Immunoglobulin variants

Abstract

The present invention provides Fc variants of a parent IgA Fc polypeptide, wherein the Fc variant exhibits altered binding to FcαR, and the Fc variant comprises at least one amino acid modification in the Fc region of the parent Fc polypeptide.

Description

Technical Field

[0001] Sequence Listing This application is electronically submitted in ASCII format and contains a sequence listing that is incorporated herein by reference in its entirety. The ASCII copy created on April 19, 2021 is named PAT058743-WO-PCT_SL.txt and is 316 kilobytes in size.

[0002] The present invention relates to Fc mutant polypeptides and antibodies having properties optimized to promote neutrophil mobilization, and engineering methods for preparing them. The Fc mutant polypeptides and antibodies may be useful for the treatment of tumors, particularly solid tumors.

Background Art

[0003] Currently, all clinically approved antibodies include the immunoglobulin IgG isotype. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism for tumor cell killing mediated by IgG antibodies that recognize and bind to Fc gamma receptors (FcγR). However, in patients with a high tumor burden, recurrence is possible, and IgG antibody therapy may lose effectiveness due to the immunosuppressive environment found in the tumor. In the tumor microenvironment, FcγR-possessing effector cells such as macrophages and natural killer cells may become insoluble due to immunosuppressive factors such as TGFβ. IgA represents an alternative isotype for antibody therapy by engaging with the Fc alpha receptor (FcαRI) expressed by bone marrow effector cells, such as neutrophils and tumor-resident myeloid suppressor cells (MDSCs). IgA is the second most abundant immunoglobulin in human serum after IgG; both monomeric IgA allotypes (IgA1 and IgA2) account for up to 25% of human serum immunoglobulins. Previously, neutrophils were not generally considered potential effector cells. However, neutrophils are the most abundant population of circulating leukocytes and have been shown to infiltrate solid tumors (Gregory & Houghton (2011) Cancer Res., 71:2411-16; Vogt Sionov et al., (2015) Cancer Microenviron., 8(3):125-58; Uribe-Querol & Rosales (2015) J.Immunol.Res., Article ID:983698; Rosales (2018) Front Physiol., 9:113). MDSCs also originate from the myeloid lineage and are one of the most common immunosuppressive cell types. IgA antibodies have been shown to effectively kill tumor cells by mobilizing neutrophils and thereby improving ADCC. Unfortunately, the use of IgA antibodies as therapeutic agents is hindered by several disadvantages and limitations, such as low expression yields and expensive purification schemes. Furthermore, production suffers from heterogeneous glycosylation. IgA has multiple glycosylation sites that may be sensitive to glycan heterogeneity. Transient expression levels for monomeric IgA have been reported to be 30-70 μg / L for human IgA1 (Lombana et al., (2019) MABS, 11:1122-38; Meyer et al., (2016) MABS, 8:87-98). [Overview of the project]

[0004] Therefore, there is still a need for IgA antibodies that can be developed as therapeutic antibodies. IgA antibodies with improved potency may offer a viable alternative to IgG therapeutic antibodies, offering the advantages of neutrophil and MDSC mobilization and effective tumor cell killing by enhanced ADCC.

[0005] The present invention provides Fc variants of IgA isotype parental Fc polypeptides that have improved binding properties to FcαRI and can be used for neutrophil recruitment and activation. The Fc variants of this disclosure include amino modifications, which may consist of amino acid insertions, amino acid deletions, and / or amino acid substitutions, either independently or in combination.

[0006] In one embodiment, the disclosure provides an Fc variant of a parent Fc polypeptide, where the Fc variant exhibits altered binding to FcαR or altered antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parent Fc polypeptide, and the Fc variant comprises at least one amino acid modification in the Fc region of the parent Fc polypeptide. In one embodiment, the amino acid modification is located at a position selected from the group consisting of: CH2.10, CH2.89, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, CH3.105, CH3.109, CH3.118 and CH3.124, and the numbering of the amino acid modification follows the IMGT numbering for the C-domain. In a preferred embodiment, the Fc variant comprises at least one amino acid modification in the Fc region of the parent Fc polypeptide, where the amino acid modification is selected from the group consisting of: A_CH2.10_S, L_CH2.89_I, G_CH2.91_Q, G_CH2.91_V, Q_CH2.94_E, N_CH2.97_H, N_CH2.97_Y, G_CH2.99_W, S_CH3.45_D, M_CH3.105_Y, E_CH3.109_D, Q_CH3.118_Y, and L_CH3.124_F, and the numbering of the amino acid modification follows the IMGT numbering for the C-domain.In further embodiments, the disclosure provides Fc variants comprising at least one amino acid modification in the Fc region, wherein the amino acid modification is: Q_CH2.94_E, N_CH2.97Y, S_CH3.45_D, M_CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D, N_CH2.97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH 2.97_Y / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.105_Y, M_CH3.105_ Y / Q_CH3.118_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3 .105_Y, Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y The amino acid modifications are selected from the group consisting of / Q_CH3.118_Y, A_CH2.10_S, L_CH2.89_I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_CH3.109_D, L_CH3.124_F, L_CH2.89_I / G_CH2.91_V / Q_CH2.94_E / N_CH2.97_Y / G_CH2.99_W, and the numbering of the amino acid modifications follows the IMGT numbering for the C-domain.

[0007] In one embodiment, the disclosure provides Fc variants of a parent Fc polypeptide, comprising amino acid modifications at positions CH2.94, CH2.97, CH3.45, CH3.105, and CH3.118. In one embodiment, the Fc variant of the parent Fc polypeptide comprises amino acid substitutions of Glu at position CH2.94, Tyr at position CH2.97, Asp at position CH3.45, Tyr at position CH3.105, or Tyr at position CH3.118. In a preferred embodiment, the Fc variant of the parent Fc polypeptide comprises amino acid substitutions Q_CH2.94_E, L_CH2.97_Y, S_CH3.45_D, M_CH3.105_Y, and Q_CH3.118_Y.

[0008] In one embodiment, the disclosure provides an Fc variant of a parent Fc polypeptide, wherein the parent Fc polypeptide is contained within human IgA1, or the parent Fc polypeptide is contained within human IgA2.

[0009] In one embodiment of the present disclosure, the Fc variant exhibits modified binding to FcαR compared to the parent Fc polypeptide. Fc variants of the parent Fc polypeptide are provided herein, where the Fc variant has an increased affinity for human FcαRI, at least 50 times greater than that of the parent Fc polypeptide, as measured by surface plasmon resonance (SPR). In one embodiment, the present disclosure provides Fc variants of the parent Fc polypeptide, where the Fc variant has an increased affinity for human FcαRI, at least about 50, about 100, about 150, about 200, about 250, or about 300 times greater than that of the parent Fc polypeptide, as measured by surface plasmon resonance. In one embodiment, the present disclosure provides Fc variants of the parent Fc polypeptide, where the Fc variant has an increased affinity for human FcαRI, at least about 300 times greater than that of the parent Fc polypeptide, as measured by SPR.

[0010] In one embodiment of this disclosure, an Fc variant exhibits modified ADCC compared to the parent Fc polypeptide. An Fc variant of the parent Fc polypeptide is provided herein, wherein the Fc variant increases antibody-dependent cell-mediated cytotoxicity (ADCC), as measured in an MDA-MB-453 cell-killing assay, by at least about 5 times compared to the parent Fc polypeptide. In one embodiment, this disclosure provides an Fc variant of the parent Fc polypeptide, wherein the Fc variant has increased efficacy in a Calu-3 cell-killing assay, at least about 2 times compared to the parent Fc polypeptide.

[0011] In another embodiment, the Disclosure provides an IgA antibody comprising a mutant Fc polypeptide, wherein the antibody has increased FcαR affinity or increased ADCC compared to an IgA antibody comprising a wild-type Fc polypeptide. In one embodiment, the Disclosure provides an IgA antibody comprising an amino acid modification at a position selected from the group consisting of CH2.10, CH2.89, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, CH3.105, CH3.109, CH3.118 and CH3.124, wherein the numbering of the amino acid modification follows the IMGT numbering for the C-domain. In preferred embodiments, the present disclosure provides IgA antibodies comprising amino acid modifications, where the amino acid modifications are selected from the group consisting of: A_CH2.10_S, L_CH2.89_I, G_CH2.91_Q, G_CH2.91_V, Q_CH2.94_E, N_CH2.97_H, N_CH2.97_Y, G_CH2.99_W, S_CH3.45_D, M_CH3.105_Y, E_CH3.109_D, Q_CH3.118_Y, and L_CH3.124_F, and the numbering of the amino acid modifications follows the IMGT numbering for the C-domain.

[0012] In further embodiments, the disclosure provides IgA antibodies comprising amino acid modifications, where the amino acid modifications are: Q_CH2.94_E, N_CH2.97Y, S_CH3.45_D, M_CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D , N_CH2.97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / M_ CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3 .118_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y , Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y / Q_C The amino acid modifications are selected from the group consisting of H3.118_Y, A_CH2.10_S, L_CH2.89_I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_CH3.109_D, L_CH3.124_F, and L_CH2.89_I / G_CH2.91_V / Q_CH2.94_E / N_CH2.97_Y / G_CH2.99_W, and the numbering of the amino acid modifications follows the IMGT numbering for the C-domain.

[0013] In one embodiment, the disclosure provides an IgA antibody comprising amino acid modifications at positions CH2.94, CH2.97, CH3.45, CH3.105, and CH3.118. In one embodiment, the IgA antibody comprises amino acid substitutions of Glu at position CH2.94, Tyr at position CH2.97, Asp at position CH3.45, Tyr at position CH3.105, or Tyr at position CH3.118. In a preferred embodiment, the IgA antibody comprises amino acid substitutions Q_CH2.94_E, L_CH2.97_Y, S_CH3.45_D, M_CH3.105_Y, Q, and CH3.118_Y.

[0014] In one embodiment, the present disclosure provides an IgA antibody comprising a mutant Fc polypeptide, wherein the antibody is a human IgA1 or IgA2 antibody.

[0015] This disclosure provides isolated nucleic acids encoding the Fc variants described herein. This disclosure optionally provides vectors containing nucleic acids operably ligated to a control sequence. This disclosure provides host cells containing the vector, and methods for generating and optionally recovering the Fc variants. This disclosure provides compositions comprising IgA antibodies containing the Fc variants described herein, and physiologically or pharmaceutically acceptable carriers or diluents.

[0016] This disclosure is intended for the therapeutic and diagnostic use of IgA antibodies, including the Fc variants disclosed herein. The Fc variants disclosed herein may also be used to construct other conjugate molecules, such as bispecific and multispecific antibodies. The IgA antibodies described herein may be used to treat a variety of indications, including but not limited to proliferative disorders such as cancer. [Brief explanation of the drawing]

[0017] [Figure 1] Fc variants with increasing concentrations in the assay described in Example 4: SEQ ID NOs: 3(■), 6(▲), 32(▼), 37(◆) and 42(

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Mode for Carrying Out the Invention

[0018] Fc variants of IgA immunoglobulins having optimized properties, and antibodies containing these Fc variants are disclosed herein. These optimized properties include enhanced binding to FcαR and modified antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parent IgA Fc polypeptide.

[0019] definition To facilitate a more readily understandable understanding of this disclosure, certain terms are specifically defined throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains.

[0020] Wherever the terms “comprise,” “comprises,” or “comprising” are used in reference to a sequence (e.g., an amino acid sequence), it should be understood that such sequence may also be limited by terms such as “consist,” “consists,” or “consisting.” As used herein, the phrase “essentially consists of” refers to a genus or species of active pharmaceutical agent contained in the method or composition, and any excipients that are inert to the intended purpose of the method or composition. In some embodiments, the phrase “essentially consists of” expressly excludes the inclusion of one or more additional activators other than the Fc variants of the Disclosure. In some embodiments, the phrase “essentially consists of” expressly excludes the inclusion of one or more additional activators other than the Fc variants of the Disclosure and a second co-administered agent.

[0021] As used herein, the term “antibody” refers to a polypeptide of the immunoglobulin family that can bind noncovalently, reversibly, and specifically to a corresponding antigen. The basic functional unit of each antibody is an immunoglobulin monomer containing only one Ig unit, which is defined herein as an “Ig monomer.” Secretory antibodies may also be dimers with two Ig units (e.g., IgA), tetramers with four Ig units, or pentamers with five Ig units (e.g., mammalian IgM). The term “antibody” includes, for example, monoclonal antibodies (e.g., full-length antibodies with an immunoglobulin Fc region). An Ig monomer is a Y-type molecule consisting of four polypeptide chains; two identical heavy chains and two identical light chains linked by disulfide bonds (Woof & Burton (2004) Nature Reviews Immunology, 4(2):89-99). Each chain contains numerous structural domains with approximately 70 to 110 amino acids, classified into two categories according to their size and function: variable or constant. The heavy chain contains one variable domain (abbreviated as VH) and three constant domains (abbreviated as CH1, CH2, and CH3). Each light chain contains one variable domain (abbreviated as VL) and one constant domain (abbreviated as CL). The immunoglobulin domain has a characteristic immunoglobulin fold in which two beta sheets are held together by interactions between conserved cysteine ​​residues and other charged amino acids, creating a "sandwich" structure. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs located from the amino acid terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain antigen-binding domains or antigen-binding sites that interact with the antigen.

[0022] The term “antibody” includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (for example, anti-Id antibodies against the antibodies of this disclosure). Antibodies may be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0023] As used herein, the term “single-specific molecule” refers to a molecule that binds to one epitope on a target antigen. In some embodiments, the single-specific molecule of this disclosure is a single-specific antibody-like molecule. In some embodiments, the single-specific molecule of this disclosure is a single-specific antibody. The term “bispecific molecule” refers to a multispecific binding molecule that binds to two different antigens. In some embodiments, the bispecific molecule of this disclosure is a bispecific antibody-like molecule. As used herein, the term “multispecific binding molecule” refers to a molecule that binds to two or more different antigens. Recognition of each antigen is generally achieved via an “antigen-binding domain.” In some embodiments, the multispecific binding molecule of this disclosure is a multispecific antibody-like molecule, for example, a bispecific antibody-like molecule.

[0024] The term "antigen-binding site" refers to the portion of an antibody that contains determinants that form an interface for binding to an antigen or its epitope. The term "antigen-binding site" can be used interchangeably with the term "antigen-binding domain." With respect to proteins (or protein mimes), the antigen-binding site typically includes one or more loops (of at least four amino acids or amino acid mimics) that form an interface for binding to the antigen polypeptide. Typically, the antigen-binding site of an antibody molecule includes at least one or two CDRs and / or hypervariable loops, or more typically, at least three, four, five, or six CDRs and / or hypervariable loops.

