WO2019242619A1

WO2019242619A1 - Fully humanized anti-lag-3 antibody and application thereof

Info

Publication number: WO2019242619A1
Application number: PCT/CN2019/091749
Authority: WO
Inventors: 胡化静; 缪小牛; 张攀; 刘军建
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2018-06-19
Filing date: 2019-06-18
Publication date: 2019-12-26
Also published as: TWI721457B; TW202016143A; CN112166125A; CN112166125B; CN110615840A

Abstract

Provided are an antibody and antibody fragment that specifically bind to LAG-3, as well as a composition containing the antibody or the antibody fragment. Also provided are nucleic acids encoding the antibody or antibody fragment thereof, and a host cell comprising same, as well as therapeutic and diagnostic applications thereof.

Description

Full-human anti-LAG-3 antibody and application thereof

Technical field

The present invention relates to novel antibodies and antibody fragments that specifically bind to LAG-3 and compositions containing said antibodies or antibody fragments. In addition, the present invention relates to a nucleic acid encoding the antibody or an antibody fragment thereof, a host cell comprising the same, and related uses. Furthermore, the invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments. In particular, the invention relates to the combination therapy of these antibodies and antibody fragments with other therapies, such as treatment modalities or therapeutic agents, such as anti-PD-1 or anti-PD-L1 antibodies.

Background technique

Lymphocyte activating gene 3 (LAG-3), also known as CD223, is a type I transmembrane protein encoded by the LAG3 gene in humans. The molecular properties and biological functions of LAG-3 have been fully characterized and described, see, for example, Sierro et al., Expert Opin Ther Targets (2011) 15 (1): 91-101. LAG-3 is a CD4-like protein expressed on the surface of T cells (especially activated T cells), natural killer cells, B cells, and plasma cell-like dendritic cells. LAG-3 has been shown to be a negative costimulatory receptor, that is, an inhibitory receptor.

It has been proposed that the binding of LAG-3 to MHC class II molecules plays a role in down-regulating antigen-dependent stimulation of CD4 + T lymphocytes (Huard et al. (1994) Eur. J. Immunol. 24: 3216-3221). The interaction between LAG3 and MHC class II is also thought to play a role in regulating dendritic cell function (Andreae et al. J Immunol 168: 3874-3880, 2002). Recent preclinical studies have also documented the role of LAG-3 in CD8 T-cell depletion (Blackburn et al. Nat Immunol 10: 29-37, 2009).

Studies have shown that CD8 + T cells that are depleted after chronic viral infection express multiple inhibitory receptors (such as PD-1, CD160, and 2B4). LAG-3 was expressed at high levels after LCMV infection and showed that blocking the PD-1 / PD-L1 pathway and blocking LAG-3 significantly reduced viral load in chronically infected mice (Blackburn et al., Nat Immunol (2009 ) 10: 29-37). It has also been shown that the combined inhibition of the PD-1 / PD-L1 pathway and LAG-3 blockers provides antitumor efficacy (Jing et al., Journal for ImmunoTherapy of Cancer (2015) 3: 2).

In view of the above-mentioned important role of LAG-3, there is a need to develop new anti-LAG-3 antibodies that regulate its activity, especially fully human antibodies. Such antibodies can be better used to treat tumors and other diseases such as infections. In addition, it is also desirable to obtain new anti-LAG-3 antibodies that can be used in combination with other therapies, such as therapeutic agents, such as anti-PD-1 or anti-PD-L1 antibodies, to treat tumors or infections.

Summary of invention

The invention provides a fully human-derived anti-human LAG-3 antibody, a coding gene and application thereof. Through genetic engineering and yeast surface display technology, the present inventors screened fully human antibodies against human LAG-3 from the human antibody library displayed on the surface of yeast, and further obtained high affinity matured on the basis of this Anti-human LAG-3 antibody. The fully human antibody molecule of the present invention can effectively block the binding of LAG-3 to major histocompatibility (MHC) class II molecules, bind to LAG-3 expressed on activated human CD4 + T cells, and administer it in vivo Inhibition of tumor growth, especially when combined with anti-PD-1 antibodies, the tumor suppression effect is particularly significant. Therefore, the antibodies of the present invention can be used for a variety of purposes, including but not limited to detecting LAG-3 protein and inhibiting tumor growth in a tumor-bearing subject.

Thus, in one aspect, the invention provides an antibody or antigen-binding fragment thereof that specifically binds LAG-3, preferably a human LAG-3 protein.

In one embodiment, an antibody or antigen-binding fragment thereof of the invention that specifically binds human LAG-3 comprises:

(i) the three complementarity determining regions HCDRs of the heavy chain variable region as shown in SEQ ID NO: 33 or 34, and the three complementarity determining regions LCDR of the light chain variable region as shown in SEQ ID NO: 39, or

(ii) 3 complementarity determining regions of the heavy chain variable regions as shown in SEQ ID NO: 35 or 36, and 3 complementarity determining regions of the light chain variable region as shown in SEQ ID NO: 40 or 41 LCDR; or

(iii) 3 complementarity determining regions of the heavy chain variable region as shown in SEQ ID NO: 37 or 38, and 3 complementarity determining regions of the light chain variable region as shown in SEQ ID NO: 42 or 43 LCDR.

In yet another aspect, the invention also provides an anti-LAG-3 antibody or antigen-binding fragment thereof having one or more of the following characteristics: (i) inhibition (e.g., competitive inhibition) of any of the antibodies listed in Table 3 and human LAG- 3 binding; (ii) epitope binding to the same or overlapping with any of the antibodies shown in Table 3; (iii) competition with any of the antibodies shown in Table 3 for binding to human LAG-3.

In some embodiments, an antibody of the invention exhibits one or more of the following biological activities:

(i) Binding to human LAG-3 with high affinity; (ii) Binding to human LAG-3 with a dissociation constant (Kd) of less than 100 × 10 ^-4 , such as 0.5 × 10 ^-4 to 50 × 10 ^-4 ; (iii) binding to human LAG-3 expressed on the cell surface with high affinity; (iv) blocking the binding of human LAG-3 to the cell surface MHCII molecule; (v) binding to activated CD4 + and / or expressing human LAG-3 CD8 + T cells; (vi) stimulate an immune response, preferably an antitumor immune response; (vii) inhibit tumor cells expressing human LAG-3, especially when used in combination with anti-PD1 antibodies. Preferably, the antibody exhibits at least two, more preferably at least three, four, five, and even more preferably all of the properties described above.

In yet another aspect, the present invention provides a nucleic acid encoding an antibody of the present invention or a fragment thereof, a vector comprising the nucleic acid, and a host cell comprising the vector. The present invention also provides a method for preparing an antibody or a fragment thereof of the present invention.

In yet another aspect, the invention provides an immunoconjugate, a multispecific antibody, and a pharmaceutical composition comprising an antibody of the invention.

The invention also provides methods of stimulating an immune response in a subject, as well as methods of preventing or treating cancer or infection.

The invention also relates to a method for detecting LAG-3 in a sample.

Brief description of the drawings

Figure 1 shows the ability of the parent antibodies (ADI-26818, ADI-26822 and ADI-26836) to bind to hLAG-3 expressed on HEK293 cells by flow cytometry. 25F7 is a control antibody.

Figure 2 shows the binding capacity of affinity-optimized anti-hLAG-3 antibodies to hLAG-3 expressed on HEK293 cells by flow cytometry. 25F7 is a control antibody.

Figure 3 shows that anti-LAG-3 antibodies block the interaction of human MHCII (HLA-DR) and LAG-3 by flow cytometry detection.

Figure 4 shows the detection of the binding capacity of anti-LAG-3 antibodies to activated human CD4 + T cells by flow cytometry detection.

Figure 5 shows the tumor suppressive effect of anti-LAG-3 antibodies in the A375-human PBMC model. ADI-31798 is an antibody of the invention. Antibody is an anti-PD-1 antibody ("Antibody D") disclosed in PCT / CN2016 / 094122.

