CN119219775A - Fc variants with reduced binding to FcRn - Google Patents

Abstract

The present application provides immunoglobulin Fc regions having reduced binding capacity to FcRn and fusion proteins comprising the same, nucleic acids encoding the same, and vectors and cells comprising the nucleic acids.

Description

Fc variants with reduced binding to FcRn

Technical Field

The invention relates to the field of biological medicine, and discloses a protein variant. In particular, the present invention discloses an immunoglobulin Fc variant having reduced binding capacity to FcRn.

Background

IgG type antibodies recognize a target antigen through two antigen binding fragments (Fab) that are linked by a hinge region to a constant crystallizable fragment (Fc) that binds to Fc receptors and induces effector functions, specific antigen binding and appropriate effector functions such that IgG type antibodies are successfully used to treat various diseases.

Humans have four IgG subtypes, of which IgG1, 2 and 4 possess a long half-life, while the key factor affecting the plasma half-life of IgG-type antibodies is a cellular receptor called neonatal Fc receptor (FcRn). FcRn is a heterodimer consisting of an N-glycosylated transmembrane MHC class I heavy chain non-covalently bound to a soluble β2-microglobulin. Two FcRn molecules can bind IgG through the symmetrical CH2-CH3 interface bound to Fc, respectively, which prevents IgG from being degraded in the lysosome, igG can be taken up by vascular endothelial cells and taken into the cells by pinocytosis, then IgG binds FcRn in the inclusion body (pH 6.0-6.5) in a pH dependent manner, while antibodies that do not bind FcRn fuse with lysosomes and are degraded by proteases in the lysosome, and IgG dissociates rapidly from FcRn in a neutral environment (pH 7.0-7.4) and reenters the circulation when IgG-FcRn complex circulates to the cell surface.

The binding capacity of the IgG to FcRn is regulated through Fc engineering, and the half life and the clearance rate of the IgG in vivo are greatly influenced. For example, fc variants that bind FcRn at acidic pH can be obtained with a longer half-life than the parent without affecting the binding to FcRn at neutral pH, one such class of engineered antibodies has three Fc substitutions (M252Y/S254T/T256E, YTE), which increases binding to human FcRn by about 10-fold at pH6.0 and plasma half-life in non-human primates by nearly 4-fold.

However, not all therapeutic antibodies are expected to possess stronger FcRn binding capacity, longer half-life and/or slower clearance rates. For example, for a class of antibodies directed against the Fc portion of an immunoglobulin, for rapid clearance of the targeted immunoglobulin after formation of a complex with the immunoglobulin by targeting the Fc portion, it is undesirable for such antibodies directed against the Fc portion of the immunoglobulin to produce further binding to FcRn, and therefore it is practically desirable to include in such antibodies directed against the Fc portion an Fc variant having reduced binding to FcRn. As another example, for topically administered drugs, it is often desirable that they be cleared rapidly in serum, so as not to cause additional toxic side effects due to systemic exposure.

Patent publications US20210238308, CN110317272a disclose Fc variants with reduced binding to FcRn, but do not disclose specific combinations of mutations. Thus, there remains a need for more engineered Fc variants with reduced FcRn binding to meet the needs of drug development of various therapeutic antibodies and the like.

Disclosure of Invention

The present invention provides for the screening of immunoglobulin Fc variants with reduced FcRn binding by protein engineering, which have a combination of amino acid mutations, which are expected to have a shorter half-life and/or faster clearance rate in mammals.

In a first aspect, the present disclosure provides an immunoglobulin Fc variant comprising a mutation at amino acids 314, 435 and 436, or comprising a mutation at amino acids 253, 254, 435 and 436, said amino acid positions being numbered using the EU index of Kabat.

In some embodiments, the immunoglobulin Fc variant is derived from IgG, e.g., the Fc variant is derived from IgG1, igG2, igG3, or IgG4. In some embodiments, the immunoglobulin Fc variant is derived from human IgG.

In some embodiments, the variant has at least one of a) reduced binding capacity to FcRn, b) increased serum clearance rate in a mammal, c) reduced serum half-life in a mammal, and d) reduced side effects due to systemic Fc exposure, as compared to a parent Fc that does not comprise the mutation.

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, L314M, L P, L314R, L314S, L V, L W or L314Y. In some preferred embodiments, the mutation at amino acid 314 is L314A, L314S, L314G, L314R, L314K, L W or L314Y. In some more preferred embodiments, the mutation at amino acid 314 is L314A, L314R, or L314W.

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

In some embodiments, the mutation at amino acid 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 254 is S254A, S254D, S E, S254G, S N or S254Q. In some more preferred embodiments, the mutation at amino acid 254 is S254D or S254E.

In some embodiments, the mutation at amino acid 435 is H435A, H435E, H435F, H435G, H I, H435K, H L, H435M, H435N, H435Q, H435S, H435T, H435V, H435W or H435Y. In some preferred embodiments, the mutation at amino acid 435 is H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H W or H435Y. In some more preferred embodiments, the mutation at amino acid 435 is H435A, H435S, H435G, H435M, H435L, H435Y or H435I. In some preferred embodiments, the mutation at amino acid 435 is H435A or H435M.

In some embodiments, the mutation at amino acid 436 is Y436A, Y436D, Y436E, Y436F, Y G, Y436H, Y436I, Y436K, Y436L, Y436N, Y436Q, Y R, Y436S, Y436T, Y V or Y436W. In some preferred embodiments, the mutation at amino acid 436 is Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q or Y436N. In some more preferred embodiments, the mutation at amino acid 436 is Y436A, Y436D, Y436E, or Y436Q.

In some embodiments, the mutations at amino acids 314, 435 and 436 are selected from the group consisting of L314A/H435M/Y436D, L A/H435M/Y436E, L W/H435M/Y436D, L W/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, L A/H435M/Y436E, L W/H435M/Y436D and L314W/H435M/Y436E, more preferably the mutations at amino acids 314, 435 and 436 are L314A/H435M/Y436D or L314W/H M/Y436D.

