Human IgG Fc region variants with improved effector functions

Abstract

The present invention relates to human IgG Fc region variants with improved effector functions and uses thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent document claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 607,591, filed December 19, 2017. The above-identified patent applications are hereby incorporated by reference in their entireties to provide continuity of disclosure.

[0002] Government Interests This invention was made with government support under P01 AI100148 awarded by the NIAID and NIH. The government has certain rights in this invention.

[0003] Technical Field The present invention relates to human IgG Fc region variants with improved effector functions and uses thereof. [Background technology]

[0004] Extensive clinical experience with numerous FDA-approved monoclonal antibodies (mAbs) for the treatment of inflammatory and neoplastic diseases strongly suggests that the therapeutic potential of antibodies is highly dependent on the interaction of the Fc region of IgG with its cognate receptor, the Fcγ receptor (FcγR), which is expressed on the surface of effector leukocytes and mediates various Fc effector functions (Nimmerjahn et al., Cancer Immun 12, 13 (2012)). For example, the therapeutic efficacy of many mAbs is associated with allelic variants of the FcγR gene that affect the receptor's ability to bind IgG (Nimmerjahn et al., Cancer Immun 12, 13 (2012) and Mellor et al., J Hematol Oncol 6, 1 (2013)). Furthermore, it has been shown that the in vivo protective activity of some therapeutic mAbs depends on Fc-FcγR interactions, and Fc region variants optimized to enhance Fcγ binding ability exhibit improved therapeutic efficacy (Goede, V. et al., N Engl J Med 370, 1101-1110 (2014)). In view of the diverse signaling activities of FcγR (Bournazos et al., Annu Rev Immunol 35, 285-311 (2017)), IgG antibodies with improved effector activity have been developed by engineering the Fc region to engage and activate specific classes of FcγR. For example, the FDA-approved anti-CD20 mAb obinutuzumab has been engineered to enhance binding to the activating FcγR FcγRIIIa and has been shown to exhibit superior therapeutic efficacy compared to anti-CD20 mAbs without Fc engineering (Goede, V. et al., N Engl J Med 370, 1101-1110 (2014)).

[0005] However, various challenges remain (Klein et al., 2012, MAbs. 4(6): 653-663). In particular, the diversity of Fc receptors and their limited expression on immune system cells have been shown to affect the range of responses associated with antibody-mediated activity. For example, the ability of antibodies to induce T cell responses has been shown to depend on the engagement of dendritic cell-activating Fc receptors, such as FcRIIA (DiLillo et al., Cell 2015). Similarly, neutrophil activation by IgG antibodies requires Fc receptors distinct from those of NK cells. Furthermore, as disclosed herein, the novel engineered IgG antibodies of the present invention have in vivo half-lives comparable to or greater than those of unmodified IgG1. Thus, there is a need for Fc variants that enable engagement of a full range of low-affinity activating receptors while minimizing engagement of the inhibitory Fc receptor, FcRIIB. Summary of the Invention

[0006] The various embodiments presented in this document address the above unmet needs and / or other needs by providing human IgG Fc region variants with improved effector functions and half-lives, and uses thereof.

[0007] In one aspect, the present invention relates to a polypeptide comprising an Fc variant of a human IgG1 Fc polypeptide. The Fc variant comprises (i) an alanine (A) at position 236, a leucine (L) at position 330, and a glutamic acid (E) at position 332, and (ii) no aspartic acid (D) at position 239. Numbering is according to the EU index of Kabat. The polypeptide or the Fc variant may further comprise a leucine (L) at position 428 and / or a serine (S) at position 434. In some embodiments, the polypeptide or the Fc variant comprises a serine (S) at position 239. In some examples, the polypeptide or the Fc variant comprises the sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[0008] The above-described polypeptides or Fc variants can be included as part of an antibody or fusion protein (e.g., fused to an Fv, sFv, or other antibody variants as described below). Accordingly, antibodies or fusion proteins comprising the above-described polypeptides or Fc variants are within the scope of the present invention. The antibody has specificity for any target molecule of interest. For example, the target molecule can be selected from the group consisting of cytokines, soluble or insoluble factors, molecules expressed on pathogens, molecules expressed on cells, and molecules expressed on cancer cells. Factors and molecules can be proteins and non-proteins such as carbohydrates and lipids. The antibody can be selected from the group consisting of chimeric antibodies, humanized antibodies, or human antibodies. The antibodies may have one or more of the following characteristics: (1) a higher binding affinity to hFcγRIIA, hFcγRIIIA, hFcRn, and / or hFcγRIIIB compared to a reference antibody having the sequence of SEQ ID NO: 1; (2) a longer serum half-life compared to a reference antibody having the sequence of SEQ ID NO: 1 or SEQ ID NO: 4; and (3) the same or better half-life compared to an antibody having the sequence of SEQ ID NO: 1. The antibodies are generally similar to the reference antibody except that the latter has a different Fc sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 4). For example, the GAALIE variant disclosed herein (SEQ ID NO: 2) unexpectedly has a longer half-life and is more stable than the GASDALIE variant (SEQ ID NO: 4).

[0009] Also within the scope of the present invention are isolated nucleic acids comprising sequences encoding the above-described polypeptides or antibodies, expression vectors comprising the nucleic acids, and host cells comprising the nucleic acids. The host cells may be used in a method for producing a recombinant polypeptide or antibody, comprising culturing the host cells in a medium under conditions that allow expression of the polypeptide or antibody encoded by the nucleic acid, and purifying the polypeptide or antibody from the cultured cells or the medium of the cells.

[0010] In another aspect, the invention provides a pharmaceutical formulation comprising (i) the above-described polypeptide, antibody, or nucleic acid, and (ii) a pharmaceutically acceptable carrier.

[0011] In another aspect, the present invention provides a method for treating a disease, such as an inflammatory disease, a neoplastic disease, or an infectious disease, comprising administering to a subject in need thereof a therapeutically effective amount of the above-described polypeptide, antibody, or nucleic acid. Also within the scope of the present invention is the use of the polypeptide, antibody, or nucleic acid in the manufacture of a medicament for treating a disease, such as an inflammatory disease, a neoplastic disease, or an infectious disease.

[0012] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. [Brief explanation of the drawings]