Figure 6 shows a comparison of the VL regions of exemplary antibodies of the invention.

Figure 7 shows a comparison of VH regions of exemplary antibodies of the invention.

Detailed description of the invention

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

The term "about" when used in conjunction with a numerical value is intended to encompass a numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

The term "and / or" should be understood to mean any one of the options or a combination of any two or more of the options.

As used herein, the term "comprising" or "including" means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps. In the present context, when the term "comprising" or "including" is used, unless otherwise indicated, the case consisting of the mentioned elements, integers, or steps is also covered. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region consisting of that particular sequence.

As used herein, the term "antibody" refers to a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an antigen. The term encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, or Chimeric antibodies or humanized antibodies, full-length antibodies, and antibody fragments are sufficient as long as they exhibit the desired antigen-binding activity.

Those skilled in the art will appreciate that "whole antibodies" (which are used interchangeably herein with "full-length antibodies", "full antibodies" and "full antibodies") comprise at least two heavy chains (H) and two light chains ( L). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a domain CL. A variable region is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to its antigen. The constant region does not directly participate in the binding of the antibody to the antigen, but displays a variety of effector functions. The light chain of an antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain. The heavy chain of an antibody can be divided into five major types depending on the amino acid sequence of the constant region of its heavy chain: IgA, IgD, IgE, IgG, and IgM, and several of these types can be further divided into subclasses, such as , IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different antibody types are called α, δ, ε, γ, and μ, respectively. The term "isotype" refers to the type of antibody determined by the constant region of the antibody heavy chain. See, for example, Fundamental Immunology, Ch. 7 (Editor Paul, W., Second Edition, Raven Press, N.Y. (1989)) (which is incorporated herein by reference in its entirety for all purposes).

The term "antigen-binding portion" of an antibody (which is used interchangeably herein with "antibody fragments" and "antigen-binding fragments") refers to a molecule that is not an intact antibody, which contains the intact antibody used to bind the intact antibody Part of the antigen. As understood by those skilled in the art, the antigen-binding portion of an antibody typically contains amino acid residues from a "complementarity determining region" or "CDR". Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of whole antibodies. Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab ', F (ab') ₂ , Fab'-SH, Fv, single-chain Fv, double-chain antibody (diabody), triple-chain antibody (triabody), four-chain antibody ( tetrabody), minibody, single domain antibody (sdAb). For a more detailed description of antibody fragments, see: Fundamental Immunology, edited by WEPaul, Raven Press, NY (1993); Shao Rongguang et al. (Editor), Antibody Drug Research and Application, People's Medical Publishing House ( 2013); Hollinger et al., PNAS USA 90: 6444-6448 (1993); Hudson et al., Nat. Med. 9: 129-134 (2003).

The terms "human antibody" or "fully human antibody" are used interchangeably herein and refer to antibodies that include variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid sequences that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or point-specific mutagenesis in vitro or somatic mutations in vivo), such as in CDRs-especially CDR3. However, as used herein, the term "human antibody" does not include antibodies in which the CDR sequences are derived from the germline of other mammalian species (eg, mice) and transplanted into human framework sequences.

As used herein, the term "recombinant human antibody" includes all human antibodies that are produced, expressed, produced, or isolated by recombinant means, for example, (a) an animal (e.g., a mouse) that has been transgenic or transchromosomic with a human immunoglobulin gene or Antibodies isolated from hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed into human antibody expression such as transfected tumors, (c) antibodies isolated from recombinant, combined human antibody libraries such as yeast display libraries, and ( d) An antibody prepared, expressed, produced or isolated by any other means, including splicing of human immunoglobulin genes to other DNA sequences. These recombinant human antibodies have framework regions and CDR regions that are derived from variable regions of human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibody can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using human Ig sequence transgenic animals), and the amino acid sequences of the VH and VL regions of the recombinant antibody thus obtained, Although derived from and related to human germline VH and VL sequences, they are not naturally found in human antibody germline libraries in vivo.

The term "monoclonal antibody" refers herein to antibodies obtained from a substantially homogeneous population of antibodies, i.e., in addition to possible variant antibodies (e.g., containing natural mutations or in the production of monoclonal antibody preparations) that are usually present in very small amounts Except for variant antibodies generated during the process, the individual antibodies constituting the population are the same and / or bind the same epitope.

The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody .

The term "humanized antibody" refers to an antibody that attaches CDR sequences derived from other mammalian species, such as mouse germline, to human framework sequences. Additional framework region modifications can be made within the human framework sequence.

An "isolated" antibody is one that has been separated from components in its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase Phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B. 848 (2007) 79-87.

The term "epitope" refers to the region of an antigen to which an antibody binds. An epitope can be formed from consecutive amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein.

The terms "lymphocyte activating gene-3" and "LAG-3" are used interchangeably and include variants, isotypes, homologs, and species homologs of LAG-3. The term "human LAG-3" refers to the human sequence LAG-3, for example, the complete amino acid sequence of human LAG-3 under Genbank accession number NP_002277. The human LAG-3 sequence may be different from the human LAG-3 of Genbank Accession No. NP_002277 in that it has, for example, a conservative mutation or a mutation in a non-conserved region, but still has substantially the same biological function, for example, in the extracellular domain It has an epitope that specifically binds to the antibody of the present invention, or has the function of binding to MHC class II molecules. Generally speaking, the human LAG-3 sequence is at least 90% identical to the human LAG-3 amino acid sequence of Genbank Accession No. NP_002277, and contains the amino acid sequence that can be identified when compared with the LAG-3 amino acid sequence of other species (rats) Is the amino acid residue of the human amino acid sequence. In some embodiments, the human LAG-3 sequence has at least 95%, even at least 96%, 97%, 98%, or 99% amino acid sequence identity with the human LAG-3 amino acid sequence of Genbank accession number NP_002277. LAG-3 proteins may also include fragments of LAG-3, such as fragments containing extracellular domains as well as extracellular domains, such as fragments that retain the ability to bind to any antibody of the invention, such as soluble LAG-3 molecules.

The term "immune response" refers to, for example, the action of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complements) produced by the above-mentioned cells or liver, which results in selective Damage, destruction, or removal of invading pathogens, cells or tissues infected with pathogens, or cancer cells.

The term "specific binding" means that an antibody selectively or preferentially binds an antigen. If in the bio-optical interferometry, the antibody is about 5x 10 ^-7 M or lower, about 1x 10 ^-7 M or lower, about 5x 10 ^-8 M or lower, about 1x 10 ^-8 M or lower, A K _{D of} about 5x 10 ^-9 M or lower, which binds to human LAG-3, then the antibody is an antibody that "specifically binds to human LAG-3". However, antibodies that specifically bind human LAG-3 can be cross-reactive with LAG-3 proteins from other species. For example, antibodies specific for human LAG-3 may, in some embodiments, cross-react with LAG-3 proteins of non-human species. In other embodiments, human LAG-3 specific antibodies can be completely specific to human LAG-3 without showing species or other types of cross-reactivity, or showing only cross-reactivity to LAG-3 of certain species Reactivity.

"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by an equilibrium dissociation constant (K _D ), which is the ratio of the dissociation rate constant and the association rate constant (k _dis and k _{on, respectively} ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

The term "high affinity" for IgG antibodies means that the antibody is at 1x 10 ^-7 M or lower, preferably 5x 10 ^-8 M or lower, more preferably about 1x 10 ^-8 M or lower, even more preferably Ground K _{D of} about 5x 10 ^-9 M or lower, binds to the target antigen. However, "high-affinity" binding can vary with antibody isotype. For example, the IgM isotype, "high affinity" refers to an antibody having 1x 10 ^-6 M or lower, preferably 1x 10 ^-7 M or less, more preferably about 1x 10 ^-8 M or less K _D .

