Fc variants with altered binding to Fc receptors

Abstract

The present invention relates to modified immunoglobulin constant region (Fc) mutant molecules with reduced binding to FcγR or C1q, which can significantly reduce undesirable ADCC and / or ADCP and / or CDC effector functions in vivo, and further relates to uses of said Fc mutant molecules, fusion proteins comprising said mutant molecules and uses thereof.

Description

[Technical field]

[0001] The present invention relates generally to the fields of immunology and antibody engineering, and specifically to variants of IgG immunoglobulins, methods for their preparation and uses. [Background technology]

[0002] With the development of therapeutic antibodies in recent years, researchers, on the one hand, continue to explore and discover new targets, and on the other hand, continue to make upgrade modifications to druggable antibodies to enhance or enhance their beneficial effects, where one of the research focuses is focused on modifications to antibody Fc region.

[0003] As is well known, the Fc region of an antibody does not have antigen-binding activity, but since it is the site where an antibody interacts with Fc receptors on the cell surface, it plays an important role in the effector functions of the antibody. Through the interaction of the Fc region with different Fc receptors, the antibody can exert various effector functions, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Regarding ADCC, ADCP, CDC effector functions, for antibodies targeted to recognize antigens expressed on tumor cells or pathogens and eliminate malignant cells, effective or even enhanced ADCC, ADCP, CDC effector functions contribute to the therapeutic efficacy of the antibody, whereas for antibodies targeted to block cell surface receptors / cytokines or immune regulation, for example, for antigens expressed on T cells, it is often necessary to reduce or even eliminate ADCC, ADCP, CDC effector functions, because antibodies of target cell surface antigens induce unwanted immune stimulation and associated effector functions as well as complement activation in immune cells, leading to adverse outcomes; this is particularly important for immune checkpoint inhibitor antibodies, where low loss of target cells is required.

[0004] Human IgG subclasses (IgG1, IgG2, IgG3, and IgG4) have different immune functions, for example, IgG antibodies of different subclasses have different ADCC activities, and compared with other subclasses, IgG1 and IgG3 have relatively strong ADCC activities, and IgG1, IgG2, IgG3, and IgG4 have antibody-dependent cellular phagocytosis (ADCP). Because therapeutic antibodies and Fc fusion constructs often cannot destroy or damage the desired local cells or tissues and need to target and activate or neutralize the target ligand functional region, there is always a real demand for Fc variants that reduce or eliminate Fc effector functions (e.g., reduce or eliminate ADCC and / or ADCP activities, reduce or eliminate CDC activity) in practical production applications, and the present application meets this demand.

[0005] According to existing literature reports, some amino acid mutations in the Fc region may have the effect of reducing or eliminating ADCC and / or ADCP activity, or reducing or eliminating CDC activity. For example, XENCOR's patent family US8734791B2, US10183999B2, US8883147B2, Roche's patent family CN103476795B, etc., many of which have the effect of reducing ADCC and CDC activity, but mutations that completely eliminate the ADCC and CDC effects are relatively rare. The present application obtains an Fc mutant molecule that completely eliminates the ADCC and CDC effects by targeted modification of the wild-type Fc amino acid sequence. Summary of the Invention

[0006] The present invention provides modified immunoglobulin constant region (Fc region) variant molecules that can be used in engineered antibodies or antibody-based therapeutics.

[0007] By including the mutations and / or combinations thereof disclosed in the present application in the Fc region, the antibodies, antibody-based therapeutics, and other molecules comprising the mutant Fc region of the present application have significantly reduced binding to FcγR or C1q compared to molecules comprising a wild-type Fc region, thereby significantly reducing undesirable ADCC and / or ADCP and / or CDC effector functions in vivo. Furthermore, the Fc mutations disclosed in the present application do not affect the ability of the antibodies, antibody-based therapeutics, and other molecules comprising the mutant Fc region of the present application to bind to FcRn, and therefore do not affect the half-life of the corresponding molecules. Thus, the present application provides Fc region polypeptide molecules having specific mutations, antibody molecules or similar structural molecules comprising said mutant Fc regions that have reduced or eliminated ADCC and / or ADCP and / or CDC effector functions, but still retain the ability to bind to FcRn.

[0008] In a first aspect, the application provides Fc variants with different modifications, which, compared to a wild-type Fc region, exhibit reduced affinity for Fc receptors and / or C1q, thus reducing or eliminating ADCC, CDC and ADCP effector function induced by the Fc variants. In one embodiment, the ADCC, CDC and ADCP effector function induced by the Fc variants is reduced by at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or even completely eliminated of the ADCC, CDC and ADCP effector function induced by the corresponding wild-type.

[0009] In one embodiment, the Fc variants provided in the application comprise one or more amino acid deletions. In one specific embodiment, the Fc variants provided in the application comprise a deletion of an amino acid at position 329 (Δ329) as numbered according to the EU index of Kabat. In one specific embodiment, the difference between the Fc variants provided in the application and the corresponding wild-type Fc is in a deletion of position 329 (Δ329) as numbered according to the EU index of Kabat. In another specific embodiment, the Fc variants provided in the application comprise a deletion of amino acids at positions 329 and 330 (Δ329 and Δ330) as numbered according to the EU index of Kabat. In one specific embodiment, the difference between the Fc variants provided in the application and the corresponding wild-type Fc is in a deletion of positions 329 and 330 (Δ329 and Δ330) as numbered according to the EU index of Kabat.

[0010] In one embodiment, the Fc variants provided in the present application comprise one or more amino acid substitutions. In one specific embodiment, the Fc variants provided in the present application comprise substitutions of amino acids at positions 234 and / or 235, numbered according to the EU index of Kabat, where L, V, F at position 234 and / or L at position 235 are replaced with A, V, L, and I, respectively. In one preferred embodiment, said Fc variant comprises L234A+L235A, V234A, or F234A+L235A. In one specific embodiment, the difference between the Fc variants provided in the present application and the corresponding wild-type Fc is L234A+L235A, V234A, or F234A+L235A.

[0011] In one embodiment, the Fc variants provided herein comprise one or more amino acid deletions and one or more amino acid substitutions. In one specific embodiment, the Fc variants provided herein comprise a deletion of an amino acid at position 329 and a substitution at position 330, numbered according to the EU index of Kabat, where position 330 is substituted with G, D or Q. In one preferred embodiment, the Fc variant comprises Δ329+A330G or Δ329+S330G. In one specific embodiment, the difference between the Fc variants provided herein and the corresponding wild-type Fc is Δ329+A330G or Δ329+S330G.

[0012] In one specific embodiment, the Fc variants provided herein comprise a deletion of the amino acid at position 329 and a substitution of the amino acids at positions 234 and 235, numbered according to the EU index of Kabat, where L, V, F at position 234 and / or L at position 235 are substituted with A, V, L, and I, respectively. In one preferred embodiment, said Fc variant comprises L234A+L235A+Δ329, V234A+Δ329, or F234A+L235A+Δ329. In one specific embodiment, the difference between the Fc variants provided herein and the corresponding wild-type Fc is L234A+L235A+Δ329, V234A+Δ329, or F234A+L235A+Δ329.

[0013] In another specific embodiment, the Fc variants provided herein comprise a deletion of amino acids at positions 329 and 330 and a substitution of amino acids at positions 234 and 235, numbered according to the EU index of Kabat, where L, V, F at position 234 and / or L at position 235 are substituted with A, V, L, and I, respectively. In a preferred embodiment, said Fc variant comprises L234A+L235A+Δ329+Δ330, V234A+Δ329+Δ330, or F234A+L235A+Δ329+Δ330. In a specific embodiment, the difference between the Fc variants provided herein and the corresponding wild-type Fc is at L234A+L235A+Δ329+Δ330, V234A+Δ329+Δ330, or F234A+L235A+Δ329+Δ330.

[0014] In another specific embodiment, the Fc variants provided in the application comprise a deletion of the amino acid at position 329 and substitutions of the amino acids at positions 234, 235, and 330, numbered according to the EU index of Kabat, where L, V, F at position 234 and / or L at position 235 are substituted with A, V, L, and I, respectively, and position 330 is substituted with G, D, or Q. In one preferred embodiment, said Fc variants comprise L234A+L235A+A330G+Δ329, L234A+L235A+S330G+Δ329, V234A+L235A+A330G+Δ329, V234A+L235A+S330G+Δ329, F234A+L235A+S330G+Δ329, or F234A+L235A+A330G+Δ329. In a specific embodiment, the difference between the Fc variants provided in the application and the corresponding wild-type Fc is L234A+L235A+A330G+Δ329, L234A+L235A+S330G+Δ329, V234A+L235A+A330G+Δ329, V234A+L235A+S330G+Δ329, F234A+L235A+S330G+Δ329, or F234A+L235A+A330G+Δ329.

