Fc variants with attenuated binding to FcRn

Abstract

Provided are an immunoglobulin Fc region having reduced binding capacity to FcRn, a fusion protein comprising the same, a nucleic acid encoding the same, and a vector and a cell comprising the nucleic acid.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and discloses a protein variant. Specifically, the present invention discloses an immunoglobulin Fc variant, which has a weakened binding ability with FcRn. Background Art

[0002] IgG antibodies recognize target antigens through two antigen-binding fragments (Fab), which are connected to a constant crystallizable fragment (Fc) through a hinge region. This fragment binds to Fc receptors and induces effector functions. Specific antigen binding and appropriate effector functions make IgG antibodies successfully used to treat various diseases.

[0003] There are four IgG subtypes in humans, among which IgG1, 2 and 4 have a longer half-life, and the key factor affecting the plasma half-life of IgG antibodies is a cell receptor called neonatal Fc receptor (FcRn). FcRn is a heterodimer composed of N-glycosylated transmembrane MHC class I heavy chains non-covalently bound to soluble β2-microglobulin. Two FcRn molecules can bind IgG by binding to the symmetrical CH2-CH3 interface in Fc, which prevents IgG from being degraded in lysosomes: IgG can be absorbed by vascular endothelial cells and enter cells through endocytosis; subsequently, IgG binds to FcRn in endosomes (pH 6.0-6.5) in a pH-dependent manner, while antibodies that are not bound to FcRn fuse with lysosomes and are degraded by proteases in lysosomes; and when the IgG-FcRn complex circulates to the cell surface, IgG quickly dissociates from FcRn in a neutral environment (pH 7.0-7.4) and re-enters the circulation.

[0004] Modulating the binding ability to FcRn through Fc engineering has an important impact on the half-life and clearance rate of IgG in vivo. For example, Fc variants that improve FcRn binding under acidic pH conditions without affecting FcRn binding under neutral pH conditions can obtain a longer half-life than the parent. One such engineered antibody has three Fc substitutions (M252Y / S254T / T256E, YTE), which improves the binding to human FcRn at pH 6.0 by about 10 times and prolongs the plasma half-life in non-human primates by nearly 4 times.

[0005] However, not all therapeutic antibodies are expected to have strong FcRn binding ability, long half-life and / or slow clearance rate. For example, for a class of antibodies targeting the Fc segment of immunoglobulins, after forming a complex with immunoglobulins by targeting the Fc segment, in order to quickly clear the targeted immunoglobulins, it is not expected that such antibodies targeting the Fc segment of immunoglobulins will further bind to FcRn, so it is desirable in practice to include Fc variants with reduced binding to FcRn in such antibodies targeting the Fc segment of immunoglobulins. For another example, for locally administered drugs, it is usually expected that they will be quickly cleared in the serum to avoid additional toxic side effects due to systemic exposure.

[0006] Patent publications US20210238308 and CN110317272A disclose Fc variants with reduced binding to FcRn, but do not disclose specific mutation combinations. Therefore, more engineered Fc variants with reduced binding to FcRn are still needed to meet the needs of drug development such as various therapeutic antibodies. Summary of the invention

[0007] The present invention screens and obtains immunoglobulin Fc variants with reduced binding to FcRn through protein engineering, which have a combination of amino acid mutations and are expected to have a shorter half-life and / or a faster clearance rate in mammals.

[0008] In a first aspect, the present disclosure provides an immunoglobulin Fc variant comprising mutations at amino acids 314, 435 and 436; or it comprises mutations at amino acids 253, 254, 435 and 436, wherein the amino acid positions are numbered using the EU index of Kabat.

[0009] In some embodiments, the immunoglobulin Fc variant is derived from IgG, for example, the Fc variant is derived from IgG1, IgG2, IgG3 or IgG4. In some embodiments, the immunoglobulin Fc variant is derived from human IgG.

[0010] In some embodiments, the variant has at least one of the following characteristics compared to a parent Fc that does not contain the mutation: a) reduced binding ability to FcRn; b) increased serum clearance rate in mammals; c) shortened serum half-life in mammals; and d) reduced side effects caused by systemic exposure of Fc.

[0011] In some embodiments, the mutation at amino acid position 314 is L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W or L314Y; preferably L314A, L314F, L314G, L314H, L314I, L314K, L314M, L314P, L314R, L314S, L314V, L314W or L314Y. In some preferred embodiments, the mutation at amino acid position 314 is L314A, L314S, L314G, L314R, L314K, L314W or L314Y. In some more preferred embodiments, the mutation at amino acid position 314 is L314A, L314R or L314W.

[0012] In some embodiments, the amino acid mutation at position 253 is I253A, I253D, I253E, I253F, I253G, I253H, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W or I253Y. In some preferred embodiments, the amino acid mutation at position 253 is I253A, I253D, I253E, I253G, I253N, I253Q or I253S. In some more preferred embodiments, the amino acid mutation at position 253 is I253E or I253D.

[0013] In some embodiments, the mutation at amino acid position 254 is S254A, S254D, S254E, S254F, S254G, S254H, S254I, S254K, S254L, S254M, S254N, S254P, S254Q, S254R, S254T, S254V, S254W or S254Y. In some preferred embodiments, the mutation at amino acid position 254 is S254A, S254D, S254E, S254G, S254N or S254Q. In some more preferred embodiments, the mutation at amino acid position 254 is S254D or S254E.

[0014] In some embodiments, the mutation at amino acid position 435 is H435A, H435D, H435E, H435F, H435G, H435I, H435K, H435L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W or H435Y. In some preferred embodiments, the mutation at amino acid position 435 is H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H435W or H435Y. In some more preferred embodiments, the mutation at amino acid position 435 is H435A, H435S, H435G, H435M, H435L, H435Y or H435I. In some preferred embodiments, the mutation at amino acid position 435 is H435A or H435M.

[0015] In some embodiments, the mutation at amino acid position 436 is Y436A, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436Q, Y436R, Y436S, Y436T, Y436V or Y436W. In some preferred embodiments, the mutation at amino acid position 436 is Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q or Y436N. In some more preferred embodiments, the mutation at amino acid position 436 is Y436A, Y436D, Y436E or Y436Q.

[0016] In some embodiments, the mutations at amino acids 314, 435 and 436 are selected from the group consisting of L314A / H435M / Y436D, L314A / H435M / Y436E, L314W / H435M / Y436D, L314W / H435M / Y436E and L314R / H435M / Y436Q. Preferably, the mutations at amino acids 314, 435 and 436 are selected from the group consisting of L314A / H435M / Y436D, L314A / H435M / Y436E, L314W / H435M / Y436D, L314W / H435M / Y436E and L314R / H435M / Y436Q. The mutation of the amino acid at position 314 is selected from the group consisting of L314A / H435M / Y436D, L314A / H435M / Y436E, L314W / H435M / Y436D and L314W / H435M / Y436E, more preferably, the mutation of the amino acid at position 314, position 435 and position 436 is L314A / H435M / Y436D or L314W / H435M / Y436D.

[0017] In some embodiments, the mutations at amino acids 253, 254, 435, and 436 are I253E / S254D / H435A / Y436D, I253E / S254D / H435M / Y436Q, or I253E / S254D / H435A / Y436A.

[0018] In some embodiments, the immunoglobulin Fc variant also exhibits a reduced ability to bind to an anti-Fc single domain antibody compared to a parent Fc that does not comprise the mutation.

[0019] In some embodiments, the immunoglobulin Fc variant further comprises a mutation that modulates the interaction of the immunoglobulin Fc variant with an Fcγ receptor.

[0020] In some embodiments, the immunoglobulin Fc variant comprises a mutation that enhances the interaction between the immunoglobulin Fc variant and FcγRIIb. In some embodiments, the mutation that enhances the interaction between the immunoglobulin Fc variant and FcγRIIb is selected from the following mutation combinations: S267A / H268A, S267A / E258A, S267A / R255A, S267A / E272A, S239D / I332E, S267E / L328F, E233D / A330R, E23 3D / P238D, G237D / P238D, P238D / H268D, P238D / P271G, P238D / A330R, E233D / P238D / A330R, E233D / P271G / A330R, G237D / H268D / P271G, G237D / P271G / A330R, S239D / A3 30L / I332E, G236A / S239D / I332E, E233D / P238D / P271G / A330R, G237D / P238D / H268 D / P271G, E233D / H268D / P271G / A330R, G237D / P238D / P271G / A330R, G237D / H268D / P In some preferred embodiments, the mutation that enhances the interaction of the immunoglobulin Fc variant with FcγRIIb is S267E / L328F or E233D / G237D / P238D / H268D / P271G / A330R.

