TW201245227A - Antibody Fc variants - Google Patents

Abstract

The invention relates to engineered polypeptides comprising Fc variants and their uses. More specifically, Fc variants are described exhibiting reduced effector function. These variants cause a benefit for a patient suffering from a disease which could be treated with an antibody for which it is desirable to reduce the effector function elicited by antibodies.

Description

201245227 VI. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to polypeptides comprising variants of the Fc region. More particularly, the present invention relates to polymorphisms containing an Fc region that has altered effector function due to substitution by one or more amino acids in the Fc region of the polypeptide. [Prior Art] Monoclonal antibodies have enormous therapeutic potential and play an important role in the current medical industry. A major trend in the pharmaceutical industry over the past decade has been the development of monoclonal antibody φ (mAb) as a therapeutic agent for many diseases such as cancer, asthma, arthritis, and multiple sclerosis. Monoclonal antibodies are produced primarily in the form of recombinant proteins in genetically engineered mammalian cell cultures. The Fc region of the antibody (i.e., the cross-domain of the antibody, the CH2 and a portion of the heavy chain end of the hinge region) is limited in variability and is involved in achieving the physiological effects elicited by the antibody. The effector functions attributed to the region of the antibody vary with the class and subclass of the antibody and include the binding of the antibody via the Fc region to a specific Fc receptor ("FcR") that triggers various biological responses on the cell. Φ These receptors typically have an intracellular domain that mediates extracellular domains that bind to the transmembrane region and mediates some signaling events within the cell. These receptor systems are expressed in a variety of immune cells, including mononuclear spheres, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, and Langerhans. , ceu), natural killer (NK) cells and tau cells. The formation of the "King complex" to recruit these effector cells to the site of binding to the antigen' usually leads to intracellular signaling events and important subsequent immune responses, such as the release of inflammatory mediators 162837.doc 201245227, B Cell activation, endocytosis, phagocytosis, and cytotoxic attack. The ability to mediate cytotoxicity and phagocytic effector functions is a potential mechanism by which antibodies disrupt cell-targeting cells. Non-specific cytotoxic cells that express FcyR recognize target cells. The cell-mediated reaction that binds to the antibody and subsequently causes lysis of the target cell is called antibody-dependent cell-mediated cytotoxicity (ADCC) (Ravetch et al., Annu Rev Immunol 19 (2001) 275-290). A cell-mediated response in which a specific cytotoxic cell recognizes a binding antibody on a target cell and subsequently causes phagocytosis of the target cell is called antibody-dependent cell-mediated phagocytosis (ADCP). In addition, the overlapping position on the Fc | region of the molecule Complement-dependent cell Toxicity (CDC) For the IgG class of Ab, ADCC and ADCP are regulated by the occlusion of the Fc region with a receptor family called the Fey receptor (F cyR). In humans, this family of proteins contains FcyRI (CD64). FcyRII (CD32), including subtypes FcyRIIA, FcyRIIB, and FcyRIIC; and FcyRIII (CD16), including subtypes FcyRIIIA and FcyRIIIB (Raghavan and Bjorkman, Annu. Rev.

Cell Dev. Biol. 12 (1996) 181-220; Abes et al., Expert · Reviews VOL 5(6), (2009) 73 5-747). FcyR is expressed on a variety of immune cells, and the formed Fc/FcyR complex recruits these cells to the site of binding to the antigen, which usually leads to signal transduction and subsequent immune responses, such as release of inflammatory mediators, B cell activation, cells Swallowing, phagocytosis and cytotoxic attack. In addition, 'FcyRI, FcyRIIA/c and FcyRIIIA are characterized by activated receptors based on the intracellular immunoreceptor tyrosine-activated motif (ITAM), whereas FcyRIIB has an inhibitory motif (ITIM) and thus has 162837.doc 201245227 Systematic. In addition, de Reys et al., Blood, 81, (1993) 1792_18 conclude that 'platelet activation induced by monoclonal antibodies (eg, CD9) and agglutination are initiated by antigen recognition followed by Fc structure involving FcyRII-receptors Domain dependent steps (see also: Taylor et al., Blood 96 (2000) 4254-4260). FQRi binds monomeric IgG with high affinity, but Fc^RIII and FcyRII are low-affinity receptors that interact with complex or agglutinated IgG. Complementary inflammatory levels are part of the innate immune response and are critical to the individual's ability to avoid infection. Another important is the complement system φ Clq. The binding of Fc to C1q mediates the process known as complement dependent cytotoxicity (cdc). C 1 q is capable of binding to a scorpion antibody, but binding to both igGs is sufficient to activate the complement cascade. Clq forms a complex with Clr and Cls serine protease to form a C1 complex of the complement pathway. In many cases, the binding mediated by the Fc region of the immunoglobulin and the stimulation of the effector function are extremely beneficial, for example for CD20 antibodies, whereas 'in some cases' may be more advantageously reduced or even Eliminate effector functions. This is especially true for antibodies that are designed to deliver drugs (eg, toxins and isotopes) to the target cells, where Fc/FcyR-mediated effector functions introduce healthy immune cells into the vicinity of the payload, thereby Lead to normal lymphoid tissue and target cell depletion (Hutchins et al, PNAS USA 92 (1995) 11980-11984; White et al., Annu Rev

Med 52 (2001) 125-145). In such cases, the use of antibodies that poorly recruit complement or effector cells can be of great benefit (see also Wu et al.

Human, Cell Immunol 200 (2000) 16-26; Shields et al, J. Biol Chem 276 (9) (2001) 6591-6604; US 6,194,551; US 162837.doc 201245227 5,885,573 and PCT Publication WO 04/029207). In other instances, for example, if the target block blocks the interaction of a widely expressed receptor with its cognate ligand, all antibody effector functions can be advantageously reduced or eliminated to reduce undesired toxicity. Similarly, where therapeutic antibodies exhibit a promiscuous binding between many human tissues, it may be sensible to limit the targeting of effector functions to multiple sets of tissues to limit toxicity. Last but not least, the reduced affinity of the antibody to the FcyRII receptor, in particular, is advantageous for antibodies that induce platelet activation and aggregation via FcYRII receptor binding, and induces platelet activation and agglutination via FcyRII receptor binding. Serious side effects. Despite the existence of certain subclasses of human immunoglobulins that lack specific effector functions, there are no known natural immunoglobulins that lack all effector functions. An alternative approach is to engineer or mutate the key residues in the Fc region responsible for effector function. See, for example, PCT Publications WO 2009/100309 (Medimmune), WO 2006/076594 (Xencor), WO 1999/58572 (Univ. Cambridge), US 2006/0134709 (Macrogenics), WO 2006/047350 (Xencor), WO 2006 /053301 (Xencor) 'US 6,737,056 (Genentech) ' US φ 5,624,821 (Scotgen Pharmaceuticals) and US 2010/0166740 (Roche).

The binding of IgG to the activated and inhibitory Fey receptor or the first component of complement (Clq) depends on the residues located in the hinge region and the CH2 domain. The two regions of the CH2 domain are critical for FcYR and complement Clq binding and have different sequences. Substitution of human IgG1 and IgG2 residues at positions 233 to 236 and IgG4 residues at positions 327, 330 and 331 significantly reduced ADCC 162837.doc 201245227 and CDC (Armour et al., Eur. J. Immunol. 29 (8) (1999) 2613-2624; Shields et al., J. Biol. Chem. 276(9) (2001) 6591-6604). Idusogie et al, J. Immunol 166 (2000) 2571-2575) mapped the C1 q binding site of rituxan and showed that Pro329Ala reduces rituximab (Rituximab) binding to Clq and activation The ability to complement. Substitution of Prola with Ala has been reported to result in reduced binding to the FcyRI, FcyRII and FcyRIIIA receptors (Shields et al, J. Biol. Chem. 276(9) (2001) 6591-6604), but this mutation has also been It exhibits binding to the wild type of FcyRI and FcyRII and has only minimal reduction in binding to the FcyRIIIA receptor (Table 1 and Table 2, Genentech, EP 1 068 241).

Oganesyan et al., Acta Cristallographica D64 (2008) 700-704 introduced the triple mutation L234F/L235E/P331S into the downstream hinge and C2H domains and showed a decrease in binding activity to human IgG1 molecules to human Clq receptors FcyRI, FcyRII and FcyRIIIA. . There is still an unmet need for antibodies that significantly reduce ADCC and/or ADCP and/or CDC. Accordingly, it is an object of the invention to identify such antibodies. Surprisingly, it has been found that mutation of the proline residue at Pro329 to glycine results in an unexpectedly strong inhibition of FcyRIIIA and FcyRIIA receptors and strong inhibition of ADCC and CDC. In addition, a combination of Pro329 and, for example, L234A and L235A (LALA) mutations resulted in unexpectedly strong inhibition of Clq, FcyRI, FcyRII and FcyRIIIA. Thus, the glycine residue appears to be unexpectedly superior to, for example, other amino acid substitutions at the .329 position (eg, propylamine) in disrupting the glycinate heart structure in the Fc/FcY receptor interface. acid). 162837.doc 201245227 SUMMARY OF THE INVENTION The present invention relates to the field of antibody variants and provides polypeptides comprising an Fc variant having reduced effector functions, such as reduced ADCC and/or Clq binding. In particular, the invention provides a polypeptide comprising an Fc variant of a wild-type human IgG Fc region comprising an amino acid substitution at the Pro329 position and at least another amino acid substitution, wherein the residues are based on Kabat The EU index is numbered, and wherein the polypeptide exhibits reduced affinity for human FcyRIIIA and/or FcyRIIA and/or FcyRI as compared to a polypeptide comprising a wild type IgG Fc region, and wherein the ADCC induced by the polypeptide is reduced to include The polypeptide of the wild type human IgG Fc region induces at least 20% of the ADCC. In a specific embodiment, the Pro 329 of the wild-type human Fc region of the above polypeptide is substituted with glycine or arginine or an amino acid residue large enough to disrupt the guanamine structure in the Fc/Fcy receptor interface. The valerine sandwich structure is formed between the Fc tryptophan 329 and the tryptophan residue Trp 87 and Trp 11〇 of FcgRIII (Sondermann et al.: Nature 406, 267-273 (July 20, 2000) ). In yet another aspect of the invention, at least one other amino acid in the Fc variant is substituted with S228P, E233P, L234A, L235A, L235E, N297A, N297D or P331S and in another embodiment, the at least one other The amino acid is substituted for L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region. In another aspect of the invention, the provided polypeptide exhibits reduced affinity for at least one further receptor comprising a population of human receptors FcyI, FcyllA and Clq as compared to a polypeptide comprising a wild type human IgG Fc region. In another aspect of the invention, the polypeptide comprises a human IgG 1 or IgG4 Fc region. In another aspect of the invention 162837.doc 201245227, the polypeptide is an antibody or an Fc fusion protein. In yet another embodiment, thrombocyte agglutination induced by a polypeptide comprising an Fc variant is reduced compared to thrombocyte agglutination induced by a polypeptide comprising a wild type human IgG Fc region. In another embodiment, the polypeptide of the invention exhibits a significantly reduced CDC compared to CDC induced by a polypeptide comprising a wild type human IgG Fc region.

In another embodiment of the invention, a polypeptide comprising an Fc variant as described above for use as a medicament is provided. In a particular embodiment, the polypeptide is an anti-CD9 antibody' characterized in that the polypeptide comprising the wild type region comprises SEQ ID ΝΟ:9 as the heavy chain variable region and the package #SEq id ΝΟ:8 as the light chain variable Area. In another aspect of the invention, 'providing an effector function of a polypeptide as described above for treating a disease, wherein the polypeptide comprising an Fc variant, is associated with an effector function induced by a polypeptide comprising a wild type human IgG Fc region It is advantageously lower than advantageously. In another embodiment, the invention provides the use of a polypeptide as described above for the manufacture of a medicament for the treatment of a disease, wherein advantageously the effector function of the polypeptide comprising an Fc variant of the wild type human IgG Fc region is The effect of the effector function induced by the polypeptide comprising the wild type human IgG Fc region was significantly reduced. In another aspect of the invention, there is provided a method of treating an individual afflicted with a disease, wherein advantageously the effector function of the polypeptide comprising the variant of the wild type human IgG Fc region and the polypeptide comprising the wild type human The polypeptide-induced effector function is significantly reduced, and the method comprises administering to the individual an effective amount of the above polypeptide. 162837.doc 201245227 A further aspect of the invention is the use of a polypeptide comprising an Fc variant of a wild-type human IgG Fc region, wherein the human IgG Fc region of the polypeptide is substituted with a glycine acid, wherein the residues are based on The EU index of Kabat is numbered, wherein the polypeptide exhibits reduced affinity for human FcyRIIIA and FcyRIIA for downregulating ADCC to at least 20% of ADCC induced by a polypeptide comprising a wild type human IgG Fc region and/or for ADCP Under the tone. Another aspect of the invention is the use of a polypeptide comprising an Fc variant of a wild-type human IgG Fc region, wherein the human IgG Fc region of the polypeptide is substituted with a glycine acid and wherein the Fc variant is in the human IgG1 Fc region L234A And at least two additional amino acid substitutions at S235P and L23 5E at L235A or the human IgG4 Fc region, wherein the residues are numbered according to the EU index of Kabat, wherein the polypeptide exhibits reduced association with human FcyRIIIA and FcyRIIA Affinity is used to downregulate ADCC to at least 20% of ADCC induced by a polypeptide comprising a wild type human IgG Fc region and/or for ADCP downregulation. Another aspect of the invention is the use of the above polypeptide, wherein the thrombocyte agglutination induced by the polypeptide is reduced as compared to a thrombocyte agglutination induced by a polypeptide comprising a wild type human Fc region, wherein the polypeptide is a platelet activating antibody . In another aspect of the invention, a method of treating an individual having a disease, wherein the individual is treated with a polypeptide, the Pro329 of the human IgG Fc region of the polypeptide is substituted with glycine, wherein the residues are based on Kabat The EU index is numbered, wherein the polypeptide is characterized by a significantly reduced binding to FcyRIIIA and/or FcyRIIA as compared to a polypeptide comprising a wild type human IgG Fc region, the method comprising administering to the individual an effective amount of the polypeptide. 162837.doc -10- 201245227 In another aspect of the invention, the polypeptide used in the method comprises at least two additional amines at L234A and L235A of the human IgG1 Fc region or at S228P and L235E of the human IgG4 Fc region. Substituted by acid. [Embodiment] Definitions In the present specification and the scope of the patent application, the numbering of residues in the immunoglobulin heavy chain is as Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Lu Institutes of Health , Bethesda, Md. (1991), the numbering of the EU index, which is hereby incorporated by reference. "EU index as in Kabat" refers to the residue number of a human IgGl EU antibody. "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or Fc receptor). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between a binding member (e.g., an antibody/F c receptor or an antibody and an antigen). The affinity of the molecule X with its partner γ • The force is usually represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are set forth below. "Affinity maturation" anti-system refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) as compared to a parent antibody that does not have an alteration, such that the affinity of the antibody to the antigen can be improved. "Amino acid modification" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include amino acid substitutions, insertions, and/or deletions. 162837.doc 201245227 A preferred amino acid modification system is substituted. "Amino acid modification" at a specified position (e.g., Fc region) refers to the insertion or deletion of a specified residue or insertion of at least one amino acid residue adjacent to a specified residue. Insertion of a "adjacent" designated residue means insertion within one to two residues. The insertion can be tied to the N-terminus or C-terminus of the specified residue. "Amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence by another different "alternative" amino acid residue. One or more of the surrogate residues may be "natural amino acid residues" (ie, encoded by the genetic code) and are selected from the group consisting of: alanine (Ala); arginine (Arg); aspartate (Asn); aspartic acid (Asp); cysteine (Cys); glutamic acid (Gin); glutamic acid (Glu); glycine (Gly); histidine (His); Leucine (lie); leucine (Leu); lysine (Lys); thiol amide (Met); phenylalanine (Phe); proline (pro); serine (Ser); Aminic acid (Thr); tryptophan (Trp); lysine (Tyr); and lysine (Val) 〇 Preferably, the replacement residue is not cysteine. The definition of amino acid substitution herein also encompasses the substitution with one or more unnatural amino acid residues. "Non-natural amino acid residues" refer to residues other than the natural amino acid residues listed above which are capable of covalently binding to her ortho-amino acid residues in the polypeptide chain. Examples of non-natural amino acid residues include norleucine, ornithine, n-decanoic acid, serranic acid, and other amino acid residue analogs, for example, as described herein.

Ellman et al., Meth. Enzym. 202 (1991) 301-336. To produce such non-natural amino acid residues, the procedures of Noren et al, Science 244 (1989) 182 and Ellman et al. (supra) can be used. Briefly, such procedures involve the chemical activation of the inhibitor I62837.doc 12 201245227 tRNA ' with non-natural amino acid residues followed by in vitro transcription and translation of rna. "Amino acid incorporation" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. Although the insertion will typically consist of the insertion of one or two amino acid residues, the present application encompasses larger "insert insertions", such as from about 3 to about 5 or even up to about 1 amino acid residues. insert. The inserted residue may be a natural or non-natural residue as disclosed above. "Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence. The term "antibody" is used herein in its broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, As long as it exhibits the desired antigen binding activity. The term "antibody variant" as used herein, refers to a variant of a wild-type antibody characterized by an alteration in the amino acid sequence of the antibody variant relative to the wild-type antibody by, for example, a specific amine in the wild-type antibody. A mutation of a base acid residue is introduced. The term "antibody effector function" or "effector function" as used herein is the function of the Fc effector domain of algG (e.g., the immunoglobulin region). This function can be achieved, for example, by binding of an Fc effector domain to an Fc receptor on immune cells having phagocytic or lytic activity or a binding of an Fc effector domain to a component of the complement system. Typical effector functions are ADCC, ADCP and CDC. "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an antigen bound by a binding antibody to an intact antibody in an intact antibody. Antibody fragment 162837.doc -13- 201245227 Examples include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, double bond antibody, direct bond antibody, single bond antibody molecule ( For example, scFv), and multispecific antibodies formed from antibody fragments. "Antibody binding to the same epitope" (as a reference antibody) means that the binding of the reference antibody to its antigen is blocked by 50° in a competitive assay. Or higher antibodies, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. An example competitive analysis is provided herein. "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which non-specific cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and macrophages) exhibit FcR. The bound antibody on the target cell is recognized and subsequently causes lysis of the target cell. Primary cells (NK cells) used to mediate ADCC exhibit only FcyRIII, while monocytes exhibit FcyRI, FcyRII, and FcyRIII. The performance of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492. The terms "antibody-dependent cellular phagocytosis" and "ADCp" refer to the application of antibody-coated cells by immunoglobulin-binding regions of the phagocytic immune cells (eg, macrophages 'adrenal spheres and dendritic cells) throughout The process of internal or partial internalization. The term "binding domain" refers to the region of a polypeptide that binds to another molecule. In the If form of FcR, the binding domain may comprise the binding of its polymorphic bond.

Part of the Fc region (eg, its ^). A useful binding domain is the extracellular domain of the μ 〇 chain. The term "binding" Fc receptor as used herein means the antibody and the antibody in (10), for example, (3) generation (8) I62837.doc 201245227 (Pharmacia Bi〇sensor AB, Uppsala, Sweden).

Binding of Fc receptors. In the BIAC〇re(R) assay, the Fc receptor is bound to the surface and the binding of the variant (e.g., an antibody variant into which the mutation has been introduced) is measured by surface plasma resonance (SPR). Binding affinity is defined by the terms ka (association rate constant of antibody from antibody/Fc receptor complex), kd (dissociation constant) and KD (kd/ka). Alternatively, the combined signal of the SPR sensing pattern and the reference reaction signal can be directly compared in terms of the resonance signal height and the dissociation property. • "Clq" is a polymorphism that includes the binding site of the fc region of immunoglobulin. Clq, together with the two serine proteases cir and cis, forms a complex of the first component of the complement dependent cytotoxicity (CDC) pathway. Human ciq is commercially available, for example, from Quidel, San Diego, Calif. The "CH2 domain" (also referred to as the "CY2" domain) of the human IgG Fc region typically extends from about the 23th amino acid to about the 340th amino acid. The reason for the difference in the CH2 structure is that it is not closely paired with another domain. Instead, two N-linked branched-chain carbohydrate chains are inserted between the two CH2 domains of the entire native IgG. It is speculated that carbohydrates can provide a domain-domain pairing alternative and help stabilize the CH2 domain (Burton, Molec. Immunol. 22 (1985) 161-206). The "CH3 domain" comprises the residue stretch portion of the C-terminus of the CH2 domain in the Fc region (i.e., from about the 341 amino acid residue of the IgG to about the 447th amino acid residue). The terms "cancer" and "cancerous" refer to or describe the physiological condition of a mammal that is typically characterized by a disorder of cell growth. Examples of cancer include (but 162837.doc -15- 201245227 is not limited to) cancer, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glial blastoma, and Cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer ( Hepatic carcinoma) and various types of head and neck cancer. As used herein, the expression "cell", "cell line" and "cell culture" are used interchangeably and all such names include progeny. Therefore, the words "transformants" and "transformed cells" include primary individual cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that the DNA content of all progeny may not be exactly the same due to intentional or unintentional mutations. Described herein are mutant progeny that have been screened for the same function or biological activity in the original transformed cell. If you want to use a different name, it can be civilized. The term "chimeric" anti-system refers to an antibody from which a portion of a heavy chain and/or a light chain is derived from a particular source or species and the remainder of the heavy and/or light linkage is derived from a different source or species. The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain has. There are five broad classes of antibodies: IgA, IgD, IgE, IgG, and IgM' and several of these classes can be further divided into subclasses (isotypes), such as 'IgG!, IgG2, igG3, IgG4, IgA, and IgA2. The heavy-chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. 162837.doc -16 - 201245227 The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (eg, At2丨丨, I131, I125, γ90, Re186, Re188, Sm153, Bi212, p32, pb2i2, and radioisotopes); chemotherapeutic agents or drugs (eg, amines) A pterin, adriamicin, vinca alkaloids (changchun new test, changchun test, etoposide), doxorubicin, melphalan , mitomycin c, chlorambucil (chl〇rambucil), φ daunorubia (daunorubiein) or other intercalating agents); growth inhibitors; enzymes and tablets thereof - such as lysozyme; antibiotics, Toxins, such as small molecule toxins or enzymatically active toxins of bacterial, vegetative, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents disclosed below. The term "complement dependent cytotoxicity" or CDC refers to a mechanism for inducing cell death wherein the Fc effector domain of the target binding antibody is activated to form a series of enzymatic reactions in the end of the cell membrane. Typically, antigen--antibody complexes (such as those on which antibody-coated target cells are coated) bind to and activate the complement component Clq' which in turn activates the complement cascade, resulting in target cell death. Complement activation can also result in the deposition of complement components on the surface of target cells. These complement components promote ADCC by combining the complement receptors on white blood cells (eg, CR3) (eg, contain!^ Variant antibodies) any condition that benefits the treatment. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose the mammal to the condition in question. In one embodiment 162837.doc • 17-201245227, the condition is cancer. "Effector function" refers to the biological activity of a region attributed to an antibody' which may vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (cdc); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); cell surface receptors (eg, B) Cell receptor) downregulation; and b cell activation. As used herein, "reduced effector function" means that a particular effector function (eg, ADCC or CDC) is reduced by at least 20% compared to a control (eg, a polypeptide having a wild-type Fc region), and "significantly reduced effector function" as used herein. Means that a particular effector function (eg, ADCC or CDC) is reduced by at least 50% compared to a control. An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result at the desired dosage over a desired period of time. The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pr 〇 230 to the carboxy terminus of the heavy chain. However, the C-terminal amide acid (Lys447) may or may not be present in the Fc region. Unless otherwise indicated herein, otherwise the numbering of amino acid residues in the Fc region or constant region is based on an EU-based system known as the number of EU days, such as Kab at et al., Sequences of