A "competitively bound antibody" to a reference antibody that binds to an antigen such as LAG-3 refers to an antibody that blocks 50% or more of the binding of the reference antibody to an antigen (such as LAG-3) in a competition test, And, in turn, a reference antibody blocks 50% or more of the antibody's binding to an antigen (eg, LAG-3) in a competition test. Exemplary competition tests are described in: "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Competitively bound antibodies may bind the same epitope region as the reference antibody, such as the same epitope, adjacent epitopes, or overlapping epitopes.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes natural sequence Fc-regions and variant Fc-regions. In one embodiment, the human IgG heavy chain Fc-region extends from Cys226 of the heavy chain or from Pro230 to the carboxy terminus. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc-region or constant region is based on the EU numbering system, also known as the EU index, such as Kabat, EA, etc. , National Institute of Health, Bethesda, MD (1991), NIH Publication 91-3242.

The term "variant" in relation to an antibody means herein that, as compared to a reference antibody, it has passed at least 1 An antibody having a target antibody region of amino acid change with 5 amino acid substitutions, deletions, and / or insertions, wherein the variant substantially retains at least one biological property (eg, antigen-binding ability) of the antibody molecule before the change. The target antibody region can be the full length of the antibody, or the heavy or light chain variable region or a combination thereof, or the heavy chain CDR region (s) or the light chain CDR region (s) or a combination thereof . An antibody region having an amino acid change relative to a reference antibody region is also referred to herein as a "variant" of the antibody region.

As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-acid-based basis in a comparison window. The "percent sequence identity" can be calculated by comparing two optimally aligned sequences in a comparison window to determine the presence of the same nucleic acid base in both sequences (for example, A, T, C, G, I ) Or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) The number of positions to get the number of matching positions, divides the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiplies the result by 100 to produce a percent sequence identity. The optimal alignment for determining the percent sequence identity can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment within the full-length sequence range being compared or within the region of the target sequence.

In the present invention, as far as the antibody sequence is concerned, the percent amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and performing optimal alignment in accordance with the Kabat numbering rule in a preferred scheme. After the alignment, the target antibody region on the reference antibody (eg, the entire variable region of the heavy or light chain, or a portion thereof, such as one or more CDR regions) is compared to the corresponding region on the candidate antibody. The percent sequence identity between the candidate antibody region and the reference antibody region is: the number of positions occupied by the same amino acid in both the candidate antibody region and the reference antibody region divided by the total number of aligned positions of the two regions (the gaps are not counted) ) And multiply by 100 to get the percentage. Therefore, in this paper, if a candidate antibody and the reference antibody have an X% sequence identity in a region corresponding to the target antibody region of the reference antibody after being compared, the candidate antibody is considered to be a reference antibody. An antibody with X% sequence identity on the target antibody region. In this context, without specifying the region of the target antibody, it will be suitable for alignment over the full length of the reference antibody sequence. In some embodiments, in the case of antibodies, sequence identity may be distributed over the entire heavy chain variable region and / or the entire light chain variable region, or the percent sequence identity may be limited to the framework region only, while corresponding CDR regions The sequence remains 100% identical.

Similarly, in terms of antibody sequences, based on the alignment, candidate antibodies with amino acid changes in the target antibody region relative to the reference antibody can be determined. Therefore, in this paper, if a candidate antibody has a comparison with a reference antibody with X amino acid changes in a region corresponding to the target antibody region (such as the CDR region) of the reference antibody, the candidate antibody is considered to be An antibody that has an X amino acid change in the target antibody region with a reference antibody. When X is 0, the candidate antibody is considered to have the same target antibody region as the reference antibody. For example, when the target antibody region is a CDR sequence, the candidate antibody is considered to be an antibody having the same CDR sequence as the reference antibody.

In the present invention, "conservative substitution" refers to an amino acid change that results in the replacement of a certain amino acid with a chemically similar amino acid. Amino acid modifications, such as substitutions, can be introduced into the antibodies of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

Conservative substitution tables that provide functionally similar amino acids are well known in the art. In a preferred aspect, the conservative substitution residues are from the conservative substitution table A below, preferably the preferred conservative substitution residues shown in Table A.

Table A

原始残基Primitive residue	示例性取代Exemplary substitution	优选的保守取代Preferred conservative substitution
Ala(A)Ala (A)	Val；Leu；IleVal; Leu; Ile	ValVal

Arg(R)Arg (R)	LVs；Gln；AsnLVs; Gln; Asn	LysLys
Asn(N)Asn (N)	Gln；His；Asp；Lys；ArgGln; His; Asp; Lys; Arg	GlnGln
Asp(D)Asp (D)	Glu；AsnGlu; Asn	GluGlu
Cys(C)Cys (C)	Ser；AlaSer; Ala	SerSer
Gln(Q)Gln (Q)	Asn；GluAsn; Glu	AsnAsn
Glu(E)Glu (E)	Asp；GlnAsp; Gln	AspAsp
Gly(G)Gly (G)	AlaAla	AlaAla
His(H)His (H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile (I)	Leu；Val；Met；Ala；Phe；正亮氨酸Leu; Val; Met; Ala; Phe; norleucine	LeuLeu
Leu(L)Leu (L)	正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe	IleIle
Lys(K)Lys (K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met (M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe (F)	Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr	TyrTyr
Pro(P)Pro (P)	AlaAla	AlaAla
Ser(S)Ser (S)	ThrThr	ThrThr
Thr(T)Thr (T)	Val；SerVal; Ser	SerSer
Trp(W)Trp (W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr (Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val (V)	Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; norleucine	LeuLeu

Aspects of the invention are further detailed in the following subsections.

I. Anti-LAG-3 antibodies of the invention

One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds LAG-3, preferably a human LAG-3 protein, especially a fully human antibody or fragment thereof. In some embodiments, the antigen-binding fragment of an antibody of the invention is an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, single chain antibodies such as scFv, (Fab') ₂ fragments, single domain antibodies , Diabody (dAb) or linear antibody.

Favorable biological properties of antibodies

In some embodiments, the anti-LAG-3 antibodies or fragments thereof of the present invention bind to human LAG-3 with high affinity, for example, the dissociation equilibrium constant (K _D ) is less than about 100 nM, less than or equal to about 50 nM, more preferably Less than or equal to about 20nM, 15nM, or 10nM, such as 0.1-20nM, more preferably less than or equal to about 5nM, 4nM, 3nM, or 2nM, preferably 0.5-3nM, and even more preferably, less than or equal to about 1nM, 0.5nM. Preferably, K _{D is} determined by using a bio-optic interferometry (for example, Fortebio affinity measurement).

In some embodiments, the antibody or LAG-3 binding fragment of the present invention, solutions of the dissociation constant (K _dis) of less than ^{100 × 10 -4, 60 × 10} -4, for example, 0.5 × 10 ^-4 to 50 × 10 ^{- 4} , preferably 1 × 10 ^-4 to 10 × 10 ^-4 , or 1 × 10 ^-4 to 6 × 10 ^-4 s ^-1 , such as about 5 × 10 ^-4 s ^-1 . In some embodiments, the binding constant (K _on ) of the anti-LAG-3 antibody molecule to human LAG-3 is greater than 0.5 × 10 ⁵ , 1 × 10 ⁵ , 2 × 10 ⁵ , 3 × 10 ⁵ , 4 × 10 ⁵ Or 5 × 10 ⁵ M ^-1 s ^-1 , for example, bound to human LAG-3 with a K _{on of} about 5 × 10 ⁵ M ^-1 s ^-1 . Preferably, K _dis and K _{on are} determined by using a bio-optic interferometry (for example, Fortebio affinity measurement).