[0013] [Figure 1] Figures 1A, 1B, 1C, and 1D (collectively "Figure 1") show the in vivo half-life of the G236A / S239D / A330L / I332E ("GASDALIE") Fc region variant in FcγR-humanized (FcR+) mice (Figures 1A and 1C) and in FcR-deficient (FcR-null) mice (Figures 1B and 1D). The S239D / I332E ("SDIE") variant is included as a control. Figures 1C and 1D show serum IgG levels of the human IgG1 Fc variants 8 days after administration to FcγR-humanized (Figure 1C) and FcR-deficient (Figure 1D) mice. [Figure 2]Figures 2A and 2B (collectively "Figure 2") show measurements of the in vivo half-life of Fc region variants in rhesus monkeys. Wild-type (WT) human IgG1 (Figure 2A) and G236A / A330L / I332E / M428L / N434S ("GASDALIE LS") (Figure 2B) Fc region variants of the 3BNC117 mAb were administered to rhesus monkeys (iv; 20 mg / kg). At different time points after administration to rhesus monkeys, IgG levels of human IgG1 were assessed by ELISA to measure the half-life (expressed in h) of the antibodies. [Figure 3] Figures 3A and 3B (collectively "Figure 3") are tables showing the binding affinity of human IgG1 Fc region variants to human FcγR (FcγRIIa H131, FcγRIIa R131, FcγRIIb, FcγRIIIa V157, FcγRIIIa F157) as measured by SPR analysis. Figure 3A shows the affinity measurements (KD(M)), and Figure 3B shows the fold increase in affinity relative to wild-type human IgG1. Variants tested: SDIE (S239D / I332E); GAIE (G236A / I332E); GAALIE (G236A / A330L / I332E); and afucosylated (lacking bisecting fucose residues on Fc-associated glycans). [Figure 4] Figure 4 is a series of SPR sensorgrams showing the binding of wild-type human IgG1 (left) and Fc region variants of GAALIE (right) to human FcγR (FcγRIIa H131, FcγRIIa R131, FcγRIIb, FcγRIIIa V157, FcγRIIIa F157). Labels indicate the concentration (μM) of the analyte (FcγR). [Figure 5]Figures 5A and 5B (collectively "Figure 5") are tables showing the binding affinity of Fc region variants of human IgG1 to mouse FcγR, as determined by SPR analysis. Figure 5A shows the affinity measurements (KD(M)), and Figure 5B shows the fold increase in affinity relative to wild-type human IgG1. Variants tested: SDIE (S239D / I332E); GAIE (G236A / I332E); GAALIE (G236A / A330L / I332E); and afucosylated (lacking bisecting fucose residues on Fc-associated glycans). [Figure 6] 6 is a series of SPR sensorgrams showing the binding of wild-type human IgG1 (left) and Fc region variants of GAALIE (right) to mouse FcγR. Labels indicate the concentration (μM) of the analyte (FcγR). [Figure 7] Figures 7A and 7B (collectively "Figure 7") are tables showing the binding affinity of Fc region variants of human IgG1 to rhesus monkey FcγR, as measured by SPR analysis. Figure 7A shows the affinity measurements (KD(M)), and Figure 7B shows the fold increase in affinity relative to wild-type human IgG1. Variants tested: SDIE (S239D / I332E); GAIE (G236A / I332E); GAALIE (G236A / A330L / I332E); and afucosylated (lacking bisecting fucose residues on Fc-associated glycans). [Figure 8] 8 is a series of SPR sensorgrams showing the binding of wild-type human IgG1 (left) and Fc region variants of GAALIE (right) to rhesus monkey FcγR. Labels indicate the concentration (μM) of the analyte (FcγR). [Figure 9] Figure 9 shows platelet depletion by Fc variants of 6A6 mAb in FcγR-humanized mice. Mice were administered Fc region variants of 6A6 mAb (SDIE (S239D / I332E); GAIE (G236A / I332E); GAALIE (G4236A / A330L / I332E)). N297A (a non-FcR-binding variant) was included as a control. Platelet counts were analyzed at the indicated time points, and values ​​represent the mean (±SEM) percentage of platelet counts relative to pre-feeding values ​​at time 0. [Figure 10] Figure 10 shows the depletion of CD4+ cells by Fc variants of GK1.5 mAb in FcγR-humanized mice. Mice were administered 100 μg i.p. of Fc region variants of GK1.5 mAb (SDIE(S239D / I332E); GAIE(G236A / I332E); GAALIE(G236A / A330L / I332E)). N297A (a non-FcR-binding variant) was included as a control. CD4+ cell counts were analyzed in the blood (A) and spleen (B) 24 hours after mAb administration. [Figure 11] Figures 11A, 11B, 11C, and 11D (collectively "Figure 11") show depletion of CD20+ B cells by Fc variants of CAT mAb in hCD20+ / FcγR-humanized mice. Mice were administered (200 μg, i.p.) Fc region variants of CAT mAb (SDIE(S239D / I332E); GAIE(G236A / I332E); GAALIE(G236A / A330L / I332E)). N297A (a non-FcR-binding variant) was included as a control. CD20+ B cell numbers and abundances were analyzed in the blood (Figures 11A and 11B) and spleen (Figures 11C and 11D) 48 hours after mAb administration. [Figure 12] Figures 12A and 12B (collectively "Figure 12") show depletion of CD20+ B cells by Fc variants of 2B8 mAb in hCD20+ / FcγR-humanized mice. Mice were intraperitoneally administered the indicated doses of wild-type human IgG1 or the GAALIE (G236A / A330L / I332E) variant of anti-CD20 mAb 2B8. CD20+ abundance (Figure 12A) and cell counts (Figure 12B) were analyzed in the blood 48 hours after mAb administration. [Figure 13]Figures 13A, 13B, and 13C (collectively "Figure 13") show the in vivo half-life of Fc region mutants in FcR-deficient (FcR null) (Figure 13A) and FcγR-humanized mice (FcR+) (Figure 13B). The Fc region mutants used for human IgG1 were SDIE (S239D / I332E), GAIE (G236A / I332E), and GAALIE (G236A / A330L / I332E). Figure 13C shows the IgG levels of human IgG1 at different time points after administration to FcγR-humanized mice. [Figure 14] Figures 14A and 14B (collectively "Figure 14") show measurements of in vivo half-life of Fc region variants in rhesus monkeys. Wild-type (WT) human IgG1 (Figure 14A) and GAALIE (G236A / A330L / I332E) (Figure 14B) Fc region variants of 3BNC117 mAb were administered to rhesus monkeys (iv; 20 mg / kg). IgG levels of human IgG1 were assessed by ELISA at different time points after administration to rhesus monkeys, and the half-life (expressed in h) of the antibodies was measured. [Figure 15] Figures 15A and 15B (collectively "Figure 15") show depletion of CD20+ B cells in rhesus monkeys by Fc variants of the 2B8 mAb. Wild-type human IgG1 or the GAALIE (G236A / A330L / I332E) variant of the anti-CD20 mAb 2B8 was administered to rhesus monkeys at 0.05 mg / kg (iv). CD20+ abundance (Figure 15A) and cell counts (Figure 15B) were analyzed in the blood at various time points before and after antibody administration. [Figure 16] Figure 16 shows the protein sequences of the constant regions of human IgG1 (wild-type and Fc region variants). The amino acid substitution positions in each variant are underlined. Residue numbering is according to the EU numbering system. [Figure 17]Figure 17 shows the Tm values ​​of various Fc region mutants measured by thermal shift assay. The human IgG1 Fc region mutants used were SDIE (S239D / I332E), GAIE (G236A / I332E), GAALIE (G236A / A330L / I332E), and GASDALIE (G236A / S239D / A330L / I332E). These mutants contained an LS mutation (M428L / N434S) that increases the affinity of human IgG1 for FcRn. [Figure 18] Figure 18 is a table showing the binding affinity of human IgG1 Fc region variants to human FcRn / β2 microglobulin at pH 6.0, as measured by SPR analysis. Affinity measurements (KD(M)) and the fold increase in affinity relative to wild-type human IgG1 are shown. The human IgG1 Fc region variants used were SDIE (S239D / I332E), GAIE (G236A / I332E), and GAALIE (G236A / A330L / I332E). These variants contained the LS mutation (M428L / N434S). [Figure 19] Figure 19 is a series of SPR sensorgrams showing the binding of Fc region variants to human FcRn / β2 microglobulin at pH 6.0. Labels indicate the concentration (μM) of the analyte (FcRn). The Fc region variants used for human IgG1 were LS (M428L / N434S), GAALIE (G236A / A330L / I332E), and GAALIE LS (G236A / A330L / I332E / M428L / N434S). [Figure 20] Figure 20 is a series of SPR sensorgrams showing the binding of Fc region variants to human FcRn / β2 microglobulin at pH 7.4. Labels indicate the concentration (μM) of the analyte (FcRn). The Fc region variants used for human IgG1 were LS (M428L / N434S), GAALIE (G236A / A330L / I332E), and GAALIE LS (G236A / A330L / I332E / M428L / N434S). [Figure 21]Figures 21A, 21B, and 21C (collectively "Figure 21") are a series of graphs showing the in vivo half-lives of Fc region variants in FcRn / FcγR-humanized mice. The Fc region variants used for human IgG1 were: LS (M428L / N434S), GAALIE (G236A / A330L / I332E), and GAALIE LS (G236A / A330L / I332E / M428L / N434S). Figures 21A and 21B show the IgG levels of human IgG1 at different time points after administration to FcRn / FcγR-humanized mice. Figure 21C shows the calculated half-lives of the Fc region variants in FcRn / FcγR-humanized mice. [Figure 22] Figure 22 is a graph showing platelet depletion by Fc variants of 6A6 mAb in FcRn / FcγR-humanized mice. Mice were administered Fc region variants of 6A6 mAb (8 μg; iv) (LS (M428L / N434S), GAALIE (G236A / A330L / I332E), and GAALIE LS (G236A / A330L / I332E / M428L / N434S)). N297A (a non-FcR-binding variant) was included as a control. Platelet counts were analyzed at the indicated time points, and values ​​represent the mean (±SEM) percentage of platelet counts relative to pre-feeding values ​​at time 0. [Figure 23]Figures 23A, 23B, 23C, and 23D (collectively "Figure 23") show that sLeA-targeting antibodies with hIgG1 Fc promote tumor clearance enhanced by engagement of activating human FcγRs. FcγR-humanized mice were inoculated intravenously with 5 x 10 B16-FUT3 tumor cells. 100 μg of anti-sLeA antibody or an isotype-matched control antibody was administered intraperitoneally on days 1, 4, 7, and 11. 14 days after inoculation, mice were euthanized, lungs were excised and fixed, and the number of metastatic lesions was counted. n≧5 / group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 23A and 23B suggest that anti-sLeA hIgG1 antibodies inhibit lung colonization of sLeA+ tumor cells. Mice were treated with 100 μg of anti-sLeA antibody (5B1-hIgG1 or 7E3-hIgG1) or an isotype-matched control antibody. Figure 23A shows a summary analysis of data obtained for all mice from a representative experiment. Figure 23B shows representative images of three excised lungs from each group. Figure 23B also suggests that the Fc-engineered anti-sLeA antibody variants exhibit excellent antitumor efficacy. Mice were treated with 100 μg of anti-sLeA antibody (clone 5B1 or 7E3, hIgG1, or hIgG1-GAALIE with G236A / A330L / I332E mutations) or an isotype-matched control antibody. Figure 23C shows a summary analysis of data obtained for all mice from two separate experiments (first experiment—■, second experiment—▲), and Figure 23D shows a representative image of a lung excised from a mouse treated with the 5B1 antibody. [Figure 24]Figures 24A, 24B, and 24C (collectively "Figure 24") show that engagement of either hFcRIIA or hFcRIIIA is necessary and sufficient for antibody-mediated tumor clearance. Figure 24A shows the relative binding affinity of hIgG1 Fc variants to human FcRs as measured by SPR studies. Figure 24B shows the 5B1-hIgG1 antibody with enhanced binding affinity to hFcRIIA, hFcRIIIA, or both, demonstrating that the antibody has superior anti-tumor efficacy. FcγR-humanized mice were inoculated intravenously with 5×10 B16-FUT3 tumor cells. 100 μg of anti-sLeA antibodies (5B1-hIgG1, 5B1-hIgG1-GA with G236A mutation, 5B1-hIgG1-ALIE with A330L / I332E mutations, or 5B1-hIgG1-GAALIE with G236A / A330L / I332E mutations) or isotype-matched control antibodies were administered intraperitoneally on days 1, 4, 7, and 11. Figure 24C shows the involvement of hFcRIIA or hFcRIIIA, which is essential for efficient tumor clearance of sLeA+ tumors. FcR-null (γ-chain KO), FcγR-humanized, hFcRIIA / IIB-transgenic, and hFcRIIIA / IIIB-transgenic mice were inoculated intravenously with 5 × 10 B16-FUT3 tumor cells. 100 μg of anti-sLeA antibody (5B1-hIgG1-GAALIE with G236A / A330L / I332E mutations) or an isotype-matched control antibody was administered intraperitoneally on days 1, 4, 7, and 11. For panels B+C, 14 days after inoculation, mice were euthanized, lungs were excised and fixed, and the number of metastatic lesions was counted. n≧6 / group. *p<0.05, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0014] This document describes human IgG Fc region variants with improved effector function and uses thereof. As described herein, antibodies or fusion proteins having IgG Fc region variants have increased binding to activating Fc receptors and have in vivo half-lives equal to or greater than those of unmodified IgG1 antibodies.

[0015] The Fc region, or constant region, of an antibody interacts with cellular binding partners to mediate antibody functions and activities, such as antibody-dependent effector function and complement activation. In IgG antibodies, the binding sites for complement Clq and Fc receptors (FcγRs) are located in the CH2 region of the Fc region. Coexpression of activating and inhibitory FcRs on different target cells regulates antibody-mediated immune responses. In addition to their involvement in the efferent phase of the immune response, FcRs are also important in regulating the activation of B cells and dendritic cells (DCs). For example, in IgG antibodies, various types of FcγRs mediate various cellular responses, such as phagocytosis by macrophages, antibody-dependent cell-mediated cytotoxicity by NK cells, and mast cell degranulation. Each FcγR exhibits different binding affinities and IgG subclass specificities. Lectin receptors also play a role. For example, DC-SIGN has been shown to play a role in the anti-inflammatory activity of Fc, including IVIG (see, e.g., US20170349662, WO2008057634, and WO2009132130).

