it is preferable that the reference antibody for use in the mouse PK assay has sufficiently strong binding affinity under the conditions of neutral pH and acidic pH. Therefore, as a result of searching for modifications that give high affinity to Hr9, H998/L63 having H998-IgG1 (SEQ ID NO: 89) as the heavy chain and L63-k0MT as the light chain was created.

Using H998/L63, the binding affinity of human IL-8 was evaluated in the same manner as in Example 9-2. Fig. 21 shows the resulting sensorgram.

Surprisingly, H998/L63 showed a slow dissociation rate under both conditions of neutral pH and acidic pH, and also showed that it has a stronger IL-8 binding affinity than Hr9. However, it is known that in the case of a protein-protein interaction having a slow dissociation rate, such as the dissociation rate constant (koff), the dissociation constant (KD), etc., cannot be accurately calculated from the limitations of BIACORE's device. Also in H998/L63, since an accurate analysis value could not be obtained, the analysis value was not shown here. However, from the results of this experiment, it was confirmed that H998/L63 has very strong binding affinity at both neutral and acidic pH, and is suitable as an antibody to be used as a comparison object in a mouse PK assay.

Example 11

Mouse PK Assay Using pH Dependent IL-8 Binding Antibody H89/L118

(11-1) Mouse PK assay using H89/L118

H89/L118 produced in Example 9 and H998/L63 produced in Example 10 were used to evaluate the rate of human IL-8 disappearance in vivo.

In vivo kinetics of human IL-8 after administration of human IL-8 and anti-human IL-8 antibodies to mice (C57BL/6J, Charles river) at the same time were evaluated. A mixed solution of human IL-8 and anti-human IL-8 antibody (10 μg/mL, 200 μg/mL, respectively) was administered to the tail vein at a single dose of 10 mL/kg. At this time, since the anti-human IL-8 antibody is present in a sufficient amount with respect to human IL-8, it is considered that almost all of the human IL-8 is bound to the antibody. Blood was collected 5 minutes after administration, 2 hours, 4 hours, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after 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 at -20°C or lower until measurement was performed.

(11-2) Measurement of human IL-8 concentration in plasma

Human IL-8 concentration in mouse plasma was measured by electrochemiluminescence. First, anti-human IL-8 antibody (in-house preparation) containing the normal region of mouse IgG was dispensed into a MULTI-ARRAY 96-well plate (Meso Scale Discovery), allowed to stand at room temperature for 1 hour, and then 5% BSA ( An anti-human IL-8 antibody immobilization plate was prepared by blocking at room temperature for 2 hours using a PBS-Tween solution containing w/v). A calibration curve sample containing 275, 91.7, 30.6, 10.2, 3.40, 1.13, or 0.377 ng/mL of human IL-8 as a plasma concentration, and a mouse plasma measurement sample diluted 25 times or more were prepared. The sample was mixed with hWS-4 and reacted overnight at 37°C. Thereafter, 50 µL of this mixture was dispensed into each well of the anti-human IL-8 antibody immobilization plate, and then stirred at room temperature for 1 hour. The final concentration of hWS-4 was prepared to be 25 μg/mL. Thereafter, Biotin Mouse Anti-Human Igκ Light Chain (BD Pharmingen) was reacted at room temperature for 1 hour, and SULFO-TAG Labeled Streptavidin (Meso Scale Discovery) was reacted at room temperature for 1 hour, and then Read Buffer T (×1 ) (Meso Scale Discovery) was dispensed, and immediately measured with SECTOR Imager 2400 (Meso Scale Discovery). Human IL-8 concentration was calculated using analysis software SOFT Max PRO (Molecular Devices) based on the response of the calibration curve.

As a result, the data on the concentration of human IL-8 in plasma are shown in Fig. 22, and the value of the human IL-8 clearance (CL) in mouse plasma is shown in Table 15.

As is clear from Fig. 22, it was shown that human IL-8 administered simultaneously with H89/L118 was surprisingly faster in disappearance from mouse plasma compared to human IL-8 administered simultaneously with H998/L63. In addition, the value of CL quantitatively indicating the rate of disappearance of human IL-8 from mouse plasma indicates that H89/L118 increased the rate of disappearance of human IL-8 by about 19 times compared to H998/L63. have.

Although not bound by a specific theory, it is also possible to consider the following from the data obtained this time. Human IL-8 administered at the same time as the antibody is mostly bound to the antibody in plasma and exists in a complex state. Human IL-8 bound to H998/L63 can exist in a state bound to the antibody due to the strong affinity of the antibody even in endosomes under acidic pH conditions. Thereafter, since H998/L63 can be returned to the plasma via FcRn in a state in which a complex with human IL-8 is formed, human IL-8 is also returned to the plasma at the same time. Therefore, most of the human IL-8 absorbed into the cells can be returned back to the plasma. That is, the rate of disappearance of human IL-8 from the plasma is significantly lowered by administration at the same time as H998/L63. On the other hand, as described above, human IL-8 absorbed into cells in a complex with H89/L118, which is a pH-dependent IL-8-binding antibody, can be dissociated from the antibody under acidic pH conditions in the endosome. . It is thought that human IL-8 dissociated from the antibody migrates to lysosomes and is degraded. Therefore, the pH-dependent IL-8-binding antibody significantly accelerated the loss of human IL-8 compared to the IL-8-binding antibody having strong binding affinity at both acidic and neutral pH, such as H998/L63. It is possible to do.

(11-3) Mouse PK assay with increased dose of H89/L118

Next, an experiment to verify the effect of changing the dosage of H89/L118 was conducted as follows. In vivo kinetics of human IL-8 after administration of human IL-8 and H89/L118 (2 mg/kg or 8 mg/kg) simultaneously to mice (C57BL/6J, Charles river) were evaluated. A mixed solution of human IL-8 (2.5 μg/mL) and anti-human IL-8 antibody (200 μg/mL or 800 μg/mL) was administered once to the tail vein at 10 mL/kg. At this time, since the anti-human IL-8 antibody is present in a sufficient amount with respect to human IL-8, it is considered that almost all of the human IL-8 is bound to the antibody. Blood was collected 5 minutes after administration, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after 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 at -20°C or lower until measurement was performed.

The measurement of the human IL-8 concentration in mouse plasma was performed in the same manner as in Example 11-2. As a result, the data on the concentration of human IL-8 in plasma are shown in Fig. 23, and the value of the human IL-8 clearance (CL) in mouse plasma is shown in Table 16.

As a result, it was confirmed that, compared with the group administered with H89/L118 at 2 mg/kg, in the group administered with 8 mg/kg of the antibody, the rate of disappearance of human IL-8 was approximately 2 times slower.

Hereinafter, on the basis of the scientific background, the inventors will describe the speculation of one of the factors that may bring about the above results, but the contents of the disclosure C are not limited to the contents of the following consideration.

Among the antibodies returned from the endosome to the plasma via FcRn, human IL-8 is bound, but a lower ratio is preferable. When paying attention to human IL-8 present in the endosome, it is preferable that the ratio of free form that does not bind to the antibody is high. When human IL-8 is administered together with an antibody that does not have pH-dependent IL-8 affinity, most (close to 100%) human IL-8 is present in a complex with the antibody in the endosome. I think that it is, and it is thought that the glass type is small (close to 0%). On the other hand, when administered together with a pH-dependent IL-8-binding antibody (for example, H89/L118), a certain proportion of human IL-8 will exist as a free form in the endosome. The ratio of the free form at this time is virtual, but it is also possible to understand as follows: [The ratio of free human IL-8 in the endosome (%)] = [Free human IL- in the endosome. 8 concentration] ÷ [total human IL-8 concentration in the endosome] × 100

The ratio of free human IL-8 in the endosome, understood as the above formula, is preferably higher, for example, 20% is preferable than 0%, 40% is preferable than 20%, and 40%. More than 60% is preferable, 80% is more preferable than 60%, and 100% is more preferable than 80%.

Therefore, there is a correlation between the ratio of free human IL-8 in the endosome and the binding affinity (KD) and/or dissociation rate constant (koff) to human IL-8 at an acidic pH. . That is, the weaker the binding affinity to human IL-8 at acidic pH and/or the higher the dissociation rate, the higher the proportion of free human IL-8 in the endosome. However, in the case of a pH-dependent IL-8-binding antibody that can bring the proportion of free human IL-8 in the endosome to close to 100%, the binding affinity is further weakened, and/or at an acidic pH. Increasing the rate of dissociation does not necessarily lead to an effective increase in the proportion of free human IL-8. For example, even if the proportion of free human IL-8 was improved from 99.9% to 99.99%, it is easily understood that the degree of improvement would not be significant.

In addition, according to the theory of general chemical equilibrium, when the anti-IL-8 antibody and human IL-8 coexist and their binding reaction and dissociation reaction reach an equilibrium state, the ratio of free human IL-8 is, It is uniquely determined by the three characters of 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, the complex is easily formed, and the ratio 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, it becomes difficult to form a complex, and the ratio of free human IL-8 increases.

Here, in this test, the rate of disappearance of human IL-8 when 8 mg/kg of H89/L118 was administered was slower than that of the case where 2 mg/kg of antibody was administered. In other words, this suggests that the ratio of free human IL-8 in endosomes administered with 8 mg/kg of the antibody was lowered compared to the case where 2 mg/kg of the antibody was administered. It is presumed that the reason for this decrease is that the antibody concentration in the endosome increased by increasing the dose of the antibody by 4 times, and the formation of a complex between IL-8 and the antibody in the endosome became easy. That is, in the administration group in which the dose of the antibody was increased, the rate of human IL-8 disappearance decreased because the proportion of free human IL-8 in the endosomes decreased. This also suggests that the size of the dissociation constant (KD) under the acidic pH condition of H89/L118 is insufficient to bring the free human IL-8 close to 100% at the time of administration of 8 mg/kg of antibody. . That is, under acidic pH conditions, if an antibody having a larger dissociation constant (KD) (weaker binding), even when 8 mg/kg of antibody is administered, free IL-8 is close to 100%, And, it is considered that human IL-8 disappearance rate equivalent to that of administration of 2 mg/kg of antibody is achieved.

Based on the above, in order to confirm whether the desired pH-dependent IL-8-binding antibody can achieve the ratio of free human IL-8 in the endosome to close to 100%, it is not particularly limited, but It is possible to verify whether there is room for an increase in the degree of the antigen-disappearing effect in the beam. For example, in certain methods, humans when using a new pH-dependent IL-8-binding antibody that has weakened the binding affinity at acidic pH than H89/L118 and/or accelerated the dissociation rate at acidic pH. The IL-8 disappearance rate was compared with the IL-8 disappearance rate when H89/L118 was used. When the above new pH-dependent IL-8-binding antibody exhibits a human IL-8 disappearance rate equivalent to H89/L118, the binding affinity and/or dissociation rate of H89/L118 at an acidic pH is within the endosome. In order to bring the ratio of free human IL-8 closer to 100%, it can be said that it is already at a sufficient level. On the other hand, when the above new pH-dependent IL-8-binding antibody exhibits a higher human IL-8 disappearance rate, the binding affinity and/or dissociation rate of H89/L118 at an acidic pH has room for improvement. It can be said that it is suggesting that there is.

Example 12

Preparation and evaluation of pH-dependent IL-8 binding antibody H553/L118

(12-1) Preparation of antibody H553/L118 having pH-dependent IL-8 binding ability

Therefore, the present inventors have attempted to produce an antibody that weakens the affinity for human IL-8 binding and/or accelerates the dissociation rate under acidic pH conditions more than H89/L118.

Based on H89/L118, amino acid modifications centered on histidine were introduced, and modified antibodies shown in Table 17 were produced in the same manner as in Example 9. Further, in the same manner as in Example 9-2, the human IL-8 binding affinity of these antibodies was measured.

Table 17 shows some of the results. Antibody H553/L118 comprising H553-IgG1 (SEQ ID NO: 90) as heavy chain and L118-k0MT as light chain, and antibody H496/L118 comprising H496-IgG1 (SEQ ID NO: 101) as heavy chain and L118-k0MT as light chain It was found that the pH dependence was further increased than that of H89/L118.

In the obtained H553/L118, two kinds of amino acid modifications, Y55H and R57P, have been introduced with respect to the heavy chain of H89/L118. On the other hand, H496/L118, in which only R57P is introduced into the heavy chain of H89/L118, has enhanced human IL-8 binding affinity at neutral pH compared to H89/L118, but human IL-8 at acidic pH The binding affinity is hardly changing. That is, the R57P modification introduced into H89/L118 does not change the human IL-8 binding affinity at acidic pH, but is a modification that enhances only the binding affinity at neutral pH. Moreover, H553/L118, into which the Y55H modification was introduced with respect to the heavy chain of H496/L118, maintains or slightly enhances the binding affinity at neutral pH, compared to H89/L118, while the binding affinity at acidic pH. The castle was degraded. That is, the combination of two kinds of amino acid modifications, Y55H and R57P, and introduction into H89/L118 retains or slightly enhances the binding affinity at neutral pH, while reducing the binding affinity at acidic pH. It made it possible to further enhance it.

(12-2) Mouse PK assay using H553/L118

Using H553/L118, evaluation of the rate of human IL-8 disappearance in mice was performed in the same manner as in Example 11-2. As a result, the data on the concentration of human IL-8 in plasma are shown in Fig. 24, and the value of the human IL-8 clearance (CL) in mouse plasma is shown in Table 18.

As a result, in the comparison of data of mice administered with 2 mg/kg of antibody, there was no significant difference between H553/L118 and H89/L118, but in comparison of data of mice administered with 8 mg/kg of antibody, It was confirmed that H553/L118 accelerated the loss of human IL-8 by 2.5 times compared to H89/L118. In another aspect, H553/L118 did not show a difference in the rate of human IL-8 disappearance between 2 mg/kg and 8 mg/kg, and a decrease in the rate of antigen disappearance due to an increase in the dose of an antibody such as H89/L118 was not observed. Did.

Although it does not specifically limit, as one of the reasons for such a result obtained, it is also possible to consider as follows. H553/L118 showed equivalent human IL-8 disappearance rate when 2 mg/kg of antibody was administered and 8 mg/kg of antibody was administered. This indicates that H553/L118 is sufficiently weak in binding to IL-8 at acidic pH, so even under the conditions of 8 mg/kg administration, the ratio of free IL-8 in the endosome can reach a level close to 100%. Can indicate that. This means that H89/L118 is capable of achieving the maximum human IL-8 loss effect at a dose of 2 mg/kg, but suggests that the effect may be attenuated at a high dose of about 8 mg/kg. I'm doing it. On the other hand, H553/L118 is also capable of achieving a maximum human IL-8 elimination effect even at a high dose of 8 mg/kg.

(12-3) Stability evaluation using H553/L118

It was shown that H553/L118 is an antibody capable of remarkably faster loss of human IL-8 than H89/L118 in mice. On the other hand, in order for this antibody to sustain the inhibitory effect of human IL-8 in vivo for a long period of time, the IL-8 neutralizing activity is stable during the period in which the administered antibody is present in vivo (for example, in plasma). It is also important to be maintained (stability in the IL-8 neutralizing activity of the antibody in question). Therefore, the stability of these antibodies in mouse plasma was evaluated by the method shown below.

Mouse plasma was collected from blood of C57BL/6J (Charles river) by a method known in the art. 200 μL of 200 mM PBS (Sigma, P4417) was added to 800 μL of mouse plasma to obtain 1 mL. In addition, 0.1% final concentration of sodium azide was added as a preservative. Further, each antibody (Hr9, H89/L118, and H553/L118) was added to the above mouse plasma so that the final concentration was 0.2 mg/mL. At this point, a part of the sample was collected and used as an initial sample. The remaining samples were stored at 40°C. When 1 week and 2 weeks elapsed from the start of storage, a part of each of the samples was collected, and a sample stored for 1 week and a sample stored for 2 weeks were used. Meanwhile, all samples were stored frozen at -80°C until each analysis.

Next, evaluation of the neutralizing activity of the anti-IL-8 antibody contained in mouse plasma against human IL-8 was performed as follows: CXCR1 and CXCR2 are known as receptors for human IL-8. PathHunter (registered trademark) CHO-K1 CXCR2 β-Arrestin Cell Line (DiscoveRx, Cat.# 93-0202C2) expresses human CXCR2 and shows chemiluminescence when a signal by human IL-8 is transmitted, It is an artificially produced cell line. Although not particularly limited, using these cells, it is possible to evaluate the neutralizing activity of the anti-human IL-8 antibody against human IL-8. When human IL-8 is added to the culture medium of the cell, 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 anti-human IL-8 antibodies are added to the culture solution together, the anti-human IL-8 antibody binds to human IL-8, thereby blocking signal transduction of human IL-8. As a result, chemiluminescence caused by the addition of human IL-8 is inhibited by the anti-human IL-8 antibody, resulting in weak chemiluminescence or no chemiluminescence compared to the case where no antibody is added. . Therefore, the stronger the human IL-8 neutralizing activity of the antibody, the weaker the degree of chemiluminescence is, and the weaker the human IL-8 neutralizing activity of the antibody, the stronger the degree of chemiluminescence.

