Fc variants that resist cleavage by immunoglobulin-degrading enzymes

Abstract

The present invention provides a molecule comprising an Fc variant, said molecule being resistant to immunoglobulin degrading enzymes compared to a molecule comprising an IgG wild-type Fc, and said molecule comprising a human IgG Fc region having a mutation, said mutation being one or more substitutions or deletions at position G237 (EU numbering), preferably said substitution being G237A. Also provided are nucleic acids encoding said molecules, vectors and host cells comprising said nucleic acids, and methods for producing said molecules.

Description

[Technical field]

[0001] The present invention relates to the biomedical field, specifically to human IgG Fc variants and molecules comprising said variants. [Background technology]

[0002] Immunoglobulin G (IgG) is the main antibody component of serum, accounting for about 75% of serum immunoglobulins. It plays a mainly protective role in the body's immunity and can effectively prevent infectious diseases. In addition to its protective role, IgG is also involved in diseases. In diseases such as rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, and immune (or spontaneous) thrombocytopenia, IgG antibodies react with the body's own molecules, and in organ transplants, IgG causes acute transplant rejection. In some diseases, such as multiple myeloma, 50% of patients have very high levels of IgG in the blood, which affects the metabolism and efficacy of therapeutic antibodies.

[0003] Research has revealed that IdeS-type immunoglobulin degrading enzymes can break down IgG in the blood, eliminating the negative effects of the body's own IgG on therapeutic antibodies. In addition, by lowering the level of IgG in the blood, the half-life of therapeutic antibodies can be extended (Abdallah, HM&A.ZXZhu.2020.Clin Pharmacol Ther 107(2):423-434). IdeS has the effect of completely cutting IgG in the human blood two hours after administration (Winstedt,L. et al.,2015.PLoS One 10(7):e0132011), making it an effective way to eliminate IgG interference. However, after administering immunoglobulin degrading enzymes such as IdeS, no other therapeutic antibodies can be used for at least four days. Therefore, to reduce the interference of circulating IgG using IdeS while at the same time enabling treatment using antibodies, i.e., to enable simultaneous administration of IdeS and therapeutic antibodies, a therapeutic antibody that can resist cleavage by IdeS is required. Summary of the Invention

[0004] The details of one or more embodiments of the invention are set forth below: Other features, objects and advantages of the invention will become apparent from the description and claims.

[0005] Item 1. A molecule comprising an Fc variant, The molecule is resistant to immunoglobulin degrading enzymes compared to a molecule comprising an IgG wild type Fc, and the molecule comprises a human IgG Fc region having a mutation, said mutation being one or more substitutions or deletions at position G237 (EU numbering), preferably said substitution being G237A, and preferably said human IgG Fc region comprises at least the sequence as set forth in SEQ ID NO: 19.

[0006] Item 2. The molecule of item 1, wherein the Fc molecule is resistant to degradation by a protease, which is capable of cleaving an IgG wild-type Fc-containing molecule between residues 236 and 237 (EU numbering).

[0007] Item 3. The molecule according to item 1 or 2, wherein the molecule is resistant to degradation by an enzyme selected from the group consisting of IdeS, IdeZ, IdeE, IdeZ2, IdeE2, IdeSORK, Xork and mutants thereof, as compared to wild-type IgG.

[0008] Item 4. The molecule according to any one of items 1 to 3, wherein the IgG of the molecule is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

[0009] Item 5. The molecule according to any one of items 1 to 4, wherein the effector function of the molecule selected from the group consisting of ADCC, ADCP, and CDC is promoted or enhanced as compared to a molecule comprising an IgG wild-type Fc.

[0010] Item 6. The method further comprises one or more substitutions in the human IgG wild-type Fc region sequence, the substitutions being S239D, I332E, G236A, S298A / E333A / K334A, S239D / I332E, S239D / A330L / I332E, G236A / S239D / I332E, G236A / A330L / I332E, G236A / S239D / A330L / I332E, F243L / R292P / Y300L, F243L / R292P / Y300L / V305I / P396L, L235V / F243L / R292P / Y3 6. The molecule according to item 5, wherein the substitutions are selected from the group consisting of: 00L / P396L, P247I / A339Q, K326A / E333A, K326W / E333S, E333A / K334A, H268F / S324T / I332E, S239D / H268F / S324T / I332E, S267E / H268F / S324T / I332E, K326A / I332E / E333A, S239D / K326A / E333A, S267E / I332E, preferably the substitutions are selected from the group consisting of: S239D, I332E, S239D / I332E.

[0011] Item 7. The molecule according to any one of items 1 to 6, further comprising an N-glycan modification, wherein the N-glycan modification has a fucose content of less than 50%, preferably less than 10%.

[0012] Item 8. The Fc molecule according to any one of items 1 to 7, further comprising an N-glycan modification, wherein the content of terminal galactose modifications in said N-glycan modifications is greater than 30%, preferably greater than 50%.

[0013] Item 9. The molecule according to any one of items 1 to 8, wherein the molecule is an antibody or an Fc fusion protein.

[0014] Item 10. The molecule according to item 9, wherein the antibody binds to an antigen on a virus, a bacterium, a tumor cell, a tumor stroma, or a tumor vasculature, preferably the antibody specifically binds to one or two antigens selected from the group consisting of COVID-19, CD38, CD20, FR5, BCMA, SLAM7, CD73, GPRCD5, CD3, most preferably the antibody specifically binds to CD38.

[0015] Item 11. The molecule according to item 9 or 10, wherein the antibody comprises at least one heavy chain and at least one light chain, the heavy chain comprising HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:2, HCDR3 as set forth in SEQ ID NO:3, or HCDR1 as set forth in SEQ ID NO:4, HCDR2 as set forth in SEQ ID NO:5, HCDR3 as set forth in SEQ ID NO:6, and the light chain comprising LCDR1 as set forth in SEQ ID NO:7, LCDR2 as set forth in SEQ ID NO:8, LCDR3 as set forth in SEQ ID NO:9, or LCDR1 as set forth in SEQ ID NO:10, LCDR2 as set forth in SEQ ID NO:11, LCDR3 as set forth in SEQ ID NO:12.

[0016] Item 12. The molecule according to any one of Items 9 to 11, wherein the antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 13 and the light chain comprises a light chain variable region represented by the amino acid sequence of SEQ ID NO: 14, or the heavy chain comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain comprises a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16.

[0017] Item 13. The molecule according to any one of items 9 to 12, wherein the epitope-binding fragment of an antibody is a Fab, Fab', F(ab')2, scFv or disulfide-linked Fv fragment.

[0018] Item 14. The molecule according to any one of items 1 to 4, wherein the molecule has an effector function selected from the group consisting of ADCC, ADCP, and CDC that is weakened and / or eliminated as compared to a molecule comprising an IgG1 wild-type Fc.

[0019] Item 15. The method further comprises one or more substitutions in the human IgG1 wild-type Fc sequence, the substitutions being N297A, N297Q, N297G, L235A, L235A / E318A, L234A / L235A, G236R / L328R, S298G / T299A, L234F / L235E / P331S, E233P / L234V / L235A. 15. The Fc molecule according to item 14, wherein the substitutions are selected from the group consisting of: / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A, preferably the substitutions are selected from the group consisting of: N297A, N297Q, N297G, L234A / L235A, L235A, L234F / L235E / P331S.

[0020] Item 16. The molecule according to any one of items 1 to 15, wherein the affinity of said molecule for FcRn is increased compared to a molecule comprising an IgG1 wild type Fc.

[0021] Item 17. The molecule according to item 16, further comprising one or more substitutions in the sequence of IgG1 wild type Fc, said substitutions being selected from the group consisting of YTEKF, YTEKF+V264A, YTEKF+V264E.