[0025] As used herein, the “complementarity-determining region” (“CDR”) refers to the hypervariable region of the VL and VH. CDRs are target protein binding sites on antibody chains that possess specificity for the target protein. Each human VL or VH contains three CDRs (CDR1-3, numbered sequentially from the N-terminus), constituting approximately 15-20% of the total variable domain. CDRs can be named according to their region and order. For example, “VHCDR1” or “HCDR1” both refer to the first CDR of the heavy chain variable region. CDRs are structurally complementary to the target protein epitope and therefore directly contribute to binding specificity. The remaining stretch of the VL or VH, the so-called framework region, exhibits less variation in amino acid sequence (Kuby (2000) Immunology, 4th ed., Chapter 4. WH Freeman & Co., New York). The location of the CDR and framework region is defined by various known definitions in the art, e.g., Kabat, Chothia, IMGT, AbM, and combined definitions (e.g., Johnson et al., (2001) Nucleic Acids Res., 29:205-206; Chothia & Lesk, (1987) J.Mol.Biol., 196:901-917; Chothia et al., (1989) Nature, 342:877-883; Chothia et al., (1992) J.Mol.Biol., 227:799-817; Lefranc, MP, (2001) Nucleic Acids Res., 29:207-209; Al-Lazikani et al., (1997) J.Mol.Biol., 273:927-748 and Kabat et al. This can be determined using (see al., (1991) Sequences of proteins of immunological interest. 5th Edition - US DHHS, NIH publication no. 91-3242, pp. 662, 680, 689).The definition of antigen-binding sites is also described in the following: Ruiz et al., (2000) Nucleic Acids Res., 28:219-221; MacCallum et al., (1996) J.Mol.Biol., 262:732-745; and Martin et al., (1989) Proc.Natl.Acad.Sci.USA, 86:9268-9272; Martin et al., (1991) Methods Enzymol., 203:121-153; and Rees et al., (1996) In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172. In combinations of Kabat and Chothia numbering schemes, in some embodiments, CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in VH, e.g., mammalian VH, e.g., human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL, e.g., mammalian VL, e.g., human VL. Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to "Kabat"). Under IMGT, the CDR region of an antibody can be determined using the IMGT / DomainGap Align program. The IMGT tool is available on the World Wide Web (www.imgt.org).

[0026] In one embodiment, the antibody includes an "antigen-binding fragment" of the antibody. Examples of such fragments include: (i) a monovalent fragment consisting of a Fab fragment, VL, VH, CL, and CH1 domains; (ii) a bivalent fragment containing an F(ab')2 fragment, two Fab fragments linked by disulfide crosslinking in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) a diabody (dAb) fragment consisting of a VH domain; (vi) a camel or camelized variable domain; and (vii) a single-chain Fv (scFv), e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) PNAS USA. See 85:5879-5883); (viii) single-domain antibodies; (ix) diabolic (Dab) antibodies (bivalent and bispecific); and (x) chimeric (e.g., humanized) antibodies that can be produced by modifying whole antibodies or de novo-synthesized antibodies using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same way as intact antibodies.

[0027] In mammals, there are two types of immunoglobulin light chains called lambda (λ) and kappa (κ). Each antibody always contains the same two light chains; in mammals, only one type of light chain, either κ or λ, exists per antibody. The approximate length of a light chain is 211–217 amino acids, and each light chain has two domains, one constant domain, and one variable domain.

[0028] There are five types of mammalian Ig heavy chains, denoted as α, δ, ε, γ, and μ. The type of heavy chain present in an antibody defines the class or isotype of the antibody: IgM, IgG, IgA, IgD, and IgE, respectively. Heavy chains vary in physicochemical, structural, and immunological properties, but each heavy chain has two domains: a variable domain and a constant domain. The variable domain contains a single Ig domain (approximately 110 amino acids long) and determines the antibody binding specificity. The constant domain is identical in all antibodies of the same isotype but differs in antibodies of different isotypes. Heavy chains γ, α, and δ have a constant region consisting of three tandem Ig domains and a hinge region for adding flexibility; heavy chains μ and ε have a constant region consisting of four immunoglobulin domains (Woof & Burton, op. cit.). The term "immunoglobulin" (Ig) is used herein interchangeably with the term "antibody."

[0029] IgG is the most abundant antibody isotype in blood (plasma), accounting for 70-75% of human immunoglobulins. IgG detoxifies harmful substances and is important in the recognition of antigen-antibody complexes by leukocytes and macrophages. In humans, IgG is further classified into four subclasses: IgG1, IgG2, IgG3, and IgG4. IgM normally circulates in the blood and accounts for about 10% of human immunoglobulins. IgM has a pentameric structure in which five basic Y-type molecules are bound together. B cells initially produce IgM in response to microbial infection / antigen invasion. Although IgM has a lower affinity for antigens than IgG, it has high avidity to antigens due to its pentameric / hexameric structure. IgM also activates cellular signaling pathways by binding to cell surface receptors. IgA is abundant in serum, nasal mucosa, saliva, breast milk, and intestinal fluid, and accounts for 25% of human immunoglobulins. IgA forms dimers (i.e., two IgA monomers bound together). IgA in breast milk protects the neonatal gastrointestinal tract from pathogens. IgA is classified into two subclasses: IgA1 and IgA2. IgD accounts for less than 1% of human immunoglobulins and may be involved in inducing antibody production in B cells, but its exact function remains unknown. IgE is present in trace amounts, accounting for less than 0.001% of human immunoglobulins. Its primary role is protection from parasites. In areas where parasitic infections are rare, IgE is primarily involved in allergies.

[0030] Immune cell activity is modulated by antibody regions known as crystallizable fragment regions or “Fc regions.” An Fc region consists of two identical polypeptide chains (each referred to herein as an “Fc domain”), with IgG and IgA containing the heavy chain's CH2 and CH3 constant domains. IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in their respective polypeptide chains. Amino acid residues in the CH2 and CH3 domains can be numbered according to the EU numbering system (Edelman et al., (1969) PNAS.USA, 63, 78-85), the “Kabat” numbering system (Kabat et al., cited above), or alternatively, using the IMGT numbering system for the C domain. The IMGT tool is available on the World Wide Web (www.imgt.org).

[0031] The Fc region binds to cell surface receptors, "Fc receptors," and complement proteins, which mediate the physiological effects of antibodies. Fc receptors are found on many cells of the immune system, such as B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells. Binding of the antibody Fc region to Fc receptors stimulates phagocytosis or cytotoxic cells, destroying microorganisms or infected cells through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. Several different types of Fc receptors (FcRs) exist, and they are classified based on the type of antibody they recognize. For example, those that bind to IgG are called Fc-gamma receptors (FcγRs), those that bind to IgA are called Fc-alpha receptors (FcαRIs), and those that bind to IgE are called Fc-epsilon receptors (FcεRs). FcR classes are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling characteristics of their respective receptors (Owen J et al., (2009) Immunology (7th ed.). New York: WH Freeman and Company. p423). FcαRI is also known as CD89, and its primary antibody ligand is IgA. This receptor has a low affinity for IgA (Kd>10). -6 It possesses M) and is found on monocytes, macrophages, neutrophils, and eosinophils. The binding of IgA to FcαRI primarily results in the induction of phagocytosis and microbial killing.

[0032] In one embodiment, the antibody comprises a full-length antibody or a full-length immunoglobulin chain. In another embodiment, the antibody comprises a full-length antibody or an antigen-binding or functional fragment of a full-length immunoglobulin chain. The antibody preparation may be monoclonal or polyclonal. The antibody may be human, humanized, CDR-grafted, or in vitro-generated antibody.

[0033] In one embodiment, antibodies or immunoglobulins can be recombinantly produced, for example, by phage display or by combinatorial methods. Phage display and combinatorial methods for antibody production are known in the art (e.g., Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication No. 92 / 18619; Dower et al., International Publication No. 91 / 17271; Winter et al., International Publication No. 92 / 20791; Markland et al., International Publication No. 92 / 15679; Breitling et al., International Publication No. 93 / 01288; McCafferty et al., International Publication No. 92 / 01047; Garrard et al., International Publication No. 92 / 09690; Ladner et al., International Publication No. 90 / 02809; Fuchs et al., (1991) Bio / Technology, 9:1370-1372; Hay et al.,(1992)Hum Antibody Hybridomas,3:81-85;Huse et al.,(1989)Science 246:1275-1281;Griffths et al.,(1993)EMBO J.,12:725-734;Hawkins et al.,(1992)J Mol Biol.,226:889-896;Clackson et al.,(1991)Nature,352:624-628;Gram et al.,(1992)PNAS,89:3576-3580;Garrard et al.,(1991)Bio / Technology,9:1373-1377;Hoogenboom et al. al., (1991) Nuc Acid Res., 19:4133-4137; and Barbas et al. (Al., (1991) PNAS, 88:7978-7982; all of their contents are incorporated herein by reference).

[0034] In one embodiment, the antibody or immunoglobulin is a fully human antibody (e.g., an antibody produced in a genetically engineered mouse to produce antibodies from a human immunoglobulin sequence, or an antibody isolated from a human), or a non-human antibody, such as a rodent (mouse or rat), goat, primate (e.g., monkey), or camel antibody. Human monoclonal antibodies can be produced using transgenic mice carrying human immunoglobulin genes, rather than mouse strains. Splenocytes from these transgenic mice immunized with the target antigen are used to produce hybridomas that secrete human monoclonal antibodies with specific affinity for epitopes from human proteins (e.g., Wood et al., International Publication No. 91 / 00906; Kucherlapati et al., International Publication No. 91 / 10741; Lonberg et al., International Publication No. 92 / 03918; Kay et al., International Publication No. 92 / 03917; Lonberg et al., (1994) Nature 368:856-859; Green et al., (1994) Nature Genet. 7:13-21; Morrison et al., (1994) PNAS USA 81:6851-6855; Bruggeman et al., (1993) Year Immunol 7:33-40; Tuaillon et al., (1993) PNAS 90:3720-3724; Bruggeman et al., (1991) Eur J Immunol 21:1323-1326).

[0035] Antibodies or immunoglobulins may have a variable region, or a portion thereof, such as a CDR, that is generated in a non-human organism, such as a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the present invention. Antibodies generated in a non-human organism, such as a rat or mouse, and then modified, for example, in a variable framework or constant region to reduce antigenicity in humans, are also within the scope of the present invention. Chimeric antibodies can be produced by recombinant DNA technologies known in the art (Robinson et al., International Publication No. 87 / 002671; Akira et al., European Patent Application Publication No. 184187A1; Taniguchi, European Patent Application Publication No. 171496A1; Morrison et al., European Patent Application Publication No. 173494A1; Neuberger et al., International Publication No. 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application Publication No. 125023A1; Better et al., (1988) Science 240:1041-1043; Liu et al., (1987) PNAS 84:3439-3443; Liu et al.,(1987),J.Immunol.139:3521-3526;Sun et al.,(1987)PNAS 84:214-218;Nishimura et al.,(1987),Canc.Res.47:999-1005;Wood et al.,(1985)Nature 314:446-449; and Shaw et al., (1988), J. Natl Cancer Inst. 80:1553-1559).

[0036] Humanized or CDR-grafted antibodies have at least one or two recipient CDRs (heavy and / or light immunoglobulin chains) replaced with donor CDRs, but generally have all three recipient CDRs. The antibody can be replaced with at least a portion of non-human CDRs, or only a portion of the CDRs can be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for the binding of the humanized antibody to the target antigen. Preferably, the donor is a rodent antibody, e.g., a rat or mouse antibody, and the recipient is a human framework or human consensus framework. Typically, the immunoglobulin providing the CDRs is referred to as the “donor,” and the immunoglobulin providing the framework is referred to as the “acceptor.” In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or consensus framework, or a sequence that is approximately 85% or more, preferably 90%, 95%, or 99% or more identical thereto.

[0037] As used herein, the term “consensus sequence” refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany (1987))). In a family of proteins, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in that family. If two amino acids occur with equal frequency, both may be included in the consensus sequence. “Consensus framework” refers to the framework region in a consensus immunoglobulin sequence.

[0038] Antibodies can be humanized by methods known in the art (see, for example, Morrison, (1985), Science 229:1202-1207; Oi et al., (1986), BioTechniques 4:214, and Queen et al., U.S. Patent Nos. 5,585,089, 5,693,761 and 5,693,762, all of which are incorporated herein by reference). Humanized or CDR-grafted antibodies can be produced by CDR grafting or CDR substitution, which can replace one, two, or all CDRs in the immunoglobulin chain. For example, see U.S. Patent No. 5,225,539; Jones et al., (1986) Nature 321:552-525; Verhoeyan et al., (1988) Science 239:1534; Beidler et al., (1988) J.Immunol. 141:4053-4060 and Winter's U.S. Patent No. 5,225,539, all of which are expressly incorporated herein by reference. Humanized antibodies in which specific amino acids are substituted, deleted, or added are also within the scope of the present invention. Criteria for selecting amino acids from a donor are described in U.S. Patent No. 5,585,089, for example, in columns 12-16 of U.S. Patent No. 5,585,089, all of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in Padlan et al., European Patent Application Publication No. 519596A1.

[0039] Methods for modifying the constant region of an antibody are known in the art. Antibodies with modified function, such as altered affinity for effector ligands, such as FcR on cells, or the C1 component of complement, can be produced by replacing at least one amino acid residue in the constant region of the antibody with a different residue (see, for example, European Patent Application Publication No. 388151A1, U.S. Patent No. 5,624,821, and U.S. Patent No. 5,648,260).

[0040] As used herein, “position” refers to the position of an amino acid in a protein sequence. Positions may be numbered sequentially or according to established formats, such as the EU index or IMGT numbering (www.imgt.org), as in Kabat. For example, when using IMGT numbering, glutamine 94 (also referred to as Gln94 or Q94) also gives its position in the Fc region to indicate whether it is found in the CH2 or CH3 domain. For example, QCH2.94, SCH3.45 represent glutamine at position 94 in the CH2 domain and serine at position 45 in the CH3 domain of the human antibody IgA1.

[0041] As used herein, "residue" refers to a position in a protein and its associated amino acid identity. For example, glutamine 94 (also known as Gln94 or Q94) is a residue in the human antibody lgA1.