This also applies to antibodies added to mouse plasma and stored for a certain period of time. If an antibody does not change its neutralizing activity by preserving it in mouse plasma, the degree of chemiluminescence will not change before and after preservation. On the other hand, in the case of an antibody whose neutralizing activity is lowered by storage in mouse plasma, the degree of chemiluminescence when the antibody after storage is used increases compared to before storage.

So, using the above cell line, it was verified whether or not the antibody preserved in mouse plasma retained its neutralizing activity. First, the cell line was suspended in AssayComplete(tm) Cell Plating 0 Reagent and seeded at a rate of 5000 cells/well in a 384 well plate. One day after the start of cell culture, a test for determining the concentration of human IL-8 to be added was performed as follows. The human IL-8 solution was gradually diluted so as to contain from 45 nM (400 ng/mL) to 0.098 nM (0.1 ng/mL) as the final human IL-8 concentration was added to the cell culture solution. Next, a detection reagent was added according to the product protocol, and the relative chemiluminescence amount was detected using a chemiluminescence detection device. As a result, in order to confirm the reactivity of cells to human IL-8, and to confirm the neutralizing activity of the anti-human IL-8 antibody, an appropriate human IL-8 concentration was determined. Here, the human IL-8 concentration was set to 2 nM.

Next, using mouse plasma to which the above-described anti-human IL-8 antibody was added, the neutralizing activity of the antibody contained therein was evaluated. Mouse plasma containing human IL-8 at the concentration determined above and the anti-human IL-8 antibody described above was added to the cell culture. Here, the amount of mouse plasma to be added was determined to be gradually included in the range of 2 μg/mL (13.3 nM) to 0.016 μg/mL (0.1 nM) as the concentration of the anti-human IL-8 antibody. Next, a detection reagent was added according to the product protocol, and the relative chemiluminescence amount was detected using a chemiluminescence detection device.

Here, when the average of the relative chemiluminescence amount of the wells to which human IL-8 and antibody are not added is 0%, and the average of the relative chemiluminescence amount of the wells to which only human IL-8 is not added is 100% Of, the relative value of the relative chemiluminescence amount at each antibody concentration was calculated.

The results of the human IL-8 inhibition assay using human CXCR2 expressing cells are shown in Fig. 25A, and the results of the initial sample (no preservation treatment in mouse plasma) are shown in Fig. 25A, and the results of the sample stored at 40°C for 1 week are shown in Fig. 25B, and 40 The results of the samples stored for 2 weeks at °C are shown in Fig. 25C, respectively.

As a result, there was no difference in human IL-8 neutralizing activity between Hr9 and H89/L118 before and after storage in mouse plasma. On the other hand, H553/L118 showed a decrease in human IL-8 neutralizing activity upon storage for 2 weeks. From this, it was found that H553/L118 is an antibody that has an unstable property in terms of IL-8 neutralizing activity, and is liable to decrease in human IL-8 neutralizing activity in mouse plasma, compared to Hr9 or H89/L118.

Example 13

Preparation of antibody with reduced immunogenicity prediction score using in silico system

(13-1) Immunogenicity prediction score of various IL-8-binding antibodies

The production of anti-drug antibodies (ADA) affects the effectiveness and pharmacokinetics of therapeutic antibodies, and sometimes leads to serious side effects. Therefore, the usefulness and efficacy of therapeutic antibodies in clinical practice is the production of ADA. May be limited by It is known that the immunogenicity of therapeutic antibodies is influenced by many factors, but there are a number of reports explaining the importance of effector T cell epitopes in therapeutic antibodies.

As in silico tools for predicting T cell epitopes, Epibase (Lonza), iTope/TCED (Antitope), and EpiMatrix (EpiVax) have been developed. Using these in silico tools, T cell epitopes in each amino acid sequence can be predicted (Walle et al., Expert Opin. Biol. Ther. 7(3):405-418(2007)), and of therapeutic antibodies. Potential immunogenicity assessment becomes possible.

Here, the immunogenicity score of each anti-IL-8 antibody was calculated using EpiMatrix. EpiMatrix mechanically designs the sequence of a peptide fragment in which the amino acid sequence of a protein for which immunogenicity is to be predicted is divided every 9 amino acids, and for them, eight kinds of major MHC Class II alleles (DRB1 ^* 0101, DRB1 ^* 0301, DRB1 ^* 0401, DRB1 ^* 0701, DRB1 ^* 0801, DRB1 ^* 1101, DRB1 ^* 1301, and DRB1 ^* 1501) by calculating the binding capacity to predict the immunogenicity of the target protein. (De Groot et al., Clin. Immunol. 131(2):189-201(2009))

The immunogenicity scores of the heavy and light chains of each anti-IL-8 antibody calculated as described above are shown in the column of "EpiMatrix score" in Table 19. Moreover, for the EpiMatrix score, the immunogenicity score corrected in consideration of the inclusion of Tregitope is shown in the column of "tReg Adjusted Epx score". Tregitope is a peptide fragment sequence mainly present in a large amount of natural antibody sequences, and is a sequence that is thought to suppress immunogenicity by activating a regulatory T cell (Treg).

In addition, for these scores, the sum of the scores of the heavy and light chains is shown in the "Total" column.

From this result, even if seeing either "EpiMatrix score" and "tReg Adjusted Epx score", the immunogenicity scores of H89/L118, H496/L118, and H553/L118 are hWS- It was found that it was lowered compared to 4.

Moreover, in EpiMatrix, in addition to taking the scores of the heavy and light chains into account, it is also possible to compare the predicted ADA incidence frequency as a whole with the actual ADA incidence frequency of various commercially available antibodies. The results of such analysis are shown in FIG. 26. On the other hand, in the relationship of the system, in Fig. 26, hWS-4 is "WS4", Hr9 is "HR9", H89/L118 is "H89L118", H496/L118 is "H496L118", and H553/L118 is "H553L118". Each is indicated.

As shown in Fig. 26, it is known that the incidence of ADA in humans of various commercial antibodies is 45% in Campath (Alemtuzumab), 27% in Rituxan (Rituximab), and 14% in Zenapax (Daclizumab). . On the other hand, the incidence of ADA predicted from the amino acid sequence of hWS-4, a known humanized anti-human IL-8 antibody, was 10.42%, but newly discovered H89/L118 (5.52%) and H496/L118 (4.67%) in this specification. ), or H553/L118 (3.45%) was significantly lowered compared to hWS-4.

(13-2) Preparation of modified antibody with reduced immunogenicity prediction score

As described above, the immunogenicity scores of H89/L118, H496/L118, and H553/L118 were lower than that of hWS-4, but as evident from Table 19, the immunogenicity score of the heavy chain was higher than that of the light chain. , In particular, the amino acid sequence of the heavy chain suggests that there is still room for improvement in terms of immunogenicity. Therefore, from the heavy chain variable region of H496, an amino acid alteration capable of lowering the immunogenicity score was searched. As a result of a thorough search, three variants of H496v1 in which alanine at position 52c was substituted with aspartic acid according to Kabat numbering, 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. Became. In addition, H1004 containing all of these three types of modifications was produced.

Table 20 shows the results of calculating the immunogenicity score in the same manner as in Example 13-1.

The three types of heavy chains H496v1, H496v2, and H496v3 including single modifications all showed a decrease in immunogenicity score compared to H496. Moreover, in H1004 containing a combination of three types of modifications, a remarkable improvement in immunogenicity score was achieved.

Here, as a light chain suitable for combination with H1004, in addition to L118, L395 was found. Therefore, in the calculation of the immunogenicity score, what was combined with both L118 and L395 was calculated. As shown in Table 20, H1004/L118 and H1004/L395, which were a combination of heavy and light chains, also showed very low immunogenicity scores.

Next, for these combinations, the frequency of occurrence of ADA was predicted in the same manner as in Example 13-1. The results are shown in Fig. 27. On the other hand, in Fig. 27, H496v1/L118 is "V1", H496v2/L118 is "V2", H496v3/L118 is "V3", H1004/L118 is "H1004L118", and H1004/L395 is "H1004L395", respectively. have.

Surprisingly, H1004/L118 and H1004/L395, which significantly reduced the immunogenicity score, also improved the predicted value of the frequency of ADA occurrence, and showed a predicted value of 0%.

(13-3) Measurement of IL-8 binding affinity of H1004/L395

An antibody H1004/L395 containing H1004-IgG1m (SEQ ID NO: 91) as a heavy chain and L395-k0MT (SEQ ID NO: 82) as a light chain was prepared. The binding affinity of H1004/L395 to human IL-8 was measured as follows using BIACORE T200 (GE Healthcare).

The running buffer was measured at each temperature using the following two types: (1) 0.05% tween20, 40mM ACES, 150mM NaCl, pH 7.4, 40°C; And (2) 0.05% tween20, 40mM ACES, 150mM NaCl, pH 5.8, 37°C.

An appropriate amount of protein A/G (PIERCE) was fixed on a sensor chip CM4 (GE Healthcare) by an amine coupling method, and the desired antibody was captured. Next, human IL-8 diluted solution and running buffer (as a reference solution) were injected, and human IL-8 was interacted with the antibody captured on the sensor chip. On the other hand, any one of the above was used as the running buffer, and each buffer was used for dilution of human IL-8. For regeneration of the sensor chip, 25 mM NaOH and 10 mM glycine-HCl, pH 1.5 were used. The KD (M) for human IL-8 of each antibody is based on the binding rate constant kon (1/Ms), which is the kinetic parameter calculated from the sensorgram obtained by the measurement, and the dissociation rate constant koff (1/s). Was calculated. BIACORE T200 Evaluation Software (GE Healthcare) was used to calculate each parameter.

Table 21 shows the measurement results. H1004/L395 with reduced immunogenicity score, compared with H89/L118, had the same KD to human IL-8 at neutral pH, but increased KD and koff at acidic pH, and in endosomes It has been shown to have a property that is easy to dissociate IL-8.

[Table 21-1]

Example 14

Preparation and evaluation of pH-dependent IL-8 binding antibody H1009/L395

(14-1) Preparation of various pH-dependent IL-8 binding antibodies

By the examination shown in Example 13, H1004/L395 having a pH-dependent IL-8 binding ability and a reduced immunogenicity score was obtained. Next, with the aim of creating a variant in which these desirable properties and stability in mouse plasma are compatible, a thorough study was conducted.

Based on H1004/L395, various modifications were introduced to prepare the following modified antibodies.

[Table 21-2]

[Table 21-3]

By combining the above 18 types of heavy chains with 2 types of light chains, a total of 36 types of antibodies were produced. About these antibodies, various evaluations were performed as shown below.

Human IL-8 binding affinity under neutral and acidic pH conditions was measured in the same manner as in the method of Example 13-3. Among the results, Table 22 shows KD at pH 7.4, KD at pH 5.8, and koff.

Next, stability evaluation in binding of IL-8 when stored in PBS by the method shown below was performed.

After dialysis of each antibody against DPBS (Sigma-Aldrich) overnight, the concentration of each antibody was prepared to be 0.1 mg/mL. At this point, a part of the antibody sample was collected and used as an initial sample. The remaining samples were stored at 50° C. for 1 week, and then collected and used as samples for thermal acceleration tests.

Next, the measurement of IL-8 binding affinity by BIACORE was performed as follows using the initial sample and the sample for thermal acceleration test.

BIACORE T200 (GE Healthcare) was used to analyze the binding amount of human IL-8 to the modified antibody. Measurement was performed at 40°C using 0.05% tween20, 40mM ACES, 150mM NaCl, pH 7.4 as a running buffer.

An appropriate amount of protein A/G (PIERCE) was fixed on a sensor chip CM4 (GE Healthcare) by an amine coupling method, and the desired antibody was captured. Next, human IL-8 diluted solution and running buffer (as a reference solution) were injected, and human IL-8 was interacted with the antibody captured on the sensor chip. Running buffer was also used for dilution of human IL-8. For regeneration of the sensor chip, 25 mM NaOH and 10 mM glycine-HCl, pH 1.5 were used. The amount of binding of human IL-8 obtained by the measurement and the amount of antibody capture when the amount of binding was obtained were extracted using BIACORE T200 Evaluation Software (GE Healthcare).

For the initial sample and the thermal acceleration test sample, the binding amount of human IL-8 per 1000 RU of the antibody capture amount was calculated. In addition, the ratio of the binding amount of human IL-8 in the sample for thermal acceleration test to the binding amount of human IL-8 in the initial sample was calculated.

The ratio of the binding amount of IL-8 between the initial sample and the sample for thermal acceleration test obtained as a result is combined with Table 22 and shown.

By the above examination, H1009/L395, an antibody containing H1009-IgG1m (SEQ ID NO: 92) as a heavy chain and L395-k0MT as a light chain, was obtained.

As shown in Table 22, in H1009/L395, human IL-8 binding affinity at neutral pH slightly increased compared to H89/L118, while the binding affinity at acidic pH decreased. In other words, the pH dependence became stronger. In addition, the stability in IL-8 binding when exposed to harsh conditions such as 50° C. in PBS was slightly increased in H1009/L395 than in H89/L118.

From these, H1009/L395 was selected as an antibody capable of stably maintaining neutralizing activity in mouse plasma while having a pH-dependent IL-8 binding ability.

(14-2) Stability evaluation of H1009/L395

Next, similarly to the method of Example 12-3, it was evaluated whether or not the IL-8 neutralizing activity of H1009/L395 was stably maintained in mouse plasma. Here, in Example 19 later, H1009/L395-F1886s described in detail was used. This antibody has the same variable region as H1009/L395, and the constant region has an alteration to enhance FcRn binding under acidic pH conditions and an alteration to reduce binding to FcγR compared to native human IgG1. . The binding of H1009/L395 to human IL-8 and the neutralizing activity of IL-8 are responsible for the variable region of this antibody, particularly the region centered on HVR, and the alteration introduced into the normal region does not affect the binding. I think.

Stability evaluation in mouse plasma was performed as follows. To 585 µL of mouse plasma, 150 µL of 200 mM phosphate buffer (pH 6.7) was added. Next, sodium azide was added as a preservative at a final concentration of 0.1%. Each antibody (Hr9, H89/L118, or H1009/L395-F1886s) was added to the above mouse plasma so that the final concentration was 0.4 mg/mL. At this point, a part of the sample was collected and used as an initial sample. The remaining samples were stored at 40°C. One week and two weeks after the start of storage, a part of each sample was taken, and a one-week storage sample and a two-week storage sample were obtained. Meanwhile, all samples were stored frozen at -80°C until each analysis.

Measurement of human IL-8 neutralizing activity using human CXCR2 expressing cells was performed in the same manner as in Example 12-3. However, the human IL-8 concentration used to confirm the neutralizing activity of the anti-human IL-8 antibody was performed at 1.2 nM this time.

The results of the human IL-8 inhibition assay obtained using the above antibodies and human CXCR2 expressing cells were obtained by preserving the results of the initial sample (no preservation treatment in mouse plasma) in FIG. 28A for 1 week at 40°C in FIG. 28B. The results of the samples are shown in Fig. 28C, respectively, of the samples stored at 40°C for 2 weeks.

As a result, surprisingly, even when H1009/L395-F1886s was stored in mouse plasma at 40° C. for 2 weeks, the neutralizing activity of human IL-8 was maintained, and IL-8 neutralizing activity was more stable than that of H553/L118. Was being maintained.

(14-3) Mouse PK assay using H1009/L395

The rate of human IL-8 disappearance in H1009/H395 mice was evaluated by the method shown below. H1009/L395, H553/L118, and H998/L63 were used as antibodies. Administration to mice, blood collection, and measurement of human IL-8 concentration in mouse plasma were performed by the method shown in Example 11.

As a result, the data on the concentration of human IL-8 in plasma are shown in Fig. 29, and the value of the human IL-8 clearance (CL) in mouse plasma is shown in Table 23.

As a result, the rate of human IL-8 disappearance in mice when H1009/L395 was administered at 2 mg/kg was about the same as that of H553/L118, and H1009/L395 contained free IL-8 in the endosome. It turns out that it is achieving close to 100%. In addition, it was found that the value of clearance (CL) quantitatively indicating the rate of human IL-8 disappearance from mouse plasma was approximately 30 times higher than that of H998/L63.