[0022] Item 18. An isolated binding polypeptide molecule comprising (i) a binding domain capable of binding to a cell or a target molecule bound to a cell, and (ii) an Fc domain having the amino acid sequence of the CH2 and CH3 constant domains of a human IgG1 heavy chain, with the exception that a substitution or deletion has occurred at residue G237 as defined by the EU numbering system, said binding molecule being capable of binding to a target molecule on a target cell, and said molecule producing measurable CDC activity or target cytotoxicity activity mediated by a required effector cell, preferably characterized in that the substitution at G237 is G237A.

[0023] Item 19. A nucleic acid encoding a molecule according to any one of items 1 to 17 or a binding polypeptide molecule according to item 18.

[0024] Item 20. An expression vector comprising the nucleic acid according to Item 19.

[0025] Item 21. A host cell comprising a nucleic acid according to item 19 or an expression vector according to item 20.

[0026] Item 22. A method for producing a molecule according to any one of items 1 to 17 or a binding polypeptide molecule according to item 18, comprising the steps of: (a) providing a host cell according to item 21; (b) culturing the host cell under conditions suitable for expression of the molecule or the binding polypeptide molecule; (c) optionally recovering said molecule or said binding polypeptide molecule from the culture of step (b). [Brief description of the drawings]

[0027] [Figure 1] Figure 1 shows the degradation of mutant antibodies by immunoglobulin degrading enzymes. M is the molecular weight symbol, 1 to 5 are antibodies 1 to 3, 5, and 6, and 6 to 13 are antibodies 1 to 3, 5, 6, wild type, wild type, and 7 after enzyme cleavage. IgG is a full-length antibody, scIgG is an antibody from which one Fc has been cut off, F(ab)2 is a Fab dimer obtained by cleavage, and Fc is an Fc monomer obtained by cleavage. [Figure 2A] FIG. 2A shows the ADCC activity of different mutant antibodies. [Figure 2B] FIG. 2B shows the ADCC activity of different mutant antibodies. [Figure 2C] FIG. 2C shows the ADCC activity of different mutant antibodies. [Diagram 3] FIG. 3 shows the ADCP activity of different mutant antibodies. [Figure 4] FIG. 4. CDC activity of different mutated antibodies. [Diagram 5] FIG. 5 shows the apoptosis-inducing activity of different mutant antibodies. [Figure 6] FIG. 6 shows the inhibition rates of different mutant antibodies against the activity of CD38 enzyme. [Figure 7]FIG. 7 shows the inhibitory effects of different antibodies on the transplanted tumor model, where Ab is the administered antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The object of the present invention is to provide an Fc mutant that is resistant to cleavage by IdeS-type immunoglobulin cleavage enzyme, which overcomes the shortcomings of the prior art. IgG, Fc fusion proteins, and Fc fragments containing the mutant are all resistant to cleavage by IdeS-type immunoglobulin cleavage enzyme, and the effect of resisting enzymatic cleavage can be obtained by only a single point mutation.

[0029] The object of the present invention is achieved by the following technical solutions: 1.Definition "Fc" and "Fc-containing protein" refer to a monomeric, dimeric, or heterodimeric protein having at least one immunoglobulin CH2 and CH3 domain. The CH2 and CH3 domains can form at least a portion of the dimeric domain of a protein (e.g., an antibody). Similar domains of other IgG subclasses can be determined by comparison of the amino acid sequence of the heavy chain or heavy chain fragment of the IgG subclass with that of human IgG1, as detailed in the following website (http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html). As used herein, "EU numbering" refers to the numbering of human IgG1 antibody residues. All amino acid positions herein use "EU numbering".

[0030] The term "wild type Fc" refers to a variant that optionally contains a mutation other than that of the reference polypeptide or the one being considered in the wild type Fc region. The term "wild type" or "WT" as used herein refers to an amino acid sequence or nucleotide sequence found in nature, which is naturally occurring (including allelic variations) and has not been intentionally modified, e.g., mutagenized, by molecular biology techniques (Vidarsson, G. et al. (2014). Front Immunol 5:520). For example, "wild type Fc region" refers to an IgG1 Fc region of SEQ ID NO: 17 (G1m1, 17 allotype), IgG1 Fc region of SEQ ID NO: 18 (G1m3 allotype), among others.

[0031] The term "half-life" refers to the period of time from when an Fc fragment appears in the serum of a recipient patient until half of it is removed from the circulation or other tissues.

[0032] The term "Fcγ receptor" or "FcγR" refers to IgG-type immunoglobulin receptors called CD64 (FcγRI), CD32 (FcγRII), and CD16 (FcγRIII), in particular the five expressed receptors (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb). In addition to human FcγRIIb (a receptor that inhibits the activation of immune cells), all of them are effector cell activating receptors (Muta T et al., Nature, 1994, 368:70-73).

[0033] The term "effector cell" refers to any cell that has an Fc receptor, for example, lymphocytes, monocytes, neutrophils, natural killer (NK) cells, eosinophils, basophils, mast cells, dendritic cells, Langerhans cells, platelets.

[0034] The term "ADCC" refers to antibody-dependent cellular cytotoxicity, an antibody-mediated cellular immune response in which effector cells expressing Fc receptors (FcR) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the main cells mediating ADCC, express only FcγRIII, whereas monocytes can express FcγRI, FcγRII, and FcγRIII. Methods for detecting ADCC include those using PBMCs, those using NK cells, those using the NK92 reporter gene, and those using the Jurkat reporter gene. ADCC is measured in the presence of blood monocytes. Methods for detecting ADCC include those using PBMCs, those using NK cells, those using the NK92 reporter gene, and those using the Jurkat reporter gene. ADCC is measured in the presence of blood monocytes.

[0035] The term "ADCP" refers to antibody-dependent cellular phagocytosis, a process in which cells engulfed by antibodies are ingested (completely or partially) by phagocytic immune cells (e.g., macrophages, neutrophils, dendritic cells) bound to the Fc effector domain of Ig. Methods for detecting ADCP include using PBMCs, using the NK92 reporter gene, and using the Jurkat reporter gene. ADCP may be measured in the presence of CD14 pos and / or CD11b pos blood monocytes.

[0036] The term "C1q" refers to the first protein in the activation pathway of the classical complement pathway; the complement protein C1q is activated by direct binding to cell surfaces or antibodies bound to cell surfaces, and also has functions such as regulating the phagocytosis of apoptotic cells, activating endothelial cells, and regulating T cells and B cells.

[0037] The term "complement-dependent cytotoxicity" or CDC refers to a cell death-inducing mechanism in which a target binds to the Fc effector domain of an antibody, activating a series of enzymatic reactions to form pores in the target cell membrane. Generally, antigen-antibody complexes, such as those on target cells engulfed by antibodies, bind to and activate complement component Clq, which then activates the complement cascade to kill the target cell. Complement activation also leads to the deposition of complement components on the surface of target cells, which aid in ADCC by binding to complement receptors (e.g., CR3) on white blood cells. A method for detecting CDC is, for example, to co-incubate target cells with complement from human blood to detect killing of the target cells.