[0016] As described herein, the biological activities of antibodies / immunoglobulins can be manipulated, altered, or controlled by introducing mutations or altering specific amino acids in the Fc region. Biological activities that can be manipulated, altered, or controlled in light of the present disclosure include, for example, one or more of Fc receptor binding, affinity for Fc receptors, specificity for Fc receptors, complement activation, signaling activity, targeting activity, effector function (such as programmed cell death or cellular phagocytosis), half-life, clearance, and transcytosis.

[0017] 1.Definition The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to describe an arrangement of amino acid residues in a polymer. A peptide, polypeptide, or protein may be composed of the standard 20 naturally occurring amino acids, as well as rare amino acids and synthetic amino acid analogs. They may be any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0018] A "recombinant" peptide, polypeptide, or protein is produced by recombinant DNA technology, i.e., from cells transformed with an exogenous DNA construct encoding the desired peptide. A "synthetic" peptide, polypeptide, or protein refers to a peptide, polypeptide, or protein prepared by chemical synthesis. The term "recombinant," when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the modification of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Fusion proteins comprising one or more of the aforementioned sequences and a heterologous sequence are within the scope of the present invention. A heterologous polypeptide, nucleic acid, or gene is derived from a foreign species or, if derived from the same species, is substantially modified from its original form. Two fused regions or sequences are heterologous to each other if they are not adjacent to each other in the naturally occurring protein or nucleic acid.

[0019] An "isolated" peptide, polypeptide, or protein is one that is separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide / protein may constitute at least 10% by dry weight of a purified preparation (or, in other words, any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%). Purity can be measured by any appropriate standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Isolated polypeptides / proteins according to the present invention can be produced by recombinant DNA technology, purified from transgenic animal sources, or produced by chemical methods. A functional equivalent of IgG Fc refers to a polypeptide derivative of IgG Fc, such as a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof, which substantially retains the activity of IgG Fc, i.e., the ability to bind to the respective receptors and elicit the respective cellular responses. The isolated polypeptide can comprise SEQ ID NO: 2 or SEQ ID NO: 3. Generally, functional equivalents are at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 (e.g., any number between 75% and 100%, inclusive, e.g., 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99%).

[0020] "Antigen" refers to a substance that elicits an immunological response or binds to the products of that response. The term "epitope" refers to the region of an antigen to which an antibody or T cell binds.

[0021] As used herein, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (such as full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. As used herein, the term "antibody" (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Thus, the term "antibody" as used in any context herein is intended to include, but is not limited to, any specific binding member, immunoglobulin class and / or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant fragments thereof, including, but not limited to, Fab, F(ab'), Fv, and scFv (single chain or related). Antibodies are understood in the art to be glycoproteins comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. The heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable regions of both the heavy and light chains comprise framework regions (FWR) and complementarity-determining regions (CDR). The four FWR regions are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) represent hypervariable regions and are arranged from the NH2-terminus to the COOH-terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The variable regions of the heavy and light chains contain the binding domains that interact with an antigen, while the constant regions, depending on the isotype, may mediate the binding of the immunoglobulin to host tissues or factors. The definition of "antibody" as used herein also includes chimeric, humanized, and recombinant antibodies, human antibodies made from transgenic non-human animals, and antibodies selected from libraries using enrichment techniques available to those skilled in the art.

[0022] As used herein, an "antibody fragment" may comprise a portion of an intact antibody, generally including the antigen-binding and variable regions of the intact antibody and / or the Fc region of the antibody that retains FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Preferably, the antibody fragment retains the entire constant region of an IgG heavy chain and includes an IgG light chain.

[0023] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., antibodies in which the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler and Milstein, Nature, 256, 495-497 (1975), incorporated herein by reference, or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567, incorporated herein by reference). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., J Mol Biol, 222, 581-597 (1991), each of which is incorporated herein by reference.

[0024] The term "monoclonal antibodies" as used herein specifically includes "chimeric" antibodies (immunoglobulins) and fragments of such antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; Morrison et al., Proc Natl Acad Sci USA, 81, 6851-6855 (1984); Neuberger et al., Nature, 312, 604-608 (1984); Takeda et al., Nature, 314, 452-454 (1985); International Patent Application No. PCT / GB85 / 00392, each of which is incorporated herein by reference).

[0025] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable region are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, the humanized antibody contains substantially all, at least one, and typically two, variable regions in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FR residues are those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321, 522-525 (1986); Riechmann et al., Nature, 332, 323-329 (1988); Presta, Curr Op Struct Biol, 2, 593-596 (1992); U.S. Patent No. 5,225,539, each of which is incorporated herein by reference.

[0026] "Human antibody" refers to any antibody with fully human sequences, such as may be obtained from human hybridomas, human phage display libraries, or transgenic mice expressing human antibody sequences.

[0027] The term "variable" refers to the fact that the sequences of certain segments of the variable (V) regions vary significantly among antibodies. The V regions mediate antigen binding and determine the specificity of a particular antibody for a particular antigen. However, variability is not evenly distributed across the 110 amino acids of the variable regions. Instead, V regions consist of relatively invariant regions called framework regions (FRs) of 15-30 amino acids separated by short, highly variable regions called "hypervariable regions," each 9-12 amino acids long. Native heavy and light chain variable regions each contain four FRs, which primarily adopt a β-sheet structure and are connected by three hypervariable regions, which form loops that connect the β-sheet structure and, in some cases, partially form a β-sheet structure. The hypervariable regions of each chain are held in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0028] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" ("CDRs").

[0029] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment comprises a dimer of one heavy-chain variable region and one light-chain variable region in tight, noncovalent association. The folding of these two regions generates six hypervariable loops (three loops each from the H and L chains), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable region (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0030] A "single-chain Fv" ("sFv" or "scFv") is an antibody fragment comprising the VH and VL antibody domains connected in a single polypeptide chain. The sFv polypeptide may further comprise a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For reviews of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

[0031] The term "diabody" refers to small antibody fragments prepared by constructing sFv fragments with a short linker (approximately 5-10 residues) between the VH and VL domains, such that interchain, rather than intrachain, pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0032] Domain antibodies (dAbs), which can be generated in fully human form, are the smallest known antigen-binding fragments of antibodies, ranging from approximately 11 kDa to approximately 15 kDa. dAbs are the robust variable regions of immunoglobulin heavy and light chains (VH and VL, respectively). They are highly expressed in microbial cell culture, exhibit favorable biophysical properties, including but not limited to solubility and temperature stability, and are well suited to selection and affinity maturation using in vitro selection systems such as phage display. dAbs are biologically active as monomers, and their small size and inherent stability allow them to be engineered into larger molecules to generate drugs with reduced serum half-life or other pharmacological activity. Examples of this technology are described, for example, in WO9425591, which describes camelid heavy-chain Ig-derived antibodies, and in US20030130496, which describes the isolation of single-domain fully human antibodies from phage libraries.

[0033] Fv and sFv are the only species with intact binding sites without constant regions. Therefore, they are suitable for reducing nonspecific binding during in vivo use. sFv fusion proteins can be constructed to fuse an effector protein to either the amino or carboxy terminus of an sFv. See, for example, Antibody Engineering, ed. Borrebaeck, supra. Antibody fragments may also be "linear antibodies," as described, for example, in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0034] As used herein, the terms "Fc fragment" or "Fc region" are used to define the C-terminal region of an immunoglobulin heavy chain. Such an Fc region is the tail region of an antibody that interacts with Fc receptors and several proteins of the complement system. The Fc region may be a native-sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is usually defined to stretch from the amino acid residue at position Cys226, or from position Pro230, to the carboxyl-terminus. A native-sequence Fc region contains an amino acid sequence identical to that of an Fc region found in nature. A variant Fc region, as will be understood by those skilled in the art, contains an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one "amino acid modification."

[0035] In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant regions of the antibody's two heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant regions (CH regions 2–4) on each polypeptide chain. The IgG Fc region contains highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is important for Fc receptor-mediated activity. The N-glycans attached to these sites are primarily core-fucosylated, complex-type biantennary structures. In addition, a small proportion of these N-glycans also contain cross-linked GlcNAc and α-2,6-linked sialic acid residues. See, e.g., US20170349662, US20080286819, US20100278808, US20100189714, US 2009004179, 20080206246, 20110150867, and WO2013095966, each of which is incorporated herein by reference.

[0036] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. A "variant Fc region" or "Fc variant" or "Fc region variant" comprises an amino acid sequence that differs from that of a native-sequence Fc region due to at least one "amino acid modification," as will be understood by those skilled in the art. Preferably, the variant Fc region comprises at least one amino acid substitution compared to the native-sequence Fc region or the Fc region of the parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about six, five, four, three, or two amino acid substitutions in the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc regions herein preferably have at least about 75 or 80% homology to the native sequence Fc region and / or the Fc region of the parent polypeptide, more preferably at least about 90% homology, more preferably at least about 95% homology, and even more preferably at least about 96%, 97%, 98%, or 99% homology. The terms "native" or "parent" refer to an unmodified polypeptide comprising an Fc amino acid sequence.

[0037] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. The Fc receptor is a protein found on the surface of certain cells that contribute to the protective functions of the immune system, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells, among others. Its name is derived from its binding specificity for the Fc region (fragment crystallizable region) of an antibody.

[0038] Some antibody functions are mediated by Fc receptors. For example, Fc receptors bind to antibodies attached to infected cells or invading pathogens. Their activity stimulates phagocytes or cytotoxic cells to destroy microorganisms or infected cells through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. It is also known in the art that the Fc region of an antibody binds to specific classes of Fc receptors and other immune molecules, such as complement proteins, ensuring that each antibody generates an appropriate immune response to a given antigen. FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are called FcγR, those for IgE are called FcεFR, and those for IgA are called FcαR. Surface receptors for immunoglobulin G exist in two distinct classes: those that activate cells upon crosslinking ("activating FcRs") and those that inhibit activation upon co-binding ("inhibitory FcRs").