[0015] In some embodiments, the Fc variants provided herein are IgG1 type Fc variants, and compared to the corresponding wild-type Fc region, the variants have a reduced or even ablated ability to bind to FcγR. In other embodiments, the Fc variants provided herein are IgG2, IgG3, and IgG4 type Fc variants, and compared to the corresponding wild-type Fc region, the variants have a reduced or even ablated ability to bind to FcγR. Preferably, the Fc variants provided herein retain the ability to bind to FcRn.

[0016] The Fc variants disclosed in the present application can be applied as platform components in any context in which there is a need to reduce or even eliminate ADCC / ADCP / CDC effector function, for example in any type of antibody molecule, or molecule with a similar antibody structure, in which it is desired to reduce or even eliminate ADCC / ADCP / CDC effector function.

[0017] In one embodiment, said Fc variants are substantially based on IgG sequences, and in a preferred embodiment, said Fc variants are substantially based on human IgG sequences, and in another embodiment, said Fc variants are substantially based on human IgG1 sequences.

[0018] In alternative embodiments, the Fc variants may further comprise other modifications, such as those known in the art to reduce immunogenicity, improve stability, solubility, function and clinical benefit.

[0019] In one specific embodiment, the Fc variant comprises the following sequence: 1) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 2) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 3, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 3) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 4) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO:5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 5) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 6) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 7) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 8, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 8) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 9) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 12, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 10) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 11) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 12) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 13) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 16, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 14) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 17, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 15) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 16) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 20, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 17) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 18) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 22, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 19) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 20) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 21) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a substance consisting of said sequence; or 22) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 27, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a substance consisting of said sequence; or 23) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 28, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a substance consisting of said sequence; or 24) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 29, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a substance consisting of said sequence; or 25) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 30, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 26) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 31, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 27) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 28) An amino acid sequence of positions 221 to 447 of SEQ ID NO: 33 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence.

[0020] In a second aspect, the invention provides a polypeptide comprising an Fc variant according to the first aspect, wherein said polypeptide has reduced or eliminated ADCC and / or ADCP and / or CDC effector function, preferably said polypeptide does not cause ADCC and / or ADCP and / or CDC effects, compared to a polypeptide comprising a wild-type Fc region. In a specific embodiment, said polypeptide simultaneously has an extended half-life. In one embodiment, the ADCC, CDC and ADCP effector function induced by a polypeptide comprising an Fc variant according to the first aspect is reduced by at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or even completely eliminated of the ADCC, CDC and ADCP effector function induced by a polypeptide comprising the corresponding wild-type.

[0021] In some embodiments, the polypeptide is an antibody molecule, preferably an IgG class antibody molecule. In another embodiment, the antibody molecule is a multispecific antibody (e.g., bispecific antibody), a humanized antibody, a chimeric antibody, or an antibody fusion. Compared to the corresponding wild-type antibody molecule, the antibody molecules comprising the Fc variants provided herein have reduced or even eliminated binding ability to FcγR, and thus reduced or eliminated ADCC and / or ADCP and / or CDC effector function.

[0022] In some embodiments, the polypeptide is a fusion protein comprising one or more fusion partners operatively linked to an Fc variant, where the fusion partner may typically be any protein or small molecule, e.g., the variable region of any antibody, the target binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain, hi one embodiment, the fusion protein is, for example, an immunoadhesin.

[0023] In one embodiment, the application provides an IgG antibody comprising an Fc variant that comprises a deletion of amino acids at position 329 (Δ329), numbered according to the EU index of Kabat. In another specific embodiment, the application provides an IgG antibody comprising an Fc variant that comprises deletions of amino acids at positions 329 and 330 (Δ329 and Δ330), numbered according to the EU index of Kabat.

[0024] In one embodiment, the application provides an IgG antibody comprising an Fc variant comprising a substitution of amino acids at positions 234 and / or 235, numbered according to the EU index of Kabat, where L, V, F at position 234 and / or L at position 235 are substituted with A, V, L, and I, respectively. In one preferred embodiment, said IgG antibody comprises L234A+L235A, V234A, or F234A+L235A.

[0025] In one embodiment, the application provides an IgG antibody comprising an Fc variant comprising a deletion of the amino acid at position 329 and a substitution at position 330, numbered according to the EU index of Kabat, where position 330 is substituted with G, D or Q. In one preferred embodiment, said IgG antibody comprises Δ329+A330G or Δ329+S330G.

[0026] In one embodiment, the application provides an IgG antibody comprising an Fc variant comprising a deletion of the amino acid at position 329 and a substitution of the amino acids at positions 234 and 235, numbered according to EU index of Kabat, where L, V, F at position 234 and / or L at position 235 are substituted with A, V, L, and I, respectively. In a preferred embodiment, said IgG antibody comprises L234A+L235A+Δ329, V234A+Δ329, or F234A+L235A+Δ329.

[0027] In one embodiment, the application provides an IgG antibody comprising an Fc variant comprising a deletion of amino acids at positions 329 and 330 and a substitution of amino acids at positions 234 and 235, numbered according to EU index of Kabat, where L, V, F at position 234 and / or L at position 235 are substituted with A, V, L, and I, respectively. In a preferred embodiment, said IgG antibody comprises L234A+L235A+Δ329+Δ330, V234A+Δ329+Δ330, or F234A+L235A+Δ329+Δ330.

[0028] In one embodiment, the application provides an IgG antibody comprising an Fc variant comprising a deletion of the amino acid at position 329 and substitutions of amino acids at positions 234, 235, 330, numbered according to the EU index of Kabat, where L, V, F at position 234 and / or L at position 235 are substituted with A, V, L, and I, respectively, and position 330 is substituted with G, D, or Q. In one preferred embodiment, said IgG antibody comprises L234A+L235A+A330G+Δ329, L234A+L235A+S330G+Δ329, V234A+L235A+A330G+Δ329, V234A+L235A+S330G+Δ329, F234A+L235A+S330G+Δ329, or F234A+L235A+A330G+Δ329.

[0029] In some embodiments, the IgG antibodies provided by the present invention are IgG1 type antibodies, and compared to the corresponding wild-type antibodies, the antibodies have a reduced or even ablated ability to bind to FcγR. In other embodiments, the IgG antibodies provided by the present invention are IgG2, IgG3, or IgG4 type antibodies, and compared to the corresponding wild-type antibodies, the antibodies have a reduced or even ablated ability to bind to FcγR. Preferably, the antibodies provided by the present invention retain the ability to bind to FcRn.

[0030] In some embodiments, the antibody molecule is an anti-claudin18.2 IgG antibody.

[0031] In one specific embodiment, an IgG antibody comprising an Fc variant provided herein comprises a heavy chain and a light chain as follows: 1) comprising a heavy chain as set forth in SEQ ID NO:2 and a light chain as set forth in SEQ ID NO:9, or 2) comprising a heavy chain as set forth in SEQ ID NO:3 and a light chain as set forth in SEQ ID NO:9; or 3) comprising a heavy chain as set forth in SEQ ID NO:4 and a light chain as set forth in SEQ ID NO:9; or 4) comprising a heavy chain as set forth in SEQ ID NO:5 and a light chain as set forth in SEQ ID NO:9; or 5) comprising a heavy chain as set forth in SEQ ID NO:6 and a light chain as set forth in SEQ ID NO:9; or 6) A heavy chain as shown in SEQ ID NO: 7 and a light chain as shown in SEQ ID NO: 9, or 7) A heavy chain as shown in SEQ ID NO: 8 and a light chain as shown in SEQ ID NO: 9, or 8) A heavy chain as shown in SEQ ID NO: 11 and a light chain as shown in SEQ ID NO: 9, or 9) A heavy chain as set forth in SEQ ID NO: 12 and a light chain as set forth in SEQ ID NO: 9, or 10) A heavy chain as set forth in SEQ ID NO: 13 and a light chain as set forth in SEQ ID NO: 9, or 11) A heavy chain as set forth in SEQ ID NO: 14 and a light chain as set forth in SEQ ID NO: 9, or 12) A heavy chain as set forth in SEQ ID NO: 15 and a light chain as set forth in SEQ ID NO: 9, or 13) A heavy chain as set forth in SEQ ID NO: 16 and a light chain as set forth in SEQ ID NO: 9, or 14) A heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO: 9, or 15) A heavy chain as set forth in SEQ ID NO: 19 and a light chain as set forth in SEQ ID NO: 9, or 16) A heavy chain as set forth in SEQ ID NO: 20 and a light chain as set forth in SEQ ID NO: 9, or 17) A heavy chain as set forth in SEQ ID NO: 21 and a light chain as set forth in SEQ ID NO: 9, or 18) A heavy chain as set forth in SEQ ID NO: 22 and a light chain as set forth in SEQ ID NO: 9, or 19) A heavy chain as set forth in SEQ ID NO: 23 and a light chain as set forth in SEQ ID NO: 9, or 20) A heavy chain as set forth in SEQ ID NO: 24 and a light chain as set forth in SEQ ID NO: 9, or 21) A heavy chain as set forth in SEQ ID NO: 25 and a light chain as set forth in SEQ ID NO: 9, or 22) A heavy chain as set forth in SEQ ID NO: 27 and a light chain as set forth in SEQ ID NO: 34, or 23) A heavy chain as set forth in SEQ ID NO: 28 and a light chain as set forth in SEQ ID NO: 34, or 24) A heavy chain as set forth in SEQ ID NO: 29 and a light chain as set forth in SEQ ID NO: 34, or 25) A heavy chain as set forth in SEQ ID NO: 30 and a light chain as set forth in SEQ ID NO: 34, or 26) A heavy chain as set forth in SEQ ID NO: 31 and a light chain as set forth in SEQ ID NO: 34, or 27) A heavy chain as set forth in SEQ ID NO: 32 and a light chain as set forth in SEQ ID NO: 34, or 28) A heavy chain as shown in SEQ ID NO: 33 and a light chain as shown in SEQ ID NO: 34.