[0038] The terms "immunoglobulin-degrading enzyme" and "IdeS-type immunoglobulin-degrading enzyme" refer to an enzyme capable of cleaving immunoglobulins. In the present invention, the immunoglobulin-degrading enzyme refers to an IgG-specific degrading protease whose enzyme cleavage site is between G236 and G237 (EU numbering) of IgG. Currently known immunoglobulin-degrading enzymes include IdeZ, IdeZ2, IdeE, and IdeE2 derived from Streptococcus zooepidemicus (Lannergard, J. & B. Guss. 2006. FEMS Microbiol Lett 262(2):230-235; Hulting, G. et al. (2009). FEMS Microbiol Lett 298(1):44-50), IdeS derived from Streptococcus pyogenes (Wenig, K. et al. (2004). Proc Natl Acad Sci USA 101(50):17371-17376), and IdeSORK derived from Streptococcus krosus. They specifically cleave immunoglobulin G (IgG) 1-4. They do not cleave other immunoglobulin classes, such as IgA, IgD, IgE, and IgM. These immunoglobulin-degrading enzymes cleave between G236 and G237 in the CH2 domain, cleaving human IgG into F(ab')2 fragments and Fc fragments. Mutants of these known enzymes can also exert the same effect, as described in CN107532158A, CN107532156A, WO2022223818A1, WO2021254479A1, and CN115443288A.

[0039] The terms "N-glycan," "N-linked glycan," "glycoprotein," "glycosylation," and "glycoform" refer to N-linked oligosaccharides, e.g., oligosaccharides that are attached to asparagine residues of a polypeptide by an asparagine-N-acetylglucosamine linkage. The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (NANA, SA including NGNA, and derivatives and analogs thereof including acetylated NANA or acetylated NGNA). In glycoengineered Pichia pastoris, sialic acid is exclusively N-acetylneuraminic acid (NANA) (Hamilton et al., Science 313(5792), 1441-1443, 2006). N-glycans share a common core pentasaccharide, Man3GlcNAc2, where "Man" refers to mannose, "Glc" refers to glucose, "NAc" refers to N-acetyl, and GlcNAc refers to N-acetylglucosamine. N-glycans differ with respect to the number of branches (antennae) that contain peripheral sugars (e.g., GlcNAc, galactose, fucose, sialic acid) that are attached to the core structure Man3GlcNAc2 ("Man3"), also called the "trimannosyl core," "core pentasaccharide," or "oligomannose core." N-glycans are classified according to their branching components (e.g., high mannose, complex, or hybrid). One N-glycan terminus can be modified with up to four sialic acids.

[0040] The term "G0" refers to a complex biantennary oligosaccharide (GlcNAc2Man3GlcNAc2) that has neither galactose nor fucose, "G1" refers to a complex biantennary oligosaccharide (GalGlcNAc2Man3GlcNAc2) that has no fucose and contains one galactosyl residue, "G2" refers to a complex biantennary oligosaccharide (Gal2GlcNAc2Man3GlcNAc2) that has no fucose and contains two galactosyl residues, and "G0F" refers to a complex biantennary oligosaccharide (Gal2GlcNAc2Man3GlcNAc2) that has no fucose and contains two galactosyl residues. "G1F" refers to a complex biantennary oligosaccharide containing one core fucose and no galactose (GlcNAc2Man3GlcNAc2F), "G1F" refers to a complex biantennary oligosaccharide containing one core fucose and one galactosyl residue (GalGlcNAc2Man3GlcNAc2F), and "G2F" refers to a complex biantennary oligosaccharide containing one core fucose and two galactosyl residues (Gal2GlcNAc2Man3GlcNAc2F).

[0041] The term "hybrid" N-glycan refers to an N-glycan having at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannosyl core and zero or more mannoses on the 1,6 mannose arm of the trimannosyl core.

[0042] IgG is the major serum immunoglobulin in blood, with a blood IgG level of about 10 g / L in healthy individuals. It is a glycoprotein consisting of two identical heavy and two light chains, each of which consists of a variable region and a constant region. IgG contains a single N-linked glycan at position Asn297 in the CH2 domain of the Fc region of each of its two heavy chains. The covalently bound complex carbohydrate consists of a one-core biantennary penta-polysaccharide containing N-acetylglucosamine (GlcNAc) and mannose. Glycosylation modification of Fc is involved in the exertion of antibody function. Knocking out fucose can significantly enhance IgG modification of antibodies. Terminal galactose modification can improve CDC function.

[0043] The term "immunoconjugate" refers to a molecule obtained by conjugating one or more therapeutic agents to a variant described in the present invention, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin or fragment thereof of bacterial, fungal, plant or animal origin), or a radioisotope.

[0044] The terms "resistant to enzymatic cleavage" and "resistant" to proteases refer to the property that an immunoglobulin IgG or an immunoglobulin antibody fragment structure comprising Fc has no ability to be cleaved by the above-mentioned immunoglobulin degrading enzymes.

[0045] The term "vector" or "expression vector" as used herein means a vector used as a vehicle to introduce a desired gene into a cell and express the desired gene in the cell. It is known to those skilled in the art that such vectors may be selected from plasmids, phages, viruses, and retroviruses. In general, vectors suitable for the present invention contain a selectable marker, appropriate restriction sites useful for cloning the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0046] As used herein, the term "transformation" is used broadly to refer to the introduction of DNA into a recipient host cell, thereby altering the genotype and resulting in the modification of the recipient cell.

[0047] Similarly, a "host cell" refers to a cell into which a vector has been introduced by transformation, said vector being constructed by recombinant DNA techniques and encoding at least one heterologous gene. Unless otherwise clearly stated, when describing methods for isolating a polypeptide from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the Fc variant. In other words, recovering the Fc variant from the "cells" may mean recovering from whole pelleted cells or from a cell culture containing both medium and suspension cells.

[0048] 2. Mutations that counteract enzyme cleavage The Fc variants of the present invention comprising the G237 mutation (EU numbering) are not degraded by enzymatic cleavage even when coexisting with the above immunoglobulin degrading enzymes. Cleavage by the immunoglobulin degrading enzymes is a structure-specific enzymatic cleavage for those containing at least the CH2 domain (Wenig, K. et al., 2004. Proc Natl Acad Sci USA 101(50):17371-17376). However, the CH2 domain contains at least a sequence starting from L235, for example, SEQ ID NO: 19 (Novarra, S. et al., 2016. MAbs 8(6):1118-1125). Thus, the Fc variants of the present invention comprise at least a CH2 domain having a G237 mutation. The G237 mutation may be one or more substitutions or deletions at the site, and the substitution is preferably G237A.

[0049] The present invention is the first to discover that a single G237 mutation alone can achieve the effect of resisting cleavage by immunoglobulin-degrading enzymes. The only reported effect is the complex combination mutation described in CN103260640B, which substitutes E233-L234-L235-G236 in the CH2 domain of antibody IgG1 for P233-V234-A235 in IgG2, and further combines it with S239D / I332E or G237A / S239D / I332E.

[0050] Other IgG isotypes than IgG1, such as IgG2, IgG3, IgG4, etc., can all be degraded by immunoglobulin degrading enzymes (Wenig, K. et al., 2004. Proc Natl Acad Sci USA 101(50):17371-17376). In some embodiments described herein, Fc fragments of the present invention may be used that are derived from different IgG subtypes, such as IgG2, IgG3, IgG4. The wild-type Fc sequences of the different allotypes IgG2 to 4 (Vidarsson, G., et al. (2014). Front Immunol 5:520) are all resistant to enzymatic cleavage after substitution or deletion at position G237.

[0051] In some embodiments described herein, amino acids may be substituted in the constant region of the antibody. For example, different IgG1 allotypes, the well-known G1m17, G1m3 or G1m1 allotypes, or combinations thereof may be used in the Fc fragments of the invention, or different IgG2-4 allotypes (Vidarsson, G. et al. (2014). Front Immunol 5:520), or combinations thereof may be used.