[0039] In mammalian species, several different classes of IgG Fc receptors have been defined: for example, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγIV in mice, and FcγRI, FcRIIA, B, C, FcRIIIA, and B in humans. FcγRI exhibits high affinity for the antibody constant region and restricted isotype specificity, while FcγRII and FcγRIII have lower affinity for the IgG Fc region but a broader isotype binding pattern (Ravetch and Kinet, 1991; Hulett and Hogarth, Adv Immunol 57, 1-127 (1994)). FcγRIV is a recently identified receptor that retains intermediate affinity and restricted subclass specificity in all mammalian species (Mechetina et al., Immunogenetics 54, 463-468 (2002); Davis et al., Immunol Rev 190, 123-136 (2002); Nimmerjahn et al., Immunity 23, 41-51 (2005)).

[0040] Functionally, there are two distinct classes of Fc receptors: activating and inhibitory receptors, which signal via tyrosine-based immunoreceptor activation motifs (ITAMs) or immunoreceptor inhibitory motifs (ITIMs), respectively (Ravetch, in Fundamental Immunology W.E. Paul, Ed. (Lippincott-Raven, Philadelphia, (2003); Ravetch and Lanier, Science 290, 84-89 (2000)). Paired expression of activating and inhibitory molecules on the same cell is key to generating a balanced immune response. Furthermore, it is recognized that the IgG Fc receptor regulates certain isotypes more tightly than others, and that individual antibodies exhibit significant differences in their affinity for these isotypes (Nimmerjahn et al., 2005).

[0041] In one embodiment of the present invention, the FcR is a native sequence human FcR. In other embodiments, an FcR, such as a human FcR, binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic regions. Activating receptor FcγRIIA contains a tyrosine-based immunoreceptor activation motif (ITAM) in its cytoplasmic region. The inhibitory receptor FcγRIIB contains a tyrosine-based immunoreceptor inhibitory motif (ITIM) in its cytoplasmic region (see review by Daron, Annu Rev Immunol, 15, 203-234 (1997); FcRs are reviewed in Ravetch and Kinet, Annu Rev Immunol, 9, 457-92 (1991); Capel et al., Immunomethods, 4, 25-34 (1994); and de Haas et al., J Lab Clin Med, 126, 330-41 (1995); Nimmerjahn and Ravetch 2006, Ravetch Fc Receptors in Fundamental Immunology, ed. William Paul 5th Ed., each of which is incorporated herein by reference).

[0042] The term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0043] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects in a subject or after administration to a subject. The carrier in a pharmaceutical composition must also be "acceptable" in the sense that it is compatible with and can stabilize the active ingredient. One or more solubilizing agents may be used as pharmaceutical carriers for delivery of the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible excipients, adjuvants, additives, and diluents to obtain a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Further suitable pharmaceutical carriers and diluents, as well as the pharmaceutical needs for their use, are described in Remington's Pharmaceutical Sciences. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Therapeutic compounds may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects (see, e.g., Berge, SM et al., (1977) J. Pharm. Sci. 66:1-19).

[0044] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. This term is intended to include toxins, such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, including radioisotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioisotopes of Lu), chemotherapeutic agents, and small molecule toxins or fragments and / or variants thereof.

[0045] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metoledopa, and uredopa; ethylenimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycin antibacterial agents (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (synthetic analogs, such as KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin, e.g., Agnew See Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicins such as dynemicin A; esperamicin;Neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, eso Rubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, pofilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine Purine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU; androgens such as calucelone, dromosalon propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; Bestravsil; bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elformitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Maytansinoids such as maytansine and ansamitocins; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark); Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triaziquone;2,2',2"-Trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included within this definition are antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and antihormonal agents that act to regulate or inhibit hormone action on tumors, such as pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0046] As used herein, "treating" or "treatment" refers to the administration of a compound or agent to a subject having or at risk of developing a disease for the purpose of curing, alleviating, ameliorating, correcting, delaying the onset of, preventing, or ameliorating the disease, symptoms of the disease, conditions secondary to the disease, or predisposition to the disease.

[0047] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the chance of developing a disease or condition in a subject who does not have the disease or condition but is at risk of or susceptible to developing the disease or condition.

[0048] "Subject" refers to humans and non-human animals. Examples of non-human animals include all vertebrates, for example, mammals such as non-human mammals, non-human primates (especially higher primates), dogs, rodents (e.g., mice or rats), guinea pigs, cats, rabbits, and non-mammals such as birds, amphibians, and reptiles. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental non-human animal or an animal suitable as a disease model.

[0049] "Effective amount" refers to the amount of active compound / drug required to provide therapeutic effect to the treated subject.As those skilled in the art will recognize, effective dose varies depending on the type of treatment, route of administration, excipient use, and the possibility of combination with other therapeutic treatments.For treating neoplastic conditions, the therapeutically effective amount of a combination is, for example, the amount that causes a decrease in tumor size, a decrease in the number of tumor foci, or a delay in tumor growth, compared with untreated animals.

[0050] As disclosed herein, several ranges of values ​​are provided. Unless the context clearly dictates otherwise, it is understood that each intermediate value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, is also specifically disclosed. Each smaller range between any stated or intermediate value in a given range and any other stated or intermediate value in the given range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded, subject to any specifically excluded limits in the stated range, and each smaller range that includes either limit, excludes both limits, or includes both limits is also encompassed within the invention. Where a given range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0051] The term "about" generally refers to plus or minus 10% of the indicated number. For example, "about 10%" indicates a range of 9% to 11%, and "about 1" means 0.9 to 1.1. Other meanings of "about" may be apparent from the context, such as rounding, so that "about 1" could mean 0.5 to 1.4, for example. 2. Polypeptides and antibodies As disclosed herein, the present invention provides isolated polypeptides having the sequence of a variant of human IgG Fc (such as hIgG1 Fc). In one embodiment, the Fc region comprises one or more substitutions of the hIgG1 Fc amino acid sequence. Non-limiting exemplary IgG1 Fc regions are provided below and in Figure 16. In the sequences, the amino acid residues at positions 236, 239, 330, 332, 428, and 434 of each sequence are shown in bold, and the positions of the amino acid substitutions are underlined. Residue numbering is according to the EU numbering system, with the first residue A corresponding to position 118 in the EU numbering system.

[0052] [ka]

[0053] The amino acid composition of the polypeptides described herein can be varied without interfering with the ability of the polypeptides to bind to their respective receptors and elicit their respective cellular responses. For example, it can contain one or more conservative amino acid substitutions. A conservative variant or functional equivalent of a peptide, polypeptide, or protein disclosed herein refers to a polypeptide derivative of the peptide, polypeptide, or protein, such as a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof, that substantially retains the activity of the parent peptide, polypeptide, or protein (such as those disclosed herein). Typically, a conservative variant or functional equivalent is at least 60% identical to the parent (e.g., any number between 60% and 100%, inclusive, e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) (e.g., SEQ ID NO: 1, 2, 3, or 4). Thus, Fc regions with one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof, as well as heavy chains or antibodies with variant Fc regions are within the scope of the invention.

[0054] As used herein, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percent identity between two sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, is a function of the number of identical positions shared by the sequences (in other words, % homology = number of identical positions / total number of positions × 100). Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0055] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as incorporated into the ALIGN program (version 2.0) using a PAM120 residue weighting table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) as incorporated into the GAP program of the GCG software package (available at www.gcg.com) using either a BLOSUM 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0056] Additionally or alternatively, the protein sequences of the present invention can be further used as a "query sequence" to perform a search against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the molecules of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see www.ncbi.nlm.nih.gov).

[0057] As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0058] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Thus, for example, a predicted non-essential amino acid residue in SEQ ID NO: 2 or SEQ ID NO: 3 is preferably replaced with another amino acid residue from the same side chain family. Alternatively, as described in the Examples below, mutations can be randomly introduced along all or part of the sequence, such as by saturation mutagenesis, to identify variants that retain activity, and the resulting variants can be screened for their ability to bind to the respective receptors and elicit the respective cellular responses. Examples of conservative amino acid substitutions other than at positions 236, 239, 330, 332, 428, and 434 can be found in U.S. Patent Nos. 9,803,023, 9,663,582, and US20170349662, the contents of which are incorporated herein by reference.

[0059] The polypeptides described in the present invention can be obtained as recombinant polypeptides. To prepare a recombinant polypeptide, the nucleic acid encoding it (e.g., SEQ ID NO: 2 or SEQ ID NO: 3) can be linked to another nucleic acid encoding a fusion partner, such as glutathione-S-transferase (GST), a 6x-His epitope tag, or an M13 Gene 3 protein. The resulting fusion nucleic acid expresses a fusion protein in a suitable host cell, which can be isolated by methods known in the art. The isolated fusion protein can be further treated, for example, by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of the present invention.

[0060] The above-described modified antibodies having Fc variants are within the scope of the present invention. Further variants of the antibody sequences with improved affinity can be obtained using methods known in the art and are included within the scope of the present invention. For example, amino acid substitution can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve the translation efficiency in an expression system for producing the antibody.

[0061] In certain embodiments, antibodies of the present invention comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences. One or more of these CDR sequences comprise specific amino acid sequences based on the preferred antibodies described herein, or conservative modifications thereof, such that the antibody retains desirable functional properties (e.g., neutralizing pathogens such as multiple HIV-1 virus strains). Similarly, antibodies of the present invention may comprise the Fc region of a preferred antibody described herein, e.g., SEQ ID NO: 2 or SEQ ID NO: 3, a section thereof, or a conservative modification thereof. One or more amino acid residues within the CDR or non-CDR regions of an antibody of the present invention may be substituted with another amino acid residue from the same side chain family, and the modified antibody may be tested for retained function using the functional assays described herein. Similarly, the variant Fc regions described herein may have one or more conservative amino acid substitutions.

[0062] Other modifications of antibodies are also encompassed within the scope of this specification. For example, the antibody can be conjugated to a cytotoxic agent, a chemotherapeutic agent, or one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. The antibody can also be encapsulated in microcapsules prepared, for example, by coacervation or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0063] In certain embodiments, the antibodies of the described invention are bispecific and can bind to two different epitopes of a single antigen. Other such antibodies can combine a first antigen-binding site with a second antigen-binding site. Bispecific antibodies can also be used to localize cytotoxic drugs to infected cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). See, e.g., WO96 / 16673, U.S. Patent No. 5,837,234, WO98 / 02463, U.S. Patent No. 5,821,337, and Mouquet et al., Nature. 467, 591-5 (2010).