In some embodiments, the antibodies or fragments thereof of the invention bind to cells expressing LAG-3 with high affinity. In one embodiment, the cells that express human LAG-3 on the surface are 293 cells, such as HEK293 cells. Preferably, the EC50 value of the antibody binding to human LAG-3 expressing HEK293 cells is less than about 50 nM, 30 nM, or 10 M, such as 0.1-10 nM, preferably less than or equal to about 8 nM, as determined by flow cytometry (eg, FACS) 5nM, 3nM, or 2nM, more preferably less than or equal to about 1.5nM, 1.2nM, or 1nM, even more preferably less than or equal to about 0.8nM, 0.6nM, 0.3M, or 0.2nM.

In some embodiments, the present invention is an antibody or fragment thereof inhibits related activity of LAG-3, for example, IC ₅₀ values of less than or equal to about 20nM, 10nM, 9nM, 8nM, 7nM, 6nM , or of 5 nM, more preferably IC ₅₀ of less than or equal to about 1-7nM, 1-5nM, 6.5nM, 6nM, 5.5nM, 5nM, 4.5nM, 4nM, 3.5nM, or 3nM. In some embodiments, the related activity of LAG-3 is the binding of MHC class II molecules to LAG-3. In some embodiments, the antibodies or fragments thereof of the invention are less than or equal to about 20 nM, 10 nM, such as 1-9 nM, such as 1-8 nM, more preferably about 1-7 nM, 1-2 nM, for example, less than or equal to 6.5 nM , 6nM, 5.5nM, 5nM, 4.5nM , 4nM, 3.5nM 3nM or the IC _50, MHCII binding molecules on the cell block human MHCII molecules of LAG-3 expression. In some embodiments, the MHC class II molecule is HLA-DR. In some embodiments, the cell is a CHO cell. In some embodiments, the inhibition of LAG-3 related activity by an antibody of the invention or a fragment thereof is measured using flow cytometry (e.g., FACS).

In some embodiments, an antibody or fragment thereof of the invention binds to activated CD4 + and / or CD8 + T cells that express human LAG-3 on the surface. Preferably, the EC50 value of the antibody binding to activated human CD4 ⁺ T cells is less than or equal to about 35 pM, 30 pM, 25 pM, 20 pM, 15 pM, or 10 pM, preferably about 1-20 pM, as determined by flow cytometry (eg, FACS). 6-15 pM, 6-10 pM, for example, less than or equal to about 12 pM, 11 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, or 5 pM. In some embodiments, flow cytometry is performed in the Accuri C6 system.

In some embodiments, an antibody or fragment thereof of the invention inhibits one or more activities of LAG-3, for example, causing one or more of the following: increased antigen-dependent stimulation of CD4 + T lymphocytes; T cell proliferation Increased expression of activated antigens (eg, CD25); increased expression of cytokines (eg, interferon-γ (IFN-γ), interleukin-2 (IL-2), or interleukin-4 (IL-4)); Increased expression of chemokines (eg, CCL3, CCL4, or CCL5); reduced suppressive activity of Treg cells; increased T cell homeostasis; increased tumor infiltrating lymphocytes; or decreased immune escape from cancer cells.

In some embodiments, the antibodies or fragments thereof of the invention inhibit the growth of tumor cells expressing human LAG-3. In one embodiment, the tumor cell is a skin cancer cell, preferably a human skin cancer cell. For example, in vivo tumor transplantation models, such as NOG mice, inhibit the growth of human skin cancer cells. In some embodiments, an antibody of the invention is used in combination with an anti-PD1 antibody to achieve a significantly better antitumor effect than when an antibody is administered alone.

Preferably, an antibody of the invention or an antigen-binding fragment thereof exhibits at least one, more preferably at least two, more preferably at least three, four, or five, even more preferably all of the properties described above.

Antibody CDR region

A "complementarity determining region" or "CDR region" or "CDR" (which is used interchangeably with the hypervariable region "HVR" herein) is an amino acid region in an antibody variable region that is mainly responsible for binding to an epitope. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.

Various protocols are known in the art for determining a CDR sequence in a given VH or VL amino acid sequence. For example, the Kabat complementarity determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interface, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. ( 1991)). And Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. A "Contact" HVR is based on an analysis of the complex crystal structures available. According to different CDR determination schemes, the residues of each HVR / CDR in these HVRs are described below.

The HVR can also be an HVR sequence located at the following Kabat residue positions according to the Kabat numbering system:

Positions 24-36 or 24-34 (LCDR1) in VL, positions 46-56 or 50-56 (LCDR2), and positions 89-97 or 89-96 (LCDR3); and positions 26-35 or in VH 27-35B (HCDR1), positions 50-65 or 49-65 (HCDR2), and positions 93-102, 94-102, or 95-102 (HCDR3).

In one embodiment, the HVR of an antibody of the invention is an HVR sequence located at the following Kabat residue position according to the Kabat numbering system:

Positions 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL, and positions 27-35B (HCDR1), 50-65 (HCDR2), and positions in VH 93-102 (HCDR3).

Positions 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL, and positions 26-35B (HCDR1), 50-65 (HCDR2), and positions in VH 95-102 (HCDR3).

HVRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (such as any of the exemplary CDRs of the invention).

Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" or "HVR" or "HVR sequence" encompasses HVR or CDR sequences determined in any of the ways described above.

Unless otherwise stated, in the present invention, when referring to the position of residues in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), it means according to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. (1991)).

In a preferred embodiment, the CDR sequences of the present invention are shown in Table 1.

In a most preferred embodiment, the CDR sequences of the invention are shown in Table 2.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the smallest overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be clear to those skilled in the art, the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Accordingly, the invention also contemplates any variant of the CDRs given herein. For example, in one CDR variant, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.

In some embodiments, the antibodies of the invention have at least one, two, three, four, five, or six CDRs that are the same as the corresponding CDRs of any of the antibodies listed in Table 3, or are variants thereof. In some embodiments, an antibody of the invention has at least one, two, or three HCDRs that are the same as the corresponding heavy chain CDRs of any of the antibodies listed in Table 3, or are variants thereof. In some embodiments, the antibodies of the invention have at least one, two, or three LCDRs that are the same as the corresponding light chain CDRs of any of the antibodies listed in Table 3, or are variants thereof. As used herein, "corresponding CDR" refers to a CDR that is located at the most similar position to the CDR of the reference antibody in the amino acid sequence of the variable region of the candidate antibody after optimal alignment. Herein, a CDR variant is a CDR that has been modified by at least one, such as 1 or 2 or 3 amino acid substitutions, deletions, and / or insertions, wherein the antigen-binding molecule comprising the CDR variant substantially remains unmodified The biological properties of the antigen-binding molecule, for example, maintain at least 60%, 70%, 80%, 90%, or 100% biological activity (eg, antigen-binding capacity). It is understood that each CDR can be modified individually or in combination. Preferably, the amino acid modification is an amino acid substitution, especially a conservative amino acid substitution, such as the preferred conservative amino acid substitutions listed in Table A. In some embodiments, amino acid substitutions preferably occur at positions corresponding to the X residues of the consensus CDR sequences provided herein (e.g., SEQ ID NO: 16, 17, 18, 19, 20, 21, 30, 31, 32). Amino acid position.

In addition, it is known in the art that the CDR3 region, independent of the CDR1 and / or CDR2 regions, alone can determine the binding specificity of an antibody to an associated antigen. And, based on a common CDR3 sequence, a variety of other antibodies can be produced with the same binding specificity. See, for example, US Patents Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905, and 5,760,185. All of these references are incorporated herein by reference.

Thus, in one embodiment, an antibody of the invention comprises CDR3 from the heavy and / or light chain variable regions of one of the antibodies shown in Table 3, wherein the antibody is capable of specifically binding human LAG-3. In yet another embodiment, the antibodies may further comprise CDR2 from the heavy and / or light chain variable regions of the same antibody, or from the heavy and / or light chain variable regions of different LAG-3 antibodies. CDR2. In yet another embodiment, the antibodies may further comprise CDR1s from the heavy and / or light chain variable regions of the same antibody, or from the heavy and / or light chain variable regions of different LAG-3 antibodies. CDR1. The activity of these antibodies can be characterized by the assay methods described herein, including the binding activity to human LAG-3, the activity to block the binding of LAG-3 to MHC class II molecules, and the activity to inhibit tumor growth.