[0052] 3. Promotion or enhancement of effector functions ADCC and the like are important functions for antibodies to exert their functions. There is a possibility that the G237 mutation may cause the weakening or loss of such effector functions (Hezareh, M. et al., 2001. J Virol 75(24):12161-12168.). In some examples described herein, the weakening of the effector functions caused by the G237 mutation can be improved by performing modifications, such as amino acid substitutions and / or glycosylation modifications, on the Fc region of the molecule. The effector functions selected from the group consisting of ADCC, ADCP, and CDC of the molecule are promoted or enhanced compared to molecules containing IgG1 wild-type Fc.

[0053] In some embodiments, the amino acid substitutions for the Fc region comprise one or more substitutions in a wild-type human IgG1 sequence, the substitutions being S239D, I332E, G236A, S298A / E333A / K334A, S239D / I332E, S239D / A330L / I332E, G236A / S239D / I332E, G236A / A330L / I332E, G236A / S239D / A330L / I332E, F243L / R292P / Y300L, F243L / R292P / Y300L / V305I / P396 L, L235V / F243L / R292P / Y300L / P396L, P247I / A339Q, K326A / E333A, K326W / E333S, E333A / K334A, H268F / S324T / I332E, S239D / H268F / S324T / I332E, S267E / H268F / S324T / I332E, K326A / I332E / E333A, S239D / K326A / E333A, S267E / I332E, preferably said substitutions are I332E, S239D / I332E.

[0054] In some embodiments, the glycosylation modification of the Fc region is characterized by having an N-glycan modification and a reduced fucose modification, wherein the N-glycan modification has a fucose content of less than 50%, preferably less than 10%, which can improve the ADCC activity of the antibody.

[0055] In some embodiments, the glycosylation modification of the Fc region is characterized by having N-glycan modifications and increased terminal galactose modifications, the N-glycan modifications having a terminal galactose modification content greater than 30%, preferably greater than 50%, which can improve the CDC activity of the antibody.

[0056] 4. Weakening or elimination of effector functions In some embodiments, amino acid substitutions in the Fc region result in the molecule having an effector function selected from the group consisting of ADCC, ADCP and CDC that is weakened and / or eliminated compared to a molecule comprising an IgG1 wild-type Fc. Said substitutions are selected from the group consisting of N297A, N297Q, N297G, L235A, L235A / E318A, L234A / L235A, G236R / L328R, S298G / T299A, L234F / L235E / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A, preferably said substitutions are selected from N297A, N297Q, N297G, L234A / L235A, L235A, L234F / L235E / P331S.

[0057] 5.Improved binding to FcRn The affinity of the Fc for FcRn controls the metabolism of antibodies in the human body, and in some embodiments, the Fc variants comprise mutations that can improve the affinity of the Fc for FcRn, such that the affinity of the molecule for FcRn is increased compared to a molecule comprising an IgG1 wild-type Fc. The mutations are preferably N434A, N434W, M252Y / S254T / T256E, M428L / N434S, M252Y / S254T / T256E / H433K / N434F, T250Q / M428L, T307Q / N434A, M252Y / V308P / N434Y, T307P / L309Q / Q311R, H285D / T307Q / A378V, L309D / Q311H / N434S, M252Y / T256D, T256D / T307Q, T256D / T307W.

[0058] There are other Fc variants that can be used in the present invention, including variants described by Dall'Acqua et al. (WF, DA et al., 2002. Journal of immunology. 169(9):5171-5180), Shan et al. (Shan, L. et al., 2016. PLoS One 11(8):e0160345), Lee et al. (Lee, CH et al., 2019. Nat Commun 10(1):5031), Mackness et al. (Mackness, BC et al., 2019. MAbs 11(7):1276-1288), Christophe et al. JPEG2024546152000001.jpg20170 and the like, but not limited to them.

[0059] In some embodiments, the Fc molecule further comprises one or more substitutions in a wild-type human IgG1 sequence, wherein the substitutions are selected from the group consisting of YTEKF, YTEKF+V264A, YTEKF+V264E.

[0060] In some embodiments, the glycosylation modification of the Fc region is characterized by having increased terminal sialic acid modifications, including N-glycan modifications, said sialic acid content being higher than 20%, preferably higher than 30%. Several amino acid substitutions in the Fc region can increase the level of sialic acid modifications, preferably the amino acid substitutions are F241A, F243A, V262E, V264A, V264E, E293DEL, E294DEL, Y300A.

[0061] 6. Antigen-binding domain The molecules of the invention are resistant to immunoglobulin degrading enzymes, which are structure-specific enzymatic cleavage at least for those containing the CH2 domain (Wenig, K. et al., 2004. Proc Natl Acad Sci USA 101(50):17371-17376), with the proviso that the CH2 domain contains at least a sequence beginning with L235, e.g., SEQ ID NO: 19 (Novarra, S. et al., 2016. MAbs 8(6):1118-1125). Thus, the invention includes any antibody or fusion protein that contains a human Fc CH2 domain. The antibody may further have altered effector function, e.g., improved ADCC, ADCP and / or CDC. In some embodiments, the effector function may be reduced.

[0062] Antibody binding domains may be produced by methods known to those skilled in the art. The structures of antibody binding domains include, but are not limited to, Fab, Fab', F(ab')2, scFv, VHH, heavy chain variable region, light chain variable region, CDR, etc.

[0063] The molecules of the present invention include a targeting moiety. The targeting moiety is selected from tumor cell surface antigens, enzymes, hormones, receptors, cytokines, immune cell surface antigens, adhesion molecules, etc. The targeting moiety may further comprise a non-proteinaceous moiety, such as a carbohydrate, lipid, lipopolysaccharide, organic molecule, metal or metal complex. Generally, the targeting molecule, if present, is linked to Fc by a linker, which may or may not be a polypeptide.

[0064] The molecules of the invention comprise a targeting moiety, which binds to an antigen on a virus, a bacterium, a tumor cell, tumor stroma, or tumor vasculature, preferably, said targeting moiety specifically binds to one or two antigens selected from the group consisting of COVID-19, CD38, CD20, FR5, BCMA, SLAM7, CD73, GPRCD5, CD3, and most preferably, said antibody specifically binds to CD38.

[0065] In one embodiment, a targeting moiety of the invention comprises at least one heavy chain and at least one light chain, said heavy chain comprising three complementarity determining regions as set forth in the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and said light chain comprising three complementarity determining regions as set forth in the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0066] In one embodiment, the heavy chain comprises a variable region as set forth in SEQ ID NO: 13, and the light chain comprises a variable region as set forth in SEQ ID NO: 15. Preferably, the light chain comprises a variable region as set forth in SEQ ID NO: 28, and the heavy chain comprises a variable region as set forth in SEQ ID NO: 29, 30.

[0067] In one embodiment, a targeting moiety of the invention comprises at least one heavy chain and at least one light chain, said heavy chain comprising three complementarity determining regions as set forth in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and said light chain comprising three complementarity determining regions as set forth in the amino acid sequences of SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0068] In one embodiment, the heavy chain comprises a variable region set forth in SEQ ID NO: 14 and the light chain comprises a variable region set forth in SEQ ID NO: 16. Preferably, the heavy chain comprises a light chain set forth in SEQ ID NO: 27, a heavy chain set forth in SEQ ID NO: 23, and a light chain set forth in SEQ ID NO: 25.

[0069] 7. Manufacturing method In one embodiment, the invention provides isolated nucleic acids encoding the Fc variants of the invention, vectors comprising said nucleic acids, and host cells comprising said nucleic acids and / or vectors. Generally, polynucleotides encoding the Fc variants disclosed herein are inserted into an expression vector for introduction into a host cell, and the host cell may be used to produce a desired amount of the Fc variant to be protected, i.e., the Fc variants of the invention. Thus, in some aspects, the invention provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising those expression vectors and polynucleotides.