[0032] In another embodiment, the polypeptides (e.g., antibodies) comprising the Fc variants provided in the present application may further comprise other modifications in the Fc region, such as other modifications known in the art to reduce immunogenicity, increase half-life, improve stability, solubility, function and clinical benefit of a polypeptide (e.g., an antibody).

[0033] In another embodiment, the antibodies comprising said Fc variants provided in the present application have heavy and light chain variable regions directed against different target antigens known in the prior art. One skilled in the art can easily graft heavy and light chain variable regions known in the prior art onto the Fc variants disclosed in the present application to obtain antibody variants with desired properties (e.g., reduced or eliminated ADCC and / or ADCP and / or CDC effector functions).

[0034] In a third aspect, the invention provides a pharmaceutical composition comprising a polypeptide according to the second aspect and a pharma- ceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises an antibody molecule having an Fc variant according to the invention. In one embodiment, the pharmaceutical composition comprises an IgG antibody having an Fc variant according to the invention. In another embodiment, the pharmaceutical composition comprises an IgG1-, IgG2-, IgG3-, or IgG4-type antibody having an Fc variant according to the invention.

[0035] In a fourth aspect, the invention provides a method of reducing or eliminating ADCC / ADCP / CDC effector function of an antibody and retaining or even increasing half-life by Fc region modifications as described herein. In one embodiment, ADCC / ADCP / CDC effector function of an antibody is reduced or eliminated by making one or more of the modifications disclosed herein to the antibody Fc region necessary to reduce or eliminate ADCC / ADCP / CDC effector function.

[0036] In a fifth aspect, the invention provides for the use of an Fc variant in the preparation of a medicament. In one embodiment, the medicament is used in immunotherapy, immune adjuvant therapy. In one embodiment, the medicament has reduced or eliminated ADCC and / or ADCP and / or CDC effector function. In one specific embodiment, for example, the medicament is a fusion protein comprising an Fc variant of the present application, e.g., an IL-2Fc fusion protein. In another specific embodiment, for example, the medicament is an antibody targeting an immune cell surface molecule comprising an Fc variant of the present application, which has reduced or eliminated ADCC / ADCP / CDC effector function. In another embodiment, the medicament is used to treat a tumor in a subject, for example, in one embodiment, the medicament activates a patient's immune cells with increased half-life without activating ADCC / ADCP / CDC function, thereby achieving an anti-tumor effect.

[0037] In a sixth aspect, the present invention provides a method of treating a disease in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition against the corresponding disease, comprising an Fc variant described herein. In one embodiment, the disease is a disease requiring immunotherapy or immune-adjuvant therapy. Removal of effector functions is advantageous for antibodies that target cell surface molecules, particularly antibodies on immune cells. In one embodiment, the present invention provides a method of treating a disease in a subject, comprising administering to the subject an effective amount of an antibody against an antigen on a corresponding immune cell, comprising an Fc variant described herein.

[0038] In a seventh aspect, the present invention provides a kit comprising an Fc variant disclosed in the present application, a polypeptide comprising said Fc variant, or an immunoglobulin molecule comprising said Fc variant. In one embodiment, the present invention not only improves the stability of the fusion protein by fusing a functional molecule (e.g., an enzyme, an antigen, a receptor, a ligand, a cytokine, etc.) to the Fc, but also provides a detection kit that can be used in non-clinical fields such as flow cytometry, immunohistochemistry, in vitro activity detection, and protein microarray detection.

[0039] The antibody molecules with specific Fc mutations provided in the present application have reduced or eliminated ADCC, ADCP and CDC effector functions, but still retain the ability to bind to FcRn, and can be used as improved antibody variants, which not only enhance the therapeutic effect of the antibody, but also ensure the safety, stability and low immunogenicity of the antibody. Surprisingly, it has been found that deleting the proline residue at position 329 in the Fc region significantly reduces the binding of the Fc region to the receptors FcγRIII, FcγRII, FcγRI and C1q, thereby significantly reducing or eliminating the ADCC, ADCP and CDC activities. Furthermore, the combination mutation of Pro329 in the Fc region with one or more selected from the group consisting of, for example, A330 deletion, A330G, L234A and L235A results in significantly reduced binding to the receptors FcγRIII, FcγRII, FcγRI and C1q, thereby significantly reducing or eliminating the ADCC, ADCP and CDC activities. [Brief description of the drawings]

[0040] [Figure 1] Crystal structures of human IgG1Fc and human CD16A (PDB:3SGJ). [Figure 2a] FIG. 2 shows the binding state of the Fc mutant of the present invention to various FcγRs. FIG. 2a shows the binding curve of the Fc mutant to CD16A (F176) (pH 7.4). [Figure 2b] Binding curve of Fc mutants and CD16A(V176) (pH 7.4) [Figure 2c] Binding curve of Fc mutants to CD16B(NA1) (pH 7.4) [Figure 2d] Binding curve of Fc mutants to CD16B(NA2) (pH 7.4) [Figure 2e] Binding curves of Fc mutants and CD32A(H167) (pH 7.4). [Figure 2f] Binding curve of Fc mutants to CD32A(R167) (pH 7.4). [Figure 2g]Binding curves of Fc variants to CD32B (pH 7.4). [Figure 2h] Binding curves of Fc variants to CD64 (pH 7.4). [Figure 2i] Binding curves of Fc mutants and FcRn at pH 6.0. [Figure 2j] Binding curves of Fc mutants and FcRn at pH 7.0. [Figure 2k] Binding and dissociation curves of Fc mutants and C1q. [Figure 3a] Figure 3 shows the binding profiles of Fc variants to various FcγRs measured based on biolayer interference technology, where the solution contains 200 nM of antibody. Figure 3a shows the binding curves of Fc variants to CD16A(V176) (pH 7.4); [Figure 3b] Binding curve of Fc mutants to CD16A(F176) (pH 7.4) [Figure 3c] Binding curve of Fc mutants to CD16B(NA1) (pH 7.4) [Figure 3d] Binding curve of Fc mutants to CD16B(NA2) (pH 7.4) [Figure 3e] Binding curves of Fc mutants and CD32A(H167) (pH 7.4). [Figure 3f] Binding curve of Fc mutants to CD32A(R167) (pH 7.4). [Figure 3g] Binding curves of Fc variants to CD32B (pH 7.4). [Figure 3h] Binding curves of Fc variants to CD64 (pH 7.4). [Figure 3i] Binding curves of Fc mutants and FcRn at pH 6.0. [Figure 4] The ADCC activity of the antibody is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description of the Invention Unless expressly indicated to the contrary, the practice of the present invention employs methods of routine chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology within the skill of the art.Descriptions of these methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Reference is made to periodicals and specialist volumes such as: Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998) Current Protocols in Immunology (QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and Advances in Immunology.

[0042] definition Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, forms, or reagents described herein, as they may be modified. In addition, the terms used in the present invention are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the claims. Unless otherwise defined, technical and scientific terms used in the present specification have the same meaning as commonly understood by those of ordinary skill in the art.

[0043] The following definitions will be used to interpret the specification, and where appropriate, terms used in the singular may also include the plural and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0044] The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values ​​from 5% less than the number or numerical value specified as the lower limit to 5% more than the number or numerical value specified as the upper limit.

[0045] The term "and / or", when used in conjunction with two or more options, should be understood to refer to any one of the options or any two or more of the options.

[0046] The term "comprise" or "include" means including the element, integer or step, but not excluding any other element, integer or step. When the term "comprise" or "include" is used herein, it also includes combinations of the other elements, integers or steps, unless otherwise specified. For example, when an antibody variable region "comprising" a specific sequence is mentioned, it is also intended to include an antibody variable region consisting of this specific sequence.