[0021] In some embodiments, the immunoglobulin Fc variant comprises mutations S267E / L328F, and (1) mutations at amino acids 314, 435, and 436, or (2) mutations at amino acids 253, 254, 435, and 436. In some specific embodiments, the immunoglobulin Fc variant comprises:

[0022] (a) mutations S267E / L328F, and mutations L314A / H435M / Y436D;

[0023] (b) mutations S267E / L328F, and mutations L314A / H435M / Y436E;

[0024] (c) mutations S267E / L328F, and mutations L314W / H435M / Y436D;

[0025] (d) mutations S267E / L328F, and mutations L314W / H435M / Y436E;

[0026] (e) mutations S267E / L328F, and mutations L314R / H435M / Y436Q;

[0027] (f) mutations S267E / L328F, and mutations I253E / S254D / H435A / Y436D; or

[0028] (g) Mutations S267E / L328F, and mutations I253E / S254D / H435A / Y436A.

[0029] In some embodiments, the immunoglobulin Fc variant comprises mutations E233D / G237D / P238D / H268D / P271G / A330R, and (1) mutations at amino acids 314, 435, and 436, or (2) mutations at amino acids 253, 254, 435, and 436.

[0030] In some specific embodiments, the immunoglobulin Fc variant comprises:

[0031] (a) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314A / H435M / Y436D;

[0032] (b) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314A / H435M / Y436E;

[0033] (c) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314W / H435M / Y436D;

[0034] (d) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314W / H435M / Y436E;

[0035] (e) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435M / Y436Q;

[0036] (f) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435M / Y436D; or

[0037] (g) Mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435A / Y436A.

[0038] In some embodiments, the immunoglobulin Fc variants of the present disclosure further comprise mutations that reduce or eliminate effector function. In some embodiments, the mutations that reduce or eliminate effector function are: D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S298G / T29 9A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A or V234A / G237A / P238S / H268A / V309L / A330S / P331S. In some preferred embodiments, the mutation that reduces or eliminates effector function is D265A / P331G.

[0039] In some embodiments, the immunoglobulin Fc variant comprises the mutation D265A / P331G, and (1) mutations at amino acids 253, 254, 435, and 436, or (2) mutations at amino acids 314, 435, and 436. In some specific embodiments, the immunoglobulin Fc variant comprises:

[0040] (a) mutations D265A / P331G, and mutations I253E / S254D / H435M / Y436Q;

[0041] (b) mutations D265A / P331G, and mutations I253A / S254A / H435M / Y436E;

[0042] (c) mutations D265A / P331G, and mutations I253E / S254D / H435M / Y436E;

[0043] (d) mutations D265A / P331G, and mutations I253A / S254A / H435A / Y436A;

[0044] (e) mutations D265A / P331G, and mutations I253A / S254A / H435M / Y436Q; or

[0045] (f) Mutation D265A / P331G, and mutation L314W / H435M / Y436E.

[0046] In a second aspect, the present disclosure provides a fusion protein comprising the immunoglobulin Fc variant described in the first aspect. In some embodiments, the fusion protein further comprises an antibody variable region.

[0047] In a third aspect, the present disclosure provides a nucleic acid molecule encoding the immunoglobulin Fc variant of the first aspect or the fusion protein of the second aspect.

[0048] In a fourth aspect, the present disclosure provides an expression vector comprising the nucleic acid molecule of the third aspect operably linked to an expression regulatory element.

[0049] In a fifth aspect, the present disclosure provides a recombinant cell comprising the nucleic acid molecule of the third aspect and / or transformed with the expression vector of the fourth aspect, and capable of expressing the immunoglobulin Fc variant or a fusion protein comprising the same.

[0050] In a sixth aspect, the present disclosure provides a composition comprising the immunoglobulin Fc variant of the first aspect, the fusion protein of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the recombinant cell of the fifth aspect.

[0051] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the specification of the present application is merely exemplary, and not restrictive. DETAILED DESCRIPTION

[0052] Definition of terms

[0053] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be understood by those skilled in the art. Reference is made to, for example, standard manuals, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd edition), Gower Medical Publishing, London, New York (1989), and the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se, which will be understood by those skilled in the art. Reference is also made to, for example, standard manuals, the above-mentioned general prior art and other references cited therein.

[0054] Unless otherwise indicated, the terms "antibody" or "immunoglobulin" used interchangeably, whether referring to heavy chain antibodies or conventional four-chain antibodies herein, are used as general terms to include full-length antibodies, their individual chains, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH / VL domains, respectively). In addition, the term "sequence" used herein (e.g., in terms of "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0055] The term "immunoglobulin" generally refers to a protein consisting essentially of one or more polypeptides encoded by immunoglobulin genes. Recognized human immunoglobulin genes include κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε and μ constant region genes, as well as numerous immunoglobulin variable region genes. In general, immunoglobulins can be heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). The NH2-terminus (about 110 amino acids) of the full-length immunoglobulin "light chain" (about 25KD, 214 amino acids) is encoded by the variable region gene, and the COOH-terminus is encoded by the κ or λ constant region gene. Natural immunoglobulins are essentially composed of two Fab molecules and an Fc region connected via an immunoglobulin hinge region.

[0056] As used herein, the term "immunoglobulin Fc region" refers to a domain derived from the C-terminal region of an immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody, typically including a hinge region and two constant domains (CH2 domain and CH3 domain). The Fc region can be a native sequence Fc region or an Fc region variant, such as the amino acid sequence shown in any one of SEQ ID NOs: 14-17, which correspond to the Fc sequences of human IgG1, IgG2, IgG3 and IgG4, respectively.

[0057] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain that is essentially composed of four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity to antibodies for antigens by having antigen binding sites.

[0058] The term "single domain antibody", "sdAb" or "nanobody" as used herein refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs). Single domain antibodies are capable of binding to an antigen alone without the need for pairing with a corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelid heavy chain antibodies (HCAbs), and their heavy chain variable domains are referred to herein as "VHHs", and camelid single domain antibodies are one of the smallest known antigen-binding antibody fragments.

[0059] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment and VHH antibody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e. "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VH domain") and the light chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VL domain"). The VHH domain specifically binds to an epitope without the need for additional antigen-binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.

[0060] For example, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used for the VHH domain of Camelidae can be numbered according to the general numbering method for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0061] Alternative methods for numbering the amino acid residues of VH domains are known in the art and may be similarly applied to VHH domains. For example, Chothia CDRs refer to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM CDRs represent a compromise between the Kabat hypervariable regions and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. The residues of the CDRs from each method are described as follows:

[0062] ring Kabat AbM Chothia Contact LCDR1 L24-L34 L24-L34 L26-L32 L30-L36 LCDR2 L50-L56 L50-L56 L50-L52 L46-L55 LCDR3 L89-L97 L89-L97 L91-L96 L89-L96 HCDR1 (Kabat numbering) H31-H35B H26-H35B H26-H32 H30-H35B HCDR1 (Chothia numbering) H31-H35 H26-H35 H26-H32 H30-H35 HCDR2 H50-H65 H50-H58 H53-H55 H47-H58 HCDR3 H95-H102 H95-H102 H96-H101 H93-H101

[0063] The CDR of an antibody can also be IMGT-CDR, which is a CDR definition method based on the IMGT antibody code, which is obtained by integrating the structural information of more than 5000 sequences. In the VH CDR code of IMGT, CDR1: 27-38; CDR2: 56-65; CDR3: 105-117.

[0064] It should be noted, however, that as is known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0065] For example, the CDRs may include "extended CDRs", such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 (LCDR3) in VL; 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.