[0070] The invention also provides a method for producing the G236A antibody. The method provides an isolated nucleic acid encoding the above polypeptide sequence, an expression vector comprising the nucleic acid, and a host cell comprising the nucleic acid. A method for producing the polypeptide of the invention is also featured. The method comprises culturing a host cell in a medium under conditions allowing expression of the polypeptide encoded by the nucleic acid, and purifying the polypeptide from the cultured cells or the medium of the cells.

[0071] A variety of expression vector systems are available for the purposes of the present invention. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus. Some also involve the use of polycistronic systems with internal ribosome binding sites. Cells in which the DNA has integrated into the chromosome may also be selected by introducing one or more markers that allow for the selection of transfected host cells. The markers may provide prototrophy for auxotrophic hosts, resistance to biocides (e.g. antibiotics) or resistance to heavy metals (e.g. copper). The selectable marker gene may be directly linked to the DNA sequence to be expressed or may be introduced into a cell by co-transformation. Other elements may be necessary for optimal synthesis of mRNA. These elements include signal sequences, splicing signals, transcription promoters, enhancers and termination signals.

[0072] More generally, once a vector or DNA sequence encoding the Fc variant has been generated, the expression vector may be introduced into a suitable host cell, i.e., transformation of the host cell is permitted. Introduction of the plasmid into the host cell may be accomplished by a variety of techniques well known to those skilled in the art, including, but not limited to, transfection (including electrophoresis, electroporation), protoplast fusion, calcium phosphate co-precipitation, fusion with cells bearing enveloped DNA, microinjection, infection with whole viruses, and the like. More preferably, the plasmid is introduced into the host by electroporation. The transformed cells are cultured under conditions suitable for production of the Fc variant, and expression of the Fc variant is measured. Exemplary measurement techniques are enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or fluorescence activated cell sorting (FACS), immunohistochemistry, and the like.

[0073] In one embodiment, the invention provides a method for producing an Fc variant of the invention, said method comprising mutating a wild-type Fc fragment of a wild-type IgG1 to obtain a corresponding Fc variant, wherein the sequence of the wild-type Fc fragment is as set forth in SEQ ID NO: 17 or 18.

[0074] In one embodiment, the mutation is selected from the group consisting of G237A, G237A / I332E, G237A / I332E / S239D, G237A / M252Y / S254T / T256E, G237A / M428L / N434S, G237A / M252Y / S254T / T256E / H433K / N434F.

[0075] In one embodiment, the Fc variants are obtained by the following procedure. (i) A nucleic acid encoding the Fc variant of the present invention is introduced into a host cell. (ii) culturing the host cells in a medium under conditions that allow expression of the Fc variant of the invention. (iii) purifying the Fc variants of the invention from the cultured cells or the cell culture medium.

[0076] In one embodiment, the host cell does not produce immunogenic NGNA or α-galactose.

[0077] In one embodiment, host cell lines for expressing the Fc variants of the invention are of mammalian origin, and one of skill in the art can determine the particular host cell most suitable for expression of the desired gene product. Exemplary host cell lines are DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR - ), SP2 / 0 (mouse myeloma), 293 (human kidney), and the like. In one embodiment, the cell line has altered glycosylation of the Fc variant expressed thereby, e.g., afucosylation (e.g., PER.C6 or FUT8 knockout CHO cell lines). In one embodiment, NS0 cells may be used. CHO cells are particularly preferred. Host cell lines may be provided by the American Tissue Culture Collection, a bioresource provider, in general, or may be obtained through literature disclosure.

[0078] In vitro production allows for scale-up to obtain large quantities of the desired Fc variant. Mammalian cell culture techniques are known in the art and include, for example, homogeneous suspension culture in bioreactors, or immobilized or entrapped cell culture, for example, in hollow fibers, microcapsules, or on agarose beads. If necessary and / or desired, the solution of the Fc variant may be purified by conventional purification methods (e.g., gel filtration, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, etc.).

[0079] Genes encoding the Fc variants of the invention may also be expressed in non-mammalian cells, such as yeast or plant cells. The most commonly used eukaryotic microorganism is Saccharomyces cerevisiae, or baker's yeast, although a variety of other strains are commonly available.

[0080] In addition to cell-based expression systems, Fc variants may be produced by cell-free or chemical synthetic methods, hi some embodiments, the Fc variants are produced by in vitro chemical synthesis.

[0081] 8. Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide or nucleic acid.

[0082] The present invention also provides a pharmaceutical preparation comprising (i) the above-mentioned polypeptide or nucleic acid and / or the above-mentioned pharmaceutical composition, and (ii) an immunoglobulin-degrading enzyme selected from the group consisting of IdeS, IdeZ, IdeZ2, IdeE, IdeE2, IdeSORK, Xork, and mutants thereof.

[0083] The pharmaceutical composition may be formulated for parenteral bolus administration (e.g., intravenous or intramuscular). The composition may be in the form of, for example, a suspension, solution or emulsion in an oily or aqueous carrier, and may contain additives such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in the form of a powder for combining with a suitable carrier (e.g., pyrogen-free water).

[0084] The present invention also provides immunoconjugates comprising the variants disclosed herein, which are conjugated (chemically linked) to one or more therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., a protein toxin, a bacterial, fungal, plant or animal derived enzymatic toxin or fragment thereof), or a radioisotope.

[0085] 9. Uses of Fc variants and pharmaceutical compositions The present invention provides a method of treating a disease using the molecules and / or pharmaceutical compositions of the present invention, wherein the disease is cancer or an autoimmune disease, and wherein the cancer is multiple myeloma.

[0086] The invention provides the use of the molecules and / or pharmaceutical compositions of the invention in the manufacture of a medicament, which is used to treat a disease, in particular a cancer or an autoimmune disease, and may also be used to induce ADCC, ADCP, apoptosis-mediated depletion of CD38 positive cells.

[0087] The present invention provides a method for treating an individual having cancer or an autoimmune disease, comprising administering to said individual an effective amount of a molecule and / or pharmaceutical composition of the present invention, and further comprising administering to a subject in need thereof a therapeutically effective amount of said polypeptide or a nucleic acid encoding said polypeptide. Also provided is the use of the polypeptide or nucleic acid in the manufacture of a medicament for treating an autoimmune disease and / or tumor, preferably the disease being a CD38-associated cancer or an autoimmune disease. Also featured is the isolated polypeptide, nucleic acid, expression vector, host cell, pharmaceutical composition or method for treating an autoimmune disease and / or tumor substantially as shown and described herein, preferably the disease being a CD38-associated cancer or an autoimmune disease. The CD38-associated cancer is a solid tumor, a B-cell lymphoma or multiple myeloma.

[0088] The invention provides a method for inducing ADCC, ADCP, apoptosis mediated depletion of CD38 positive cells in an individual, the method comprising administering to the individual an effective amount of a molecule and / or pharmaceutical composition of the invention to induce ADCC, ADCP, apoptosis mediated depletion of CD38 positive cells.

[0089] The Fc variants of the present invention have the following advantages: 1) they are resistant to cleavage by immunoglobulin degrading enzymes, 2) they can be used simultaneously with immunoglobulin degrading enzymes, and 3) they have improved bioavailability when used simultaneously with immunoglobulin degrading enzymes.

[0090] As long as it is in line with common knowledge in the art, each of the above preferred conditions can be freely combined to obtain each of the preferred embodiments of the present invention.

[0091] The reagents and raw materials used in the present invention may all be commercially available products.