Proteins of Immunological Interest, 5th Edition, Public Health

Service, National Institutes of Health, Bethesda, MD (1991). 162837.doc • 18· 201245227 The "variant Fc region" comprises an amino acid sequence which differs from the "natural" or "wild-type" sequence Fc region by at least one "amino acid modification" as defined herein. Preferably, the 'variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or to the Fc region of the parent polypeptide, eg, from about 1 to the native sequence Fc region or the Fc region of the parent polypeptide. Substitution of about 1 amino acid, and preferably from about 1 to about 5 amino acid substitutions. The variant Fc region herein will preferably have at least the native sequence Fc region and/or the Fc region of the parent polypeptide. It is about 80% homologous, and optimally has at least about 90% homology with it, more preferably at least about 95% homology thereto. The term "Fc-variant" as used herein refers to a polypeptide comprising a modification in the Fc domain. The Fc variant system of the present invention is defined by the amino acid modification constituting it. Thus, 'exemplary' P329G is an Fc variant in which glyceryl acid is substituted for valeric acid at position 329 relative to the parent Fc polypeptide, wherein the numbering is based on the EU index. The characteristics of the wild type amino acid may not be specified, and in this case, the above variant is referred to as P329G. For all locations discussed in this disclosure, the numbers are based on the EU index. The EU index or EU index as in the Kabat or EU numbering scheme refers to the number of the EU antibody (Edelman et al, Proc Natl Acad Sci USA 63 (1969) 78-85, which is incorporated herein by reference). Modifications may be the addition, deletion or substitution of hydrazines which may include natural amino acids and non-natural amino acids. Variants may comprise an unnatural amino acid. Examples include U.S. Patent No. 6,586,207; WO 98/48032; WO 03/073238; US 2004/0214988 Al; WO 05/35727 A2; WO 05/74524 A2; Chin, JW et al., Journal of the American Chemical Society 124 ( 2002) 9026-9027; Chin, JW and Schultz, PG, 162837.doc • 19_ 201245227

ChemBioChem 11 (2002) 1135-1137; Chin, JW et al, PICAS United States of America 99 (2002) 11020-11024; and Wang, L. and Schultz, PG, Chem. (2002) 1-10, these patents And the literature is fully incorporated herein by reference. The term "polypeptide comprising an Fc region" refers to a polypeptide such as an antibody or immunoadhesin (see definition below) which comprises an Fc region. The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. Preferred FcR is the native sequence human FcR. Furthermore, preferred FcR lines bind to IgG antibody (gamma receptors) and include receptors for the FcyRI, FcyRII and FcyRIII subclasses φ, including dual gene variants and alternative splicing forms of such receptors. The FcyRII receptor includes FcyRIIA ("activated receptor") and FcyRIIB ("inhibitory receptor") having similar amino acid sequences, which are predominantly different in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See DaSron, M., Annu Rev. Immunol. 1 5 (1997) 203-234 for a review). FcR is reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492; Capel et al, Immunomethods 4 (1994) 25-34; and de Haas et al, J. Lab. Clin. Med. 1995) 330-41. The term "FcR" as used herein encompasses other FcRs, including those who will be identified in the future. The term also includes the negative transfer of maternal IgG to the neonatal receptor FcRn in the fetus (Guyer et al, J. Immunol. 117 (1976) 587 and Kim et al, J. Immunol 24 (1994) 249). 162837.doc -20· 201245227 As used herein, "IgG Fc ligand" means a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include, but are not limited to, FcyR, FcYR, FcyR, FcRn, Clq, C3, mannan-binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcyR. Fc ligands also include the fc receptor homolog (FcRH) 'a family of Fc receptors homologous to FcyR (Davis et al, Immunological Reviews 190 (2002) 123-136, which is fully incorporated by way of use | In this article). An Fc ligand can include a molecule that is not found to bind to Fc. Specific IgG Fc ligand systems FcRn and Fey receptors. As used herein, "Fc ligand" means a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc/Fc ligand complex. As used herein, "Fey receptor", "FcyR" ("FeyR" or "FegammaR") means any member of a family protein that binds to the Fc region of an IgG antibody and is encoded by the FeyR gene. In humans, this family includes, but is not limited to, FcyRI (CD64), including subtypes FcYRIA 'FcyRIB and FcyRIC; FcyRII (CD32), including subtypes FcyRIIA (including allotypes H131 and R131), FcyRIIB (including FcyRIIB-l And FcyRIIB-2) and FcyRIIc; and FcyRIII (CD16), including subtypes FcyRIIIA (including allotypes V158 and F158) and FcyRIlIb (including allotypes FCYRnB-NAl and FcyRIIB-NA2) (Jefferis et al., Immun〇l Lett 82) (2002) 57-65 'which is fully incorporated herein by reference, as well as any undiscovered human FcYR or FeyR subtype or allotype. FcyR can be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FeyR includes, but is not limited to, FcyRI (CD64), FcyRII (CD32), 162837.doc 21 201245227 MRIII (CD16) and FcYRm-2 (CD16_2), and any undiscovered mouse FcyR or FcyR subtype or allotype . As used herein, "FcRn" or "neonatal Fc receptor" means a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn can be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, functional FcRn proteins comprise two polypeptides commonly referred to as heavy and light chains. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise indicated herein, the FcRn4 FcRn protein refers to a complex of an FcRn heavy chain and beta-2-microglobulin. As used herein, "wild-type or parent polypeptide" means an unmodified polypeptide that is subsequently modified to produce a variant. A wild-type polypeptide can be a variant or engineered form of a native polypeptide or a natural polypeptide. A wild-type polypeptide may refer to a composition comprising the parent polypeptide itself or an amino acid sequence encoding the same. Thus, as used herein, "wild-type immunoglobulin" means an unmodified immunoglobulin polypeptide modified to produce a variant, and "wild-type antibody" as used herein means unmodified, modified to produce a variant antibody. Antibody. It should be noted that "wild-type antibodies" include commercially available antibodies produced by known recombinants as outlined below. The term "fragment crystallizable (Fc) polypeptide" is the part of an antibody molecule that interacts with an effector molecule and a cell. It contains the C-terminal portion of the immunoglobulin heavy chain. The term "framework" or "FRj" refers to a variable domain residue other than a hypervariable region (HVR) residue. The FR of a variable domain is typically composed of four FR domains FR1, FR2, FR3, and FR4. The HVR and FR sequences usually appear in the following sequences in VH (or VL): FR1-H1(L1)-FR2-162837.doc •22· 201245227 H2(L2)-FR3-H3(L3)-FR4. "Full length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a native antibody or having a heavy chain comprising an Fc region as defined herein. The "functional Fc region" has the "effector function" of the native sequence Fc region. Exemplary "effector functions" include Clq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); sputum action; cell surface receptors (eg, B cell receptor; BCR) under the adjustment. Such effector functions typically require that the Fc region be combined with a binding domain (e. g., an antibody variable domain) and can be assessed using, for example, various assays disclosed herein. The "hinge region" is usually defined as extending from the human IgGl Glu216 to

Pro230 (Burton, Molec. Immunol. 22 (1985) 161-206). The hinge region and IgG1 sequence of other IgG isotypes can be aligned by placing the first and last cysteine residues in the same position to form an inter-heavy chain S-S bond. The "downstream hinge region" of the Fc region is generally defined as the stretch portion of the Φ residue immediately adjacent the c-terminus of the hinge region, i.e., residues 233 to 239 of the Fc region. "Homology" is defined as the percentage of residues in an amino acid sequence variant that are identical after alignment of the sequence and introduction of a gap (if desired) to achieve a maximum % homology. Methods and computer programs for comparison are well known in the art. One such computer program is "Align 2" designed by Genentech, Inc., which was filed on December 10, 1991 with user documentation in United States Copyright Office, Washington, D.C. 20559. The terms "host cell", "host cell line" and "host cell culture 162837.doc • 23· 201245227" are used interchangeably and refer to cells into which exogenous nucleic acids are introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny and parental cells may not be exactly the same, but may contain mutations. Described herein are mutant progeny that have been screened or selected for use in the original transformed cell with the same function or biological activity. "Human antibody" is an amino acid sequence having an amino acid sequence corresponding to an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody profile or other sequence encoding a human antibody. The definition of this human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. "Human effector cells" are white blood cells that exhibit one or more FcRs and perform effector functions. Preferably, the cells exhibit at least FqRni and perform ADCC effector functions. Examples of human leukocytes that modulate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, mononuclear spheres, cytotoxic T cells, and neutrophils; of which pBMC and NK cells are preferred. Effector cells can be isolated from their natural source, such as blood or PBMC as described herein. A "humanized" anti-system refers to a chimeric antibody comprising an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (eg, CDRs) correspond to non-human HVRs, And all or substantially all of the FRs of the FRs corresponding to the human antibodies may optionally comprise at least a portion of the antibody constant region derived from the human antibody of 162837.doc -24 - 201245227. An antibody that is "humanized" (for example, a non-human antibody) refers to an antibody that has been humanized. The term "hypervariable region" or "hvr" as used herein, refers to a sequence in an antibody variable domain region that is hyperdenatured and/or forms a structurally defined loop ("hypervariable loop"). Typically, the native four-bond antibody contains 6 HVRs; 3 are located in VH (HI, H2, H3), and 3 are located in V1 (1^1, [2, [3]). 1^11 typically contains amino acid residues from the hypervariable loop and/or from the "mutual complement determination region" (CDR), which has the highest sequence variability and/or participates in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101. (H3) (Chothia and Lesk, J. Mol. Biol. 196 (1987) 901-917). An exemplary 〇011 (0011-1^1, €01^2, €011-L3, CDR-H1, CDR-H2, and CDR-H3) occurs at 24-34 of the amino acid residue L1, 50- of L2. 56, 89-97 of L3, 31-35B of H1, 50-65 of H2 and 95-102 of H3 (Kabat et al. 'Sequence of Proteins of φ Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) In addition to CDR1 in VH, CDRs typically comprise an amino acid residue that forms a hypervariable loop. The CDR also contains a "specificity determining residue" or "SDR" which is a residue that contacts the antigen. SDR is contained within the region of the CDR called the abbreviated-CDR or a-CDR. Illustrative &-0〇11 (ugly - 〇11-1^1, &-CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) Appears at 31-34 of amino acid residue L1, 50-55 of L2, 89-96 of L3, 162837.doc -25·201245227 HI 31-35B, H2 50-58 and H3 95-102 (See Almagro and Fransson, Front. Biosci. 13 (2008) 1619-1633). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al. (see above). "Immune complex" refers to a relatively stable structure formed when at least one target molecule and at least one polypeptide comprising a heterologous Fc region bind to each other to form a larger molecular weight complex. Examples of immune complexes are antigen-antibody aggregates and target molecule-immunoadhesin aggregates. The term "immune complex" as used herein, unless otherwise indicated, refers to an ex vivo complex (i.e., other than the form or environment in which it can be found in nature). However, the immune complex can be administered to a mammal to, for example, assess the clearance of the immune complex in the lactating animal. An "immunoconjugate" is an antibody that is conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents. "Individual (subject or subject)" is a mammal. Mammals include, but are not limited to, domestic animals (eg, 'bovine, sheep, cat, dog, and horse), primates (eg, humans and non-human primates, such as monkeys), rabbits, and rodents ( For example, mice and rats). In some embodiments, the system is human. The "separated" anti-system is separated from its natural environment components. In some embodiments, the antibody is purified to have a purity greater than 95% or 99%, such as by, for example, electrophoresis (eg, SDS_PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange) Or as determined by reverse phase HpLC). A review of methods for assessing antibody purity [for example, see Flatman et al.,] 162837.doc •26- 201245227

Chromatogr. B 848 (2007) 79-87 「 An "isolated" polypeptide is a polypeptide that has been identified and isolated and/or recovered from its natural environmental components. The contaminating component of its natural environment interferes with the antibody's use or therapeutic use, and may include enzymes, hormones, and other protein cryo- or non-proteinaceous solutes. In a preferred embodiment, the polypeptide can be purified () to greater than 95% by weight of the polypeptide, as determined by the Lowry method, and optimally greater than 99% by weight of the polypeptide, (2) to the use of a spin-on sequence The analyzer acquires at least 15 N-terminal or internal amino acid sequence residues, or # to homogeneity, such as by using SDS_PAGE under reduced or non-reducing conditions using Comassie Mue or (preferably) measured by silver staining. Since at least one polypeptide natural environmental component should not be present, the isolated antibody comprises a heavy, and in situ, polypeptide within the cell. Typically, however, the isolated polymorph will be prepared by at least one purification step. "Separated" nucleic acid refers to a nucleic acid molecule isolated from a natural environmental component. The isolated nucleic acid comprises a nucleic acid molecule contained in a cell which usually contains a nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural staining position. "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding an antibody heavy and light chain (or a fragment thereof), including the (4) nucleic acid molecule in a single vector or a separate vector, and present in a host cell - This (etc.) nucleic acid molecule at or at multiple positions. The word "label" as used herein, refers to a detectable compound or composition that is coupled directly or indirectly to a polypeptide. The label may itself be detectable (e.g., 'radioisotope label or fluorescent label), and ❹ ❹ ❹ 162 837.doc • 27· 201245227 Form, catalyzes the chemical alteration of a detectable substrate or composition. The term "ligand binding domain" as used herein, refers to any natural cell surface receptor or any region or derivative thereof that retains at least one of the qualitative ligand binding capabilities of a corresponding natural receptor. In a particular embodiment, the subject is pleated from the surface of a cell having an extracellular domain homologous to a member of the immunoglobulin supergene family. Not a member of the immunoglobulin supergene family but is defined by other receptors that are specifically covered by the system, and in particular receptors, hematopoietic and receptors with alanine kinase activity (receptor tyrosine kinase) NGF receptor superfamily members and cell adhesion molecules, eg, (E _,) selectin - as used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homologous antibodies, ie, comprising the population The individual antibodies are identical and/or bind to the same epitope, with the possible exception of the variant antibody, for example, containing a natural mutation or produced during the production of a monoclonal antibody preparation, such variants typically being present in minor amounts. With respect to a plurality of antibody preparations which usually include different antibodies against different determinants (epitopes), each of the individual antibody preparations is directed against a single determinant β on the antigen. Therefore, the modifier "individual j indicates The antibody properties obtained from a population of substantially homologous antibodies, and should not be construed as requiring production of antibodies by any particular method. For example, monoclonal antibodies used in the present invention can be made by multiple (four) techniques. (but not limited to) hybridoma method, recombinant DNA method, celestial display method, and methods for preparing monoclonal antibodies using methods for transgenic animals containing all or part of human immunoglobulin loci, and other exemplary methods As used herein, a "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a 162837.doc 201245227 radiolabel. Naked antibodies can be present in pharmaceutical formulations. "Native antibody" refers to a natural immunoglobulin molecule having a different structure. For example, a natural IgG anti-system of about 150,000 daltons of a heterotetrameric glycoprotein, which is composed of two disulfide bonds. The same light chain and two identical heavy chains. Each heavy chain from the N-terminus to the C-terminus in turn has a variable region (VH) (also known as a variable heavy chain domain or a heavy chain variable domain) and three constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each light chain in turn has a variable region (VL) (also known as a variable light chain domain or a light φ chain variable domain) and a constant light chain (CL) domain. . The light chain of the antibody can be assigned to one of two types, called kappa type and human type, based on the amino acid sequence of the antibody constant domain and the variable domain. The "native sequence Fc region" contains an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. The native sequence human Fc region includes the native sequence human IgGl Fc region (non-A and A allotype); the native sequence human igG2 Fc region; the native sequence human igG3 Fc region; and the native sequence human lgG4 Fc region and its natural variants. ® is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if the DNA of the pre-sequence or the secretion leader sequence is expressed in the form of a protein prior to the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; if the promoter or enhancer affects the transcription of the coding sequence, then Operabically linked to the sequence; or operably linked to the coding sequence if the ribosome binding site is positioned to facilitate translation. Generally, "operably linked" means that the ligated DNA sequences are contiguous and, in the case of a secretory leader sequence, contiguous and in reading phase. However, 162837.doc •29· 201245227, enhancers are not necessarily contiguous. The connection is achieved by engagement at a convenient restriction site. If this site is not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice. The term "package insert" is used to mean a specification that is typically included in a commercial package of a therapeutic product, containing indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings relating to the use of such therapeutic products. News. As used herein, it refers to a site in a protein sequence. The locations can be numbered sequentially or according to a confirmation format (eg, an Eu index for antibody numbers). The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of an amino acid residue (containing a natural or unnatural amino acid residue) and are not limited to a minimum length of 0. Therefore, the definition includes peptides and oligos. , dimers, multimers, and the like. This definition covers both full length proteins and their fragments. These terms also include polymorphic post-translational modifications, including (eg, m-based, (iv) acidified, acetylated, and acidified. "Peptide" herein also refers to a modified protein, such as a single § native sequence. Or deleting, adding and substituting a plurality of amino acid residues, which is difficult to maintain the egg. For example, the serine residue can be taken to eliminate the mono-reactive caspase (IV) or to remove The sulfur bond may be substituted with an amino acid to eliminate the cleavage site. The (4) may be deliberate, such as by mutagenesis, or may be carried out by chance, for example, via host mutation, which may be due to polymerase chain reaction. (PCR) amplification produces protein or error. The terms "wild-type" and "wild-type (human), Fe-region" are used herein. 162837.doc 201245227 refers to a polypeptide and an Fc region, respectively, which comprise a lack of the Fc disclosed herein. The amino acid sequence of one or more of the region modifications, since such modifications have not been introduced and are used, for example, as a control. The wild-type polypeptide may comprise a pre-existing amino acid sequence modification (eg, addition, Deleted and/or substituted) native sequence Fc region Fc region. The term "pharmaceutical formulation" refers to a formulation that is presented in a form that allows for the biological activity of the active ingredient contained therein to be effective and that does not contain additional components that are unacceptable to the individual to which the formulation is administered. Pharmaceutically acceptable carrier J means a pharmaceutical preparation which is non-toxic to the individual in addition to the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives. A polypeptide having "altered" FcR binding affinity or ADCC activity has increased or decreased FcR binding activity and/or ADCC activity compared to a parent polypeptide or a polypeptide comprising a native sequence Fc region. "Indicating increased FcR A polypeptide variant that binds to binds at least one FcR with an affinity that is superior to the parent polypeptide. A polypeptide variant that "displays a reduced binding to an FcR" binds at least one FcR with an affinity that is inferior to the pro-accepting polypeptide. Such display is reduced The variant that binds to the FcR may have a very low or no appreciable binding to the FcR as compared to the native sequence IgG Fc region 'eg, 〇 2〇% of the binding to the FCR, As determined by the examples herein. A polypeptide that binds to an FcR with "affinity lower than the parent polypeptide" is substantially less than the parent when the amount of the polypeptide variant and the parent polypeptide in the binding assay is substantially the same. The binding affinity of the antibody binds to a polypeptide of any one or more of the above identified FcRs. For example, a polypeptide variant having reduced FcR binding affinity 162837.doc 31 201245227 can exhibit FcR binding affinity compared to the parent polypeptide. Decrease to 20/23 to about 1/100', such as from about 5/6 to about 1/5 〇, wherein μ binding affinity is determined as described, for example, in the examples herein. Compared to parental or wild-type polypeptides A polypeptide comprising an FC variant with an antibody-dependent cell-mediated cytotoxicity (adcc) in the presence of a human effector cell, the amount of the polypeptide variant and the parent antibody used in the assay At substantially the same time, ADCC is mediated with substantially lower effectiveness in vitro or in vivo. Typically, such variants will be identified using in vitro ADCC assays as disclosed herein, but encompass other assays or methods for determining ADCC activity in, for example, animal models and the like. Preferably, the variant system modulates ADCC in an in vitro assay as disclosed herein, such that the parent is about 2/3 to about 丨/丨〇 〇 (e.g., about 1/2 to about 1/50). A "receptor" is a polypeptide that is capable of binding at least one ligand. Preferred receptor systems have cell surface receptors having an extracellular ligand binding domain and optionally other domains (e.g., transmembrane domains, intracellular domains, and/or membrane anchors). The receptor to be evaluated in the assays described herein may be an intact receptor or a fragment or derivative thereof (e.g., a fusion protein comprising a binding domain fused to a receptor of one or more heterologous polypeptides). A receptor for the property may be present in the cell or isolated and optionally applied to an assay plate or some other solid phase. The term "receptor binding domain" is used to designate any natural ligand (including cell adhesion molecules) of the receptor or any region or derivative of this natural ligand that retains at least one of the qualitative receptor binding capabilities of the natural ligand. This definition specifically includes the binding sequence of the ligand of the above receptor. 162837.doc -32- 201245227 The term "remediation, 「 "t .. ^ %'" (and its grammatical variants, such as "treat" or "treating") as used herein refers to the clinical course of the natural processes of individuals undergoing treatment. Can be used for preventive purposes or during the course of clinical pathology: Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological outcome of the disease, preventing metastasis, reducing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving the pre-amp; In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of the disease.