[0047] The term "antibody" is used herein in the broadest sense to encompass a wide variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, complete antibodies, or antibody fragments that exhibit the desired antigen-binding activity. Complete antibodies usually contain at least two full-length heavy chains and two full-length light chains, although in some cases they may contain fewer chains, e.g., naturally occurring antibodies in camels may contain only heavy chains.

[0048] As used herein, the terms "binding" and "specific binding" mean that the binding action of an antibody is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The binding ability of an antibody to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or biofilm layer optical interference technology (ForteBio), or other conventional binding assays known in the art. For example, in SPR, an antibody binds to approximately 1×10 -7 KD below 1×10 -8 KD below 1×10 -9 KD below 1×10 -10 KD below 1×10 -11 An antibody "specifically binds to BCMA or CD3" if it binds to BCMA or CD3 with a KD of 0.1 or less. However, an antibody that specifically binds to human BCMA or CD3 may have cross-reactivity with BCMA or CD3 proteins from other species. For example, an antibody specific for human BCMA or CD3 may, in some embodiments, cross-react with cynomolgus BCMA or CD3. Methods for measuring cross-reactivity include those described in the Examples and standard assays known in the art, such as optical interference bioassays or flow cytometry techniques.

[0049] The terms "Kabat variable domain residue numbering system", "Kabat numbering system" or "Kabat amino acid position numbering system" and variations thereof refer to the numbering system for antibody heavy or light chain variable domains according to Kabat et al. (See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The Kabat numbering system is typically applied to residues in the variable domain of an antibody (approximately light chain residues 1-107 and heavy chain residues 1-113).

[0050] The terms "EU numbering system", "as EU index of Kabat" or "EU index" are generally applied to residues in constant regions of immunoglobulin heavy chains (see, e.g., Kabat et al., supra). Unless otherwise specified herein, residue numbering in antibody variable domains herein is according to the residue numbering of the Kabat numbering system, and residue numbering in antibody constant domains is according to the residue numbering scheme of the EU numbering system.

[0051] An "effector function" of an antibody refers to a biological activity attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) that varies with the antibody isotype. Examples of antibody effector functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, and the like.

[0052] The term "effector cell" refers to a cell that expresses one or more FcRs and exerts an effector function, e.g., a cell that expresses FcγRIIIA and exerts an ADCC effector function, and in one embodiment, the cell that mediates ADCC function is, for example, a NK cell, a peripheral blood mononuclear cell, a monocyte, a cytotoxic T cell, a neutrophil cell. Effector cells may be derived from their natural environment, such as blood.

[0053] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated immune response in which Fc receptors present on the surface of some cytotoxic cells recognize antibodies bound on target cells, allowing the cytotoxic cells to specifically bind to the antigen-bearing target cells and activate effector cells of the immune system to lyse the target cells. The typical ADCC effect is mediated by natural killer cells (NK), and macrophages, neutrophils, and eosinophil cells can also mediate ADCC effects. For example, eosinophil cells can kill some specific parasites by ADCC effects.

[0054] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to a cellular response in which an antibody bound to a target cell binds to FcγRIIIa on the surface of the macrophage, inducing activation of the macrophage, which then causes the target cell to be internalized and degraded by acidification in the phagosome. ADCP can also be mediated by FcγRIIa and FcγRI, but to a lesser extent.

[0055] The complement system is a part of the innate immune system that consists of a series of proteins. The proteins of the complement system, called "complements" and abbreviated as C1, C2, C3, etc., are a group of heat-labile, activated enzymatic proteins present in serum and tissue fluids of humans and vertebrates. C1q is the first component of the complement-dependent cytotoxicity (CDC) pathway and can bind six antibodies, but only two IgGs are required to activate the complement cascade.

[0056] The term "complement-dependent cytotoxicity" or "CDC" refers to complement-mediated cytotoxicity, in which the Fc effector domain of an antibody that binds to a target activates a series of complement cascade reactions that form holes in the target cell membrane, thereby killing the target cell.

[0057] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The Fc region of a human IgG heavy chain is usually defined as the segment from the amino acid residue at Cys226 or Pro230 to the carboxyl terminus thereof, and the lysine residue at the C-terminus of the Fc region at position 447 (according to the EU numbering system) may be present or absent. Thus, a complete antibody composition may include a population of antibodies in which all K447 residues have been removed, a population of antibodies in which the K447 residue has not been removed, or a mixture of antibodies with and without the K447 residue.

[0058] In some embodiments, the Fc region of an immunoglobulin comprises two constant domains, namely, CH2 and CH3, and in some further embodiments, the Fc region of an immunoglobulin comprises three constant domains, namely, CH2, CH3 and CH4.

[0059] Binding of IgG to Fcγ receptors or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are important for binding of FcγR and complement C1q and have unique sequences in IgG2 and IgG4. Substitution of residues at positions 233-236 in human IgG1 and IgG2, and at positions 327, 330, and 331 in human IgG4, have been shown to significantly reduce ADCC and CDC activity (Armour et al., Eur. J. Immunol. 29(8), 1999, 2613-2624; Shields et al., J. Biol. Chem. 276(9), 2001, 6591-6604). Furthermore, Idusogie et al. found that alanine substitutions at different positions, including K322, significantly reduced complement activation (Idusogie EE et al., J. Immunol 164(8), 2000, 4178-84).

[0060] Similar terms such as "functional Fc region" and "functional Fc region" may be used interchangeably and refer to an Fc region that has an effector function of a wild-type Fc region.

[0061] Similar terms such as "mutated Fc region," "Fc mutant," "Fc region with mutation," "mutated Fc region," "Fc region mutant," "Fc mutant," "mutant Fc region" and "mutant Fc region" may be used interchangeably and refer to an Fc region that contains at least one amino acid modification that distinguishes it from a native sequence / wild-type Fc region.

[0062] In some embodiments, the variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by one or more amino acid substitutions, deletions, or additions. In some embodiments, the variant Fc region has at least one amino acid deletion compared to the Fc region of a wild-type IgG. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the Fc region of a wild-type IgG. In some embodiments, the variant Fc region has one or more amino acid substitutions and one or more amino acid deletions in the Fc region of a wild-type antibody. In some embodiments, the variant Fc region has at least one or two amino acid deletions of the Fc region described herein. In some embodiments, the variant Fc region has at least one, two, three or more amino acid substitutions of the Fc region described herein and at least one, two Fc region deletions described herein. In some embodiments, the variant Fc region has at least one, two, three or more amino acid substitutions of the Fc region described herein and at least one, two Fc region deletions described herein. In some embodiments, the variant Fc region has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the wild-type and / or parent Fc region.

[0063] "Fc receptor" or "FcR" refers to a molecule that binds to an antibody Fc region. In some embodiments, the FcR is a naturally occurring human FcR. In some embodiments, the FcR is a receptor that binds to IgG antibodies, i.e., FcγR, and includes the three receptors FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), as well as allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA and FcγRIIB, and FcγRIII receptors include FcγRIIIA and FcγRIIIB.

[0064] Depending on the receptor function, FcγR can be classified into activating receptors (FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIIIB, also called CD64, CD32A, CD32C, CD16A, CD16B) and inhibitory receptors (FcγRIIB, CD32B). Activating receptors contain an immunoreceptor tyrosine-based activation motif (or ITAM) in their cytoplasmic domain, which transmits activation signals and promotes the action of cell activation, whereas inhibitory receptors contain an immunoreceptor tyrosine-based inhibitory motif (or ITIM) in their cytoplasmic domain, which inhibits the action of cell activation. The effector functions of activating FcγR mainly include ADCC, ADCP, and antigen presentation, whereas the effector functions of inhibitory FcγR mainly include inhibition, scavenging, etc. FcγRIIB is the only inhibitory FcγR expressed in humans and mice, and in antibodies that directly target tumors, expression of FcγRIIB is associated with reduced therapeutic efficacy of the antibody.

[0065] Different FcγRs have different cellular expression profiles, for example, FcγRIIIA (CD16A) is all expressed on cells such as macrophages, monocytes, and natural killer cells (NK cells). The FcγRIIIA receptor is the only receptor expressed on NK cells that can mediate ADCC function.

[0066] The term "FcR" further includes neonatal receptor (FcRn), which is an IgG antibody receptor located on the surface of the cell membrane. FcRn transfers maternal IgG to the fetus and is responsible for regulating the steady state of immunoglobulin in vivo. FcRn can bind to the Fc portion of IgG to prevent IgG molecules from being dissolved by lysosomes, has the effect of extending the in vivo half-life of IgG, and can be involved in the in vivo transport, maintenance, and distribution metabolism of IgG.