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

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

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

In some embodiments, the immunoglobulin Fc variant comprises a mutation that enhances the interaction of the immunoglobulin Fc variant with fcyriib. In some embodiments, the mutation that enhances the interaction of the immunoglobulin Fc variant with fcyriib is selected from the group consisting of mutation combination ：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/A330R、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/P271G/A330R、G237D/H268D/P271G/A330R、E233D/G237D/H268D/P271G/A330R、E233D/P238D/H268D/P271G/A330R、G237D/P238D/H268D/P271G/A330R or E233D/G237D/P238D/H268D/P271G/a330R. In some preferred embodiments, the mutation that enhances the interaction of the immunoglobulin Fc variant with FcgammaRIIB is S267E/L328F or E233D/G237D/P238D/H268D/P271G/A330R.

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

(a) Mutation S267E/L328F, mutation L314A/H435M/Y436D;

(b) Mutation S267E/L328F, mutation L314A/H435M/Y436E;

(c) Mutation S267E/L328F, mutation L314W/H435M/Y436D;

(d) Mutation S267E/L328F, mutation L314W/H435M/Y436E;

(e) Mutation S267E/L328F, mutation L314R/H435M/Y436Q;

(f) Mutation S267E/L328F, mutation I253E/S254D/H435A/Y436D, or

(G) The mutation S267E/L328F and the mutation I253E/S254D/H435A/Y436A.

In some embodiments, the immunoglobulin Fc variant comprises a mutation E233D/G237D/P238D/H268D/P271G/a330R, and (1) a mutation at amino acids 314, 435, and 436, or (2) a mutation at amino acids 253, 254, 435, and 436.

In some specific embodiments, the immunoglobulin Fc variant comprises:

(a) The mutation E233D/G237D/P238D/H268D/P271G/A330R and the mutation L314A/H435M/Y436D;

(b) The mutation E233D/G237D/P238D/H268D/P271G/A330R and the mutation L314A/H435M/Y436E;

(c) The mutation E233D/G237D/P238D/H268D/P271G/A330R and the mutation L314W/H435M/Y436D;

(d) The mutation E233D/G237D/P238D/H268D/P271G/A330R and the mutation L314W/H435M/Y436E;

(e) The mutation E233D/G237D/P238D/H268D/P271G/A330R and the mutation I253E/S254D/H435M/Y436Q;

(f) The mutation E233D/G237D/P238D/H268D/P271G/A330R, the mutation I253E/S254D/H435M/Y436D, or

(G) The mutation E233D/G237D/P238D/H268D/P271G/A330R, and the mutation I253E/S254D/H435A/Y436A.

In some embodiments, the immunoglobulin Fc variants of the present disclosure further comprise mutations that reduce or eliminate effector function. In some embodiments, the mutation that reduces or eliminates effector function is ：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. In some preferred embodiments, the mutation that reduces or eliminates effector function is D265A/P331G.

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

(a) Mutation D265A/P331G, mutation I253E/S254D/H435M/Y436Q;

(b) Mutation D265A/P331G and mutation I253A/S254A/H435M/Y436E;

(c) Mutation D265A/P331G, mutation I253E/S254D/H435M/Y436E;

(d) Mutation D265A/P331G and mutation I253A/S254A/H435A/Y436A;

(e) The mutation D265A/P331G and the mutation I253A/S254A/H435M/Y436Q, or

(F) Mutation D265A/P331G and mutation L314W/H435M/Y436E.

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

In a third aspect, the present disclosure provides a nucleic acid molecule encoding an immunoglobulin Fc variant according to the first aspect or a fusion protein according to the second aspect.

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.

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

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

Other aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the descriptions in the specification of the present application are intended to be illustrative, and not limiting.

Detailed Description

Definition of terms

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

The terms "antibody" or "immunoglobulin" are used interchangeably herein, unless otherwise indicated, to refer to either a heavy chain antibody or a conventional four chain antibody, and are used as general terms to include full length antibodies, individual chains thereof, and all portions, domains, or fragments thereof (including but not limited to antigen binding domains or fragments, e.g., VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" (e.g. in terms of "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) as used herein is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the context requires a more defined interpretation.

The term "immunoglobulin" generally refers to a protein consisting essentially of one or more polypeptides encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA 1 and IgA 2), gamma (IgG 1, igG2, igG3, igG 4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. In general, an immunoglobulin may be a heterotetrameric glycoprotein of about 150,000 daltons, which consists of two identical light chains (L) and two identical heavy chains (H). The NH ₂ -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 kappa or lambda constant region gene. A natural immunoglobulin essentially consists of two Fab molecules and an Fc region linked via an immunoglobulin hinge region.

As used herein, the term "immunoglobulin Fc region" refers to a domain derived from the C-terminal region of an immunoglobulin heavy chain that can be produced by papain digestion of an intact antibody, typically comprising a hinge region and 2 constant domains (CH ₂ and CH ₃ domains). The Fc region may be a native sequence Fc region or a variant of an Fc region, such as the amino acid sequence shown in any of SEQ ID NOs 14-17, which correspond to the Fc sequences of human IgG1, igG2, igG3 and IgG4, respectively.

The term "immunoglobulin variable domain" as used herein refers to an immunoglobulin domain consisting essentially 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", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be expressed as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity to an antigen to an antibody by having an antigen binding site.

The term "single domain antibody", "sdAb" or "nanobody" as used herein refers to a single antigen-binding polypeptide having three Complementarity Determining Regions (CDRs). The single domain antibodies are capable of binding to the antigen alone without pairing with the corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelidae heavy chain antibodies (hcabs), and their heavy chain variable domains are referred to herein as "VHHs", camelidae single domain antibodies being one of the smallest known antigen binding antibody fragments.