"Shaw variant protein" or "protein variant" "variant" as used herein means a protein that differs from the parent protein by modification of at least one amino acid. A protein variant may refer to the protein itself, or a composition comprising the protein, or an amino-based sequence thereof. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, for example from about i to about 70 amino acid modifications, and preferably from about 1 to about 5 amine groups compared to the parent. Acid modified. Preferably, the protein variant sequences herein will have at least about 8 〇〇/〇 homology with the parent protein sequence, and preferably at least about 90% homology' more preferably at least about 95% homology. A variant protein may refer to a variant protein itself, a composition comprising the variant of the protein, or a DNA sequence encoding the same. Thus, as used herein, "antibody variant" or "variant antibody" means an antibody that differs from the parent antibody by modification with at least one amino acid. 'IgG variants' or 'variant IgGs' as used herein means at least An antibody modified with an amino acid that is different from the parental IgG, and as used herein, "immunoglobulin variant" or "variant immunoglobulin" means different from the parent immunoglobulin sequence by modification of at least one amino acid. Immunoglobulin sequence The term "variable region" or "variable domain" refers to a domain involved in binding an antibody to an antigen in an antibody heavy or light chain 162837.doc-33·201245227. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) typically have similar structures, each of which contains four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co. (2007) p. 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind to a particular antigen can be isolated by screening a complementary VL or VH domain library, respectively, using a VH or VL domain from an antibody that binds to the antigen. See, for example, Portolano et al., J. Immunol. 150 (1993) 880-887; Clarkson Lu, et al, Nature 352 (1991) 624-628. The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are included in the genome of the host cell into which they are introduced. Certain vectors are capable of directing the performance of the operably linked nucleic acid. Such vectors are referred to herein as "expression vectors." The present application relates to polypeptides comprising a combination of amino acid modifications that modulate binding to an Fc receptor, particularly a Fcy receptor. DETAILED DESCRIPTION The invention herein relates to methods of making polypeptides comprising Fc variants. Parental, "initial", "non-variant" or wild-type polypeptides are prepared using commercially available techniques for generating polypeptides or antibodies of the Fc region of the package. In a preferred embodiment of the invention, exemplary methods of parent polypeptide-based antibodies and antibody production are set forth in more detail in the following sections. Alternatively, the parent polypeptide may be any other polypeptide comprising a region, such as an immunoadhesin. Preparation of Immunoadhesin 162837.doc -34 - 201245227 The method is set forth in more detail below. In an alternative embodiment, the 'variant 1 region (Fc variant) can be generated according to the methods disclosed herein and this Fc variant An antibody variable domain or binding domain that can be fused to a heterologous polypeptide of choice, such as a receptor or ligand. The wild type polypeptide comprises an Fc region. Typically, the Fc region of a wild-type polypeptide will comprise a native or wild-type sequence Fc region, and preferably a human native sequence Fc region (human Fc region). However, the Fc region of a wild-type polypeptide may have one or more pre-existing amino acid sequence changes or modifications from the native sequence Fc region. For example, the Clq or Fey binding activity of the Fc region can be altered in advance (other types of Fc region modifications are set forth in more detail below). In yet another embodiment, the parent polypeptide Fc region is "conceptual" and, although it is not actually present, antibody engineering can determine the desired variant Fc region amino acid sequence and generate a polypeptide or code comprising the sequence. DNA of the amino acid sequence of the variant Fc region is desired. However, in a preferred embodiment of the invention, a nucleic acid encoding an Fc region of a wild-type polypeptide can be used and the nucleic acid sequence can be altered to generate a variant nucleic acid sequence encoding an Fc region variant. The DNA encoding the amino acid sequence variant of the starting polypeptide is prepared by a variety of methods known in the art. These methods include, but are not limited to, the site (or oligonucleotide) of the DNA encoding the polypeptide prepared by early preparation. Preparation by mutagenesis, PCR mutagenesis and cassette mutagenesis. Site-directed mutagenesis is a preferred method of preparing substituted variants. This technique is well known in the art (see, for example, Carter et al, Nucleic Acids Res. 13 (1985) 4431-4443 and Kunkel et al, Proc. Natl. Acad. Sci. USA 82 (1985) 488). Briefly, in the case of site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing the oligonucleotide encoding the desired mutation to the single strand of the starting DNA by first 162837.doc -35 - 201245227. Thereafter, a DNA polymerase was used to synthesize the entire second strand in which a hybrid oligonucleotide was used as a primer and a single strand of the starting DNA was used as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA. PCR mutagenesis is also suitable for the preparation of amino acid sequence variants of the starting polypeptide. See Higuchi, PCR Protocols, Academic Press (1990) pp. 177-183; and Vallette et al, Nuc. Acids Res. 17 (1989) 723-733. Briefly, 'When a small amount of template DNA is used as a starting material in PCR, primers that differ slightly in sequence from the corresponding regions in the template DNA can be used to generate a relatively large number of specific DNA fragments, which are only The primer sequence is different from the template sequence at a position different from the template. Another method of preparing variants is based on box mutagenesis by Wells et al.

Gene 34 (1985) 315-323 describes the technology. One embodiment of the invention encompasses a polypeptide comprising an Fc region of an antibody comprising the addition, substitution or deletion of at least one amino acid residue to the Fc region, thereby reducing or eliminating affinity for at least one Fc receptor. "Body or ligand interactions" include, but are not limited to, Fc receptors (eg, FqRi, FqRIIA, FqRIIIA), complement protein CIq, and other molecules (eg, proteins A and G). These interactions are critical for a variety of effector functions and downstream signaling events, including but not limited to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular uptake (ADCp), and complement dependence. Cytotoxicity (CDC). Thus, in certain embodiments, the amino acid sequence is the same as the polypeptide comprising the Fc variant of the invention but is not included in the addition, substitution or deletion of at least one amino acid residue in the region 162837.doc • 36 - 201245227 In contrast to polypeptides (also referred to herein as "wild-type polypeptides"), the variants of the invention have reduced or removed affinity for Fc receptors responsible for effector function. In certain embodiments, a polypeptide comprising an FC variant of the invention comprises at least one or more of the following properties: reducing or removing an effector (ADCC and/or CDC and/or ADCP) function, reducing or removing Fc Receptor binding 'reduces or removes binding to ciq or reduces or removes toxicity ❶ More specifically, the anti-CD20 (same as GA101 or GA), anti-CD9 (same as TA) and Anti-selectin (pSel) antibodies have reduced affinity for Fc receptors (eg, FcyRI, FcyRII, FcyRIIIA) and/or complement protein Clq. In one embodiment, an antibody of the invention comprises an Fc region comprising a addition, substitution or deletion of at least one amino acid residue at the P329 position, wherein the numbering system of the site is as in Kabat et al., NIH Publication 91 (1991) 3242, National Technical Information Service, Springfield, VA The numbering system for the EU index. In a particular embodiment, the polypeptide of the invention comprises a φ Fc variant of a wild-type human Fc polypeptide comprising an amino acid substitution at the Pro329 position, wherein the numbering of the residues in the igG Fc region is as defined by the EU index in Kabat Numbering. In another embodiment, the variant comprises at least another amino acid substitution. In another embodiment, a polypeptide comprising an Fc variant of a wild-type human Fc polypeptide has an amino acid substitution, deletion or addition which disrupts or reduces the valerine sandwich structure in the region of the Fc polypeptide and the Fey receptor and/or Or the function in the interface. In another embodiment, 'Pr 329 is substituted with an amino acid less than or greater than the proline. In another embodiment, the substituted amino acid system Gly, Ala or 162837.doc -37- 201245227

Arg. In still another aspect of the invention, the pr〇329 of the Fc polypeptide is substituted with glycine. In another embodiment, the polypeptide comprising an Fc variant has at least one other amino acid substitution, addition or deletion. In another embodiment, the variants exhibit reduced affinity to a human Fc receptor (FcYR) and/or a human complement receptor as compared to a polypeptide comprising a wild type Fc polypeptide. In another embodiment, the Fc variant comprising a polypeptide exhibits reduced affinity for a human Fe receptor (FcyR) & / human complement receptor as compared to a polypeptide comprising a wild type human Fc region. In yet another embodiment, the affinity to at least one of FqRI, FcyRII, Fc^RIIIA is reduced, in another embodiment 'degrading affinity for FcYRI and FcyRIIIA, and in another embodiment' The affinity of FcyRI, FcyRII and FcyRIIIA is decreased, and in another aspect of the invention, the affinity with the FcyRI receptor, FcyRIIIA receptor and Clq is lowered, and in another aspect of the invention, FcyRI, FcyRII are made. The affinity of FcyRIIIA and Clq receptors is reduced. In another embodiment, the ADCC induced by the polypeptide comprising the Fc variant is reduced and, in a preferred embodiment, the ADCC is reduced to at least 2% of the ADCC induced by the polypeptide comprising the wild type Fc polypeptide. In another aspect of the invention, 'reducing or removing ADCC and CDC induced by a polypeptide comprising a wild-type Fc polymorphism and 'in another aspect' comprising a polypeptide of the above-described Fc variant and comprising a wild-type Fc polypeptide The polypeptide exhibits reduced ADCC, CDC and ADCP compared to the peptide. In one embodiment, at least one other amino acid substitution in the polypeptide comprising an Fc variant is selected from the group consisting of: S228P, E233P, L234A, 162837.doc -38 - 201245227 L235A, L235E, N297A, N297D or P331S . In a certain aspect of the invention, the polypeptide comprising an Fc variant comprises an antibody. In another aspect of the invention, the polypeptide comprising an Fc variant comprises a human IgGl or IgG4 Fc region. In another aspect of the invention, the variant system Ig(}1 or IgG4 antibody) In another embodiment of the invention, the polypeptide comprising the Pro329 Fc variant further comprises an increased stability of the antibody in the Fc region Addition, substitution or deletion of at least one amino acid residue in question. In another aspect of the invention, 'the affinity of the polypeptide comprising the above Fc variant with the Fen receptor is only slightly changed' and, for example, does not exceed 10-20% of the affinity of the polypeptide of the wild-type Fc polypeptide. In one embodiment, the addition, substitution or deletion of an amino acid residue in the polypeptide comprising the variant is in position 228 of the Fc region and/or Position 235, wherein the numbering system of the ten-crossing region is the numbering system of the EU index as described in Kabat et al. In a specific embodiment, the 228 position of the polypeptide comprising the Fc variant is serine and/or The leucine acid at position 235 is substituted with another amino acid. In a particular embodiment, the polypeptide comprising an Fc variant of the invention comprises a region comprising an amino acid substitution at position 228, wherein the serine residue is Proline acid substitution. In a specific embodiment, the pc of the invention is included The variant polypeptide comprises a region comprising an amino acid substitution at position 235, wherein the leucine residue is substituted with glutamic acid. In a particular embodiment, the polypeptide comprising the Fc variant comprises a triple mutation: 162837.doc - 39· 201245227 Amino acid substitution at position P329, S228P and L235E mutation (P329/SPLE). In yet another embodiment, the polypeptide comprising the FC variant comprises a human igG4 region. In one embodiment, the 'amino acid residue Addition, substitution or deletion is in position 234 and/or 235 of the Fc region where the numbering system of the constant region is the numbering system of the eu index as described in Kabat et al. In a particular embodiment, the Fc is included The leucine acid at position 234 and/or the leucine acid at position 235 in the polypeptide of the variant are substituted with another amino acid. In a particular embodiment, the polypeptide comprising an Fc variant of the invention comprises an amine group at position 234 Acid-substituted Fc region 'where the leucine residue is substituted with alanine. In a particular embodiment, the polypeptide comprising an FC variant of the invention comprises an Fc region comprising an amino acid substitution at position 23 5, wherein leucine The residue is substituted with serine. In a particular embodiment, the polypeptide comprising an Fc variant of a wild-type human Fc polypeptide comprises a triple mutation: an amino acid substitution at the Pro329 position, a L234A and a L235A mutation (P329/LALA). In yet another embodiment, the above polypeptide comprises a human IgG 1 region. Although preferred alterations bind to FcyR', Fc region variants with altered binding affinity to the nascent receptor (FcRn) are also contemplated herein. Fc region variants with improved affinity for FcRn are expected. The body has a longer serum half-life and these molecules will have useful applications in methods of treating mammals where the long half-life of the administered polypeptide is desired, for example, to treat a chronic disease 162837.doc • 40·201245227 disease or condition. Conversely, Fc region variants with reduced FcRn binding affinity are expected to have a shorter half-life, and such molecules can be administered, for example, to a mammal, where it may be advantageous to shorten the circulation time, such as for in vivo diagnostic imaging or for blood. For peptides with toxic side effects when circulating for a long time, etc. It is expected that Fc region variants with reduced FcRn binding affinity are less likely to cross the placenta and are therefore useful for treating diseases or conditions in pregnant women. Fc region variants having altered binding affinity to FcRn include those comprising an Fc region amino acid modification at any one or more of the following amino acid positions: 238, 252, 253, 254, 255, 256, 265, 272, 286 '288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360 , 362, 376, 378, 380, 382, 386, 388,

400, 413, 415, 424, 433, 434, 435, 436, 439 or 447. Those exhibiting reduced binding to FcRn will typically comprise an Fc region amino acid modification at any one or more of the following amino acid positions: 252, 253, 254 '255' 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447; and their increased binding to FcRn will typically be in the following amino acid positions Either or more of the Fc region amino acid modification: 238 bits, 256 bits, 265 bits, 272 bits, 286 bits, 303 bits, 305 bits, 307 bits, 311 bits, 312 bits, 317 bits, 340 bits 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434. In another embodiment, an antibody of the invention can be in any of the categories 162837.doc -41 - 201245227 (such as, but not limited to, IgG, IgM, and IgE) » In certain embodiments, the invention An antibody against members of the system IgG class. In a particular embodiment, the antibodies of the invention belong to the IgGl, IgG2 or IgG4 subclass. In another embodiment, the antibodies of the invention belong to the IgGl subclass and comprise the following amino acid substitutions: P3MG and/or L234A and L235A of the Fc region. In an alternative embodiment, the antibodies of the invention belong to the IgG4 subclass. In a specific embodiment, the antibody of the invention belongs to

The IgG4 subclass and comprises the following amino acid substitutions: P329G and/or S228P and L23 5E in the Fc region. In certain embodiments, a modified antibody of the invention can be produced by combining a variable domain or a fragment thereof with an Fc domain comprising one or more of the amino acid substitutions disclosed herein. In other embodiments, a modified antibody of the invention can be produced by introducing one or more of an amino acid-substituted residue into the Fc domain to modify an antibody comprising the fc domain. Reduced Binding to Fc Ligands It is understood by those skilled in the art that antibodies of the invention may have FcyR&/ or Clq binding properties that are altered (relative to unmodified antibodies) (examples of binding properties include (but are not limited to) Binding specificity, equilibrium dissociation constant (Kd), dissociation and association rate ("and U", binding affinity and/or affinity, respectively) and some changes are expected more or less. Definition of equilibrium dissociation constant (KD) ^ Those skilled in the art will be able to determine the most important kinetic parameters for a given antibody application. For example, reducing the binding to one or more positive regulators (eg, FeYRIIIA) / or enhance the binding to inhibitory Fc receptors (such as 'F_IB) (4) will be suitable to reduce ADCC activity. Therefore, the ratio of binding affinity (for example, equilibrium dissociation often 162837.doc • 42 · 201245227 number (kd)) Indicates that the ADCC activity of the antibody of the present invention is increased or decreased. Further, the modification that reduces the binding to C1 q is suitable for reducing or eliminating CDC activity. The affinity and binding properties of the Fc region of the ligand can be determined by the industry. Determination of a variety of in vitro assays (based on biochemical or immunological assays) of Fc-FqR interactions (ie, specific binding of the Fc region to FcyR), including but not limited to equilibrium methods (eg, enzyme-linked Immunosorbent assay (ELISA) or radioimmunoassay (RIA) or kinetics (eg, BIACORE® analysis) and other methods such as indirect binding assay, competitive inhibition assay, fluorescence resonance transfer (FRET), gel Electrophoresis and chemical chromatography (eg, gel filtration) and other methods may utilize labeling and/or various detection methods on one or more of the components being examined, including but not limited to, color, fluorescence , luminescent or isotope labeling. For a detailed description of binding affinities and kinetics, see Paul, W E,

Immunology, 4th edition, Lippincott-Raven, Philadelphia (1999) » In one aspect of the invention, a polypeptide comprising an Fc variant of the wild-type human Fc region exhibits a reduced expression compared to a polypeptide comprising a wild-type polypeptide. Affinity of the human Fc receptor (FcYR) and/or human complement receptor, the variant comprising an amino acid substitution at the Pro329 position and at least another amino acid substitution. In one aspect, the polypeptide comprising an Fc variant of the invention exhibits an affinity for an Fc receptor of at most 1/2, or at most 1/3, or at most 1/5 of the affinity of the wild-type Fc polypeptide to the Fc receptor, Or at most 1/7, or at most 1/1〇, or at most 1/2〇, or at most 1/30, or at most 1/4〇' or at most 1/5〇, or at most 1/6〇, or at most 1 /70, or up to 1/8 inch, or up to 1/9 inch, or up to 1/1 inch, or up to 162837.doc -43- 201245227 more than 1/200. In one aspect, a polypeptide comprising an Fc variant of the invention exhibits reduced binding affinity to one or more Fc receptors including, but not limited to, FcyRI (CD64) compared to an unmodified antibody. ), including subtypes

FcyRIA, FcyRII and FcyRIII (CD 16, including subtype FcYRIIlA).

In one aspect, a polypeptide comprising an Fc variant of the invention exhibits reduced binding affinity to FcyRI (CD64) FcyRIIA and FcyRIIIA as compared to an unmodified antibody. In one aspect, a polypeptide comprising an Fc variant of the invention exhibits reduced binding affinity to FcYRnA and FcyRIIIA as compared to an unmodified antibody. In one aspect, a polypeptide comprising an Fc variant of the invention exhibits reduced binding affinity to FcyRI (CD64) and FcyRniA2 as compared to an unmodified antibody. In one aspect of the invention, a polypeptide comprising an Fc variant of the invention that exhibits reduced binding affinity to an Fc receptor also exhibits reduced affinity for the ciq receptor. In certain aspects, a polypeptide comprising a & variant of the invention does not comprise a concomitant increase in binding to a FeyRI player compared to a wild-type polypeptide. In certain aspects of the invention 'the polypeptide comprising the variant has reduced human receptor & gamma 相比 compared to the polymorphism comprising the wild type & polypeptide and comprises the human receptor FWIA, FcyIIIB and Clq At least the group's affinity for the receptor. In other aspects of the invention, the inclusion of the variant is reduced in comparison to a polypeptide comprising a wild-type Fc polypeptide and is associated with a human receptor ancestor and comprises 162837.doc 201245227 human receptors FcyllA, FcYIIIB and Clq The affinity of the two other receptors in the group. In other aspects of the invention, a polypeptide comprising an Fc variant has reduced affinity for human FcyRIA, FcYIIIA, FcyllA, FqlllB and Clq as compared to a polypeptide comprising a wild type Fc polypeptide. In another aspect of the invention, a polypeptide comprising an Fc variant has reduced affinity for the human receptors FqRIA, FcylllA, FcyllA, FcyIIIB and Clq as compared to a polypeptide comprising a wild type Fc polypeptide. In one aspect of the invention, a polypeptide comprising an Fc variant of the invention exhibits reduced affinity for FcyRI or FcyRIIA relative to an unmodified antibody. In one aspect of the invention, the polypeptide comprising an Fc variant exhibits an affinity for FcyRI or FcyRIIA of at least 1/2, or at most 1/3, or at most 1/5 of the affinity of the wild-type polypeptide to FqR1 or FcyRIIA, Or at most 1/7, or at most 1/10, or at most 1/20, or at most 1/30, or at most 1/40, or at most 1/50, or at most 1/6〇, or at most 1/7〇, Or at most 1/80, or at most 1/90, or at most ι/loo, or at most 1/200. In one aspect of the invention, a polypeptide comprising an Fc variant exhibits an affinity for FqRi or ® FcYRHA that is at least 90%, at least 80%, at least 70%, at least 60% less than the affinity of the wild-type polypeptide to FcyRI or FcyRIIA, At least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5%. In one aspect of the invention, a polypeptide comprising an Fc variant of the invention exhibits reduced affinity for FcyRIIIA relative to an unmodified antibody. In one aspect, the polypeptide comprising an Fc variant of the invention exhibits an affinity for FcyRIIIA of at least 1/2, or at most 162837.doc •45·201245227 1/3, or at most 1/% of the affinity of the wild-type polypeptide to FcyRIIIA. 5, or at most 1/7, or at most 1/1〇, or at most 1/2〇, or at most 1/30, or at most 1/40, or at most 1/5〇, or at most 1/6〇, or at most 1/70, or up to 1/80, or up to 1/9 inch, or up to 1/1 inch, or up to 1/200 inch. In one aspect of the invention, the polypeptide comprising the variant of the invention is expressed The affinity of FqRIIIA is at least 90%, at least 80%, at least 70%, at least 60%, at least 5%, at least 40 less than the affinity of the wild-type polypeptide to FcyRIIIA. /. At least 30%, at least 20%, at least 1%, or at least 5〇/〇. It is understood in the art that the F1-58V dual gene variant of FqRIIIA has altered binding properties to antibodies. In one embodiment, a polypeptide comprising an Fc variant of the invention binds to the FqRIIIA receptor with reduced affinity relative to a wild-type polypeptide. In one aspect, the polypeptide comprising an Fc variant of the invention exhibits an affinity to FCYRIIIA (FI 58V) of at least 1/2, or at most 1/3, or at most "5, or the affinity of the wild-type polypeptide to FcyRIIIA, or Up to 1/7, or up to 1/10, or up to 1/2 inch, or up to 1/3 inch, or up to 1/40, or up to 1/50, or up to 1/60, or up to 1/7 inch, Or at most 1/80, or at most 1/9〇, or at most ^〇〇, or at most 1/2〇〇. In one aspect of the invention, a polypeptide comprising a variant of the invention exhibits reduced affinity for the Clq receptor relative to an unmodified antibody. In one aspect, a polypeptide comprising an FC variant of the invention exhibits an affinity for the Clq receptor of at most 1/2, or at most 1/3, or at most 1/5 of the affinity of the wild-type polypeptide to the Clq receptor, or Up to 1/7, or up to 1/1 inch, or up to 1/2 inch, or up to 1/30, or up to 1/4 inch, or up to 1/5 inch, or up to 1/6 inch, or up to 1/ 7〇, or up to 1/80, or up to 1/9〇, or up to 1/1〇〇, or to 162837.doc -46- 201245227 more than 1/200. In one aspect of the invention, a polypeptide comprising an Fc variant of the invention exhibits an affinity for C1 q that is at least 90 〇/〇, at least 80%, at least 70%, at least 6〇 less than the affinity of the wild-type polypeptide to C1 q. %, at least 50%, at least 40%, at least 30%, at least 20%, at least 1%, or at least 5〇/0. In one aspect of the invention, a polypeptide comprising an Fc variant of the invention exhibits at least 90% less affinity than a wild-type polypeptide for a human FcyRI, FcyRIIA, FcyRIIIA, FcyRIIIA (FI 58V) or c 1 q receptor, at least 80 %, at least 70%, at least 60°/. At least 50%, at least 40%, at least 30°/. , at least 2 %, at least 1 %, or at least 5%. In another aspect of the invention, the polypeptide comprising an Fc variant of the invention exhibits an affinity for FcyRI, FcyRIIA, FcyRIIIA, FcyRIIIA (FI 58V) and/or Clq receptor of between about 10 nM and 100 nM, respectively. 10 nM to 1 μΜ, 1〇〇nM to about 100 μΜ, or about 100 nM to about 10 μΜ, or about 100 nΜ to about 1 μΜ, or about 1 nΜ to about 100 μΜ, or about 10 ηΜ to about 100 μΜ Or, between about 1 μΜ and about 100 μΜ, or between about 10 μΜ and about 100 μΜ. In certain embodiments, a polypeptide comprising an Fc variant of the invention exhibits an affinity for FcyRI, FcyRIIA, FcyRIIIA, FcyRIIIA (F1-58V) or Clq receptors greater than 100 nM, 500 nM, 1 μΜ, greater than 5 μΜ, greater than 10 μΜ, greater than 25 μΜ, greater than 50 μΜ or greater than 100 μΜ. In another aspect of the invention, a polypeptide comprising an Fc variant of the invention exhibits increased affinity for FcyRIIB as compared to a wild-type polypeptide. In another aspect, the polypeptide comprising an Fc variant of the invention exhibits no change or increase in affinity to FcyRIIB of at least 2 fold, or at least 3 fold, or 162837.doc •47·201245227 at least 5 Multiple, or at least 7 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 Multiple, or at least 100 times, or at least 200 times. In another aspect, the polypeptide comprising an Fc variant of the invention exhibits an increase in affinity to the FcyRIIB receptor to at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50 of the wild-type polypeptide. %, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%. In another aspect of the invention, the variant of the invention exhibits an affinity to the FcyRI, FcyRIIA, FcyRIIIA or FcyRIIIA (FI 58V) or Clq receptor of less than 10 0 μΜ, less than 50 μΜ, less than 10 μΜ, less than 5 μΜ Less than 2.5 μΜ, less than 1 μΜ, or less than 100 ηΜ, or less than 10 ηΜ. Reduced Effector Function In certain aspects of the invention, a polypeptide comprising an Fc variant of the invention modulates effector function compared to a polypeptide comprising a wild-type Fc polypeptide. In another aspect of the invention, the adjustment is an adjustment of ADCC and/or ADCP and/or CDC. In yet another aspect of the invention, the downregulation effect is reduced or decreased. In another aspect of the invention, this is the modulation of ADCC and in another aspect of the invention, the regulation is ADCC down-regulated. In another aspect, the regulation is ADCC and CDC down-regulated. In another embodiment, this is only the ADCC down-regulation. In another embodiment, the ADCC and CDC and/or ADCP are down-regulated. In another aspect of the invention, a polypeptide comprising an Fc variant of the invention downregulates or reduces ADCC/CDC and ADCP. In yet another aspect of the invention, the decrease or down-regulation of ADCC or CDC or ADCP induced by a polypeptide comprising an Fc variant is achieved for inclusion of wild type 162837.doc -48 - 201245227