[0067] IgG1 to IgG4 subclasses have different abilities to bind to Fc receptors, and for different FcγRs, IgG1 and IgG3 are universal ligands, bind to all FcγRs, and have strong ADCC effects. IgG2 or IgG4 is usually also called the "inactive" IgG subclass, and the immune activation effect can be avoided by using both subclasses. In fact, many monoclonal antibodies choose IgG4 as the antibody backbone to avoid the ADCC effect. However, IgG2 and IgG4 are not completely "inactive" and can bind to the activating FcγRIIa-H131 and FcγRI, respectively, which can initiate the activation of neutrophils.

[0068] Furthermore, in practical situations, it is necessary to reduce the ADCC effect caused by the IgG1 subclass, and in the prior art, the "LALA" (L234A+L235A) mutation of IgG1 is widely used, which can reduce the binding affinity of the antibody Fc region to FcγR by 100-fold. The Fc mutant obtained in the present application has a lower binding affinity to FcγR compared to the "LALA" mutation, and therefore has a stronger reduction in the ADCC effect.

[0069] In the context of ADCC / ADCP / CDC, "reduced ADCC and / or ADCP and / or CDC effector function" or "reduced ADCC / ADCP / CDC effector function" and similar expressions refer to a reduction in the numerical value of ADCC and / or ADCP and / or CDC effector function caused by an Fc variant, a polypeptide comprising an Fc variant, etc. described herein, compared to the numerical value of ADCC and / or ADCP and / or CDC effector function caused by the corresponding wild type, which is high enough that one of skill in the art would consider it to be statistically significant in the corresponding biological context. For example, in some embodiments, the reduction in the two numerical values ​​is, for example, more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, or even more than about 100%.

[0070] An "amino acid substitution" refers to the replacement of at least one amino acid residue present in a given amino acid sequence with another, different, "substitute" amino acid residue.

[0071] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid within the same class, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the table below:

[0072] [ka]

[0073] They are grouped according to their common side chain properties: (1) hydrophobic: Ile, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions involve exchanging a member of one of these classes for another.

[0074] An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0075] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be exactly identical to the parent cell in nucleic acid content and may contain mutations. As used herein, includes mutant progeny that have the same function or biological activity as screened or selected from the primary transformed cell.

[0076] The sequence identity between sequences is calculated as follows.

[0077] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.

[0078] A mathematical algorithm can be used to realize the sequence comparison and identity percentage calculation between two sequences. In one preferred embodiment, the identity percentage between two amino acid sequences is determined by the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm integrated in the GAP program of the GCG software package (available at http: / / www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the identity percentage between two nucleotide sequences is determined by the GAP program of the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise noted) employs a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0079] The percentage identity between two amino acid or nucleotide sequences may also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) integrated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0080] Antibody target antigen Antibodies comprising the Fc variants provided by the present invention can target any antigen, including but not limited to proteins, subunits, domains, motifs and / or epitopes belonging to target antigens such as, for example, cytokines, membrane-bound factors, enzymes, receptors, ligands, pathogens and their toxins, viral particles, tumor-associated factors, signal transduction pathway member molecules, etc. Suitable antigens will vary depending on the intended application. For anti-cancer therapy, it is desirable to have targets that have restricted expression in cancer cells. Some targets that have proven particularly suitable for antibody therapy are those that have signal transduction functions. Other therapeutic antibodies exert their action by blocking receptor signal transduction by inhibiting binding between the receptor and its cognate ligand.

[0081] Many antibodies that have been approved or are in development for clinical trials may benefit from the Fc variants of the present invention. Thus, the variable regions of these antibodies can be integrated with the Fc variants disclosed in the present application to form products with improved and superior properties. In one embodiment, the Fc variants disclosed in the present application can be incorporated into humanized, affinity matured, engineered antibodies, for example, by fusion to their heavy chain variable regions.

[0082] Other modifications of the Fc region Using various methods already disclosed in the prior art, the Fc variants provided herein, fusion proteins (e.g., antibodies) comprising the Fc variants, and the like, can be further modified, for example, to reduce immunogenicity and improve stability, solubility, function, and other clinical benefits, including, but not limited to, modifications at positions 252, 254, and 256, which may extend serum half-life. EXAMPLES

[0083] Working Example The present invention is further described by the following examples, which are given by way of illustration rather than limitation, and it should be understood that various modifications may be made by those skilled in the art.

[0084] Unless expressly indicated to the contrary, the practice of the present invention employs methods of routine chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology within the skill of the art.

[0085] Example 1. Design of Fc variants Considering that the interaction between the antibody Fc region and the Fc receptor may have adverse effects, it may be necessary to remove the effector function (e.g., ADCC, CDC) of the Fc region in the use of a monoclonal antibody. For example, in the case of an IgG1 subclass antibody, the ADCC function is mainly realized by binding to FcγRIIIA via the Fc region. In the case of an IgG1 subclass antibody that needs to reduce the ADCC and CDC functions, the Fc region must be remodeled to reduce the binding to the Fcγ receptor. Therefore, in this example, based on the co-crystal structure of human IgG1Fc and FcγR, the important site of the interaction between human IgG1Fc and FcγR was examined, and the corresponding Fc mutant was designed to reduce the interaction with FcγR.

[0086] The crystal structures (PDB:3SGJ) of human IgG1Fc and human CD16A (FcγRIII) are shown in FIG. 1, where two segments of Fc, P232-V240 and N325-E333, constitute the binding site of the CD16A epitope, and amino acid P329 of Fc interacts with amino acids W90 and W113 of CD16A through a π bond, and is a key amino acid in their mutual binding interface. Based on these key segments and amino acids, the inventors designed Fc mutants as shown in Table 1 based on the intermolecular action interface.

[0087] In this example, the IgG1 monoclonal antibody (Fcmut-01) against human claudin18.2 protein obtained through internal screening at Innovent Biologics, Inc. was used as an example to carry out the relevant design and research. That is, based on Fcmut-01 as the parent and control, a series of mutants (Fcmut-02 to Fcmut-024) with different mutations in Fc shown in Table 1 were obtained. Fcmut-25 is Herceptin (Genentech), and Fcmut-26 to Fcmut32 are different modifications made to the Fc of Herceptin.

[0088] [Table 1]

[0089] Example 2: Expression and purification of antibodies with Fc mutations Plasmid construction: According to conventional experimental methods, the nucleotide sequences of the heavy chain Fc mutant and the light chain were obtained and cloned into the pcDNA3.1 vector to obtain the respective plasmids.

[0090] Protein expression and purification Preparation of transient transfection plasmid: 1 / 10 (calculated by transfection volume) of serum-reduced medium Opti-MEM TM (Gibco, product number 31985-070), add the plasmid mixture (50ug / 50mL, heavy chain to light chain mass ratio is 1:1), and mix the plasmid-containing Opti-MEM TM The medium was filtered into a new 50 mL centrifuge tube, and the filtered PEI (1 g / L, Polγsciences) was added to the centrifuge tube (mass ratio (plasmid:PEI)=1:3), mixed uniformly, and allowed to stand for 20 minutes.

[0091] Cell transfection: The DNA / PEI mixture obtained above was poured lightly and flexibly into Expi293 cells (Gibco), mixed evenly, and transfected at 37°C and 8% CO2 for 14 hours. Then, 0.1% valproic acid sodium salt (VPA) (2.2M, Sigma) was added, 2.5% glucose (200g / L, Sigma) and 2.5% Feed solution (1g / L Phγtone Peptone + 1g / L Difco Select Phγtone) were added in the volume of the transfected cells, and the cells were again cultured at 37°C and 8% CO2 for 7 days.

[0092] Purified product: The cell culture after cultivation was centrifuged at 4000 rpm for 50 minutes, the supernatant was collected, and the supernatant was purified using a prepacked column Hitrap Mabselect Sure (GE, 11-0034-95). The specific operation is as follows: before purification, the packed column was equilibrated with 5 column volumes of equilibration solution (20 mM Tris, 150 mM NaCl, pH 7.2), the collected supernatant was passed through the column, and the packed column was washed with 10 column volumes of equilibration solution to remove non-specifically bound proteins, and the packed material was washed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate was collected. The pH of the eluate was adjusted to 6.0 with 2 M Tris, and the concentration was measured to obtain the purified antibody product.

[0093] The collected antibody product was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), then further isolated and purified with superdex200 increase (GE, 10 / 300GL, 10245605), the elution peak of the monomer was collected, and the column equilibration and elution buffer was PBS (Gibco, 70011-044).