"VHH domain", also known as heavy chain single domain antibodies, VHH domains, VHH antibody fragments and VHH antibodies, is the variable domain (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)). of an antigen-binding immunoglobulin known as a "heavy chain antibody" (i.e., an "antibody lacking a light chain") the term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain (which is referred to herein as a "VH domain") found in conventional 4-chain antibodies, as well as the light chain variable domain (which is referred to herein as a "VL domain") found in conventional 4-chain antibodies. The VHH domain specifically binds to the epitope without the need for additional antigen binding domains (this is in contrast to VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain). VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain.

As shown in FIG. 2 of Riechmann and Muyldermans, J.Immunol. Methods 231,25-38 (1999), amino acid residues used for the VHH domain of the family Camelidae may be numbered according to the general numbering of the VH domain 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)).

Alternative methods of numbering amino acid residues of VH domains are known in the art, which may also be similarly applied to VHH domains. For example, chothia CDR refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)). AbM CDR represents Kabat hypervariable region and Chothia structural loop compromise, and in the Oxford Molecular's AbM antibody modeling software. The "Contact" CDR is based on an analysis of the crystal structure of the available complex. Residues from CDRs of each method are described as follows:

Ring(s)	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

The CDRs of an antibody may also be IMGT-CDRs, a CDR-defining means based on IMGT antibody coding obtained by combining structural information of more than 5000 sequences. In VH CDR encoding of IMGT, CDR1:27-38, CDR2:56-65, CDR3:105-117.

It should be noted, however, that 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 Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence or the actual sequence may contain more amino acid residues than the Kabat numbering allows), as is well known in the art for VH domains and VHH domains. This means that in general, numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

For example, CDRs may include "extended" CDRs, e.g., 24-36 or 24-34 (LCDR 1), 46-56 or 50-56 (LCDR 2) and 89-97 or 89-96 (LCDR 3) in VL, 26-35 (HCDR 1), 50-65 or 49-65 (HCDR 2) and 93-102, 94-102 or 95-102 (HCDR 3) in VH.

Methods for obtaining VHH binding to a specific antigen or epitope 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.,JBiol Chem.,287(2012)13713-13721;Deffar et al.,African Journal of Biotechnology Vol.8(12),pp.2645-2652,17June,2009 and WO94/04678.

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

Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in a reference sequence as compared to the other sequence. In the case of a substitution, the substitution will preferably be a conservative amino acid substitution, which refers to the replacement of an amino acid residue with another amino acid residue of similar chemical structure, with little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g., conservative amino acid substitutions are preferably those in which one amino acid in the following groups (i) - (v) is substituted by another amino acid residue in the same group, (i) smaller aliphatic nonpolar or weakly polar residues: ala, ser, thr, pro and Gly, (ii) polar negatively charged residues and (uncharged) amides thereof: asp, asn, glu and Gln, (iii) polar positively charged residues: his, arg and Lys, (iv) larger aliphatic nonpolar residues: met, leu, ile, val and Cys, and (v) aromatic residues: phe, tyr and Trp. Particularly preferred conservative amino acid substitutions are Ala with Gly or Ser, arg with Lys, asn with Gln or His, asp with Glu, cys with Ser, gln with Asn, glu with Asp, gly with Ala or Pro, his with Asn or Gln, ile with Leu or Val, leu with Ile or Val, lys with Arg, gln or Glu, met with Leu, tyr or Ile, phe with Met, leu or Tyr, ser with Thr, thr with Ser, trp with Tyr, tyr with Trp or Phe, val with Ile or Leu.

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 deletion, truncation, inactivation, disruption, substitution, or translocation of an amino acid or nucleotide. In the present application, the amino acid mutation may be an amino acid substitution, the term "substitution" generally means the replacement of at least one amino acid residue in a predetermined parent amino acid sequence with a different "replacement" amino acid residue. The one or more substituted amino acid residues may be "naturally occurring amino acid residues" (i.e., encoded by the genetic code). AxxxB is generally used herein to represent mutations in amino acids, such as "S267E", where a represents a parent amino acid residue, B represents a substituted amino acid residue, xxx represents the amino acid residue numbering of the mutation, as numbering herein using the EU index of Kabat, unless otherwise indicated.

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 mimics of the corresponding naturally occurring amino acid, 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 modification. Polypeptides, proteins and fusion proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of the native protein, or molecules having the deletion, addition and/or substitution of one or more amino acids of the native sequence.

In the present application, the term "nucleic acid" molecule generally refers to any length of isolated form of nucleotide, deoxyribonucleotide or ribonucleotide or analog thereof, either isolated from the natural environment or synthesized.

In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.

In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that may or may not contain a plasmid or vector comprising a nucleic acid molecule as described herein, or that is capable of expressing an antigen binding protein as described herein. The cell may comprise progeny of a single host cell. The daughter cells may not necessarily be identical in morphology or in genome to the original parent cells due to natural, unexpected or deliberate mutation, but are capable of expressing the antibodies or antigen-binding fragments thereof of the present application. The cells may be obtained by transfecting cells in vitro using the vectors of the present application. The cells may be prokaryotic cells (e.g., E.coli) or eukaryotic cells (e.g., yeast cells, e.g., COS cells, chinese Hamster Ovary (CHO) cells, heLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell.

As used herein, "cytokine" means a generic term for proteins released by one cell population that act on another cell as intercellular modulators.

Detailed Description

Immunoglobulin Fc variants

In one aspect, the invention relates to an immunoglobulin Fc variant comprising mutations at amino acids 314, 435 and 436, said amino acid positions being numbered using the EU index of Kabat.

In another aspect, the invention also relates to an immunoglobulin Fc variant comprising mutations at amino acids 253, 254, 435 and 436, said amino acid positions being numbered using the EU index of Kabat.

In some embodiments, the immunoglobulin Fc variants of the invention have at least one of a) reduced binding capacity to FcRn, b) increased serum clearance rate in a mammal, c) reduced serum half-life in a mammal, and d) reduced side effects due to systemic Fc exposure, as compared to a parent Fc that does not comprise the mutation. In some embodiments, the immunoglobulin may be IgG, igA, igM, igD or IgE. In some preferred embodiments, the immunoglobulin is IgG.