Although not particularly limited, the effect of increasing the rate of disappearance of human IL-8 can be interpreted as follows. In general, in a living body where the antigen concentration is maintained substantially constant, the rate of production and disappearance of the antigen are also maintained substantially constant. When an antibody is administered in this state, even when the rate of antigen production is not affected, the rate of antigen disappearance can be changed by the antigen forming a complex with the antibody. In general, since the rate of disappearance of the antigen is greater than that of the antibody, in such a case, the rate of disappearance of the antigen formed in the complex with the antibody decreases. When the rate of disappearance of the antigen decreases, the concentration of the antigen in the plasma increases, but the degree of increase at that time can also be defined by the ratio of the rate of disappearance when the antigen is alone and the rate of disappearance when the antigen is formed into a complex. That is, when the rate of disappearance at the time of formation of the complex is lowered to 1/10 compared to the rate of disappearance at the time of antigen alone, the concentration of the antigen in the plasma of the living body to which the antibody has been administered can rise to about 10 times that of before administration of the antibody. Here, it is also possible to use the clearance CL as these vanishing rates. That is, it can be considered that the increase in antigen concentration (accumulation of antigen) occurring after administration of the antibody to a living body is defined by the antigen CL in each state before and after administration of the antibody.

Here, the fact that there was a difference of about 30-fold in the CL of human IL-8 when H998/L63 and H1009/L395 were administered is the concentration of human IL-8 in plasma that occurs when these antibodies are administered to humans. It suggests that there can be a difference of about 30 times in the degree of ascent. Moreover, the fact that there is a 30-fold difference in the concentration of human IL-8 in plasma means that the amount of antibody required to completely block the biological activity of human IL-8 in each situation is also about 30-fold. It becomes that it can be said. In other words, H1009/L395 is an extremely small amount of antibody, about one half of that of H998/L63, and it is possible to block the biological activity of IL-8 in plasma. In addition, when each of H1009/L395 and H998/L63 is administered to humans at the same dose, H1009/L395 is stronger, and on the other hand, it becomes possible to block the biological activity of IL-8 over a longer period of time. In addition, in order to block the biological activity of IL-8 over a long period of time, it is necessary to stably maintain the IL-8 neutralizing activity. As shown in Example 14, it is clear from experiments using mouse plasma that H1009/L395 can maintain its human IL-8 neutralizing activity over a long period of time. It has also been shown that H1009/L395, which has these special properties, is an antibody having an excellent effect from the viewpoint of an effect of neutralizing IL-8 in vivo.

Example 15

Evaluation of binding to extracellular matrix using pH-dependent IL-8 binding antibody H1009/L395

The human IL-8 disappearance effect, which was 30 times superior to that of H1009/L395, shown in Example 14, was a remarkable effect. It is known that the rate of antigen disappearance upon administration of a pH-dependent antigen-binding antibody depends on the rate at which the antibody-antigen complex is absorbed into cells. That is, when the antigen-antibody complex is formed, when the rate at which the pH-dependent antigen-binding antibody is absorbed into the cell increases as compared to when the complex is not formed, the effect of the pH-dependent antigen disappearance may increase. Methods of enhancing the rate at which antibodies are absorbed into cells include methods of imparting FcRn-binding ability to antibodies under neutral pH conditions (WO2011/122011), methods of enhancing the binding ability of antibodies to FcγR (WO2013/047752), and multivalent A method using the promotion of the formation of a complex containing an antibody and a multivalent antigen (WO 2013/081143) is included.

However, in the normal region of H1009/L395, the technique was not used. In addition, it is known that IL-8 forms a homodimer, but since H1009/L395 recognizes the formation surface of a homodimer of human IL-8, it is clear that human IL-8 bound to H1009/L395 becomes a monomeric state. Is becoming. Therefore, this antibody does not form a multivalent complex.

That is, for H1009/L395, the above technique was not used, but H1009/L395 showed a 30-fold effect of human IL-8 disappearance.

Therefore, the inventors made the following considerations as one of the factors that may bring about the above characteristics of the pH-dependent IL-8-binding antibody typified by H1009/L395. However, the following is only one of the possibilities that the inventors have speculated based on the technical background, and the contents of the disclosure C are not limited to the following considerations.

Human IL-8 is a protein having a high isoelectric point (pI), and the theoretical isoelectric point calculated by a known method is approximately 10. That is, under conditions of neutral pH, human IL-8 is a protein having a charge biased toward the positive charge side. The pH-dependent IL-8-binding antibody represented by H1009/L395 is also a protein having a charge biased toward the positively charged side, and the theoretical isoelectric point of H1009/L395 is approximately 9. That is, the complex formed by binding of H1009/L395, which is a protein having a high isoelectric point and rich in positive charge, with human IL-8 having a high isoelectric point, has an isoelectric point higher than that of H1009/L395 alone.

As shown in Example 3, the increase in the isoelectric point of the antibody, including increasing the number of positive charges of the antibody, and/or reducing the number of negative charges, results in non-specific uptake of the antibody-antigen complex into cells. It is also possible to think of it as an increase. The isoelectric point of the complex formed between the anti-IL-8 antibody and human IL-8 having a high isoelectric point is higher than the isoelectric point of the anti-IL-8 antibody alone, and this complex can be more easily absorbed into the cell.

As described above, affinity for the extracellular matrix is also one of the factors that may affect intracellular uptake. Therefore, it was verified whether the binding properties of the antibody alone to the extracellular matrix and the complex of human IL-8 and the antibody were different.

Evaluation of the amount of antibody binding to the extracellular matrix by the ECL (electrochemiluminescence) method

Using TBS (Takara, T903), the extracellular matrix (BD Matrigel basement membrane matrix/manufactured by BD Co.) was diluted to 2 mg/mL. The diluted extracellular matrix was dispensed into MULTI-ARRAY 96well Plate, High bind, Bare (Meso Scale Discovery: manufactured by MSD), and 5 µL per well were dispensed, and solidified overnight at 4°C. Thereafter, blocking was performed using a 20 mM ACES buffer containing 150 mM NaCl, 0.05% Tween20, 0.5% BSA, and 0.01% NaN _{3, pH 7.4.}

In addition, the antibody used for evaluation was adjusted as follows. For antibody samples added alone, each antibody was diluted to 9 μg/mL using Buffer 1 (20 mM ACES buffer containing 150 mM NaCl, 0.05% Tween20, and 0.01% NaN ₃ , pH 7.4), and then final concentration. It was prepared by further diluting to ₃ μg/mL with Buffer 2 (150 mM NaCl, 0.05% Tween20, 0.1% BSA, and 20 mM ACES buffer containing 0.01% NaN 3, pH 7.4).

On the other hand, in the antibody sample added as a complex with human IL-8, human IL-8 at a molar concentration of 10 times that of the antibody was added to the antibody sample, and then the antibody concentration was 9 μg/mL using Buffer-1. After each dilution, it was further diluted with Buffer-2 so that the final antibody concentration was 3 μg/mL, respectively. On the other hand, at this time, the human IL-8 concentration was about 0.6 μg/mL. In order to form a complex, it was shaken for 1 hour at room temperature.

Next, to the plate from which the blocking solution was removed, the antibody alone or the antibody solution as a complex was added, followed by shaking at room temperature for 1 hour. Thereafter, the antibody alone or the solution of the complex was removed, Buffer-1 containing 0.25% glutaraldehyde was added, allowed to stand for 10 minutes, and washed with DPBS (manufactured by Wako Pure Chemical Industries, Ltd.) containing 0.05% Tween20. . The ECL detection antibody was prepared by converting Goat anti-human IgG (gamma) (manufactured by Zymed Laboratories) into Sulfo-Tag using Sulfo-Tag NHS Ester (manufactured by MSD). The antibody for ECL detection was diluted with Buffer-2 so as to be 1 μg/mL, added to the plate, and shaken for 1 hour at room temperature under light shielding. The antibody for ECL detection was removed, and a solution obtained by diluting MSD Read Buffer T (4x) (manufactured by MSD) twice with ultrapure water was added, and then the amount of light emission was measured using SECTOR Imager 2400 (manufactured by MSD).

The results are shown in Fig. 30. Interestingly, all of the anti-IL-8 antibodies such as H1009/L395 showed almost no binding to the extracellular matrix with the antibody alone (-IL8), but formed a complex with human IL-8 (+hIL8), Bound to the extracellular matrix.

The property that the anti-IL-8 antibody obtains affinity for the extracellular matrix by binding to human IL-8 as described above is unknown. In addition, although not limited, it is also possible to increase the rate of disappearance of IL-8 more efficiently by combining such a property with a pH-dependent IL-8-binding antibody.

Example 16

Mouse PK assay using FcRn non-binding antibody

In mice, whether or not a pH-dependent IL-8-binding antibody forms a complex with human IL-8 and increases the absorption of the complex into cells was confirmed using the method shown below.

First, an antibody variant having a variable region of H1009/L395 and an Fc region that lacks binding affinity to various Fc receptors was prepared. Specifically, substitution of isoleucine at position 253 with alanine and serine at position 254 with aspartic acid for H1009-IgG1, which is a heavy chain, as a modification to delete the binding ability to human FcRn under acidic pH conditions. Provided on. In addition, as a modification to delete the binding to mouse FcγR, leucine at position 235 was substituted with arginine, glycine at position 236 with arginine, and serine at position 239 with lysine. H1009-F1942m (SEQ ID NO: 93) was prepared as a heavy chain containing these four modifications. Further, H1009/L395-F1942m containing H1009-F1942m as the heavy chain and L395-k0MT as the light chain was produced.

Since the antibody having this Fc region lacks the FcRn binding affinity under acidic pH conditions, the transition from the endosome to the plasma does not occur. Therefore, such an antibody is quickly lost from plasma in vivo compared to an antibody containing a native Fc region. At this time, after the antibody containing the native Fc region is absorbed into the cell, only a portion of the antibodies that did not receive salvage by FcRn migrate to the lysosome and decompose, but the Fc does not contain binding affinity for FcRn. In the case of an antibody containing a region, all of the antibody absorbed into the cell is degraded into lysosomes. That is, in the case of an antibody containing such a modified Fc region, it may be considered that the rate of disappearance of the administered antibody from the plasma is the same as the rate of absorption into cells. That is, also by measuring the rate of disappearance from the plasma of an antibody having a deficient binding affinity for FcRn, it is possible to confirm the rate at which the antibody is absorbed into cells.

Therefore, it was verified whether or not the uptake of the complex formed by H1009/L395-F1942m and human IL-8 into cells increased compared to the introduction of H1009/L395-F1942m alone. Specifically, when the antibody was administered alone or when a complex with human IL-8 was formed and administered, it was verified whether or not the rate of disappearance of the antibody from the plasma was changed.

Human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories, Methods Mol Biol. 602:93-104 (2010)), when only anti-human IL-8 antibody was administered And, in each case where human IL-8 and anti-human IL-8 antibody were administered at the same time, the in vivo kinetics of the anti-human IL-8 antibody was evaluated. Each of the anti-human IL-8 antibody solution (200 μg/mL) and the mixed solution of human IL-8 (10 μg/mL) and anti-human IL-8 antibody (200 μg/mL) was added to the tail vein at a single dose of 10 mL/kg. Administered. At this time, since the anti-human IL-8 antibody is present in a sufficient amount with respect to human IL-8, it is considered that almost all of the human IL-8 is bound to the antibody. Blood was collected 5 minutes after administration, 2 hours, 7 hours, 1 day, and 2 days after 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 at -20°C or lower until measurement was performed.

The concentration of anti-human IL-8 antibody in mouse plasma was measured according to the electrochemiluminescence method. First, on a Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery) blocked overnight at room temperature using a PBS-Tween solution containing 5% BSA (w/v), Anti-Human Kappa Light Chain Goat IgG Biotin (IBL) Was reacted at room temperature for 1 hour to prepare an anti-human antibody immobilization plate. A calibration curve sample containing an anti-human IL-8 antibody at concentrations of 3.20, 1.60, 0.800, 0.400, 0.200, 0.100, and 0.0500 μg/mL in plasma and a sample for measuring mouse plasma diluted 100 times or more were prepared. Each sample was mixed with human IL-8, then dispensed into each well of an anti-human antibody immobilization plate in 50 µL, and stirred at room temperature for 1 hour. The final concentration of human IL-8 was prepared to be 333 ng/mL.

Thereafter, to the plate, an anti-human IL-8 antibody (in-house preparation) containing a constant region of mouse IgG was added and reacted at room temperature for 1 hour. In addition, anti-mouse IgG (BECKMAN COULTER) ruthenized with SULFO-TAG NHS Ester (Meso Scale Discovery) was added to the plate and reacted for 1 hour, and then Read Buffer T (×1) (Meso Scale Discovery) was added to the plate. And immediately measured with SECTOR Imager 2400 (Meso Scale Discovery). The concentration of the anti-human IL-8 antibody was calculated using the analysis software SOFTmax PRO (Molecular Devices) based on the response of the calibration curve.

As a result, the antibody concentration in mouse plasma obtained as a result is shown in Fig. 31, and the clearance of the antibody under each condition is shown in Table 24.

It was found that the rate of uptake into cells of the complex of H1009/L395-F1942m and human IL-8 was at least 2.2 times higher than that of H1009/L395-F1942m. On the other hand, the expression "at least 2.2 times" here is one of the possible values of 5 times, 10 times, or 30 times, and the following reasons are also mentioned. Since the rate of disappearance of human IL-8 from mouse plasma is very fast compared to the rate of disappearance of H1009/L395-F1942m, the ratio of H1009/L395-F1942m bound to human IL-8 in plasma is quickly after administration. It will deteriorate. That is, even when administered simultaneously with human IL-8, not all of H1009/L395-F1942m present in plasma are in a state of binding to human IL-8, but rather, already at about 7 hours after administration, Most of them exist as a glass type. Since the absorption rate is evaluated under such conditions, the intracellular absorption rate of the complex of H1009/L395-F1942m and human IL-8 is actually 5 times or 10 times that of H1009/L395-F1942m. , Or even if it is increased by 30 times, since only a part of it is reflected in the result in this experimental system, there is a possibility that it will appear as an effect of about 2.2 times. That is, from the results obtained here, it was found that the rate of absorption of the complex of H1009/L395 and IL-8 into the cells is higher than the actual rate of absorption of H1009/L395 into the cells in vivo. The effect is not limited at all to a value of 2.2 times.

Although it does not specifically limit, The following analysis can also be performed from the knowledge obtained so far. When H1009/L395, which is a pH-dependent IL-8-binding antibody, forms a complex with human IL-8, the complex has a higher isoelectric point and is biased toward positive charge as compared to the case where the antibody alone exists. At the same time, the affinity of the complex to the extracellular matrix is greater than that of the antibody alone. Properties such as an increase in the isoelectric point or enhancement of binding to the extracellular matrix can be considered as factors that promote the uptake of antibodies into cells in vivo. Moreover, from experiments using mice, it was shown that the rate of absorption of the complex of H1009/L395 and human IL-8 into cells was increased by 2.2 times or more than that of H1009/L395. From the above, consistent with the theoretical explanation, the properties in vitro, and the phenomenon in vivo, H1009/L395 forms a complex with human IL-8, and absorption into the cell is promoted, resulting in the loss of human IL-8. It supports the hypothesis that it is increasing significantly.

Until now, some antibodies to IL-8 have been reported, but when the complex with IL-8 is formed, the increase in binding affinity to the extracellular matrix and the increase in the uptake of the complex into cells are now There is no report until.

In addition, based on the fact that an increase in the uptake of anti-IL-8 antibodies into cells is seen when a complex with IL-8 is formed, anti-IL-8 antibodies that form a complex with IL-8 in plasma are rapidly It is also possible to consider that a free antibody that is absorbed into cells and does not form a complex with IL-8 is likely to remain in the cell without being absorbed. In this case, when the anti-IL-8 antibody is pH dependent, once the anti-IL-8 antibody absorbed into the cell dissociates the IL-8 molecule from within the cell, it returns to the extracellular area and binds to a separate IL-8 molecule. Since it becomes possible, it is also possible to think that the increase in absorption into cells during complex formation has an additional effect of more strongly removing IL-8. In other words, it is considered to be another embodiment of Initiation C to select an anti-IL-8 antibody with increased binding to the extracellular matrix or an anti-IL-8 antibody with increased intracellular uptake.

Example 17

Immunogenicity prediction using in silico system of pH-dependent IL-8 binding antibody H1009/L395

Next, for H1009/L395, the immunogenicity score and the frequency of occurrence of ADA were predicted in the same manner as in Example 13-1. The results are shown in Table 25 and Fig. 32. In addition, in FIG. 32, H1009/L395 is indicated as "H1009L395".

From the results of Table 25, it is shown that H1009/L395 has a low immunogenicity score to the same extent as H1004/L395. In addition, from the result of FIG. 32, the predicted ADA occurrence frequency in H1009/L395 was 0%, and this was also the same as in H1004/L395.

From the above, the immunogenicity predicted in H1009/L395 was significantly lowered compared to hWS-4, which is a known anti-human IL-8 antibody. From this, it is thought that H1009/L395 has extremely low immunogenicity in humans and is capable of stably maintaining anti-IL-8 neutralizing activity over a long period of time.