In yet another aspect, considering that the antigen-binding specificity is mainly provided by the CDR1, 2 and 3 regions, in some embodiments, the VHCDR1, 2 and 3 sequences and the VLCDR1, 2 and 3 sequences can be "mixed and matched" (ie It is possible to mix and match CDRs from different antibodies that bind to the same LAG-3 antigen, although each antibody preferably contains VH (CDR1, 2 and 3 and VL (CDR1, 2 and 3)) to produce other LAG-3 binding of the present invention. molecule. The binding of such "mixed and matched" antibodies to LAG-3 can be tested using binding assays known in the art (eg, ELISA, SET, Biacore) and those described in the examples. When VH CDR sequences are mixed and matched, the CDR1, CDR2 and / or CDR3 sequences from a particular VH sequence are preferably replaced with structurally similar CDR sequences. Also, when VL CDR sequences are mixed and matched, the CDR1, CDR2 and / or CDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequences. "Mixing and matching" of the CDRs can be performed between the antibodies shown in Table 3 of the present invention. In addition, those skilled in the art will understand that the structurally similar CDR sequences of the antibodies shown herein can also be replaced by one or more VH and / or VL CDR region sequences from other different antibodies to generate other antibody.

Thus, in some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region comprising a heavy chain complementarity determining region 3 (HCDR3), said HCDR3:

(i) is the same as the HCDR3 of the heavy chain variable region of any of the antibodies listed in Table 3; or

(ii) HCDR3 (i) contains at least 1 and no more than 3 (preferably 1-2 or more preferably 1) amino acid changes (preferably substitutions, more preferably conservative substitutions). Preferably, the HCDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 6, 9, 12, 15, 18, and 21.

In some embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, and the antibody has a heavy chain complementarity determining region 3 (HCDR3) and a light chain complementarity determining region 3 ( LCDR3):

(i) the HCDR3 and LCDR3 of the heavy and light chain variable region sequences of any of the antibodies listed in Table 3; or

(ii) A total of at least 1 and no more than 3 (preferably 1-2 or more preferably 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) with respect to HCDR3 and LCDR3 of (i). Preferably, the HCDR3 and LCDR3 comprise an amino acid sequence combination selected from the group consisting of: SEQ ID NO: 3/24, 6/24, 18/24, 9/25, 9/26, 9/30, 12/28, 15 / 29 and 21/32 amino acid sequences.

In one embodiment, an antibody of the invention or an antigen-binding fragment thereof comprises a heavy chain variable region (VH), wherein the VH comprises:

(i) the HCDR1, HCDR2 and HCDR3 sequences contained in the VH sequence of any of the antibodies listed in Table 3; or

(ii) with respect to the sequence of (i), a total of at least one and no more than 10 or 5, 4, 3, 2, 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) are included on the three CDR regions; sequence.

In another embodiment, an antibody of the invention or an antigen-binding fragment thereof comprises a light chain variable region (VL), wherein the VL comprises:

(i) the LCDR1, LCDR2 and LCDR3 sequences contained in the VL sequence of any of the antibodies listed in Table 3; or

In another embodiment, an antibody of the invention or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the antibody comprises:

(i) six CDR sequences identical to the six CDR sequences contained in the VH and VL sequences of any of the antibodies listed in Table 3; or

(ii) with respect to the sequence of (i), a total of 6 CDR regions comprising at least one and no more than 20, 10 or 5, 4, 3, 2, 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) sequence.

In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises:

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 sequence from the heavy chain variable region of SEQ ID NO: 33 , CDR2 sequence, and CDR3 sequence, and the light chain variable region comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence from the light chain variable region of SEQ ID NO: 39.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 sequence from the heavy chain variable region of SEQ ID NO: 34 , CDR2 sequence, and CDR3 sequence, and the light chain variable region comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence from the light chain variable region of SEQ ID NO: 39.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 sequence from the heavy chain variable region of SEQ ID NO: 35 , CDR2 sequence, and CDR3 sequence, and the light chain variable region comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence from the light chain variable region of SEQ ID NO: 40.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 sequence from the heavy chain variable region of SEQ ID NO: 36 CDR2 sequence, and CDR3 sequence, and the light chain variable region comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence from the light chain variable region of SEQ ID NO: 41.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 sequence from the heavy chain variable region of SEQ ID NO: 37 CDR2 sequence, and CDR3 sequence, and the light chain variable region comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence from the light chain variable region of SEQ ID NO: 42.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 sequence from the heavy chain variable region of SEQ ID NO: 38 , CDR2 sequence, and CDR3 sequence, and the light chain variable region comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence from the light chain variable region of SEQ ID NO: 43.

In one embodiment, an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH comprises

(i) a heavy chain HCDR combination selected from the following amino acid sequence combinations (in the order HCDR1, HCDR2, and HCDR3, respectively):

SEQ ID NOs: 1/2/3, SEQ ID NOs: 4/5/6, SEQ ID NOs: 16/17/18, SEQ ID NOs: 7/8/9, SEQ ID NOs: 10/11/12, SEQ ID NOs: 13/14/15, and SEQ ID NOs: 19/20/21; or

(ii) a heavy chain HCDR combination selected from the following amino acid sequence combinations (in the order HCDR1, HCDR2, and HCDR3, respectively):

SEQ ID NOs: 70/2/71, SEQ ID NOs: 72/5/73, SEQ ID NOs: 74/8/75, SEQ ID NOs: 76/11/77, and SEQ ID NOs: 78/14/79 ;or

(iii) a variant of the HCDR combination of (i) or (ii) comprising a total of at least one and no more than 10 or 5, 4, 3, 2, 1 amino acid changes on the three CDR regions (Amino acid substitutions are preferred, conservative substitutions are preferred).

In another embodiment, an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises:

(i) a light chain LCDR combination selected from the following amino acid sequence combinations (in the order LCDR1, LCDR2, and LCDR3, respectively):

SEQ ID NOs: 22/23/24, SEQ ID NOs: 31/23/24, SEQ ID NOs: 22/23/25, SEQ ID NOs: 22/23/26, SEQ ID NOs: 31/23/30, SEQ ID NOs: 27/23/28, SEQ ID NOs: 27/23/29, and SEQ ID NOs: 31/23/32;

(ii) a variant of the LCDR combination of (i), said variant comprising a total of at least one and no more than 10 or 5, 4, 3, 2, 1 amino acid changes (preferably amino acid substitutions) on said three CDR regions , Preferably conservative substitutions).

In another embodiment, an anti-LAG-3 antibody or antigen-binding fragment thereof of the invention comprises:

(i) a combination of heavy and light chain CDRs selected from the following amino acid sequence combinations (in the order HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, respectively):

SEQ ID NOs: 1/2/3/22/23/24, SEQ ID NOs: 4/5/6/22/23/24, SEQ ID NOs: 16/17/18/31/23/24, SEQ ID NOs: 7/8/9/22/23/25, SEQ ID NOs: 7/8/9/22/23/26, SEQ ID NOs: 7/8/9/31/23/30, SEQ ID NOs: 10/11/12/27/23/28, SEQ ID NOs: 13/14/15/27/23/29, and SEQ ID NOs: 19/20/21/31/23/32; or

(ii) a heavy and light chain CDR combination selected from the following amino acid sequence combinations (in the order HCDR1, HCDR2, and HCDR3, LCDR1, LCDR2, and LCDR3, respectively):

SEQ ID NOs: 70/2/71/22/23/24, SEQ ID NOs: 72/5/73/22/23/24, SEQ ID NOs: 74/8/75/22/23/25, SEQ ID NOs: 74/8/75/22/23/26, SEQ ID NOs: 74/8/75/31/23/30, SEQ ID NOs: 76/11/77/27/23/28, and SEQ ID NOs : 78/14/79/27/23/29;

(ii) a variant of the combination of the heavy and light chain CDRs of (i), said variant comprising at least one and no more than 10 or 5, 4, 3, 2, 1 amino acids on said six CDR regions Changes (preferably amino acid substitutions, preferably conservative substitutions).