In some embodiments, the immunoglobulin Fc variant is derived from IgG, igA, igM, igD or the Fc of IgE. In some preferred embodiments, the immunoglobulin Fc variant is derived from an Fc of an IgG.

In some embodiments, the immunoglobulin Fc variant is obtained by making mutations at amino acids 314, 435 and 436, or comprises mutations at amino acids 253, 254, 435 and 436, on the basis of wild-type or engineered IgG, igA, igM, igD or IgE (preferably IgG).

In some embodiments, the immunoglobulin may be of human, mouse, rat, rabbit or monkey origin, e.g., may be an IgG of mammalian origin as described above, including IgG1, igG2, igG3 and IgG4, e.g., may be an IgG1 of human origin.

In some embodiments, the mutation at amino acid 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, L314 62314H, L314 858 314K, L314M, L314P, L314S, L314V, L W or L314Y. In some preferred embodiments, the mutation at amino acid position 314 may be L314A, L314S, L314G, L314R, L K, L314W or L314Y. In some more preferred embodiments, the mutation at amino acid 314 may be L314A, L W or L314R.

In some embodiments, the mutation at amino acid 435 may be H435A, H435D, H435E, H435F, H435G, H I, H435 37435L, H M, H435N, H435Q, H435S, H435V, H435W or H435Y. In other embodiments, the mutation at amino acid 435 may be H435A, H435F, H435G, H435I, H435L, H435M, H435S, H435V, H435W or H435Y. In some preferred embodiments, the mutation at amino acid 435 may be H435A, H435S, H435G, H435M, H435L, H435Y or H435I. In some more preferred embodiments, the mutation at amino acid 435 may be H435A or H435M.

In some embodiments, the mutation at amino acid 436 may be Y436A, Y436D, Y436F, Y G, Y436H, Y436I, Y436K, Y436L, Y436N, Y436Q, Y436S, Y436T, Y V or Y436W. In other embodiments, the mutation at amino acid 436 may be Y436S, Y436A, Y436G, Y436D, Y436E, Y436Q or Y436N. In some preferred embodiments, the mutation at amino acid 436 may be Y436A, Y436D, Y436E or Y436Q.

In some embodiments, the mutation at amino acid 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 253 may be I253A, I253D, I253E, I253G, I253N, I253Q or I253S. In some preferred embodiments, the mutation at amino acid 253 may be I253E or I253D.

In some embodiments, the mutation at amino acid 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 254 may be S254A, S254D, S E, S254G, S N or S254Q. The mutation at amino acid 254 may be S254D or S254E.

In some specific embodiments, the immunoglobulin Fc variant may comprise the following combination of amino acid mutations:

In some embodiments, an immunoglobulin Fc variant comprising the foregoing mutations also exhibits reduced binding capacity with an anti-immunoglobulin Fc antibody (hereinafter abbreviated as anti-Fc antibody) as compared to the parent Fc not comprising the foregoing mutations. The anti-immunoglobulin Fc antibody refers to a therapeutic antibody capable of specifically binding to an immunoglobulin Fc segment, blocking the interaction between the Fc segment and FcRn, thereby producing a biological effect.

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

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

In some embodiments, CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO. 1 are selected from any of 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 a VHH as set forth in SEQ ID NO. 1, and in some embodiments iFc106 has an amino acid sequence as set forth in SEQ ID NO. 1.

In another aspect, the immunoglobulin Fc variant may further comprise a mutation that modulates the interaction of the immunoglobulin Fc variant with an fcγ receptor. For example, the fcγreceptor can be fcγri (CD 64), fcγriia (CD 32 a), fcγriib (CD 32 b), fcγriiia (CD 16 a) and/or fcγriiib (CD 16 b). In some embodiments, the immunoglobulin Fc variant comprises a mutation that enhances the interaction of the immunoglobulin Fc variant with fcyriib.

The mutation that enhances interaction with fcyriib may be any known mutation or combination of mutations in the art, such as described in Rena Liu,Antibodies 2020,9,64;Chu,S.Y.,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. et al.

In some embodiments, the immunoglobulin Fc variant further comprises a mutation ：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 selected from one or more of the following that enhances interaction with fcyriib.

In some embodiments, the immunoglobulin Fc variant further comprises a combination of mutations ：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/A330R、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/P271G/A330R、G237D/H268D/P271G/A330R、E233D/G237D/H268D/P271G/A330R、E233D/P238D/H268D/P271G/A330R、G237D/P238D/H268D/P271G/A330R and E233D/G237D/P238D/H268D/P271G/a330R that enhance interaction with fcyriib, selected from the group consisting of.

In some embodiments, the immunoglobulin Fc variant further comprises a mutation selected from the group consisting of P238D, S267E, L328F, A R, S E/L328F, G237D/P238D/H268D/P271G/A330R or E233D/G237D/P238D/H268D/P271G/A330R that enhances interaction with FcgammaRIIB. 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 fcyriib.

In some specific embodiments, the immunoglobulin Fc variant comprises a combination of mutations that enhance interaction with fcyriib and the aforementioned amino acid mutations, e.g., the immunoglobulin Fc variant may comprise a combination of mutations ：EF(S267E/L328F)/L314A/H435M/Y436D、EF/L314A/H435M/Y436E、EF/L314A/H435M/Y436Q、EF/L314W/H435M/Y436E、EF/L314W/H435M/Y436D、EF/L314W/H435M/Y436Q、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、V12/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 selected from the group consisting of.

In some embodiments, the immunoglobulin Fc variant may further comprise 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), among others. In particular, the mutation that reduces or eliminates effector function reduces or eliminates binding between an immunoglobulin and fcyri, fcyriia, fcyriib, fcyriiia, fcyriiib, and/or C1 q.