Example 18

Philippine monkey PK assay using H89/L118 variant with enhanced FcRn binding capacity under acidic pH conditions

As described in the examples so far, the pH-dependent IL-8-binding antibody H1009/L395 is an antibody having very excellent properties when it has a natural IgG1 as a constant region. However, it can also be used as an antibody containing an amino acid substitution in a constant region, for example, an antibody containing an Fc region in which FcRn binding at an acidic pH is enhanced as illustrated in Example 5. Therefore, it was confirmed using H89/L118 that the Fc region having enhanced FcRn binding at acidic pH also functions in a pH-dependent IL-8-binding antibody.

(18-1) acid in pH FcRn Combine Augmented Of H89/L118 Fc domain Change Preparation of antibody

For the Fc region of H89/L118, various FcRn binding enhancement modifications described in Example 5-1 were introduced. Specifically, with respect to the Fc region of H89-IgG1, modifications used in F1847m, F1848m, F1886m, F1889m, F1927m, and F1168m were introduced to produce the following modifications: (a) H89-IgG1m as heavy chain (SEQ ID NO: 94) and H89/L118-IgG1 with L118-K0MT as light chain; (b) H89/L118-F1168m including H89-F1168m as heavy chain (SEQ ID NO: 95) and L118-K0MT as light chain; (c) H89/L118-F1847m with H89-F1847m as heavy chain (SEQ ID NO: 96) and L118-K0MT as light chain; (d) H89/L118-F1848m with H89-F1848m as heavy chain (SEQ ID NO: 97) and L118-K0MT as light chain; (e) H89/L118-F1886m including H89-F1886m as heavy chain (SEQ ID NO: 98) and L118-K0MT as light chain; (f) H89/L118-F1889m including H89-F1889m as heavy chain (SEQ ID NO: 99) and L118-K0MT as light chain; And (g) H89/L118-F1927m comprising H89-F1927m as a heavy chain (SEQ ID NO: 100) and L118-K0MT as a light chain.

Philippine monkey PK assay using these antibodies was carried out by the method shown below.

(18-2) Philippine monkey PK assay of antibodies containing novel Fc region variants

The in vivo kinetics of the anti-human IL-8 antibody after administration of the anti-human IL-8 antibody to Philippine monkeys was evaluated. The anti-human IL-8 antibody solution was administered intravenously at a single dose of 2 mg/kg. 5 minutes after administration, 4 hours, 1 day, 2 days, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 Blood was collected after days and after 56 days. The collected blood was immediately centrifuged at 4° C. and 15,000 rpm for 10 minutes to obtain plasma. The separated plasma was stored in a freezer set at -60°C or lower until measurement was performed.

The concentration of anti-human IL-8 antibody in the plasma of Philippine monkeys was measured according to the electrochemiluminescence method. First, Anti-hKappa Capture Ab (Antibody Solutions) was dispensed into a MULTI-ARRAY 96-well plate (Meso Scale Discovery) and stirred at room temperature for 1 hour. Thereafter, an anti-human antibody immobilization plate was prepared by blocking at room temperature for 2 hours using a PBS-Tween solution containing 5% BSA (w/v). A calibration curve sample 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 μg/mL in plasma and a sample for measurement of Philippine monkey plasma diluted 500 times or more were prepared. After 50 µL of the solution was dispensed into each well of the human antibody immobilization plate, it was stirred at room temperature for 1 hour. Thereafter, to the plate, Anti-hKappa Reporter Ab and Biotin conjugate (Antibody Solutions) were added and reacted at room temperature for 1 hour. In addition, SULFO-TAG Labeled Streptavidin (Meso Scale Discovery) was added and reacted at room temperature for 1 hour, and then Read Buffer T (× 1) (Meso Scale Discovery) was dispensed into the plate, and immediately SECTOR Imager 2400 (Meso Scale Discovery) The measurement was carried out. The concentration of the anti-human IL-8 antibody was calculated using the analysis software SOFT Max PRO (Molecular Devices) based on the response of the calibration curve.

The half-life (t1/2) and clearance (CL) of each antibody obtained as a result are shown in Table 26, and the change of antibody concentration in the plasma of Philippine monkeys is shown in Fig. 33, respectively.

From the above results, it was confirmed that any of the Fc region variants have long-term plasma retention compared to the antibody having the native IgG1 Fc region. In particular, H89/L118-F1886m showed the most desirable blood dynamics.

Example 19

Fc region with reduced binding ability to FcγR

It is known that native human IgG1 has its Fc region bound to Fcγ receptors (hereinafter referred to as FcγR) on various immune system cells, and exhibits effector functions such as ADCC and ADCP with respect to target cells.

On the other hand, IL-8 is a soluble cytokine, and anti-IL-8 antibodies used as pharmaceuticals are expected to exhibit pharmacological action by neutralizing their function mainly at sites where IL-8 is excessively present. . The site in which such IL-8 is present in excess is not particularly limited, but, for example, an inflammation site may be assumed. In general, it is known that various immune cells gather and are activated in such an inflammatory site. Transmitting an unintended activation signal through an Fc receptor to these cells, or inducing an activity such as ADCC or ADCP against unintended cells is not necessarily desirable. Therefore, although it is not particularly limited, from the viewpoint of safety, it may be considered that the anti-IL-8 antibody to be administered in vivo preferably has a low affinity for FcγR.

(19-1) Preparation of modified antibody with reduced binding to FcγR

For the purpose of lowering the binding ability to various human and Philippine monkey FcγR, amino acid modifications were further introduced into the Fc region of H1009/L395-F1886m. Specifically, for the heavy chain H1009-F1886m, according to EU numbering, L at position 235 to R, G at position 236 to R, and S at position 239 to K, respectively, by substituting H1009-F1886s (sequence Number: 81) was produced. Similarly, for H1009-F1886m, L at position 235 according to EU numbering is substituted with R, and G at position 236 is substituted with R, and the region from position 327 to position 331 according to EU numbering is further human native IgG4 sequence. Substituting with one of, H1009-F1974m (SEQ ID NO: 80) was prepared. H1009/L395-F1886s and H1009/L395-F1974m were produced as antibodies having L395-k0MT as these heavy and light chains.

(19-2) Confirmation of affinity for various human FcγR

Next, the affinity of H1009/L395-F1886s or H1009/L395-F1974m to human or Philippine monkey soluble FcγRIa or FcγRIIIa was confirmed by the method shown below.

An assay was performed for the binding of soluble FcγRIa or FcγRIIIa in human or Philippine monkeys using BIACORE T200 (GE Healthcare) of H1009/L395-F1886s or H1009/L395-F1974m. Soluble FcγRIa and FcγRIIIa of human and Philippine monkeys, respectively, were prepared as molecular types to which a His tag was attached by a method known to those skilled in the art. An appropriate amount of rProtein L (BioVision) was fixed on the sensor chip CM4 (GE Healthcare) by an amine coupling method, and the desired antibody was captured. Next, soluble FcγRIa or FcγRIIIa and a running buffer (as a reference solution) were injected to interact with the antibody captured on the sensor chip. On the other hand, HBS-EP+ (GE Healthcare) was used as a running buffer, and HBS-EP+ was also used for dilution of soluble FcγRIa or FcγRIIIa. For regeneration of the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 20°C.

The results are shown in Fig. 34. Here, in the order of human FcγRIa, human FcγRIIIa, Philippine monkey FcγRIa, Philippine monkey FcγRIIIa, it is described as hFcγRIa, hFcγRIIIa, cynoFcγRIa, cynoFcγRIIIa. While H1009/L395-F1886m was shown to bind to any FcγR, it was confirmed that H1009/L395-F1886s and H1009/L395-F1974m did not bind to any FcγR.

(19-3) Mouse IL-8 disappearance assay of Fc variant

Next, for H1009/L395-F1886s and H1009/L395-F1974m, the rate of human IL-8 disappearance in mice and the plasma retention of the antibody were confirmed by the experiments shown below. On the other hand, here, in order to evaluate including the effect of increasing the antibody dosage of H1009/L395-F1886s, the dosage is 2 mg/kg, 5 mg/kg, and 10 mg/kg for H1009/L395-F1886s. It evaluated using 3 points of.

In human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 32 +/+ mouse, Jackson Laboratories, Methods Mol. Biol. 602:93-104 (2010)), human IL-8 and anti-human IL- 8 The in vivo kinetics of human IL-8 after simultaneous administration of antibodies was evaluated. A mixed solution of human IL-8 (10 μg/mL) and anti-human IL-8 antibody (200 μg/mL, 500 μg/mL, or 1000 μg/mL) was administered to the tail vein at a single dose of 10 mL/kg. At this time, the anti-human IL-8 antibody is present in a sufficient amount with respect to human IL-8, so it is considered that almost all of the human IL-8 is bound to the antibody. Blood was collected 5 minutes after administration, 2 hours, 4 hours, 7 hours, 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after 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 at -20°C or lower until measurement was performed.

The measurement of the human IL-8 concentration in mouse plasma was performed in the same manner as in Example 11. As a result, the data on the concentration of human IL-8 in plasma are shown in Fig. 35, and the value of the human IL-8 clearance (CL) in mouse plasma is shown in Table 27.

First, in the comparison of the administration group of 2 mg/kg, it was found that H1009/L395 containing the Fc region of native IgG1 and H1009/L395-F1886s containing the modified Fc region have equivalent human IL-8 disappearance effects. .

Next, when the antibody dose of H1009/L395-F1886s was changed, there was a slight difference in the plasma IL-8 concentration at the time point 1 day after administration, but the human IL-8 clearance value was 2 mg/dose. There was no significant difference between kg and 10 mg/kg. This strongly suggests that the antibody containing the variable region of H1009/L395 exhibits a sufficient IL-8 elimination effect even when administered at a high dose.

(19-4) Philippine monkey PK assay of Fc variant

Next, using H1009/L395-F1886s or H1009/L395-F1974m, the plasma retention of antibodies in Philippine monkeys was verified by the method shown below.

The in vivo kinetics of the anti-human IL-8 antibody was evaluated when an anti-human IL-8 antibody was administered to Philippine monkeys alone, or when both human IL-8 and anti-human IL-8 antibodies were administered simultaneously. An anti-human IL-8 antibody solution (2 mg/mL) or a mixed solution of human IL-8 (100 μg/kg) and an anti-human IL-8 antibody (2 mg/kg) was administered intravenously at a single dose of 1 mL/kg. 5 minutes after administration, 4 hours, 1 day, 2 days, 3 days, 7 days, 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days After, and 56 days later, blood was collected. The collected blood was immediately centrifuged at 4° C. and 15,000 rpm for 10 minutes to obtain plasma. The separated plasma was stored in a freezer set at -60°C or lower until measurement was performed.

The measurement of the anti-human IL-8 antibody concentration in the plasma of Philippine monkeys was performed by the method of Example 18. As a result, the data of the anti-human IL-8 antibody concentration in plasma are shown in Fig. 36, and the half-life (t1/2) and clearance (CL) values of the anti-human IL-8 antibody from the plasma of Philippine monkeys are shown in Table 28. Shown in.

First, compared with Hr9 and H89/L118 having an Fc region of native human IgG1, it was shown that H1009/L395-F1886s having an improved Fc region had a significantly longer plasma retention.

Furthermore, even when H1009/L395-F1886s was administered simultaneously with human IL-8, the plasma concentration transition was equivalent to that when the antibody was administered alone. Although it does not specifically limit, From this, it is also possible to consider as follows. As described above, it has been shown that the uptake of the complex of H1009/L395 and human IL-8 into cells is higher than that of H1009/L395 alone. In general, it is thought that high molecular weight proteins are absorbed into cells in a nonspecific or receptor-dependent manner, then migrate to lysosomes, and are degraded by various degrading enzymes present in the lysosome. Therefore, if the rate of absorption of the protein into cells increases, it is considered that the plasma retention property will also deteriorate. However, in the case of an antibody, since it has the property of being returned to the plasma by FcRn present in the endosome, as long as the salvage by FcRn is sufficiently functioning, even when the intracellular absorption rate is increased, in the plasma It is thought that it will not affect retention. Here, even when H1009/L395-F1886s was administered to Philippine monkeys at the same time as human IL-8, no effect on plasma retention was observed. This indicates the possibility that H1009/L395-F1886s is sufficiently salvaged by FcRn and returned to plasma even if the intracellular absorption rate of the antibody is increased.

Moreover, H1009/L395-F1974m, which is another kind of Fc modification, also exhibited the same plasma retention as H1009/L395-F1886s. As described above, these Fc modifiers are modifications for lowering the binding ability to various FcγRs, and different ones are introduced, but it has been shown that they do not affect the plasma retention of the antibody itself. From the above, both H1009/L395-F1886s and H1009/L395-F1974m showed that plasma retention in Philippine monkeys was remarkably improved compared to the antibody having the Fc region of native IgG1, and was extremely good.

As shown from the above examples, H1009/L395 contains the characteristics that the pH-dependent IL-8 binding ability and the complex with IL-8 is rapidly absorbed into the cell, thereby in vivo in human IL-8. It was realized for the first time as an antibody that significantly increases the rate of disappearance of. Moreover, the binding affinity of this antibody to IL-8 under neutral pH conditions is also elevated compared to the known hWS-4 antibody, and this antibody is more strongly human under neutral pH conditions such as in plasma. It is possible to neutralize IL-8. In addition, it is an antibody that has excellent stability under conditions in plasma and does not decrease IL-8 neutralizing activity even after in vivo administration. In addition, H1009/L395 produced based on Hr9 whose production amount was significantly improved compared to hWS-4 is an antibody suitable for production from the viewpoint of production amount. Moreover, in predicting the immunogenicity of in silico, the immunogenicity shows a very low score, and the score is considerably low even when compared with some of the known hWS-4 antibodies and existing commercially available antibodies. In other words, it is expected that H1009/L395 can be safely used over a long period of time without generating ADA in humans. Therefore, H1009/L395 is an antibody showing improvement from various viewpoints compared to a known anti-human IL-8 antibody, and is very useful as a pharmaceutical product.

H1009/L395 having an Fc region of native IgG is sufficiently useful as described above, but a variant of H1009/L395 containing an Fc region with improved function is also appropriately used as an antibody with improved usefulness. It is possible. Specifically, it is possible to increase FcRn binding under acidic pH conditions, prolong retention in plasma, and sustain the effect over a longer period of time. In addition, the modified variant containing the Fc region into which the modification to decrease the binding ability to FcγR has been introduced is for high safety treatment to avoid the occurrence of unintended immune cell activation or cytotoxic activity in the administered body. It is possible to use it as an antibody. As such an Fc variant, it is particularly preferable to use F1886s or F1974m implemented in this specification, but it is not limited to these Fc variants, and as long as the Fc variant has the same function, treatment containing other modified Fc regions A lysed antibody is also used as an aspect of initiation C.

As a result, the antibodies of initiation C containing H1009/L395-F1886s and H1009/L395-F1974m, which were produced by conducting intensive studies by the present inventors, strongly inhibit the biological activity of human IL-8 over a long period of time and safely. It is possible to maintain the state. In the present specification, levels that cannot be achieved with conventional anti-IL-8 antibodies are realized, and these antibodies of Initiation C are expected to be used as anti-IL-8 antibody drugs with extremely high degree of completion.

Example 20

Anti-agent IXa/X factor bispecific antibody

The humanized anti-agent IXa/X factor bispecific antibody disclosed in WO2012/067176 binds to human factor IXa and factor X and induces blood coagulation activity. In this example, F8M (Q499-z121/J327-z119/L404-k: H chain (SEQ ID NO: 330)/H chain (SEQ ID NO: 330) which is a humanized anti-agent IXa/X factor bispecific antibody described in WO2012/067176 331) A common L chain (SEQ ID NO: 332)) is used, and F8M contains two different H chains and two identical common L chains. F8M was produced according to the method described in Examples of WO2012/067176.

(20-1) Preparation of anti-agent IXa/X factor bispecific antibody

As an anti-agent IXa/X factor bispecific antibody based on F8M, the following three types of antibodies were prepared according to the method of Reference Example 2: (a) F8M-F1847mv1 (SEQ ID NO: 323) and F8M- as heavy chains. F8M-F1847mv, a conventional antibody comprising F1847mv2 (SEQ ID NO: 324) and F8ML as light chain (SEQ ID NO: 325); (b) F8M-F1868mv1 (SEQ ID NO: 326) and F8M-F1868mv2 (SEQ ID NO: 327) as the heavy chain and F8ML (SEQ ID NO: 325) as the light chain, a conventional antibody, F8M-F1868mv; And (c) F8M-F1927mv1 (SEQ ID NO: 328) and F8M-F1927mv2 (SEQ ID NO: 329) as the heavy chain and F8ML (SEQ ID NO: 325) as the light chain.

The heavy chain sequence contains the same Fc variant sequence as described in Example 5 above with respect to the enhancement of FcRn binding and reduction of rheumatoid factor binding as follows.