[0066] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0067] In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain of the present invention) can bind to. Its specificity can be determined based on the affinity and / or avidity of the antigen binding protein. The affinity represented by the dissociation equilibrium constant (KD) of the antigen and the antigen binding protein is a measure of the binding strength between the epitope and the antigen binding site on the antigen binding protein: the smaller the KD value, the stronger the binding strength between the epitope and the antigen binding protein (or, affinity can also be expressed as an association constant (KA), which is 1 / KD). As will be appreciated by those skilled in the art, affinity can be measured in a known manner, depending on the specific antigen of interest. Avidity is a measure of the binding strength between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing it) and a related antigen. Avidity is related to the following two: the affinity between the antigen binding site on its antigen binding protein, and the number of related binding sites present on the antigen binding protein.

[0068] Amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence compared to another sequence. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution, which refers to the replacement of an amino acid residue by another amino acid residue of similar chemical structure and which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art. For example, conservative amino acid substitutions are preferably substitutions in which an amino acid within the following groups (i) to (v) is replaced by another amino acid residue within the same group: (i) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.

[0069] As used herein, "mutation" or "amino acid mutation" generally includes any type of change or modification to a sequence (nucleic acid or amino acid sequence), including the deletion, truncation, inactivation, destruction, substitution or translocation of an amino acid or nucleotide. In the present application, an amino acid mutation may be an amino acid substitution, and the term "substitution" generally refers to replacing at least one amino acid residue in a predetermined parent amino acid sequence with a different "replacement" amino acid residue. The one or more replaced amino acid residues may be "naturally occurring amino acid residues" (i.e., encoded by a genetic code). AxxxB is generally used herein to represent an amino acid mutation, such as "S267E", wherein A represents the parent amino acid residue, B represents the replaced amino acid residue, and xxx represents the numbering of the mutated amino acid residue, and if not otherwise specified, the EU index of Kabat is used herein for numbering.

[0070] As used herein, the terms "polypeptide," "protein," or "fusion protein" are used interchangeably and generally refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term may also include modified amino acid polymers, for example, by the addition of sugar residues to form glycoproteins or by phosphorylation. Polypeptides, proteins, and fusion proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may include molecules having the amino acid sequence of a native protein, or molecules having deletions, additions, and / or substitutions of one or more amino acids of the native sequence.

[0071] In this application, the term "nucleic acid" molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, either isolated from their natural environment or artificially synthesized.

[0072] In the present application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0073] In the present application, the term "cell" generally refers to a plasmid or vector that may contain or already contain a nucleic acid molecule described in the present application, or an individual cell, cell line or cell culture that can express the antigen-binding protein described in the present application. The cell may include the progeny of a single host cell. Due to natural, unexpected or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but can express the antibody or its antigen-binding fragment described in the present application. The cell can be obtained by using the vector described in the present application to transfect cells in vitro. The cell can be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell or a myeloma cell). In some cases, the cell can be a mammalian cell. For example, the mammalian cell can be a CHO-K1 cell.

[0074] As used herein, "cytokine" is intended to be a general term for proteins released by one cell population that act on another cell as intercellular regulators. DETAILED DESCRIPTION OF THE INVENTION

[0076] Immunoglobulin Fc variants

[0077] In one aspect, the present invention relates to an immunoglobulin Fc variant comprising mutations at amino acids 314, 435 and 436, wherein the amino acid positions are numbered using the EU index of Kabat.

[0078] On the other hand, the present invention also relates to an immunoglobulin Fc variant comprising mutations at amino acids 253, 254, 435 and 436, wherein the amino acid positions are numbered using the EU index of Kabat.

[0079] In some embodiments, the immunoglobulin Fc variant of the present invention has at least one of the following characteristics compared to a parent Fc that does not contain the mutation: a) reduced binding ability to FcRn; b) increased serum clearance rate in mammals; c) shortened serum half-life in mammals; and d) reduced side effects caused by systemic exposure of Fc. In some embodiments, the immunoglobulin may be IgG, IgA, IgM, IgD or IgE. In some preferred embodiments, the immunoglobulin is IgG.

[0080] In some embodiments, the immunoglobulin Fc variant is derived from the Fc of IgG, IgA, IgM, IgD or IgE. In some preferred embodiments, the immunoglobulin Fc variant is derived from the Fc of IgG.

[0081] In some embodiments, the immunoglobulin Fc variant is obtained by performing mutations at amino acids at positions 314, 435 and 436 on the basis of wild-type or artificially modified IgG, IgA, IgM, IgD or IgE (preferably IgG); or it comprises mutations at amino acids at positions 253, 254, 435 and 436.

[0082] In some embodiments, the immunoglobulin may be derived from human, mouse, rat, rabbit or monkey, for example, it may be IgG derived from the above mammals, including IgG1, IgG2, IgG3 and IgG4; for example, it may be IgG1 derived from human.

[0083] In some embodiments, the mutation at amino acid position 314 may be L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W or L314Y. In other embodiments, the mutation at amino acid position 314 may be L314A, L314F, L314G, L314H, L314I, L314K, L314M, L314P, L314R, L314S, L314V, L314W or L314Y. In some preferred embodiments, the mutation at amino acid position 314 may be L314A, L314S, L314G, L314R, L314K, L314W or L314Y. In some more preferred embodiments, the mutation at amino acid position 314 may be L314A, L314W or L314R.

[0084] In some embodiments, the mutation at amino acid position 435 may be H435A, H435D, H435E, H435F, H435G, H435I, H435K, H435L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W or H435Y. In other embodiments, the mutation at amino acid position 435 may be H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H435W or H435Y. In some preferred embodiments, the mutation at amino acid position 435 may be H435A, H435S, H435G, H435M, H435L, H435Y or H435I. In some more preferred embodiments, the mutation at amino acid position 435 may be H435A or H435M.

[0085] In some embodiments, the mutation at amino acid position 436 may be Y436A, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436Q, Y436R, Y436S, Y436T, Y436V or Y436W. In other embodiments, the mutation at amino acid position 436 may be Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q or Y436N. In some preferred embodiments, the mutation at amino acid position 436 may be Y436A, Y436D, Y436E or Y436Q.

[0086] In some embodiments, the mutation at amino acid position 253 may be I253A, I253D, I253E, I253F, I253G, I253H, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W or I253Y. In other embodiments, the mutation at amino acid position 253 may be I253A, I253D, I253E, I253G, I253N, I253Q or I253S. In some preferred embodiments, the mutation at amino acid position 253 may be I253E or I253D.

[0087] In some embodiments, the mutation at amino acid position 254 may be S254A, S254D, S254E, S254F, S254G, S254H, S254I, S254K, S254L, S254M, S254N, S254P, S254Q, S254R, S254T, S254V, S254W or S254Y. In other embodiments, the mutation at amino acid position 254 may be S254A, S254D, S254E, S254G, S254N or S254Q. The mutation at amino acid position 254 may be S254D or S254E.

[0088] In some specific embodiments, the immunoglobulin Fc variant may contain the following amino acid mutation combinations:

[0089]

[0090]

[0091] In some embodiments, the immunoglobulin Fc variant comprising the aforementioned mutations also exhibits a weakened ability to bind to an anti-immunoglobulin Fc antibody (hereinafter referred to as anti-Fc antibody) compared to a parent Fc that does not comprise the aforementioned mutations. The anti-immunoglobulin Fc antibody refers to a therapeutic antibody that can specifically bind to the immunoglobulin Fc segment, block the interaction between the Fc segment and FcRn, and thus produce a biological effect.

[0092] In some embodiments, the anti-Fc antibody is an anti-Fc single domain antibody.

[0093] The anti-Fc single domain antibody can be obtained by immunizing camels, wherein the representative single domain antibody is iFc106, which can specifically bind to human IgG1-Fc and effectively block the binding of Fc to FcRn, thereby achieving the effect of reducing the level of pathogenic IgG in the body. iFc106 comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1.

[0094] In some embodiments, the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NO: 2-4, SEQ ID NO: 5-7, SEQ ID NO: 8-10, SEQ ID NO: 11-13. In some embodiments, iFc106 comprises the VHH shown in SEQ ID NO: 1; in some embodiments, the amino acid sequence of iFc106 is as shown in SEQ ID NO: 1.