The mutation that reduces or eliminates effector function may be any mutation or combination of mutations known in the art, such as described in Esohe E.I.,J Immunol2000;164:4178-4184;Hutchins,J.T.,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,S.Y.,Mol.Immunol.2008,45,3926-3933;Sazinsky,S.L.,Proc.Natl.Acad.Sci.USA 2008,105,20167-20172;Oganesyan,V.,Acta Crystallogr.Sect.D Biol.Crystallogr.2008,64Pt 6,700-704;An,Z.,MAbs 2009,1,572-579;Moore,G.L.,Methods 2019,154,38-50;Schlothauer,T.,Protein Eng.Des.Sel.2016,29,457-466;Strohl,W.,US20150337053;Engelberts,P.J.,EBioMedicine 2020,52,102625, et al.

In some embodiments, the immunoglobulin Fc variant may further comprise a mutation ：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 selected from the group consisting of reducing or eliminating effector function. Preferably, the immunoglobulin Fc variant may further comprise a mutation selected from the group consisting of D265A, P G, D265A/P331G (abbreviated AG hereinafter) or L234F/L235E/P331S that reduces or eliminates effector function.

In some specific embodiments, the immunoglobulin Fc variant comprises a combination of mutations that reduce or eliminate effector function and the aforementioned amino acid mutations, e.g., the immunoglobulin Fc variant may comprise a combination of mutations ：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 selected from the group consisting of.

Fusion proteins

In another aspect, the invention also provides a fusion protein comprising an immunoglobulin Fc variant according to the invention. In some embodiments, the fusion protein further comprises a second polypeptide selected from the group consisting of 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.

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

In some embodiments, the fusion protein wherein the second polypeptide is linked directly to an immunoglobulin Fc variant, or via a linker. In some embodiments, the linker may be a flexible polypeptide linker.

In some embodiments, the fusion protein wherein the second polypeptide is an antibody variable region. The antibody variable region may target any antigen. the target antigen includes a membrane-binding factor including a transmembrane receptor, e.g., 17-IA, 4-1BB, 4Dc, 6-keto-PGF 1a, 8-iso-PGF 2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE-2, activin A, activin AB, activin B, activin C, activin RIA, activin RIAALK-2, activin RIBALK-4, activin RIIA, activin RIIB, ADAM, ADAM, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressee, 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 stimulating factor (BlyS)、BACE、BACE-1、Bad、BAFF、BAFF-R、Bag-1、BAK、Bax、BCA-1、BCAM、Bcl、BCMA、BDNF、b-ECGF、bFGF、BID、Bik、BIM、BLC、BL-CAM、BLK、BMP、BMP-2BMP-2a、BMP-3 osteoblast 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-DNA, BTC, complement factor 3 (C3), C3a, C4, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-related 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、CCR4、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、CD56、CD61、CD64、CD66e、CD74、CD80(B7-1)、CD89、CD95、CD123、CD137、CD138、CD140a、CD146、CD147、CD148、CD152、CD164、CEACAM5、CFTR、cGMP、CINC、 botulinum (Clostridiumbotulinum) toxin, bacillus perfringens (Clostridiumperfringen) 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, DNAzyme, dpp, DPPIV/CD26, dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, eot, eosinophil chemokine 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-19, FGF-2, FGF3, FGF-8, FGFR-3, fibrin, FL, FLIP, flt-3, flt-4, follicle stimulating hormone, fractal (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 (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, GRO, 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/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, HIVIIIBgp V3loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, hrk, human cardiac myoglobulin, 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) -alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4/beta 1, Integrin alpha 4/beta 7, integrin alpha 5 (alpha V), integrin alpha 5/beta 1, integrin alpha 5/beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, 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-1bp1, LBP, LDGF, LECT, 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, MCAM, 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-11、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 (Muc 1), MUC18, mullerian (Mullerian) inhibitor, mug, muSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, neuzyme, neurotrophin-3, neurotrophin-4 or neurotrophin-6, Neurturin, neuronal Growth Factor (NGF), NGFR, NGF-beta, 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, PF, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), plGF, PLP, PP, proinsulin, relaxin, protein C, PS, PSA, PSCA, prostate Specific Membrane Antigen (PSMA), PTEN, PTHrp, ptk, PTN, R, RANK, RANKL, RANTES, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory Syncytial Virus (RSV) F, RSVFgp, ret, rheumatoid factors, 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 alpha/beta), tdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, T-cell receptor, Testis PLAP-like alkaline phosphatase, tfR, TGF, TGF-alpha, TGF-beta pan specificity (PANSPECIFIC), TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIB, TGF-beta RIII TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, 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)、TNFRSF10C(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(LTbRTNFRIII、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, TL 2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAKApo-3 ligand, DR3 ligand), TNFSF13 (APRILTALL), TNFSF13B (BAFFBLYS, TALL, THANK, TNFSF 20), TNFSF14 (LIGHTHVEM ligand, LTg), TNFSF15 (TL 1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL 6), TNFSF1A (TNF-a linker (Conectin), DIF, TNFSF 2), TNFSF1B (TNF-bLTa, TNFSF 1), TNFSF3 (LTbTNFC, p 33), TNFSF4 (OX 40 ligand gp34, TXGP 1), TNFSF5 (CD 40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD 27 ligand CD 70), TNFSF8 (CD 30 ligand CD 153), TNFSF9 (4-1 BB ligand CD137 ligand), TNFSF5 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD 27 ligand CD 70), TP-1, t-PA, tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, metastasis receptor, TRF, trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, lewis Y-associated carbohydrate-expressing tumor-associated antigen, TWEAK, TXB2, ung, uPAR, uPAR-1, urokinase, VCAM-1, VECAD, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigens, VLA-1, VLA-4, VNR integrin, von Willebrand (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.