[0094] Example 3. Measurement of affinity between Fc variants and Fc receptors 1. Measurement of affinity of antibodies with Fc variants using SPR method When the binding of the antibody having the Fc mutant obtained in the present invention to human FcγR was measured by surface plasmon resonance (SPR), the equilibrium dissociation constant (KD) of each antibody mutant with FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and FcRn was specifically detected. According to the principle of SPR, when a polarized beam is incident on the end face of a prism at a certain angle, a surface plasmon wave is generated at the interface between the prism and the gold film, which causes the resonance of free electrons in the metal film, i.e., surface plasmon resonance. When analyzing, first, a layer of biomolecule recognition film is fixed on the surface of the sensing chip, and then the sample to be measured flows over the chip surface. If molecules that can interact with the biomolecule recognition film on the chip surface are present in the sample, it will cause a change in the refractive index of the gold film surface, which will ultimately lead to a change in the SPR angle. By detecting the change in the SPR angle, information such as the affinity and kinetic constant of the analyte can be obtained.

[0095] In this example, the KD of the antibody with the Fc region mutation obtained in Example 2 and human FcγR was measured by Biacore (Cγtiva, T200), and the specific method is as follows: Histidine tag-containing FcγR and FcRn proteins (see Table 2 below for information on each Fc receptor) were captured on the chip surface coupled with anti-histidine antibody, and then the affinity and kinetic constants were obtained by detecting the binding and dissociation between the chip surface protein and the antibody in the mobile phase. This method includes the preparation of the chip and the detection of affinity. The measurement process used 10x HBS-EP+ (BR-1006-69, Cγtiva) diluted 10 times as the experiment buffer.

[0096] [Table 2]

[0097] Based on the manufacturer's specifications, the specific method is as follows: Chip preparation: Using an amino coupling kit (BR-1006-33, Cγtiva) and a histidine capture kit (28995056, Cγtiva), the anti-histidine antibody in the histidine capture kit was coupled to the surface of a CM5 chip (29-1496-03, Cγtiva), and after coupling, 1 M ethanolamine was injected to block the remaining activated sites.

[0098] Affinity detection: Each cycle includes receptor capture, binding to a fixed concentration of antibody with the Fc region mutation of the present application, and regeneration of the chip. Gradient-diluted solutions of each antibody variant (dilution gradient is 0 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM when binding to FcγRs, and dilution gradient is 0 nM, 50 nM, 100 nM, 200 nM, 400 nM, 800 nM, 1600 nM when binding to FcRn) were each completed in each affinity detection cycle, from low to high concentration. In each cycle, the antibody variant solution was flowed over the chip surface at a flow rate of 30 μL / min, with a binding time of 60 seconds and a dissociation time of 60 seconds. Finally, the chip was regenerated using 10 mM Glγcine pH 1.5 (BR-1003-54, Cγtiva). The obtained data were analyzed using the Biacore T200 analysis software (version number 3.1) using the analytical 1:1 binding or steady state analysis model to obtain the corresponding results.

[0099] Table 3 below shows the affinity data for each Fc receptor for each antibody mutant in this study, and Figure 2 shows the fitting curves for the corresponding molecules.

[0100] These results indicate that the IgG1 wild-type Fcmut-01 antibody, which was used as a control antibody in this study, has high affinity for CD64 and K D The K value was 1.07E-09, and the antibody bound to both H167 and R167 subtypes of CD32A. DThe affinity values ​​for two CD16A subtypes, F176 and V176, were 1.57E-07 and 2.44E-08, respectively, whereas weak binding was observed for CD16B (NA1), CD16B (NA2), and CD32B.

[0101] Except for mutant Fcmut-05, which has a relatively weak affinity (3.06E-07) for CD16A(V176), none of the antibody molecules with other Fc mutations bound to CD16A(F176), CD16A(V176), CD16B(NA1), CD16B(NA2), CD32A(H167), CD32A(R167), or CD32B. For CD64, which has strong binding to wild-type IgG1, mutants Fcmut-06 / 07 / 08 all did not bind to CD64, but the binding of Fcmut-02 / 03 / 04 / 05 to CD64 was also significantly reduced, weakening by 8.7-fold, 12.9-fold, 14.02-fold, and 50.75-fold, respectively, compared to the control Fcmut-01. Similar results were also observed for Fcmut-26 to Fcmut-32, and compared to Fcmut-25, none of the antibody molecules with other Fc mutations other than Fcmut-29 bound to CD16A(F176), CD16A(V176), CD16B(NA1), CD16B(NA2), CD32A(H167), CD32A(R167), or CD32B.

[0102] F176 and V176 are the major genotypes of CD16A, and the crystal structure has revealed that the 176th amino acid of CD16A is located in the Fc binding region, and therefore has a significant effect on the affinity of CD16A to Fc. Since the Fc variants obtained in the present application do not substantially interact with the F176 and V176 genotypes of CD16A, it has been shown that the Fc variants obtained in the present application do not substantially interact with all functional CD16A, and thus do not induce ADCC effects. Therefore, protein molecules (e.g., antibodies) containing the Fc variants of the present application do not substantially have ADCC effector functions.

[0103] It is known that the Fc region of an antibody does not bind to FcRn under neutral pH conditions, but only under acidic conditions, which was the main mechanism by which antibody molecules have a relatively long half-life. As can be seen from the detection, the control antibody Fcmut-01 has an affinity for FcRn of 3.86E-07 at pH 6.0, but does not bind to FcRn at pH 7.0, and the antibody molecules with Fc mutations obtained in this study are consistent with the control antibody Fcmut-01 under the same conditions (Figure 2i, Figure 2j), indicating that the Fc mutants obtained in this study do not affect binding to FcRn.

[0104] FcRn is expressed in various cells in vivo, and the binding of FcRn to the Fc region is pH-dependent, binding only at a weakly acidic pH around pH 6.0, but not at a neutral pH, thereby extending the half-life of the corresponding antibody. The experimental data of the present application show that the Fc region mutations of the antibody do not affect the binding of the antibody molecule to FcRn, and therefore FcRn can still extend the in vivo half-life of the antibody mutants of the present application.

[0105] The antibody mutant obtained in this application does not bind to CD32B, and it has been demonstrated that the therapeutic effect of an antibody can be enhanced by mutating the Fc region of the antibody.

[0106] The "LALA" mutation (Fcmut-05), which has been widely used in the prior art, reduced the binding affinity of the antibody Fc region to FcγR by 100-fold. The Fc mutant obtained in the present application has a lower binding affinity to FcγR compared to the "LALA" mutation, and therefore has a stronger reduced ADCC effect.

[0107] Because wild-type IgG2 and IgG4 have weak or no ADCC effects, many antibody molecules that are desired to avoid the ADCC effect have selected Fc regions of IgG2 or IgG4 subtypes during production. However, in some cases, it is still desirable to further reduce the ADCC effector function of IgG2 or IgG4 subtypes. For this reason, the applicant made corresponding mutations to the corresponding sites of the Fc regions of IgG2 and IgG4 subtypes (see Table 1) and then further explored the circumstances in which it binds to various FcRs. The data showed that the Fc mutants of IgG2 or IgG4 subtypes containing the corresponding mutations of the present application weakened binding to FcγR compared to wild-type Fc.

[0108] [Table 3] NB: Do not combine

[0109] 2. Measurement of the binding of the Fc variants of the present invention to Fc receptors using biolayer interference technology Biolayer interferometry (BLI) was used to measure the binding affinity (KD) of the Fc variants of the invention for human Fc receptors.

[0110] Half an hour before the start of the experiment, depending on the number of samples, an appropriate number of HIS1K sensors (18-5120, Sartorius) were immersed in SD buffer (1xPBS, 0.1% BSA, 0.05% Tween®-20), and when binding to FcRn, the buffer used in the experiment was changed to 10 mM HEPS, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 6.0. Antibodies were diluted to 200 nM and Fc receptors (see Table 2 for sample information) were diluted to 100 nM.

[0111] 200 μL of SD buffer, 200 μL of labeled antibody solution, and 200 μL of Fc receptor were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a Fortebio Octet Red96e, and the plate was laid out and the sensor position was selected according to the sample position. The instrument setting parameters were as follows: run steps: equilibration baseline 120 s, immobilization Fc receptor 100 s, equilibration baseline 120 s, antibody binding 60 s, and dissociation 60 s, the rotation speed was 1000 rpm, and the temperature was 30 °C. After the experiment was completed, the KD value was analyzed using the ForteBio Octet analysis software.

[0112] The results are shown in Table 4 and Figure 3 below. Compared with Fcmut-25, none of the antibody molecules with other Fc mutations except Fcmut-29 bound to CD16A(F176), CD16A(V176), CD16B(NA1), or CD16B(NA2). The mutant Fcmut-26 / 27 / 28 / 29 / 30 / 31 / 32 mutants did not bind to CD32A H167, CD32A R167, or CD32B. None of the mutant Fcmut-30 / 31 / 32 mutants bound to CD64, but the binding of Fcmut- / 26 / 27 / 28 / 29 to CD64 was also significantly reduced.