[0095] On the other hand, the immunoglobulin Fc variant may further comprise a mutation that regulates the interaction of the immunoglobulin Fc variant with an Fcγ receptor. For example, the Fcγ receptor may be FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a) and / or FcγRIIIb (CD16b). In some embodiments, the immunoglobulin Fc variant comprises a mutation that enhances the interaction of the immunoglobulin Fc variant with FcγRIIb.

[0096] The mutation that enhances the interaction with FcγRIIb can be any mutation or combination of mutations known in the prior art, such as those described in Rena Liu, Antibodies 2020, 9, 64; Chu, SY, Mol. Immunol. 2008, 45, 3926-3933; Zhang, D., J. Biol. Chem. 2016, 291, 27134-27146; Dahan, R., Cancer Cell 2016, 29, 820-831; Mimoto, F., Protein Eng. Des. Sel. 2013, 26, 589-598; Robert L. Shields, J. Biol. Chem. 2001, 276: 6591-6604.

[0097] In some embodiments, the immunoglobulin Fc variant further comprises one or more mutations selected from the following that enhance interaction with FcγRIIb: E233D, G237D, P238D, T265A, H268D, K290A, R255A, E258A, S267A, S267E, P271G, E272A, N276A, D280A, H285A, N286A, T307A, L309A, N315A, K326A, L328E, L328F, A330R, P331A, S337A, A378Q, E430A, H268A, R301A, R301M, K322A, K334R and K334N.

[0098] In some embodiments, the immunoglobulin Fc variant further comprises a combination of mutations that enhance interaction with FcγRIIb selected from the following: S267A / H268A, S267A / E258A, S267A / R255A, S267A / E272A, S239D / I332E, S267E / L328F, E233D / A330R, E23 3D / P238D, G237D / P238D, P238D / H268D, P238D / P271G, P238D / A330R, E233D / P238D / A330R, E233D / P271G / A330R, G237D / H268D / P271G, G237D / P271G / A330R, S239D / A3 30L / I332E, G236A / S239D / I332E, E233D / P238D / P271G / A330R, G237D / P238D / H268 D / P271G, E233D / H268D / P271G / A330R, G237D / P238D / P271G / A330R, G237D / H268D / P : The models with the same specifications include E233D / G237D / H268D / P271G / A330R, E233D / P238D / H268D / P271G / A330R, G237D / P238D / H268D / P271G / A330R and E233D / G237D / P238D / H268D / P271G / A330R.

[0099] In some embodiments, the immunoglobulin Fc variant further comprises a mutation selected from the following that enhances interaction with FcγRIIb: P238D, S267E, L328F, A330R, S267E / L328F, G237D / P238D / H268D / P271G / A330R or E233D / G237D / P238D / H268D / P271G / A330R. Preferably, the immunoglobulin Fc variant further comprises a mutation selected from S267E / L328F (hereinafter abbreviated as EF) or E233D / G237D / P238D / H268D / P271G / A330R (hereinafter abbreviated as V12) that enhances interaction with FcγRIIb.

[0100] In some specific embodiments, the immunoglobulin Fc variant comprises a combination of a mutation that enhances the interaction with FcγRIIb and the aforementioned amino acid mutations, for example, the immunoglobulin Fc variant may comprise a combination of mutations selected from the following: EF(S267E / L328F) / L314A / H435M / Y436D, EF / L314A / H435M / Y436E, EF / L314A / H435M / Y436Q, EF / L314W / H435M / Y436E, EF / L314W / H435M / Y436D, EF / L314A / H435M / Y436E 36Q、EF / L314R / H435M / Y436E、EF / L314R / H435M / Y436D、EF / L314R / H435M / Y436Q、EF / I253E / S254D / H435A / Y436D、EF / I253E / S254D / H435A / Y436E、EF / I253E / S254D / H435A / Y436Q、EF / I253E / S254D / H435A / Y436A、EF / I253E / S254D / H435M / Y436D、EF / I253E / S254D / H435M / Y436E, EF / I253E / S254D / H435M / Y436Q, V12 / L314A / H435M / Y436D, V12 / L314A / H435M / Y436E, V12 / L314A / H435M / Y436Q, V12 / L314W / H435M / Y436E、V12 / L314W / H435M / Y436D、V12 / L314W / H435M / Y436Q、V12 / L314R / H435M / Y436E、V12 / L314R / H435M / Y436D、V1 2 / L314R / H435M / Y436Q, V12 / I253E / S254D / H435A / Y436D, V12 / I253E / S254D / H435A / Y436E, V12 / I253E / S254D / H435A / Y436Q, V12 / I253E / S254D / H435A / Y436A, V12 / I253E / S254D / H435M / Y436D, V12 / I253E / S254D / H435M / Y436E, or V12 / I253E / S254D / H435M / Y436Q.

[0101] In some embodiments, the immunoglobulin Fc variant may also include a mutation that reduces or eliminates effector function. The effector function may be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutation that reduces or eliminates effector function weakens or eliminates the binding between the immunoglobulin and FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb and / or C1q.

[0102] The mutation that weakens or eliminates the effector function can be any mutation or combination of mutations known in the prior art, such as Esohe EI, J Immunol 2000; 164: 4178-4184; Hutchins, JT, Proc. Natl. Acad. Sci. USA 1995, 92, 11980-11984; Xu D., Cell Immunol. 2000, 200, 16-26; Hezareh, M., J. Virol. 2001, 75, 12161-12168; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457-466; Chu, SY, Mol. Immunol. 2008, 45, 3926-3933; Sazinsky, SL, Proc. Natl. Acad. Sci. USA 2008,105,20167-20172;Oganesyan,V.,ActaCrystallogr.Sect.D Biol.Crystallogr.2008,64Pt 6,700-704;An,Z.,MAbs 2009,1,572-579;Moore,GL,Methods 2019,154,38-50;Schlothauer,T.,ProteinEng.Des.Sel.2016,29,457-466;Strohl,W.,US20150337053;Engelberts,PJ,EBioMedicine 2020,52,102625,etc.

[0103] In some embodiments, the immunoglobulin Fc variant may further comprise a mutation selected from the group consisting of D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S298G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A or V234A / G237A / P238S / H268A / V309L / A330S / P331S. Preferably, the immunoglobulin Fc variant may also comprise a mutation selected from the group consisting of D265A, P331G, D265A / P331G (hereinafter abbreviated as AG) or L234F / L235E / P331S that reduces or eliminates effector function.

[0104] In some specific embodiments, the immunoglobulin Fc variant comprises a combination of a mutation that reduces or eliminates effector function and the aforementioned amino acid mutations, for example, the immunoglobulin Fc variant may comprise a combination of mutations selected from the following: AG / L314W / H435M / Y436E, AG / L314W / H435M / Y436D, AG / L314A / H435M / Y436Q, AG / L314A / H435M / Y436E, AG / L314A / H435M / Y436D, AG / L314A / H435M / Y436Q, AG / I253E / S254D / H435M / Y436Q, AG / I253E / S254D / H435M / Y436D, AG / I253E / S254D / H435M / Y436E, AG / I253A / S254A / H435M / Y436E , AG / I253A / S254A / H435M / Y436D, AG / I253A / S254A / H435M / Y436Q, AG / I253A / S254A / H435A / Y436A, or AG / I253A / S254A / Y436E.

[0105] Fusion Protein

[0106] On the other hand, the present invention also provides a fusion protein, the fusion protein comprises the immunoglobulin Fc variant of the present invention. In some embodiments, the fusion protein further comprises a second polypeptide, the second polypeptide is selected from an antibody variable region, a receptor target binding region, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine and / or any other biologically active protein, protein domain or polypeptide.

[0107] In some embodiments, in the fusion protein, the second polypeptide is fused to the N-terminus or C-terminus of the immunoglobulin Fc variant.

[0108] In some embodiments, in the fusion protein, the second polypeptide is directly connected to the immunoglobulin Fc variant, or is connected via a linker. In some embodiments, the linker can be a flexible polypeptide linker.