[0042] As used herein, "modification" or "mutation" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, the change resulting from a sequence alteration involving one or more amino acid residues / positions. Typical modifications include substitution of one or more residues (or at the said position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more amino acids adjacent to the said one or more residues / positions, deletion of the said one or more residues / positions, inversion of the said one or more residues / positions, and duplication of the said one or more residues / positions. An amino acid "substitution" or variation thereof refers to the substitution of one or more existing amino acid residues in a given (starting) amino acid sequence with one or more different amino acid residues. Generally and preferably, a modification results in a change in at least one physicobiochemical activity of the variant polypeptide compared to the polypeptide containing the starting or parent (or "wild-type") amino acid sequence. For example, in the case of an antibody or Fc isomer, the altered physicobiochemical activity may be binding affinity, binding ability, and / or binding effect to a target molecule.

[0043] As used herein, “mutant polypeptide,” “polypeptide variant,” or “variant” means a polypeptide sequence that differs from a parent polypeptide sequence by at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or a modified version of a WT polypeptide. A mutant polypeptide may refer to the polypeptide itself, a composition containing the polypeptide, or an amino sequence encoding the polypeptide. Preferably, the mutant polypeptide has at least one amino acid modification compared to the parent polypeptide, for example, about 1 to 10 amino acid modifications compared to the parent, preferably about 1 to about 5 amino acid modifications. The mutant polypeptide sequences described herein will have at least about 80% homology, preferably at least about 90% homology, and more preferably at least about 95% homology with the parent polypeptide sequence. In one embodiment, the mutant polypeptide sequence described herein will have at least about 85% homology, preferably at least about 90% homology, and more preferably at least about 95% homology with the parent IgA CH2 polypeptide sequence. In one embodiment, the mutant polypeptide sequence described herein will have at least about 90% homology, preferably at least about 95%, and more preferably at least about 97% homology, with the parent IgA CH3 polypeptide sequence. In a preferred embodiment, the mutant polypeptide sequence described herein will have at least about 85% homology, preferably at least about 90%, and more preferably at least about 95% homology, with the parent IgA CH2 polypeptide sequence, and at least about 90% homology, preferably at least about 95%, and more preferably at least about 97% homology, with the parent IgA CH3 polypeptide sequence. Thus, as used herein, “Fc mutant” or “mutant Fc” means an Fc sequence that differs from the parent Fc sequence by at least one amino acid modification. An Fc mutant may consist only of an Fc region or may exist in the context of an antibody, an Fc fusion, isolated Fc, an Fc fragment, or another polypeptide substantially encoded by Fc.An Fc variant may refer to the polypeptide itself, a composition containing an Fc variant polypeptide, or an amino acid sequence encoding an Fc variant. As used herein, “Fc polypeptide variant” or “variant Fc polypeptide” means an Fc polypeptide that differs from the parent Fc polypeptide by at least one amino acid modification. As used herein, the term “parent Fc polypeptide” means the starting Fc polypeptide from which the amino acid modification is performed. The parent Fc polypeptide may be a wild-type Fc polypeptide or an allele variation of a wild-type Fc polypeptide. The parent Fc polypeptide may also be an Fc polypeptide that has already undergone amino acid modification. As used herein, “protein variant” or “variant protein” means a protein that differs from the parent protein by at least one amino acid modification. As used herein, “antibody variant” or “variant antibody” means an antibody that differs from the parent antibody by at least one amino acid modification. As used herein, “IgA variant” or “variant IgA” means an antibody that differs from the parent IgA by at least one amino acid modification. The parent IgA may be isotype IgA1 or IgA2. As used herein, "immunoglobulin variant" or "mutant immunoglobulin" means an immunoglobulin sequence that differs from the parent immunoglobulin sequence by at least one amino acid modification.

[0044] In this specification, “wild-type” or “WT” means a naturally occurring amino acid or nucleotide sequence, including allelic variations. WT proteins, polypeptides, antibodies, immunoglobulins, IgA, etc., have an amino acid or nucleotide sequence that has not been intentionally modified.

[0045] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine (K), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), non-loading side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)), nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)).

[0046] The terms “identical percentage” or “identity percentage” in relation to two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are identical. Two sequences are “substantially identical” if, when compared over a comparison window or designated region using one of the following sequence comparison algorithms or by manual alignment and visual inspection and aligned for maximum correspondence, the two sequences have a specified percentage of the same amino acid residues or nucleotides (i.e., 60% identity compared to a designated region, or, if not designated, compared to the entire sequence; optionally, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity). Optionally, identity resides over a region of at least approximately 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 nucleotides or more (or 20, 50, or 200 amino acids or more). The “identity percentage” or “sequence identity percentage” in this disclosure can be calculated by (i) comparing two optimally aligned sequences (nucleotides or proteins) over a comparison window, (ii) determining the number of positions in both sequences where identical nucleic acid bases (for nucleotide sequences) or amino acid residues (for proteins) occur to obtain the number of match positions, (iii) dividing the number of match positions by the total number of positions in the comparison window, and then (iv) multiplying this quotient by 100% to obtain the identity percentage. If the “identity percentage” is calculated with respect to a reference sequence without a specific comparison window, the identity percentage is determined by dividing the number of match positions in the alignment region by the total length of the reference sequence. Therefore, for the purposes of this disclosure, if two sequences (query and subject) are optimally aligned (allowing gaps in their alignment), the “identity percentage” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window), and then multiplying that by 100%.

[0047] For sequence comparison, typically one sequence serves as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage for the test sequence relative to the reference sequence based on the program parameters.

[0048] As used herein, the term “comparison window” includes reference to one segment of the number of consecutive positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, in which the sequences can be compared to the same number of consecutive positions of the reference sequence after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol., 48:443, the similarity search method of Pearson & Lipman (1988) PNAS. USA, 85:2444, by computer execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[0049] Two examples of algorithms suitable for determining sequence identity percentage and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., (1977) Nuc. Acids Res, 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves initially identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the adjacent word score threshold (Altschul et al., (1990), op. cit.). These initial adjacent word hits serve as seeds to initiate a search for longer HSPs that contain them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matched residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Word hit extension in each direction stops if the cumulative alignment score falls by amount X from its maximum achieved value; if the cumulative score becomes 0 or less due to the accumulation of one or more negative-scoring residue alignments; or if the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, word length (W) 11, expected value (E) or 10, M=5, N=-4, and comparison of both strands.For amino acid sequences, the BLASTP program uses a word length of 3 and an expected value (E) of 10 as defaults, while the BLOSUM62 scoring matrix (see Henikoff & Henikoff, (1989) PNAS.USA, 89:10915) uses an alignment (B) of 50, an expected value (E) of 10, M=5, N=-4, and a comparison of both strands as defaults.

[0050] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) PNAS.USA, 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, if the smallest sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is considered similar to the reference sequence.

[0051] The percentage of identity between two amino acid sequences can also be determined using the algorithm by E. Meyers and W. Miller (Comput.Appl.Biosci.4:11-17(1988)) incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, gap length penalty 12, and gap length penalty 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the aforementioned algorithm by Needleman & Wunsch incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either the Blossom62 matrix or the PAM250 matrix, along with gap weights 16, 14, 12, 10, 8, 6, or 4, and length weights 1, 2, 3, 4, 5, or 6.

[0052] Aside from the sequence identity ratio described above, another indicator that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibody produced against the polypeptide encoded by the second nucleic acid, as described below. Therefore, the polypeptide is typically substantially identical to a second polypeptide where the two peptides differ only in conservative substitutions. Another indicator that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize with each other under stringent conditions, as described below. Yet another indicator that two nucleic acid sequences are substantially identical is that their sequences can be amplified using the same primers.

[0053] The term “nucleic acid” is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof, either in single-stranded or double-stranded form. This term encompasses known nucleotide analogs or nucleic acids containing modified skeletal residues or bonds, which are synthetic, spontaneously occurring, and unspontaneously occurring, and which have similar binding properties to the reference nucleic acid and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramides, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0054] Unless otherwise specified, a particular nucleic acid sequence implicitly includes not only the explicitly indicated sequence but also its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, as detailed below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., (1991) Nucleic Acid Res., 19:5081; Ohtsuka et al., (1985) J Biol Chem., 260:2605-2608; and Rossolini et al., (1994) Mol Cell Probes, 8:91-98). As used herein, the term “optimized nucleotide sequence” means a nucleotide sequence that has been modified to encode an amino acid sequence using preferred codons in a producing cell, in this case Chinese hamster ovary cell (CHO). The optimized nucleotide sequence is artificially engineered to retain the complete amino acid sequence initially encoded by the starting nucleotide sequence, also known as the “parent” sequence. In certain embodiments, the optimized sequence described herein is artificially engineered to have preferred codons in CHO mammalian cells.

[0055] As used herein, "C-terminus" refers to the carboxyl-terminal amino acid of a polypeptide chain having a free carboxyl group (-COOH). As used herein, "N-terminus" refers to the amino-terminal amino acid of a polypeptide chain having a free amine group (-NH2).

[0056] As used herein, the terms “operatably bound” or “functionally bound” refer to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, this refers to a functional relationship between a transcriptional regulatory sequence and a sequence being transcribed. For example, a promoter or enhancer sequence is operatably bound to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operatably bound to a sequence being transcribed are physically contiguous to the sequence being transcribed; i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequence to which they enhance transcription.

[0057] The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This also applies to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence implicitly includes its conservatively modified variants.

[0058] As used herein, “parent polypeptide,” “parent protein,” “precursor polypeptide,” or “precursor protein” means an unmodified polypeptide that is subsequently modified to produce a mutant. A parent polypeptide may be a naturally occurring polypeptide, or a mutant or modified version of a naturally occurring polypeptide. A parent polypeptide may refer to the polypeptide itself, a composition containing the parent polypeptide, or an amino acid sequence encoding the parent polypeptide. Therefore, as used herein, “parent Fc polypeptide” means an Fc polypeptide modified to produce a mutant, and as used herein, “parent antibody” means an antibody modified to produce a mutant antibody. In some embodiments, the “parent” is a wild-type protein.

[0059] As used herein, the term "in vivo half-life" refers to the half-life of the molecule of interest or its variant circulating in the blood of a given mammal.

[0060] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. In preferred embodiments, the subject is human. Unless otherwise noted, the terms "patient" and "subject" are used interchangeably herein.

[0061] When used herein, the terms “patients requiring treatment” or “subjects requiring treatment” include, for example, subjects who would benefit from the administration of the molecules or pharmaceutical compositions of this disclosure used in detection, diagnostic procedures and / or treatment, such as mammalian subjects.

[0062] As used herein, the terms “treatment” or “to treat” are defined herein as the application or administration of the Fc isomers according to this disclosure, or a pharmaceutical composition comprising such Fc isomers, to a subject having a particular disease (e.g., arthritis), symptoms associated with that disease, or predisposition to the disease (if applicable), and the purpose is to cure (if applicable), prevent (if applicable), delay the onset, reduce the severity, alleviate, improve, improve the disease, improve the disease, reduce or improve any associated symptoms or predisposition to the disease. The terms “treatment” or “to treat” include treating patients suspected of having a disease and patients with the disease or diagnosed with a disease or medical condition, and include suppression of clinical relapse. The phrase “to reduce prognosis” means delaying the onset, development or progression of a disease, infection or disorder.

[0063] The terms “therapeutic tolerance,” “therapeutic effective dose,” or “therapeutic effective dose” are interchangeable to refer to a sufficient amount to produce the desired outcome (i.e., reduction of disease activity, reduction of disease progression, reduction of disease signs and / or symptoms, etc.). In some embodiments, the therapeutic tolerance does not induce or cause any undesirable side effects. The therapeutic tolerance can be determined by administering a low dose first, and then gradually increasing the dose until the desired effect is achieved. The “preventive effective dose” and “therapeutic effective dose” of the molecules of this disclosure may prevent the onset of disease symptoms (where applicable) or reduce their severity, respectively.

[0064] As used herein, “selecting” and “being selected” with respect to a patient are used to mean that a particular patient is specifically selected from a larger patient group due to a particular patient meeting certain criteria. Similarly, “selectively treating a patient” refers to providing treatment to patients who are specifically selected from a larger patient group due to a particular patient meeting certain criteria. Similarly, “selectively administering” refers to administering a drug to patients who are specifically selected from a larger patient group due to a particular patient meeting certain criteria.

[0065] Unless otherwise specifically stated or made clear from the context, the term “about” in relation to a number, when used herein, is understood to mean within the normal tolerance range in the art, for example, within two standard deviations of the mean. Thus, “about” may be within + / - 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.05%, or 0.01% of the stated value, preferably within + / - 10% of the stated value. When the term “about” is used before a number or a list of numbers, it applies to each number in that series; for example, the phrase “about 1 to 5” should be interpreted as “about 1 to about 5,” or for example, the phrase “about 1, 2, 3, 4” should be interpreted as “about 1, about 2, about 3, about 4,” and so on.

[0066] The term "substantially" does not exclude "completely"; for example, a composition that "substantially does not" Y does not have to completely possess Y. Where necessary, the term "substantially" may be excluded from the definitions in this disclosure.

[0067] The term "co-administered" refers to the simultaneous presence of two activators in the blood of an individual. Activators (e.g., additional therapeutic agents) co-administered with the antibodies and antigen-binding fragments of this disclosure may be delivered simultaneously or sequentially.

[0068] Various aspects of this disclosure are described in further detail in the following sections and subsections.

[0069] Fc variant of the present invention In addition to their ability to bind to antigens, a key characteristic of antibodies is their ability to mobilize immune effector functions. Humoral immune response engagement is primarily governed by interaction with C1q and the initiation of the complement cascade (Meyer et al., (2014) MABS, 6(5):1133-44). Cellular immune responses largely arise from interactions between antibodies and Fc gamma receptors (FcγR). Intracellular signaling via activating receptors is modulated via phosphorylation of immune receptor tyrosine-based activation motifs (ITAMs), which lead to effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and inflammation through the induction of cytokine secretion.