[0092] The present invention will be further described in the following examples, but the present invention is not limited to the scope of the examples. In the following examples, the test methods for which no specific conditions are specified are carried out according to the usual methods and conditions or selected according to the product instructions.

[0093] Example 1: Protein Production The base sequences encoding the amino acid sequences other than IdeS in Table 1 were synthesized and constructed in the pcDNA3.1 vector, which is a mammalian cell expression vector.

[0094] The heavy and light chains were mixed in a 1:2 ratio and introduced into ExpiCHO-S cells and fucose-unmodified CHO cells, respectively, by transient transformation. The liquid was harvested 6 to 8 days after transfection, and the expression supernatant was centrifuged at 9500 rpm for 20 minutes to collect the supernatant. It was purified with Protein A to limit endotoxin to less than 1 EU / mg. The heavy chains and their mutants with different numbers were co-expressed with the light chain to obtain wild-type antibody, antibodies 1 to 6, where the heavy chains corresponded to SEQ ID NOs: 20 to 26, respectively, and the light chain sequence used was SEQ ID NO: 27. Antibody 7 corresponded to the light chain of SEQ ID NO: 28 and the heavy chain of SEQ ID NO: 29.

[0095] The expression of the IdeS enzyme. A polynucleotide sequence encoding IdeS (SEQ ID NO: 31) was synthesized through codon optimization, and the N-terminal signal peptide sequence MRKRCYSTSAAVLAAVTLFVLSVDRGVIA (SEQ ID NO: 32) was added. After the sequence was synthesized, it was inserted into the pET32a expression vector, and the correct sequence was confirmed, and a recombinant plasmid for expression was obtained. The mutant recombinant plasmid was introduced into E. coli BL21 Star (DE3) by electrotransformation and inoculated onto an LB agarose plate containing 100 μg / mL ampicillin. It was cultured overnight at 37 ° C until colonies appeared. A single colony was picked and inoculated into 200 μL of LB medium containing 100 μg / mL ampicillin, and cultured overnight at 37 ° C, 250 rpm. The overnight culture was inoculated into 1 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37 ° C for 4 hours, after which 0.1 mM IPTG was added and cultured at 30 ° C overnight. Overnight cultures were centrifuged and the supernatants were collected. SDS-PAGE was used to assess the concentration of mutant proteins in mutant-expressing supernatants.

[0096] The supernatant was purified using ion exchange chromatography followed by hydrophobic chromatography to limit endotoxin to less than 1 EU / mg, finally obtaining pure protein. [Table 1] JPEG2024546152000003.jpg219170

[0097] Example 2: Detection of Mutants Resistant to Enzymatic Cleavage The cleavage effect of different mutants by IdeS was evaluated by SDS-PAGE. IdeS was added to 50 μL reaction systems containing 2 mg / mL of different mutants at a ratio of 1:50 to initiate the cleavage reaction, and the reaction was carried out at 37°C for 30 min. The sample was mixed with an equal amount of non-reducing 2×SDS loading buffer, and then incubated in a water bath at 75°C for 5 min, and the cleavage products were detected by SDS-PAGE.

[0098] As shown in the results of cleavage (FIG. 1), antibodies 1 to 3, 5, and 7 were all able to oppose the cleavage, antibody 6 partially opposes the cleavage, and the wild type did not oppose the cleavage.

[0099] Example 3: Detection of ADCC activity The ADCC activity of each mutant was detected using reporter genes and PBMCs, respectively. In the reporter gene method, Daudi cells were used at a density of 1 × 10 6 The antibody was adjusted to cells / mL and plated at 25 μL / well on a 96-well plate. The test antibody was diluted to 20 μg / mL in 1640 medium and further diluted 7 times in a 6-fold gradient to obtain 8 concentration gradients of the drug. 25 μL of the diluted drug was added to the 96-well plate. 1640 medium was used as a negative control (NC). The 96-well plate was placed in a carbon dioxide incubator at 37°C and incubated for 45 minutes. Cells containing a reporter gene were added and incubated in a carbon dioxide incubator for 6 hours. After the incubation, 75 μL of firefly luciferase substrate solution (Rhinogen®) was added to each well, shaken on a microplate shaker to mix uniformly, and left at room temperature for 5 minutes. The luminescence (relative light unit, RLU) value was read using a microplate reader, and the data was analyzed using a 4-parameter logistic regression model in the software Graphpad.

[0100] For the method using PBMCs, Calcein-AM (Biyuntian, Cat. No. C2012) was used to introduce the dye into the target cells by co-incubating with Daudi cells for 20 min, and the final concentration of calcein was 4 μM. After incubation, the cells were seeded into 96-well plates and 2 × 10 4 The test antibodies were diluted in 1640 medium to a concentration range of 2000 to 0.02 ng / mL and added to a 96-well plate. 5 × 10 5PBMC cells were added to the medium, and the final ratio of PBMC cells to Daudi cells was 25:1. 1640 medium was used as the negative control (NC) and total control. After incubation in a humidified incubator at 37°C for 4 hours, the medium was centrifuged, the supernatant was separated, and the fluorescence value was measured at an excitation wavelength of 490 nm and an emission wavelength of 515 nm. For the total control, the cells were directly lysed with 0.1% Triton X-100 without centrifugation, and the supernatant was separated after centrifugation and the fluorescence value was measured. The target cell lysis rate was calculated from the fluorescence value.

[0101] As shown in the results (Figure 2A), antibody 1 has weakened ADCC activity compared to the wild type. Antibody 2 has no ADCC activity, and antibodies 3 and 5 have enhanced ADCC activity. EC 50 The EC values ​​for antibodies 1, 3, and 5 were 0.6, 1.3, and 3.9 times higher than the wild type, respectively. For the non-fucose-modified antibody sample (Figure 2B), the EC 50 When comparing the values, antibody 3-Fucose - , Antibody 5-Fucose - The activity folds of the wild-type antibody and antibody 3-Fucose were 12.7 and 23.2, respectively, which were much higher than the control. - , EC of antibody 7 50 The values ​​were 2.072 ng / mL, 0.6578 ng / mL, and 2.095 ng / mL, respectively (Figure 2C). - The activity folds of antibody 3-Fucose and antibody 7 relative to the wild type were 3.1 and 1.0, respectively. - was described as antibody 8.

[0102] Example 4: Detection of mutant stability The thermostability of the antibody was detected for the mutant proteins using Unchained Labs' high-throughput protein stability analysis system Uncle. The purified mutant samples were loaded at 1.0 mg / mL for detection. The temperature range was set to 25-95°C using the Tm&Tagg with optional DLS program, and one parallel well was provided for the sample. The thermostability was analyzed by analyzing the result parameters Tm (melting temperature of the protein) and Tagg266 (aggregation temperature of the protein). [Table 2]

[0103] As shown in the results (Table 2), the Tm value of each antibody was 50° C. or higher. Antibodies 1, 2, and 3 had Tm values ​​of 60° C. or higher and were more stable. The change in Tm value due to G237A alone was negligible.

[0104] Example 5: Detection of ADCP activity Antibody 8, antibody 7 and control were diluted 5-fold from 10 μg / mL to give 7 concentrations. Romas cells were labeled with CFSE. Monocytes were stimulated to obtain macrophages (MΦ), and 8 × 10 5 The cells were plated at 100 cells / well (50 μL). 50 μL of the diluted sample was added to the macrophages and incubated for 15 min in a 37°C, 5% CO2 incubator. The cells were then diluted at a ratio of 1:5 (the amount of cells containing macrophages was 8 × 10 5 The amount of Romas cells per well was 4 x 10 6 The labeled Romas cells (cells / well) were added, plated in a 48-well cell plate, and incubated at 37°C in a 5% CO2 incubator for 2.5 hours. After staining with CD11b-APC, flow cytometry detection was performed.