(20-2) Philippines For monkeys Monoclonal Antibody F8M - F1847mv , F8M -F1868mv, and the pharmacokinetic test of F8M-F1927mv

The pharmacokinetics of monoclonal antibodies F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were evaluated, respectively, after a single intravenous bolus administration at a dose of 0.6 mg/kg to male Philippine monkeys. Plasma concentrations of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were determined by sandwich ELISA. Pharmacokinetic parameters were calculated using WinNonlin ver 6.4 software. As shown in Table 30, the half-life of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv were 29.3 days, 54.5 days, and 35.0 days, respectively. On the other day, a PK test of F8M at a dose of 6 mg/kg was performed using Philippine monkeys, and the half-life was found to be 19.4 days. It was found that the half-life of F8M-F1847mv, F8M-F1868mv, and F8M-F1927mv was longer than that of F8M. This suggests that the half-life of the anti-agent IXa/X factor bispecific antibody could be extended by the same Fc region sequence modification as mentioned in Example 5 above.

Example 21

Evaluation of clearance of IgE from plasma using pI-raising Fab variant

In order to increase the clearance of human IgE, in this Example, the pI elevation substitution in the Fab portion of the antibody was evaluated using a pH-dependent antigen-binding antibody. The method of increasing pi by adding amino acid substitutions to the antibody variable region is not particularly limited, but can be carried out, for example, by the method described in WO2007/114319 or WO2009/041643. As the amino acid substitution to be introduced into the variable region, it is preferable to increase the number of amino acids having a negative charge (for example, aspartic acid and glutamic acid) while increasing the amino acids having a positive charge (for example, arginine and lysine). In addition, amino acid substitution may be introduced at any position in the antibody variable region. Although not particularly limited, as a site for introducing an amino acid substitution, a position where an amino acid side chain can be exposed on the surface of an antibody molecule is preferable.

(21-1) Preparation of antibodies with elevated pi by alteration of amino acids in variable regions

The tested antibodies are summarized in Tables 32 and 33.

Heavy chain Ab1H003 (SEQ ID NO: 144, also called H003) was prepared by introducing substitution H32R that raises pi into Ab1H (SEQ ID NO: 38). By introducing each substitution shown in Table 32 into Ab1H according to the method shown in Reference Example 1, another heavy chain modified product was also prepared. All heavy chain variants were expressed with Ab1L (SEQ ID NO: 39) as light chains. The pH-dependent binding profile of this antibody is summarized in Table 5 (Ab1).

Similarly, the present inventors also evaluated the substitution in the light chain that raises pI.

Light chain Ab1L001T (SEQ ID NO: 164, also called L001) was prepared by introducing substitution G16K that increases pi into Ab1L. By introducing each substitution shown in Table 33 into Ab1L according to the method shown in Reference Example 1, other light chain modified variants were also prepared. All light chain variants were expressed together with Ab1H as a heavy chain.

(21-2) pI Increase Variant Used, To BIACORE By human FcγRIIb Combination Assay

Regarding the antibody containing the produced Fc region variant, a binding assay between a soluble human FcγRIIb and an antigen-antibody complex was performed using BIACORE T200 (GE Healthcare). Soluble human FcγRIIb (NCBI accession NM_004001.3) was prepared as a molecular type to which His tag was attached by a method known in the art. An appropriate amount of anti-His antibody was immobilized on the sensor chip CM5 (GE Healthcare) by an amine coupling method using His capture kit (GE Healthcare), and human FcγRIIb was captured. Next, an antibody-antigen complex and a running buffer (as a reference solution) were injected to interact with human FcγRIIb captured on a sensor chip. As a running buffer, 20mM N-(2-acetamide)-2-aminoethanesulfonic acid, 150mM NaCl, 1.2mM CaCl ₂ , and 0.05% (w/v) Tween20, pH 7.4 were used, and soluble human FcγRIIb was diluted. Each buffer was also used. For regeneration of the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 25°C. Analysis is performed based on the binding (RU) calculated from the sensorgram obtained by the measurement, and expressed as a relative value when the binding amount of Ab1H/Ab1L (original Ab1) is 1.00. BIACORE T100 Evaluation Software (GE Healthcare) was used to calculate the parameters.

The results of the SPR analysis are summarized in Table 32 and Table 33. In some variants, it has been shown that the binding of BIACORE to human FcγRIIb immobilized on a sensor chip is enhanced.

An antibody produced by introducing an alteration that raises pI into the variable region is an antibody in which the variable region has a greater positive charge compared to before introducing the alteration. Therefore, it is thought that the Coulomb interaction between the variable region (positive charge) and the surface of the sensor chip (negative charge) is strengthened by amino acid alteration that increases pI. Moreover, since such an effect is expected to occur in the same manner on the cell membrane surface having a negative charge, they are also expected to exhibit an effect of accelerating the rate of absorption into cells in vivo.

Here, compared with the binding of the original Ab1 to hFcγRIIb, when the binding of the variant to hFcγRIIb is about 1.2 times or more, it was shown that it has a strong charging effect on the binding of the antibody to hFcγRIIb on the sensor chip.

Among the pI rising heavy chain variants, antibodies having Q13K, G15R, S64K, T77R, D82aN, D82aG, D82aS, S82bR, E85G, or Q105R substitutions (according to Kabat numbering) alone or in combination have stronger binding to hFcγRIIb. Indicated. It is estimated that this single amino acid substitution in the heavy chain or a combination of these substitutions has a strong charging effect on the binding to hFcγRIIb on the sensor chip. Therefore, by introducing an alteration that increases the pI in the heavy chain variable region of the antibody, at one or more positions that are expected to exhibit an effect of accelerating the rate of absorption or the rate of absorption into cells in vivo, for example, , According to Kabat numbering, 13th, 15th, 64th, 77th, 82a, 82b, 85th, and 105th may be included. The amino acid substitution introduced at such a position may be asparagine, glycine, serine, arginine, or lysine, preferably arginine or lysine.

Among the pi-raising light chain variants, G16K, Q24R, A25R, S26R, E27R, Q37R, G41R, Q42K, S52K, S52R, S56K, S56R, S65R, T69R, T74K, S76R, S77R, Q79K substitution (according to Kabat numbering) were performed. Antibodies, either alone or in combination, show stronger binding to human FcγRIIb. It is estimated that this single amino acid substitution in the light chain or a combination of these substitutions has a strong charging effect on the binding to human FcγRIIb on the sensor chip. Therefore, at one or a plurality of positions that are expected to exhibit an effect of accelerating the rate of absorption or the rate of absorption into cells in vivo by introducing an alteration that increases the pI in the light chain variable region of the antibody, for example , According to Kabat numbering, 16th, 24th, 25th, 26th, 27th, 37th, 41st, 42nd, 52nd, 56th, 65th, 69th, 74th, 76th, 77th, And 79th place. The amino acid substitution introduced at such a position may be arginine or lysine.

(21-3) Cell uptake of antibody containing pI-raising Fab region variant

In order to evaluate the rate of intracellular uptake into hFcγRIIb-expressing cell lines using the produced antibody containing the Fab region variant, the same assay as in (4-5) was performed, except that the amount of antigen introduced was Ab1H. It was expressed as a relative value when the value of /Ab1L (original Ab1) was set to 1.00.

The quantitative results of cell introduction are summarized in Tables 32 and 33. In some Fc variants, strong fluorescence derived from antigens in cells was observed. Here, when the fluorescence intensity of the antigen absorbed into the cells of the variant is about 1.5 times or more compared to the fluorescence intensity of the original Ab1, it was shown that it has a strong charging effect on the antigen absorbed into the cell.

Among the pI-raising heavy chain variants, antibodies having P41R, G44R, T77R, D82aN, D82aG, D82aS, S82bR, or E85G substitution (according to Kabat numbering) alone or in combination showed stronger antigen uptake into cells. It is estimated that this single amino acid substitution in the heavy chain or a combination of these substitutions has a strong charging effect on the uptake of the antigen-antibody complex into cells. Therefore, at one or more positions that are expected to cause the absorption of the antigen-antibody complex into the cell more quickly or more frequently by introducing a modification that raises the pI in the heavy chain variable region of the antibody, for example, , According to Kabat numbering, the 41st, 44th, 77th, 82a, 82b, or 85th place may be included. The amino acid substitution introduced at such a position may be asparagine, glycine, serine, arginine, or lysine, preferably arginine or lysine.

Among the pi-raising light chain variants, G16K, Q24R, A25R, A25K, S26R, S26K, E27R, E27Q, E27K, Q37R, G41R, Q42K, S52K, S52R, S56R, S65R, T69R, T74K, S76R, S77R, or Q79K substitution Antibodies having either alone or in combination (according to Kabat numbering) showed stronger antigen uptake into cells. It is estimated that this single amino acid substitution in the light chain or a combination of these substitutions has a strong charging effect on the uptake of the antigen-antibody complex into cells. A variant having 4 or more amino acid substitutions tends to exhibit a stronger charging effect than a variant having fewer amino acid substitutions. At one or more positions that are expected to cause the absorption of the antigen-antibody complex into cells more quickly or at a higher frequency by introducing an alteration that raises the pi into the light chain variable region of the antibody, for example, Kabat 16th, 24th, 25th, 26th, 27th, 37th, 41st, 42nd, 52nd, 56th, 65th, 69th, 74th, 76th, 77th, or 79th according to numbering The stomach may be included. The amino acid substitution introduced at such a position may be glutamine, arginine, or lysine, preferably arginine or lysine.

Although not bound by a specific theory, this result can be explained as follows. The antigen and antibody added to the cell culture solution form an antigen-antibody complex in the culture solution. The antigen-antibody complex binds to human FcγRIIb expressed on the cell membrane through the Fc region of the antibody, and is receptor-dependently absorbed into cells. Since the antibody used in this experiment binds to the antigen in a pH-dependent manner, it is possible for the antibody to dissociate from the antigen in endosomes (acidic pH conditions) within the cell. Dissociated antigens are transported and accumulated in lysosomes, so they fluoresce in cells. Therefore, the fact that the fluorescence intensity in the cell is strong is considered to indicate that the intracellular absorption of the antigen-antibody complex is occurring more rapidly or at a higher frequency.

(21-4) Evaluation of clearance of human IgE in a mouse simultaneous administration model

Several pH-dependent antigen-binding anti-IgE antibodies (original Ab1, Ab1H/Ab1L013, Ab1H/Ab1L014, Ab1H/Ab1L007) were tested in a mouse co-administration model to evaluate their ability to accelerate the clearance of IgE from plasma. . In the simultaneous administration model, C57BL6J mice (Jackson Laboratories) were each administered with an anti-IgE antibody and IgE previously mixed by single intravenous administration. To all groups, 0.2 mg/kg of IgE containing 1.0 mg/kg of anti-IgE antibody was administered. The total concentration of IgE in plasma was determined by anti-IgE ELISA. First, anti-human IgE (clone 107, MABTECH) was dispensed into a microwell plate (Nalge nunc International), and left to stand at room temperature for 2 hours or at 4° C. overnight to prepare an anti-human IgE antibody-immobilized plate. The calibration curve sample and the sample were mixed with an excess amount of anti-IgE antibody (prepared in-house) to form an immune complex having a uniform structure. These samples were added to an anti-human IgE antibody immobilization plate, and left still at 4°C overnight. Next, these samples were reacted sequentially with human GPC3 core protein (prepared in-house), biotinylated anti-GPC3 antibody (prepared in-house), and Streptavidin Poly HRP80 Conjugate (Stereospecific Detection Technologies) over 1 hour. Thereafter, SuperSignal (registered trademark) ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific) was added. Chemiluminescence was read using SpectraMax M2 (Molecular Devices). The concentration of human IgE was calculated using SOFTmax PRO (Molecular Devices). 37 shows the temporal profile of the plasma concentration of IgE in C57BL6J mice.

After administration of the pi-raising pH-dependent antigen-binding Fab variant, the total IgE concentration in plasma was lower than that of the original Ab1. These results indicate that the antigen-antibody immune complex of the pH-dependent antigen-binding variant having a high pI can more strongly bind to cell membrane receptors such as FcγR, thereby increasing cellular uptake of the antigen-antibody immune complex. have. The antigen absorbed by the cell can be effectively released from the antibody inside the endosome, thereby accelerating the loss of IgE. The IgE concentration of mice treated with Ab1H/Ab1L007, which showed a weak effect in the in vitro test, was higher than that of antibodies containing other pI-raising Fab variants. These results show that the sensitivity of the in vitro system using fluorescence intensity by InCell Analyzer 6000 may be higher than that of the in vitro BIACORE system with regard to estimation of the evaluation of the clearance of the antigen from plasma in vivo. It also suggests.

Example 22

Evaluation of clearance of C5 from plasma using pI-raising Fab variant

In order to increase the clearance of human IgE, in this Example, the pI elevation substitution in the Fab portion of the antibody was evaluated using a pH-dependent antigen-binding antibody.

(22-1) Preparation of C5 [expression and purification of recombinant human C5]

Recombinant human C5 (NCBI GenBank accession number: NP_001726. 2, SEQ ID NO: 207) was transiently expressed using the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA). After diluting the purified medium expressing human C5 with an equivalent amount of milliQ water, it was applied to a Q-sepharose FF or Q-sepharose HP anion exchange column (GE healthcare, Uppsala, Sweden), and then elution by a NaCl gradient was performed. Did. After pooling the fraction containing human C5, the salt concentration and pH were adjusted to 80 mM NaCl and pH 6.4, respectively. The obtained sample was applied to an SP-sepharose HP cation exchange column (GE healthcare, Uppsala, Sweden), and eluted by a NaCl gradient. Fractions containing human C5 were pooled and provided on a CHT ceramic Hydroxyapatite column (Bio-Rad Laboratories, Hercules, CA, USA). Next, the human C5 eluate was applied to a Superdex 200 gel filtration column (GE healthcare, Uppsala, Sweden). Fractions containing human C5 were pooled and stored at -150°C. For the test, either recombinant human C5 prepared in-house or human C5 derived from plasma (CALBIOCHEM, Cat#204888) was used.

Expression and purification of recombinant Philippine monkey C5 (NCBI GenBank accession number: XP_005580972, SEQ ID NO: 208) were performed in the same manner as in the human counterpart.

(22-2) Preparation of synthetic calcium library

The gene library of the antibody heavy chain variable region used as the synthetic human heavy chain library was composed of 10 types of heavy chain libraries. The germline frameworks VH1-2, VH1-69, VH3-23, VH3-66, VH3-72, VH4-59, VH4-61, VH4-b, VH5-51, and VH6-1 For this purpose, it was selected based on the frequency of germline lines in the repertoire of human B cells and the biophysical properties of the V gene family. Synthetic human heavy chain libraries mimic the antibody repertoire of human B cells to have diversity in antibody binding sites.

The gene library of the antibody light chain variable region is designed to have a calcium-binding motif, and is diversified in positions that can contribute to antigen recognition according to the antibody repertoire of human B cells. The design of a gene library of antibody light chain variable regions exhibiting features for calcium-dependent binding to antigens is described in WO 2012/073992.

(de Heard et al., Meth. Mol. Biol. 178:87-100 (2002)), a combination of a heavy chain variable region library and a light chain variable region library was inserted into a phagemid vector to construct a phage library. A trypsin digestion site was introduced into the linker region between the Fab and pIII protein of the phagemid vector. For the preparation of Fab display phage, a modified M13KO7 helper phage having a trypsin digestion site between the N2 and CT domains of gene III was used.

(22-3) Isolation of calcium-dependent anti-C5 antibody

The phage display library was diluted with TBS containing BSA and CaCl ₂ at a final concentration of 4% and 1.2 mM, respectively. As a panning method, conventional magnetic bead selection was applied with reference to a general protocol (Junutula et al., J. Immunol. Methods 332(1-2):41-52(2008), D'Mello et al. 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, beads coated with NeutrAvidin (Sera-Mag SpeedBeads coated with NeutrAvidin) or beads coated with Streptavidin (Dynabeads M-280 Streptavidin) were used. Human C5 (CALBIOCHEM, Cat#204888) was labeled with EZ-Link NHS-PEG4-Biotin (PIERCE, Cat No. 21329).

For the first round of phage selection, the phage display library was incubated with biotinylated human C5 (312.5 nM) for 60 minutes at room temperature. Thereafter, the phage showing the bound Fab variant was captured using magnetic beads.

After incubating for 15 minutes at room temperature with beads, the beads were _{washed three times with 1 mL of TBS containing 1.2 mM CaCl 2} and 0.1% Tween20, and 1 mL of TBS _{containing 1.2 mM CaCl 2} was used. Washed twice. For 15 minutes, the phage was eluted by resuspending the beads in TBS containing 1 mg/mL trypsin. The eluted phage was infected with ER2738, and rescued using a helper phage. The rescued phage was precipitated with polyethylene glycol, and BSA and CaCl ₂ were resuspended in TBS containing 4% and 1.2 mM final concentration, respectively, and used for the next round of panning.