[0070] While many antibody-based therapies have shown clinical and commercial success, these treatments are often effective only in small populations of patients. To date, all commercially available antibodies belong to the IgG class, primarily IgG1, which mobilizes immune effector function via FcγRIII (CD16). A typical response involves the activation of natural killer (NK) cells that display FcγRIII on their cell surface via the Fc portion of the IgG antibody, triggering ADCC. However, NK cells are the sole component of the innate immune system, and the activation of other forms of leukocytes could be used to enhance the IgG-triggered ADCC response. The IgA class of antibodies engages FcαRI, which is widely expressed on neutrophils. Neutrophils constitute the largest proportion of innate effector cells found in circulation, and their activation triggers both ADCC and ADCP. Furthermore, they have been shown to infiltrate solid tumors (Gregory & Houghton (2011), op. cit.). However, because IgG antibodies do not bind to FcαRI, most commercially available antibody-based therapies cannot activate neutrophils. Currently, IgA-based therapeutic antibodies have not been commercially developed compared to IgG antibodies, due to the recognized shortcomings of this class of antibodies. However, we have shown that by modifying the Fc region, it is possible to generate IgA antibodies with improved binding to FcαRI. This improved affinity, up to 1000-fold, has been shown to translate directly into increased potency in cell-based assays. Therefore, when used therapeutically, less antibody is required, and the frequency of antibody administration can be reduced, which is advantageous for the patient.

[0071] Generally, as outlined above, Fc variants include amino acid modifications in the CH2 and / or CH3 domains of the Fc region. An Fc variant comprises one or more amino acid modifications from the parent Fc polypeptide, where the amino acid modifications may provide one or more optimized properties, but in some cases, the variant exhibits substantially identical biological properties. Properties that can be optimized include, but are not limited to, enhanced or reduced affinity for FcαRIs. In one embodiment, the Fc variant of the present invention is modified to have enhanced affinity for human FcαRIs. In a preferred embodiment, the Fc region of the Fc variant is affinity-mature, and thereafter, amino acid modifications are made to the CH2 and / or CH3 domains to enhance the binding of the Fc region to its target FcαRI. Such types of modifications can improve binding and / or dissociation dynamics to the target antigen. This optimized property is expected to provide an Fc variant with enhanced therapeutic properties in humans, such as enhanced effector function and higher anticancer efficacy.

[0072] As used herein, “higher affinity,” “improved affinity,” “enhanced affinity,” or “better affinity” than the parent Fc polypeptide means that the Fc variant has a significantly higher equilibrium binding constant (K) than the parent Fc polypeptide when the amounts of the mutant and the parent polypeptide are essentially the same in the binding assay. A ) or a low equilibrium dissociation constant (K D This means that it binds to the Fc receptor. For example, an Fc variant with improved Fc receptor binding affinity may, as measured by surface plasmon resonance, show an affinity approximately 10 to 100 times, for example, at least 50 times, that of the parent Fc polypeptide. For example, an Fc variant of the parent Fc polypeptide may have an increased affinity for human FcαRI of at least 50, 100, 150, 200, 250, or 300 times that of the parent Fc polypeptide, as measured by surface plasmon resonance.

[0073] The Fc receptor selectivity or specificity of a given Fc variant will offer different properties depending on whether it constitutes an antibody, an Fc fusion, or an Fc variant having a bound fusion or conjugate partner.

[0074] The Fc variants of the present invention may include modifications that modulate interactions with Fc receptors other than FcαRI, including but not limited to FcγRs and / or FcRn.

[0075] The Fc variant of the present invention has a different amino acid sequence from its parent IgA Fc region due to at least one amino acid modification. Therefore, the Fc variant of the present invention has at least one amino acid modification compared to the parent. Alternatively, the Fc variant of the present invention may have more than one amino acid modification compared to the parent, for example, about 1 to 10 amino acid modifications compared to the parent, preferably 1 to 5 amino acid modifications, 1 to 4 amino acid modifications, 1 to 3 amino acid modifications, or 1 to 2 amino acid modifications. Therefore, the sequence of the Fc variant and the sequence of the parent Fc polypeptide are substantially homologous or identical. For example, the variant Fc variant sequence described herein will have about 80% homology (including identity) with the parent Fc variant sequence, preferably at least about 90% homology, and most preferably at least about 95, 96, 97, 98 and 99% identity.

[0076] In one embodiment, one or more amino acid insertions, deletions, or substitutions are made. All of these substitutions may be made to an IgA molecule, for example, IgA1 or IgA2, particularly IgA2. Preferably, in one embodiment, the amino acid substitutions may be made at positions within the Fc region of CH2.10, CH2.89, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, CH3.105, CH3.109, CH3.118 and / or CH3.124, and the numbering of the amino acid modifications follows the IMGT numbering for the C-domain. These amino acid substitutions (and any possible combination of substitutions, insertions, and deletions) include, but are not limited to, A_CH2.10_S, L_CH2.89_I, G_CH2.91_Q, G_CH2.91_V, Q_CH2.94_E, N_CH2.97_H, N_CH2.97_Y, G_CH2.99_W, S_CH3.45_D, M_CH3.105_Y, E_CH3.109_D, Q_CH3.118_Y, and / or L_CH3.124_F, and the numbering of amino acid modifications follows the IMGT numbering for the C-domain.In preferred embodiments, amino acid substitutions or combinations thereof include: Q_CH2.94_E, N_CH2.97Y, S_CH3.45_D, M_CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D, N_CH2.97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y, N _CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y / S_C H3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.9 4_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y / Q_CH3.118_ Y, A_CH2.10_S, L_CH2.89_I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_CH3.109_D, L_CH3.124_F, L_CH2.89_I / G_CH2.91_V / Q_CH2.94_E / N_CH2.97_Y / G_CH2.99_W may be included but are not limited to these, and the numbering of amino acid modifications follows the IMGT numbering for the C-domain.

[0077] In one embodiment, amino acid substitutions can be made at the Fc region positions of CH2.94, CH2.97, CH3.45, CH3.105, and CH3.118. In one embodiment, amino acid substitutions can be made at the Fc region positions of Glu at position CH2.94, Tyr at position CH2.97, Asp at position CH3.45, Tyr at position CH3.105, or Tyr at position CH3.118. In a preferred embodiment, amino acid substitutions can be made at the Fc region positions of Q_CH2.94_E, L_CH2.97_Y, S_CH3.45_D, M_CH3.105_Y, and Q_CH3.118_Y.

[0078] Functionally, variants that result in increased binding to FcαRI find specific applications in several embodiments. Fc variants may contain more than one protein chain. That is, Fc variants may find applications in antibodies or Fc fusions that are monomers or oligomers containing mono- or hetero-oligomers.

[0079] Fc fusions, antibody fusions, and antibody conjugates The Fc polypeptides and antibodies of the present invention may have diverse structures, including but not limited to antibody fragments, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimes”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and each of the respective fragments. The present invention includes mutant Fc polypeptides and antibodies (e.g., antibodies or antibody-like molecules) or fragments thereof, which are recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugates) to heterologous proteins or polypeptides (or fragments thereof, preferably polypeptides of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to produce fusion proteins. Methods for fusion or conjugation of proteins, polypeptides, or peptides to antibodies or antibody fragments are known in the art. For example, see U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. 307434 and 367166; International Publication Nos. 1996 / 04388 and 1991 / 06570; Ashkenazi et al., (1991) PNAS.USA 88:10535-10539; Zheng et al., (1995) J.Immunol. 154:5590-5600; and Vil et al., (1992) PNAS.USA 89:11337-11341.

[0080] Additional fusion proteins can be generated through techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to modify the activity of the molecules or fragments thereof of this disclosure (e.g., molecules or fragments thereof with higher affinity and lower dissociation rates). See, in general, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patent et al., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama (1998) Trends Biotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol. 287:265-76; and Lorenzo & Blasco (1998) Biotechniques, 24(2):308-313 (each of these patents and publications is incorporated herein by reference as a whole). The molecules or fragments thereof described herein can be modified before recombination by subjecting them to error-prone PCR, random nucleotide insertion, or random mutagenesis by other means. The polynucleotides encoding the fragments of these molecules can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0081] Furthermore, the variant Fc polypeptides and antibodies of this disclosure can be fused to marker sequences, such as peptides, to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide (SEQ ID NO: 81), for example, a tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), many of which are commercially available. For example, as described in Gentz ​​et al., (1989) PNAS.USA 86:821-824, hexa-histidine (SEQ ID NO: 81) provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, hemagglutinin ("HA") tags (Wilson et al., (1984) Cell 37:767) corresponding to epitopes derived from influenza hemagglutinin protein, and "flag" tags.

[0082] In other embodiments, the variant Fc polypeptides and antibodies of this disclosure are conjugated into diagnostic or detection agents. Such molecules may be useful for monitoring the onset, development, progression and / or severity of a disease or disorder as part of a clinical trial procedure, or for prognosis, e.g., for determining the effectiveness of a particular treatment. Such diagnostics and detections may involve substances capable of detecting the molecule, such as, but not limited to, various enzymes, e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, e.g., streptavidin / biotin and avidin / biotin; and fluorescent materials, e.g., umbelliferone, fluorescein, fluorescein isothiocyanate (fluorescein). Isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, e.g., luminol (not limited to these); bioluminescent materials, e.g., luciferase, luciferin, and aequorin (not limited to these); radioactive materials, e.g., iodine (131I, 125I, 123I, and 121I), carbon (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), galium This can be achieved by coupling positron-emitting metals, including 68Ga, 67Ga, palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin, as well as various positron-emitting metals used in positron emission tomography, and non-radioactive paramagnetic metal ions.

[0083] This application further encompasses the use of variant Fc polypeptides and antibodies of the present disclosure conjugated to the therapeutic portion. For example, the therapeutic portion may be a cytotoxin, e.g., a cell proliferation inhibitor or cytotoxic agent, a therapeutic agent, or a radioactive metal ion, e.g., an alpha emitter. Examples of cytotoxins or cytotoxic agents include any agent that is harmful to cells.

[0084] Furthermore, mutant Fc polypeptides and antibodies can be conjugated to a therapeutic or drug moiety that modifies a given biological response. For example, the drug moiety may be a protein, peptide, or polypeptide having the desired biological activity. Examples of such proteins include toxins, such as abrin, lysine A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins, such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agents, anti-angiogenic agents; or biological response modifiers, such as lymphokines.

[0085] For further considerations on cytotoxins, linker types, and methods for conjugating mutant Fc polypeptides and antibodies, see also Saito et al., (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al., (2003) Cancer Immunol. Immunother. 52:328-337; Payne (2003) Cancer Cell 3:207-212; Allen (2002) Nat. Rev. Cancer, 2:750-763; Pastan & Kreitman (2002) Curr. Opin. Investig. Drugs, 3:1089-1091; Senter & Springer (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0086] The variant Fc polypeptides and antibodies of this disclosure can also be conjugated with radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to artificially engineered immunoglobulins for diagnostic or therapeutic use include, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are well-established in the art. See, for example, Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med. Biol. 26(8):943-50, which are incorporated as a whole by reference, respectively.

[0087] Techniques for conjugating the therapeutic portion of a mutant Fc polypeptide with an antibody, such as an antibody or antibody-like molecule, are publicly known, for example, Arnon et al., “Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy”, Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review”, Monoclonal Antibodies 84: Biological and Clinical Applications, Pinchera et al. al. (eds.), pp. 475-506 (1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy”, Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al. al., (1982) Immunol. Rev. 62:119-58.

[0088] Mutant Fc polypeptides and antibodies can also be attached to solid carriers, which are particularly useful for immunoassays or the purification of target antigens. Examples of such solid carriers, but not limited to, include glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0089] The Fc variants of the present invention may comprise one or more modifications that provide reduced or enhanced intracellular translocation of the Fc variant. In one embodiment, the Fc variants of the present invention may be used or combined with additional modifications to reduce intracellular translocation of the Fc variant, which occurs via interaction with one or more Fc ligands. This property may be expected to enhance effector function and potentially reduce the immunogenicity of the Fc variants of the present invention. Alternatively, the Fc variants of the present invention may be used directly or combined with additional modifications to enhance intracellular translocation of the Fc variant, which occurs via interaction with one or more Fc ligands.

[0090] In preferred embodiments, modifications are made to improve the biophysical properties of the Fc variants of the present invention, including but not limited to stability, solubility, and oligomeric state. Modifications may include, for example, substitutions that provide more favorable intramolecular interactions in the Fc variant to provide higher stability, or substitution of exposed nonpolar amino acids with polar amino acids for higher stability. To further optimize the Fc variants of the present invention, several optimization targets and methods available for additional modification operations are described in U.S. Patent No. 10 / 379,392, incorporated herein by reference. The Fc variants of the present invention may also be combined with additional modifications that reduce oligomeric state or size to enhance tumor penetration or to increase in vivo clearance rates as desired. Other modifications to the Fc variants of the present invention include those that enable specific formation or homodimer or homomultimer molecules. Such modifications include, but are not limited to, manipulated disulfides and chemical modifications or aggregation methods that can provide mechanisms for generating covalent homodimers or homomultimers. For example, methods and compositions for manipulating such molecules are described in Kan et al., (2001) J. lmmunol., 166:1320-1326; Stevenson et al., (2002) Recent Results Cancer Res. 159:104-12; U.S. Patent No. 5,681,566; Caron et al., (1992), J. Exp. Med. 176:1191-1195 and Shapes (1992) J. lmmunol. 148(9):2918-22 (all incorporated herein by reference). Additional modifications to the variants of the present invention include those that enable the formation of specific molecules or homodimers, homopolymers, bifunctional, and / or polyfunctional molecules. Such modifications include, but are not limited to, one or more amino acid substitutions in the CH3 domain, which reduce homodimer formation and increase heterodimer formation.For example, methods and compositions for manipulating such molecules are described in Atwell et al., 1997, J.Mol.Bioi.270(1):26-35 and Carter et al., 2001, J.lmmunol.Methods 248:7-15 (both incorporated herein by reference). Additional modifications include modifications in the hinge and CH3 domain, which reduce the tendency to form dimers.

[0091] Preparation of Fc mutant polypeptides Antibodies and fragments thereof containing the mutant Fc polypeptide disclosed herein can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as standard somatic cell hybridization as described in Kohler & Milstein, (1975) Nature 256:495.

[0092] The animal system used for preparing hybridomas is the mouse system. Hybridoma production in mice is a known procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0093] Chimeric or humanized antibodies can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the target mouse hybridoma and artificially manipulated using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences. For example, to produce a chimeric antibody, the mouse variable region can be conjugated to the human constant region using methods known in the art (see, e.g., U.S. Patent No. 4,816,567, Cabilly et al.). To produce a humanized antibody, the mouse CDR region can be inserted into the human framework using methods known in the art. See, e.g., U.S. Patent No. 5,225,539, Winter, and U.S. Patents No. 5,530,101; U.S. Patent No. 5,585,089; U.S. Patents No. 5,693,762, and U.S. Patent No. 6,180,370, Queen et al.