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions HCDRs of the variable region of the heavy chain, and three complementarity determining regions LCDR of the variable region of the light chain, wherein

(i) HCDR1 contains the amino acid sequence shown in SEQ ID NO: 1 or 4 or 16 or 70 or 72; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 2 or 5 or 17; HCDR3 contains SEQ ID No: 3 or 6 Or the amino acid sequence shown in 18 or 71 or 73, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 22 or 31, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 contains SEQ ID No: 24 The amino acid sequence of;

(ii) HCDR1 contains the amino acid sequence shown in SEQ ID NO: 7 or 74, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 8, HCDR3 contains the amino acid sequence shown in SEQ ID NO: 9 or 75, and LCDR1 contains SEQ ID The amino acid sequence shown by NO: 22 or 31, LCDR2 contains the amino acid sequence shown by SEQ ID NO: 23, and LCDR3 contains the amino acid sequence shown by SEQ ID NO: 25 or 26 or 30; or

(iii) HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10 or 13 or 19 or 76 or 78, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11 or 14 or 20, and HCDR3 contains SEQ ID No: 12 or 15 Or the amino acid sequence shown in 21 or 77 or 79, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 27 or 31, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 contains SEQ ID No: 28 or 29 Or the amino acid sequence shown in 32.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 1; the HCDR2 sequence of SEQ ID NO: 2; the HCDR3 sequence of SEQ ID NO: 3; the sequence of SEQ ID NO: 22 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 24.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 70; the HCDR2 sequence of SEQ ID NO: 2; the HCDR3 sequence of SEQ ID NO: 71; the sequence of SEQ ID NO: 22 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 24.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 4; the HCDR2 sequence of SEQ ID NO: 5; the HCDR3 sequence of SEQ ID NO: 6; the SEQ ID NO: 22 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 24.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 72; the HCDR2 sequence of SEQ ID NO: 5; the HCDR3 sequence of SEQ ID NO: 73; the sequence of SEQ ID NO: 22 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 24.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NQ: 7; the HCDR2 sequence of SEQ ID NO: 8; the HCDR3 sequence of SEQ ID NO: 9; the sequence of SEQ ID NO: 22 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 25.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 74; the HCDR2 sequence of SEQ ID NO: 8; the HCDR3 sequence of SEQ ID NO: 75; the sequence of SEQ ID NO: 22 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 25.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 7; the HCDR2 sequence of SEQ ID NO: 8; the HCDR3 sequence of SEQ ID NO: 9; the sequence of SEQ ID NO: 22 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 26.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 74; the HCDR2 sequence of SEQ ID NO: 8; the HCDR3 sequence of SEQ ID NO: 75; the sequence of SEQ ID NO: 22 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 26.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 10; the HCDR2 sequence of SEQ ID NO: 11; the HCDR3 sequence of SEQ ID NO: 12; the sequence of SEQ ID NO: 27 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 28.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 76; the HCDR2 sequence of SEQ ID NO: 11; the HCDR3 sequence of SEQ ID NO: 77; the SEQ ID NO: 27 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 28.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 13; the HCDR2 sequence of SEQ ID NO: 14; the HCDR3 sequence of SEQ ID NO: 15; the sequence of SEQ ID NO: 27 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 29.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises: the HCDR1 sequence of SEQ ID NO: 78; the HCDR2 sequence of SEQ ID NO: 14; the HCDR3 sequence of SEQ ID NO: 79; the SEQ ID NO: 27 LCDR1 sequence; LCDR2 sequence of SEQ ID NO: 23; LCDR3 sequence of SEQ ID NO: 29.

Antibody variable region

A "variable region" or "variable domain" is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to its antigen. The heavy chain variable region (VH) and light chain variable region (VL) can be further divided into hypervariable regions (HVR, also known as complementarity determining regions (CDRs)) with a more conservative region (i.e., framework (FR)). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some cases, a single VH or VL domain is sufficient to confer antigen-binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains (see, for example, Portolano, S. et al., J. Immunol. 150 (1993 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

It is known in the art that one or more residues in one or both of the two variable regions (ie, VH and / or VL) can be modified, for example, to one or more CDR regions and / or to one Or multiple framework regions are subject to residue modification, especially conservative residue substitution, and the modified antibody still substantially retains at least one biological property (eg, antigen-binding ability) of the previous antibody molecule. For example, residues in the CDR regions can be mutated to improve one or more binding properties (e.g., affinity) of the antibody. The antibody-binding or other functional properties of the mutated antibody can be assessed in in vitro or in vivo assays. Preferably, conservative substitutions are introduced. Preferably, no more than one, two, three, four or five residues are introduced into the CDR region. In addition, framework region residues can be mutated, for example, to improve the properties of the antibody. For example, one or more framework residues can be "backmutated" to corresponding germline sequence residues.

CDR grafting is another modification of antibody variable region known in the art. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants can be constructed that mimic the properties of known antibodies. In this antibody variant, CDR sequences from known antibodies are grafted onto the framework regions of different antibodies with different properties. Thus, in one embodiment, the invention relates to an anti-LAG-3 antibody, or an antigen-binding fragment thereof, comprising heavy and light chain CDR sequences from one of the antibodies of Table 3, but with different framework region sequences . Framework region sequences for replacement can be obtained from public DNA databases, including germline antibody gene sequences, or from LAG-3 antibody sequences reported in published literature. For example, germline DNA encoding human heavy and light chain variable region genes can be obtained from the GenBank database. The antibody protein sequence can be compared with the protein sequence in the database using a sequence similarity search tool such as Gapped BLAST. Preferably, the framework sequence used for substitution has structural similarity to the framework sequence of the antibody of the present invention selected for change, for example, having sequence identity of at least 80%, 85%, 90%, or 95%, 96%, 97 %, 98%, 99% or more framework sequences.

In yet another embodiment, VH from an exemplary antibody of the invention (one of the antibodies shown in Table 3) and other different anti-LAG-3 antibodies (preferably, the other antibody shown in Table 3) can be "mixed and matched" And VL sequences to generate other antibodies of the invention that bind LAG-3. When mixing and matching these strands, it is preferred to replace VH sequences from a specific VH / VL pair with structurally similar VH sequences. Also, VL sequences from a particular VH / VL pair are preferably replaced with structurally similar VL sequences. Such "mixed and matched" antibodies can be tested for binding to LAG-3 using binding assays known in the art (eg, ELISA, and other assays described in the Examples section).

Therefore, in one embodiment, the antibody of the invention comprises a heavy chain variable region VH sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 34, 35, 36, 37, and 38, or Consists of the amino acid sequence. In yet another embodiment, an antibody of the invention comprises a variant of said VH sequence.

In another embodiment, an antibody of the invention comprises a light chain variable region VL sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42 and 43 or by Amino acid sequence composition. In yet another embodiment, an antibody of the invention comprises a variant of said VL sequence.

In yet another embodiment, an antibody of the invention comprises heavy and light chain variable region VH / VL sequence pairs, wherein the VH sequence (i) comprises a group selected from SEQ ID NOs: 33, 34, 35, 36, 37, and 38 The amino acid sequence shown, or consists of the amino acid sequence, or (ii) is a variant of the VH sequence of (i); and the VL sequence comprises (i) selected from SEQ ID NOs: 39, 40, 41, 42 and The amino acid sequence shown in 43, or consists of the amino acid sequence, or (ii) is a variant of the VL sequence of (i).