[0064] Methods for producing bispecific antibodies are known in the art. The traditional method for producing full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, for example, Millstein et al., Nature, 305:537-539 (1983)). Similar procedures are disclosed, for example, in WO93 / 08829, Traunecker et al., EMBO J., 10:3655-3659 (1991), and also see Mouquet et al., Nature. 467, 591-5 (2010). Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical conjugation. See, for example, Brennan et al., Science, 229: 81 (1985).

[0065] Typically, the antibodies used or described in the present invention can be produced using conventional hybridoma technology or can be made recombinantly using vectors and methods available in the art. Human antibodies can also be produced by in vitro activated B cells (see, e.g., U.S. Patent Nos. 5,567,610 and 5,229,275). General methods in molecular genetics and genetic engineering useful in the present invention are described in the latest edition of Molecular Cloning: A Laboratory Manual (Sambrook et al., Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Lab., 2012), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991, Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutscher et al. (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis et al. 1990, Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney, 1987, Liss, Inc., New York, NY), and Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ (Murray, The Humana Press Inc., Clifton, NJ) Reagents, cloning vectors, and kits for genetic manipulation are available from commercial vendors such as BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0066] Other techniques known in the art for selecting antibodies from libraries using enrichment techniques, including, but not limited to, phage display, ribosome display (Hanes and Pluckthun, 1997, Proc. Nat. Acad. Sci. 94: 4937-4942), bacterial display (Georgiou et al., 1997, Nature Biotechnology 15: 29-34), and / or yeast display (Kieke et al., 1997, Protein Engineering 10: 1303-1310), can be utilized as an alternative to the previously discussed techniques for selecting single chain antibodies. Single chain antibodies are selected from libraries of single chain antibodies generated directly using filamentous phage technology. Phage display technology is disclosed in U.S. Patent Nos. 5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255; 6,140,471; 6,225,447; 6,291,650; 6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081 and other U.S. counterpart patent applications, or applications that rely on priority applications of GB 9206318 filed May 24, 1992; (Vaughn et al. 1996, Nature Biotechnology 14: Single chain antibodies are known in the art (see, for example, techniques by Cambridge Antibody Technology (CAT)), as disclosed in, for example, "Single chain antibodies" (see also, for example, "Single chain antibodies" (see, for example, "Single chain antibodies"), and are known in the art (see, for example, techniques by Cambridge Antibody Technology (CAT)). Single chain antibodies can be engineered and constructed using available recombinant DNA techniques, such as DNA amplification methods (e.g., PCR), or optionally using the cDNA of the respective hybridoma as a template.

[0067] Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies without producing endogenous immunoglobulins. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into the germline mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all GenPharm); U.S. Patent No. 5,545,807; and WO 97 / 17852. The animals can be genetically engineered to produce human antibodies comprising the polypeptides of the described invention.

[0068] Any known monoclonal antibody can benefit from the Fc region variants and modifications disclosed herein by fusing its antigen-binding section to the Fc region / region variants described herein. Examples of known therapeutic monoclonal antibodies include any of the following non-limiting antibodies: 3F8, 8H9, abagovomab, abciximab, abirulumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afacevicumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, aniflomab, anru Kinzumab, apolizumab, arcitumomab, ascribacumab, acelizumab, atezolizumab, atinumab, atlizumab, atlizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezolotoxumab, biciromab, bimagumab, bimekizumab, bivatuzumab mertansine, bleselumab, blinatumomab, brontovetomab, brozozumab, bococizumab, brazikumab ...bleselumab, bleselumab, bleselumab, bleselumab, bleselumab, bleselumab, bleselumab, bleselumab, bleselumab, bleselumab, bleselumab, bleselum Ntuximab vedotin, briakinumab, brodalumab, brolucizumab, brontixumab, burosumab, cabilalizumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, carotuximab, catomaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, sergituzumab amnaleukin, certolizumab pegol, cetuximab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenolipid Cimabu, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine, conatumab, concizumab, CR6261, crenezumab, clotedomab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumab, dectrecumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, delrotuximab biotin, detumomab, dinutuximab, dilidabumab, domagrozumab, dolimomab alitoxin, dorozitumab,Durigotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, ergemtumab, ertuzumab, ersilimomab, emactuzumab, emibetuzumab, emicizumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enotimab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxumab, etaracizumab, etroli Ibuprofen, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faretuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fivatuzumab, ficlatuzumab, fititumumab, filibumab, framvotumab, fretikumab, fontolizumab, foralumab, foravirumab, frezolimumab, furanumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, dilentuximab, glenbutamumab Vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, igovomab, IMAB362, imalumab, inciromab, ingatumab, incratumab, indatuximab ravtansine, indusatumab vedotin, inebilizumab, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumab, isatuximab, itolizumab, ixekizumab, keliximab, labetuzumab, lampalizumab, lanadelumab, Landgro Zumab, laprituximab entansine, lebrikizumab, remaresomab, lendalizumab, lenzilumab, lerdelimumab, lexatumab, ribivirumab, rifatuzumab vedotin, ligelizumab, rilotumab satetraxetan, lintuzumab, lirilumab, roderucizumab, lokivetmab, lorvotuzumab mertansine, lucatumab, lurizumab pegol, lumiliximab, lumutuzumab, MABp1, maptamumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, milatuzumab, minletumomab,Mirvetuximab soravtansine, mitumomab, mogamulizumab, monalizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, naratuximab emtansine, narunatumab, natalizumab, nabixizumab, nabixizumab Bumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obilutoxaximab, obinutuzumab, occaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, ontuximab Ibuprofen, opisinumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, otleruzumab, oxelumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuximab, patec lisumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placuramab, prosalizumab, pogalizumab, polatuzumab vedotin, ponezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO 140, Kirizumab, Racotumomab, Ladretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Linucumab, Risankizumab, Rituximab, Ribabatumumab pegol, Lobatumumab, Loredomab, Romosozumab, Rontalizumab, Robalpituzumab Tecilline, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Sapelizumab, sarilumab, satumomab pendetide, secukinumab, serivantomab, cetoxaximab, sevirumab, SGN-CD19A, SGN-CD33A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan,Tadocizumab, talizumab, tamtubetomab, tanezumab, taplitumomab paptox, talexuzumab, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, ticilimumab, tigatuzumab, tildrakizumab, timolumab, tisotumab vedotin, Tnx-650, tocilizumab, toralizumab, tosatokizumab, tositumomab, tobetumab, traloximab, trastuzumab, trastuzumab entansine, TRBS07, tregalizumab, tremelimumab, trevog lumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadatuximab butarilin, bundletuzumab vedotin, vanticizumab, vanucizumab, bapaliximab, varlilumab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, bisilizumab, bovalilizumab, volociximab, borsetuzumab mafodotin, votumumab, xentuzumab, zalutumumab, zanolimumab, zatuximab, diralimumab, zolimomab alitox, and combinations thereof.

[0069] The targets may include any of the following non-limiting targets: β-amyloid, 4-1BB, 5AC, 5T4, α-fetoprotein, angiopoietin, AOC3, B7-H3, BAFFc-MET, c-MYC, C242 antigen, C5, CA-125, CCL11, CCR2, CCR4, CCR5, CD4, CD8, CD11, CD18, CD125, CD140a, CD127, CD1 5, CD152, CD140, CD19, CD2, CD20, CD22, CD23, CD25, CD27, CD274, CD276, CD28, CD3, CD30, CD33, CD37, CD3 8, CD4, CD40, CD41, CD44, CD47, CD5, CD51, CD52, CD56, CD6, CD74, CD80, CEA, CFD, CGRP, CLDN, CSF1R, CSF2, CTGFCTLA-4, CXCR4, CXCR7, DKK1, DLL3, DLL4, DR5, EGFL7, EGFR, EPCAM, ERBB2, ERBB3, FAP, FGF23, FGFR1, GD2, GD3 , GDF-8, GPNMB, GUCY2C, HER1, HER2, HGF, HIV-1, HSP90, ICAM-1, IFN-α, IFN-γ, IgE, CD221, IGF1, IGF2, IGHE, IL-1 IL2, IL-4, IL-5, IL-6, IL-6R, IL-9, IL-12 IL-15, IL-15R, IL-17, IL-13, IL-18, IL-1 beta, IL-22, IL-23, IL23A, integrin, ITGA2, IGTB2, Lewis Y antigen, LFA-1, LOXL2, LTA, MCP-1, MIF, MS5A1, MUC1, MUC16, MSLN, myostatin, MMP superfamily, NCA-90, NFG, NOGO-A, Notch1, NRP1, OX-40, OX-40L, P2X superfamily, PCSK9, PD-1, PD-L1, PDCD1, PDGF-R, RANKL, RHD, RON, TRN4, serum albumin, SDC1, SLAMF7, SIRPα, SOST, SHP1, SHP2, STRAP1, TAG-72, TEM1, TIGIT, TFPI, TGF-β, TNF-α, TNF superfamily, TRAIL superfamily, Toll-like receptors, WNT superfamily, VEGF-A, VEGFR-1, VWF, cytomegalovirus (CMV), respiratory syncytial virus (RSV), hepatitis B, hepatitis C, influenza A hemagglutinin, rabies virus, HIV virus, herpes simplex virus, and combinations thereof. Other targets or antigens can be found in U.S. Patent No. 9,803,023, U.S. Patent No. 9,663,582, and U.S. Patent No. 20170349662, the contents of which are incorporated herein by reference.

[0070] 3. Nucleic acid Another aspect of the present invention features an isolated nucleic acid comprising a sequence encoding the above-described polypeptide, protein, or antibody. A nucleic acid refers to a DNA molecule (e.g., cDNA or genomic DNA), an RNA molecule (e.g., mRNA), or a DNA or RNA analog. The DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA. An "isolated nucleic acid" refers to a nucleic acid whose structure is not identical to that of any naturally occurring nucleic acid or any fragment of a naturally occurring genomic nucleic acid. Thus, the term covers, for example, (a) DNA having the sequence of a portion of a naturally occurring genomic DNA molecule, but not both adjacent to the coding sequences that flank that portion of the molecule in the genome of a naturally occurring organism; (b) a nucleic acid incorporated into a prokaryotic or eukaryotic vector or genomic DNA in such a manner that the resulting molecule is not identical to the naturally occurring vector or genomic DNA; (c) another molecule, such as a cDNA, a genomic fragment, a fragment generated by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The above nucleic acids can be used to express the polypeptides, fusion proteins, or antibodies of the invention. For this purpose, the nucleic acid may be operably linked to appropriate regulatory sequences to generate an expression vector.