[0094] In one embodiment, the antibody of this disclosure or a fragment thereof containing an Fc isomer described herein is a human monoclonal antibody. Such human monoclonal antibodies can be produced using transgenic or transchromosomal mice that carry a portion of the human immune system rather than a mouse lineage. Examples of these transgenic and transchromosomal mice are referred to herein as HUMAB mice and KM mice, respectively, and collectively referred to herein as "human Ig mice."

[0095] HUMAB mice (Medarex, Inc.) contain human immunoglobulin gene miniloci encoding unreorganized human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg, et al., (1994) Nature 368(6474):856-859). Therefore, mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGκ monoclonals (as outlined in Lonberg et al., (1994) op. cit.; Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg & Huszar, (1995) Intern. Rev. Immunol. 13:65-93; and Harding & Lonberg, (1995) Ann. NYAcad. Sci. 764:536-546). Preparation and use of HUMAB mice, and the genomic modifications carried by such mice, are described in Taylor et al., (1992) Nucleic Acids Research 20:6287-6295; Chen et al., (1993) International Immunology 5:647-656; Tuaillon et al., (1993) PNAS USA 94:3720-3724; Choi et al., (1993) Nature Genetics 4:117-123; Chen et al., (1993) EMBO J.12:821-830; Tuaillon et al., (1994) J.Immunol.152:2912-2920; Taylor et al., (1994) Int.Immun., 579-591; and Fishwild et al. Further details are provided in al., (1996) Nature Biotec., 14:845-851, all of which are incorporated herein by reference in their entirety.Furthermore, U.S. Patent No. 5,545,806; U.S. Patent No. 5,569,825; U.S. Patent No. 5,625,126; U.S. Patent No. 5,633,425; U.S. Patent No. 5,789,650; U.S. Patent No. 5,877,397; U.S. Patent No. 5,661,016; U.S. Patent No. 5,814,318; U.S. Patent No. 5,874,299; and U.S. Patent No. 5,770,429; all by Lonberg & Kay; U.S. Patent No. 5,545,807, Surani et al. See also International Publication Nos. 92 / 103918, 93 / 12227, 94 / 25585, 97113852, 98 / 24884, and 99 / 45962, all by Lonberg & Kay; and International Publication No. 01 / 14424, Korman et al.

[0096] In another embodiment, human antibodies can be generated using mice carrying a transgene and a human immunoglobulin sequence on a transchromosome, for example, mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice are referred to herein as "KM mice" and are described in detail in International Publication No. 2002 / 43478 (Ishida et al).

[0097] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate human antibodies. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963 (Kucherlapati et al).

[0098] Furthermore, alternative transchromosomal animal lines expressing human immunoglobulin genes are available in the art and can be used to produce human antibodies. For example, mice carrying both human heavy chain and human light chain transchromosomes, referred to as "TC mice," can be used; such mice are described in Tomizuka et al., (2000) PNAS USA 97:722-727. In addition, cattle carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al., (2002) Nature Biotechnology 20:889-894) and can be used to produce human antibodies useful in this application.

[0099] Human monoclonal antibodies or fragments thereof can also be prepared using phage display methods for screening libraries of human immunoglobulin genes. Such phage display methods for isolating human antibodies are established in the art or are described in the following examples. For example, see U.S. Patent No. 5,223,409; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571,698 (Ladner et al); U.S. Patent No. 5,427,908 and U.S. Patent No. 5,580,717 (Dower et al); U.S. Patent No. 5,969,108 and U.S. Patent No. 6,172,197 (McCafferty et al); and U.S. Patent No. 5,885,793; U.S. Patent No. 6,521,404; U.S. Patent No. 6,544,731; U.S. Patent No. 6,555,313; U.S. Patent No. 6,582,915 and U.S. Patent No. 6,593,081 (Griffiths et al).

[0100] Human monoclonal antibodies or fragments thereof that are useful in this disclosure can also be prepared using SCID mice in which human immune cells have been reconstituted to produce a human antibody response upon immunization. Such mice are described, for example, in U.S. Patent No. 5,476,996 and U.S. Patent No. 5,698,767 (Wilson et al).

[0101] Human monoclonal antibodies or fragments thereof prepared according to the present invention as described below may be further modified to improve one or more binding properties (e.g., affinity) of the antibody or fragment thereof against a target receptor by introducing mutations in amino acid residues within the VH, VL, CH1, CL, CH2, and CH3 domains (a process known as "affinity maturation"). Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on receptor binding or other functional properties of interest may be evaluated by in vitro or in vivo assays as described herein and provided in the examples. Thus, in one embodiment, this disclosure relates to affinity-matured antibodies or fragments thereof, particularly to affinity-matured Fc regions. Mutations may be amino acid substitutions, additions, or deletions. For example, the Fc variants of this disclosure are affinity-matured Fc regions in which one, two, three, four, or five or more residues within the CH2 and / or CH3 domains are modified. All of these substitutions may occur in an IgA molecule, e.g., IgA1 or IgA2, particularly in IgA2. In preferred embodiments, amino acid substitutions may be made at the positions of CH2.10, CH2.89, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, CH3.105, CH3.109, CH3.118 and / or CH3.124 in the Fc region, and the numbering of the amino acid modifications follows the IMGT numbering for the C-domain. These amino acid substitutions (and any possible combination of substitutions, insertions, and deletions) include, but are not limited to, A_CH2.10_S, L_CH2.89_I, G_CH2.91_Q, G_CH2.91_V, Q_CH2.94_E, N_CH2.97_H, N_CH2.97_Y, G_CH2.99_W, S_CH3.45_D, M_CH3.105_Y, E_CH3.109_D, Q_CH3.118_Y, and / or L_CH3.124_F, and the numbering of amino acid modifications follows the IMGT numbering for the C-domain.In preferred embodiments, amino acid substitutions or combinations thereof in affinity-mature Fc mutants of the present invention include: Q_CH2.94_E, N_CH2.97Y, S_CH3.45_D, M_CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_ D, N_CH2.97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / M_ CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3. 118_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q _CH2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y / Q_CH3. 118_Y, A_CH2.10_S, L_CH2.89_I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_CH3.109_D, L_CH3.124_F, L_CH2.89_I / G_CH2.91_V / Q_CH2.94_E / N_CH2.97_Y / G_CH2.99_W may be included but are not limited to these, and the numbering of amino acid modifications follows the IMGT numbering for the C-domain.

[0102] Nucleic acids and expression systems The present invention also encompasses nucleic acids encoding polypeptide chains of Fc variants described herein. Nucleic acid molecules of the present disclosure include both single-stranded and double-stranded DNA and RNA, as well as their corresponding complementary sequences. Nucleic acid molecules of the present disclosure include full-length genes or cDNA molecules and combinations thereof. Nucleic acids of the present disclosure are derived from human resources, but may also be derived from non-human species.

[0103] "Isolated nucleic acid" refers to nucleic acid isolated from adjacent gene sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acid isolated from naturally occurring resources. For example, in the case of nucleic acid synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, it is understood that the nucleic acid resulting from such processes is isolated nucleic acid. Isolated nucleic acid molecules refer to nucleic acid molecules in the form of individual fragments or as components of a larger nucleic acid construct. In one preferred embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is preferably derived from DNA or RNA that has been isolated at least once in a substantially pure form and in an amount or concentration that allows for the identification, manipulation, and recovery of its constituent nucleotide sequences by standard biochemical methods (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and / or constructed in the form of internal untranslated sequences typically found in eukaryotic genes, or in open reading frames uninterrupted by introns. The untranslated DNA sequence may be located at 5' or 3' from the open reading frame, in which case it does not interfere with the manipulation or expression of the coding region.

[0104] Variant sequences, such as libraries of variant sequences, can be prepared as outlined herein by site-directed mutagenesis of nucleotides in polypeptide-encoding DNA using cassettes or PCR mutagenesis or other techniques known in the art, and subsequently by expressing the recombinant DNA in cell culture.

[0105] An "optimized nucleotide sequence" means that the nucleotide sequence has been modified to encode an amino acid sequence using preferred codons in a producing cell, such as a Chinese hamster ovary cell (CHO). The optimized nucleotide sequence is artificially engineered to completely retain the amino acid sequence initially encoded by the starting nucleotide sequence, also known as the "parent" sequence.

[0106] The disclosure also provides expression systems and constructs in the form of plasmids, expression vectors, transcriptions, or expression cassettes comprising at least one of the above polynucleotides. Furthermore, the disclosure provides host cells comprising such expression systems or constructs.

[0107] In one embodiment, the present invention provides a method for preparing an antibody or fragment thereof comprising a mutant Fc region as described herein, the method comprising: (a) culturing host cells comprising nucleic acids encoding mutant heavy chain and light chain polypeptides, wherein the cultured host cells express the mutant polypeptides; and (b) recovering the antibody or fragment thereof from the host cell culture.

[0108] The expression vectors used in this disclosure can be constructed from a starting vector, for example, a commercially available vector. After constructing the vector and inserting a nucleic acid molecule encoding the polypeptide chain of the artificially engineered immunoglobulin into the appropriate site in the vector, the completed vector can be inserted into a host cell suitable for amplification and / or polypeptide expression. Transformation of the expression vector into selected host cells can be achieved by known methods, e.g., translocation, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated translocation, or other known techniques. The method selected depends in part on the type of host cell to be used. These methods and other suitable methods are known to those skilled in the art and are described, for example, Sambrook et al., 2001, cited above.

[0109] Typically, expression vectors used in host cells contain sequences for plasmid maintenance and for the cloning and expression of exogenous nucleotide sequences. Such sequences are collectively referred to as “flanking sequences” and in one embodiment, typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding the polypeptide to be expressed, and a selection marker element.

[0110] Host cells, when cultured under appropriate conditions, can be used to express Fc mutants, which can then be recovered from the culture medium (if the host cells secrete them into the medium) or directly from the host cells producing them (if they do not secrete them). The selection of appropriate host cells depends on various factors, such as the desired expression level, polypeptide modifications desirable or required for activity (e.g., glycosylation or phosphorylation), and the ease of folding into biologically active molecules. Host cells may be eukaryotes or prokaryotes.

[0111] Mammalian cell lines available as expression hosts are known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC) and any cell lines used in expression systems known in the art, which can be used to produce polypeptides containing the artificially engineered immunoglobulins of this disclosure. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the desired artificially engineered immunoglobulin. Host cells that can be used include prokaryotes, yeasts, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli (E. coli) or bacteria. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Suitable mammalian host cell lines include COS-7 cells, L cells, Cl27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, or their derivatives and related cell lines grown in serum-free medium, HeLa cells, BHK cell lines, CVIIEBNA cell lines, human embryonic kidney cells, e.g., 293, 293EBNA, or MSR293, human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, primary explants, HL-60, U937, HaK, or Jurkat cells. Optionally, if polypeptide use is desired in various signal transduction or reporter assays, mammalian cell lines, e.g., HepG2 / 3B, KB, NIH3T3, or S49, can be used for polypeptide expression. Alternatively, polypeptides can be produced in lower eukaryotes, e.g., yeast, or in prokaryotes, e.g., bacteria. Suitable yeasts include budding yeast (S. cerevisiae), fission yeast (S. pombe), Kluyveromyces strains, Candida strains, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Salmonella typhimurium (S. typhimurium), or any bacterial strain capable of expressing heterologous polypeptides.When artificially engineered immunoglobulins are produced in yeast or bacteria, it is sometimes desirable to modify the resulting product, for example, by phosphorylation or glycosylation at the appropriate site, to obtain a functional product. Such covalent bonding can be achieved using known chemical or enzymatic methods.

[0112] In further embodiments, the Fc variants of the present invention include modifications that remove protease sites. These may include, for example, protease sites that reduce product yield and protease sites that degrade the administered protein in vivo.

[0113] In preferred embodiments, Fc variants are purified or isolated after expression. Proteins can be isolated or purified by a variety of methods known in the art. Standard purification methods include ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and chromatographic techniques, including reversed phase, performed at atmospheric pressure or under high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoresis, immunological, precipitation, dialysis, and isoelectric focusing techniques. Ultrafiltration and dialysis techniques, combined with protein concentration, are also useful. As is known in the art, a variety of native proteins bind to Fc and antibodies, and these proteins can be utilized in the present invention for the purification of Fc variants. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies, as well as the target antigens of the antibodies. Purification can often be made possible by specific fusion partners. For example, Fc variants can bind to glutathione resin when a GST fusion is used, or to Ni when a His tag is used. +2 When affinity chromatography or flag tagging is used, purification may be performed using immobilized anti-flag antibodies. For general guidance on appropriate purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994 (incorporated herein by reference in its entirety).

[0114] Pharmaceutical composition and administration Pharmaceutical compositions comprising the Fc variants of this disclosure are provided herein. The Fc variants may be incorporated in antibody form, for example, as monospecific, bispecific, or multispecific antibodies, in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0115] To prepare a pharmaceutical or sterile composition containing the Fc variant of this disclosure, the molecule is mixed with a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable" means that it is approved by a federal or state regulatory agency, or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, more specifically, in humans. The term "pharmaceutical composition" means a mixture of at least one active ingredient (e.g., the Fc variant of this disclosure) and at least one pharmaceutically acceptable excipient, diluent, or carrier. "Pharmaceutical" means a substance used in a medical procedure.

[0116] Pharmaceutical compositions for therapeutic and diagnostic agents can be prepared, for example, by mixing them with physiologically acceptable carriers, excipients, or stabilizers in the form of lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Oral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; see Weiner & Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0117] The selection of a drug regimen for a therapeutic agent depends on several factors, such as the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix. In some embodiments, the drug regimen maximizes the amount of therapeutic agent delivered to the patient while aligning with an acceptable level of side effects. Therefore, the amount of biopharmaceutical delivered depends in part on the specific entity and the severity of the disease being treated. Guidelines for selecting appropriate doses of antibodies, cytokines, and small molecules are available (e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) New Engl.J.Med.344:783-792;Beniaminovitz,et al.(2000)New Engl.J.Med.342:613-619;Ghosh,et al.(2003)New Engl.J.Med.348:24-32;Lipsky,et al.(2000)New See Engl.J.Med.343:1594-1602).