[0113] Fc-25 is an antibody that binds to HER2, whose constant region is the wild-type IgG1 sequence, and the results showed that deletion of P329 can reduce the binding of the molecule to the Fc gamma receptor, but binding to FcRn (pH 6.0) is not affected.

[0114] [Table 4a] [Table 4b] [Table 4c] [Table 4d] [Table 4e] Table 4f [Table 4g] [Table 4h] [Table 4i]

[0115] Experimental Example 4. Measurement of affinity of Fc variants of the present invention for C1q using biolayer interference technology Since the strength of the affinity between an antibody's Fc and C1q directly determines whether or not the antibody has CDC effector function, in this example, the affinity of each Fc mutant for C1q was measured to determine whether the Fc mutant has CDC effector function.

[0116] The binding affinity (KD) of the antibody of the present invention for human C1q was measured using biolayer interferometry (BLI). The affinity measurement by BLI was performed by a conventional method (Estep, P. et al., High throughput solution based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p270-8).

[0117] First, biotin and antibody were mixed at a molar ratio of 3:1 using EZ-LinkTM Sulfo-NHS-LC-Biotin (21327, Thermo Scientific), then allowed to stand to perform biotin labeling. Unlabeled biotin was removed by centrifugation, and the antibody was replaced with an equal volume of PBS solution.

[0118] Half an hour before the start of the experiment, an appropriate number of SA sensors (Foretbio, 18-5019) were immersed in SD buffer (1xPBS, 0.1% BSA, 0.05% Tween®-20) depending on the number of samples. Antibodies were diluted to approximately 100 nM, and C1q (A099, Complement Technology) was diluted to 40 nM.

[0119] 200 μL of SD buffer, 200 μL of labeled antibody solution, and 200 μL of C1q antigen were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a Fortebio Octet Red96e, and the plate was laid out and the sensor position was selected according to the sample position. The instrument setting parameters were as follows: run steps: baseline, sample addition ~3 nm, baseline, association (Kon) and dissociation (Kdis), the run time of each step depended on the binding and dissociation rate of the sample, the rotation speed was 1000 rpm, and the temperature was 30 °C. After the experiment was completed, the KD values ​​were analyzed using ForteBio Octet analysis software, and the coordinate axis data was exported and plotted using Graphpad Prism8 software.

[0120] The results are shown in Table 5 and Figure 2k. Only Fcmut-01 was able to bind to C1q with an affinity of 2.02E-08, while Fcmut-2~Fcmut-8 all did not bind to C1q. The Fc mutants in this study were shown to be able to weaken or block the binding of Fc to C1q, thus reducing or eliminating the CDC effector function of the antibody. Compared to the corresponding wild-type control, Fcmut-10~Fcmut-16, Fcmut-18~Fcmut-24 also did not bind to C1q. Also, compared to Fcmut-25, Fcmut-26~Fcmut-32 also did not bind to C1q.

[0121] [Table 5]

[0122] Experimental Example 5. Measurement of affinity between the antibody of the present invention and the antigen by biolayer interference technology Affinity detection was performed using the same BLI method as described in Example 4, with the following exceptions.

[0123] Half an hour before the start of the experiment, an appropriate number of AHC (Foretbio, 18-5060) sensors were immersed in SD buffer (1xPBS, 0.1% BSA, 0.05% Tween®-20) depending on the number of samples. Antibodies and Claudin18.2 (cp0007, Genscript) were each diluted to 100 nM.

[0124] 200 μL of SD buffer, 200 μL of antibody, and 200 μL of Claudin18.2 antigen were added to a 96-well black polystyrene microplate (Greiner, 655209) respectively. Detection was performed using Fortebio Octet Red96e, the plate was laid out according to the sample position, and the sensor position was selected. The instrument setting parameters were as follows: run steps: baseline, sample addition, baseline, association (Kon) and dissociation (Kdis) were performed, the run time of each step depended on the binding and dissociation rate of the sample, the rotation speed was 1000 rpm, and the temperature was 30 °C. After the experiment was completed, the KD value was analyzed using ForteBio Octet analysis software.

[0125] The results are shown in Table 6. The KD values ​​of Fcmut-02 to 08 and the antigen are similar to the KD values ​​of Fcmut-01 and the antigen, indicating that the specific mutations in the Fc region disclosed in the present invention do not affect the affinity of the corresponding antibody to its antigen, and thus that antibodies containing the Fc variants of the present invention retain their own antigen affinity.

[0126] [Table 6]

[0127] Example 6. Antibody-mediated ADCC effect One of the major effector functions mediated by FcγR is antibody-dependent cell-mediated cytotoxicity (ADCC), which is mediated by FcγRIIIA (CD16A) on NK cells and macrophages. After studying the affinity of the antibodies with Fc mutations obtained in this application to Fc receptors in the above example, in this example, we continued to examine the ADCC effector functions of the antibody mutants.

[0128] In this example, the Jurkat-ADCC NF-AT luciferase effector cell line (hereinafter referred to as ADCC effector cells) from Promega was used to detect the activation state of the NF-AT signal, thereby detecting the ADCC activity of the antibody. The specific experimental procedure is as follows.

[0129] 1) Cell preparation Cell counting was performed on DANG-18.2 cells (CLS Cell Lines Service) that surface-overexpress human claudin18.2 and ADCC effector cells: the supernatants of DANG-18.2 cells and ADCC effector cells were removed by centrifugation, the cells were washed twice with PBS solution, and the cells were resuspended in detection medium (1640 medium containing 5% low IgG serum (Gibco)) to obtain a concentration of ADCC effector cells of 6 × 10 6 Adjust the concentration of DANG-18.2 cells to 1 x 10 cells / mL. 6 The concentration was adjusted to 1 / mL.

[0130] 2) Plating: Target cells DANG-18.2 were plated in 96-well plates at 25 μL per well.

[0131] 3) Serially diluted antibodies of the present invention were added: the starting concentrations of each antibody sample with Fc region mutation and the control sample were as shown in Table 7, and 3-fold dilutions were performed to obtain a total of 10 dilution gradients, each of which was added sequentially to the well plate at 25 μL per well.

[0132] [Table 7]

[0133] 4) ADCC effector cells were added to each well plate at 25 μL per well.

[0134] 5) Incubate in a 37°C incubator for 12 hours.

[0135] 6) Remove the 96-well plate, leave it at room temperature for 10 minutes, and add thawed Luciferase test reagent (Bio-Glo) to each well. TM 75 μL of Luciferase assay reagent was added. Detection was performed using a microplate reader, and a concentration-dependent curve was fitted using GraphPad software.

[0136] As shown in Figure 4, the control antibody Fcmut-01 has the Fc region of a wild-type IgG1 monoclonal antibody, and can effectively activate the NF-AT signal of ADCC effector cells by binding to an antigen on target cells (DANG-18.2), thereby activating the downstream signaling pathway of ADCC, showing that the antibody has excellent ADCC killing ability. However, other antibodies with Fc region mutations obtained in this application showed very weak or almost no ADCC effect, specifically, mutant Fcmut-05 (having L234A and L235A mutations) showed very weak ADCC activity at a concentration 10 times higher than that of the control Fcmut-01, while the other mutants had no ADCC activity even at a concentration 10 times higher than that of Fcmut-01, showing that the ADCC effector function of the antibody molecules with Fc region mutations obtained in this application was substantially eliminated, and this result was also consistent with the affinity data for CD16A of each antibody molecule in Example 3.

[0137] According to conventional technology, it is known that performing LALA mutation in the Fc region significantly reduces the ADCC effect of an antibody. However, the results of this example show that the Fc mutant obtained in the present application has a greater reducing effect on the ADCC effect than LALA mutation.

[0138] The antibody mutants obtained by the present application making mutation modifications (e.g., deletion, substitution) to one or more amino acids at positions 329, 330, 234, or 235 of the antibody Fc region substantially eliminate the ADCC effector function, thereby indicating that the positions of said amino acids are important for the ADCC / ADCP effector function of the antibody. If the target antibody needs to avoid the ADCC / ADCP effector function in practical requirements, those skilled in the art can choose to make corresponding modifications to one or more amino acids at positions 329, 330, 234, or 235 of the Fc region according to the contents disclosed in the present application, thereby achieving practical technical effects. Specifically, deletion of the amino acid at position 329 in the antibody Fc region, deletion of amino acids from positions 329 to 330, deletion of the amino acid at position 329, and substitution of the amino acid at position 320, as well as the combined application of the above modifications with the LALA modification (L234A+L235A), can be considered, thereby obtaining an antibody molecule that eliminates ADCC / ADCP effector function.