[0109] In some embodiments, in the fusion protein, the second polypeptide is an antibody variable region. The antibody variable region can target any antigen. The target antigen includes a membrane-bound factor, and the membrane-bound factor includes a transmembrane receptor, such as: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxy-dG, A1 adenosine receptor, A33, ACE, ACE-2, activator protein, activator protein A, activator protein AB, activator protein B, activator protein C, activator protein RIA, activator protein RIAALK-2, activator protein RIBALK-4, activator protein RIIA, activator protein RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS 4. ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bc l, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2BMP-2a, BMP-3 osteogenic protein, BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-D NA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2,CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, C CR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD5 6. CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin Clostridium botulinum toxin, Clostridium perfringen toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, DNA enzyme, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM,EphrinB2 / EphB4, EPO, ERCC, endothelial cell selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2 、GCSF、GD2、GD3、GDF、GDF-1、GDF-3(Vgr-2)、GDF-5(BMP-14、CDMP-1)、GDF-6(BMP-13、CDMP-2)、GDF-7(BMP-12、CDMP-3)、GDF-8(myostatin)、GDF-9、GDF-15(MIC-1)、GDNF、GDNF、GFAP、GFRa-1、GFR-α1、GFR-α2、GFR-α3、GITR、glucagon、Glut4、glycoprotein IIb / IIIa(GPIIb / IIIa)、GM-CSF、gp130、gp72、GR O, growth hormone releasing factor, hapten (NP-caporNIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV)gH envelope glycoprotein, HCMVUL, hematopoietic growth factor (HGF), HepBgp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSVgD glycoprotein, HGFA, high molecular weight melanoma associated antigen (HMW-MAA), HIVgp120, HIVIIIBgp120V3loop, HLA, HLA-DR, HM1.24, HM FGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R,IL-23, interferon (INF)-α, INF-β, INF-γ, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, M CAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC(HLA-DR), MIF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-1 1. MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Mullerian inhibitory substance, Mug, MuS K, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, neurotrophin-4 or neurotrophin-6, Neurturin, neuron growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PD-1, PD-L1, PDGF, PDGF, PDK-1,PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptor (e.g., T-cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β pan-specific (PanSpecific), TGF-βRI (ALK-5 )、TGF-βRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymus Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAILR1Apo-2, DR4), TNFRSF10B (TRAILR2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10 C (TRAILR3DcR1, LIT, TRID), TNFRSF10D (TRAILR4DcR2, TRUNDD), TNFRSF11A (RANKODFR, TRANCER), TNFRSF11B (OPGOCIF, TR1), TNFRSF12 (TWEAKRFN14 ), TNFRSF13B(TACI), TNFRSF13C(BAFFR), TNFRSF14(HVEMATAR, HveA, LIGHTR, TR2), TNFRSF16(NGFRp75NTR), TNFRSF17(BCMA), TNFRSF18(GITRAITR),TNFRSF19(TROYTAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNFRICD120a, p55-60), TNFRSF1B(TNFRIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(L TbRTNFRIII, TNFCR), TNFRSF4 (OX40ACT35, TXGP1R), TNFRSF5 (CD40p50), TNFRSF6 (FasApo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BBCD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAILR2TNFRH2), TNFRST23 (DcTRAILR1TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAILApo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAKApo-3 ligand, DR3 ligand), TNFSF13 (APRILTALL2) 、TNFSF13B(BAFFBLYS, TALL1, THANK, TNFSF20)、TNFSF14(LIGHTHVEM ligand, LTg)、TNFSF15(TL1A / VEGI)、TNFSF18(GITR ligand AITR ligand, TL6)、TNFSF1A(TNF-a connexin (Conectin), DIF, TNFSF2)、TNFSF1B(TNF-bLTa, TNFSF1)、TNFSF3(LTbTNFC, p33)、TNFSF4(OX40 ligand gp34, TXGP1)、TNFSF5(CD40 ligand CD154, gp39, HIGM1, I MD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM,VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth factors.

[0110] In some embodiments, in the fusion protein, the second polypeptide is a cytokine, which can be fused with the immunoglobulin Fc variant to provide many desired properties. For example, the cytokine can be: growth hormone, such as human growth hormone, N-methionyl human growth hormone, bovine growth hormone; parathyroid hormone, thyroxine; insulin; proinsulin; glucagon; glucagon-like peptides, such as GLP-1, GLP-2; glucose-dependent insulinotropic polypeptide; amylin; YY peptide; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor, such as FGF19, FGF21, FGF23; prolactin; placental lactogen; tumor necrosis factor-α and tumor necrosis factor-β; Mullerian inhibitory substance; mouse gonadotropin-related peptides; inhibins; activins; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs), such as TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, interferon-β and interferon-γ; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15.

[0111] Nucleic acids, vectors, recombinant cells

[0112] In another aspect, the present invention relates to a nucleic acid molecule encoding an immunoglobulin Fc variant of the present invention or comprising a fusion protein thereof. The nucleic acid of the present invention may be RNA, DNA or cDNA. According to one embodiment of the present invention, the nucleic acid of the present invention is a substantially isolated nucleic acid.

[0113] The nucleic acid of the present invention can also be in the form of a vector, can be present in a vector and / or can be a part of a vector, such as a plasmid, a cosmid or a YAC. The vector can be an expression vector in particular, that is, a vector that can provide immunoglobulin Fc variants or contain its fusion protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system) expression. The expression vector usually comprises at least one nucleic acid of the present invention, which is operably connected to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common sense for those skilled in the art to select the elements and their sequences for expression in a specific host. The specific examples of the regulatory elements and other elements useful or necessary for the expression of immunoglobulin Fc variants of the present invention or containing its fusion protein, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0114] The nucleic acids of the invention can be prepared or obtained by known means (e.g. by automated DNA synthesis and / or recombinant DNA technology) based on the information on the amino acid sequences of the polypeptides of the invention given herein, and / or can be isolated from suitable natural sources.

[0115] In another aspect, the present invention relates to a recombinant host cell expressing or capable of expressing one or more immunoglobulin Fc variants of the present invention or comprising a fusion protein thereof and / or comprising a nucleic acid or vector of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.

[0116] Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0117] Suitable fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0118] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0119] However, the present invention can also use amphibian cells, insect cells, and the like, and any other cells known in the art for expressing heterologous proteins.

[0120] The immunoglobulin Fc variants of the present invention or fusion proteins comprising the same can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0121] Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. are known in the art. Similarly, protein separation and purification techniques suitable for use in the methods of producing the immunoglobulin Fc variants of the present invention or fusion proteins thereof are well known to those skilled in the art.

[0122] Composition

[0123] In another aspect, the present invention relates to a composition comprising the immunoglobulin Fc variant, fusion protein, nucleic acid molecule, expression vector, or recombinant cell described herein.

[0124] In some embodiments, the composition is a pharmaceutical composition, which comprises the immunoglobulin Fc variant, fusion protein, nucleic acid molecule, expression vector, or recombinant cell described in the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0125] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0126] In this article, pharmaceutically acceptable carrier refers to a drug carrier that helps to administer immunoglobulin Fc variants, fusion proteins, nucleic acid molecules, expression vectors, or recombinant cells to the human body and / or facilitates their absorption or function. For example: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium / magnesium stearate, polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0127] For example, a pharmaceutical composition comprising the immunoglobulin Fc variant, fusion protein, nucleic acid molecule, expression vector, or recombinant cell of the present invention may comprise a pharmaceutically acceptable diluent or a sustained-release matrix, in which the immunoglobulin Fc variant, fusion protein, nucleic acid molecule, expression vector, or recombinant cell of the present invention is embedded.

[0128] The present invention also provides the following non-limiting embodiments:

[0129] 1. An immunoglobulin Fc variant comprising mutations at amino acids 314, 435 and 436; or, comprising mutations at amino acids 253, 254, 435 and 436; the amino acid positions are numbered using the EU index of Kabat.

[0130] 2. The immunoglobulin Fc variant according to embodiment 1, which has at least one of the following characteristics compared to the Fc before mutation: a) weakened binding ability to FcRn; b) increased serum clearance rate in mammals; c) shortened serum half-life in mammals; d) reduced side effects caused by systemic exposure of Fc.

[0131] 3. The immunoglobulin Fc variant according to embodiment 1 or 2, wherein the immunoglobulin is IgG, IgA, IgM, IgD and IgE; preferably IgG.

[0132] 4. The immunoglobulin Fc variant according to any one of embodiments 1-3, wherein the immunoglobulin is of human, mouse, rat, rabbit or monkey origin.