[0118] The determination of the appropriate dose is made by a clinician using, for example, parameters or factors known, suspected, or predicted to affect the treatment in the art. Generally, the dose is started at a somewhat lower amount than the optimal dose and then gradually increased until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic measures include, for example, the symptoms of inflammation or the level of inflammatory cytokines produced.

[0119] The actual dose levels of the active ingredients in the pharmaceutical compositions of this disclosure can be varied to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient. The selective dose level depends on various pharmacokinetic factors, such as the activity of the particular composition of this disclosure or its ester, salt, or amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors known in the medical field.

[0120] Pharmaceutical compositions comprising the Fc variants of this disclosure may be provided by continuous infusion or by doses at intervals of, for example, one day, one week, or one to seven times per week. Doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation.

[0121] The preferred dose of the therapeutic agent containing the Fc variant of this disclosure is approximately the same as that of the antibody or polypeptide on a mole / kg body weight basis. The dose administered to the subject may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more.

[0122] For therapeutic agents containing the Fc variants of this disclosure, the dose administered to a patient may range from approximately 0.0001 mg / kg to approximately 100 mg / kg patient body weight.

[0123] When administering a series of doses, they can be given, for example, almost daily, almost weekly, or almost monthly. The doses may continue until, for example, disease progression, adverse events occur, or other times determined by the physician.

[0124] The effective dose for a particular patient may vary depending on factors such as the condition being treated, the patient's overall health, the method, route and dose of administration, and the severity of side effects (see, for example, Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0125] If necessary, therapeutic agents comprising the Fc variants of this disclosure may be incorporated into a composition comprising a solubilizer and a local anesthetic for reducing pain at the injection site, such as lidocaine. Furthermore, transpulmonary administration may be used, for example, by the use of a formulation having an inhaler or nebulizer and an aerosolizing agent. For example, see U.S. Patent No. 6,019,968, U.S. Patent No. 5,985,320, U.S. Patent No. 5,985,309, U.S. Patent No. 5,934,272, U.S. Patent No. 5,874,064, U.S. Patent No. 5,855,913, U.S. Patent No. 5,290,540, and U.S. Patent No. 4,880,078; and see International Publication No. 92 / 19244, International Publication No. 97 / 32572, International Publication No. 97 / 44013, International Publication No. 98 / 31346, and International Publication No. 99 / 66903, each of which is incorporated herein by reference as a whole.

[0126] Therapeutic agents comprising the Fc variants of this disclosure may also be administered via one or more routes of administration using one or more of the various methods known in the art. As will be recognized by those skilled in the art, the route and method of administration will vary depending on the desired outcome. Selective routes of administration for antibodies include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. Parenteral administration may typically refer to methods of administration other than intestinal and topical administration by injection, and includes, but are not limited to, intravenous, intramuscular, intra-arterial, intra-shearing, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Alternatively, the compositions of this disclosure may be administered via parenteral routes, such as topical, epidermal, or mucosal administration routes, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0127] Therapeutic agents containing the Fc variants of this disclosure can be administered via any of the above routes, for example, using injection devices, injection pens, vials and syringes, pre-filled syringes, auto-injectors, infusion pumps, patch pumps, infusion bags and needles. When therapies containing the Fc variants of this disclosure are administered in a controlled-release or sustained-release system, controlled-release or sustained-release can be achieved using a pump (see Langer, op. cit.; Sefton, 1987, CRC Crit.Ref Biomed.Eng.14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl.J.Med.321:574). The controlled release or sustained release of the therapeutic agents of this disclosure can be achieved using polymer materials (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen & Ball (eds.), Wiley, New York (1984); Ranger & Peppas (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., (1985) Science 228:190; During et al., (1989) Ann. Neurol. 25:351; Howard et al. See also al., (1989) J. Neurosurg., 7(1):105; U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; International Publication No. 99 / 15154; and International Publication No. 99 / 20253.Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of leaching impurities, storage stable, sterile, and biodegradable. Controlled-release or sustained-release systems can be positioned close to the prophylactic or therapeutic target, and therefore only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, op. cit., vol. 2, pp. 115-138 (1984)).

[0128] A controlled-release system is discussed in an overview by Langer (Science (1990) 249:1527-1533). A sustained-release formulation containing one or more Fc variants of this application can be produced using any technique known to those skilled in the art. For example, see U.S. Patent No. 4,526,938, International Publication No. 91 / 05548, International Publication No. 96 / 20698, Ning et al., (1996) Radiotherapy & Oncology 39:179-189; Song et al., (1995) PDA Journal of Pharm Sci & Tech., 50:372-397; Cleek et al., (1997) Pro.Int'l.Symp.Control.Rel.Bioact.Mater. 24:853-854; Lam et al., (1997) Proc.Int'l.Symp.Control Rel.Bioact.Mater., 24:759-760, each of which is incorporated herein by reference as a whole.

[0129] When a pharmaceutical composition containing the Fc variant of this disclosure is administered topically, it may be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, liquid, emulsion, or other form known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-spray topical dosage forms, they contain a carrier or one or more excipients suitable for topical application, and in some examples, a viscous to semi-solid or solid form with a dynamic viscosity greater than that of water is typically used. Suitable formulations, but are not limited to, include liquids, suspensions, emulsions, creams, ointments, powders, liniments, and ointments, which may be sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties, such as osmotic pressure. Other suitable topical dosage forms include spray aerosol formulations in which the active ingredient is packaged in a mixture of a pressurized volatile substance (e.g., a gas propellant, e.g., Freon) in combination with a solid or liquid inert carrier, or in a squeeze bottle. Moisturizers or humidifiers may be added to the pharmaceutical composition and dosage form if desired. Examples of such additional components are known in the art.

[0130] When a pharmaceutical composition containing the Fc variant of this disclosure is administered intranasally, it may be formulated in aerosol form, spray, mist, or droplet form. In particular, prophylactic or therapeutic agents for use according to this disclosure can be conveniently delivered in the form of an aerosol spray offering from a pressurized pack or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dose unit can be determined by providing a valve for delivering a measured amount. Capsules and cartridges (e.g., composed of gelatin) for use in inhalers or sprayers may be formulated containing the compound and a suitable powder base, e.g., a powder mixture of lactose or starch.

[0131] The pharmaceutical compositions comprising the Fc variants of this disclosure may also be administered to patients on a regular basis.

[0132] In some embodiments, pharmaceutical compositions comprising the Fc variants of the present disclosure may be formulated to ensure appropriate in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present disclosure cross the BBB, they may be formulated (optionally) in liposomes, for example. For methods of producing liposomes, see, for example, U.S. Patent No. 4,522,811; U.S. Patent No. 5,374,548; and U.S. Patent No. 5,399,331. Liposomes may contain one or more moieties that are selectively transported into specific cells or organs and thus improve targeted drug delivery (see, for example, Ranade (1989) J. Clin. Pharmacol. 29:685). Examples of targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016, Low et al.); mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (Bloeman et al., (1995) FEBS Lett., 357:140; Owais et al. (1995) Antimicrob. Agents Chemother., 39:180); surfactant protein A receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); and p120 (Schreier et al (1994) J. Biol. Chem. 269:9090; Keinaenen & Laukkanen (1994) FEBS See also Lett., 346:123-6; Killion & Fidler (1994) Immunomethods, 4:273.

[0133] This application also provides protocols for the co-administration or treatment of patients using pharmaceutical compositions comprising the Fc variant of the Disclosure in combination with other therapeutic agents or therapeutic agents. Methods of co-administration or treatment using additional therapeutic agents, such as cytokines, steroids, chemotherapeutic agents, antibiotics, or radiation, are known in the art (see, for example, Hardman et al., (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th sup.th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective dose of the treatment may reduce symptoms by at least 10%, at least 20%, at least about 30%, at least 40%, or at least 50%.

[0134] In some embodiments, the pharmaceutical compositions of this disclosure further comprise one or more additional therapeutic agents.

[0135] In addition to the treatment regimens described above, patients may be subjected to surgery and other forms of physical therapy.

[0136] therapeutic use Therapeutic or pharmaceutical compositions comprising the Fc variants of this disclosure are useful for treating, preventing, or improving disorders or conditions involving abnormal cell proliferation, which are referred to herein as “proliferative disorders or conditions.” In one embodiment, this disclosure provides a method for treating a proliferative disorder or condition. In one embodiment, the subject of treatment is a human.

[0137] Examples of cell proliferation disorders or pathological conditions that can be treated, prevented, or improved using therapeutic or pharmaceutical compositions comprising the Fc variants of this disclosure include, but are not limited to, cancer. As used herein, the term "cancer" means all types of malignant growth or carcinogenic processes, metastatic tissues, or malignant transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion.

[0138] In specific embodiments, administration of a therapeutic or pharmaceutical composition comprising the Fc variant of the present disclosure to a subject by the method described herein achieves one, two, or three or more results: (1) reduction of tumor or neoplasm growth; (2) reduction of tumor formation; (3) eradication, removal, or control of primary, local, and / or metastatic cancer; (4) reduction of metastatic spread; (5) reduction of mortality; (6) increase of survival rate; (7) increase of survival time; (8) increase of patients in remission; (9) decrease of hospitalization rate; (10) decrease of hospitalization duration; and (11) maintenance such that tumor size does not increase by more than 10%, or more than 8%, or more than 6%, or more than 4%; preferably, tumor size does not increase by more than 2%.

[0139] In specific embodiments, administration of a therapeutic or pharmaceutical composition comprising the Fc variant of the present disclosure to a subject with cancer (in some embodiments, an animal model of cancer) by the method described herein inhibits or reduces tumor growth by at least about 2 times, preferably at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 7 times, or at least about 10 times compared to tumor growth in a subject with cancer (in some embodiments, an animal model of the same cancer) administered with a negative control, as measured using assays known in the art. In another embodiment, administration of a therapeutic or pharmaceutical composition comprising the Fc variant of the present disclosure to a subject with cancer (in some embodiments, an animal model of cancer) by the method described herein inhibits or reduces tumor growth by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% compared to tumor growth in a subject with cancer (in some embodiments, an animal model of the same cancer) administered with a negative control, as measured using assays known in the art.

[0140] Examples of cancerous disorders include, but are not limited to, solid tumors, hematological malignancies, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignant tumors of various organ systems, such as those affecting the liver, lungs, breasts, lymphatic system, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., kidneys, urothelial cells), prostate, and pharynx, such as sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas). Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small intestine cancers, and esophageal cancers. Squamous cell carcinomas include, for example, malignant tumors of the lungs, esophagus, skin, head and neck region, oral cavity, anus, and cervix. In one embodiment, the cancer is melanoma, for example, advanced melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of this disclosure.

[0141] Exemplary cancers whose growth can be inhibited using therapeutic or pharmaceutical compositions comprising the Fc variants of this disclosure include cancers that typically respond to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer), and epithelial carcinoma. Furthermore, refractory or recurrent malignancies can be treated using the combination therapies described herein.

[0142] Other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastroesophageal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue cancer, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia Examples include hematological diseases, chronic or acute leukemia including chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodysplastic syndrome, kidney or ureteral cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, neuroblastoma, tumor angiogenesis, spinal axial tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos (e.g., mesothelioma), and combinations of the above cancers.

[0143] In specific embodiments, the cancers include breast cancer, neuroblastoma, lymphoma, colon cancer, pancreatic ductal adenocarcinoma, melanoma, renal cell carcinoma, bladder cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, and multiple myeloma.

[0144] Combination therapy With respect to additional therapeutic agents, "administered in combination" means that two (or more) different treatments are delivered to the subject during the course of the subject's suffering due to the disorder. In some embodiments, the delivery of one treatment still occurs when the delivery of the second treatment begins, resulting in overlap in administration. This is referred to as "simultaneous" or "simultaneous delivery." In other embodiments, the delivery of one treatment is completed before the delivery of the other treatment begins. This is referred to as "sequential delivery." In some embodiments of either case, the treatments are more effective for combination administration. Additional therapeutic agents in combination therapy of the present disclosure may also be administered periodically. Combination cycling therapy involves the administration of the first treatment for a set period followed by the administration of the second treatment for a set period, and repetition of this sequential administration.

[0145] Therapeutic or pharmaceutical compositions comprising the artificially engineered immunoglobulins described herein can be administered in combination with one or more other therapeutic agents, such as anticancer agents, cytokines, or antihormone agents, to treat and / or manage cancer. Other therapeutic agents that can be used in combination with therapeutic or pharmaceutical compositions comprising the artificially engineered immunoglobulins described herein include, but are not limited to, small molecules, synthetic drugs, peptides (including cyclic peptides), polypeptides, proteins, nucleic acids (e.g., DNA and RNA nucleotides, for example, but not limited to antisense nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding biologically active proteins, polypeptides, or peptides), antibodies, synthetic or natural inorganic molecules, mimics, and synthetic or natural organic molecules.

[0146] Non-limiting examples of one or more other therapeutic methods that can be used in addition to the therapeutic or pharmaceutical composition comprising the artificially engineered immunoglobulin described herein include, but are not limited to, chemotherapy, radiotherapy, cytotoxic agents, chemotherapeutic agents, cytokines, kinase inhibitors, low-dose gemcitabine, 5-fluorouracil, and cytokine modulators. In particular, one or more other therapeutic methods that can be used in addition to the therapeutic or pharmaceutical composition comprising the artificially engineered immunoglobulin of this disclosure include, in particular, immuno-oncological approaches that perturb the tumor microenvironment, such as recombinant IL-2, recombinant IL-15, recombinant IL-12, recombinant IL-21, anti-IL1β, anti-TGFβ, anti-CD39, anti-CD73, anti-CTLA4, anti-PD(L)1, anti-TIM3, HDAC inhibitors, HIF1a inhibitors, and anti-angiogenic agents, such as anti-VEGF.

[0147] kit This disclosure also encompasses kits for treating patients with cell proliferation disorders. Such kits include a therapeutically effective dose of a therapeutic or pharmaceutical composition containing the Fc variant described herein. Furthermore, such kits may include means for administering the therapeutic or pharmaceutical composition containing the Fc variant described herein (e.g., auto-injectors, syringes and vials, pre-filled syringes, pre-filled pens) and instructions for use. These kits may contain additional therapeutic agents (listed below) for treating cell proliferation disorders. Such kits may also include instructions for administering the therapeutic or pharmaceutical composition containing the Fc variant described herein for treating patients. Such instructions may provide dosages, routes of administration, regimens, and total duration of treatment for use with respect to the therapeutic or pharmaceutical composition containing the Fc variant described herein.