After the first round of panning, phages were selected for calcium dependence in which the antibody more strongly binds to C5 in the presence of calcium ions. In the second and third rounds, panning was performed in the same manner as in the first round except that 50 nM (second round) or 12.5 nM (third round) of biotinylated antigen was used, and 0.1 mL of elution buffer (50 mM MES) was used. , 2 mM EDTA, 150 mM NaCl, pH 5.5) was used to perform final elution, followed by contact with 1 μL of 100 mg/mL trypsin to select calcium dependence. After selection, the selected phage clones were converted to IgG form.

The binding ability of the converted IgG antibody to human C5 was evaluated under the following two different conditions: 1.2 _{mM CaCl 2} -pH 7.4 (20 mM MES, at 30°C) using the Octet RED384 system (Pall Life Sciences). Binding and dissociation in 150mM NaCl, 1.2mM CaCl ₂ ), and binding and dissociation in 1.2mM CaCl ₂ -pH 7.4 (20mM MES, 150mM NaCl, 1.2mM CaCl ₂ ) and _{3μM CaCl 2} -pH 5.8 (20mM MES, 150mM Dissociation in NaCl, 3 μM CaCl _{2 ).} 25 pH-calcium dependent antigen binding clones were isolated. The sensorgrams of these antibodies are shown in FIG. 38.

(22-4) Identification of anti-C5 bispecific antibodies

From the clones isolated in Example B-3, nine pH- or calcium-dependent anti-C5 antibody clones were selected for further analysis (CFP0008, 0011, 0015, 0016, 0017, 0018, 0019, 0020, 0021). In order to improve properties such as physicochemical properties of the antibody, several amino acid substitutions were introduced into the CFP0016 heavy chain variable region according to a method known to those skilled in the art. Instead of CFP0016, this variant CFP0016H019 of CFP0016 was used for further analysis. Table 34 shows the amino acid sequences of the VH and VL regions of these nine antibodies. In this table, the names given in parentheses indicate an abbreviation.

A full-length gene having a nucleotide sequence encoding an antibody heavy and light chain was synthesized and prepared according to a method known to those skilled in the art. Vectors expressing heavy and light chains were prepared by inserting the obtained extranuclear gene fragment into a vector for expression in mammalian cells. The obtained expression vector was sequenced according to a method known to those skilled in the art. For expression of the antibody, the prepared extranuclear gene was transiently transfected into the FreeStyle293-F cell line (Thermo Fisher Scientific). Purification of the expressed antibody from the purification medium was performed using rProtein A Sepharose Fast Flow (GE Healthcare), according to a method known to those skilled in the art.

(22-5) Preparation and characterization of pH-dependent anti-C5 bispecific antibody

A bispecific antibody that recognizes two different epitopes of C5 was produced by a combination of CFP0020 and CFP0018. The bispecific antibody is prepared in the form of an IgG having two types of clones having different Fabs at each binding site of the antibody, and prepared using a method known to those skilled in the art. In this bispecific IgG antibody, the two types of heavy chains contain different heavy chain constant regions (G1dP1, SEQ ID NO: 227 and G1dN1, SEQ ID NO: 228) so that heterodimers of the two types of heavy chains are efficiently formed. do. An anti-C5 bispecific antibody comprising the binding site of the anti-C5 MAb "X" and the anti-C5 MAb "Y" is indicated by "X//Y".

By introducing several amino acid substitutions into the heavy and light chain CDRs by methods well known to those skilled in the art, the present inventors obtained a 20//18 light chain covalent variant, which was "optimized 20//18" (two heavy chain CFP0020H0261 -G1dP1, consisting of SEQ ID NO: 229 and CFP0018H0012-G1dN1, SEQ ID NO: 230, and a common light chain CFP0020L233-k0, SEQ ID NO: 231).

The reaction rate parameter of optimization 20//18 for recombinant human C5 was evaluated at 37° C. under two different conditions using a BIACORE T200 instrument (GE Healthcare) (for example, (A) binding at pH 7.4. And dissociation, and (B) binding at pH 7.4, dissociation at pH 5.8). By amine coupling method on Series SCM4 (GE Healthcare, catalog number BR-1005-34), protein A/G (Pierce, catalog number 21186) or anti-human IgG (Fc) antibody (included in Human Antibody Capture Kit; GE) Healthcare, catalog number BR-1008-39) was fixed. After capturing the anti-C5 antibody to the immobilized molecule, human C5 was injected. Running buffers used were ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl ₂ , 0.05% Tween 20). Using BIACORE T200 Evaluation software, version 2.0 (GE Healthcare), by fitting a 1:1 binding-RI (no bulk effect adjustment) model and a sensorgram, reaction rate parameters under both pH conditions were determined. The reaction rate parameters, the rate of binding (ka), the rate of dissociation (kd), and the binding affinity (KD) at pH 7.4, and the rate of dissociation (kd) determined only by calculations in the dissociation phase of each pH condition, It is shown in Table 35. Optimization 20//18 showed faster dissociation at pH 5.8 compared to the dissociation rate at pH 7.4 for human C5.

(22-6) Preparation of antibodies with elevated pi by alteration of amino acids in variable regions

The tested antibodies are summarized in Tables 36 and 37.

Heavy chain CFP0020H0261-001-G1dP1 (also called 20H001, SEQ ID NO: 232) was prepared by introducing substitution P41R/G44R that raises pi into CFP0020H0261-G1dP1 (SEQ ID NO: 229). Similarly, by introducing substitution T77R/E85R that raises pi into CFP0018H0012-G1dN1 (SEQ ID NO: 230), heavy chain CFP0018H0012-002-G1dN1 (also called 18H002, SEQ ID NO: 251) was prepared. Other heavy chain variants were also prepared by introducing each of the substitutions shown in Table 36 into CFP0020H0261-G1dP1 and CFP0018H0012-G1dN1, respectively, according to the method shown in Reference Example 1. Both of the heavy chain variant CFP0020H0261-G1dP1 variant and the CFP0018H0012-G1dN1 variant were expressed together with CFP0020L233-k0 (SEQ ID NO: 231) as a light chain to obtain a bispecific antibody.

Similarly, the present inventors also evaluated the substitution in the light chain that raises pI. The light chain CFP0020L233-001-k0 (SEQ ID NO: 271, also referred to as 20L233-001) was prepared by introducing substitution G16K that increases pI into CFP0020L233-k0. By introducing each substitution shown in Table 37 into CFP0020L233-k0 according to the method shown in Reference Example 1, other light chain modified variants were also prepared. All light chain variants were expressed together with CFP0020H0261-G1dP1 and CFP0018H0012-G1dN1 as heavy chains to obtain bispecific antibodies.

(22-7) pI Increase Variant Used, To BIACORE By human FcγRIIb Combination Assay

Regarding the antibody containing the produced Fc region variant, the binding assay of the soluble hFcγRIIb and the antigen-antibody complex was performed using BIACORE T200 (GE Healthcare). Soluble hFcγRIIb was prepared as a molecular type to which His tag was attached by a method known in the art. An appropriate amount of anti-His antibody was fixed on the sensor chip CM5 (GE Healthcare) by the amine coupling method using His capture kit (GE Healthcare), and hFcγRIIb was captured. Next, an antibody-antigen complex and a running buffer (as a reference solution) were injected to interact with hFcγRIIb captured on the sensor chip. As the running buffer, 20mM N-(2-acetamide)-2-aminoethanesulfonic acid, 150mM NaCl, 1.2mM CaCl ₂ , and 0.05% (w/v) Tween20, pH 7.4 were used, and the soluble hFcγRIIb was diluted. Each buffer was used. For regeneration of the sensor chip, 10 mM glycine-HCl, pH 1.5 was used. All measurements were carried out at 25°C. Analysis is performed based on the binding (RU) calculated from the sensorgram obtained by the measurement, and it is expressed as a relative value when the binding amount of CFP0020H0261-G1dP1/CFP0018H0012-G1dN1/CFP0020L233-k0 (original Ab2) is 1.00. BIACORE T100 Evaluation Software (GE Healthcare) was used to calculate the parameters.

The results of the SPR analysis are summarized in Tables 36 and 37. In some variants, it has been shown that the binding to hFcγRIIb immobilized on BIACORE's sensor chip is increased. Here, compared with the binding of the original Ab2 to hFcγRIIb, when the binding of the variant to hFcγRIIb is about 1.2 times or more, it was shown that it has a strong charging effect on the binding of the antibody to hFcγRIIb on the sensor chip.

Among the pi-raising heavy chain variants, antibodies with L63R, F63R, L82K, or S82bR substitutions (according to Kabat numbering) showed stronger binding to hFcγRIIb. It is estimated that this single amino acid substitution in the heavy chain or a combination of these substitutions has a strong charging effect on the binding to hFcγRIIb on the sensor chip. Therefore, at one or more positions that are expected to exhibit an effect of accelerating the rate of absorption or the rate of absorption into cells in vivo by introducing an alteration that increases the pI in the heavy chain variable region of the antibody, for example, , The 63rd, 82nd, or 82b place according to Kabat numbering may be included. The amino acid substitution introduced at such a position may be arginine or lysine.

Among the pi-raising light chain variants, antibodies with G16K, Q27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R, or Q79K substitution (according to Kabat numbering) showed stronger binding to hFcγRIIb. It is estimated that this single amino acid substitution in the light chain or a combination of these substitutions has a strong charging effect on the binding to human FcγRIIb on the sensor chip. Therefore, at one or a plurality of positions that are expected to exhibit an effect of accelerating the rate of absorption or the rate of absorption into cells in vivo by introducing an alteration that increases the pI in the light chain variable region of the antibody, for example , According to Kabat numbering, the 16th, 27th, 41st, 52nd, 56th, 65th, 69th, 74th, 76th, 77th, or 79th place may be included. The amino acid substitution introduced at such a position may be arginine or lysine. A variant having 4 or more amino acid substitutions tends to exhibit a stronger charging effect than a variant having fewer amino acid substitutions.

(22-8) Cell uptake of antibodies containing pI-raising Fab region variants

The following assay was performed in order to evaluate the rate of intracellular uptake into hFcγRIIb-expressing cell lines using the produced antibody containing the Fab region variant.

Using a known method, a MDCK (Madin-Darby canine kidney) cell line that normally expresses hFcγRIIb was constructed. Using these cells, the intracellular uptake of the antigen-antibody complex was evaluated. Specifically, human C5 was labeled according to a previously established protocol using Alexa555 (Life Technologies), and an antigen-antibody complex was formed in the culture medium at an antibody concentration of 10 mg/mL and an antigen concentration of 10 mg/mL. For the culture plate of the above hFcγRIIb normal expression MDCK cells, after incubating for 1 hour by adding a culture medium containing an antigen-antibody complex, the fluorescence intensity of the antigen absorbed into the cells was quantified using InCell Analyzer6000 (GE healthcare). did. The amount of introduced antigen was expressed as a relative value when the original Ab2 value was set to 1.00.

The quantitative results of cell uptake are summarized in Tables 36 and 37. In some heavy chain variants and light chain variants, strong antigen-derived fluorescence in cells was observed. Here, when the fluorescence intensity of the antigen absorbed into the cells of the variant is about 1.5 times or more compared to the fluorescence intensity of the original Ab2, it was shown that it has a strong charging effect on the antigen absorbed into the cell.

Among the pi-raising heavy chain variants, antibodies with G8R, L18R, Q39K, P41R, G44R, L63R, F63R, Q64K, Q77R, T77R, L82K, S82aN, S82bR, T83R, A85R, or E85G substitution (according to Kabat numbering) , Showed stronger antigen uptake into cells. It is estimated that this single amino acid substitution in the heavy chain or a combination of these substitutions has a strong charging effect on the uptake of the antigen-antibody complex into cells. Therefore, at one or more positions that are expected to cause the absorption of the antigen-antibody complex into the cell more quickly or more frequently by introducing a modification that raises the pI in the heavy chain variable region of the antibody, for example, , 8th, 18th, 39th, 41st, 44th, 63rd, 64th, 77th, 82nd, 82a, 82b, 83th, or 85th according to Kabat numbering may be included. The amino acid substitution introduced at such a position may be asparagine, glycine, serine, arginine, or lysine, preferably arginine or lysine.

Among the pI-elevating light chain variants, antibodies with G16K, Q27R, S27R, G41R, S52R, S56R, S65R, T69R, T74K, S76R, S77R, or Q79K substitution (according to Kabat numbering) have stronger antigen uptake into cells. Indicated. It is estimated that this single amino acid substitution in the light chain or a combination of these substitutions has a strong charging effect on the uptake of the antigen-antibody complex into cells. A variant having 4 or more amino acid substitutions tends to exhibit a stronger charging effect than a variant having fewer amino acid substitutions. As shown in Example 21(21-3), the combination of 42K substitution and 76R substitution is effective for IgE antibodies. However, in the case of the C5 antibody, the amino acid at position 42 in Kabat numbering is already lysine, so the present inventors can observe the charging effect of 42K/76R by a single substitution of 76R. The fact that the variant having the 76R substitution had a strong charging effect also in the C5 antibody indicates that the combination of 42K/76R has a strong charging effect regardless of the antigen. Therefore, at one or more positions that are expected to cause the absorption of the antigen-antibody complex into the cell more quickly or more frequently by introducing a modification that raises the pi into the light chain variable region of the antibody, for example , According to Kabat numbering, the 16th, 27th, 41st, 52nd, 56th, 65th, 69th, 74th, 76th, 77th, or 79th place may be included. The amino acid substitution introduced at such a position may be arginine or lysine.

(22-9) Evaluation of C5 clearance in a mouse simultaneous administration model

Several anti-C5 bispecific antibodies (original Ab2, 20L233-005, 20L233-006, and 20L233-009) were tested in a mouse co-administration model to evaluate their ability to accelerate the clearance of C5 from plasma. In the simultaneous administration model, C57BL6J mice (Jackson Laboratories) were each administered with an anti-C5 bispecific antibody and C5 previously mixed by single intravenous administration. All groups were administered 0.1 mg/kg of C5 with 1.0 mg/kg of anti-C5 bispecific antibody. The total concentration of C5 in plasma was determined by anti-C5 ECLIA. First, anti-human C5 mouse IgG was dispensed onto an ECL plate and left to stand at 5°C overnight to prepare an anti-human C5 mouse IgG immobilized plate. The calibration curve samples and samples were mixed with anti-human C5 rabbit IgG. These samples were added to an anti-human C5 mouse IgG immobilization plate, and left standing at room temperature for 1 hour. Next, these samples were reacted with HRP-conjugated anti-rabbit IgG (Jackson Immuno Research). After the plate was incubated for 1 hour at room temperature, an anti-HRP antibody conjugated with a sulfo tag was added. ECL signals were read using Sector Imager 2400 (Meso Scale discovery). The concentration of human C5 was calculated from the ECL signal in the calibration curve using SOFTmax PRO (Molecular Devices). 39 shows the temporal profile of the plasma concentration of C5 in C57BL6J mice.

All bispecific antibodies with pi raising substitutions investigated in this test showed rapid C5 clearance from plasma compared to the original Ab2. Therefore, it is suggested that amino acid substitutions at T74K/S77R, S76R/Q79K, and Q37R of the light chain accelerate the disappearance of the C5 antibody immune complex also in vivo. Moreover, the C5 loss of 20L233-005 and 20L233-006 was faster than that of 20L233-009, which was consistent with in vitro imaging and BIACORE analysis. These results were tested under either the Invitro system using the fluorescence intensity of the InCell Analyzer 6000 or the Invitro BIACORE system described above, and the position considered to not contribute to the clearance of the antigen from the plasma in vivo. Even in, suggests that by the use of a more sensitive in vivo system, it can be found that it is contributing to it. These results show that the sensitivity of the in vitro system using fluorescence intensity by InCell Analyzer 6000 may be higher than that of the in vitro BIACORE system with regard to estimation of the evaluation of the clearance of the antigen from plasma in vivo. It also suggests.

Example 23

Evaluation of clearance of IgE from plasma using pi-raising Fc variant

In order to increase the clearance of human IgE or human C5, a pH-dependent antibody was used to evaluate the pI elevation substitution in the Fc portion of the antibody. The method of adding an amino acid substitution to the antibody constant region in order to increase pI is not particularly limited, but can be carried out, for example, by the method described in WO2014/145159.

(23-1) Preparation of antibodies with elevated pi by single amino acid alteration in the normal region

The tested antibodies are summarized in Table 38. Heavy chain Ab1H-P1394m (SEQ ID NO: 307) was prepared by introducing substitution Q311K that raises pi into Ab1H. By introducing each substitution shown in Table 38 into Ab1H according to the method shown in Reference Example 1, another heavy chain modified product was also prepared. All heavy chain variants were expressed together with Ab1L as the light chain.