[0133] 5. The immunoglobulin Fc variant according to any one of embodiments 1-4, wherein the immunoglobulin is IgG1, IgG2, IgG3 or IgG4; preferably IgG1 of human origin.

[0134] 6. According to the immunoglobulin Fc variant described in embodiment 1, the mutation at amino acid position 314 is selected from: L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W or L314Y; preferably It is L314A, L314F, L314G, L314H, L314I, L314K, L314M, L314P, L314R, L314S, L314V, L314W or L314Y; more preferably it is L314A, L314S, L314G, L314R, L314K, L314W or L314Y; even more preferably it is L314A, L314W or L314R.

[0135] 7. According to the immunoglobulin Fc variant described in embodiment 1, the mutation at amino acid position 435 is selected from: H435A, H435D, H435E, H435F, H435G, H435I, H435K, H435L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W or H435Y; preferably H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H435W or H435Y; more preferably H435A, H435S, H435G, H435M, H435L, H435Y or H435I; even more preferably H435A or H435M.

[0136] 8. According to the immunoglobulin Fc variant described in embodiment 1, the mutation at amino acid position 436 is selected from: Y436A, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436Q, Y436R, Y436S, Y436T, Y436V or Y436W; preferably Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q or Y436N; more preferably Y436A, Y436D, Y436E or Y436Q.

[0137] 9. According to the immunoglobulin Fc variant described in embodiment 1, the mutation at amino acid position 253 is selected from: I253A, I253D, I253E, I253F, I253G, I253H, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W or I253Y; preferably I253A, I253D, I253E, I253G, I253N, I253Q or I253S; more preferably I253E or I253D.

[0138] 10. According to the immunoglobulin Fc variant described in embodiment 1, the mutation at amino acid position 254 is selected from: S254A, S254D, S254E, S254F, S254G, S254H, S254I, S254K, S254L, S254M, S254N, S254P, S254Q, S254R, S254T, S254V, S254W or S254Y; preferably S254A, S254D, S254E, S254G, S254N or S254Q; more preferably S254D or S254E.

[0139] 11. The immunoglobulin Fc variant according to any one of embodiments 1-10, which further exhibits a reduced ability to bind to an anti-Fc single domain antibody compared to a parent Fc not comprising the mutation.

[0140] 12. According to the immunoglobulin Fc variant of embodiment 11, the anti-Fc single-domain antibody comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1.

[0141] 13. According to the immunoglobulin Fc variant described in embodiment 12, the CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1 are selected from any one of the following groups: SEQ ID NO: 2-4 (according to the Kabat system numbering), SEQ ID NO: 5-7 (according to the AbM system numbering), SEQ ID NO: 8-10 (according to the Chothia system numbering), SEQ ID NO: 11-13 (according to the IMGT system numbering).

[0142] 14. The immunoglobulin Fc variant according to embodiment 12 or 13, wherein the anti-Fc single domain antibody comprises the VHH shown in SEQ ID NO: 1.

[0143] 15. The immunoglobulin Fc variant according to any one of embodiments 1-14, further comprising a mutation that regulates the interaction of the immunoglobulin Fc variant with an Fcγ receptor.

[0144] 16. The immunoglobulin Fc variant according to embodiment 15, comprising a mutation that enhances the interaction of the immunoglobulin Fc variant with FcγRIIb.

[0145] 17. The immunoglobulin Fc variant according to embodiment 16, wherein the mutation that enhances the interaction between the immunoglobulin Fc variant and FcγRIIb is selected from the following mutation combinations: S267A / H268A, S267A / E258A, S267A / R255A, S267A / E272A, S239D / I332E, S267E / L328F, E233 D / A330R, E233D / P238D, G237D / P238D, P238D / H268D, P238D / P271G, P238D / A330R, E2 33D / P238D / A330R, E233D / P271G / A330R, G237D / H268D / P271G, G237D / P271G / A330R, S 239D / A330L / I332E, G236A / S239D / I332E, E233D / P238D / P271G / A330R, G237D / P238D / H268D / P271G, E233D / H268D / P271G / A330R, G237D / P238D / P271G / A330R, G237D / H26 8D / P271G / A330R, E233D / G237D / H268D / P271G / A330R, E233D / P238D / H268D / P271G / A 330R, G237D / P238D / H268D / P271G / A330R or E233D / G237D / P238D / H268D / P271G / A330R.

[0146] 18. The immunoglobulin Fc variant according to any one of embodiments 1-14, further comprising a mutation that reduces or eliminates effector function.

[0147] 19. The immunoglobulin Fc variant according to embodiment 18, comprising a mutation selected from the group consisting of: D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R , S298G / T299A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S 267K, L234A / L235A / P329G, L234F / L235E / D265A or V234A / G237A / P238S / H268A / V309L / A330S / P331S.

[0148] 20. A fusion protein comprising the immunoglobulin Fc variant of any one of embodiments 1-19.

[0149] 21. According to the fusion protein of embodiment 20, it also comprises a second polypeptide, and the second polypeptide is selected from an antibody variable region, a receptor target binding region, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine and / or any other protein or protein domain with biological activity.

[0150] 22. According to the fusion protein of embodiment 21, the second polypeptide is directly connected to the immunoglobulin Fc variant or connected through a flexible polypeptide chain.

[0151] 23. A nucleic acid molecule encoding the immunoglobulin Fc variant of any one of embodiments 1-19 or the fusion protein of any one of embodiments 20-22.

[0152] 24. An expression vector comprising the nucleic acid molecule of embodiment 23 operably linked to an expression control element.

[0153] 25. A recombinant cell comprising the nucleic acid molecule of embodiment 23 and / or transformed with the expression vector of embodiment 24, and capable of expressing the immunoglobulin Fc variant or a fusion protein comprising the same.

[0154] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0155] Example

[0156] Example 1 Screening for Fc variants with reduced binding to FcRn

[0157] Variants of human IgG1-Fc were screened, and some Fc variants were introduced with S267E / L328F mutations (hereinafter abbreviated as FcEF) or E233D / G237D / P238D / H268D / P271G / A330R mutations (hereinafter abbreviated as FcV12) that enhance the binding activity of Fc to FcγRIIb (CD32b).

[0158] The binding kinetics of Fc variants to human FcRn were detected by biomembrane interferometry (BLI) technology. Under pH 6.0 conditions, human FcRn protein was directly immobilized on the HIS1K sensor, and IgG1Fc and its variants (at concentrations of 2000nM, 3000nM and 10000nM) were added to bind to the immobilized human FcRn protein. The binding ability was judged by the response value, and a response value <0.1 was judged as no binding. The results are shown in Tables 1-3. Table 1 shows the response values ​​of Fc or Fc variants binding to FcRn at a concentration of 2000nM. Table 2 shows the response values ​​of Fc or Fc variants binding to FcRn at a concentration of 3000nM. Table 3 shows the response values ​​of Fc or Fc variants binding to FcRn at a concentration of 10000nM. Numbers + single letters are used to indicate the mutation site and the amino acid after mutation. Mutations at adjacent positions are marked with only the number of the starting position. For example, "Fc-314A-435MD" represents an Fc variant containing the L314A / H435M / Y436D mutation combination, and the same below.

[0159] Table 1. Binding of Fc or Fc variants to FcRn

[0160]

[0161]

[0162] Table 2. Binding of Fc or Fc variants to FcRn

[0163]

[0164] Table 3. Binding of Fc or Fc variants to FcRn

[0165]

[0166]

[0167] As can be seen from the results, the mutation combination containing L314 / H435 / Y436 significantly weakened the binding ability of Fc to FcRn. When the mutation combination containing L314 / H435 / Y436 was combined with other additional mutations (S267E / L328F mutations or E233D / G237D / P238D / H268D / P271G / A330R mutations that enhance the binding activity of Fc to FcγRIIb (CD32b), its ability to weaken FcRn binding was maintained. In addition, the mutation combination of I253 / S254 / H435 / Y436 can achieve similar effects.