[0148] The phrase “means of administration” is used to refer to any available method for systemically administering a drug to a patient, including, but not limited to, pre-filled syringes, vials and syringes, injection pens, auto-injectors, IV infusions and bags, infusion pumps, patches, infusion bags and needles. Using such devices, a patient may self-administer the drug (i.e., administer the drug without the assistance of a physician), or a healthcare professional may administer the drug. Various embodiments of the present invention are shown below. 1. An Fc variant of a parent Fc polypeptide, wherein the Fc variant exhibits modified binding to FcαR or modified antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parent Fc polypeptide, the Fc variant comprises at least one amino acid modification in the Fc region of the parent Fc polypeptide, the amino acid modification being located at a position selected from the group consisting of: CH2.10, CH2.89, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, CH3.105, CH3.109, CH3.118 and CH3.124, and the numbering of the amino acid modification follows the IMGT numbering for the C-domain. 2. The Fc variant described in 1 above, wherein the at least one amino acid modification is selected from the group consisting of: A_CH2.10_S, L_CH2.89_I, G_CH2.91_Q, G_CH2.91_V, Q_CH2.94_E, N_CH2.97_H, N_CH2.97_Y, G_CH2.99_W, S_CH3.45_D, M_CH3.105_Y, E_CH3.109_D, Q_CH3.118_Y, and L_CH3.124_F, and the numbering of the amino acid modification follows the IMGT numbering for the C-domain. 3. The above-mentioned at least one amino acid modification is: Q_CH2.94_E, N_CH2.97_Y, S_CH3.45_D, M_CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D, N_CH2.97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y / Q_CH3.118_Y, A_CH2.10_S, L_CH2.89_I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_CH3.109_D, L_CH3.124_F, and L_CH2.89_I / G_CH2.91_V / Q_CH2.94_E / N_CH2.97_Y / G_CH2.99_W An Fc variant according to 1 or 2 above, selected from the group consisting of the above, wherein the numbering of the amino acid modification follows the IMGT numbering for the C-domain. 4. An Fc variant according to any one of 1 to 3 above, wherein the parent Fc polypeptide is contained within human IgA. 5. An Fc variant according to any one of 1 to 4 above, wherein the parent Fc polypeptide is contained within human IgA2. 6. The Fc mutant according to any one of 1 to 5 above, wherein the Fc mutant has an increased affinity for human FcαRI, measured by surface plasmon resonance, which is at least about 50 times that of the parent Fc polypeptide. 7. The Fc variant according to any one of 1 to 3 above, wherein the parent Fc polypeptide contains human IgG1. 8. The Fc variant according to 7, wherein the Fc variant has an increased affinity for human FcαRI, measured by surface plasmon resonance, which is at least about 300 times that of the parent Fc polypeptide. 9. The Fc variant according to any one of 1 to 8 above, wherein the Fc variant increases antibody-dependent cell-mediated cytotoxicity by at least about 5 times compared to the parent Fc polypeptide, as measured in an MDA-MB-453 cell killing assay. 10. The Fc variant according to any one of 1 to 9 above, wherein the Fc variant has increased efficacy in a Calu-3 cell killing assay at least about twice that of the parent Fc polypeptide. 11. An IgA antibody containing an Fc variant, wherein the antibody has increased FcαR affinity or increased antibody-dependent cell-mediated cytotoxicity compared to an IgA antibody containing a parent Fc polypeptide. 12. The IgA antibody as described in 11 above, wherein the antibody comprises an amino acid modification at a position selected from the group consisting of CH2.10, CH2.89, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, CH3.105, CH3.109, CH3.118 and CH3.124, and the numbering of the amino acid modification follows the IMGT numbering for the C-domain. 13. The IgA antibody described in 12 above, wherein the antibody is a human IgA1 or IgA2 antibody. 14. An IgA antibody according to any one of items 11 to 13 above, wherein the antibody binds to a tumor antigen. 15. A pharmaceutical composition comprising an Fc variant described in any of items 1 to 10 above or an antibody described in any of items 11 to 14 above, in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers. 16. The pharmaceutical composition according to 15, further comprising one or more additional activators. 17. An Fc variant according to any of items 1 to 10 above, or an antibody according to any of items 11 to 14 above, for use in the treatment of cell proliferation disorders or conditions. 18. The Fc variant or antibody for use as described in 17 above, wherein the cell proliferative disorder or condition is selected from the group consisting of: breast cancer, neuroblastoma, lymphoma, pancreatic ductal adenocarcinoma, melanoma, renal cell carcinoma, bladder cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, and multiple myeloma. 19. An isolated nucleic acid molecule encoding an Fc variant as described in any of items 1 to 10 above, or an antibody as described in any of items 11 to 14 above. 20. A cloning or expression vector comprising one or more nucleic acid sequences as described in item 19 above, which is suitable for recombinant production of an Fc variant as described in any of items 1 to 10 above or an antibody as described in any of items 11 to 14 above. 21. Host cells containing one or more of the cloning or expression vectors described in item 20 above. 22. A method for preparing an Fc mutant according to any of items 1 to 10 above or an antibody according to any of items 11 to 13 above, comprising: culturing the host cells described in item 21 above; purifying the Fc mutant or antibody from the host cell culture; and recovering the Fc mutant or antibody from the host cell culture. [Examples]

[0149] The following embodiments are provided for further illustration of the present disclosure, but are not intended to limit its scope. Other variants of the present disclosure will be readily apparent to those skilled in the art and are included by the appended claims.

[0150] All constructs derived from the amino acid sequences generated according to Example 1 were expressed in mammalian systems, purified (Example 4), and assessed for binding to human FcαRI and rat FcαR using surface plasmon resonance (SPR) (Example 3). Finally, the functionality of the artificially engineered immunoglobulins was assessed by cell-based assays using human fresh isolated PMN (Example 4). All examples were performed using Fc variants of an antibody format containing VH and VL domains that recognize the antigen HER2, and an artificially engineered hinge and Fc region based on IgA2. Sequence ID 1 is the full-length heavy chain sequence of an anti-HER2-binding antibody having a VH domain that binds to HER2, and a hinge and constant domain from IgG1. Sequence ID 2 is the full-length heavy chain sequence of an anti-HER2-binding antibody having a VH domain that binds to HER2, and a hinge and constant domain from the m2 allotype of IgA2 (Lombana et al., (2019) MABS, 11:1122-38). Sequence ID 4 is the light chain sequence of an anti-HER2 binding antibody having a VL domain that binds to HER2 and a constant domain (CL) derived from IgA2.

[0151] Example 1: IgA2 Fc affinity maturation for hFcαRI 1.1 IgA2 Fc Library Design In silico analysis was performed on the IgA1 Fc / hFcαRI complex using the crystal structure database PDB 1OW0. This was because IgA1 has a similar structure to IgA2, differing only in its hinge region. All residues located in close proximity to hFcαRI were considered potentially involved in the IgA1 Fc / hFcαRI interaction and were classified into two categories: (i) residues from the "core" interface region (LCH2.15, LCH2.15.1, MCH3.105, ECH3.109, PCH3.113, LCH3.114, ACH3.115, FCH3.116, QCH3.118, where residues are numbered according to the IMGT numbering for the C-domain) and (ii) residues from the "shell" region surrounding the core (QCH2.94, NCH2.97, HCH2.98, RCH3.1, ECH3.3, RCH3.40, LCH3.42, SCH3.45, ECH3.45.2, where residues are numbered according to the IMGT numbering for the C-domain). Using a trinucleotide-specific mutagenesis (TRIM)-based approach (Virnekaes et al, (1994) Nucleic Acids Res., 22:5600-5607; Knappik et al., (2000) J Mol Biol., 296:57-86), we diversified both groups of residues to generate two libraries, L1 and L2, corresponding to the "shell" and "core" regions, respectively.

[0152] A third library (EP library) was generated using error-prone PCR of the IgA2 Fc domain (Gram et al., (1992) PNAS USA, 89:3576-3580).

[0153] 1.2 Library screening using yeast display All three IgA2 Fc libraries were screened using yeast display technology (Boder et al., (1997) Nature Biot., 15:553-557). Briefly, IgA Fc was displayed on the yeast cell membrane via α-aglutinin (Aga1 / Aga2) protein heterodimers, with IgA2 Fc fused to the N-terminus of the Aga2 protein. Four sorting rounds were performed:

[0154] (i) During the first sorting round, all three libraries were grown at 20°C for 2 days with shaking in selective medium containing 1% raffinose and 2% galactose to induce IgA2 Fc expression on the surface of yeast cells. Each culture was pelletized, the supernatant was removed, and the pellet was washed once with PBSM (Gibco, Waltham, MA) containing 1% BSA (bovine serum albumin) and 2 mM EDTA. After resuspending the pellet in 20 ml of PBSM, 100 μl each of streptavidin and antibiotin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were added. The cells were incubated at room temperature for 1 hour with rotation. MACS was then performed. Beads were removed using an LS column (Miltenyi). The library was then pelleted and resuspended in 20 ml of PBSM + 50 nM biotinylated FcαRI (CD89) (SEQ ID NO: 5), and incubated at room temperature for 1 hour with shaking. The cells were then pelleted, the supernatant was removed, washed once in PBSM, and then resuspended in 20 ml of PBSM + 100 μl of streptavidin microspheres (Miltenyi). The library was incubated on ice for 5 minutes with occasional shaking, then the cells were pelleted, the supernatant was removed, resuspended in 20 ml of PBSM, and then separated on a MACS LS column (Miltenyi). The column was washed once with 5 ml of PBSM, then the bound cells were eluted with selective medium, and the final volume was 10 ml in selective medium. The cells were grown overnight at 30°C with shaking.

[0155] (ii) For the second sorting round, the first round output from each of the three libraries was grown for 24 hours at 20°C in selective medium containing 1% raffinose and 2% galactose to induce IgA expression. The libraries were pelletized, washed once in PBSF (PBS (Gibco) + 0.1% bovine serum albumin), and resuspended in PBSF. Each library was divided into two samples; the first sample was prepared as 25 nM biotinylated FcαRI in PBSF, and the second sample as 10 nM biotinylated FcαRI in PBSF. Rabbit anti-myc-tag Dylight 488 (Rockland, Limerick, PA) was added to each at a final dilution of 1:100, and the samples were incubated at room temperature for 1.5 hours with rotation. Samples were pelletized, washed once with PBSF, and then incubated with PBSF + 1:100 final streptavidin Dylight 633 (Invitrogen, Waltham, MA) for 5 minutes while rotating. The samples were then pelletized, washed once, resuspended in PBSF, filtered through a 40 μm strainer, and analyzed and sorted using flow cytometry on a FACS Aria cell sorter (Becton Dickinson Biosciences, San Jose, CA). 10 nM FcαRI samples were sorted for L1 and L2 libraries, and 25 nM FcαRI samples were sorted for EP libraries. In each case, yeast showing the top 1–2% signal was gated, harvested, and grown overnight in selective medium at 30°C.

[0156] (iii) For the third sorting round, cultures from the second sorting round were inoculated into selective medium + 1% raffinose + 2% galactose and grown overnight at 20°C to induce IgA expression. Cells were prepared and sorted from each of the three libraries as in the second round, except that chicken anti-myc tag FITC (Genetex, Irvine, CA) and Neutravidin Dylight 633 (Invitrogen) were used as detection reagents. Biotinylated FcαRI was used at 5 nM for the EP library, 2 nM for the L1 library, and 1 nM for the L2 library. In each case, yeast showing the top 1-2% of signals was gated, harvested, and grown overnight in selective medium at 30°C.

[0157] (iv) The fourth sorting round was completed for the EP and L1 libraries only. Cultures from the third sorting round were inoculated into selective medium with 1% raffinose and 2% galactose and grown overnight at 20°C to induce IgA expression. Cells were prepared and sorted from each library as in the second round, except that mouse anti-c myc Dylight 488 (Invitrogen) and streptavidin cy5 (Invitrogen) were used as detection reagents. Biotinylated FcαRI was used at 2 nM for the EP library and at 1 nM for the L1 library. In each case, yeast showing the top 1-2% of signals was gated, harvested, and grown overnight in selective medium at 30°C.

[0158] 1.3 Hotspot identification, translation in full-length immunoglobulins, and SPR validation for hFcαRI Plasmids were purified from the third (L2 library) and fourth (EP and L1 library) round cultures, transformed in Escherichia coli (E. coli), plated on selective agar plates, grown overnight at 37°C, and sent to Genewiz (South Plainfield, NJ) for Sanger sequencing (Sanger et al (1975) J Mol Biol., 94(3):441-8; Sanger et al (1977) PNAS USA., 74(12):5463-7). Top clones were selected based on their frequency of occurrence, and these were used to identify mutations that enhance IgA2 / hFcαRI interaction. The IgA2 residue positions are shown in Table 1.

[0159] [Table 1]

[0160] The identified mutations were incorporated as single point mutations or in combination into full-length IgA2 immunoglobulin containing SEQ ID NO: 2 and transiently expressed in HEK293 cells (as described in Example 2). The same mutations were also incorporated into IgG1 isotype immunoglobulins containing artificially modified IgG1 Fc capable of binding to hFcαRI, which have SEQ ID NOs: 3 and 6. The test mutation sets are presented in Table 2 (based on SEQ ID NO: 2), Table 4 (based on SEQ ID NO: 3), and Table 6 (based on SEQ ID NO: 6).

[0161] Fc mutants were purified and assessed using surface plasmon resonance (SPR) measured against hFcαRI to evaluate the effect of specified mutations on immunoglobulin affinity for hFcαRI. Interestingly, all mutations had a limited or no effect on the expression yield and aggregation tendency of each immunoglobulin. SPR data and aggregate content after capture are shown in Tables 3, 5, and 7.

[0162] Finally, lead candidates were selected based on SPR and aggregation data, and SPR experiments were repeated using a wider range of hFcαRI concentrations. The use of a suitable concentration range allowed for more accurate measurement of the interaction between the artificially engineered immunoglobulin and the hFcαRI. The results are shown in Tables 8, 9, and 10.

[0163] [Table 2]

[0164] [Table 3]

[0165] [Table 4]

[0166] [Table 5]

[0167] [Table 6]

[0168] [Table 7]

[0169] [Table 8]

[0170] [Table 9]

[0171] [Table 10]

[0172] 1.4 Conversion of affinity maturation to improve alpha effector function in in vitro assays Lead candidates possessing homodimer Fc (SEQ ID NOs: 32, 37, and 42) were selected for their improved binding ability to hFcαRI. These were then tested in a PMN killing assay according to the procedure described in Example 4. Efficacy (EC) 50 The results for the immunoglobulin concentration required to produce 50% of the maximum effect of immunoglobulin and efficacy (Emax; the maximum expected effect of immunoglobulin) are shown in Figures 1 to 4.