In a preferred embodiment, an antibody of the invention comprises a heavy chain variable region and a light chain variable region sequence pair selected from:

(a) a VH sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 33, and a VL sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 39;

(b) a VH sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 34, and a VL sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 39;

(c) a VH sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 35, and a VL sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 40;

(d) a VH sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 36, and a VL sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 41;

(e) a VH sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 37, and a VL sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 42;

(f) a VH sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 38, and a VL sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 43.

In one embodiment, the variant of the VH sequence is at least 80%, 85% in amino acid sequence compared to the reference VH sequence (preferably over the full length or over the three regions of CDR1, 2 and 3). %, 90%, 92%, 95%, 97%, 98%, 99% or higher identity. In one embodiment, the variant of the VH sequence is at least one and no more than the reference VH sequence (preferably, over the full length or over the three regions of CDR1, 2 and 3) in the amino acid sequence. 30, 10, or 5, 4, 3, 2, 1, 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions). Preferably sequence differences do not occur in the CDR regions.

In a preferred embodiment, the variant of the VL sequence is at least 80% in amino acid sequence compared to the reference VL sequence (preferably over the full length or in the three regions of CDR1, 2 and 3) , 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity. In a preferred embodiment, the variant of the VL sequence is in amino acid sequence, compared to the reference VL sequence, (preferably over the full length or over the three regions of CDR1, 2 and 3), comprising at least one and No more than 30, 10, or 5, 4, 3, 2, 1, 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions). Preferably sequence differences do not occur in the CDR regions.

In a preferred embodiment, an antibody of the invention comprises a heavy chain variable region and a light chain variable region VH / VL sequence pair, said VH / VL sequence pair comprising an amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 33/39, 34/39, 35/40, 36/41, 37/42, and 38/43, or consist of the amino acid sequence pair. The invention also provides variants of the antibody, such as variants having at least 95-99% identity on VH, VL, or VH and VL, or comprising no more than 10 amino acid changes.

In any of the above embodiments, preferably, the heavy chain variable region of the antibody variant contains no more than 10, preferably no more than 1 CDR regions on one or more CDR (preferably all 3 CDRs) regions relative to the reference antibody. 5 (eg, 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).

In any of the above embodiments, it is preferred that the light chain variable region VL of the antibody variant contains no more than 10, preferably not more than 1 CDR regions relative to a reference antibody, on one or more CDRs (preferably all 3 CDRs). More than 5 (e.g., 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).

In any of the above embodiments, preferably, the antibody of the present invention or an antigen-binding fragment thereof comprises a combination of CDR sequences selected in the order (HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, respectively):

SEQ ID NOs: 1/2/3/22/23/24, SEQ ID NOs: 4/5/6/22/23/24, SEQ ID NOs: 16/17/18/31/23/24, SEQ ID NOs: 7/8/9/22/23/25, SEQ ID NOs: 7/8/9/22/23/26, SEQ ID NOs: 7/8/9/31/23/30, SEQ ID NOs: 10/11/12/27/23/28, SEQ ID NOs: 13/14/15/27/23/29, and SEQ ID NOs: 19/20/21/31/23/32.

Antibody heavy and light chains

In some embodiments, an antibody of the invention comprises a heavy chain Fc region, such as an Fc region of the IgG1, IgG2, or IgG4 isotype. In one embodiment, an antibody of the invention contains an IgG4-Fc region with a serine to proline mutation (S228P) at amino acid residue position 228 (EU numbering). In yet another preferred embodiment, an antibody of the invention comprises an IgG4-PAAFc moiety. The IgG4-PAA Fc portion has a serine to proline mutation (S228P) at position 228, a phenylalanine to alanine mutation at position 234 (EU number), and a leucine at position 235 (EU number) To alanine mutation. The S228P mutation is a mutation in the hinge region of the constant region of tumors, which can reduce or eliminate heterosulfide heterodisulfide bridges. The F234A and L235A mutations can further reduce the effector function of the human IgG4 isotype (which already has low effector function). In some embodiments, the antibodies of the invention contain an IgG4-PAA Fc portion with the heavy chain C-terminal lysine (des-Lys) removed. In some embodiments, an antibody of the invention comprises a kappa light chain constant region, such as a human kappa light chain constant region.

In a further preferred embodiment, the Fc region comprises the amino acid sequence of SEQ ID NO: 68, or the amino acid sequence of SEQ ID NO: 68 contains at least one, two or three, but not more than 20, 10 Or an amino acid sequence of 5 amino acid changes, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 68.

In a preferred embodiment, an antibody of the invention comprises a light chain constant region. In a preferred embodiment, the light chain constant region is a human kappa light chain constant region. In a further preferred embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 69, or the amino acid sequence of SEQ ID NO: 69 contains at least one, two or three, but no more than 20, 10 Amino acid sequence with one or five amino acids changed, or a sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 68.

In some preferred embodiments, the antibody of the invention comprises a heavy chain, and the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 44, 45, 46, 47, 48, and 49, or at least one relative thereto, Two or three, but no more than 20, 10, or 5 amino acid sequence changes or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% Or higher amino acid sequence. Preferably, the amino acid changes do not occur in the CDR regions, and more preferably, they do not occur in the variable regions.

In some preferred embodiments, the antibody of the invention comprises a light chain, and the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 63, 64, 65, 66, and 67, or at least one, two relative to it. Or three, but no more than 20, 10, or 5 amino acid sequence changes, or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more Highly identical amino acid sequences. Preferably, the amino acid changes do not occur in the CDR regions, and more preferably, they do not occur in the variable regions.

In a preferred embodiment, an antibody of the invention comprises a heavy chain sequence and / or a light chain sequence selected from:

(a) a heavy chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 44, and / or a light chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 50;

(b) a heavy chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 45, and / or a light chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 50;

(c) a heavy chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 46, and / or a light chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 51;

(d) a heavy chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 47, and / or a light chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 52;

(e) a heavy chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 48, and / or a light chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 53;

(d) a heavy chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 49, and / or a light chain sequence or a variant thereof comprising the amino acid sequence of SEQ ID NO: 54,

Wherein the variant contains at least one, two or three, but no more than 20, 10 or 5 amino acid altered amino acid sequences compared to the corresponding reference sequence, or has at least 80%, 85%, 90% , 92%, 95%, 97%, 98%, 99% or higher amino acid sequence identity. Preferably, the amino acid changes do not occur in the CDR regions, and more preferably do not occur in the variable regions.

In one embodiment, residue modifications are made in the constant region of the antibody to, for example, alter the properties of the antibody, such as effector function.

Exemplary antibody sequences

The invention provides fully human antibodies that specifically bind to LAG-3 (eg, human LAG-3) as isolated and characterized in the Examples. The VH and VL sequences of the antibody variable regions of these exemplary antibodies of the invention are listed in Table 3 below. Exemplary CDR sequences of the antibodies are listed in Tables 1 and 2 below.

Antibody variants

In one aspect, the invention provides variants of any of the antibodies described herein, particularly the exemplary antibodies listed in Table 3. In one embodiment, the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen-binding capacity) of the pre-modified antibody. In some embodiments, the alteration does not cause the antibody variant to lose binding to the antigen, but optionally can confer properties such as increased antigen affinity and different effector functions.