[0168] According to reports in the prior art (reference US20210238308), L314, H435, etc. are key positions for the binding of Fc to FcRn. Non-conservative single-point mutations at the L314 and H435 positions will significantly weaken the binding ability of Fc to FcRn, such as L314A, L314R, H435A, H435M, etc. However, according to our experimental results, it was found that when the amino acid mutations at these two positions were combined, or combined with other mutations respectively, the binding ability of the Fc variant to FcRn was significantly retained, indicating that the effect of the combination of amino acid mutations at these positions is difficult to predict.

[0169] Using a similar detection method as described above, the effect of the above mutation combination in other fusion proteins was tested, the N-terminus of the protein had a target binding portion, and the FcγRIIb binding enhancement mutation was replaced with a mutation (D265A / P331G, hereinafter abbreviated as ag) that reduced or eliminated the effector function in Fc. The binding kinetics of the Fc variants to human FcRn were detected by biomembrane interferometry (BLI) technology. Response values ​​<0.1 were judged as no binding, and the results are shown in Table 4.

[0170] Table 4. Binding of Fc variants to FcRn

[0171]

[0172]

[0173] It can be seen that the binding of Fc variants comprising the L314 / H435 / Y436 or I253 / S254 / H435 / Y436 mutation combinations to FcRn is stably weakened.

[0174] Example 2 Identification of the interaction between Fc variants and anti-Fc single domain antibodies

[0175] By immunizing camels and panning phage libraries, multiple anti-IgG1-Fc single-domain antibodies were obtained, among which the iFc106 amino acid sequence is shown in SEQ ID NO: 1.

[0176] iFc106 (SEQ ID NO: 1)

[0177] QVQLQESGGGSVQSGGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVAAICSDGKTTYADSVKGRFTISQDGAKNTLYLQMNSLKPEDTAMYYCAAGVKQPRYSGGYYVIWDGDFTYWGQGTQVTVSS

[0178] iFc106 can specifically bind to human IgG1-Fc and effectively block the binding of Fc to FcRn, thereby achieving the effect of reducing the level of pathogenic IgG in the body.

[0179] Table 5. CDR regions of iFc106 anti-IgG1-Fc single domain antibody

[0180]

[0181] The biomembrane interferometry (BLI) technique was used to detect the binding kinetics of the anti-IgG1-Fc single domain antibody iFc106 to human IgG1-Fc. Under the conditions of pH 7.4 and pH 6.0, human IgG1-Fc was immobilized on the AHC sensor, and then the Fc single domain antibody was diluted to multiple concentrations and bound to the immobilized human IgG1-Fc. The equilibrium dissociation constant (KD) was calculated using Octet data analysis software. The test results showed that the KD values ​​of iFc106 under pH 7.4 and pH 6.0 were 3.809E-09 and 2.71E-09, respectively.

[0182] 5μg / mL IgG1-Fc was used to coat a 96-well plate. The anti-Fc single domain antibody was diluted 3-fold to 11 concentration points starting from 6500nM, and added to the wells for incubation with biotin-mFcRn (mouse FcRn). Then, Streptavidin (HRP) secondary antibody was added for incubation. Finally, the OD value was read after 8 minutes of color development with high-sensitivity TMB colorimetric solution. The data was processed by Softmax Pro software. The test results showed that the EC50 of the anti-IgG1-Fc single domain antibody iFc106 for blocking IgG1-Fc and FcRn was 6.41nM.

[0183] After the anti-IgG1-Fc single-domain antibody iFc106 forms a complex with its target IgG1, in order to quickly clear IgG1, it is not desired that the complex further binds to FcRn, so the Fc variant with reduced binding to FcRn is desired when constituting the iFc106-Fc fusion protein. However, if the iFc106 antigen-binding portion can also interact with the Fc variant fused to the anti-IgG1-Fc single-domain antibody iFc106, the stability and therapeutic effect of the anti-IgG1-Fc single-domain antibody may be reduced. Therefore, it is further expected that no additional interaction occurs between the aforementioned Fc variant with reduced FcRn binding and the single-domain antibody iFc106.

[0184] The binding ability between the aforementioned Fc variants with reduced binding to FcRn and the anti-IgG1-Fc single domain antibody was tested: 96-well plates were coated with 5 μg / mL iFc106-Chis, Fc and Fc variants were diluted to multiple concentrations as shown in Tables 6 to 8, added to the wells for incubation, and then the secondary antibody was added for incubation, and finally the OD value was read after color development with a high-sensitivity TMB colorimetric solution for 30 minutes. The results are shown in Tables 6-8.

[0185] Table 6. Binding of Fc or Fc variants to anti-IgG1-Fc single domain antibodies

[0186]

[0187] Table 7. Binding of Fc or Fc variants to anti-IgG1-Fc single domain antibodies

[0188]

[0189] Table 7 continued. Binding of Fc or Fc variants to anti-IgG1-Fc single domain antibodies

[0190]

[0191] Table 8. Binding of Fc variants to anti-IgG1-Fc single domain antibodies

[0192]

[0193] It can be seen that the L314 / H435 / Y436 and I253 / S254 / H435 / Y436 mutation combinations obtained in Example 1 both showed weakened binding activity to the anti-IgG1-Fc single domain antibody.

[0194] Example 3 Identification of the ability of Fc variants to bind to FcγRIIb

[0195] The changes in the binding ability of Fc and its variants to FcγR, especially FcγRIIb, were investigated before and after the introduction of mutations that weakened FcRn binding. The binding kinetics of Fc and its variants to human FcγR were detected by biomembrane interferometry (BLI) technology. The human FcγR protein was directly immobilized on the HIS1K sensor, and then Fc and its variants were diluted to multiple concentrations and bound to the immobilized human FcγR protein. The equilibrium dissociation constant (KD) was calculated using Octet data analysis software, or the strength of the binding ability was judged by the response value. The results are shown in Tables 9-11.

[0196] Table 9. Binding of Fc and Fc variants to FcγR

[0197]

[0198]

[0199] Table 10. Binding of Fc and Fc variants to FcγR

[0200]

[0201] Table 11. Binding of Fc and Fc variants to FcγR

[0202]

[0203] It can be seen that after the introduction of the EF or V12 mutation combination, the resulting Fc variant has an increased ability to bind to CD32b (FcγRIIb), while its binding to CD16a and CD32a becomes weaker. After further adding the mutation of the present invention that weakens FcRn binding, the above-mentioned trend of binding ability to FcγR is maintained.

[0204] Information on sequences used in the present disclosure is shown below.

[0205] Amino acid sequence of iFc106 (SEQ ID NO: 1)

[0206] QVQLQESGGGSVQSGGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVAAI

[0207] CSDGKTTYADSVKGRFTISQDGAKNTLYLQMNSLKPEDTAMYYCAAGVKQP

[0208] CDR1, CDR2 and CDR3 sequences of RYSGGYYVIWDGDFTYWGQGTQVTVSSiFc106 according to different definition schemes

[0209] Amino acid sequence of IgG1-Fc (SEQ ID NO: 14)

[0210] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0211] Amino acid sequence of IgG2-Fc (SEQ ID NO: 15)

[0212] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0213] Amino acid sequence of IgG3-Fc (SEQ ID NO: 16)

[0214] ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0215] Amino acid sequence of IgG4-Fc (SEQ ID NO: 17)

[0216] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 。

Claims

1. An immunoglobulin Fc variant comprising mutations at amino acids 314, 435 and 436; or comprising mutations at amino acids 253, 254, 435 and 436, wherein the amino acid positions are numbered using the EU index of Kabat. 2 . The immunoglobulin Fc variant according to claim 1 , wherein the immunoglobulin Fc variant is derived from IgG, for example, the Fc variant is derived from IgG1, IgG2, IgG3 or IgG4. The immunoglobulin Fc variant according to claim 2 , wherein the immunoglobulin Fc variant is derived from human IgG.

4. The immunoglobulin Fc variant according to any one of claims 1 to 3, which has at least one of the following characteristics compared to a parent Fc not comprising the mutation: a) weakened binding ability to FcRn; b) increased serum clearance rate in mammals; c) shortened serum half-life in mammals; and d) reduced side effects caused by systemic exposure of Fc.

5. The immunoglobulin Fc variant according to any one of claims 1 to 4, wherein the mutation at amino acid position 314 is L314A, L314D, L314E, L314F, L314G, L314H, L314I, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W or L314Y ; Preferably it is L314A, L314F, L314G, L314H, L314I, L314K, L314M, L314P, L314R, L314S, L314V, L314W or L314Y; More preferably it is L314A, L314S, L314G, L314R, L314K, L314W or L314Y; Most preferably it is L314A, L314R, or L314W.