In some embodiments, the second polypeptide is a cytokine that can be fused to an immunoglobulin Fc variant to provide a number of desired properties, e.g., the cytokine can be a growth hormone such as human growth hormone, N-methionyl human growth hormone, bovine growth hormone; parathyroid hormone, thyroxine, insulin, proinsulin, glucagon-like peptides such as GLP-1, GLP-2, glucose-dependent insulinotropic polypeptides, amylin, YY peptides, relaxin, 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 prolactin, tumor necrosis factor-alpha and tumor necrosis factor-beta, miylor inhibitor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factors such as NGF-beta, platelet growth factor, transforming Growth Factor (TGF) such as TGF-alpha and TGF-beta, insulin-like growth factor-I and TGF-II, erythropoietin (EPO), osteoinductive factor, interferons such as interferon-alpha, interferon-beta and interferon-CSF, colony Stimulating Factor (CSF), granulocyte-CSF-granulocyte-CSF (CSF), granulocyte-macrophage (CSF) And granulocyte-CSF (G-CSF), interleukins (IL), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15.

Nucleic acid, vector, recombinant cell

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

The nucleic acids of the invention may also be in the form of a vector, may be present in and/or may be part of a vector, such as a plasmid, cosmid, or YAC. The vector may in particular be an expression vector, i.e. a vector that provides for the expression of an immunoglobulin Fc variant or fusion protein comprising the same in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The choice of the element and its sequence for expression in a particular host is common knowledge to the skilled person. Specific examples of regulatory elements and other elements useful or necessary for expression of the immunoglobulin Fc variants or fusion proteins comprising the same of the invention, e.g., promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

The nucleic acids of the invention may be prepared or obtained by known means (e.g. by automated DNA synthesis and/or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptides of the invention given herein and/or may be isolated from a suitable natural source.

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

Suitable bacterial cells include cells of gram-negative bacterial strains, such as E.coli (ESCHERICHIA COLI) strains, proteus (Proteus) strains and Pseudomonas (Pseudomonas) strains, and gram-positive bacterial strains, such as Bacillus (Bacillus) strains, streptomyces (Streptomyces) strains, staphylococcus (Staphylococcus) strains and Lactococcus (Lactobacillus) strains.

Suitable fungal cells include cells of species of Trichoderma (Trichoderma), neurospora (Neurospora) and Aspergillus (Aspergillus), or of Saccharomyces (Saccharomyces, e.g. Saccharomyces (Saccharomyces cerevisiae)), schizosaccharomyces (Schizosaccharomyces, e.g. Schizosaccharomyces pombe (Schizosaccharomyces pombe)), pichia (Pichia), e.g. Pichia pastoris (Pichia pastoris) and Pichia methanolica (Pichia methanolica)) and Hansen (Hansenula).

Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, heLa cells, COS cells, and the like.

However, the present invention may also be used with amphibian cells, insect cells, and any other cells known in the art for expressing heterologous proteins.

The immunoglobulin Fc variants of the invention or fusion proteins comprising the same may be produced in cells as described above in an intracellular manner (e.g., in the cytoplasm, in the periplasm or in inclusion bodies) followed by isolation from the host cell and optionally further purification, or they may be produced in an extracellular manner (e.g., in the medium in which the host cell is cultured) followed by isolation from the medium and optionally further purification.

Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, and the like are known in the art. Similarly, protein isolation and purification techniques suitable for use in methods of making immunoglobulin Fc variants or fusion proteins comprising the same of the present invention are well known to those skilled in the art.

Composition and method for producing the same

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

In some embodiments, the composition is a pharmaceutical composition comprising an immunoglobulin Fc variant, fusion protein, nucleic acid molecule, expression vector, or recombinant cell of the invention, and optionally a pharmaceutically acceptable carrier or excipient.

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.

Herein, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates administration of an immunoglobulin Fc variant, fusion protein, nucleic acid molecule, expression vector, or recombinant cell to a human and/or facilitates its absorption or function. For example, diluents, excipients such as water, fillers such as starch, sucrose, etc., binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone, humectants such as glycerin, disintegrants such as agar-agar, calcium carbonate, and sodium bicarbonate, absorption enhancers such as quaternary ammonium compounds, surfactants such as cetyl alcohol, adsorption carriers such as kaolin and saponite, lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, etc. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

For example, a pharmaceutical composition comprising an immunoglobulin Fc variant, fusion protein, nucleic acid molecule, expression vector, or recombinant cell of the invention may comprise a pharmaceutically acceptable diluent or a slow release matrix into which the immunoglobulin Fc variant, fusion protein, nucleic acid molecule, expression vector, or recombinant cell of the invention is embedded.

The invention also provides the following non-limiting embodiments:

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, said amino acid positions being numbered using the EU index of Kabat.

2. The variant of an immunoglobulin Fc according to embodiment 1, which variant has at least one of the following characteristics a) reduced binding capacity to FcRn, b) increased serum clearance rate in a mammal, c) reduced serum half-life in a mammal, d) reduced side effects due to systemic Fc exposure compared to Fc prior to mutation.

3. The immunoglobulin Fc variant according to embodiment 1 or 2, wherein the immunoglobulin is IgG, igA, igM, igD and IgE, preferably IgG.

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

5. The immunoglobulin Fc variant according to any of embodiments 1-4, wherein the immunoglobulin is IgG1, igG2, igG3 or IgG4, preferably human-derived IgG1.

6. The immunoglobulin Fc variant according to embodiment 1, wherein 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 L314A, L314F, L314G, L314I, L314M, L314P, L314R, L314S, L314V, L314W or L314Y, more preferably L314A, L314S, L314G, L314R, L314K, L W or L314Y, even more preferably L314A, L W or L314R.

7. The immunoglobulin Fc variant according to embodiment 1, wherein the mutation at amino acid 435 is selected from the group consisting of: H435A, H435D, H435E, H435F, H435G, H435I, H K, H435L, H435N, H435Q, H435S, H435T, H435V, H W or H435Y; preferably H435A, H435F, H435G, H435I, H435L, H435L, H435L, H435L, H435W or H435Y, more preferably H435L, H435L, H435L, H435L, H435L, H435Y or H435I, even more preferably H435A or H435M.