A decrease in the value of 0%, 2.5%, 5%, ' 10%, 20%, 50% or 75°/〇 observed for the induction of ADCC or CDC or ADCP by the polypeptide of the Fc region, respectively. In another aspect of the invention, the modulatory lineage of ADCC induced by a polypeptide comprising an Fc variant of the invention is reduced such that the EC50 of the Fc variant comprises a polypeptide of a wild type Fc polypeptide > /10. In another aspect, the variants of the invention do not have any substantial ADCC and/or CDC and/or ADCP in the presence of human effector cells as compared to a polypeptide comprising a wild type Fc polypeptide. In another aspect of the invention, a polypeptide comprising an Fc variant of the invention exhibits an effector function that reduces (eg, decreases by at least 20%) or significantly decreases (eg, decreases by at least 50%), which can be ADCC (downregulated), Reduction in CDC and / or ADCP. Reduced ADCC Activity In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/consumption of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcyR binding ability (and thus may lack ADCC activity), but retains FcRn binding ability. Primary cells (NK cells) used to mediate ADCC exhibited only FcyRIII, while mononuclear spheres exhibited FcyRI, FcyRII, and FcyRIII. The FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro analysis of ADCC activity to assess molecules of interest are set forth in U.S. Patent No. 5,500,362 (for example, see Hellstrom, I. et al., Proc_ Nat'l Acad. Sci. USA 83 (1986) 7059- 7063) and Hellstrom, I. et al., Proc. Nat'l Acad. 162837.doc -49- 201245227

Sci. USA 82 (1985) 1499-1502; US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays can be used (see, for example, ACTITM non-radioactive cytotoxicity assays for flow cytometry (CellTechnology, Mountain View, CA), and CytoTox 96® non-radioactive cytotoxicity assays (Promega) , Madison, WI)). Useful effector cells for such analysis include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal animal models (e.g., Clynes et al, Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656. Revealer). Clq binding assays can also be performed to confirm that the antibody does not bind Clq and thus lacks CDC activity. See, for example, the Clq and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC analysis can be performed (see, for example, Gazzano-Santoro et al, J. Immunol. Methods 202 (1996) 163; Cragg, MS et al, Blood 101 (2003) 1045-1052; and Cragg, MS and

Glennie, M.J., Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance/half life assays can also be performed using methods known in the art (see, for example, Petkova, S. B. et al, Int'l. Immunol. 18(12) (2006) 1759-1769). A polypeptide comprising an Fc variant of the invention is characterized by an in vitro functional assay that determines the function of one or more FcYR-mediated effector cells. In certain embodiments, the antibodies of the invention have binding properties in their in vitro-based assays in an in vivo model (eg, as described and disclosed herein) 162837.doc -50 - 201245227 and effectors Cell-like functional binding properties and effector cell function. However, the invention does not exclude variants of the invention that do not exhibit the desired phenotype but exhibit the desired phenotype in vivo in an in vitro assay. In one embodiment, the polypeptide comprising the Fc variant of the invention is unmodified. The wild-type fc polypeptide exhibits reduced ADCC activity compared to the wild-type fc polypeptide. In another aspect, the polypeptide comprising an Fc variant of the invention exhibits at most 1/2, or at most 1/3, or at most 1/5 or at most 1/1 〇 or at most 1 of the unmodified antibody of the ADCC activity. /5 or up to 1/100. In another embodiment, an antibody of the invention exhibits a decrease in ADCC activity of at least 1%, or at least 20%, or at least 30%/〇, or at least 40%, or at least 50%, or At least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 1%. In yet another aspect of the invention, the decrease or down-regulation of ADCC induced by a polypeptide comprising an Fc variant reaches 〇% of the value observed for the induction of ADCC or CDC or ADCP by a polypeptide comprising a wild-type FC region, respectively. , 2.5%, 5%, 10%, 20°/. , 50°/. Or 75°/. The reduction. In certain embodiments, a polypeptide comprising an Fc variant of the invention does not have detectable adcC activity. In a specific embodiment, the reduction and/or removal of ADCC activity is attributable to a decrease in the affinity of the polypeptide comprising the Fc variant of the invention to the FC ligand and/or receptor. In a particular embodiment of the invention, the downregulation of ADCC is reduced in potency such that the Fc variant comprises a fragmented EC5 野生 wild-type Fc polypeptide of about 1/1 〇 or less. In another aspect, a polypeptide comprising a variant of the invention modulates ADCC and/or CDC and/or ADCP. In a particular aspect, the variants of the invention exhibit reduced CDC and ADCC and/or ADCP activity. I62837.doc 201245227 Reduced CDC activity The complement activation pathway is initiated by binding of the first component of the complement system (C1q) to a molecule (eg, an antibody) complexed with a homologous antigen. CDC analysis can be performed to assess complement activation, for example, as Gazzano-Santoro et al., j

As described in Immunol. Methods 202 (1996) 163. The binding properties of different variants to Clq can be analyzed by ELISA sandwich-type immunoassay. The half-maximal reaction antibody concentration determines the EC5Q value. This readout value is reported as the coefficient of variation of the sample and reference material as measured by the relative difference from the reference standard measured on the same plate. In one embodiment, a polypeptide comprising an Fc variant of the invention exhibits reduced affinity for Clq relative to a wild-type polypeptide. In another embodiment A, the polypeptide comprising an Fc variant of the invention exhibits an affinity for the Clq receptor of up to 1/2, or at most 1/3' or at most 1/5, or at most 1/7 of the wild-type polypeptide, Or at most 1/10, or at most 1/; 2〇, or at most 1/3〇, or at most 1/4〇, or at most 1/50, or at most 1/60, or at most 1/70, or at most" 8〇, or up to 1/90, or up to 1/1〇〇, or up to 1/2〇〇. In another embodiment of the invention, the polypeptide comprising the variant of the invention exhibits an affinity for Clq that is at least 90%, at least 80%, at least 70%, at least 60%, at least less than the affinity of the wild-type polypeptide. 50% 'at least 40 〇 / 〇, at least 30%, at least 20%, at least 〇 %, or at least 5%. In another embodiment, the variant of the invention exhibits an affinity for C1 q of from about 1 〇〇 nM to about 100 μΜ, or from about 1 〇〇 nM to about 1 μμΜ, or from about 100 nM to about 1 μΜ. Or, from about 1 Μ 约 to about 1 〇〇 μΜ, or from about 1 〇ηΜ to about 100 μΜ ' or from about 1 μΜ to about 1 μμΜ, or from about 1 μμΜ to about 1 μμΜ. At some 162837.doc ^

S 201245227 In some embodiments, a polypeptide comprising an Fc variant of the invention exhibits an affinity for cIq greater than 1 μΜ, greater than 5 μΜ, greater than 10 μΜ, greater than 25 μΜ, greater than 50 μΜ or greater than 10 μ μΜ. In one embodiment, a polypeptide comprising an Fc variant of the invention exhibits reduced iCDC activity compared to a wild-type Fc polypeptide. In another embodiment, the polypeptide comprising an Fc variant of the invention exhibits at most 1/2, or at most 1/3, or at most 1/s or at most 1/1 〇 or at most 1/5 of the CDC-active wild-type polypeptide. 〇 or up to 1/100. In another embodiment, a polypeptide comprising a variant of the invention exhibits a decrease in CDC activity of at least 10° relative to a wild-type polypeptide relative to φ. Or at least 20%, or at least 30%, or at least 4%, or at least 5%, or at least. Or at least 70%, or at least 80%, or at least 9%, or at least 1%, or at least 200%, or at least 300%, or at least 4%, or at least 5%. In certain aspects, a polypeptide comprising an Fc variant of the invention does not exhibit detectable cDc = sex. In particular embodiments, the reduction and/or removal of CDC activity can be attributed to a decrease in the affinity of the Fc variant-containing polypeptide to the Fc ligand and/or receptor. # REDUCED ANTIBODIES RELATED TO INFLUENCE The industry should understand that biological therapy can have adverse toxicity problems associated with directing the immune system to recognize and attack the complex nature of undesired cells and/or silk markers. When attack identification and/or targeting does not occur, results such as adverse toxicity may occur if treatment is required. For example, antibody staining of non-dried tissues can indicate potential toxicity problems. In one aspect, a polypeptide comprising an Fc variant of the invention exhibits reduced staining of non-dried tissue compared to a wild-type polypeptide. In another aspect, the polypeptide comprising 162837.doc •53·201245227 of the present month Fe variant exhibits at least 1/2, or at most 1/3, of the stained wild-type FC polypeptide of the reduced non-tissue tissue, or Up to 1/5, or up to 7 or up to 1/10, or up to 1/20, or up to 1/30, or up to _, or up to 1/50, "up to 1/6", < up to 1/7〇 , or at most 1/80, or at most 1/90, or at most "100, or at most 1/200. In another embodiment, a variant of the invention exhibits a reduced non-targeted tissue staining reduction of at least 10%, or at least 20%, or at least, or to: > 40 / / relative to a wild-type Fc polypeptide. Or at least 5%, or at least 6%, or at least, or at least 8%, or at least 90%, or at least 100%, or at least 200. /. , or at least 300%, or at least 400%, or at least 5%. In one embodiment, a polypeptide comprising a F c variant of the invention exhibits reduced antibody-related toxicity as compared to a wild-type polypeptide. In another embodiment, the polypeptide comprising an Fc variant of the invention exhibits at most 1/2, or at most 1/3, or at most 1/5, or at most 1/7, or at most 1/1 of the toxic wild-type polypeptide. 〇, or up to 1/20, or up to 1/30, or up to 1/4 inch, or up to 1/5 inch, or up to 1/60' or up to 1/70, or up to 1/8 inch, or up to 1 /9〇, or up to 1/100, or up to 1/200. In another aspect, a polypeptide comprising an Fc variant of the invention exhibits a reduced toxicity of at least 1%, or at least 2〇/〇, or at least 30 relative to a wild-type polypeptide. /. , or at least 4 〇〇 /. , or at least 5%, or at least 60/. , or at least 70 ° /. , or at least 8%, or at least 9%, or at least 100/. Or at least 200%, or at least 3%, or at least 4%, 500%. Thrombosis agglutination In one aspect of the invention, the wild-type polypeptide induces platelet activation and / / 162837.doc 54 · 201245227 or platelet aggregation, and variants thereof (ie, comprising the polypeptide of the variant) exhibit reduced or even removed thrombus Cell activation and/or agglutination. In another aspect of the invention, the wild type polypeptide filaments are directed to a platelet protein. In another aspect, the anti-system CD9 antibody. In another embodiment, the CD9 antibody has a mutation at the P329G position and/or at L234A/L235A or S228p/L23_0; 329G/LALA, P329G/SPLE). In yet another embodiment, the antibody is characterized by SEQ ID NOs: 8-14. It is understood in the industry that biological therapy may have the undesirable effect of thrombocyte agglutination. In vitro and in vivo assays can be used to measure thrombocyte agglutination. It is assumed that the in vitro analysis reflects the situation in the living body. In one aspect, a polypeptide comprising an Fc variant of the invention exhibits reduced thrombocyte agglutination in an in vitro assay as compared to a wild-type polypeptide. In another aspect, the polypeptide comprising an Fc variant of the invention exhibits at most 1/2, or at most 1/3, at most 1/5, or at most of the reduced thrombocyte agglutination wild-type polypeptide in an in vitro assay. 1/7, or at most 1/1〇' or at most 1/2〇, or at most 1/30, or at most 1/4〇, or at most ^0, or at most 1/6〇, or at most ♦ 1/70, Or at most 1/80, or at most 1/9 〇, or at most 1/1 〇〇, or at most 1/200 ^ In another embodiment, the polypeptide comprising the variant of the invention is in vitro relative to the wild-type polypeptide Reduced assayed cell agglutination in the assay is reduced by at least 10%, or by at least 20%, or by at least 30°/. , or at least 4%, or at least 50%, or at least 6 inches. /0, or at least 70%, or at least 8%, ge to | 90%, or at least 100%, or at least 2%, or at least 3%, or to +400%, or at least 500%. In another aspect, a polypeptide comprising an Fc variant of the invention exhibits reduced thrombocyte agglutination in vivo as compared to wild-type polypeptide 162837.doc • 55-201245227. In another aspect, the variant of the invention exhibits at most 1/2, or at most 1/3, or at most 1/5, or at most 1/7 of the reduced thrombocyte agglutination wild-type Fc polypeptide in an in vivo assay. Or at most 1/10, or at most 丨/2〇, or at most 1/3〇, or at most 1M0, or at most 1/50, or at most 1/60, or at most 1/7〇, or at most 1/80, Or at most 1/90, or at most ι/1()0, or at most 1/2〇〇. In another embodiment, a polypeptide comprising an Fc variant of the invention exhibits a reduced thrombocyte agglutination reduction of at least %, or at least 20%, or at least 30%, or at least 40 in vivo in an in vivo assay relative to a wild-type polypeptide. %, or at least 50%, or at least 600/. Or at least 70%, or at least 80%, or at least 90%, or at least 100°/. Or at least 200%, or at least 300%, or at least 400%, or at least 500%. Internalizing Antibodies The variants of the invention can bind to an internalizable cell surface antigen to further transport the antibody into the cell. Following intracellular, the variant can be released into the cytosol 'targeting a specific compartment' or recycled to the cell surface. In some embodiments, a variant of the invention binds to an internalized cell surface antigen. In other embodiments, an antibody of the invention can be targeted to a particular organelle or compartment of a cell. In other embodiments, the variants of the invention may be recycled to the cell surface or periphery after internalization. In a specific embodiment, the antibody of the invention is specific for P-selectin, CD9, CD19, CD81, CCR5 or CXCR5, IL17a or 11-33. Antibody Preparation 162837.doc • 56· 201245227 In a preferred embodiment of the invention, a polypeptide-based antibody comprising an Fc region is modified according to the teachings herein. The technique for producing antibodies is based on: antigen selection and preparation. If the polypeptide is an antibody, it is directed against the antigen of interest. Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal having the disease or condition produces a therapeutic benefit in the mammal. However, antibodies directed against non-polypeptide antigens (e.g., tumor-associated glycolipid antigens; see U.S. Patent No. 5,091,178) are also contemplated. • if the antigen is a polypeptide, it may be a transmembrane molecule (eg receptor) or a ligand (eg growth factor). Example antigens include molecules such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth Hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; heart anti-trypsin, tamsin A-bond, temsin B-chain; proinsulin; follicle stimulating hormone; calcitonin Luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor (TF) and von Willebrands factor; anticoagulant factors such as protein c; atrial natriuretic factor; Pulmonary surfactant; plasminogen activator, such as urokinase or human urine or tissue plasminogen activator (t_PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor alpha and tumor necrosis Factor_β; enkephalinase; RANTES (normal tau cell expression and secretion factor regulated by activation); human macrophage inflammatory protein (Μιρ-1_α); serum albumin, such as human serum albumin MueUerian inhibiting substance; relaxin A• chain; relaxin B_chain; relaxinogen 'mouse gonadotropin-related peptide; microbial protein, such as bupropion 162837.doc -57- 201245227 enzyme; DNase; IgE; cytotoxic T-lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptor, hormone or growth factor receptor; A or D; rheumatoid factor; neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4 'neurotrophin_5 or neurotrophin-6 (NT-3 , NT-4, NT-5 or NT-6), or nerve growth factor, such as NGF-β, small plate-derived growth factor (PDGF); fibroblast growth factor, such as aFGF and bFGF; epidermal growth factor (EGF) Transforming growth factor (TGF), such as TGF-α and TGF-隹β, including TGF-βΙ, TGF-P2, TGF-p3, TGF-P4 or TGF-P5; Tengdao-like growth factor and insulin-like growth Factor (IGF-I &IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein; 0 proteins, such as 匸04, 匸08, €019 and €020; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic protein (BMP); interferons, such as interferon-α, interferon-β and interference -γ; colony-stimulating factor (CSF), such as 'M-CSF, GM-CSF and G-CSF; interleukin (IL), for example, IL-1 to IL-10; superoxide dismutase; surface film Egg white; decay accelerating factor; viral antigen 'eg part of AID S envelope; transporter; homing receptor; addressin; regulatory protein; integrin, such as CDlla, CDllb, CDllc, CD18, ICAM, VLA- 4 and VCAM; tumor associated antigens, such as the HER2, HER3 or HER4 receptor; and fragments of any of the polypeptides listed above. Preferred molecular targets for antibodies encompassed by the invention include CD proteins such as CD4, CD8, CD19, CD20 and CD34; members of the ErbB receptor family, 162837.doc-58-

S 201245227 eg EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Macl, pl50.95, VLA-4, ICAM-1, VCAM, α4/β7 integrin and αν/β3 Integrins, including their alpha or beta subunits (eg, anti-O11 & anti-〇18 or anti-11115 antibodies); growth factors such as VEGF; tissue factor (TF); alpha interferon (alpha-IFN) Interleukins, such as IL-8; IgE; blood group antigen; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, and the like. A soluble antigen or a fragment thereof conjugated to other molecules as appropriate may be used as an immunogen for generating an antibody. For transmembrane molecules such as receptors, fragments thereof (e.g., the extracellular domain of the receptor) can be used as the immunogen. Another option is to use cells that express transmembrane molecules as immunogens. Such cells may be derived from a natural source (e. g., a cancer cell line) or may be a cell that has been transformed by a recombinant technique to express a transmembrane molecule. Those skilled in the art will recognize other antigens and the forms in which they can be used to prepare antibodies. Multiple antibodies are preferably produced in multiple animals by multiple subcutaneous (sc) injections or intraperitoneal (ip) injections of the associated antigen Φ and adjuvants. Usefully, difunctional reagents or derivatizing agents can be used (eg 'maleimide benzylidenesulfonyl succinimide acetal (coupled via cysteine residues), N-hydroxysuccinimide (via an lyophilic acid residue), glutaraldehyde, succinic anhydride, s〇Cl2 or carbodiimide (wherein R and R1 are different alkyl groups) to associate the relevant antigen with an immunogenic protein in the immunized species (for example, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor). 162837.doc •59- 201245227 Immunization of animals against antigens, immunogenic conjugates or derivatives is performed as follows: (for example) 丨00 (four) or 5 protein or conjugate (for rabbits or mice, respectively) ) Combined with 3 volumes of Freund's complete adjuvant' and the solution was injected intradermally into multiple sites. One month later, the animals were boosted by a multi-site subcutaneous injection with an initial amount (for example) of 1/10 of the peptide or conjugate in Freund's complete adjuvant. After 7 days to 14 days, the blood of the animals was taken and the serum was subjected to antibody titer analysis. Animals are boosted until the titer reaches a steady state. Preferably, the animal is boosted with a conjugate coupled to a different protein and/or the same antigen coupled via a different crosslinking reagent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Also, agglutinating agents such as alum are suitable for enhancing the immune response. The monoclonal antibody monoclonal antibody can be prepared by the hybridoma method first described by Kohler et al., Nature, 256 (1975) 495, or can be prepared by the recombinant DNA method (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal (e.g., hamster or macaque) is immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Another option is to immunize lymphocytes in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (e.g., polyethylene glycol) to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) pp. 59-103). The hybridoma cells thus prepared are sown in a suitable medium and grown therein at 162837.doc • 60·201245227, which preferably contains one or more substances which inhibit the growth or survival of the unfused parental bone tumor cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium used for hybridomas usually includes hypoxanthine, aminopterin, and thymidine (HAT medium). ), these substances prevent the growth of HGPRT-deficient cells. Preferred myeloma cell lines are effective for fusion, support for selected antibody-producing cells, stable production of antibodies, and sensitivity to media such as HAT medium. Preferred myeloma cell lines are, in particular, murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors (purchased from Salk Institute Cell Distribution Center, San Diego, Calif. USA) and SP-2. Or X63-Ag8-653 cells (purchased from the American Type Culture Collection, Rockville, Md. USA.). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J., Immunol. 133 (1984) 3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, New York (1987) pp. 51-63). The culture medium for growing hybridoma cells is analyzed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, these pure lines can be subjected to colonization by a limiting dilution procedure and 182837.doc-61 · 201245227 can be grown and grown by standard methods. (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) pp. 59-103). The medium suitable for this purpose includes, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals. The monoclonal antibodies secreted by the meptoline can be separated from the culture medium, ascites or serum by a conventional immunoglobulin purification program, such as protein A-Sepharose, hydroxyapatite. Stone chromatography, gel electrophoresis, dialysis or affinity chromatography. The DNA encoding the monoclonal antibody can be easily isolated and sequenced using a conventional procedure (for example, by using an oligonucleotide probe capable of specifically binding to a gene encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells are used as a preferred source of this DNA. After isolation, the DNA can be immediately placed in a performance vector and subsequently transfected into a host cell that does not produce immunoglobulin (eg, E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells). Or myeloma cells) to achieve synthesis of monoclonal antibodies in recombinant host cells. Recombinant production of antibodies will be described in more detail below. In yet another embodiment, the antibody or antibody fragment can be isolated from an antibody phage library generated using the technique set forth in McCafferty, J. et al, Nature 348 (1990) 552-554. Clackson et al, Nature 352 (1991) 624-628 and Marks et al, J. Mol. Biol. 222 (1991) 58 1-597 describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe group recombination and in vivo recombination (Waterhouse by chain reorganization (Marks et al, Bio/Technology 10 (1992) 779-783), and as a strategy for constructing maximal phage libraries 162837.doc • 62- 201245227 Et al., Nuc. Acids. Res. 21 (1993) 2265-2266) to generate high affinity (nM range) human antibodies. Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma technology for isolation of monoclonal antibodies. DNA can also be modified, for example, by substituting homologous murine sequences with coding sequences for human heavy and light chain constant domains (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci) USA, 81 (1984) 6851-6855) or covalently conjugates all or part of the φ coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Typically, such non-immunoglobulin polypeptides can be substituted for the constant domain of an antibody or a variable domain thereof that can replace an antigen combining site of an antibody to produce a chimeric bivalent antibody comprising a pair An antigen has a specific antigen combining site and another antigen combining site that is specific for a different antigen. Antibody Affinity In certain embodiments, the dissociation constant (Kd) of an antibody provided herein is y ΜμμΜ, $1 〇〇nM, £i〇nM, $1 nM, $0.1 nM, S0.01 nM, or $0.001 nM (eg, I〇-8 m or less, for example, ΙΟ·8 M to l〇·13 M, for example, l〇_9 M to ΙΟ·〗 3 m). In one embodiment, 'Kd' is measured by radiolabeled antigen or Fc receptor binding assay (RIA). This assay is performed using the Fab format of the antibody of interest and its antigen as described in the analysis below. The solution binding affinity of the Fab to the antigen is measured by the following method: using a minimum concentration of the (1251) labeled antigen to equilibrate the Fab in the presence of a titrated series of unlabeled antigens, and then using 162837.doc -63·201245227 A plate coated with an anti-Fab antibody captures the bound antigen (see, for example, Chen et al, J. Mol. Biol. 293 (1999) 865-881). To establish the conditions for this assay, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 pg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then at room temperature. Blocked with 2% (w/v) bovine serum albumin in PBS for 2 to 5 hours (about 23 ° C). In a non-adsorbing plate (Nunc No. 269620), 100 pM or 26 pM [1251]-antigen is mixed with serial dilutions of the Fab of interest (eg, consistent with the evaluation of the anti-VEGF antibody Fab-12, see Presta et al. , Cancer Res. 57 (1997) 4593-4599). The Fab of interest is then incubated overnight; however, incubation can be continued for a longer period of time (e.g., about 65 hours) to ensure equilibrium is achieved. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., up to one hour). The solution was then removed and the plate was washed 8 times using 0.1 ° / 〇 polysorbate 20 ( butyl \\ ^ 1 ^ -20 @) in PBS. While the plates were dry, 150 μΐ/well scintillant (MICROSCINT-20tm; Packard) was added and the plates were counted on a TOPCOUNTtm gamma counter (Packard) for 10 minutes. The concentration of each Fab that yields a maximum combination of less than or equal to 20% is selected for competitive binding analysis. According to another embodiment, Kd is measured by surface plasmon resonance analysis using a fixed antigen or BIACORE®-2000 or BIACORE®-3000 (BIAcore, Piscataway, NJ) at 25 °C. The Fc receptor CM5 wafer was implemented in about 10 reaction units (RU). Briefly, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were used according to the supplier's instructions. I62837.doc -64- 201245227 Activated carboxymethylated dextran biosensor wafer (CM5, BIACORE). The antigen was diluted to 5 pg/ml (about 0.2 μM) using 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μΐ/min to achieve about 1〇 reaction unit (RU) of the coupled protein. After the antigen was injected, 1 μ of ethanolamine was injected to block the unreacted group. For kinetic measurements, two-fold serial dilutions of Fab (〇·78 nM to 500 nM) were injected at 25 ° C at a flow rate of approximately 25 μΐ/min to a 0.05% polysorbate 20 (TWEEN) -20 tm) surfactant in PBS (PBST). A simple one-to-one Langmuir binding model φ (version 3.2 BIAC0RE positive evaluation software) was used to calculate the association rate and dissociation rate (4) by simultaneously combining the association and dissociation sensing patterns. The equilibrium dissociation constant (Kd) is calculated as a ratio, for example, see Chen et al, J. Mol Biol. 293 (1999) 865-881. If the upper rate exceeds 106 S-1 as measured by the surface plasma resonance analysis described above, the upper rate can be determined by using a fluorescence quenching technique at 25 ° C in the presence of increasing concentrations of antigen. Increasing or decreasing the fluorescence emission intensity of the 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) (excitation wavelength = 295 nm; Lu emission wavelength = 34 〇 nm '16 nm band pass), such as Spectrophotometer in a spectrophotometer, such as a stop-flow device with a stirring cuvette (Aviv