[0105] As shown in the results (Figure 3), antibodies 8 and 7 had strong phagocytic effects, indicating that the mutations did not have a negative effect on the ADCP activity of the antibodies.

[0106] Example 6: Detection of CDC activity Antibody 3 was diluted 5-fold from 100 μg / mL in 1640 medium containing 0.5% BSA to obtain a concentration of 10 in 9 increments. The diluted samples were added at 40 μL / well to a 96-well cell plate containing 30 μL of Daudi cells (5 × 10 4 cells / well) and the cell plate was incubated for 1 hour at 4° C. Serum from healthy donors was diluted 2-fold in 1640 medium containing 0.5% BSA and added to the sample wells at 40 μL / well and incubated for 2 hours at 37° C. The remaining cell viability was detected by Celltiter-Glo™.

[0107] As shown in the results (Figure 4), antibody 8 had no CDC activity, just like the wild-type antibody, and the mutations did not change the CDC activity properties. On the other hand, antibody 7 retained CDC activity, which was consistent with the report on wild-type antibodies (Michael Karl Bauer, CD38: A Unique Target In Multiple Myeloma, 2014).

[0108] Example 7: Detection of apoptosis-inducing ability Daudi cells were inoculated and the cell density was adjusted, followed by addition of 750 μL / well (2 × 10 5The cells were plated in a 24-well plate at 1000 μg / well (cells / well). The antibody to be tested was diluted to 3-4 μg / mL in 1640 medium supplemented with 10% serum (Sigma-Aldrich) and 2 mM L-glutamine, and further diluted 10-fold, five times to obtain six concentration gradient samples. 250 μL of the diluted sample was added to the 24-well plate, and 1640 medium was used as a negative control (NC). The 24-well plate was placed in a 5% CO2 incubator at 37°C for 20 hours, after which it was transferred to a 1.5 mL EP tube and centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, 200 μL of PBS was added to resuspend the cells, then the cells were transferred to a 96-well V-bottom well plate and centrifuged to remove the supernatant. After staining using the Annexin V-FITC kit and incubation for 10-20 minutes, flow cytometer detection was performed and the percentage of apoptotic cells was analyzed with GraphPad Prism.

[0109] As shown in the results (Figure 5), the ability of antibody 8 to induce apoptosis in cells was close to that of the wild-type antibody. The mutations did not affect the apoptosis induction ability, while antibody 7 retained the apoptosis induction ability.

[0110] Example 8: Detection of binding activity The affinity of the wild-type antibody and antibody 3 for the target CD38 and for human FcRn under acidic conditions was detected using the BLI method. As shown in the results (Table 3), the affinity of antibody 8 and the wild-type antibody for the two proteins and for CD38 was within 3-fold, indicating that the mutations did not affect the binding activity of the Fab. [Table 3]

[0111] Example 9: Detection of inhibition of enzyme activity Antibody 8 and wild-type antibody were diluted to 40 nM, and CD38 protein was diluted to 10 nM in 20 mM Tris-HCl, pH 7.0. The diluted CD38 and antibody were mixed 1:1, and reacted for 15 minutes at room temperature in the dark, with the dilution solution without antibody as a blank control. After the reaction was completed, 100 μL of NGD (nicotinamide guanine dinucleotide sodium salt) was added to a final concentration of 80 μM, and incubated at room temperature in the dark for 60 minutes, followed by detection. The fluorescence signal at an excitation wavelength of 300 nm and an emission wavelength of 410 nm was measured to determine the inhibitory effect of enzyme activity. The lower the RFU value, the higher the inhibitory effect.

[0112] As shown in the results (Figure 6), the ability of antibody 8 to inhibit CD38 enzyme activity was almost identical to that of the wild-type antibody, indicating that the mutation did not affect the inhibitory function of Fab against CD38 enzyme activity. Antibody 7 had almost no activity inhibition.

[0113] Example 10: Animal Pharmacokinetics The in vivo activity of the mutants of the present invention was evaluated using the Daudi mouse model. The study was designed with seven groups, which were group 1 to group 7 from the top as shown in Figure 7. Specifically, cells in the logarithmic growth phase were harvested and 5 × 10 cells were inoculated into the right axilla of each huPBMC-NOG-Dko mouse under aseptic conditions. 6 Daudi cells were subcutaneously inoculated at a dose of 0.1 mL per mouse. Approximately 10 days after cell inoculation, the tumor volume increased to 200 mm 3When the tumor volume reached about 100%, 56 animals were screened according to tumor volume and randomly divided into 7 groups, with 8 animals per group. On the day of grouping, animals in groups 3, 4, 6, and 7 were administered IVIg by a single intraperitoneal injection (volume 20 mL / kg, dose 1 g / kg), and 24 hours later, animals in groups 4 and 7 were administered IdeS by a single intravenous injection (volume 10 mL / kg, dose 5 mg / kg). Two hours after IdeS administration, antibodies were administered intravenously (volume 10 mL / kg, dose 2 mg / kg). The day of the first antibody administration was set as day 1, and on days 8 and 15, antibodies were administered intravenously to animals in groups 2 to 7, and the corresponding volume of saline was administered intravenously to the vehicle control group. Tumor volumes were measured twice a week after the start of administration.

[0114] As shown in the test results (Figure 7), administration of antibody 8 alone had the same effect as administration of the wild-type antibody alone. In the presence of IgG (IVIg), the tumor therapeutic effect of the antibody was suppressed. The combination of antibody 8 with IgA reduced the inhibition of IgG on the therapeutic effect of the antibody, and significantly enhanced the therapeutic effect of the therapeutic antibody. The wild-type antibody did not have this effect.

[0115] The applicant clearly states that, although the method of the present invention is described in detail in the above examples of the present invention, the present invention is not limited to the above detailed methods, that is, the present invention is not necessarily implemented according to the above detailed methods. Any improvements of the present invention, equivalent replacement of each raw material of the product of the present invention and addition of auxiliary ingredients, selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention, and should be obvious to those skilled in the art. [Table 4] JPEG2024546152000007.jpg225170JPEG2024546152000008.jpg225170JPEG2024546152000009.jpg191170

Claims

1. 1. A molecule comprising an Fc variant, comprising:

1. A molecule comprising an IgG Fc region comprising at least one of the sequences set forth in SEQ ID NO: 19, said molecule being resistant to immunoglobulin degrading enzymes compared to a molecule comprising an IgG wild type Fc, and said molecule comprising a human IgG Fc region having a mutation, said mutation being one or more substitutions or deletions at position G237 (EU numbering), preferably said substitution being G237A.

2. 2. The molecule of claim 1, wherein the Fc molecule is resistant to degradation by a protease, the protease being capable of cleaving an IgG wild-type Fc-containing molecule between residues 236 and 237 (EU numbering).

3. 3. The molecule of claim 1 or 2, wherein the molecule is resistant to degradation by an enzyme selected from the group consisting of IdeS, IdeZ, IdeE, IdeZ2, IdeE2, IdeSORK, Xork and mutants thereof, compared to wild-type IgG.

4. The molecule of any one of claims 1 to 3, wherein the IgG of the molecule is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

5. The molecule of any one of claims 1 to 4, wherein an effector function selected from the group consisting of ADCC, ADCP, and CDC of the molecule is enhanced or increased compared to a molecule comprising an IgG wild-type Fc.