6. According to the immunoglobulin Fc variant of any one of claims 1-4, the amino acid mutation at position 253 is I253A, I253D, I253E, I253F, I253G, I253H, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W or I253Y; preferably I253A, I253D, I253E, I253G, I253N, I253Q or I253S; more preferably I253E or I253D.

7. The immunoglobulin Fc variant according to any one of claims 1-4 and 6, wherein the mutation at amino acid position 254 is S254A, S254D, S254E, S254F, S254G, S254H, S254I, S254K, S254L, S254M, S254N, S254P, S254Q, S254R, S254T, S254V, S254W or S254Y; preferably S254A, S254D, S254E, S254G, S254N or S254Q; more preferably S254D or S254E.

8. The immunoglobulin Fc variant according to any one of claims 1 to 7, wherein the mutation at amino acid position 435 is H435A, H435D, H435E, H435F, H435G, H435I, H435K, H435L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W or H435Y; preferably H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H435W or H435Y; more preferably H435A, H435S, H435G, H435M, H435L, H435Y or H435I; more preferably H435A or H435M.

9. The immunoglobulin Fc variant according to any one of claims 1 to 8, wherein the mutation at amino acid position 436 is Y436A, Y436D, Y436E, Y436F, Y436G, Y436H, Y436I, Y436K, Y436L, Y436M, Y436N, Y436Q, Y436R, Y436S, Y436T, Y436V or Y436W; preferably Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q or Y436N; more preferably Y436A, Y436D, Y436E or Y436Q.

10. The Fc variant according to any one of claims 1-5 and 8-9, wherein the mutations at amino acids 314, 435 and 436 are selected from the group consisting of: L314A / H435M / Y436D, L314A / H435M / Y436E, L314W / H435M / Y436D, L314W / H435M / Y436E and L314R / H435M / Y436Q. The mutations at amino acids at positions 314, 435 and 436 are selected from the group consisting of: L314A / H435M / Y436D, L314A / H435M / Y436E, L314W / H435M / Y436D and L314W / H435M / Y436E, more preferably, the mutations at amino acids at positions 314, 435 and 436 are L314A / H435M / Y436D or L314W / H435M / Y436D.

11. The Fc variant according to any one of claims 1-4 and 6-9, wherein the mutations at amino acids 253, 254, 435 and 436 are I253E / S254D / H435A / Y436D, I253E / S254D / H435M / Y436Q or I253E / S254D / H435A / Y436A.

12. The immunoglobulin Fc variant according to any one of claims 1 to 11, which further exhibits a reduced ability to bind to an anti-Fc single domain antibody compared to a parent Fc not comprising the mutation.

13. The immunoglobulin Fc variant according to any one of claims 1 to 12, further comprising a mutation that regulates the interaction of the immunoglobulin Fc variant with an Fcγ receptor. The immunoglobulin Fc variant according to claim 13, comprising a mutation that enhances the interaction of the immunoglobulin Fc variant with FcγRIIb.

15. The immunoglobulin Fc variant according to claim 14, wherein the mutation that enhances the interaction between the immunoglobulin Fc variant and FcγRIIb is selected from the following mutation combinations: S267A / H268A, S267A / E258A, S267A / R255A, S267A / E272A, S239D / I332E, S267E / L328F, E233D / A330R, E233D / P238D, G237D / P238D, P238D / H268D, P238D / P271G, P238D / A330R, E233D / P238D / A330R, E233D / P271G / A3 30R, G237D / H268D / P271G, G237D / P271G / A330R, S239D / A330L / I332E, G236A / S239D / I332E, E233D / P238D / P271G / A330R, G237D / P238D / H268D / P271G, E233D / H268D / P271G / A330R, G237D / P238D / P 271G / A330R, G237D / H268D / P271G / A330R, E233D / G237D / H268D / P271G / A330R, E233D / P238D / H268 D / P271G / A330R, G237D / P238D / H268D / P271G / A330R or E233D / G237D / P238D / H268D / P271G / A330R, preferably, the mutation that enhances the interaction of the immunoglobulin Fc variant with FcγRIIb is S267E / L328F or E233D / G237D / P238D / H268D / P271G / A330R.

16. The immunoglobulin Fc variant according to claim 14, wherein the immunoglobulin Fc variant comprises mutations S267E / L328F, and (1) mutations at amino acids 314, 435, and 436, or (2) mutations at amino acids 253, 254, 435, and 436.

17. The immunoglobulin Fc variant according to claim 16, wherein the immunoglobulin Fc variant comprises: (a) mutations S267E / L328F, and mutations L314A / H435M / Y436D; (b) mutations S267E / L328F, and mutations L314A / H435M / Y436E; (c) mutations S267E / L328F, and mutations L314W / H435M / Y436D; (d) mutations S267E / L328F, and mutations L314W / H435M / Y436E; (e) mutations S267E / L328F, and mutations L314R / H435M / Y436Q; (f) mutations S267E / L328F, and mutations I253E / S254D / H435A / Y436D; or (g) Mutations S267E / L328F, and mutations I253E / S254D / H435A / Y436A.

18. The immunoglobulin Fc variant according to claim 14, wherein the immunoglobulin Fc variant comprises mutations E233D / G237D / P238D / H268D / P271G / A330R, and (1) mutations at amino acids at positions 314, 435, and 436, or (2) mutations at amino acids at positions 253, 254, 435, and 436.

19. The immunoglobulin Fc variant according to claim 18, wherein the immunoglobulin Fc variant comprises: (a) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314A / H435M / Y436D; (b) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314A / H435M / Y436E; (c) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314W / H435M / Y436D; (d) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations L314W / H435M / Y436E; (e) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435M / Y436Q; (f) mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435M / Y436D; or (g) Mutations E233D / G237D / P238D / H268D / P271G / A330R, and mutations I253E / S254D / H435A / Y436A.

20. The immunoglobulin Fc variant according to any one of claims 1 to 19, further comprising a mutation that reduces or eliminates effector function.

21. The immunoglobulin Fc variant according to claim 20, wherein the mutations that reduce or eliminate effector function are: D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S298G / T29 9A, L234F / L235E / P331S, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236del / S267K, L234A / L235A / P329G, L234F / L235E / D265A or V234A / G237A / P238S / H268A / V309L / A330S / P331S, preferably D265A / P331G.

22. The immunoglobulin Fc variant according to claim 20, wherein the immunoglobulin Fc variant comprises the mutations D265A / P331G, and (1) mutations at amino acids 253, 254, 435, and 436, or (2) mutations at amino acids 314, 435, and 436.

23. The immunoglobulin Fc variant according to claim 22, wherein the immunoglobulin Fc variant comprises: (a) mutations D265A / P331G, and mutations I253E / S254D / H435M / Y436Q; (b) mutations D265A / P331G, and mutations I253A / S254A / H435M / Y436E; (c) mutations D265A / P331G, and mutations I253E / S254D / H435M / Y436E; (d) mutations D265A / P331G, and mutations I253A / S254A / H435A / Y436A; (e) mutations D265A / P331G, and mutations I253A / S254A / H435M / Y436Q; or (f) Mutations D265A / P331G, and L314W / H435M / Y436E.

24. A fusion protein comprising the immunoglobulin Fc variant according to any one of claims 1 to 23. The fusion protein of claim 24 , further comprising an antibody variable region.

26. A nucleic acid molecule encoding the immunoglobulin Fc variant according to any one of claims 1 to 23 or the fusion protein according to claim 24 or 25.

27. An expression vector comprising the nucleic acid molecule of claim 26 operably linked to an expression control element.

28. A recombinant cell comprising the nucleic acid molecule of claim 26 and / or transformed with the expression vector of claim 27, and capable of expressing the immunoglobulin Fc variant or a fusion protein comprising the same.

29. A composition comprising the immunoglobulin Fc variant of any one of claims 1-23, the fusion protein of claim 24 or 25, the nucleic acid molecule of claim 26, the expression vector of claim 27, or the recombinant cell of claim 28.

Images