[0071] A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. A vector can be capable of autonomous replication or can be integrated into a host DNA. Examples of vectors include plasmids, cosmids, or viral vectors. The vector contains a nucleic acid in a form suitable for expression in a host cell. Preferably, the vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed.

[0072] "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and / or inducible sequences. The manipulation of expression vectors may depend on factors such as the choice of host cell to be transformed, the desired protein or RNA expression level, etc. The expression vector can be introduced into host cells to produce the polypeptide of the present invention. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that initiates mRNA at a high frequency.

[0073] Any of the above polynucleotides, or biologically equivalent polynucleotides available to those skilled in the art for the same intended purpose, can be inserted into an appropriate expression vector and combined with other DNA molecules to form a "recombinant DNA molecule" that expresses the receptor. These vectors can be composed of DNA or RNA; for most cloning purposes, DNA vectors are preferred. Conventional vectors include plasmids, modified viruses, bacteriophages, and cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA. Determining the appropriate vector for a particular application is well within the skill of the artisan.

[0074] Various mammalian expression vectors can be used to express the above-mentioned IgG Fc in mammalian cells. As described above, an expression vector can be a DNA sequence necessary for the transcription of cloned DNA and the translation of its mRNA in a suitable host. The vector can be used to express eukaryotic DNA in various hosts, such as bacteria, blue-green algae, plant cells, insect cells, and animal cells. Specially designed vectors allow for the shuttling of DNA between hosts, such as bacteria-yeast or bacteria-animal cells. A properly constructed expression vector must contain an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of useful restriction enzyme sites, high copy number capability, and an active promoter. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids, or viruses. Commercially available mammalian expression vectors that may be suitable include, but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38, and pLITMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC 37565).

[0075] Host cells containing the above-described nucleic acids are also within the scope of the present invention. Examples include bacterial cells (e.g., E. coli cells, insect cells (e.g., using baculovirus expression vectors)), yeast cells, or mammalian cells. See, for example, Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. To produce a polypeptide of the present invention, host cells can be cultured in a medium under conditions that allow expression of the polypeptide encoded by the nucleic acid of the present invention, and the polypeptide can be purified from the cultured cells or the cell medium. Alternatively, the nucleic acid of the present invention can be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and T7 polymerase.

[0076] All naturally occurring, genetically engineered, and chemically synthesized IgG Fc can be used to practice the invention disclosed herein. IgG Fc obtained by recombinant DNA technology can have the same amino acid sequence as SEQ ID NO: 2 or SEQ ID NO: 3, or a functionally equivalent amino acid sequence. The term "IgG Fc" also covers chemically modified versions. Examples of chemically modified IgG Fc include IgG Fc that has undergone structural changes, additions, or deletions of carbohydrate chains, and IgG Fc to which compounds such as polyethylene glycol have been conjugated.

[0077] The function and efficacy of the polypeptides / proteins / antibodies thus produced can be verified using animal models as described below. A statistically significant increase in in vivo half-life, increased affinity to FcγR receptors (e.g., FcγRIIA, FcγRIIIA, or FcγRIIIB), FcRn, and / or enhanced cytotoxic activity indicates that the polypeptides / proteins / antibodies are candidates for treating the diseases mentioned below. A skilled artisan will be able to combine and adapt various research tools without undue experimentation. Once purified and tested by standard methods or according to the assays and methods described in the Examples below, the polypeptides / proteins / antibodies can be included in pharmaceutical compositions for treating diseases as described below.

[0078] 4. Composition Compositions comprising a suitable carrier and one or more of the above-described agents, such as IgG Fc variants, related proteins, or related antibodies, are within the scope of the present invention. The compositions may be pharmaceutical compositions comprising a pharmaceutically acceptable carrier, or cosmetic compositions comprising a cosmetically acceptable carrier.

[0079] Any of the compositions in the above-mentioned forms can be used to treat the diseases described herein.Effective amount refers to the amount of active compound / medicine that is required to give the treated subject a therapeutic effect.As those skilled in the art will recognize, effective amount varies depending on the type of disease to be treated, route of administration, excipient use and the possibility of being used in combination with other therapeutic treatments.

[0080] The pharmaceutical compositions of the present invention can be administered parenterally, orally, nasally, rectally, topically, or bucally. As used herein, the term "parenteral" refers to subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

[0081] Sterile injectable compositions can be solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, including, but not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, fixed oils are conventionally used as solvents or suspending media (e.g., synthetic mono- or diglycerides). Fatty acids, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, and their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as, but not limited to, carboxymethylcellulose or similar dispersants. Other commonly used surfactants, including, but not limited to, TWEENS and SPANS, and other similar emulsifiers and bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0082] Compositions for oral administration can be orally acceptable dosage forms such as capsules, tablets, emulsions, and aqueous suspensions, dispersions and solutions.For tablets, commonly used carriers include but are not limited to lactose and cornstarch.Lubricants, such as but not limited to magnesium stearate, are also typically added.For oral administration in capsule form, useful diluents include but are not limited to lactose and dry cornstarch.When aqueous suspension or emulsion is orally administered, active ingredient can be suspended or dissolved in the oil phase combined with emulsifier or suspending agent.If necessary, certain sweeteners, flavorings or colorings can be added.

[0083] Pharmaceutical compositions for topical administration according to the described invention can be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils. Alternatively, topical formulations can be in the form of patches or bandages impregnated with the active ingredient, which may optionally contain one or more excipients or diluents. In some preferred embodiments, the topical formulations include substances that enhance absorption or penetration of the active agent through the skin or other affected areas. The topical compositions are useful for treating inflammatory skin conditions, including, but not limited to, eczema, acne, rosacea, psoriasis, contact dermatitis, and responses to poison ivy.

[0084] A topical composition comprises a safe and effective amount of a dermatologically acceptable carrier suitable for application to the skin. A "cosmetically acceptable" or "dermatologically acceptable" composition or ingredient refers to a composition or ingredient suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic reaction, or the like. The carrier allows the active agent and any optional ingredients to be delivered to the skin at an appropriate concentration. Thus, the carrier may function as a diluent, dispersant, solvent, or the like to ensure that the active material is applied to the selected target at the appropriate concentration and is uniformly dispersed. The carrier may be solid, semi-solid, or liquid. The carrier may be in the form of a lotion, cream, or gel, particularly one with a sufficient thickness or yield point to prevent the active material from settling. The carrier may be inert or may have dermatological benefits. It should also be physically and chemically compatible with the active ingredients described herein and should not unduly impair the stability, efficacy, or other use benefits associated with the composition. The topical composition may be a cosmetic or dermatological product in any form known in the art for topical or transdermal application, such as a solution, aerosol, cream, gel, patch, ointment, lotion, or foam.

[0085] 5.Treatment method The above-mentioned agents can be administered to subjects for the prophylactic and therapeutic treatment of various diseases, such as neoplastic diseases, inflammatory diseases, and infectious diseases. For example, the agents can be used to treat viral or bacterial infections, metabolic or autoimmune diseases, or cancer or other cell proliferative diseases.

[0086] A. Neoplastic diseases In one aspect, the invention relates to treating a subject in vivo using the above-described agents so that the growth and / or metastasis of a cancerous tumor is inhibited. In one embodiment, the invention provides a method of inhibiting the growth of tumor cells and / or limiting the metastatic spread thereof in a subject, comprising administering to the subject a therapeutically effective amount of the above-described agent.

[0087] Non-limiting examples of cancers that are preferred for treatment include chronic or acute leukemias such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, breast cancer, ovarian cancer, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), colon cancer, and lung cancer (e.g., non-small cell lung carcinoma). Additionally, the present invention includes refractory or recurrent malignant tumors whose growth can be inhibited using the antibodies of the present invention. Examples of other cancers that may be treated using the methods of the present invention include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers such as those induced by asbestos, and combinations of these cancers.

[0088] The above-mentioned treatment can also be combined with standard cancer treatment. For example, it can be effectively combined with chemotherapy regimen. In these cases, it may be possible to reduce the dose of chemotherapy agent administered (Mokyr, M. et al. (1998) Cancer Research 58: 5301-5304).

[0089] Other antibodies that can be used to activate host immune responsiveness can be used in combination with the agents of the present invention. These include molecules that target the surface of dendritic cells, activating DC function and antigen presentation. For example, anti-CD40 antibodies can effectively replace T cell helper activity (Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in combination with the multispecific molecules of the present invention (Ito, N. et al. (2000) Immunobiology 201 (5) 527-40). Similarly, antibodies targeting T cell costimulatory molecules such as CTLA-4 (e.g., U.S. Patent No. 5,811,097), CD28 (Haan, J. et al. (2014) Immunology Letters 162:103-112), OX-40 (Weinberg, A. et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine 3: 682-685 (1997)), and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266), or antibodies targeting PD-1 (U.S. Patent No. 8,008,449), PD-1L (U.S. Patent Nos. 7,943,743 and 8,168,179), can provide enhanced levels of T cell activation. In other examples, the multispecific molecules of the invention may be used in combination with anti-tumor antibodies such as, by way of example, Rituxan (rituximab), Herceptin (trastuzumab), Beksar (tositumomab), Zevalin (ibritumomab), Campath (alemtuzumab), Lymphoside (epratuzumab), Avastin (bevacizumab), and Tarceva (erlotinib).

[0090] B. Inflammatory diseases The described invention provides a method for treating inflammatory diseases in a subject. The term "inflammatory disease" refers to diseases characterized by abnormal or unwanted inflammation, such as autoimmune diseases. Autoimmune diseases are diseases characterized by chronic activation of immune cells under non-activating conditions. Examples include psoriasis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, lupus, type I diabetes, primary biliary cirrhosis, and transplantation.