6. The present invention further comprises one or more substitutions in a human IgG wild-type Fc region sequence, the substitutions being S239D, I332E, G236A, S298A / E333A / K334A, S239D / I332E, S239D / A330L / I332E, G236A / S239D / I332E, G236A / A330L / I332E, G236A / S239D / A330L / I332E, F243L / R292P / Y300L, F243L / R292P / Y300L / V305I / P396L, L235V / F243L / R292P / Y300L 6. The molecule of claim 5, wherein the substitutions are selected from the group consisting of: / P396L, P247I / A339Q, K326A / E333A, K326W / E333S, E333A / K334A, H268F / S324T / I332E, S239D / H268F / S324T / I332E, S267E / H268F / S324T / I332E, K326A / I332E / E333A, S239D / K326A / E333A, S267E / I332E, preferably the substitutions are selected from the group consisting of S239D, I332E, S239D / I332E.

7. The molecule according to any one of claims 1 to 6, further comprising an N-glycan modification, wherein said N-glycan modification has a fucose content of less than 50%, preferably less than 10%.

8. The Fc molecule of any one of claims 1 to 7, further comprising an N-glycan modification, wherein the content of terminal galactose modifications in said N-glycan modification is greater than 30%, preferably greater than 50%.

9. The molecule according to any one of claims 1 to 8, wherein the molecule is an antibody or an Fc fusion protein.

10. 10. The molecule of claim 9, wherein the antibody binds to an antigen on a virus, a bacterium, a tumor cell, tumor stroma, or tumor vasculature, preferably the antibody specifically binds to one or two antigens selected from the group consisting of COVID-19, CD38, CD20, FR5, BCMA, SLAM7, CD73, GPRCD5, CD3, and most preferably the antibody specifically binds to CD38.

11. The molecule of claim 9 or 10, wherein the antibody comprises at least one heavy chain and at least one light chain, the heavy chain comprising HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 2, HCDR3 set forth in SEQ ID NO: 3, or HCDR1 set forth in SEQ ID NO: 4, HCDR2 set forth in SEQ ID NO: 5, and HCDR3 set forth in SEQ ID NO: 6, and the light chain comprising LCDR1 set forth in SEQ ID NO: 7, LCDR2 set forth in SEQ ID NO: 8, LCDR3 set forth in SEQ ID NO: 9, or LCDR1 set forth in SEQ ID NO: 10, LCDR2 set forth in SEQ ID NO: 11, and LCDR3 set forth in SEQ ID NO:

12.

12. 12. The molecule of any one of claims 9 to 11, wherein the antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 13 and the light chain comprises a light chain variable region represented by the amino acid sequence of SEQ ID NO: 14, or wherein the heavy chain comprises a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15 and the light chain comprises a light chain variable region represented by the amino acid sequence of SEQ ID NO:

16.

13. The epitope-binding fragment of the antibody may be Fab, Fab', F(ab') ２ 13. The molecule of any one of claims 9 to 12, which is a Fv fragment, scFv or a disulfide-linked Fv fragment.

14. 5. The molecule of any one of claims 1 to 4, wherein the effector function of said molecule selected from the group consisting of ADCC, ADCP, CDC is weakened and / or eliminated compared to a molecule comprising an IgG1 wild-type Fc.

15. The human IgG1 wild-type Fc sequence further comprises one or more substitutions, the substitutions being N297A, N297Q, N297G, L235A, L235A / E318A, L234A / L235A, G236R / L328R, S298G / T299A, L234F / L235E / P331S, E233P / L234V / L235A / G2 15. The Fc molecule of claim 14, wherein the substitutions are selected from the group consisting of: 36del / S267K, L234A / L235A / P329G, L234F / L235E / D265A, and preferably the substitutions are selected from the group consisting of: N297A, N297Q, N297G, L234A / L235A, L235A, L234F / L235E / P331S.

16. 16. The molecule of any one of claims 1 to 15, wherein the affinity of the molecule for FcRn is increased compared to a molecule comprising an IgG1 wild type Fc.

17. 17. The molecule of claim 16, further comprising one or more substitutions in the IgG1 wild-type Fc sequence, said substitutions being selected from the group consisting of YTEKF, YTEKF+V264A, YTEKF+V264E.

18. 1. An isolated binding polypeptide molecule comprising: (i) a binding domain capable of binding to a cell or a target molecule bound to a cell; and (ii) an Fc domain having the amino acid sequence of the CH2 and CH3 constant domains of a human IgG1 heavy chain, with the exception that a substitution or deletion has occurred at residue G237 as defined by the EU numbering system, said binding molecule being capable of binding to a target molecule on a target cell, and said molecule producing measurable CDC activity, or target cytotoxicity activity mediated by a required effector cell, preferably characterized in that the substitution at G237 is G237A.

19. An immunoconjugate comprising a molecule according to any one of claims 1 to 17 or a binding polypeptide molecule according to claim 18 and a cytotoxic agent.

20. 19. One or more isolated nucleic acids encoding a molecule according to any one of claims 1 to 17 or a binding polypeptide molecule according to claim 18.

21. An expression vector comprising the nucleic acid of claim 20.

22. 22. A host cell comprising a nucleic acid according to claim 20 or an expression vector according to claim 21.

23. A method for producing a molecule according to any one of claims 1 to 17 or a binding polypeptide molecule according to claim 18, comprising the steps of: (a) culturing the host cell of claim 22 under conditions suitable for expression of said molecule or said binding polypeptide molecule; (b) optionally recovering said molecule or said binding polypeptide molecule from the culture of step (a).

24. 24. A molecule or binding polypeptide molecule produced by the method of claim 23.

25. 20. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 17, a binding polypeptide molecule according to claim 18 or an immune complex according to claim 19 and an immunoglobulin degrading enzyme, said immunoglobulin degrading enzyme being capable of cleaving IgG molecules between residues 236 and 237 (EU numbering), preferably said immunoglobulin degrading enzyme being IdeS, IdeZ, IdeE, IdeSORK, Xork and variants of these enzymes.

26. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 17, a binding polypeptide molecule according to claim 18 or an immunoconjugate according to claim 19, and a pharmaceutical carrier.

27. A molecule according to any one of claims 1 to 17, a binding polypeptide molecule according to claim 18, an immunoconjugate according to claim 19 or a pharmaceutical composition according to claim 25 or 26 as a medicament, preferably said medicament being used for treating a disease, more preferably said disease being cancer or an autoimmune disease, most preferably said cancer being multiple myeloma.

28. 20. Use of a molecule according to any one of claims 1 to 17, a binding polypeptide molecule according to claim 18, an immunoconjugate according to claim 19 or a pharmaceutical composition according to claim 25 or 26 in the manufacture of a medicament, said medicament being used to treat a disease, in particular cancer or an autoimmune disease.

29. Use of a molecule according to any one of claims 1 to 17, a binding polypeptide molecule according to claim 18, an immunoconjugate according to claim 19 or a pharmaceutical composition according to claim 25 or 26 in the manufacture of a medicament, wherein said medicament is used to induce ADCC, ADCP, apoptosis mediated depletion of CD38 positive cells.

30. A method for treating an individual having cancer or an autoimmune disease, comprising administering to said individual an effective amount of a molecule according to any one of claims 1 to 17, a binding polypeptide molecule according to claim 18, an immunoconjugate according to claim 19 or a pharmaceutical composition according to claim 25 or 26.

31. 27. A method for inducing ADCC, ADCP, apoptosis mediated depletion of CD38 positive cells in an individual, comprising administering to said individual an effective amount of a molecule according to any one of claims 1 to 17, a binding polypeptide molecule according to claim 18, an immunoconjugate according to claim 19 or a pharmaceutical composition according to claim 25 or 26 to induce ADCC, ADCP, apoptosis mediated depletion of CD38 positive cells.

32. The invention described herein.

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