[0173] All test candidates were active and demonstrated improved efficacy compared to parental IgA2 in the SK-BR-3 PMN killing assay (Figure 1).

[0174] Furthermore, a significant improvement in efficacy of up to 2-fold was demonstrated in a PMN killing assay using Calu-3 cells, which are known to have a lower HER2 receptor density than SK-BR-3 cells (Figure 2) (described in Example 4). In addition, a significant improvement in efficacy was demonstrated for the variant with SEQ ID NO: 42 in a PMN killing assay using MDA-MB-453 cells, which are also known to express lower levels of HER2 receptors (Figure 3). This improvement in efficacy was approximately 7-fold.

[0175] Finally, the affinity-matured variant with SEQ ID NO: 42 was tested in a PMN killing assay using MDA-MB-175 cells, a known minimal HER2-expressing cell line, and showed no killing even at extremely high concentrations (Figure 4). This observation highlights the safe profile of the test candidate.

[0176] Mutation sets were applied to heterodimer Fc to yield anti-complementary proteins including SEQ ID NOs. 7-8, SEQ ID NOs. 80-8, SEQ ID NOs. 7-9, or SEQ ID NOs. 80-9 (Figure 5). The tested candidate SEQ ID NOs. 80-8 and SEQ ID NOs. 80-9 showed better killing properties against SK-BR-3 cells compared to their parent immunoglobulins and IgA2 (Figures 5A and 5C) in PMN killing assays, but did not show any effect in mediating the γ response in PBMC killing assays (Figures 5B and 5D).

[0177] Example 2: Expression and purification of artificially manipulated proteins Nucleic acid sequences encoding the heavy and light chains were synthesized at Geneart (LifeTechnologies) and cloned into mammalian expression vectors using restriction enzyme-ligation-based cloning technology. The resulting plasmids were co-transferred into HEK293T cells. Briefly, for transient expression of immunoglobulins (IgG, IgA, and engineered immunoglobulins), equivalent amounts of the light chain and their respective engineered heavy chain vectors were co-transferred into suspension-compatible HEK293T cells using polyethyleneimine ((PEI), reference catalog no. 24765, Polysciences, Inc.). Typically, 100 ml of cells in a suspension at a density of 1 to 2 million cells per ml were transfused with DNA containing 50 μg of expression vector encoding the engineered heavy chain and 50 μg of expression vector encoding the light chain. Next, the recombinant expression vector was introduced into host cells, and the cells were further cultured for 7 days to produce constructs that could be secreted into culture medium (HEK, serum-free medium) supplemented with 0.1% pluronic acid, 4 mM glutamine, and 0.25 μg / ml antibiotic.

[0178] Next, the produced constructs were purified from the cell-free supernatant using immunoaffinity chromatography. Anti-kappa LC resin (KappaSelect, GE Healthcare Life Sciences) equilibrated in PBS buffer pH 7.4 was incubated with filtered medium using a liquid chromatography system (Aekta pure chromatography system, GE Healthcare Life Sciences). After washing the resin with PBS pH 7.4, the constructs were eluted with elution buffer (50 mM citrate, 90 mM NaCl, pH 2.7).

[0179] After capture, the eluted protein was pH-neutralized using 1M TRIS pH 10.0 solution and polished using size exclusion chromatography (HiPrep Superdex 200 16 / 60, GE Healthcare Life Sciences). Finally, the purified protein was compounded in PBS buffer pH 7.4.

[0180] Aggregation tendencies were measured using analytical size exclusion chromatography (Superdex 200 Increase 3.2 / 300GL, GE Healthcare Life Sciences) after the capture and pH neutralization steps.

[0181] Example 3: SPR measurement for human Fcα receptor (hFcαRI) and rat Fcα receptor (rFcαR) A direct binding assay was performed to characterize the binding of Fc variant antibodies with the light chain of SEQ ID NO: 4 to human FcαRI or rat FcαR.

[0182] Dynamic binding affinity constants (KD) were measured at room temperature using a BIAcore® T200 instrument (GE Healthcare, Glattbrugg, Switzerland). Proteins were diluted in running buffer 10 mM NaP, 150 mM NaCl, 0.05% Tween 20, pH 7.6. Manipulated immunoglobulins were captured using a streptavidin sensor chip (Sensor Chip SA, GE Healthcare Life Sciences) immobilized with biotinylated anti-κ light chain scFv, and recombinant human hFcαRI or recombinant rat FcαR were used as analytes.

[0183] To function as a reference, one flow cell did not capture any immunoglobulins. Binding data were collected by subsequent injections of the analyte dilution series on the reference and by measurements of the flow cell. A zero-concentration sample (running buffer only) was included to allow for dual referencing during data evaluation. For data evaluation, the dual-reference sensorgrams were analyzed by applying a 1:1 binding model analysis to generate the equilibrium dissociation constant (KD). The results for rFCαR are summarized in Table 11.

[0184] This experiment demonstrated that the candidate Fc variant cross-reactive with human / rat FcαRI, which is a desirable characteristic for candidate testing in in vivo disease models.

[0185] [Table 11]

[0186] Example 4: Antibody-dependent cytotoxicity (ADCC) assay method Blood samples from healthy donors were collected from freshly drawn peripheral blood according to the Swiss Human Research Act (Basel Tissue Donor Program - Prevomed). After lysing of erythrocytes with ACK lysis buffer, polymorphonuclear cells and peripheral blood mononuclear cells (PMNs and PBMCs) were isolated by ficoll-paque gradient. PMNs were used to characterize the alpha effector function of artificially engineered immunoglobulins, while PBMCs were used to characterize the gamma effector function.

[0187] Effector cells (freshly isolated PMN or PBMC cells) were added to HER2-expressing target cells (SK-BR-3, Calu-3, MDA-MB-453, or MDA-MB-175 cells, purchased from American Type Culture Collection, Rockville MD) in an effector-to-target ratio of 20:1. SK-BR-3 is a breast cancer cell lineage that overexpresses HER2. Calu-3 and MDA-MB-453 are lung and breast cancer cell lines that overexpress HER2 at lower levels compared to SK-BR-3, respectively (Cheung et al., 2019). MDA-MD-175 is a breast cancer cell lineage that expresses the lowest amount of HER2 (Crocker et al., 2005). PMN cell killing was not observed with any of the candidate Fc variants, indicating a favorable safety profile for low-HER2-expressing cell lines.

[0188] The immunoglobulin construct was added at the indicated concentration, the combination was gently mixed, and then centrifuged at 260 × g for 4 minutes without disruption to promote the co-localization of target and effector cells. The assay was then incubated in a standard tissue culture incubator in 5% CO2 at 37°C for 18 hours. After 18 hours, the supernatant was used for LDH release measurement using Cytotox96 reagent (Promega) according to the manufacturer's instructions. Absorbance at 490 nm was read on a Biotek Synergy HT plate reader. Data were analyzed and graphed using GraphPad Prism 6.0.

[0189] Example 5: Improvement of pharmacokinetic (PK) properties of artificially modified immunoglobulins compared to IgA 5.1 Material production of artificially engineered immunoglobulins Nucleic acids encoding anti-HER2 artificially manipulated immunoglobulin heavy chain variants having sequences SEQ ID NOs: 1, 2, 7, 8, 40, 80, 82, 83, and 84 were synthesized at Geneart (Life Technologies) and cloned into mammalian expression vectors using restriction enzyme-ligation-based cloning technology. Selective N-glycosylation sites were removed by substitution of specific Asp residues with Ala residues. The resulting plasmid encoding the heavy chain was simultaneously transfused into a mammalian expression system along with the plasmid encoding the light chain (SEQ ID NO: 4). For the HEK293T expression cell line, expression was carried out according to the procedure described in Example 2. For the CHO-S expression cell line (Thermo), the following procedure was used. Briefly, for transient protein expression, the expression vector was transfused into suspension-compatible CHO-S cells using the Expifectamine CHO transfusion agent (Thermo). Typically, 400 ml of cells in a suspension at a density of 6 million cells per ml were transfused with DNA containing 400 μg of an expression vector encoding an artificially engineered protein. The recombinant expression vector was then introduced into host cells for 7 days in culture medium (ExpiCHO expression medium (Thermo) supplemented with ExpiCHO feed and enhancer reagents) for further secretion. The expression construct was then purified from the cell-free supernatant according to the procedure described in Example 2. Measured immunoglobulin concentrations in serum were plotted as a function of time and are shown in Figures 6 and 7. The generated materials are listed in Tables 12 and 13.

[0190] [Table 12]

[0191] [Table 13]

[0192] Consistent with the construct design, the engineered immunoglobulins having SEQ ID NOs. 7-8 and 8-80 bound to CD89 while retaining binding to FcRn, and exhibited improved PK properties and improved half-lives compared to IgA immunoglobulin, as shown in Figure 6. Furthermore, as shown in Figure 6, affinity-matured mutants having SEQ ID NOs. 8-80 exhibited the same PK as the parent construct having SEQ ID NOs. 8. This indicates that affinity maturation to CD89 impairs the PK properties of the engineered immunoglobulins.

[0193] The data shown in Figure 7 illustrates how the N-glycosylation pattern affects immunoglobulin PK. The PK properties of the manipulated immunoglobulins were improved compared to IgA, and this improvement was further enhanced when individual N-glycosylation sites CH2.84.4 were removed (SEQ ID NO: 83).

[0194] 5.1 Mouse Test Male CD1 mice were obtained from Charles River Laboratories. Upon arrival, all mice were housed in a pathogen-free animal facility at room temperature of 21°C under a standard 12-hour light / 12-hour dark cycle with continuous feeding and watering. All mice received a single intravenous (IV) injection of IgG or IgA or artificially engineered immunoglobulin (3 mg / kg) produced and purified as described above. Each compound was injected into three mice. Blood samples were collected at various time points after injection via the saphenous vein into serum separation tubes. The blood was allowed to coagulate at ambient temperature for at least 20 minutes. Coagulated samples were maintained at room temperature until centrifugation, which was started within 1 hour of collection time. Each sample was centrifuged at a relative centrifugal force of 1500–2000 × g at 2–8°C for 5 minutes. Within 20 minutes after centrifugation, serum was separated from the blood samples and transferred into labeled 2.0 mL polypropylene conical-bottom microcentrifuge tubes. Only animals that were clearly healthy and showed no obvious abnormalities were used in the study. All animal work performed was reviewed and approved by Novartis' Institutional Animal Care and Use Committee.

[0195] 5.2 Immunoglobulin ELISA for pharmacokinetic studies Immunoglobulin levels were measured by serial sandwich ELISA. For IgA administration, wells of Nunc Maxisorp microtiter plates were coated overnight at 4°C with goat anti-human IgA (Southern Biotech, catalog number 2053-01). For IgG and artificially engineered immunoglobulin administration, wells of Roche StreptaWell microtiter plates were coated for 1 hour at room temperature with biotinylated SB goat anti-human IgG (Southern Biotech, catalog number 2049-08). After 1 hour incubation with blocking buffer (PBS, 0.5% bovine serum albumin (BSA)), samples diluted in the same blocking buffer were added to the blocked plates and incubated at room temperature for 2 hours. After incubation, horseradish peroxidase conjugate goat anti-human IgA (SouthernBiotech, catalog no. 2053-05) or horseradish peroxidase conjugate goat anti-human IgG (SouthernBiotech, catalog no. 2049-05) was added and incubated at room temperature for 1 hour. The plate was then incubated with substrate solution (BM Blue POD substrate TMB, Roche, catalog no. 11484281001) and the reaction was stopped with 0.5 M sulfuric acid. Absorbance was measured at 450 nm using a plate reader and subtracted at 650 nm. Between steps, the plate was washed three times with washing buffer (0.05% Tween-20 in PBS).

[0196] Sequence information Table 14 lists the amino acid sequences (SEQ ID NOs) of the full-length heavy and light chains containing the variant Fc region described in the Examples, which are used to generate complete antibodies. The Fc variants, full-length heavy chains, light chains, or complete antibodies described herein can be produced using conventional recombinant protein production and purification processes.

[0197] All sequences (sequence numbers) referenced herein are found in Table 14. Reference numbers refer to internal sequence reference numbers. Throughout the text of this application, in the event of any inconsistency between the text of this application (e.g., Table 14) and the sequence listings, the text of this application shall prevail.

[0198] [Table 14]

[0199] [Table 15]

[0200] [Table 16]

[0201] [Table 17]

[0202] [Table 18]

[0203] [Table 19]

[0204] [Table 20]

[0205] [Table 21]

[0206] [Table 22]

[0207] Table 23

Claims

1. An Fc variant of a parent Fc polypeptide contained within human IgA2 having at least 90% sequence identity with SEQ ID NO: 2, wherein the Fc variant exhibits modified binding to FcαR or modified antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the parent Fc polypeptide, and the Fc variant comprises at least one amino acid modification in the Fc region of the parent Fc polypeptide, wherein the at least one amino acid modification is: N_CH2.97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y / Q_CH3.118_Y, Selected from the group consisting of, The numbering of the amino acid modification is in accordance with the IMGT numbering for the C-domain, and the Fc variant is an Fc variant that, as measured by surface plasmon resonance, has an increased affinity for human FcαRI that is at least 50 times greater than that of the parent Fc polypeptide contained in Sequence ID No.

2.

2. The Fc variant according to claim 1, wherein the Fc variant increases antibody-dependent cell-mediated cytotoxicity by at least five times compared to the parent Fc polypeptide, as measured in an MDA-MB-453 cell killing assay.

3. The Fc variant according to claim 1 or 2, wherein the Fc variant has at least twice the increased efficacy in a Calu-3 cell killing assay compared to the parent Fc polypeptide.

4. An isolated nucleic acid molecule encoding the Fc variant according to any one of claims 1 to 3.

5. A cloning or expression vector comprising one or more nucleic acid sequences according to claim 4, the vector being suitable for recombinant production of an Fc variant according to any one of claims 1 to 3.

6. A host cell comprising one or more cloning or expression vectors according to claim 5.

7. A method for preparing an Fc mutant according to any one of claims 1 to 3, comprising: culturing a host cell according to claim 6; purifying the Fc mutant or antibody from the host cell culture; and recovering the Fc mutant or antibody from the host cell culture.

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