[0091] Other examples of inflammatory diseases that can be treated by the methods of the present invention include asthma, myocardial infarction, stroke, inflammatory skin diseases (e.g., dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, necrotizing vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic myositis, polymyositis, dermatomyositis, and eosinophilic fasciitis), acute respiratory distress syndrome, fulminant hepatitis, hypersensitivity lung disease (e.g., hypersensitivity pneumonitis, eosinophilic pneumonia, delayed hypersensitivity, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis), and allergic rhinitis. Additional examples also include myasthenia gravis, juvenile-onset diabetes, glomerulonephritis, autoimmune thyroiditis, ankylosing spondylitis, systemic sclerosis, acute and chronic inflammatory diseases (e.g., systemic anaphylaxis or hypersensitivity reactions, drug allergies, insect sting allergies, allograft rejection, and graft-versus-host disease), and Sjogren's syndrome.

[0092] Subjects who are to be treated for inflammatory disease can be identified by standard diagnostic techniques for the disease. If necessary, the subject can be tested for the level or proportion of one or more cytokines or cells in a test sample obtained from the subject by methods known in the art. If the level or proportion is below a threshold (obtainable from a normal subject), the subject is a candidate for the treatment described herein. To confirm the suppression or treatment, the level or proportion of one or more of the above cytokines or cells in the subject after treatment can be evaluated and / or verified.

[0093] C. Infectious disease The present invention also relates to the treatment of infectious diseases using the above-mentioned agents that target antigens on or in pathogens. Examples of infectious diseases herein include diseases caused by pathogens such as viruses, bacteria, fungi, protozoa, and parasites. Infectious diseases can be caused by adenovirus, cytomegalovirus, dengue fever, Epstein-Barr, hantavirus, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus (HIV), human papillomavirus (HPV), influenza, measles, mumps, papovavirus, polio, respiratory syncytial virus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis, etc. Infectious diseases can also be caused by bacteria such as Bacillus anthracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, diphtheria, Escherichia coli, Legionella, Helicobacter pylori, Mycobacterium rickettsiae, Mycoplasma necessaria, pertussis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus aureus, Staphylococcus aureus, Vibrio cholerae, Yersinia pestis, and the like. Infectious diseases can also be caused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Penicillium marneffei, and the like. Infectious diseases can also be caused by protozoa and parasites such as chlamydia, coccidioides, leishmania, malaria, rickettsia, trypanosoma, and the like.

[0094] The therapeutic methods may be performed in vivo or ex vivo, alone or in combination with other drugs or therapies. A therapeutically effective amount may be administered in one or more administrations, applications or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0095] The agents can be administered in vivo or ex vivo, alone or in combination with other drugs or therapies (i.e., cocktail therapy), for co-administration. As used herein, the term "co-administration" or "co-administered" refers to the administration of at least two agents or therapies to a subject. In some embodiments, co-administration of two or more agents / therapies occurs simultaneously. In other embodiments, a first agent / therapy is administered before a second agent / therapy. Those skilled in the art will understand that the formulations and / or routes of administration of the various agents / therapies used may vary.

[0096] In the in vivo approach, a compound or agent is administered to a subject. Generally, the compound or agent is suspended in a pharmaceutically acceptable carrier (such as, but not limited to, saline), and then administered orally or by intravenous injection, or injected or implanted subcutaneously, intramuscularly, intrathecally, intraperitoneally, rectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonary.

[0097] The required dosage is determined by the choice of route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other medications being administered; and the attending physician's judgment. Suitable dosages range from 0.01 to 100 mg / kg. Variations in the required dosage should be anticipated, taking into account the variety of compounds / agents available and the differing efficiencies of various administration routes. For example, oral administration is expected to require higher dosages than administration by intravenous infusion. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art. Encapsulation of the compound in an appropriate delivery vehicle (e.g., polymeric microparticles or implantable devices) can enhance delivery efficiency, particularly for oral delivery. [Example]

[0098] 6. Working Example Example 1 This example describes the materials and methods used in Examples 2-3 below.

[0099] Materials and Methods Mouse strains All mouse in vivo experiments were performed in accordance with federal law and institutional guidelines and were approved by the Rockefeller University Institutional Animal Care and Use Committee. Mice were bred and maintained at the Rockefeller University Center for Comparative Biological Sciences. The following strains were used in the experiments: (i) FcγR-deficient mice (FcγR), previously developed and characterized in Smith, P et al. Proc Natl Acad Sci USA 109, 6181-6186 (2012); null (ii) the FcγR humanized mouse (mFcγRα) generated and extensively characterized in Smith, P et al. Proc Natl Acad Sci USA 109, 6181-6186 (2012) null , Fcgrl - / - , hFCGR1A + , hFCGR2A + , hFCGR2B + , hFCGR3A + , hFCGR3B + (iii) FcγR / FcRn humanized mouse (mFcγRα null , Fcgr1 - / - , Fcgrt - / - , hFCGR1A + , hFCGR2A + , hFCGR2B + , hFCGR3A + , hFCGR3B + , hFCGRT + ) were generated by crossing FcγR-humanized mice with FcRn-humanized mice (developed in Petkova, SB et al. Int Immunol 18, 1759-1769); (iv) FcγR / CD20-humanized mice (mFcγRα null , Fcgrl - / - , hFCGR1A + , hFCGR2A + , hFCGR2B + , hFCGR3A + , hFCGR3B + , hCD20 + ).

[0100] Surface plasmon resonance (SPR) analysis The binding affinities of the human IgG1 Fc region variants to FcγR and FcRn were determined by surface plasmon resonance (SPR) using a previously described protocol (Wang, T. T. et al., Science 355, 395-398 (2017) and Li, T. et al., Proc Natl Acad Sci USA 114, 3485-3490, (2017)). All experiments were performed on a Biacore T200 SPR system (GE Healthcare) in HBS-EP 1000 at 25°C. + The assay was performed in buffer (pH 7.4 for FcγR and pH 6.0 for FcRn). Recombinant Protein G (Thermo Fisher) was immobilized on a surface FcM5 sensor chip (GE Healthcare) using amine coupling chemistry at a concentration of 500 resonance units (RU). Human IgG1 Fc variants were captured on the Protein G-bound surface (250 nM injected at 20 μl / min for 60 seconds), and recombinant human, rhesus, or mouse FcγR ectoregions (7.8125–2000 nM; Sino Biological) or human FcRn / β2 microglobulin (1.95–500 nM; Sino Biological) were injected through the flow cell at a flow rate of 20 μl / min. The association time was 60 seconds, followed by a 600-second dissociation phase. At the end of each cycle, the sensor surface was regenerated with 10 mM glycine, pH 2.0 (50 μl / min; 40 s). Background binding to an immobilized blank flow cell was subtracted, and affinity constants were calculated using a 1:1 Langmuir binding model with BIAcore T200 evaluation software (GE Healthcare).

[0101] In vivo cytotoxicity model platelets, CD4 + T cells, and hCD20 +B cell depletion experiments were performed in FcγR-humanized and FcγR / FcRn-humanized mice using previously described protocols (Smith, P et al. Proc Natl Acad Sci USA 109, 6181-6186 (2012) and Wang, TT et al. Science 355, 395-398 (2017)). For B cell depletion experiments in rhesus macaques, rhesus macaques were administered (iv) 0.05 mg / kg of wild-type human IgG1 or the GAALIE (G236A / A330L / I332E) variant of anti-CD20 mAb 2B8. + The abundance and cell numbers were analyzed in the blood by flow cytometry at various time points before and after antibody administration.

[0102] Antibody expression, purification, and analysis Antibodies were produced by transient transfection of HEK293T or Expi293 cells as previously described in Bournazos, S. et al. Cell 158, 1243-1253 (2014). Antibodies were purified using Protein G Sepharose 4 Fast Flow or MabSelect SuRe LX affinity purification medium (GE Healthcare). Purified proteins were dialyzed against PBS and sterile filtered (0.22 μm). Purity was assessed by SDS-PAGE and Coomassie staining and estimated to be >90%. Protein Tm was measured using the Protein Thermal Shift Dye Kit (ThermoFisher) according to the manufacturer's instructions associated with a QuantStudio 6K Flex real-time thermal cycler.

[0103] Quantification of serum IgG levels Serum concentrations of human IgG1 variants were quantified using neutravidin-coated plates (5 μg / ml, overnight) coated with biotinylated goat anti-human IgG (absorbed mouse IgG, Jackson Immunoresearch) for mouse serum samples and CaptureSelect™ for rhesus monkey plasma samples. TM Plates were incubated with either Human IgG-Fc PK Biotin Conjugate or Human IgG-Fc PK Biotin Conjugate. After incubation (60 min at room temperature), plates were blocked for 2 h with PBS + 2% (w / v) BSA + 0.05% (v / v) Tween 20. Serially diluted serum samples (1:3 starting with a 1:10 dilution) were incubated for 1 h. IgG binding was detected using goat anti-human IgG (Fcγ-specific, 1 h; 1:5000; Jackson Immunoresearch). Plates were developed using a TMB (3,3',5,5'-tetramethylbenzidine) two-component peroxidase substrate kit (KPL), and the reaction was stopped by adding 1 M phosphoric acid. Absorbance at 450 nm was immediately recorded using a SpectraMax Plus spectrophotometer (Molecular Devices), and background absorbance from negative control samples was subtracted.

[0104] Example 2 We developed an Fc region variant (termed GASDALIE) containing specific mutations (G236A / S239D / A330L / I332E) in the amino acid backbone of human IgG1. This variant exhibits selectively enhanced binding to the activating human FcγRs, FcγRIIa and FcγRIIIa (Smith, P., DiLillo, DJ, Bournazos, S., Li, F., and Ravetch, JV. Mouse model recapitulating human Fcgamma receptor structural and functional diversity. Proc Natl Acad Sci USA 109, 6181-6186 (2012)). In various models of antibody-mediated protection against bacterial and viral infections, the GASDALIE Fc region variant of the protective mAb demonstrated significantly improved protective activity compared to wild-type human IgG1. See Smith, P. et al., Proc Natl Acad Sci USA 109, 6181-6186 (2012); Bournazos, S. et al., Cell 158, 1243-1253 (2014); Bournazos, S. et al., J Clin Invest 124, 725-729 (2014); and DiLillo, DJ et al., Nat Med 20, 143-151 (2014).