8. The immunoglobulin Fc variant according to embodiment 1, wherein the mutation at amino acid position 436 is selected from the group consisting of Y436A, Y436D, Y436E, Y436G, Y436H, Y436I, Y436L, Y436M, Y N, Y436Q, Y436 6278 436S, Y436T, Y V or Y436W, preferably Y436S, Y436A, Y436G, Y436D, Y436E, Y Q or Y436N, more preferably Y436A, Y436D, Y436E or Y436Q.

9. The immunoglobulin Fc variant according to embodiment 1, wherein the mutation at amino acid 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, I N, I253Q or I253S, more preferably I253E or I253D.

10. The immunoglobulin Fc variant according to embodiment 1, wherein 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, S E, S254G, S N or S254Q, more preferably S254D or S254E.

11. The immunoglobulin Fc variant of any of embodiments 1-10, further exhibiting reduced ability to bind to an anti-Fc single domain antibody as compared to a parent Fc that does not comprise the mutation.

12. The immunoglobulin Fc variant according to embodiment 11, wherein the anti-Fc single domain antibody comprises CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO. 1.

13. The immunoglobulin Fc variant according to embodiment 12, wherein CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO. 1 are selected from any of SEQ ID NO. 2-4 (numbering according to the Kabat system), SEQ ID NO. 5-7 (numbering according to the AbM system), SEQ ID NO. 8-10 (numbering according to the Chothia system), SEQ ID NO. 11-13 (numbering according to the IMGT system).

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

15. The immunoglobulin Fc variant of any of embodiments 1-14, further comprising a mutation that modulates the interaction of the immunoglobulin Fc variant with an fcγ receptor.

16. The immunoglobulin Fc variant of embodiment 15, comprising a mutation that enhances the interaction of the immunoglobulin Fc variant with fcyriib.

17. The immunoglobulin Fc variant of embodiment 16, wherein the mutation that enhances the interaction of the immunoglobulin Fc variant with fcyriib is selected from the group consisting of mutation combination ：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/A330R、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/P271G/A330R、G237D/H268D/P271G/A330R、E233D/G237D/H268D/P271G/A330R、E233D/P238D/H268D/P271G/A330R、G237D/P238D/H268D/P271G/A330R or E233D/G237D/P238D/H268D/P271G/a330R.

18. The immunoglobulin Fc variant of any of embodiments 1-14, further comprising a mutation that reduces or eliminates effector function.

19. The immunoglobulin Fc variant of embodiment 18, comprising a mutation ：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 selected from the group consisting of.

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

21. The fusion protein of embodiment 20, further comprising a second polypeptide selected from the group consisting of 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 or protein domain.

22. The fusion protein of embodiment 21, wherein the second polypeptide is linked directly to an immunoglobulin Fc variant, or via a flexible polypeptide chain.

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.

24. An expression vector comprising the nucleic acid molecule of embodiment 23 operably linked to an expression regulatory element.

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 fusion protein comprising the same.

Without intending to be limited by any theory, the following examples are meant to illustrate the various aspects of the present application and are not intended to limit the scope of the present application.

Examples

Example 1 screening for Fc variants with reduced binding to FcRn

Variants of human IgG1-Fc were screened, and part of the Fc variants had introduced either the S267E/L328F mutation (hereinafter abbreviated FcEF) or the E233D/G237D/P238D/H268D/P271G/A330R mutation (hereinafter abbreviated FcV) which enhanced Fc binding to FcgammaRIIB (CD 32 b).

The binding kinetics of Fc variants to human FcRn were examined by biofilm interference (BLI) techniques. At pH6.0, human FcRn protein was immobilized directly onto HIS1K sensor, and IgG1Fc and its variants (concentrations of 2000nM, 3000nM and 10000 nM) were added, respectively, to bind to immobilized human FcRn protein. The binding ability was judged to be strong or weak by the response value, and the response value <0.1 was judged to be no binding, and the results are shown in tables 1 to 3. Table 1 shows the response values for Fc or Fc variant binding to FcRn at a concentration of 2000 nM. Table 2 shows the response values for Fc or Fc variant binding to FcRn at a concentration of 3000 nM. Table 3 shows the response values for Fc or Fc variant binding to FcRn at a concentration of 10000 nM. The mutation site and the amino acid after mutation are indicated by a number + single letter, and the mutation at the adjacent position is marked with only the number at the starting position, e.g. "Fc-314A-435MD" represents an Fc variant comprising a combination of L314A/H435M/Y436D mutations, as follows.

TABLE 1 binding of Fc or Fc variants to FcRn

TABLE 2 binding of Fc or Fc variants to FcRn

TABLE 3 binding of Fc or Fc variants to FcRn

As can be seen from the results, the combination of mutations comprising L314/H435/Y436 significantly impaired Fc binding to FcRn. When the combination of mutations comprising L314/H435/Y436 was combined with other additional mutations (the S267E/L328F mutation or the E233D/G237D/P238D/H268D/P271G/A330R mutation that enhanced Fc binding activity to FcgammaRIIB (CD 32 b)), the ability to impair FcRn binding was maintained. In addition, a similar effect can be achieved with a combination of mutations of I253/S254/H435/Y436.

According to prior art reports (cf. US 20210238308), L314, H435 etc. are key positions for Fc binding to FcRn, and non-conservative single point mutations at positions L314 and H435 greatly impair Fc binding to FcRn, e.g. L314A, L314R, H435A, H M etc., whereas according to our experimental results it was found that when amino acid mutations at these two positions are combined, or combined with other additional mutations separately, the Fc variant binding to FcRn is significantly preserved, indicating that the effect of the combined amino acid mutations at these positions is less predictable.

Using a similar assay as described above, the effect of the above described combination of mutations in other fusion proteins with a target binding moiety at the N-terminus was tested, substituting the mutation in Fc that increased FcgammaRIIB binding with a mutation that reduces or eliminates effector function (D265A/P331G, abbreviated ag below). The binding kinetics of Fc variants to human FcRn were examined by biofilm interference (BLI) techniques. The response value <0.1 was judged to be unbound, and the results are shown in table 4.