Instruments) or 8000-Series SLM-AMINCOtm Spectrophotometer (ThermoSpectronic) ° In certain embodiments, anti-system antibody fragments are provided herein. Antibody fragments include, but are not limited to, Fab, Fab', Fab, -SH, F(ab')2, Fv and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al, Nat. Med. 9 (2003) 129-134. For a review of the 162837.doc-65-201245227 scFv fragment, see, for example, piuckthUn, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed. (Springer-Verlag, New York), pp. 269-315 (1994); See also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising a salvage receptor binding epitope residue and having an extended in vivo half-life, see U.S. Patent No. 5,869,046. A double-stranded anti-system has two antigen-binding sites which are bivalent or bispecific antibody fragments. See, for example, EP 0 404 097; WO 1993/01161; φ Hudson et al, Nat. Med. 9 (2003) 129-134; and Hollinger et al., Proc. Natl. Acad. Sci_ USA 90 (1993) 6444-6448 . Tri-chain antibodies and four-chain antibodies are also described in Hudson et al, Nat. Med. 9 (2003) 129-134. A single domain anti-system comprises antibody fragments that comprise all or a portion of a heavy chain variable domain or all or a portion of a light chain variable domain in an antibody. In certain embodiments, a single domain anti-system human single domain antibody (Domantis ' Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1). Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein, chimeric and humanized antibodies at a certain In some embodiments, the anti-system chimeric antibodies provided herein. Some of the chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and 162,837.doc 66.201245227

Mornson et al., proc. Natl. Acad. Sci. USA, 81 (1984) 685 1-6855). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate animal (e.g., a monkey)) and a human constant region. In yet another example, a "class-switching" antibody that has been altered in the class or subclass of the anti-system from the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, a chimeric anti-system humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental φ non-human antibody. Typically, a humanized antibody comprises one or more variable domains, wherein the HVR (e.g., CDR) (or a portion thereof) is derived from a non-human antibody' and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody also optionally includes at least a portion of the human constant region. In some embodiments, some of the FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody that produces an HVR residue) to, for example, restore or improve antibody specificity or affinity. Humanized antibodies and methods for their preparation are reviewed, for example, in Almagr(R) and Luransson, Front. Biosci. 13 (2008) 1619-1633, and further described in, for example, the following: Rieehmann et al, Nature 332 (1988). 323-329; Queen et al., Proc. Nat'l Acad. Sci. USA 86 (1989) 10029-10033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al. , Methods 36 (2005) 25-34 (Explaining SDR (a-CDR) Transplantation); Padlan, Mol. Immunol. 28 (1991) 489-498 (Explaining "Surface Resurfacing"); Dall'Acqua et al., Methods 36 ( 2005) 43-60 (Explanation 162837.doc -67- 201245227 "FR Reorganization"); and Osbourn et al, Methods 36 (2005) 61-68 and Klimka et al, Br. J. Cancer, 83 (2000) 252_260 ( Explain the "guided selection" method of FR restructuring. Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" approach (see, for example, Sims et al, J. Immunol. 151 (1993) 2296); The framework region of the consensus sequence of the human antibody of a particular subgroup of the bond or heavy bond variable region (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA, 89 (1992) 4285; and Presta et al, J Immunol., 151 (1993) 2623); human maturation (via somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13 (2008) 1619-1633); The framework regions obtained from screening FR libraries (see, for example, Baca et al, J. Biol. Chem. 272 (1997) 10678-10684 and Rosok et al,] Biol. Chem. 271 (1996) 2261 1-22618) . Human Antibodies In certain embodiments, anti-system human antibodies are provided herein. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are outlined in Van Dijk Avan de Winkel, Curr. Opin. Pharmacol. 5 (2001) 368-74 and Lonberg, Curr. Opin. Immunol. 20 (2008) 450-459 by administering immunogenic to transgenic animals Human antibodies are prepared which have been modified to produce intact human antibodies or intact antibodies with human variable regions that are stimulated by antigenicity. Such animals typically contain all or part of the human immunoglobulin locus, which can be substituted for the endogenous 162837.doc-68 - 201245227 immunoglobulin locus, or present extrachromosomally or randomly integrated into the animal chromosome. In such transgenic mice, the endogenous immunoglobulin loci are generally not activated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23 (2005) 1117-1125. See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, the disclosure of which is incorporated herein by reference in its entirety by U.S. Pat.

HUMAB® technology; U.S. Patent No. 7,041,870, which describes K-M

MOUSE® technology; and US Patent Application Publication No. US 2007/0061900, which describes VELOCIMOUSE® technology). Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions. Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor, J. Immunol., 133 (1984) 3001; Brodeur et al., Monoclonal Antibody Production

Techniques and Applications, Marcel Dekker, ‘New York, (1987) pp. 51-63; and Boerner et al, J. Immunol., 147 (1991) 86. Human antibodies produced by human B cell hybridoma technology are also described in Li et al, Proc. Natl. Acad. Sci. USA, 103 (2006) 3557-3562. Other methods include those described in, for example, U.S. Patent No. 7,189,826 (which describes the production of a single human IgM antibody from a hybridoma cell line) and Ni, Xiandai Mianyixue, 26(4) (2006) 265-268 (explaning humans) - Human hybridoma). Human hybridoma technology (Trioma technology) is also described in the following literature: Vollmers and Brandlein, 162837.doc •69- 201245227

Histology and Histopathology 20 (3) (2005) 927-937; and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology 27(3) (2005) 185-91. Human antibodies can also be produced by isolating Fv pure line variable domain sequences selected from human phage display libraries. These variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are set forth below. Antibodies derived from libraries The antibodies of the invention can be isolated by screening antibodies having one or more desired activities from a combinatorial library. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding properties. Such methods are reviewed, for example, in Hoogenboom, HR et al, Methods in Molecular Biology 178 (2002) 1-37 (Edited by O'Brien et al., Human Press, Totowa, NJ, 2001) and further illustrated in (for example) In the following literature: McCafferty, J. et al, Nature 348 (1990) 552-554; Clackson et al, Nature 352 (1991) 624-628; Marks et al, J. Mol. Biol, 222 (1992) 581-597 Marks and Bradbury, Methods in Molecular Biology 248 161-175 (Lo edit, Human Press, Totowa, NJ, 2003); Sidhu et al, J. Mol. Biol. 338(2) (2004) 299-310; Lee et al. Human, J. Mol. Biol. 340(5) (2004) 1073-1093; Fellouse, Proc. Natl. Acad. Sci. USA 101(34) (2004) 12467-12472; and Lee et al., J. Immunol. Methods 284(1-2) (2004) 119-132 o In some phage display methods, the VH and VL gene profiles are separately selected by polymerase chain reaction (PCR) 162837.doc -70- 201245227 and Randomly recombined into a phage library, the antigen-binding bacillus can then be screened as described in Winter et al., Ann. Rev. Immunol., 12 (1994) 433-455. Phage typically display antibody fragments which are single-chain Fv (scFv) fragments or Fab fragments. A library from an immunogen source can provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the native profile can be colonized (e.g., from humans) to provide a single source of antibodies to a number of non-autologous and autoantigens that are free of any immunity, as by Griffiths et al., EMBO J, 12 (1993). ) φ 725-734 stated. Finally, a natural library can also be produced synthetically by selecting an unrearranged V-gene fragment from a stem cell and encoding a highly variable CDR3 region using a PCR primer containing a random sequence and achieving in vitro rearrangement. As described by Hoogenboom and Winter, J. Mol. Biol., 227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007 /0160598, 2007/0237764, No. 2007/〇292936 and No. 2009/0002360. An antibody or antibody fragment isolated from a human antibody library can be considered as a human antibody or a human antibody fragment herein. Multispecific Antibodies In certain embodiments, anti-system multispecific antibodies, such as bispecific antibodies, are provided herein. A multispecific anti-system against at least two different sites, with a "sigma σ-specific monoclonal antibody. In certain embodiments, one of the binding specificities is for a specific antigen and the other is for Any other anti-162837.doc •71 · 201245227 original. In certain embodiments, the bispecific antibody can bind to two different epitopes of the antigen. Bispecific antibodies can also be used to localize the cytotoxic agent to performance. A cell to which an antibody binds to an antigen. A bispecific antibody can be produced as a full length antibody or antibody fragment. Techniques for preparing a multispecific antibody include, but are not limited to, recombinantly expressing two immunoglobulin heavy chains having different specificities. Light chain pairs (see Miistein and Cuello, Nature 305 (1983) 537, WO 93/08829, and Traunecker et al, EMBO J. 1 (1991) 3655), and "knob-in-hole" Modification (see, for example, U.S. Patent No. 5,731,168). Also, a multispecific antibody can be prepared by engineering an electrostatic traction effect for preparing an antibody Fc-heterodimer molecule (w〇2009/089004 A1); making two or more antibodies or fragments Cross-linking (see, for example, U.S. Patent No. 4,676,980, and Brennan et al, Science 229 (1985) 81); production of bispecific antibodies using leucine zippers (see, for example,

Kostelny et al, J. Immunol, 148(5) (1992) 1547-1553); using "double-stranded antibody" techniques for the preparation of bispecific antibody fragments (eg 'see' Hollinger et al., Proc. Natl. Acad. Sci. USA, 90 Lu (1993) 6444_6448), and using a single bond Fv (sFv) dimer (see, for example, Gruber et al, J. Immun〇l. 152 (1994) 5368); and preparation of trispecific Sex antibodies are described, for example, in Tutt et al, j. Immunol. 147 (1991) 60. Also included herein are antibodies (including "octopus antibodies") that have been engineered to have two or more functional antigen binding sites (see, for example, US 2006/0025576 A1). 162837.doc -72-

S 201245227 The antibodies or fragments herein also include "dual-acting FAb" or "DAF" containing an antigen-binding site that binds to a specific antigen and another different antigen (see, for example, US 2008/0069820). Antibody Variants Having Altered Binding Affinity to Antigens In certain embodiments, it may be desirable to improve the binding affinity of an antibody to an antigen and/or other biological properties. The amino acid sequence of the antibody is prepared by introducing a suitable modification into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct has desirable properties, such as antigen binding. Substitutions, Insertions, and Deletion Variants In certain embodiments, polypeptides comprising an Fc variant are provided that additionally have one or more amino acid substitutions in addition to the Fc moiety. Sites of interest for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown under the heading "Conservative substitutions" in Table i. Further substantial changes are provided under the heading "Example Substitutions" in Table 1 and are further described below with reference to the amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and screened for products having the desired activity, e.g., retained/improved antigen binding or reduced immunogenicity. Table 1: Example of initial residues Substituting conservative substitutions Ala (A) Val ; Leu ; lie — Val " _~~ Arg(R) Lys ; Gin ; Asn Lys Asn (N) Gin ; His ; Asp, Lys ; Arg "GliT Asp (D) Glu ; Asn Glu 162837.doc •73· 201245227 Initial Residues Substitutively Substituted Conservative Substituted Cys(C) Ser; Ala Ser Gln(Q) Asn ; Glu Asn Glu (E) Asp ; Gin Asp Gly(G) Ala Ala His (H) Asn; Gin; Lys; Arg Arg He (I) Leu; Val; Met; Ala; Phe; ortho-leucine Leu Leu (L) ortho-amine; Met ; Ala ; Phe lie Lys ( K ) Arg ; Gin ; Asn Arg Met ( M ) Leu ; Phe ; lie Leu Phe ( F ) Trp ; Leu ; Val ; lie ; Ala ; Tyr Tyr Pro (P) Ala Ala Ser ( S) Thr Thr Thr(T) Val ; Ser Ser Trp(W) Tyr ; Phe Tyr Tyr(7) Trp ; Phe ; Thr ; Ser Phe Val(V) lie ; Leu ; Met ; Phe ; Ala ; Grouping of side chain properties: (1) Hydrophobic residues: lie, Met, Ala, Val, Leu, lie; (2) Neutral hydrophilic residues: Cys, Ser, Thr, Asn, Gin; (3) Acidic residues Base: Asp, Glu; (4) Test residue: His , Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic residues: Trp, Tyr, Phe. Non-conservative substitutions require the exchange of members of one of these categories into another category. One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, the resulting variants selected for further study will have modifications (eg, improvements) and/or substantial retention of the parent antibody relative to certain biological properties (eg, enhanced affinity, reduced immunogenicity). Certain biological properties of the generation of antibodies. An exemplary substitutional system affinity matured antibody, which can be conveniently produced, for example, using 162837.doc-74-201245227 based on an affinity development technology (e.g., as described herein). Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for specific biological activities (e. g., binding affinity). Changes (e. g., substitutions) can be made to the HVR to, for example, improve antibody affinity. Such changes may be at HVR "hot spots" (i.e., residues encoded by codons that occur during high frequency mutations during somatic cell maturation) (see, for example, Chowdhury, Methods Mol. Biol. 207 (2008) 179- Performed in 196), and/or SDR (a-CDR), where the resulting variant or VL binding affinity is tested. Affinity maturation by self-level library construction and re-selection is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178 (2002) 1.37 (Edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the selection for maturation by any of a variety of methods (eg, error-prone j>CR, strand shuffling or oligonucleotide-directed mutagenesis) In the GM. A secondary library is then established. The library is then screened to identify the other side of the introduction of diversity of any antibody variant β with the desired affinity. • The method involves an HVR-directed method in which a tHVR residue (e.g., 4 to 6 residues at a time) is randomized. HVR residues involved in antigen binding can be specifically identified (e.g., using alanine scanning mutagenesis or modeling). In particular, it is usually dry to CDR-H3 and CDR-L3. In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs as long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) can occur in HVR. These changes 162837.doc -75· 201245227 can be located outside the HVR "hot spot" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered or contains no more than one, two or three amino acid substitutions. A method suitable for identifying residues or regions on an antibody that can serve as a target for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells Science, 244 (1989) 1081-1085. In this method, a residue or set of residues (eg, charged residues such as arg, asp, his, lys, and glu) has been identified and consists of a neutral or negatively charged amino acid (eg, alanine) Or polyalanine) replacement to determine if it affects the interaction of the antibody with the antigen. Other substitutions can be introduced at the position of the amino acid which is sensitive to the initial substitution display. Alternatively or additionally, the contact point between the antibody and the antigen is identified by the crystal structure of the antigen-antibody complex. The contact residues and adjacent residues that serve as replacement candidate residues can be targeted or excluded. Variants can be screened to determine if they contain the desired properties. Amino acid sequence insertions include fusion at the amino- and/or carboxy terminus (within the range of one residue to a polypeptide containing one hundred or more residues), and insertion of a single or multiple amino acids within the sequence Residues. Examples of the terminal insertion include an antibody having an N-terminal thiopurine residue. Other insertional variants of the antibody molecule include enzymes (e.g., for use in ADEPT) or fusions of the polypeptide that extend the serum half-life of the antibody to the N-terminus or C-terminus of the antibody. Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of an antibody glycosylation site can be facilitated by altering the amino acid sequence to create or remove one or more glycosylation sites. 162837.doc -76 - 201245227. If the antibody contains an Fc region, the carbohydrate to which it is attached may change. Natural antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide, which is typically attached to the region by an N-linkage (: 112 domain

Asn297. See, for example, Wright et al., TIBTECH 15 (1997) 26-32. The vouchers may include various carbohydrates such as mannose, N-ethyl glucosamine (GlcNAc), galactose, and sialic acid, as well as rocks of GlcNAc attached to the "backbone" of the biantennary sugar-supplying structure. Alginose. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to produce antibody variants having certain improved properties. Further provided are polypeptides comprising an FC variant having a sialylated oligosaccharide, e.g., wherein the differential sialylation is attached to the antibody! ^ Fc core of the zone coupon sugar. Such polypeptides may have increased sialylation and/or reduced ADCC function. Examples of such antibody variants are set forth, for example, in Kanek〇 et al.

Science 313 (2006) 670-673. Cysteine-engineered antibody variants In certain embodiments, it may be desirable to produce a cysteine-engineered antibody, such as a "thio-mab" in which one or more residues of the antibody are celiac-resistant Substituted. In a particular embodiment, the substituted residue is present at the accessible site of the antibody. By substituting the residues with cysteine, the reactive thiol group is thus located at the accessible site of the antibody and can be used to couple the antibody to other moieties (eg, drug moiety or linker-drug moiety) To generate an immunoconjugate as described further herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 of the light chain (Kabat ed. 162837.doc • 77·201245227), heavy chain A118 (EU number) ) and the s4〇〇 (EU number) of the heavy-key fc area. The cysteine-modified antibody can be produced as described in, for example, U.S. Patent No. 7,521,541. Anti-Zhao Derivatives In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Portions suitable for derivatizing antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrole Pyridone, poly-fluorene, 3-dioxolane, polyfluorene, 3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and Dextran or poly(η-vinyl.pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyoxyethylated polyol (for example, glycerin), poly Vinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer may have any sub-amount and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, it can be the same or different molecules. In general, the number and/or type of polymers used in the derivatization can be determined based on considerations including, but not limited to, the following: whether to modify the specific properties or functions of the antibody, whether the antibody derivative will be used to define the conditions The next treatment. In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Pr 〇c chest) VIII...^ 162837.doc • 78 · 201245227 USA 102 (2005) 1 1600-11605). The radiation can have any wavelength and includes, but is not limited to, light shots of the following wavelengths: it does not harm normal cells, but heats the non-proteinaceous portion to a temperature that kills cells adjacent to the antibody-non-proteinaceous portion. Recombinant Methods and Compositions Recombinant methods and compositions can be used to produce antibodies, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody variant described herein is provided. The nucleic acid may encode an amino acid sequence comprising φ VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (e. g., the light chain and/or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. In yet another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (eg, has been transformed with such a substance): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amine group comprising the VH of the antibody An acid sequence, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method of making an antibody variant is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as set forth above, and optionally from a host cell (or host cell) under conditions suitable for expression of the antibody Medium) recovers antibodies. To produce an antibody variant for recombination, the nucleic acid encoding the antibody (e.g., as described above, 162,837. doc. 79, 201245227) is isolated and inserted into one or more vectors for further colonization and/or expression in the host cell. This nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Suitable host cells for the selection or expression of vectors encoding the antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For the performance of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton,

Methods in Molecular Biology 248 (2003) 245-254 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), which describes the performance of antibody fragments in E. coli). After performance, the antibody can be isolated as a soluble fraction from the bacterial cell paste and can be further purified. In addition to prokaryotes, 'eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable colonization or expression hosts for vectors encoding antibodies, including glycosylation pathways that have been "humanized" to produce partial or complete human glycosylation. A fungal and yeast strain of a model antibody. See Gerngross, Nat. Biotech. 22 (2004) 1409-1414 and Li et al, Nat. Biotech. 24 (2006) 210-215. Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains that can be used in combination with insect cells have been identified, particularly for transfection of sPodoPtera frugiperda cells. 162837.doc -80 · 201245227 Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, the disclosure of which is incorporated herein by reference. Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed by SV40; human embryonic kidney line (293 or kidney cell (BHK)); mouse support cells (TM4 cells such as, for example) Mather, Biol. Reprod. 23 (1980) 243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK) Buffalo rat liver cell (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumors (MMT 060562); TRI cells, such as (for example) Mather et al, Annals NY Acad, Sci. 383 (1982) 44-68; MRC 5 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 (1980) 4216); and myeloma cell lines, such as Y〇, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular

Biology 248 (2003) 255-268 (Edited by B.K.C. Lo, Humana

Press, Totowa, NJ) ° Analysis The antibodies provided herein can be identified by various assays known in the art, 162837.doc -81 - 201245227 Screening or characterizing their physical/chemical properties and/or biological activity. Binding analysis and other analysis In one aspect, for example, the antigen binding activity of the antibody of the present invention is tested by a known method (e.g., elisa, western blot, etc.). In an exemplary competition assay, a fixed antigen is incubated in a solution comprising a first labeled antibody that binds to an antigen, eg, a second unlabeled antibody, wherein the second unlabeled antibody is tested for binding to a shawl antibody This force of the antigen. The second antibody can be present in the hybridoma supernatant. As a control, the antigen is fixed in a solution containing the first labeled antibody but not the second unlabeled antibody. Excess unbound antibody is removed after incubation under conditions which permit the binding of the first antibody to the antigen, and the amount of the label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen is substantially reduced relative to the control sample in the test sample, this indicates that the second antibody competes with the first antibody for binding to the antigen (see Harlow and Lane (1988) Antib〇dies: a

Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Immunoconjugates are also provided for use in immunoconjugates comprising the antibodies herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (eg For example, a protein toxin, an enzymatically active toxin of a bacterium, a fungus, a plant, or an animal source, or a fragment thereof, or a radioisotope. In one embodiment, the immunoconjugate is an antibody 'drug conjugate 162837.doc • 82· 201245227 (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to (maytansinoid) (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatin, such as the monothiol auristatin drug moiety DE and DF (MMAE and MMAF) ( See U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298; dolastatin; calicheamicin or a derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739) , 116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al, Cancer Res. 53 (1993) 3336-3342; and Lode et al, Cancer Res. 58 ( 1998) 2925-2928); anthracycline, such as daunomycin or doxorubicin (see Kratz et al, Current Med. Chem. 13 (2006) 477-523; Jeffrey et al. Human, Bioorganic & Med. Chem. Letters 16 (2006) 358-362; Torgov et al, Bioconj. Chem. 16 (2005) 717-721; Nagy et al, Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik et al, Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King et al, J. Med. Chem. 45 (2002) 4336-4343; and U.S. Patent No. 6,630,579 Aminopterin; vindesine; yew house, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel ; trichothecene; and CC1065. In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or a fragment thereof, 162837.doc-83-201245227, the enzymatically active toxin or fragment thereof including, but not limited to, diphtheria A-chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from pseudomonaz nas aerugin〇sa), ricin A chain, abrin toxin A chain, box root toxin A chain, α · Ascagalococcus, Aleurites fordii protein, Dianthus protein, Phytolaca americana protein (PAPI ' pApiI & pAp_s), Momordica charantia inhibitor, Diarrhea, Croton toxin, Saponin (sapaonaria officinalis) inhibitor, leucotoxin, mitogellin, restrict 〇cin, phenomycin, enomycin, and trichothecium Trichothecene In another embodiment, an immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioactive conjugates. Examples include the radioisotopes of At211, I131, I125, Y9〇 'Rem, Rem 'Sml53, Bi212, p32, pb212 & Lu. § When using conjugates for detection, it may include radioactive atoms for scintillation studies such as tc99m or 1123; or spins for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) Labels such as ang-123 (again), ang-131 'indium-111, fluoro-19, carbon-13, nitrogen-15, oxygen-17, ruthenium, manganese or iron. A conjugate of an antibody to a cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-pyridyldithio)propionic acid N-succinimide (SPDP), 4-(N- Malay-branched imine oxime) cyclohexan _ι_carboxylic acid succinic acid imine (SMCC), subunit sulfur heterocyclic sputum pit (it), imiline vinegar bifunctional 162837.doc - 84- 201245227 Organisms (eg hexamethylenediamine dimethyl HCl), active esters (eg diammonium iodide suberate), aldehydes (eg glutaraldehyde), bis-azido compounds (eg double) p-Azidobenzoyl) hexamethylenediamine), bis-diazo derivatives (eg bis-(p-diazophenyl)-ethylenediamine), diisocyanates (eg toluene 2,6-) Diisocyanate) and bis-active fluorine compound (for example, 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238 (1987) 1098. The carbon-14-labeled 1-isothiocyanate benzyl-3-methyldiethylidamine pentaacetic acid (MX-DTPA) is used as an exemplary chelating agent for coupling radionucleotides to antibodies. See WO 94/11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in the cell. For example, an acid labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing linker (Chari et al. 'Cancer Res· 52 (which can be used) can be used. 1992) 127-131; U.S. Patent No. 5,208,020). The immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared using the following cross-linking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, thio-EMCS, thio-GMBS, thio-KMUS, thio-MBS, thio-SIAB, thio-SMCC and thio-SMPB, and SVSB ((4-ethylene) The sulfone) amber sulfonate benzoate), the above reagents are commercially available (for example, available from Pierce Biotechnology, Inc., Rockford, ILV, USA). Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the antibody variants provided herein can be used to detect antigens bound to the antibody in a biological sample 162837.doc ^85-201245227 presence. The term "detection" as used herein encompasses quantitative or deterministic detection. In certain embodiments the biological sample comprises cells or tissues. In one embodiment, antibody variants for use in diagnostic or detection methods are provided. In yet another aspect, a method of detecting the presence of an antigen bound by the antibody variant in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an antibody described herein under conditions that permit binding of the antibody to the antigen, and detecting the presence or absence of a chimera between the antibody and the antigen. This method can be an in vitro or in vivo method. In one embodiment, an antibody variant is used to select an individual suitable for antibody therapy, e.g., wherein the antibody to which the antibody binds selects a biomarker for the patient. Exemplary conditions that can be diagnosed using the antibodies of the invention include cancer, cardiovascular disease, neuronal disorders, and diabetes. In certain embodiments, a labeled antibody variant is provided. Labels include, but are not limited to, directly detected labels or moieties (eg, fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive) and = indirectly detected by, for example, enzymatic or molecular phase 1 Part (eg enzyme or φ ligand). Exemplary labels include, but are not limited to, radioisotopes up, C, I, Η and I, fluorophores (such as rare earth chelates or fluorescent yellow and its derivatives), rhodamine and its derivatives , tannin, umbelliferone 'luciferase (eg, firefly luciferase and bacterial luciferase) (U.S. Patent No. 4,737,456), looxin, 2,3-dihydropyridazine, Horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase (eg, glucose oxidase, galacto 162837.doc -86 · 201245227 sugar oxidation Enzymes and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase, which are oxidized with hydrogen peroxide, such as HRP, lactoperoxidase, or microperoxidase) Enzyme coupling of dye precursors), biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like. Pharmaceutical Formulations The pharmaceutical formulations of the antibody variants described herein are prepared by mixing the antibody of the desired purity with one or more pharmaceutically acceptable optional carriers φ (Remington's Pharmaceutical Sciences, 16th Edition, 〇s〇i , a. Edit (1980)) Prepared as a lyophilized formulation or as an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include buffering agents such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; (e.g., octadecyldimethylbenzylammonium hydride; gasified hexamethylene diammonium; benzalkonium chloride, benzethonium chloride; benzalkonium or methanol; a benzoic acid ester, such as p-benzoic acid methyl or propyl p-hydroxybenzoate; catechol; m-diphenol; cyclohexanol, 3 · pentanol; and m-cresol; low Molecular weight (less than about 1 残 residue) polypeptide, protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acid, such as glycine, glutamine Acid, aspartame, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin, chelating agents such as EDTA; sugars such as sucrose, Mannitol, trehalose or sorbitol; salt formation counterbalance Sub, such as sodium; metal complex (eg 162837.doc •87- 201245227

Zn-protein complex); and/or a nonionic surfactant such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble pH 2 〇 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International). Certain exemplary HASEGPs and methods of use (including rHuPH20) are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968, in one aspect, sHASEGP with one or more additional glycosaminoglycans (eg, Chondroitinase) combination. Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006/044908, the latter including a histidine acid acetate buffer. The formulations herein may also contain more than one active ingredient for the particular indication being treated, preferably with complementary agents that do not adversely affect each other. These active ingredients are suitable for effective purposes. The amount exists in a combined form. The active ingredient may also be separately incorporated into a microcapsule (for example, hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules) prepared by, for example, coacervation or interfacial polymerization, a glial drug delivery system. (eg, liposomes, albumin microspheres 'microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remingt〇n, s pharmaceuticd

Sciences, 16th ed., 〇s〇i, A. ed. (1980). A sustained release preparation can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of 162837.doc-88 - 201245227 solid hydrophobic polymers containing antibodies in the form of shaped articles, for example, films or microcapsules. Formulations intended for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile membrane. Methods of Treatment and Compositions Any of the polypeptides provided herein can be used in a method of treatment. In a particular aspect of the invention, the polypeptide of the invention is used to treat a disease. In a more specific aspect, the disease advantageously reduces the effector function of the variant by at least 50 compared to the polypeptide comprising the polypeptide. /〇. In a particular aspect, a polypeptide of the invention is used to produce a medicament for treating a disease wherein the effector function of the polypeptide is advantageously significantly reduced as compared to the wild type. In yet another embodiment, a polypeptide of the invention is used to produce a medicament for treating a disease wherein the effector function of the polypeptide is advantageously reduced by at least 20% compared to the wild-type Fc polypeptide. A further aspect is a method of treating an individual having a disease, wherein the effector function of the variant is advantageously significantly reduced as compared to a wild-type Fc polypeptide comprising administering to the individual an effective amount of a polypeptide of the invention. Significant reduction in effector function is an effector function that reduces at least 50% of the effector function induced by the wild-type polypeptide. Such diseases are, for example, all diseases that should not destroy stem cells by, for example, ADCC, ADCP or CDC. Moreover, this is the case for antibodies that are designed to deliver drugs (eg, 'toxins and isotopes') to target cells, where Fc/FqR-mediated effector functions introduce healthy immune cells into the vicinity of the payload' This results in depletion of normal lymphoid tissues as well as target cells 162837.doc -89 - 201245227 (Hutchins et al, pNAs USA 92 (1995) 11980-11984; White et al, Annu Rev Med 52 (2001) 125-145). In such cases, the use of antibodies that poorly recruit complement or effector cells can be of great benefit (see, for example, Wu et al., Cell Immunol 200).

(2000) 16-26; Shields et al, J. Biol Chem 276 (9) (2001) 6591-6604; US 6,194,551; US 5,885,573 and PCT Publication WO 04/029207) In other cases 'for example, If the target blocks the interaction of a widely expressed receptor with its cognate ligand, then all antibody effector functions can be advantageously reduced or eliminated to reduce undesired toxicity. Similarly, where therapeutic antibodies exhibit promiscuous binding between many human tissues, it may be sensible to limit the targeting of effector functions to multiple sets of tissues to limit cytotoxicity. Similarly, for agonistic antibodies, such antibodies are extremely beneficial if they exhibit reduced effector function. Therapies that can be treated with polymorphic variants are numerous and include cancer (eg, where antibody variants bind to HER2 receptor, angiogenin is affected) Body or vascular endothelial growth factor (VEGF)); allergic conditions, such as asthma (with anti-IgE antibodies); and LFA-1 - mediated disorders (eg, wherein the polypeptide system is resistant to lfa-1 or anti-ICAM-1 antibodies), Neurological and metabolic disorders. If the antibody binds to the HER2 receptor, the condition is preferably a cancer that exhibits HER2', e.g., characterized by a benign or malignant tumor that overexpresses the HER2 receptor. Such cancers include, but are not limited to, breast cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioma blastoma, cervical cancer, ovarian cancer, bladder cancer, hepatocellular carcinoma, Colon cancer, colon straight 162837.doc •90- 201245227 广子吕内膜, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer (hepatic carcin〇ma) and various types of head and neck cancer The antibody or antibody system is administered by any suitable means, including non-subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and (if desired for intra-P immunosuppressive therapy). Infusion includes intramuscular, intravenous, intraperitoneal, or subcutaneous administration. In addition, antibody variants are suitable for administration by pulse infusion, especially in descending doses of polypeptide variants. Preferably, 'injection The dosage to be administered is optimally administered by intravenous or subcutaneous injection, depending in part on the length of administration. For the prevention or treatment of the disease, the appropriate dose of the polypeptide or antibody variant will depend on the disease to be treated. _, the severity and progress of the disease, the purpose of administering the polypeptide variant is the prevention or treatment, the prior therapy, the clinical history of the patient and the response to the polypeptide variant, and the decision of the attending physician. The polypeptide variant is suitable for ~ Under- or _ series of treatments for patients. Depending on the type and severity of the disease, 'about i ^ ^ / " to 15 mg / kg (eg (m. 20 mg / kg) polymorphism or antibody changes The initial dose of the patient to be administered to the patient' is administered, for example, by one or more separate administrations, or by continuous infusion. Depending on the above factors, a typical daily dose may range from about i Mg/kg to 1 〇〇. Within the range of mg/kg or higher. For repeated administrations over several days or longer, depending on the condition, treatment continues until the desired suppression of the symptoms of the disease occurs. However, other Dosage regimen. The progress of this therapy can be readily monitored by conventional techniques and assays. In certain embodiments, the invention provides antibody variants or polypeptides for use in a method of treating a subject having cancer 162837. doc 201245227 The method includes administering to the individual effective Antibody variants In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In yet another embodiment, the invention provides for An antibody variant that inhibits angiogenesis, inhibits cell proliferation, or depletes B cells in an individual, comprising administering to the individual an effective amount of an antibody variant to inhibit angiogenesis, inhibit cell proliferation, or deplete B cells. The "individual" of one is preferably human.

In yet another aspect, the invention provides for the use of an antibody variant or polypeptide in the manufacture or preparation of a medicament. In one embodiment, the agent is for the treatment of cancer or an inflammatory disease. In yet another embodiment, the medicament is for use in a method of treating cancer, diabetes, a neuronal disorder, or an inflammatory disorder, comprising administering an effective amount to an individual having cancer, diabetes, a neuronal disorder, or an inflammatory disorder. Pharmacy. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In still another embodiment, the agent inhibits angiogenesis, inhibits cell proliferation, or consumes B cells.

In still another embodiment, the agent is for use in a method of inhibiting angiogenesis, inhibiting cell proliferation, or consuming B cells in an individual, comprising administering to the subject an effective amount of an agent to inhibit angioplasty, 匕 3 ° &quot ; B cells are consumed. The "individual" which inhibits cell proliferation or any of the above embodiments may be human. The present invention, for use in a pharmaceutical formulation, which comprises any of the anti-social agents herein, is for example used in the above-described method of treatment 162837.doc-92·201245227. Wherein the 'pharmaceutical formulation comprises any of the antibody variants provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the antibody variants provided herein and At least one additional therapeutic agent, for example, as described below. The present month antibody can be used in therapy alone or in combination with other agents. By way of example, the antibody of the invention can be co-administered with at least one additional therapeutic agent. The combination therapy encompasses combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and administered separately (in which case, prior to administration of the additional therapeutic agent and/or adjuvant, Simultaneously and/or after administration of an antibody of the invention, the antibody of the invention may also be used in combination with radiation therapy. The antibody of the invention (and any additional therapeutic agent) may be administered by any suitable means, including parenteral 'intrapulmonary and Intranasal, And (if desired for topical treatment) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intra-abdominal or subcutaneous administration. It can be administered by any suitable route, for example by Injections, such as intravenous or subcutaneous injections, depend in part on the length of administration. This article covers various dosing regimens, including (but not limited to) single or multiple doses at different time points, with concentrated doses. And pulse infusion. The formulation, administration and administration of the antibodies of the invention should be carried out in a manner consistent with good medical practice. In this case, considerations include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient. Conditions, causes of illness, route of administration of the drug, method of administration, timing of administration, and other factors known to the medical practitioner. The antibody need not (but optionally) be associated with one or more agents currently used to prevent or treat the disorder in question The amount of the antibody present in the formulation, the type of condition or treatment, and the types discussed above. Depending on other factors, the agents are usually administered in the same dosages and in the administration routes described herein, or from about 1% to 99% of the dosages described herein, or empirically/clinically determined to be any suitable dosage and any Routes of Use. For the prevention or treatment of a disease, a suitable dose of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) should be directed at the type of disease to be treated, the type of antibody, and the severity of the disease. The degree and duration of the disease, the administration of the anti-system for prophylactic or therapeutic purposes, prior treatment, the clinical history of the patient, the response to the antibody, and the judgment of the attending physician. The antibody is suitable for one-time or a series of treatments. Depending on the type and severity of the disease, an antibody of about i ^^/4 to 15 mg/kg (eg, 1 mg/kg to 1 mg/kg) may be the initial candidate dose for administration to the patient. , whether by, for example, by one or more separate administrations, or by continuous infusion. Depending on the above factors, a typical daily dose may range between about i _ to 1 〇〇 mg/kg or higher. For repeated administrations over several days or longer, depending on the condition, treatment will usually continue until the desired repression of the symptoms of the disease occurs. An exemplary dose of antibody will range from about 5 mg/kg to about 10 mg/kgi. Thus, one or more doses (any combination) of about 5 mg/kg 2.0 mg/kg, 4.0 mg/kg or 1 mg/kg can be administered to the patient. Such doses can be administered intermittently, such as once a week or every three weeks (e.g., such that the patient receives about two to about two (or, for example, about six doses of the antibody). A higher loading dose can be administered at the beginning, followed by one or more lower doses. However, other dosage regimens can be used. This treatment 162837.doc -94- 201245227 The progress of the method can be easily monitored by conventional techniques and analysis. It will be appreciated that the immunoconjugates of the invention may be used in place of or in conjunction with the antibodies of the invention to administer any of the above formulations or methods of treatment. Articles of Manufacture In another aspect of the invention, an article of manufacture containing materials for treating, preventing, and/or diagnosing the above conditions is provided. The article comprises a container and a label or package insert located on or associated with the container. Suitable containers include (1) bottles, vials, syringes, solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. The container is loaded with itself or with another. "A composition that is effective to treat, prevent, and/or diagnose a condition" and may have a sterile access channel (eg, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle) The combination: at least one active agent is an antibody of the invention. The label or package insert indicates that the composition is for treating a selected condition. In addition, the preparation may comprise (4) a composition containing the composition - Xuan Zhan, Li The composition comprises the antibody of the present invention; and (b) the second cytotoxic agent or the phytotherapy agent Z in which the composition is contained. ', the medium I composition contains again - : 丨, therapeutic agent. The article of this embodiment of the invention may further be used to treat a package insert for a particular condition. Alternatively - or alternatively, the sigma 5 4 container comprises a medicinal product: Γ: the step comprises a second (or third) container, the _), the dish salt buffer salt, the salt water spray and the dextrose solution 1 further U "Liquid (RlngW fine) Other materials expected, including commercial and user considerations of the syringe. ^ 'Punching, thinner, transition, needle 162837.doc -95· 201245227 It should be understood that any of the above products The immunoconjugates of the invention may be included in place of or in combination with antibody variants. Polypeptides Non-therapeutic Uses The antibody variants of the invention may be used as affinity purification agents. In this method, well known in the art. Method of immobilizing an antibody variant on a solid phase (e.g., Sephadex resin or filter paper). Contacting the immobilized polypeptide variant with a sample containing the antigen to be purified, and thereafter washing the carrier with a suitable solvent, which will remove the sample. Substantially all substances other than the antigen to be purified bound to the immobilized antibody variant. Finally, the carrier is washed with another suitable solvent (eg, glycine buffer 'pH 5.0') which will be released from the polypeptide. Antigens. Antibody variants can also be used in diagnostic assays, for example, to detect the expression of an antigen of interest in a particular cell, tissue or serum. For diagnostic applications, antibody variants are typically labeled with a detectable moiety. The mark 'usually can be divided into the following categories: (a) Radioisotopes such as 35$, 14c, 125j, and 13丨1. Can be used, for example, in Coligen et al., Current Protocols in Immunology 'Vol. 1 and 2 , edited by Wiley-Interscience, New Ruy York, Ν·Υ·, Pubs. (1991) Techniques for labeling polypeptide variants with radioisotopes' and using scintillation counting to measure radioactivity. (b) Fluorescent labels can be used, For example, rare earth chelates (ruthenium chelates) or luciferins and their derivatives, rose red and its derivatives, tannin, lisane, phycoerythrin and Texas Red Fluorescent labels can be coupled to polypeptide variants using, for example, the techniques disclosed in Current Protocols in Immunology (see above). Fluorescence can be quantified using a fluorometer. 162837.doc •96- 201245227 (C ) Various enzyme-substrate labels are used and a review of some of these labels is provided in U.S. Patent No. 4,275,149. Enzymes typically catalyze chemical changes in chromogenic matrices, which can be measured using various techniques. For example, enzymes can catalyze The color of the substrate can be measured by spectrophotometry. Alternatively, the enzyme can change the fluorescence or chemiluminescence of the substrate. The technique for quantifying fluorescence changes is described above. Chemiluminescence is due to chemical reactions. The light is excited electronically and can then be emitted (eg, using a chemiluminometer) or supplied to the fluorescent acceptor. Examples of enzyme labels include luciferase (for example, firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydropyridazinedione, malate dehydrogenation Enzymes, urease, peroxidases (eg horseradish peroxidase (HRPO)), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, sugar oxidase (eg glucose oxidase) , galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase and the like. Techniques for coupling enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-

Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (edited by J. Langone & H. Van Vunakis), Academic Press, New York, 73: 147-166 (1981). Examples of enzyme-substrate combinations include, for example: (1) horseradish peroxidase (HRPO), which is treated with catalase, wherein the catalase oxidizes the dye precursor (eg, phenyl Amine (POD) or 3,3',5,5'-tetraphenylbenzidine hydrochloride (butyl 1^8)); (ii) a phosphatase (AP), which is p-nitrophenyl phosphate Ester as a hair color by I62837.doc -97- 201245227; and ((1)) β-D-galactosidase (p-D_Gal), which has a chromogenic receptor (for example, p-nitrophenyl-β-D -galactosidase) or fluorescent receptor 4_methylumbelliferyl fluorenyl-β-D-galactosidase. Many other enzyme-substrate combinations are available to those skilled in the art. For a general review of combinations such as 泫, see U.S. Patent Nos. 4,275,149 and 4,318,980. Sometimes, the label is indirectly coupled to the polypeptide variant. Those skilled in the art should be aware of the various technologies that accomplish this. For example, a polypeptide variant can be coupled to biotin and any of the above three broad classes of labels can be coupled to avidin, or vice versa. Biotin selectively binds to avidin and, therefore, the label can be coupled to the polypeptide variant in this indirect manner. Another option is to 'couple the polypeptide to a small hapten (eg, digoxin) for the in-coupling of the tag to the polypeptide variant and to antagonize any of the different types of labels described above A hapten polypeptide variant (eg, a new antibody against the mantle) is coupled. Thus, a coupling between a tag and a polypeptide variant can be achieved. In another embodiment of the invention, the antibody variant is not required to be labeled and its presence can be detected using a labeled antibody that binds to the polypeptide variant. The antibody variants of the invention can be used in any known analytical method, such as competitive binding assays, direct and indirect sandwich structure assays, and immunosuppression assays. Zola, Monoclonal Antibodies: A Manual of Techniques, (1987) pp. 147-158 ' CRC Press. Antibody variants can also be subjected to in vivo diagnostic assays. Typically, multiple 162837.doc · 98· 201245227 peptide variants are labeled with a radionuclide (eg, inIn, 99Tc, 14C, mI, 25l, 3H, 32P, or 35S) such that the antigen or cell expressing it can be used with immunostaining Visualize to locate. Although the invention has been described in some detail by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention. BRIEF DESCRIPTION OF THE SEQUENCE LISTING: SEQ ID ΝΟ: 1 Human K light chain SEQ ID NO: 2 Human λ light chain SEQ ID NO: 3 Human IgG1 (Caucasian allotype) • SEQ ID NO: 4 Human IgGl (African American Human (Afroamerican allotype) SEQ ID NO: 5 Human IgG1 LALA-mutant (Caucasian allotype) SEQ ID NO: 6 Human IgG4 SEQ ID NO: 7 Human IgG4 SPLE· mutant, which represents the kappa light chain, λ Exemplary human sequences of light chains, IgGl and IgG4, which can be used as a basis for generating variants of the invention. • In sequence Id No 3-5 (human IgGl allotype sequence), according to the Kabat EU index, the P329 region is located at position 212, and the P329 region of sequences Id Nos 6 and 7 can be found at position 209. SEQ ID NO: 8 mAb 40A746.2.3 κ light chain SEQ ID NO: 9 mAb 40A746.2.3 wild type IgG 1 heavy chain SEQ ID NO: 10 rnAb 40A746.2.3 IgGl P329G heavy chain SEQ ID NO: 11 The heavy chain of IgG1 LALA/P329G of mAb 40A746.2.3 162837.doc -99- 201245227 SEQ ID NO: 12 mAb The heavy chain of IgG4 SPLE of 40A746.2.3 SEQ ID NO: 13 mAb The weight of IgG4 SPLE/P329G of 40A746.2.3 Chain SEQ ID NO: 14 mAb Example of Heavy Chain of IgGl LALA of 40A746.2.3 The following seven examples are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be practiced in accordance with the general description provided above. Although the invention has been described in some detail by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety. Example 1 Antibodies For the experiments described below, antibodies against CD9 (see SEQ ID 8-14), P-selectin (sequences described in WO 2005/100402) and CD20 (synonym: GA101, described in Sequence in EP 1 692 182). All variants used herein, such as selectin P329G, P329A, P329R SPLE, LALA 'P329G/LALA, P329G/SPLE variant; CD9; CD20 (GA101) and CD20 (GA101) • Glycoengineered binding antibodies (named according to EU The numbering is generated using PCR-based mutagenesis. IgG molecules were visualized in the HEK-EBNA or HEK293 (CD9 Fc variant) system and purified using protein A and size exclusion chromatography. Example 2 Determination of Binding Affinity of Different Fey Receptors and Immunoglobulins