[0105] More importantly, when the therapeutic activity of a GASDALIE variant of the anti-CD20 mAb was evaluated in a mouse model of CD20+ lymphoma, it was found that this variant not only exhibited improved cytotoxic activity against CD20+ lymphoma cells, but also promoted the induction of long-term T cell memory responses that conferred protection against subsequent lymphoma challenge (DiLillo, DJ et al. Cell 161, 1035-1045 (2015)). Mechanistic studies revealed that enhanced cytotoxicity during primary lymphoma challenge was mediated through enhanced engagement of FcγRIIIa with effector leukocytes, such as monocytes and macrophages, whereas cross-linking of FcγRIIa on dendritic cells promoted dendritic cell maturation and the induction of T cell memory responses that mediated protection during secondary challenge (DiLillo, DJ et al. Cell 161, 1035-1045 (2015)). Collectively, these studies demonstrated the improved therapeutic activity of GASDALIE Fc region variants achieved through selectively enhanced binding to human FcγRIIa and FcγRIIIa.

[0106] Despite improved Fc effector function, GASDALIE variants exhibited significantly shorter half-lives in vivo, primarily in FcγR-humanized mice and, to a lesser extent, in mouse strains lacking all classes of FcγR (Figure 1). This effect may have been due to increased affinity for FcγR and reduced protein stability in vivo. GASDALIE Fc region variants exhibited very short half-lives in vivo in non-human primates, even when combined with Fc region mutations (e.g., LS:M428L / N434S) that increase affinity for FcRn and extend half-life (Figure 2).

[0107] The present inventors have developed an Fc region variant (termed GAALIE) that exhibits all the characteristics of the GASDALIE variant, including increased affinity for FcγRIIa and FcγRIIIa and enhanced cytotoxic activity in several mAb-mediated cytotoxicity models, but unexpectedly maintains biological half-life. In the following study, the present inventors identified Fc region variants (afucosylated variants and S239D / I332E variants) that have already been evaluated in humans and exhibit increased binding affinity to FcγR without significantly compromising in vivo stability and half-life. Goede, V. et al. N Engl J Med 370, 1101-1110 (2014); Zalevsky, J. et al. Blood 113, 3735-3743 (2009); and Woyach, JA et al. Blood 124, 3553-3560 (2014).

[0108] The GAALIE variant (G236A / A330L / I332E) was characterized for its affinity to all classes of FcγR in humans, rhesus monkeys, and mice (Figures 3-8), and for its cytotoxic effector activity in platelet, CD4+ T cell, and B cell depletion models in FcγR-humanized mice (Figures 9-12). The half-life of the GAALIE variant was evaluated in FcγR-humanized mice, FcγR-deficient mice, and rhesus monkeys, and was found to exhibit a physiological half-life (Figures 13-14). Furthermore, the in vivo cytotoxicity of the GAALIE variant was evaluated in non-human primates (rhesus monkeys) in a model of mAb-mediated depletion of CD20+ B cells (Figure 15).

[0109] Example 3 To further extend the in vivo half-life of the GAALIE variants, they were combined with mutations that increased affinity for FcRn without affecting FcγR binding (Zalevsky, J. et al., Nat Biotechnol 28, 157-159 (2010) and Grevys, A. et al., J Immunol 194, 5497-5508 (2015)). These mutations included M428L and N434S (LS variant, Zalevsky, J. et al., Nat Biotechnol 28, 157-159 (2010)). The amino acid sequences of the resulting Fc region variants are shown in Figure 16. The protein melting temperatures and FcRn-binding affinities of the FcγR / FcRn-enhanced variants were measured (Figures 17 to 20). Furthermore, the in vivo half-lives of these variants were evaluated in FcRn / FcγR-humanized mice (Figure 21). As expected, GAALIE LS (G236A / A330L / I332E / M428L / N434S) exhibited a prolonged half-life in a model of mAb-mediated platelet depletion in FcγR / FcRn-humanized mice, indicating prolonged and enhanced Fc effector activity (Figure 22).

[0110] Example 4 To mimic the interaction of antibodies designed for clinical use with human FcRs, B16-FUT3 cells were inoculated into FcγR-humanized mice, a strain that harbors transgenes for all human FcγRs but lacks all mouse FcRs (Smith, P. et al., Proc Natl Acad Sci USA 109, 6181-6186 (2012)), resulting in the recapitulation of the cellular expression pattern of human FcRs in a fully immunocompetent mouse background. B16 tumor-bearing mice were treated with sLeA-targeting antibodies, clones 5B1 and 7E3, expressing the hIgG1 subclass. Both clones, 5B1 and 7E3, demonstrated comparable therapeutic efficacy (Figure 23A), resulting in a significant reduction in the number of metastatic foci in the lungs. As observed with chimeric human-mouse antibodies (data not shown), engineering 5B1-hIgG1 with an Fc mutation (N297A) that abolishes its ability to engage human FcRs results in a loss of therapeutic efficacy of the sLeA-targeted antibody (data not shown).

[0111] In light of the above-mentioned role of FcR activation in mediating antibody-induced tumor clearance, we sought to increase the therapeutic efficacy of sLeA-targeting antibodies by increasing their affinity with activating FcRs. To do so, we redesigned a hIgG1 sLeA-targeting antibody by introducing three point mutations (G236A / A330L / I332E) ("GAALIE"). The GAALIE point mutation significantly enhanced the affinity of the sLeA-targeting antibody with two activating human FcRs, hFcγRIIA and hFcγRIIIA, while reducing binding to the inhibitory receptor hFcRIIB without suppressing binding affinity to sLeA. The redesigned 5B1 and 7E3 antibody variants exhibited superior antitumor activity compared to the parent antibody possessing the wild-type hIgG1 Fc portion (Figure 24B). These findings support the conclusion that the involvement of FcR activation is a critical step in the process of efficient antibody-mediated tumor clearance.

[0112] Example 5 Although engagement of the activating receptor hFcγRIIA was insufficient to mediate tumor clearance, engagement of hFcγRIIIA alone was necessary and sufficient for antibody-mediated tumor clearance in some tumor models. In this study, we aimed to determine whether these findings also apply to carbohydrate-targeting antibodies. We compared the antitumor activity of three Fc variants with enhanced affinity for either hFcγRIIA (GA), hFcγRIIIA (ALIE), or both (GAALIE) in mice bearing FcγR-humanized tumors (Figure 24A). The affinities of GA and ALIE hIgG1 Fc variants for different human FcRs have been reported (9, 34, 35); the GALLIE Fc variant exhibited reduced affinity for hFcRIIB and higher affinity for hFcRIIA and hFcRIIIA, and demonstrated superior ADCC function compared to the parent hIgG1, while exhibiting an in vivo half-life comparable to that of hIgG1 (data not shown).

[0113] All three Fc variants exhibited comparable antitumor potency, significantly higher than that of the wild-type parent human IgG1 antibody (Figure 24B). To confirm these findings, we compared the antitumor activity of the Fc variant 5B1-hIgG1-GAALIE (with enhanced affinity for both activating FcRs) in several recombinant mouse strains expressing human FcRs. Figure 24C shows that the 5B1-hIgG1-GAALIE variant exhibits significant but comparable antitumor activity not only in FcγR-humanized mice, but also in hFcγRIIA-only and hFcγRIIIA-only mice. As expected, tumor clearance was not observed in FcR-null mice. NK depletion did not substantially inhibit the antitumor activity of this sLeA-targeting antibody (data not shown), suggesting that tumor cell depletion is primarily mediated by effector cells expressing hFcγRIIIA and hFcRγIIA, such as macrophages.

[0114] The foregoing examples and descriptions of the preferred embodiments should be construed as illustrative, not limiting, of the present invention as defined by the claims. As will be readily understood, numerous variations and combinations of the features described above can be utilized without departing from the invention as set forth in the claims. Such variations are not to be considered a departure from the scope of the present invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. 1. An antibody comprising a polypeptide comprising an Fc variant of a human IgG1 Fc polypeptide, said Fc variant comprising alanine (A) at position 236, leucine (L) at position 330, glutamic acid (E) at position 332, serine (S) at position 239, leucine (L) at position 428, and serine (S) at position 434, numbering according to the EU index of Kabat; An antibody having said Fc variant, wherein the antibody has an extended half-life compared to an antibody having the wild-type IgG1 Fc sequence of SEQ ID NO:

1.

2. The antibody of claim 1, wherein the Fc variant comprises the sequence of SEQ ID NO:

3.

3. The antibody of claim 1 , which has specificity for a target molecule.

4. The antibody of claim 3, wherein the target molecule is selected from the group consisting of a cytokine, a soluble factor, a molecule expressed on a pathogen, a molecule expressed on a cell, and a molecule expressed on a cancer cell.

5. The antibody of claim 1 , which is selected from the group consisting of a chimeric antibody, a humanized antibody, and a human antibody.

6. (1) The antibody described in claim 1, which has a higher binding affinity to hFcγRIIA, hFcγRIIIA, hFcRn, or / and hFcγRIIIB compared to an antibody having the sequence of SEQ ID NO:

1.

7. A nucleic acid comprising a sequence encoding the antibody of any one of claims 1 to 6.

8. An expression vector comprising the nucleic acid of claim 7.

9. A host cell comprising the nucleic acid of claim 7.

10. 10. A method for producing an antibody, comprising culturing the host cell of claim 9 in a culture medium under conditions that allow expression of the antibody encoded by the nucleic acid, and purifying the antibody from the cultured cells or the culture medium of the cells. 。

11. A pharmaceutical formulation comprising: (i) the antibody of any one of claims 1 to 6; and (ii) a pharmaceutically acceptable carrier.

12. A pharmaceutical formulation comprising: (i) the nucleic acid of claim 7; and (ii) a pharmaceutically acceptable carrier.

13. Use of the antibody of any one of claims 1 to 6 in the manufacture of a medicament for treating an inflammatory disease.

14. Use of an antibody according to any one of claims 1 to 6 in the manufacture of a medicament for treating a tumor disease.

15. Use of an antibody according to any one of claims 1 to 6 in the manufacture of a medicament for treating an infectious disease.

16. 10. Use of the nucleic acid of claim 7 in the manufacture of a medicament for treating an inflammatory disease.

17. 10. Use of the nucleic acid according to claim 7 in the manufacture of a medicament for treating a tumor disease.

18. 10. Use of the nucleic acid of claim 7 in the manufacture of a medicament for treating an infectious disease.

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