TABLE 4 binding of Fc variants to FcRn

It can be seen that the Fc variants comprising the combination of L314/H435/Y436 or I253/S254/H435/Y436 mutations stably decreased binding to FcRn.

Example 2 identification of interactions between Fc variants and anti-Fc Single Domain antibodies

Through immunization of camels and panning of phage libraries, a plurality of anti-IgG 1-Fc single domain antibodies are obtained, wherein the iFc amino acid sequence is shown as SEQ ID NO. 1.

iFc106(SEQ ID NO:1)

QVQLQESGGGSVQSGGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVAAICSDGKTTYADSVKGRFTISQDGAKNTLYLQMNSLKPEDTAMYYCAAGVKQPRYSGGYYVIWDGDFTYWGQGTQVTVSS

IFc to iFc can specifically bind to human IgG1-Fc and effectively block Fc binding to FcRn, thereby achieving the effect of reducing pathogenic IgG levels in vivo.

TABLE 5 CDR regions of iFc106 anti-IgG 1-Fc single domain antibodies

The binding kinetics of the anti-IgG 1-Fc single domain antibody iFc to human IgG1-Fc was detected using biological membrane interferometry (BLI) techniques. Human IgG1-Fc was immobilized onto an AHC sensor at pH 7.4 and pH 6.0, then Fc single domain antibodies were diluted to multiple concentrations and bound to immobilized human IgG1-Fc, and equilibrium dissociation constants (KD) were calculated using Octet data analysis software. The detection results show that the KD values of iFc106 under the conditions of pH 7.4 and pH 6.0 are 3.809E-09 and 2.71E-09 respectively.

The 96-well plate was coated with 5. Mu.g/mL IgG1-Fc, the anti-Fc single domain antibody was diluted 3-fold from 6500nM to 11 concentration spots, incubated with biotin-mFcRn (mouse FcRn) in the well, then STREPTAVIDIN (HRP) secondary antibody was added for incubation, finally the OD was read by developing with high sensitivity TMB chromogenic solution for 8min, and the data were processed by Softmax Pro software. The results showed that anti-IgG 1-Fc single domain antibody iFc had an EC50 of 6.41nM for IgG1-Fc blocking with FcRn.

After the anti-IgG 1-Fc single domain antibody iFc forms a complex with its target IgG1, further binding of the complex to FcRn is undesirable for rapid clearance of IgG1, and thus Fc variants with reduced binding to FcRn are desirable when making up iFc-Fc fusion proteins. However, if iFc antigen-binding portion also interacts with the Fc variant fused to the anti-IgG 1-Fc single domain antibody iFc, the stability and therapeutic efficacy of the anti-IgG 1-Fc single domain antibody may be reduced. Thus, it is further desirable that no additional interactions occur between the aforementioned Fc variants with reduced FcRn binding and the single domain antibody iFc106,106.

The binding capacity between the aforementioned Fc variants with reduced FcRn binding and anti-IgG 1-Fc single domain antibodies was tested by coating 96-well plates with 5 μg/mL iFc-106-Chis, diluting Fc and Fc variants to multiple concentrations as shown in tables 6-8, incubating in the wells, then adding secondary antibody, and finally reading the OD value with high sensitivity TMB chromogenic solution for 30 min. The results are shown in tables 6-8.

TABLE 6 binding of Fc or Fc variants to anti-IgG 1-Fc Single-domain antibodies

TABLE 7 binding of Fc or Fc variants to anti-IgG 1-Fc Single-domain antibodies

TABLE 7 binding of Fc or Fc variants to anti-IgG 1-Fc Single Domain antibodies

TABLE 8 binding of Fc variants to anti-IgG 1-Fc Single Domain antibodies

As can be seen, the combination of the L314/H435/Y436 and I253/S254/H435/Y436 mutations obtained in example 1 all exhibited reduced binding activity to the anti-IgG 1-Fc single domain antibody.

Example 3 identification of the ability of Fc variants to bind fcyriib

The change in the binding capacity of Fc and its variants to fcγr, especially fcγriib, before and after introduction of mutations with reduced FcRn binding was examined. The binding kinetics of Fc and its variants to human fcγr were detected by biofilm interference (BLI) techniques. The human fcγr proteins are immobilized directly onto the HIS1K sensor, and then Fc and its variants are diluted to multiple concentrations and the immobilized human fcγr proteins are bound. The equilibrium dissociation constant (KD) was calculated using the Octet data analysis software, or the binding capacity was judged by the response value, and the results are shown in tables 9 to 11.

TABLE 9 binding of Fc and Fc variants to FcγR

TABLE 10 binding of Fc and Fc variants to FcγR

TABLE 11 binding of Fc and Fc variants to FcγR

It can be seen that upon introduction of either EF or V12 mutation combinations, the resulting Fc variants have an increased binding capacity to CD32b (fcyriib) and a weaker binding to CD16a, CD32 a. The trend of the aforementioned fcγr binding ability was maintained after further addition of the FcRn binding attenuating mutation of the present invention.

The following shows information of sequences used in the present disclosure.

IFc106 amino acid sequence (SEQ ID NO: 1)

QVQLQESGGGSVQSGGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVAAI

CSDGKTTYADSVKGRFTISQDGAKNTLYLQMNSLKPEDTAMYYCAAGVKQP

RYSGGYYVIWDGDFTYWGQGTQVTVSSIFC106 CDR1, CDR2 and CDR3 sequences according to different definition schemes

IgG1-Fc amino acid sequence (SEQ ID NO: 14)

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

IgG2-Fc amino acid sequence (SEQ ID NO: 15)

ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

IgG3-Fc amino acid sequence (SEQ ID NO: 16)

ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

IgG4-Fc amino acid sequence (SEQ ID NO: 17)

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK .

Claims (29)

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.