Biacore T100 instrumentation by surface plasma resonance (SPR) (GE 162837.doc • 100- 201245227

Healthcare) The binding affinity of different pcyRs for immunoglobulins was measured at 25 °C. Fully establish the BIAcore® system to study molecular interactions. It allows continuous monitoring of ligand/analyte binding and thus determination of association rate constant (ka), dissociation rate constant (kd) and equilibrium constant (Kd). The change in refractive index indicates the change in mass on the surface caused by the interaction of the immobilized ligand with the analyte in the injected solution. If the molecule binds to a fixed ligand on the surface, the mass will increase, and if dissociated, the mass will decrease. For 1:1 interactions, no difference in results should be observed if the binding molecules are injected onto the surface or fixed to the surface. Therefore, different environments (using the FcY receptor as a ligand or analyte, respectively) are used depending on the solubility and availability of the ligand or corresponding analyte. For FcyRI, 10,000 resonance units were identified by immobilization of the polyhistidine sequence (Five His monoclonal antibody 'Qiagen Hilden, Cat. No. 34660) at pH 4.5 using an amine coupling kit supplied by GE Healthcare and a CM5 wafer ( The capture system of RU) captures FcyRI at a concentration of 5 gg/ml with a 60 sec pulse at a flow rate of 5 μΐ/min. Different concentrations of antibodies in the range of 〇nM to 100 nM were passed through the flow cells at a flow rate of 30 μΐ/min and maintained at 298 K for 120 sec to record the association period. The dissociation period was monitored for up to 240 sec and The sample solution is converted to running buffer to trigger. The surface was regenerated by performing a 2 min wash with a glycine acid (pH 2) solution at a flow rate of 30 ml/min. For all experiments, HBS-P+ buffer (1 mM HEPES, pH 7.4, 150 mM NaCl, 0·05 °/〇 (v/v) surfactant P20) supplied by GE Healthcare was selected. The bulk refractive index difference was corrected by subtracting the response obtained from the surface of the 162837.doc 201245227 without the capture of FcyRI. The blank injection (= double reference) is also subtracted. The equilibrium dissociation constant (KD) defined as ka/kd was determined by using a BIA evaluation software package analysis to obtain a sensory curve obtained using several different concentrations. The data was fitted according to a suitable binding model. For FcyRIIA and FcyRIIIAV158, the loooo resonance unit (RU) of the individual antibody to be tested was immobilized on a CM5 wafer by using an amine coupling kit (pH 4_5, supplied at a concentration of 10 pg/ml) supplied by GE.

Different concentrations of FcyRIIA and · FcyRIIIA in the range of 0 nM to 12800 nM were passed through the flow cells at a flow rate of 5 μΐ/min and maintained at 298 K for 120 sec to record the association period. The dissociation phase was monitored for up to 240 sec and triggered by conversion from the sample solution to running buffer. By using 3 mM

The surface was regenerated by a 2 min wash with a NaOH/1 M NaCl solution at a flow rate of 30 ml/min. For all experiments, 'HBS-P+ buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05 ° (v/v) surfactant P2 供给) supplied by GE Healthcare was selected. The difference in the body angulation rate is corrected by subtracting the reaction obtained from the surface of the non-captured antibody. Blank injection (= double reference) is also subtracted. The equilibrium dissociation constant (Kd) was determined by using a BIA evaluation software package to analyze the sensorgram curves obtained using several different concentrations. Data fitting was performed according to a suitable binding model using a steady state fit. For FcyRIIB, a total of 10000837 was identified by using the amine coupling kit supplied by ge Healthcare and the CM5 wafer at pH 4.5 to fix the polyhistidine sequence (Five His antibody, Qiagen Hilden, catalog number 34660). Doc -102· 201245227 Vibration unit (RU) capture system. FcyRIIB was captured at a concentration of 5 pg/ml with a pulse of 120 sec at a flow rate of 5 μΐ/min. Different antibodies were passed through the flow cells at a flow rate of 1340 nM at a flow rate of 5 μΐ/min and maintained at 298 K for 60 sec to record the association period. The dissociation phase is monitored for up to 120 sec and triggered by conversion from the sample solution to running buffer. The surface was regenerated by applying a 0-5 min strip with a solution of glycine (pH 2.5) at a flow rate of 30 ml/min. For all experiments, 'HBS_P + buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 〇·〇5°/.(v/v) _surfactant P20) supplied by GE Healthcare was selected. The refractive index difference of the body is corrected by subtracting the reaction obtained from the surface of the non-captured FqRIIB. Also subtracted from the blank injection (= double reference) ° Because FcyRIIB has a very low intrinsic affinity with wild-type IgG1, it is impossible to calculate the affinity' but to assess the qualitative binding. The following table summarizes the introduction of mutations into the Fc portion for pairing with FcYRI, FcyRIIA, FcyRIIB And the effect of the combination of FcYRIIIAV1-58 (A) and the effect on ADCC (not using (BLT) and using target cells (ADCC)) and the binding of φ Clq Table 2A:

FcyRI FcyRIIaR131 FcyRIIIAV158 FcyRIIB WT IgGl ++(5 nM) Ή· (2 μΜ) + (0.7 μΜ) -Η-IgG4 SPLE - +/- CIO μΜ) -(>20 μΜ) + IgGl P329G ++(6 nM ) -(>20 μΜ) -(>20 μΜ) - IgGl P329A ge ++(8 nM) + (4.4 μΜ) + (1.8 μΜ) + IgGl P329G LALA - -(>20 μΜ) -(&gt ;20 μΜ) - IgGl P329G ge ++ (10 nM) -ί>20 μΜ) -(>10 μΜ) *++ 'For ge IgGl 30 nM 162837.doc • 103· 201245227 Table 2B: FcyRI FcyRII FcyRIII ~~Clq~~ Not used; Cell ADCC cells - APcr ADCC1""""" Analysis Biacore Biacore Biacore CDC Clq BLT P329G P329R + ndndndndnd -- 1 LALA IgGl P329G/LALA — nd - Ndndndnd·nd u nd IgG4 SPLE ndnd -- significantly reduced/inactive relative to wt, - reduced relative to wt, + equivalent to wt interaction, nd not determined / no result. The following results were obtained in more detail: with FcyRI Affinity has introduced P329G, P329A, SPLE and LALA mutations into the Fc polypeptides of P-selectin, CD20 and CD9 antibodies, and Measured with the Biacore system and the amount of binding affinity of FcyRI. Although the antibody with the P329G mutation still binds to FcyRl (Figures la and lb), the introduction of the triple mutations P329G/LALA and P329G/SPLE, respectively, produced antibodies that were barely detectable (Figure lb). LALA or SPLE mutations reduce the binding to the receptor to a greater extent than P329G alone, but less than in combination with P329G (Figures la and lb). Therefore, the combination of P329G with LALA or SPLE mutations is more effective than either the P329G mutation alone or the double mutation LALA or SPLE. The kd value of the CD20 IgG1 wild type antibody was 4.6 nM and the kd value of the P329G mutant of the same antibody was 5.7 nM, but for the mutant P329G/LALA, the binding of the antibody to the FcyRI receptor could hardly be detected and could not be determined. Kd value. Regardless of whether CD9 or CD20 or P_selectin is tested, the antibodies themselves have little effect on binding affinity. I62837.doc • 104· 201245227 Affinity with FcyRIIA The P329G, SPLE and LALA mutations were introduced into the Fc polypeptide of the CD9 antibody and the Biacore system was used to measure the binding affinity to the FcyRIIA-R13 1 receptor. The degree of binding is corrected, for example, the captured mAb represents 100 RU. Therefore, it is expected to be no more than about 20 RU for a 1:1 stoichiometry. Figure lc shows that binding to the FcyRIIA receptor is significantly reduced by introducing LALA, SPLE/P329G, P329G and LALA/P329G mutations into the Fc variant. Unlike the binding to the FcyR1 receptor, the introduction of the P329G mutation alone can more or less significantly block binding to the receptor to the extent similar to the triple mutation P329G/LALA (Fig. lc). Affinity with the FqRIIB receptor has been separately introduced into the Fc polypeptide of CD9 and P-selectin antibodies and the binding affinity to the FcyRIIB receptor was measured using the Biacore system. . Panel Id shows a significant decrease in binding to the FcyRIIB receptor for LALA and the triple mutant P329G/LALA 'P329G/SPLE. • Affinity with the FcyRIIIA receptor P329G, LALA, SPLE, P329G/LALA and SPLE/P329G mutations have been introduced into the Fc polypeptide of CD9 and the binding affinity to the FcyRIIIA-V158 receptor was measured using the Biacore system. The P329G mutation and the triple mutation P329G/LALA reduced the binding to the FcyRIIIA receptor most significantly to an almost undetectable extent. P329G/SPLE also produced a significantly reduced binding affinity, and the mutations SPLE and LALA only slightly reduced the binding affinity to the FcyRIIIA receptor, respectively (Fig. le). 162837.doc -105- 201245227 Example 3

C1Q ELISA The binding properties of different polypeptides comprising Fc variants to Clq were analyzed by ELIS A sandwich structured immunoassay. Each variant was coupled to a hydrophobic Maxisorp 96-well plate at 8 concentrations between 1 μ μβ/ιη丨 and 〇 pg/ml. This conjugate stimulates the antibody complex's high affinity binding of this Clq molecule before k. After washing the strips, the pattern is allowed to allow C 1 q to bind. After the step, the bound C1 q molecule was detected by a plurality of rabbit anti-hC 1 q antibodies. The enzyme-labeled anti-rabbit-Fey specific antibody was added after the next washing step. The immunological response is visualized by the addition of a substrate that is converted to a colored product by an enzyme. The absorbance measured by photometric measurement is proportional to the amount of dq bound to the antibody to be studied. Calculate the E of the variant-Clq interaction (:5◦ value. Plot the concentration of the absorption unit obtained from the color reaction on the antibody. The half-maximal reaction antibody concentration determines the ECS0 value. Report this readout value on the same plate. The relative difference between the measured and the reference standard and the coefficient of variation of the sample and the reference. The p329G mutation introduced into the P-selectin or CD2〇 antibody significantly reduces the binding to c 1 q, which is similar to the SPLE mutation (Fig. 2) Table 3 summarizes the EC 5 〇 values of the binding of the calculated variant to the Clq receptor. Clq belongs to the complement activating protein and plays a major role in the activation of the classical complement pathway, which leads to the formation of membrane attack complexes. C1q is also involved in other immunological processes, such as enhancing swallowing, clearing apoptotic cells, or neutralizing viruses. Therefore, it is expected that mutants that reduce binding to Clq (such as P329G and SPLE and most likely also include The triple mutation of the above single mutation) significantly reduced the above functions of dq. 162837.doc •106· 201245227 Table 3: Antibody EC50 value P-selectin IgGlwt 1.8 - _GA101 IgGl wt 2.4 P-selectin IgGl_P329G 2 .7 P-selectin IgG4 SPLE 3.0 GA101 leGl P329G 5.5 GA101 I2G4 SPLE >10 Example 4 ADCC without target cells, BLT assay The antibody to be tested (CD20 (GA101) will be tested in a suitable 96-well flat bottom plate at 4 °C. And CD9) were plated overnight in PBS. After washing the plate with PBS, the remaining binding sites were blocked with PBS/l°/〇BSA solution for 1 h at RT » simultaneously, effector cells were harvested (transfected) 100 μΐ/well AIM V medium in the wells after seeding the NK-92 cell line expressing low or high affinity human FcyRIII and discarding the buffered water bottle solution The maximum esterase release of effector cells was determined using 100 μΐ/well saponin buffer (0.5% saponin + 1% BSA in PBS). The cells were incubated at 37 ° C, 5% in the incubator. Incubate for 3 h at C02. After 3 h, mix 20 μΐ/well supernatant with 180 μΐ/well BLT substrate (0.2 mM BLT + 0.11 mM DTNB in 0.1 Μ Tris-HCL, pH 8.0) at 37 ° Incubate for 30 min at C, then read at 405 nm in a microplate counter. Determine the percent esterase release, which will release the maximum (saponin- Cell therapy) is set to 100% and is strongly induced (set to 0% release ab & uncoated). Wild type CD20 antibody (GA101 wt (1) the unstimulated cells) displayed the cytolytic activity. The LALA variant showed a significant decrease in esterase release, while the P329G and P329G/LALA variants did not show any ADCC activity (Fig. 3a). Figure 3b shows 162837.doc • 107-201245227 shows that not only does the exchange of G at the P329 position result in significantly reduced cell lytic activity, but the same is true for the exchange of P329 for R329 (CD20 antibody). Therefore, arginine appears to disrupt the function of the glycine sandwich structure in the antibody, which is similar to glycine. The ADCC, which is observed here to have a significant decrease in the P329G mutant, is most likely due to a significant decrease in binding to the FcyRIIA and FcyRIIIA receptors (see Figure lc and Figure). Example 5

ADCC using target cells used human peripheral blood mononuclear cells (PBMC) as effector cells and was prepared using Histopaque-1077 (Sigma Diagnostics, St. Louis, M063 178 USA) and essentially according to the manufacturer's instructions. Briefly, venous blood was collected from volunteers using a heparinized syringe. Dilute the blood with PBS (without 〇&++ or]^§++) at 1: -.75-1.3 and layer it on Histopaque-1077. Gradiently at room temperature (RT) without interruption Centrifuge at 400 X g for 30 min. The intermediate phase containing PBMC was collected and washed with PBS (50 ml/cell from two gradients) and harvested by centrifugation at 300 X g for 10 minutes at RT. After the pellet was resuspended in PBS, the PBMCs were counted and washed again by centrifugation at 200 X g for 1 minute at RT. The cells are then resuspended in a suitable medium for subsequent procedures. For PBMC and NK cells, the ratio of effector to target for ADCC analysis was 25:1 and 10:1, respectively. Appropriate concentrations of effector cells were prepared in AIM-V medium to add 50 ml to each well of a round bottom 96-well plate. The target cell line was grown in human B lymphoma cells (e.g., Raji cells) in DMEM containing 10 〇/〇 FCS. Target cells were washed in PBS, counted and resuspended at 0.3 million/ml 162837.doc •108- 201245227 In AIM-V, 3 cells were added in 1 μ ml per microwell. The antibody was diluted in AIM-V. 5 〇mi was added to the pre-plated target cells and allowed to bind to the target at RT for 1 min. Then, effector cells were added and the plate was incubated at 37 ° C for 4 hours in a humidified atmosphere containing 5% C 2 . Depletion of target cells was assessed by measuring the release of lactate dehydrogenase (LDH) from damaged cells using a cytotoxicity test kit (R〇ehe Diagnostics, Rotkreuz, Switzerland). After 4 hours of incubation, the plates were centrifuged at 80 〇 X g. Transfer 100 ml of supernatant from each well to a new clear flat 96 well plate. Add 100 ml of color receptor buffer from the kit to each φ well. The Vmax value of the color reaction was determined using an s〇FTmax PRO software (Molecular Devices, Sunnyvale, CA 94089, USA) at 490 nm for at least 1 〇 min in an ELISA reader. Spontaneous LDH release containing only dry and effector cells but no antibody-containing wells was measured. The maximum release of wells containing only target cells and 1% Trit〇n X-100 was determined. The percent elimination of specific antibody mediation was calculated as follows: ((x-SR)/(MR-SR)*100' where the average of Vmax at the X-specific antibody concentration' is the average of the spontaneous release Vmax of the SR system and MR The average value of Vmax for maximal release. The efficacy of recruiting immune-effector cells depends on the type of Fc variant, as measured by classical ADCC analysis. Here, 'using human NK92 cell line transfected with human FCgRina As an effector and using CD20-positive Raji cells as target cells. As can be found in Figure 4a, the ADCC of glycine acid instead of the GA101 (CD20) Fc variant of lysine (P329G) was significantly reduced, and the same double mutant P329G The degree of ADCC reduction was similar in /LALA. In contrast, ADCC reduction was less significant for LALA mutations. For better variants of 162837.doc -109- 201245227, variants were also produced in the form of sugar modifications. To enhance ADCC potential. It can be observed that the parental molecule (GA101 (CD20)) shows strong ADCC, as expected. The ADCC potential of the LALA form is significantly impaired. The P329G mutant significantly reduces ADCC; much larger than GA101 (CD20) Antibody variant P329A (fig 4b) Example 6 Complement activity The target cells were counted, washed with PBS, resuspended in AIM-V (Invitrogen) at 1 million cells/ml, and 50 ml cells/well were plated in flat-bottom 96-well plates. Antibody dilutions were prepared in AIM-V and added to the cells in 50 ml. The antibody was allowed to bind to the cells for 10 minutes at room temperature. Human serum complement (Quidel) was just thawed, diluted 3 fold with AIM-V and 50 ml Add to the wells. Rabbit complement (Cedarlane Laboratories) was prepared as described by the manufacturer, diluted 3 fold with AIM-V and added to the wells at 50 ml. As a control, the complement source was heated at 55 °C for 30 min. , then added for analysis. The assay plates were incubated for 2 h at 37 ° C. Cell depletion was determined by measuring LDH release. Briefly, the plates were centrifuged at 300 X g for 3 min. The ml supernatant was transferred to a new 96-well plate and 50 ml of analytical reagent from the cytotoxic kit (Roche) was added. Kinetic measurements using an ELISA reader were used to determine the Vmax corresponding to the LDH concentration in the supernatant. Maximum release was determined by incubating cells in the presence of 1% Triton X-100. Analysis of different Fc changes To mediate CDC on SUDH-L4 target cells. Non-glycoengineered GA101 molecules showed significant induction of CDC. LALA variants showed activity only at the highest concentration, while P329G and P329G/LALA variants did not 162837.doc-110- 201245227 shows any CDC activity (Fig. 5a). Furthermore, the LALA variant of the glycoengineered GA101 molecule and the P329G and Ρ329 variants did not show any CDC activity (Fig. 5b). Example 7 Carbohydrate Spectrum of Human IgG1 The carbohydrate profile of a human IgG1 antibody containing a mutation in Fc was analyzed by MALDI/TOF-MS in a cationic mode (neutral sugar collection), and the mutations were designed to eliminate the FcY receptor The combination. φ Treatment of human (h) with sialidase (QA-Bio) according to the manufacturer's instructions

IgGl variants to remove terminal sialic acid. The hlgG1 neutral oligosaccharide is then released by PNGase F (QA-Bio) digestion as previously described (Ferrara, c. et al, Biotech. Bioeng. 93 (2006) 851-861). The water-crushing compound profile was analyzed in a cationic mode by mass spectrometry (Autoflex, Bruker Daltonics GmbH) as previously described (Ferrara, C. et al., Biotech, Bioeng. 93 (2006) 851-861). The carbohydrate profile of human IgG 1 neutral Fc-related glycans is characterized by three major m/z peaks, which can be assigned to have 0 (GO), 1 (G1) or 2 (G2) a fucosylated complex oligosaccharide of a terminal galactose residue. The carbohydrate profile of the hlgG1 containing the mutation in the Fc was analyzed and compared to those obtained against the wild type antibody, which were designed to eliminate binding to the Fc receptor. An IgG variant (P329G, LALA, P329A, P329G/LALA) containing one of the mutations showed a similar carbohydrate profile to the wild-type antibody, wherein the Fc-related glycan-based fucosylation complex recruited sugar (data not shown). Mutations in Fc can affect the degree of galactosylation and terminal sialylation of terminal galactose 162837.doc 201245227, as observed by replacing amino acid 241, 243, 263, 265 or 301 with alanine ( Lund, J. et al, j. Immunol. 157 (1996) 4963-4969) Figure 6a shows the relative percentage of galactosylation of the different hlgGl Fc-variants described herein. When the antibodies were expressed in different hosts, minor variations were observed' but significant differences in terminal galactosylation were not observed. Figure 6b indicates the variability in the galactosylation content of wild type and IgGl-P329G/LALA of 4 different antibodies, wherein the amount of galactosylation when 4 different V-domains were expressed in Hek293 EBNA cells was compared. φ Example 8 Antibody-induced platelet aggregation in whole blood analysis Whole blood platelet aggregation analysis was performed using a Multiplate instrument from Dynabyte. First, 20 ml of blood was drawn from a normal human donor and transferred to a water worm (Dynabyte Medical, MP0601). A microcell impedance device (Dynabead, MP0021) was inserted into the Multiplate instrument for analysis. Then, 175 μ! 〇 9〇/. NaCl is added to the microchannel. The antibody was added to the microwell to obtain the final test Lu concentration. 175 μΐ human blood was then added and incubated for 3 min at 37 °C. The impedance analysis was automatically started at 37 ° C for 6 min. The data was analyzed by quantifiing the underlying curve as a measure of platelet aggregation. CD9 antibodies have been shown to induce platelet activation and platelet aggregation (Worthington et al, Br. J_ Hematol 74 (2) (1990) 216-222). Platelet aggregation induced by binding of antibodies to platelets has previously been shown to involve binding to FcyRIIA (de Reys et al, Blood 81 (1993) 162837. doc 112 201245227 1792-1800). As shown above, the mutations introduced into the CD9 antibody, LALA, P3 29G, P3 29G/LALA and P329G/SPLE, significantly reduced the binding of the CD9 antibody to the FcyRIIA receptor (Fig. lc). Activation by CD9 antibody (data not shown by Ca efflux measurement) and platelet aggregation was abolished by introducing a P329G and LALA triple mutation into the antibody such that FcyRIIA binding was significantly reduced compared to the wild type antibody (see Figure 7a). And 7b). Murine IgG1 induced platelet aggregation at low antibody concentrations (0.1-1 pg/ml). At higher concentrations (3·30 gg/ml), excessive puncture of φ-excited platelets leads to silencing of the agglutination reaction. Donor variability was observed for chim-hu-IgG4-SPLE. The data for chim-hu-IgG4-SPLE responders at higher antibody concentrations are shown in Figure 6a, and the chim-hu-IgG4-SPLE non-responder data is shown in Figure 6b. For antibody variants chim-hu-IgGl-LALA, chim-hu-IgG-WT-P3 29G, chim-hu-IgG 1 -LALA-P329G, chim-hu-IgG4-SPLE-P329G, chim-hu-IgG4- For SPLE-N297Q, none of the blood samples showed any agglutination reaction. Control: spontaneous agglutination of untreated blood samples (background); ADP induction (ADP) and coagulation # enzyme analogue induction (TRAP6) platelet aggregation. Isotype control: murine IgGl (murine isotype) and human IgG4-SPLE (hu-IgG4-SPLE isotype). One of these data may be understood to be that the reduced binding of the CD9 antibody with the triple mutation to the FcyRII A receptor is responsible for the reduced platelet aggregation for these types of mutant antibodies. Therefore, in principle, thrombocyte agglutination which is a toxic side effect of antibody treatment can be prevented by introducing the above mutation capable of reducing binding to the FcyRIIA receptor into the Fc portion of the antibody. 162837.doc -113- 201245227 [Simple diagram of the diagram] Figure 1