Claims

1. An Fc variant comprising one or more amino acid modifications compared to a human wild-type Fc region, said modifications being selected from positions 329, 234, 235, or 330, numbered according to the EU index of Kabat.

2. The Fc variant of claim 1, comprising the following modifications: 1) a deletion of amino acid 329 (Δ329) as numbered according to the EU index of Kabat; or 2) containing a deletion of amino acids at positions 329 and 330 (Δ329 and Δ330) as numbered according to the EU index of Kabat; or 3) the modifications Δ329+A330G or Δ329+S330G, numbered according to the EU index of Kabat; or 4) the modifications L234A+L235A+Δ329, V234A+Δ329 or F234A+L235A+Δ329, numbered according to the EU index in Kabat; or 5) the modifications L234A+L235A+Δ329+Δ330, V234A+Δ329+Δ330 or F234A+L235A+Δ329+Δ330, numbered according to the EU index in Kabat; or 6) The modifications L234A+L235A+A330G+Δ329, L234A+L235A+S330G+Δ329, V234A+L235A+A330G+Δ329, V234A+L235A+S330G+Δ329, F234A+L235A+S330G+Δ329 or F234A+L235A+A330G+Δ329 numbered according to the EU index in Kabat.

3. The Fc variant of claim 1, which is an IgG1 type, an IgG2 type, or an IgG4 type Fc variant.

4. 2. The Fc variant of claim 1 comprising the sequence: 1) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or consisting of said sequence; or 2) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 3, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or consisting of said sequence; or 3) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or consisting of said sequence; or 4) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or consisting of said sequence; or 5) a sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or consisting of said sequence; or 6) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or a sequence consisting of said sequence; or 7) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 8, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or a sequence consisting of said sequence; or 8) A sequence of amino acids 221 to 447 of SEQ ID NO: 11 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or a sequence consisting of said sequence; or 9) A sequence of amino acids 221 to 447 of SEQ ID NO: 12 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 10) A sequence of amino acids 221 to 447 of SEQ ID NO: 13 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or a sequence consisting of said sequence; or 11) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or a sequence consisting of said sequence; or 12) A sequence of amino acids 221 to 447 according to the EU index of SEQ ID NO: 15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or a sequence consisting of said sequence; or 13) A sequence of amino acids 221 to 447 of SEQ ID NO: 16 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 14) A sequence of amino acids 221 to 447 of SEQ ID NO: 17 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 15) A sequence of amino acids 221 to 447 of SEQ ID NO: 19 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 16) A sequence of amino acids 221 to 447 of SEQ ID NO: 20 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 17) A sequence of amino acids 221 to 447 of SEQ ID NO: 21 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 18) A sequence of amino acids 221 to 447 of SEQ ID NO: 22 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 19) A sequence of amino acids 221 to 447 of SEQ ID NO: 23 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 20) A sequence of amino acids 221 to 447 of SEQ ID NO: 24 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 21) A sequence of amino acids 221 to 447 of SEQ ID NO: 25 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 22) A sequence of amino acids 221 to 447 of SEQ ID NO: 27 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 23) A sequence of amino acids 221 to 447 of SEQ ID NO: 28 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 24) A sequence of amino acids 221 to 447 of SEQ ID NO: 29 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 25) A sequence of amino acids 221 to 447 of SEQ ID NO: 30 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity thereto, or a sequence consisting of said sequence; or 26) A sequence of amino acids 221 to 447 of SEQ ID NO: 31 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or a sequence consisting of said sequence; or 27) A sequence of amino acids 221 to 447 of SEQ ID NO: 32 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even higher identity thereto, or a sequence consisting of said sequence; or 28) An amino acid sequence of positions 221 to 447 of SEQ ID NO: 33 according to the EU index, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even higher identity thereto, or a sequence consisting of said sequence.

5. A polypeptide comprising an Fc variant according to any one of claims 1 to 4.

6. The polypeptide of claim 5 which is an antibody molecule.

7. The polypeptide of claim 6, wherein the antibody molecule comprises a heavy chain and a light chain as follows: 1) comprising a heavy chain as set forth in SEQ ID NO:2 and a light chain as set forth in SEQ ID NO:9; or 2) comprising a heavy chain as set forth in SEQ ID NO:3 and a light chain as set forth in SEQ ID NO:9; or 3) comprising a heavy chain as set forth in SEQ ID NO:4 and a light chain as set forth in SEQ ID NO:9; or 4) comprising a heavy chain as set forth in SEQ ID NO:5 and a light chain as set forth in SEQ ID NO:9; or 5) A heavy chain as set forth in SEQ ID NO:6 and a light chain as set forth in SEQ ID NO:9; or 6) A heavy chain as shown in SEQ ID NO: 7 and a light chain as shown in SEQ ID NO: 9; or 7) A heavy chain as shown in SEQ ID NO: 8 and a light chain as shown in SEQ ID NO: 9; or 8) A heavy chain as set forth in SEQ ID NO: 11 and a light chain as set forth in SEQ ID NO: 9; or 9) A heavy chain as set forth in SEQ ID NO: 12 and a light chain as set forth in SEQ ID NO: 9; or 10) A heavy chain as set forth in SEQ ID NO: 13 and a light chain as set forth in SEQ ID NO: 9; or 11) A heavy chain as set forth in SEQ ID NO: 14 and a light chain as set forth in SEQ ID NO: 9; or 12) A heavy chain as set forth in SEQ ID NO: 15 and a light chain as set forth in SEQ ID NO: 9; or 13) A heavy chain as set forth in SEQ ID NO: 16 and a light chain as set forth in SEQ ID NO: 9; or 14) A heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO: 9; or 15) A heavy chain as set forth in SEQ ID NO: 19 and a light chain as set forth in SEQ ID NO: 9; or 16) A heavy chain as set forth in SEQ ID NO: 20 and a light chain as set forth in SEQ ID NO: 9; or 17) A heavy chain as set forth in SEQ ID NO: 21 and a light chain as set forth in SEQ ID NO: 9; or 18) A heavy chain as set forth in SEQ ID NO: 22 and a light chain as set forth in SEQ ID NO: 9; or 19) A heavy chain as set forth in SEQ ID NO: 23 and a light chain as set forth in SEQ ID NO: 9; or 20) A heavy chain as set forth in SEQ ID NO:24 and a light chain as set forth in SEQ ID NO:9; or 21) A heavy chain as set forth in SEQ ID NO: 25 and a light chain as set forth in SEQ ID NO: 9; or 22) A heavy chain as set forth in SEQ ID NO: 27 and a light chain as set forth in SEQ ID NO: 34; or 23) A heavy chain as set forth in SEQ ID NO: 28 and a light chain as set forth in SEQ ID NO: 34; or 24) A heavy chain as set forth in SEQ ID NO: 29 and a light chain as set forth in SEQ ID NO: 34; or 25) A heavy chain as set forth in SEQ ID NO: 30 and a light chain as set forth in SEQ ID NO: 34; or 26) A heavy chain as set forth in SEQ ID NO: 31 and a light chain as set forth in SEQ ID NO: 34; or 27) A heavy chain as set forth in SEQ ID NO: 32 and a light chain as set forth in SEQ ID NO: 34; or 28) A heavy chain as shown in SEQ ID NO: 33 and a light chain as shown in SEQ ID NO:

34.

8. A pharmaceutical composition comprising an Fc variant according to any one of claims 1 to 4, or a polypeptide comprising an Fc variant according to any one of claims 1 to 4, and a pharma- ceutically acceptable carrier.

9. 13. Use of the Fc variant of any one of claims 1 to 4 in reducing or eliminating ADCC, ADCP or CDC effector function.

10. A nucleic acid molecule encoding an Fc variant according to any one of claims 1 to 4, or a polypeptide comprising an Fc variant according to any one of claims 1 to 4.

11. A vector comprising the nucleic acid molecule of claim 10.

12. 11. A host cell comprising a nucleic acid molecule according to claim 10 or a vector comprising a nucleic acid molecule according to claim 10.

13. The pharmaceutical composition according to claim 8 for treating a tumor in a subject.

14. A kit comprising an Fc variant according to any one of claims 1 to 4, or a polypeptide comprising an Fc variant according to any one of claims 1 to 4.

15. A method for preparing an Fc variant according to any one of claims 1 to 4, or a polypeptide comprising an Fc variant according to any one of claims 1 to 4, comprising expressing a nucleic acid molecule or vector in a host cell under suitable conditions, wherein said nucleic acid molecule encodes an Fc variant according to any one of claims 1 to 4 or encodes a polypeptide comprising an Fc variant according to any one of claims 1 to 4, and said vector comprises a nucleic acid molecule encoding an Fc variant according to any one of claims 1 to 4 or encoding a polypeptide comprising an Fc variant according to any one of claims 1 to 4; Optionally, the method further comprises recovering the expressed Fc variant or polypeptide.

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