(23-2) pI Increase Fc domain Variant Using an antibody containing, To BIACORE Human FcγRIIb binding assay

In order to evaluate the charging effect of the antigen-antibody complex formed by the use of the antibodies shown in Table 38 on FcRγRIIb binding, an FcRγRIIb binding assay was performed in the same manner as described in Example 21 (21-2). Table 38 shows the assay results. Here, compared with the binding of the original Ab1 to hFcγRIIb, when the binding of the variant to hFcγRIIb is about 1.2 times or more, it was shown that it has a strong charging effect on the binding of the antibody to hFcγRIIb on the sensor chip.

Among the pI-elevating variants having a single amino acid substitution from the original Ab1, the antigen-antibody complex produced by several modifications, such as P1398m, P1466m, P1482m, P1512m, P1513m, and P1514m, has the strongest binding to hFcγRIIb. Indicated. This single amino acid substitution in D413K, Q311R, P343R, D401R, D401K, G402R, Q311K, N384R, N384K, or G402K is estimated to have a strong charging effect on the binding to hFcγRIIb on the sensor chip. Therefore, in a position where it is expected to exhibit an effect of accelerating the rate or rate of absorption into cells in vivo by introducing a single alteration that increases the pi in the constant region or the Fc region of the antibody, for example, EU 311th, 343th, 384th, 401st, 402th, or 413th may be included according to numbering. The amino acid substitution introduced at such a position may be arginine or lysine.

(23-3) Cell uptake of an antibody containing a pI-raising Fc region variant

In order to evaluate the intracellular uptake of the antigen-antibody complex formed by the antibodies shown in Table 38, a cell imaging assay was performed in the same manner as described in Example 21 (21-3). Table 38 shows the assay results. Here, when the fluorescence intensity of the antigen absorbed into the cells of the variant is about 1.5 times or more compared to the fluorescence intensity of the original Ab1, it was shown that it has a strong charging effect on the antigen absorbed into the cell.

Antigen-antibody complex produced by several modifications such as P1398m, P1466m, P1469m, P1470m, P1481m, P1482m, P1483m, P1512m, P1513m, and P1653m among the pI-elevating variants having a single amino acid substitution from the original Ab1. Showed stronger antigen uptake into cells. This single amino acid substitution in D413K, Q311R, N315K, N384R, Q342K, P343R, P343K, D401R, D401K, or D413R is estimated to have a strong charging effect on the uptake of antigen-antibody complexes into cells. Therefore, at a position where it is expected to cause the absorption of the antigen-antibody complex into cells more quickly or at a higher frequency by introducing a single alteration that raises the pi into the constant region or the Fc region of the antibody, for example, 311, 315, 342, 343, 384, 401, or 413 according to EU numbering can be included. The amino acid substitution introduced at such a position may be arginine or lysine.

(23-4) Evaluation of clearance of human IgE in a mouse simultaneous administration model

Several pH-dependent antigen-binding anti-IgE antibodies (original Ab1, P1466m, P1469m, P1470m, P1480m, P1482m, P1512m, P1653m) were tested in a mouse co-administration model to demonstrate their ability to accelerate the clearance of IgE from plasma. Evaluated. The assay was carried out in the same manner as described in Example 21 (21-4). 40 shows the time profile of the plasma concentration in C57BL6J mice.

After administration of the pI-raising pH-dependent antigen-binding variant (single amino acid substitution only) excluding P1480m, the total IgE concentration in plasma was lower than that of the original Ab1. P1480m, which showed a weak effect in either of the in vitro tests, did not accelerate the loss of IgE. Moreover, the total concentration of IgE in plasma in the mice treated with the mutant having a high pI that does not involve pH-dependent antigen binding was significantly higher than that of the pH-dependent antigen-binding mutant having a high pI (data not shown). These results indicate that the cellular uptake of the antigen-antibody immune complex is increased by the introduction of a modification that increases pI. The antigen absorbed into cells in a complex state with a pH-dependent antigen-binding antibody can be effectively released from the antibody inside the endosome, thereby accelerating the loss of IgE. These results were tested under the in vitro BIACORE system described above and contributed to it by the use of the more sensitive in vivo system, even at the substitutional position that is thought to not contribute to the clearance of the antigen from the plasma in vivo. It suggests that it can be found that it is doing. These results show that the sensitivity of the above in vitro system using fluorescence intensity by InCell Analyzer 6000 may be higher than that of the above in vitro BIACORE system with regard to estimation of the evaluation of the clearance of antigen from plasma in vivo. Also suggests.

Reference Example 1

Construction of an expression vector of an IgG antibody substituted with an amino acid

Using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), by inserting an extranuclear gene fragment containing a variant produced by the method described in the accompanying instructions into an animal cell expression vector, the desired H chain expression vector and L chain expression vector Was produced. The base sequence of the obtained expression vector was determined by a method known to those skilled in the art.

Reference Example 2

Expression and purification of IgG antibodies

Expression of the antibody was performed using the following method. HEK293H strain (Invitrogen) derived from human fetal kidney cancer cells was suspended in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen), and a dish for adherent cells at a cell density of ^{5 to 6×10 5 cells/mL (diameter} 10 cm, CORNING) in each dish, 10 mL each, and _{cultured in a CO 2} incubator (37° C., 5% CO ₂ ) overnight, and then aspirated off the medium, and CHO-S-SFM-II (Invitrogen) medium 6.9 mL was added. The prepared extranuclear gene was introduced into cells by lipofection. After the obtained culture supernatant was collected, the cells were removed by centrifugation (about 2000 g, 5 minutes, room temperature), and sterilized by passing through a 0.22 μm filter MILLEX (registered trademark)-GV (Millipore) to obtain a culture supernatant. . The antibody was purified from the obtained culture supernatant by a method known in the art using rProtein A SepharoseTM Fast Flow (Amersham Biosciences). The concentration of the purified antibody was measured for absorbance at 280 nm using a spectrophotometer. From the obtained value, the antibody concentration was calculated using the extinction coefficient calculated by the method described in Pace et al., Protein Science 4: 2411-2423 (1995).

Reference Example 3

Preparation of soluble human IL-6 receptor

Recombinant soluble human IL-6 receptor as an antigen was prepared as follows. Mullberg et al., J. Immunol. 152: 4958-4968 (1994), a CHO cell line that normally expresses the soluble human IL-6 receptor consisting of the 1st to 357th amino acid sequence on the N-terminal side was constructed by a method known to those skilled in the art. The soluble human IL-6 receptor was expressed by culturing the CHO strain. From the culture supernatant of the obtained CHO strain, the soluble human IL-6 receptor was purified by two steps of Blue Sepharose 6FF column chromatography and gel filtration column chromatography. The fraction eluted as the main peak in the final process was used as the final refined product.

SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA <120> IL-8-BINDING ANTIBODIES 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 80 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 Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 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 80 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 Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 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 80 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 Thr Leu Leu Phe Ser Trp Ala Ser Ile Arg Asp Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Ala Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 65 70 75 80 Ile Ser Asp Leu Gln Ala Glu Asp Ala Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Arg Ala Pro Ser Phe Gly Gln Gly Thr Lys Leu Gln Ile Lys 100 105 110 <210> 5 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 6RL#9-IgG1 <400> 5 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 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 Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 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 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 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 Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 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 80 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 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 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 80 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 Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 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 80 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 Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 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 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 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 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 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 80 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 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> 12 <211> 433 <212> PRT <213> Homo Sapian <400> 12 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 Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 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 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 115 120 125 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 130 135 140 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 145 150 155 160 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 Val Glu Pro Lys Ser Cys Asp Lys 195 200 205 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 210 215 220 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 225 230 235 240 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 245 250 255 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 260 265 270 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 275 280 285 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 290 295 300 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 305 310 315 320 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 325 330 335 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 340 345 350 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 355 360 365 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 370 375 380 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 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 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 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 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr 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 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 20 25 30 Glu Met His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 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 80 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 Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 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 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 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 Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 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 Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45 Pro Arg 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 80 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 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 Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 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 80 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 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Ser Cys 210 215 220 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 290 295 300 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 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 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 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 Ala Lys Thr Leu Ala 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 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Glu Ser Pro Leu 85 90 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 80 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 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 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 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 Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 19 <211> 326 <212> PRT <213> Homo Sapian <400> 19 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 Asn Phe Gly Thr Gln Thr 65 70 75 80 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 Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 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 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 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 80 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 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 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 Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 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 80 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 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 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 80 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 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 1 5 10 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 80 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 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 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 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 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 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> 25 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> VH3(High_pI)-IgG1 <400> 25 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 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 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 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 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 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 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 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 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 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 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 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 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 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 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 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 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 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 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 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> 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 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 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 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 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> 29 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> VH3(High_pI)-F1180 <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 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 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 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 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 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 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 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 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 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 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 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 Tyr His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 31 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> VH3(High_pI)-F11 <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 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 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 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 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 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 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> 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 Ser Ala Ser Val Gly 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 Pro Gly Lys Ala Pro Glu 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 Glu Ala 65 70 75 80 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 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 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 80 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 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> 34 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H54 <400> 34 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 Asp Asp 20 25 30 Gln Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Tyr 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 Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala 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 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> 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 80 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 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> 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 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 Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 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 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 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 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 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 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr 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 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 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 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 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 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> 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 Ser Val Gly 1 5 10 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 80 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 Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 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 80 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 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 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 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 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 Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Asn Ile Tyr Ser Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 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 80 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 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 Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Ala Ser 1 5 10 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 80 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 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 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 Phe Glu Leu Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly 1 5 10 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 80 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 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> 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 Lys Asp Asn 1 5 10 15 Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser Pro Leu Lys 20 25 30 <210> 45 <400> 45 000 <210> 46 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> VH3-IgG1m <400> 46 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 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 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 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 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 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 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 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 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 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> 48 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> VH3-LS <400> 48 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 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 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 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 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 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 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 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 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 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 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 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 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 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 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 <400> 54 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 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 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 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 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu 420 425 430 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 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 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 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 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 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 <210> 56 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> OHBH-IgG1 <400> 56 Gln Val Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr Glu 1 5 10 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 80 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 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> 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 Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Ser Ser 20 25 30 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 80 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 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 Val Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr Glu 1 5 10 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 80 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 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 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 <220> <223> OHBH-N434A <400> 59 Gln Val Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr Glu 1 5 10 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 80 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 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 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 Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr Glu 1 5 10 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 80 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 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 Ala His Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445 <210> 61 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> OHBH-F1848m <400> 61 Gln Val Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr Glu 1 5 10 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 80 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 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 Ala His Val Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445 <210> 62 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> OHBH-F1886m <400> 62 Gln Val Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr Glu 1 5 10 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 80 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 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 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 Gln Val Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr Glu 1 5 10 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 80 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 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 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 Thr Leu Lys Glu Ser Gly Gly Arg Leu Val Lys Pro Thr Glu 1 5 10 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 80 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 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 Leu His Glu 420 425 430 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 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 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 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 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 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 <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 65 70 75 <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> <223> HVR-H2 of WS4 <400> 68 Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Arg Glu Tyr Ser Ala Ser 1 5 10 15 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 <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 10 <210> 75 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 of L395 <400> 75 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 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 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 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 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 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 80 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 115 120 <210> 79 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of L395 <400> 79 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 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 80 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 <400> 80 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 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 80 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 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 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> 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 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 80 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 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 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 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 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 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 80 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 <220> <223> hWS4H-IgG1 <400> 83 Glu 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 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 80 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 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 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 <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 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 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 80 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 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> 85 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> Hr9-IgG1 <400> 85 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 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 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 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 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> 86 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H89-IgG1 <400> 86 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 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 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 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 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> 87 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> L63-k0MT <400> 87 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 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 80 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 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 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 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 80 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> 89 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H998-IgG1 <400> 89 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 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 80 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 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 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> 90 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H553-IgG1 <400> 90 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 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 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 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 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 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> 91 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H1004-IgG1m <400> 91 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 Asp Asn Gly Tyr 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 80 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 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 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> 92 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H1009-IgG1m <400> 92 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 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 80 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 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 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 <212> PRT <213> Artificial Sequence <220> <223> 1009-F1942m <400> 93 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 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 80 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 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 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> 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 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 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 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 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 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 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 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 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 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 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 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 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 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 Thr Thr Arg Lys Glu Leu Ser Leu 435 440 445 Ser Pro 450 <210> 97 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H89-F1848m <400> 97 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 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 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 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 Ser Pro 450 <210> 98 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H89-F1886m <400> 98 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 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 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 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 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> <223> H89-F1889m <400> 99 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 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 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 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 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 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 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 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 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 Leu His Glu Ala Leu His Ala His Tyr Thr Arg Lys Glu Leu Ser Leu 435 440 445 Ser Pro 450 <210> 101 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> H496-IgG1 <400> 101 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 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 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 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 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 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> 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 Val Lys 1 5 10 15 Gly <210> 104 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 <400> 104 Asp Arg Ile Ala Val Ala Asp Tyr 1 5 <210> 105 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> HVR-L1 <400> 105 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 106 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 <400> 106 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 107 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 107 Gln Gln Tyr Gly Ser Ser Phe Thr 1 5 <210> 108 <211> 5 <212> PRT <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 <210> 112 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 <400> 112 Gly Thr Phe Ile Arg Ala Thr 1 5 <210> 113 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 113 Gln Gln Phe Gly Arg Ser Pro Leu Thr 1 5 <210> 114 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HVR-H1 <400> 114 Ser Tyr Gly Met Leu 1 5 <210> 115 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> HVR-H2 <400> 115 Asp Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 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 <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 Tyr Trp Thr 1 5 <210> 121 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HVR-H2 <400> 121 Glu Val Ile His His Gly Ser Thr Asn Tyr Ser Pro Ser Leu Lys Ser 1 5 10 15 <210> 122 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-H3 <400> 122 Gly Gly Ala Ala Ala Ala Leu Asp Ser 1 5 <210> 123 <211> 17 <212> PRT <213> Artificial Sequence <220> <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> 124 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 <400> 124 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 125 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 125 Gln Gln Tyr Tyr Ser Thr Pro Phe Thr 1 5 <210> 126 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HVR-H1 <400> 126 Ser Tyr Trp Met His 1 5 <210> 127 <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 Thr Ser Thr Leu Gln Pro 1 5 <210> 131 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 131 Leu Gln Tyr Asp Asn Leu Trp Thr 1 5 <210> 132 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HVR-H1 <400> 132 Asn Tyr Trp Ile Val 1 5 <210> 133 <211> 17 <212> PRT <213> 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> 134 <211> 10 <212> PRT <213> Artificial Sequence <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 Ser 1 5 10 <210> 136 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HVR-L2 <400> 136 Tyr Ala Thr Arg Leu Ala Asp 1 5 <210> 137 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HVR-L3 <400> 137 Leu Gln His Gly Glu Ser Pro Pro Thr 1 5 <210> 138 <211> 5 <212> PRT <213> Artificial Sequence <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 <210> 142 <211> 7 <212> PRT <213> 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 Thr 1 5 <210> 144 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H003 <400> 144 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 Arg 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 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 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 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> 145 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H005 <400> 145 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 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 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 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 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> PRT <213> Artificial Sequence <220> <223> H010 <400> 146 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 Arg 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 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 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 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 80 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 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 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 Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 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 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 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 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 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 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 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> H016 <400> 150 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 80 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 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 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 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 80 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 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 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 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 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 Arg 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 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 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> 153 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H027 <400> 153 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 Arg Val Tyr Leu 65 70 75 80 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 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 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 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 Arg Val Tyr Leu 65 70 75 80 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 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 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 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 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 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 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> 156 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H030 <400> 156 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 Arg Val Tyr Leu 65 70 75 80 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 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 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> 157 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H031 <400> 157 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 Arg Val Tyr Leu 65 70 75 80 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 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 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> 158 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H032 <400> 158 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 80 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 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 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 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 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 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 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 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> 160 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H035 <400> 160 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Arg 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 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 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 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 <213> Artificial Sequence <220> <223> H039 <400> 161 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 Lys 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 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 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> 162 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> H041 <400> 162 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 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 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 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> 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 80 Gln Ile Asp 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 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 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> 164 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L001 <400> 164 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Lys 1 5 10 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 80 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> 165 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L002 <400> 165 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 166 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L003 <400> 166 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 167 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L004 <400> 167 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 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 65 70 75 80 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> 168 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L005 <400> 168 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 80 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> 169 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L006 <400> 169 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 170 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L007 <400> 170 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser 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 65 70 75 80 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> 171 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L008 <400> 171 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 80 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> 172 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L009 <400> 172 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 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 65 70 75 80 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> 173 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L010 <400> 173 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 80 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> 174 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L011 <400> 174 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 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 80 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> 175 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L012 <400> 175 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 80 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> 176 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L013 <400> 176 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 Lys Ile Ser Arg Leu Gln Pro 65 70 75 80 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> 177 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L014 <400> 177 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 Arg Ser Leu Lys Pro 65 70 75 80 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> 178 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L015 <400> 178 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Lys 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 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 80 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> 179 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L016 <400> 179 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 180 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L017 <400> 180 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 181 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L018 <400> 181 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 65 70 75 80 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> 182 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L019 <400> 182 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 183 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L020 <400> 183 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 184 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L021 <400> 184 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 185 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L022 <400> 185 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 186 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L023 <400> 186 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 187 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L024 <400> 187 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Lys 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 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 80 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> 188 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L025 <400> 188 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 65 70 75 80 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> 189 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L026 <400> 189 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 190 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L028 <400> 190 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 191 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L029 <400> 191 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 192 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L030 <400> 192 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 193 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L032 <400> 193 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 Arg Ser Leu Lys Pro 65 70 75 80 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> 194 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L033 <400> 194 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 195 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L034 <400> 195 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 Arg 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 80 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> 196 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L035 <400> 196 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 197 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L036 <400> 197 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 198 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L037 <400> 198 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 Lys Ile Ser Arg Leu Gln Pro 65 70 75 80 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> 199 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L038 <400> 199 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 200 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L039 <400> 200 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 Arg Arg Leu Lys Pro 65 70 75 80 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> 201 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L040 <400> 201 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 80 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> 202 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L041 <400> 202 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 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 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 65 70 75 80 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> 203 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L061 <400> 203 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 80 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> 204 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> L062 <400> 204 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 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 Thr Glu Tyr Ala Leu Thr Ile Ser Ser Leu Lys Pro 65 70 75 80 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> 205 <400> 205 000 <210> 206 <400> 206 000 <210> 207 <211> 1676 <212> PRT <213> Artificial Sequence <220> <223> Recombinant human C5 <400> 207 Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile Phe Leu Gly Lys Thr 1 5 10 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 80 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 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 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 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 Gly Thr Arg Glu Lys Phe 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 Ala Tyr Ser Pro Gly Gln Thr Val Ser Leu Asn Met 580 585 590 Ala Thr Gly Met Asp Ser Trp Val Ala Leu Ala Ala Val Asp Ser Ala 595 600 605 Val Tyr Gly Val Gln Arg Gly 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 Asn 645 650 655 Ala Asn Ala Asp Asp Ser Gln Glu Asn Asp Glu Pro Cys Lys Glu Ile 660 665 670 Leu Arg Pro Arg Arg Thr Leu Gln Lys Lys Ile Glu Glu Ile Ala Ala 675 680 685 Lys Tyr Lys His Ser Val Val Lys Lys Cys Cys Tyr Asp Gly Ala Cys 690 695 700 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 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 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 Leu Ile Glu Lys Gln Lys Leu Lys Lys Lys Leu Lys Glu Gly Met 1040 1045 1050 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 Glu Thr Thr Ala Tyr Ala Leu Leu Thr Ser Leu Asn Leu Lys Asp 1250 1255 1260 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 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 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 Glu Asp Ile Phe Leu Asn Gly Cys 1670 1675 <210> 208 <211> 1676 <212> PRT <213> Artificial Sequence <220> <223> cynomolgus monkey C5 <400> 208 Met Gly Leu Leu Gly Ile Leu Cys Phe Leu Ile Phe Leu Gly Lys Thr 1 5 10 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 80 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 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 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 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 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 Tyr 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 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 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 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 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 Ser Arg 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 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 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 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 Glu Ser Ser Ser Gly Ser Ser His Ala Val Met Asp Ile Ser Leu 1415 1420 1425 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 Ala Leu Gln Ile Lys Tyr Asn Phe Thr 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 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 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 80 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 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> CFP0011H <400> 211 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 <400> 212 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 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 80 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 Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 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 80 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 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 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 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 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 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 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 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 80 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 <212> PRT <213> Artificial Sequence <220> <223> CFP0017H <400> 217 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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> 218 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> CFP0017L <400> 218 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 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 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 219 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H <400> 219 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 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 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 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 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 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 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 80 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 <213> Artificial Sequence <220> <223> CFP0020H <400> 223 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 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 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 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 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 80 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 Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Thr Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala 50 55 60 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 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 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 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 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asp Ser Tyr Pro Leu 85 90 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 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 80 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 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 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 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 Lys Ser Leu Ser Leu Ser Pro 325 <210> 228 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> G1dN1 <400> 228 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 80 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 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 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 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 Ser 65 70 75 80 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 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 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> 230 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-G1dN1_optimized 18 heavy chain <400> 230 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 231 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-k0_optimized 20//18 light chain <400> 231 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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 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 <210> 232 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-001-G1dP1 <400> 232 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 Arg Gly Lys Arg 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 80 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 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 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> 233 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-002-G1dP1 <400> 233 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 Arg Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Arg 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 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 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> 234 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-003-G1dP1 <400> 234 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 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 80 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 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 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> <223> CFP0020H0261-005-G1dP1 <400> 235 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Arg 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 Ser 65 70 75 80 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 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 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 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 Arg 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 80 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 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 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> 237 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-009-G1dP1 <400> 237 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 Lys 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 80 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 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 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> 238 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-013-G1dP1 <400> 238 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 Arg 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 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 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 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> 239 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-018-G1dP1 <400> 239 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 Arg 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 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 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 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> 240 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-019-G1dP1 <400> 240 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 Ser 65 70 75 80 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 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 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 <210> 241 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-020-G1dP1 <400> 241 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 Ser 65 70 75 80 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 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 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> 242 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-021-G1dP1 <400> 242 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 Ser 65 70 75 80 Leu Lys Leu Gly 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 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 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> 243 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-022-G1dP1 <400> 243 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 Ser 65 70 75 80 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 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 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> 244 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-023-G1dP1 <400> 244 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 Ser 65 70 75 80 Leu Lys Leu Ser Ser Arg 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 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 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> 245 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-025-G1dP1 <400> 245 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 Ser 65 70 75 80 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 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 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 Ser Pro 450 <210> 246 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-026-G1dP1 <400> 246 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 Ser 65 70 75 80 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 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 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 <210> 247 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-032-G1dP1 <400> 247 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 Ser 65 70 75 80 Leu Lys Lys 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 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 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> 248 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-035-G1dP1 <400> 248 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 Ser 65 70 75 80 Leu Lys Leu Ser Arg Val Arg 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 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 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> 249 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-036-G1dP1 <400> 249 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 Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Arg 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 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 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> 250 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CFP0020H0261-037-G1dP1 <400> 250 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 Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Gly 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 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 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> 251 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-002-G1dN1 <400> 251 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Arg Ala Tyr 65 70 75 80 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 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 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> 252 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-003-G1dN1 <400> 252 Gln Val Gln Leu Val Gln Ser Arg Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 253 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-005-G1dN1 <400> 253 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Arg Ser 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 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 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 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 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 254 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-008-G1dN1 <400> 254 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Arg 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 80 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 225 230 235 240 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 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 255 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-009-G1dN1 <400> 255 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 80 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 225 230 235 240 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 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 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Arg 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 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 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 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 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 258 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-016-G1dN1 <400> 258 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Arg 50 55 60 Lys 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 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 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 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> <223> CFP0018H0012-018-G1dN1 <400> 259 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Arg 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 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 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 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 Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 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 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 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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Asn 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 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 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> 262 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-021-G1dN1 <400> 262 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 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 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 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 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 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 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 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> 264 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-023-G1dN1 <400> 264 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 265 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-024-G1dN1 <400> 265 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 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> 266 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-025-G1dN1 <400> 266 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 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> 267 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-026-G1dN1 <400> 267 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 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> 268 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-027-G1dN1 <400> 268 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 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 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 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 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 445 <210> 269 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CFP0018H0012-032-G1dN1 <400> 269 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Lys 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 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 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 <213> Artificial Sequence <220> <223> CFP0018H0012-037-G1dN1 <400> 270 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 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 Gln Gly Arg Val Thr Ile Thr Arg Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 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 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 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> 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 Ser Val Lys 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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 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 <210> 272 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-002-k0 <400> 272 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 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 80 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 <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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 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 <210> 274 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-004-k0 <400> 274 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 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 80 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 <210> 275 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-005-k0 <400> 275 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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 80 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 <210> 276 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-006-k0 <400> 276 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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 80 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 <210> 277 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-007-k0 <400> 277 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 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 80 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 <210> 278 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-008-k0 <400> 278 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 Lys Lys 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 80 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 <210> 279 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-009-k0 <400> 279 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 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 80 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 <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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 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 <210> 281 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-011-k0 <400> 281 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 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 80 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 <210> 282 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-012-k0 <400> 282 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 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 80 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 <210> 283 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-013-k0 <400> 283 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 Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 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 <210> 284 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-016-k0 <400> 284 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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 80 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 <210> 285 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-017-k0 <400> 285 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 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 Lys Ile Ser Arg Leu Gln Pro 65 70 75 80 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 <210> 286 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-018-k0 <400> 286 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 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 80 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 <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 Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 80 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 <210> 288 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-021-k0 <400> 288 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 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 80 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 <210> 289 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-022-k0 <400> 289 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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 80 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 <210> 290 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-023-k0 <400> 290 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 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 80 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 <210> 291 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-024-k0 <400> 291 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 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 80 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 <210> 292 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-025-k0 <400> 292 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 Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 80 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 <210> 293 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-027-k0 <400> 293 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 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 80 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 <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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 80 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 <210> 295 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-029-k0 <400> 295 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 Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Lys Ile Ser Arg Leu Gln Pro 65 70 75 80 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 <210> 296 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-031-k0 <400> 296 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 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 80 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 <210> 297 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-032-k0 <400> 297 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 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 80 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 <210> 298 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-034-k0 <400> 298 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 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 80 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 <210> 299 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-036-k0 <400> 299 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 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 Thr Ile Arg Ser Leu Lys Pro 65 70 75 80 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 <210> 300 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-037-k0 <400> 300 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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 80 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 <210> 301 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-038-k0 <400> 301 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 Gly 50 55 60 Arg Gly Ser Gly Arg Asp Phe Thr Leu Thr Ile Arg Ser Leu Lys Pro 65 70 75 80 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 <210> 302 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-040-k0 <400> 302 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 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 80 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 <210> 303 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-041-k0 <400> 303 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 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 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 <210> 304 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-043-k0 <400> 304 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 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 80 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 <210> 305 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> CFP0020L233-044-k0 <400> 305 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 Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro 65 70 75 80 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 <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 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 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Lys Pro 65 70 75 80 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 <210> 307 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1394m <400> 307 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 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 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 Lys 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> 308 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1398m <400> 308 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 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 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 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 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 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 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 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 385 390 395 400 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> 310 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1468m <400> 310 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 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 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 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 <210> 311 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1469m <400> 311 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 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 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 Lys 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> 312 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1470m <400> 312 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 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 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 Arg 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> 313 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1471m <400> 313 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 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 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 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 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> 314 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1480m <400> 314 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 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 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 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 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1481m <400> 315 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 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 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 Lys 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> 316 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1482m <400> 316 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 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 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 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 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> 317 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1483m <400> 317 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 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 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 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 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 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 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 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 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> <223> Ab1H-P1513m <400> 319 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 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 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 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 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 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 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 Arg 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> 321 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1515m <400> 321 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 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 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 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 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 322 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Ab1H-P1653m <400> 322 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 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 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 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 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 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 80 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 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 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 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 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 80 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 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 Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 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 Met His Glu Ala Leu His 420 425 430 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 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 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 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 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> 326 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> F8M-F1868mv1 <400> 326 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 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 80 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 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 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 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 Thr Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440 445 <210> 327 <211> 444 <212> PRT <213> 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 Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr 65 70 75 80 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 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 Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 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 Thr Thr Arg Glu Glu Leu Ser Leu Ser Pro 435 440 <210> 328 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> F8M-F1927mv1 <400> 328 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 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 80 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 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 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 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 <212> PRT <213> Artificial Sequence <220> <223> F8M-F1927mv2 <400> 329 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 Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr 65 70 75 80 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 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 Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 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 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 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 80 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 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 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 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 Lys 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 Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 <210> 331 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> J327-z119 <400> 331 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 Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr 65 70 75 80 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 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 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 Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 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 Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro 435 440 <210> 332 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> L404-k <400> 332 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 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 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu 85 90 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

Claims (26)

8. An isolated anti-IL-8 antibody that binds to human IL-8, which comprises at least one amino acid substitution in at least one of the following (a) to (f) IL-8 antibody:
(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 method according to claim 1,
Comprising the amino acid substitution of the tyrosine at position 9 of the amino acid sequence of SEQ ID NO: 68, the arginine at position 11 of the amino acid sequence of SEQ ID NO: 68, and the tyrosine at position 3 of the amino acid sequence of SEQ ID NO: Antibody. The method according to claim 1 or 2, wherein
An alanine at position 6 of the amino acid sequence of SEQ ID NO: 68 and an amino acid substitution of the glycine at position 8 of the amino acid sequence of SEQ ID NO: 68. 4. The method according to any one of claims 1 to 3,
An asparagine at position 1 of the amino acid sequence of SEQ ID NO: 71, a leucine at position 5 of the amino acid sequence of SEQ ID NO: 71, and an amino acid substitution at position 1 of glutamine at the amino acid sequence of SEQ ID NO: Antibody. 5. The method according to any one of claims 1 to 4,
An anti-IL-8 antibody comprising an Fc region having at least one property selected from the following properties (a) to (f):
(a) the binding affinity for the FcRn of the Fc region at the acidic pH is greater than the binding affinity for the FcRn of the native Fc region;
(b) the binding affinity of the Fc region for the existing ADA is lower than the binding affinity for the native ADA of the native Fc region;
(c) the plasma half-life of the Fc region is greater than the plasma half-life of the native Fc region;
(d) the clearance in the plasma of the Fc region is lower than the clearance in the plasma of the native Fc region; And
(e) the binding affinity of the Fc region for the effector receptor is lower than the binding affinity for the effector receptor of the native Fc region; And
(f) the binding to the extracellular matrix is increased. 6. The method of claim 5,
The Fc region is located at one or more positions selected from the group consisting of 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440 according to the EU numbering An anti-IL-8 antibody comprising an amino acid substitution. The method according to claim 6,
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. 8. The method of claim 7,
Wherein the Fc region comprises amino acid substitutions of L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E. 8. The method of claim 7,
Wherein the Fc region comprises amino acid substitutions of L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E. 9. An isolated nucleic acid encoding the anti-IL-8 antibody of any one of claims 1 to 9. 12. A vector comprising a nucleic acid according to claim 10. 12. A host cell comprising the vector of claim 11. A method for producing an anti-IL-8 antibody, which comprises culturing the host cell of claim 12. 14. The method of claim 13,
8. A method for producing an anti-IL-8 antibody, comprising the step of isolating the antibody from the culture supernatant. 10. A pharmaceutical composition comprising an anti-IL-8 antibody according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier. A method of producing an anti-IL-8 antibody, wherein the binding activity to IL-8 comprises a pH-dependent variable region,
(a) a step of evaluating the binding of an IL-8 antibody and an extracellular matrix,
(b) a step of selecting an anti-IL-8 antibody having a high binding to an extracellular matrix,
(c) culturing a host comprising a vector comprising a nucleic acid encoding said antibody, and
(d) a step of isolating the antibody from the culture solution
RTI ID = 0.0 &gt; IL-8 &lt; / RTI &gt; Amino acid substitution at one or more positions selected from the group consisting of 235, 236, 239, 327, 330, 331, 428, 434, 436, 438 and 440 according to EU numbering Lt; RTI ID = 0.0 &gt; IL-8 &lt; / RTI &gt; 18. The method of claim 17,
An anti-IL-8 antibody comprising an Fc region having at least one property selected from the following properties (a) to (f):
(a) the binding affinity for the FcRn of the Fc region at the acidic pH is greater than the binding affinity for the FcRn of the native Fc region;
(b) the binding affinity of the Fc region for the existing ADA is lower than the binding affinity for the native ADA of the native Fc region;
(c) the plasma half-life of the Fc region is greater than the plasma half-life of the native Fc region;
(d) the clearance in the plasma of the Fc region is lower than the clearance in the plasma of the native Fc region;
(e) the binding affinity of the Fc region for the effector receptor is lower than the binding affinity for the effector receptor of the native Fc region; And
(f) the binding to the extracellular matrix is increased. The method according to claim 17 or 18,
Comprising 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 according to EU numbering. Antibody. 20. The method of claim 19,
According to EU numbering, (a) L235R, G236R, S239K, M428L, N434A, Y436T, Q438R, and S440E; Or (b) an Fc region comprising one or more amino acid substitutions selected from the group consisting of L235R, G236R, A327G, A330S, P331S, M428L, N434A, Y436T, Q438R, and S440E. 20. An isolated nucleic acid encoding an anti-IL-8 antibody according to any one of claims 17 to 20. A vector comprising the nucleic acid according to claim 21. 29. A host cell comprising the vector of claim 22. A method for producing an anti-IL-8 antibody, which comprises culturing the host cell according to claim 23. 25. The method of claim 24,
20. A method of producing an anti-IL-8 antibody, further comprising the step of isolating the anti-IL-8 antibody according to any one of claims 17 to 20 from a host cell culture. 20. A pharmaceutical composition comprising an anti-IL-8 antibody according to any one of claims 17 to 20, and a pharmaceutically acceptable carrier.

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