Biacore T100 instrument (GE) by surface plasma resonance (SPR)

Healthcare) The binding affinity of different FcYRs for immunoglobulins was measured at 25 °C. a) Test FcyRI binding affinity of the following antibodies: GA101 (GA) antibody variants (IgGl-P329G, IgG4-SPLE and IgGl-LALA mutations) and 卩_Selectin (PS) antibody variants (IgGl-P329G, IgGl-LALA) And IgG4-SPLE) as well as wild-type antibodies. b) Test FcyRI binding affinity of the following antibodies: CD9 antibody variant (IgGl-wild type, 1§01-?3290, 1 layer 01-1^^^, 1§04-SPLE, IgGrP329G/LALA, IgG4-SPLE/ P329G) and wild type antibodies. c) Test FcyRIIA (R131) binding affinity of the following antibodies: CD9 antibody variants (IgGl-wild type, 18〇1-卩3290, 18〇1-1^!^, 18〇4-SPLE, IgGl-P329G/LALA , IgG4-SPLE/P329G) and wild type antibodies. The corrected response is shown to vary with receptor concentration. d) Test FcyRIIB binding affinity of the following antibodies: CD9 (herein referred to as "TA") antibody variants (IgGl-wild type, IgG4-SPLE/P329G, IgGl-LALA, IgGl-LALA/P329G) and P-selectin (pSel) antibody variant (IgG4-wild type, IgG4-SPLE) and wild type antibody. e) Test FcyRIIIA-V158 binding affinity of the following antibodies: CD9 antibody variants (IgGl-wild type, IgG4-SPLE, IgGl-LALA, IgG4-SPLE/P329G, IgGl-P329G, IgGl-LALA/P329G) and 162837.doc 114 201245227 Wild type antibody. The corrected response is shown to vary with receptor concentration. Figure 2 Testing Clq binding of the following antibodies: P-selectin (PS) antibody variants (IgGl wild type, P329G, IgG4-SPLE) and CD20 (GA) antibody variants (IgGl-wild type, P329G and IgG4-SPLE) . Figure 3 The efficacy of recruiting immune-effector cells depends on the type of Fc variant. Fc variants were plated on ELISA plates and human NK92 effector cells transfected with human FcyRIIIA were added. Esterase analysis was used to measure the induction of cytolytic activity of activated NK cells. a) Analysis of CD20 (GA101) antibody variants (wild type, LALA, P329G, P329G/LALA). b) Analysis of CD20 (GA101) antibody variants (introduction of P329R or P329G mutations). . All variants were generated in the form of a glycoengineering to have a strong signal for any effector cell recruitment function. Figure 4 The efficacy of recruiting immune-effector cells depends on the type of Fc variant, as measured by classical ADCC analysis. A human NK92 cell line transfected with human FycRIIIA was used as an effector and CD20-positive Raji cells were used as target cells. Testing different glycoengineered CD20 antibodies (GA101 G(2) and non-glycoengineered CD20 antibody (GA101) variants (introducing P329G, P329A or LALA mutations) ° a) Non-glycoengineered CD20 antibodies: P329G, LALA, respectively And P329G/LALA mutations were introduced into the antibodies, respectively. 162837.doc • 115· 201245227 b) Non-glycoengineered CD20 antibodies: P329G, P329A and LALA mutations have been introduced into antibodies, respectively. Figure 5 Complement dependent cytotoxicity (CDC) analysis. The efficacy of different Fc variants of non-glycoengineered and sugar modified CD20 (GA101) antibodies in mediating CDC on SUDH-L4 target cells was analyzed. a) Non-glycoengineered CD20: P329G, LALA and P329G/LALA mutations have been introduced into antibodies, respectively. b) Non-glycoengineered CD20: P329G, P329A and LALA mutations have been introduced into antibodies, respectively. Figure 6 a) Carbohydrate profile of Fc-related glycans of human IgGl variants. The percentage of galactosylation of Fc-related oligosaccharides containing hlgG1 with LALA, P329G, P329A or P329G/LALA mutations differs only slightly from the percentage of galactosylation of wild-type antibodies. b) Relative galactosylation: Four different IgGs introduced with IgGl P329G/LALA mutations. The amount of galactosylation when four different V-domains were expressed in Hek293 EBNA cells was compared. Figure 7. Antibody-induced platelet aggregation in a whole blood assay. Murine IgGl induces platelet aggregation, as determined for both donors, which differ in their antibody concentration dependent response. a) Donor A, b) Donor B. 162837.doc 201245227 Sequence Listing

<1>0> Swiss company Rozcyliya <120> Antibody Fc variant <130> 27397 FT <140> 101110910 <141> 2012-03-28 <150> 11160251.2 <151> 2011 -03-29 <160> 14 <170> Patentln version 3.5 <210> 1 <211> 107 <212> PRT <213> Homo sapiens <400>

Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 15 10 15 • Gin 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 Gin Trp Lys Val Asp Asn Ala Leu Oln 35 40 45

Ser Gly Asn Scr Gin Glu Ser Val Tlir Glu Gin Asp Ser Lys Asp Ser 50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Tlir Leii Ser Lys Ala Asp Tyr 01 u 65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser 85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

• <210> 2 <211> 105 <212> PRT Homo sapiens <400> 2

Gin Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 15 10 15

Glu Leu Gin Ala Asn Lys Ala Thr Leu Val Cys Leu lie 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 Gin Ser Asn Asn Lys 50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gin Trp Lys Ser 65 70 75 80 162837 · Sequence Listing.doc 201245227

His Arg Ser Tyr Ser Cys Gin Val Thr His Glu Gly Ser Thr Val Clu 85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 3 <211> 330 <212> PRT <213> Homo sapiens <400> 3

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr G】n Thr 65 70 75 80

Tyr lie 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 lie 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 Tr, 145 150

15S

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175

Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220

Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 -2- 162837 · Sequence Listing.doc 201245227

Leu Thr Lys Asji Gin Va] Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255

Pro Ser Asp He Ala Val Glu Trp Giu Ser Asn Gly Gin Pro GIu 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 Gin Gin 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

Gin Lys Ser Leu Ser Leu Scr Pro Gly Lys 325 330

<210> 4 <211> 330 <212> PRT <213> Homo sapiens <400> 4

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 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 Gin Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 75 80

Tyr lie 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 He 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 162837 - Sequence Listing.doc 201245227

Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220

Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240

Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 25S

Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin 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 Va丨 Asp Lys Ser Arg Trp Gin Gin 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

Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 5 <211> 330 <212> PRT <213> Homo sapiens <400>

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 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 Va] Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser 50 55 60

Leu Ser Ser Val Va] Thr Val Pro Ser Ser Ser Leu G]y Thr Gin Thr 65 70 75 80

Tyr He 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 -4-

162837 - Sequence Listing.doc 201245227

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125

Lys Pro Lys Asp Thr Leu Met lie 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 Asti Ala Lys Thr Lys Pro Arg Glu 165 170 175

Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly 210 215 220

Gin Pro Arg Glu Pro Gin Val rryr Thr Leu Pro Pro Ser Arg Asp Qlu 225 230 235 240

Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255

Pro Ser Asp IU Ala Val Glu Trp Glu Ser Asn Gly Gin 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 Gin Gin 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

Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 6 <211> 327 <212> PRT <213> Homo sapiens <400>

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 15 10 15

Ser Thr Set Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

Phe Pro Glu Pro Val TTir Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 162837 · Sequence Listing.doc 201245227

Gly Val His Thr Phe Pro Ala Val Leu Gin 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 Tfar Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140

Asp Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp 145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe 165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Va] Leu His Gin 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 lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg 210 215 220 01u Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys 225 230 235 240

Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 lie Ala Val Glu Trp Glu Ser Asn Gly Gin 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

Are Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser 290 295 300

Cys Ser Val Met His Glu Ala Leu His Ass His Tyr Thr Gin Lys Ser 305 310 315 320

Leu Ser Leu Ser Leu Gly Lys 325 <210> 7 162837 - Sequence Listing.doc 201245227 <211> 327 <212> PRT <213> Homo sapiens <400>

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 15 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 Gin 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 Pro Cys Pro Ala Pro 100 105 110

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125

Asp Thr Leu Met lie Ser Arg Ihr Pro Glu Val Thr Cys Val Val Val 130 135 140

Asp Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp 145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe 165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 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 He Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg 210 215 220

Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys 225 230 235 240

Asn Gin Val Ser Leu Thr Cys Lju Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255

He Ala Val Glu Trp Glu Ser Asn Gly Gin 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 162837 - Sequence Listing.doc 201245227 275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser 290 295 300

Cys Ser Val Met His Glu Ala Lea His Ass His Tyr Thr Gin Lys Ser 305 310 315 320

8th <211>

Asp lie Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 15 10 15

Asp Arg Va] Thf lie Ser Cys Ser Ala Ser Gin Gly Ik Ser Asn Tyr 20 25 30

Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Aso Val Lys Lea Leu lie 35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr lie Ser Asn Leu Glu Pro 65 70 75 80

Glu Asp lie Ala Thr Tyr Tyr Cys Gin Gin Tyr Ser Lys Leu Pro Tyr 85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg Thr Val Ala Ala 100 105 110

Pro Ser Vai Phe lie Phe Pro Pro Ser Asp Glu Gin 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 Gin Trp Lys Val Asp Asn Ala Leu Oln Ser Gly Asn Ser Gin 145 150 155 160

Glu Ser Val Thr Glu Gin 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 Gin Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 162837 - Sequence Listing.doc 201245227

Phe Asn Arg Gly Glu Cys 210 <210> 9 <211> 449 <212> PRT <2I3> Artificial Sequence <220><223> 8201_HC_IGG1-PEP <400>

Asp Val Gin Leu Gin Glu Ser Oly Pro Gly Leu Val Lys Pro Ser Gin 15 10 IS

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser lie Thr Ser Asp 20 25 30

Tyr Ala Trp Asn Trp lie Arg Gin Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45

Met Gly Tyr lie Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg He Ser lie Thr Arg Asp Thr Ser Lys Asn Gin Phe Phe 65 70 75 80

Leu Gin Leu Ser Ser Val Thr Thr Olu Asp Thr Ala Thr Tyr Phe Cys 85 90 95

Glu Gly Gly Asn Tyr Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gin Gly 100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125

Pro Uu 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

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Va] Pro Ser 180 】85 190

Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 • 9- 162837 - Sequence Listing, doc 201245227

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie 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 Asa 275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300

Val Ser Va] Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335

Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Olu Pro Gin Val Tyr Thr 340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380

Ser Asn Gly Gin 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 Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445

Lys <210> 10 <211> 449 <212> PRT <213> artificial sequence <220><223> 8206_HC_IGG1-P329G-PEP <400>

Asp Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser lie Thr Ser Asp 20 25 30 •10·

162837 - Sequence Listing.doc 201245227

Tyr Ala Trp Asn Trp lie Arg Gin Phe Pro Gly Asa Lys Leu OIu Trp 35 40 45

Met Gly Tyr lie Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg lie Ser lie Thr Arg Asp Thr Ser Lys Asn Gin Phe Phe 65 70 75 80

Leu Gin Leu Ser Ser Vat Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95

Glu Gly Gly Asn Tyr Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gin Gly 100 105 110

Thr Leu Val Thr Val Ser Ala 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

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190

Ser Ser Leu Gly Thr Gin Thr Tyr lie 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 lie Ser 245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Olu 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300

Vala Leu Asn Gly Lys Glu 305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro lie Glu Lys 325 330 335 -11 - 162837 - Sequence Listing.doc 201245227

Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr 340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380

Ser Asn Gly Gin 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 Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445

Lys <210>11 <211> 449 <212> PRT <213> artificial sequence <220><223> 8207_HC_IGG1-LAU-P329G-PEP <400>

Asp Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 ' 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser lie Thr Ser Asp 20 25 30

Tyr Ala Trp Asn Trp lie Arg Gin Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45

Met Gly Tyr lie Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg lie Ser lie Thr Arg Asp Thr Ser Lys Asn Gin Phe Phe 65 70 75 80

Leu Gin Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95

Glu Gly Gly Asn Tyr Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gin Gly 100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 •12· 162837-Sequence List.doc 201245227

Pro Leu Ala Pro Scr Ser Lys Ser Thr Ser Gly Gly 130 135 140

Thr Ala Ala Leu

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 ISO 155

Val Ser Vel

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170

Pro Ala Val Leu 175

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185

Thr Val Pro Ser 190

Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val 195 200

Asn His Lys Pro 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220

Scr Cys Asp Lys

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235

Ala Gly G,y

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250

Leu Met He Ser 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265

Ser His Glu Asp 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280

Glu Val His Asn 285

Ala Lys Thr Lys Pro Arg Olu Glu Gin Tyr Asn Ser 290 295 300

Thr Tyr Arg Val

Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 305 310 315

Asn Gly Lys Glu 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala 325 330

Pro He Glu Lys 335

Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 340 345

Gin Val Tyr Thr 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin 355 360

Val Scr Leu Thr 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala 370 375 380

Va! Glu Trp Glu

Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395

Pro Pro Val Leu 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410

Thr Val Asp Lys 415

Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser 420 425

Val Met His Glu 430 162837 - Sequence Listing.doc -13· 201245227

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445

Lys <210> 12 <211> 446 <212> PRT <213> artificial sequence <220><223> 8202_HC_IGG4-SPLE-PEP <400>

Asp Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser lie Thr Ser Asp 20 25 30

Tyr Ala Trp Asn Trp lie Arg Gin Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45

Met Gly Tyr lie Ser Tyr Ser Gly Phe Thr Asn Tyr Asn Pro Ser Leu 50 55 60

Lys Ser Arg lie Ser lie Thr Arg Asp Thr Ser Lys Asn Gin Phe Phe 65 70 75 80

Leu Gin Leu Ser Ser Val Thr Thr G]u Asp Thr Ala Thr Tyr Phe Cys 85 90 95

Glu Gly Gly Asn Tyr Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gin Gly 100 105 110

Thr Leu Val Thr Val Ser Ala 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 T; 145 150 155 1(

Asn Ser Gly Ala Leu Tbr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

Gin Ser Ser Gly Leu Tyr Ser Leu Scr Ser Val Val Thr Val Pro Ser 180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205

Ser Asn Thr Lys Va] Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 162837 - Sequence Listing.doc -14- 201245227

Cys Pro Pro Cys Pro Ala Pro GIu Phe Glu Gly Gly Pro Ser Val Phe 225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro 245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val 260 265 270

Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285

Lys Pro Arg Glu Glu Gin Phe Asn Scr Thr Tyr Arg Val Val Ser Val 290 295 300

Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320

Lys Val Ser Asn Lys Gly Lea Pro Ser Ser lie Glu Lys Thr lie Ser 325 330 335

Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 340 345 350

Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Lea Val 355 360 365

Lys Gly Phe Tyr Pro Scr Asp He Ala Val Glu Trp Glu Ser Asn Gly 370 375 380

Gin Pro Glu Asn Asn Tyr Lys rrhr 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

Gin Glu Gly Asn Vai Phe Scr Cys Scr Val Met His Glu Ala Leu His 420 425 430

Asn His Tyr Thr Gin Lys Sei Uu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 13 <211> 446 <212> PRT <213>Artificial Sequence<220><223> 8208JC„IGG4- SPLE-P329G-PEP <400> 13

Asp Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser lie Thr Ser Asp 20 25 30

Tyr Ala Trp Asn Trp lie Arg Gin Phe Pro Gly Asn Lys Leu Glu Trp -15· 162837 - Sequence Listing.doc 201245227 35 40 45

Met Gly Tyr lie Ser Tyr Ser Gly Phe Tbr Asn Tyr Asn Pro Ser Leu SO 55 60

Lys Ser Arg lie Ser lie Thr Arg Asp Thr Ser Lys Asn Gin Phe Phe 65 70 75 80

Leu Gin Leu Ser Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95

Glu Gly Gly Asn Tyr Arg Tyr Ser Tip Phe Pro Tyr Trp Gly Gin Gly 100 105 110

Thr Leu Val Thr Val Ser Ala 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

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205

Ser Asn Thr Lys Va] Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe 225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro 245 250 255

Glu Val Thr Cys Va] Val Val Asp Val Ser G]n Glu Asp Pro Glu Val 260 265 270

Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Tbr 275 280 285

Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300

Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320

Lys Val Ser Asn Lys Gly Leu Gly Ser Ser lie Glu Lys Thr lie Ser 325 330 335 -16- 162837 - Sequence Listing.doc 201245227

Lys Ala Lys Qly Gin Pro Arg Glu Pro Gin Val Tyi Thr Leu Pro Pro 340 345 350

Scr Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 355 360 365

Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp G.u Scr Asn Gly

Gin Pro G!u Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Scr Asp 385 390 395 400

Gly Ser Phe Phe Leu Tyr Scr Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415

Gin Glu Gly Asn Val Phe Scr Cys Ser Val Met His Glu Ala Leu His 420 425 430

Asn His Tyr Thi Gin Lys Scr Leu Scr Leu Scr Leu Gly Lys 435 440 445

<211> 449 <212> PRT <213> artificial sequence <220><223> mSJiCJQGlJMA^E? <400>

Asp Val Gin Leu OIn Glu Ser Qly Pro Gly Leu Val Lys Pro Ser Gin 15 10 15

Ser Leu Ser Leu Thr Cys TTir Val Thr Gly Tyr Set 丨le Thr Ser Asp 20 25 30

Tyr Ala Trp Asn Trp lie Arg Gin Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45

Met Gly Tyr lie Ser Tyr Ser Gly Phe Thi Asn Tyr Asn Pro Ser Leu 50 55 60

Lys Scr Arg lie Ser lie Thr Arg Asp Thr Ser Lys Asn OIn Phe Phe 65 70 75 80

Leu Gin Leu Scr Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95

Glu Gly Gly Asti Tyr Arg Tyr Ser Trp Phe Pro Tyr Trp Gly Gin Gly 100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Qly Pro Ser Val Phe 115 120 125

Pro Leu Ala Pro Ser Scr Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro QIu Pro Val Thr Val Ser Trp -17- 162837 - Sequence Listing.doc 201245227 145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175

Glo Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190

Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro 195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Va】G1 u Pro Lys Ser Cys Asp Lys 210 215 220

Thr H5s Thr Cys Pro Pro Cys Pro Ala Pro GIu Ala Ala Gly Gly Pro 225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie 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 Gin Tyr Asn Ser Thr Tyr Arg Val 290 295 300

Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu 305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys 325 330 335

Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Olu Pro Gin Val Tyr Thr 340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr 355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu 370 375 380

Ser Asn Gly Gin 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 Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445

Lys -18 - 162837 · Sequence Listing, doc

Claims (1)

201245227 VII. Patent Application Range: 1 'A polypeptide comprising an Fc variant of a wild type human IgG Fc region comprising an amino acid substitution at the Pro329 position and at least another amino acid substitution 'where such residues The basis is numbered according to the EU index of Kabat, and wherein the polypeptide exhibits reduced affinity to human FcyRIIIA and/or FcyRIIA and/or FcYRI as compared to a polypeptide comprising a wild type IgG Fc region, and wherein the polypeptide is induced by the polypeptide ADCC has been reduced to at least 20% of the ADCC induced by the polypeptide comprising the wild type human IgG Fc region. 2. The polypeptide of claim 1, wherein the wild type human Fc is substituted with glycine or arginine or an amino acid residue large enough to disrupt the sandwich of the valyanate sandwich in the Fc/Fcy receptor interface. Pro329. The polypeptide of any one of claims 1 or 2, wherein the at least one other amino acid is substituted with S228P, E233P, L234A, L235A, L235E, N297A, N297D or P331S. The polypeptide of any one of claims 1 to 2, wherein the at least one other amino acid is substituted with L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region. 5. The polypeptide of any one of claims 1 to 2, wherein the affinity for at least one further receptor than the population comprising the human receptors Fcyl, FcyllA and Clq is greater than the affinity of the polypeptide comprising the wild type human IgG Fc region reduce. The polypeptide of any one of claims 1 to 2, wherein the polypeptide comprises a human IgG1 or IgG4 Fc region. 7. The polypeptide according to any one of the preceding claims, wherein the polypeptide is an antibody or an Fc fusion protein. 162837. doc 201245227. The polypeptide of any one of the claims (1), wherein the thrombocyte induced by the polypeptide The agglutination ratio is reduced by the thrombocyte agglutination induced by the polypeptide comprising the wild type human IgG. 9. The polypeptide of any one of clause 2, wherein the CDC induced by the polypeptide is significantly reduced compared to the CDC induced by the polypeptide comprising the wild type human IgG Fc region. 10. The polypeptide of any one of claims 1 to 2 for use as a medicament. The polypeptide of any one of claims 1 to 2, wherein the polypeptide is an anti-(:1)9 antibody, characterized in that the polypeptide comprising the wild-type Fc region comprises SEQ m N〇:9 as a heavy chain. The variable region and contains redundant () id NO: 8 as a light chain variable region. 12. The polypeptide of any one of claims 1 to 2 for use in the treatment of a disease, wherein the effector function of the polypeptide is advantageously significantly reduced compared to the effector function induced by the polypeptide comprising the wild type human IgG Fc region. . 13. The use of a polypeptide according to any one of claims 1 to 12 for the manufacture of a medicament for the treatment of a disease, wherein the effector function of the polypeptide comprising an Fc variant of the wild type human IgG Fc region This effector function is advantageously significantly reduced compared to the polymorphism induced by the inclusion of the wild-type human IgG Fc region. 14. Use of a polypeptide comprising an Fc variant of a wild-type human IgG Fc region, wherein the human IgG Fc region of the polypeptide is substituted with a glycine acid, wherein the residues are numbered according to the EU index of Kabat, Wherein the polypeptide exhibits reduced affinity for human FcyRIIIA and FcyRIIA, downregulating ADCC to at least 20% of the ADCC induced by the polypeptide comprising the wild type human IgG Fc region, and/or for downregulating ADCP. The use of the polypeptide of claim 14, wherein the Fc variant comprises at least two additional amino acid substitutions at the L234A and L235A of the human IgG1 Fc region or at S228P and L235E of the human IgG4 Fc region. 16_ Use of claim I4 or 15 wherein the thrombocyte agglutination induced by the polypeptide is reduced by agglutination of the thrombus induced by a polypeptide comprising a wild-type human Fc region, wherein the polypeptide is a platelet-activating antibody. 17. Use of a polypeptide of the Human IgG Fc region of Pro329 via Gly for the manufacture of a medicament for treating an individual having a disease, wherein the residues are numbered according to the EU index of Kabat, wherein The polypeptide is characterized in that its binding to FcyRIIIA and FcyRIIA is significantly reduced in binding to the polypeptide comprising the wild type human IgG Fc region. 18. The use of claim 17, wherein the polypeptide comprises at least two additional amino acid substitutions at the L234A and L235A of the human IgG1 Fc region or at S228P and L235E of the human IgG4 Fc region. 162837.doc