It can be understood that the variable region of the heavy chain or the variable region of the light chain of the antibody, or each CDR region can be changed individually or in combination. In some embodiments, there are no more than one, two, three, four, five, six, seven, eight, nine amino acid changes in one or more or all three heavy chain CDRs Or 10. Preferably, the amino acid is changed to an amino acid substitution, preferably a conservative substitution. In some embodiments, there are no more than one, two, three, four, five, six, seven, eight, nine amino acid changes in one or more or all three light chain CDRs Or 10. In some embodiments, there are no more than one, two, three, four, five, six, seven, eight, nine, or ten amino acid changes in one or more or all six CDRs Each. Preferably, the amino acid is changed to an amino acid substitution, preferably a conservative substitution. In some embodiments, the antibody variant and the reference antibody have at least 80%, 85%, 90%, or 95% or 99% or higher amino acid identity on the region of the target antibody sequence. For example, in one embodiment, an antibody of the invention has at least 90%, 91%, 92%, 93% on 3 heavy chain CDR regions compared to a reference antibody (such as one of the antibodies listed in Table 3), 94%, 95%, 96%, 97%, 98%, or 99% or higher identity. In one embodiment, an antibody of the invention has at least 90%, 91%, 92%, 93%, 94% of the three light chain CDR regions compared to a reference antibody (eg, one of the antibodies listed in Table 3). , 95%, 96%, 97%, 98%, or 99% or higher identity. In another embodiment, an antibody of the invention has at least 90%, 91%, 92%, 93%, 94% of the 6 CDR regions compared to a reference antibody (eg, one of the antibodies listed in Table 3), 95%, 96%, 97%, 98%, or 99% or higher identity. In yet another embodiment, an antibody of the invention has at least 80%, 85%, 90%, 91%, 92% of the heavy chain variable region compared to a reference antibody (such as one of the antibodies listed in Table 3). , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In yet another embodiment, an antibody of the invention has at least 80%, 85%, 90%, 91%, 92% of the light chain variable region compared to a reference antibody (e.g., one of the antibodies listed in Table 3). , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. In yet another embodiment, an antibody of the invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.

In addition, changes can be made to the Fc region of the antibody. Changes in the Fc region can be made alone or in combination with changes to the framework and / or CDR regions described above. The Fc region can be altered, for example, to alter one or more functions of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. In addition, the antibodies of the invention can also be chemically modified (e.g., linked to PEG) or their glycosylation pattern can be changed.

In certain embodiments, the Fc region may comprise an Fc-region having one or more amino acid substitutions that increase ADCC activity, for example, substitutions at positions 298, 333, and / or 334 of the Fc-region (EU numbering of residues) . In some embodiments, changes can also be made to the Fc-region to cause altered (ie, increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC) (see, for example, US 6,194, 551, WO99 / 51642 and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184).

In other embodiments, changes can be made to the Fc to increase or decrease its degree of glycosylation and / or change its glycosylation pattern. Addition or deletion of a glycosylation site of an Fc can be conveniently achieved by altering the amino acid sequence in order to create or remove one or more glycosylation sites. For example, one or more amino acid substitutions can be made to eliminate one or more glycosylation sites, thereby eliminating glycosylation at that site. Antibodies with altered types of glycosylation can be made, such as low or no fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisected GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. The present invention also contemplates antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region. These antibody variants may have improved CDC function.

In certain embodiments, the invention also contemplates antibody variants that have some but not all effector functions, which makes them ideal candidates for certain applications in which the in vivo half-life of the antibody is important, but Certain effector functions (such as complement and ADCC) are unnecessary or harmful. For example, the Fc region may contain mutations that eliminate or reduce effector functions, such as a human IgGl Fc region with mutations P329G and / or L234A and L235A, or a human IgG4 Fc region with mutations P329G and / or S228P and L235E.

In certain embodiments, it may be desirable to produce a cysteine engineered antibody, such as a "thio MAb", wherein one or more residues of the antibody are replaced with cysteine residues. For example, the number of cysteine residues in the hinge region of an antibody can be altered to, for example, facilitate assembly of the light and heavy chains or increase or decrease the stability of the antibody. Residues are, for example, U.S. Patent No. 5,677,425.

In certain embodiments, the antibodies provided herein can be further modified to contain a non-proteinaceous moiety. Suitable antibody-derived moieties include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG) to, for example, increase the half-life of an antibody (eg, serum). Methods for PEGylation of proteins are known in the art and can be applied to antibodies of the invention. See for example EP 0154 316 and EP 0401384.

II. Polynucleotides, vectors and hosts

The present invention provides a nucleic acid encoding any of the above anti-LAG-3 antibodies or fragments thereof. A vector comprising the nucleic acid is also provided. In one embodiment, the vector is an expression vector. A host cell comprising the nucleic acid or the vector is also provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells). In another embodiment, the host cell is prokaryotic.

In one aspect, the invention provides a nucleic acid encoding any of the above anti-LAG-3 antibodies or fragments thereof. The nucleic acid may comprise a nucleic acid encoding an amino acid sequence of a light chain and / or heavy chain variable region of an antibody, or a nucleic acid comprising an amino acid sequence of a light and / or heavy chain of an antibody. An exemplary nucleic acid sequence encoding an antibody heavy chain variable region comprises at least 80%, 85%, 90%, 91%, 92% of a nucleic acid sequence selected from SEQ ID NO: 57, 58, 59, 60, 61 or 62. %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleic acid sequences, or comprise a nucleic acid sequence selected from 57, 58, 59, 60, 61, or 62. Exemplary nucleic acid sequences encoding the variable region of an antibody light chain include at least 80%, 85%, 90%, 91%, 92% of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 50, 51, 52, 53, or 54 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleic acid sequences, or include a nucleic acid sequence selected from the group consisting of SEQ ID NO: 50, 51, 52, 53, or 54. When expressed from a suitable expression vector, the polypeptide encoded by these polynucleotides can show LAG-3 antigen-binding ability.

Also provided in the present invention are polynucleotides that encode at least one CDR region and generally all three CDR regions from the heavy chain VH or light chain VL sequences of an antibody that binds LAG-3 as described above. In some further embodiments, the polynucleotide encodes a complete or substantially complete variable region sequence of the heavy and / or light chain of the LAG-3 binding antibody described above.

As will be apparent to those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences.

In a preferred embodiment, the nucleic acid of the invention encoding an antibody further comprises a nucleotide sequence encoding a heavy chain Fc region, such as the Fc region sequence shown in SEQ ID NO: 68 or a sequence substantially identical thereto.

In a preferred embodiment, a nucleic acid of the invention encoding an antibody further comprises a nucleotide sequence encoding a light chain constant region sequence, such as the sequence shown in SEQ ID NO: 69 or a sequence substantially identical thereto.

Existing sequences encoding antibodies or binding fragments thereof that bind to LAG-3 (for example, the sequences shown in SEQ ID NO: 57-67) can be adopted by methods well known in the art, either by de novo solid phase DNA synthesis or by PCR mutagenesis. To produce these polynucleotide sequences.

In one embodiment, one or more vectors comprising a nucleic acid of the invention are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector of the invention is a pTT5 expression vector.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing a vector encoding an antibody include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. , Pages 245-254, which describe the expression of antibody fragments in E. coli). After expression, the antibodies can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for use in making antibodies or antigen-binding fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, a mammalian cell line adapted to suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293HEK or 293 cells, such as, for example, Graham et al., J. Gen Virol. 36:59 (1977) As described in)). Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 216 (1980)); and myeloma cell lines such as Y0 , NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003) .

III. Preparation of antibodies

In one embodiment, a method of making an anti-LAG-3 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for antibody expression, as provided above, and The antibody is optionally recovered from the host cell (or host cell culture medium). To recombinantly produce an anti-LAG-3 antibody, a nucleic acid encoding an antibody (such as the antibody described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids are easy to isolate and sequence using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains).

IV. Assay

The anti-LAG-3 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

In one aspect, the antibodies of the invention are tested for their antigen-binding activity. For example, binding to human LAG-3 can be determined by methods known in the art, such as ELISA, Western blot, etc., or exemplary methods disclosed in the examples herein. For example, the assay can be performed using flow cytometry in which the antibody reacts with a cell line expressing human LAG-3, such as HEK293 cells expressing human LAG-3 on the cell surface after transfection. Other cells are also suitable for flow cytometry, including activated CD4 + T cells expressing native LAG-3. Alternatively, the antibody binding, including binding kinetics (e.g., K _D value), using recombinant LAG-3 protein, in a biological assay of optical interference. In some embodiments, a bio-optic interferometry (e.g., Fortebio affinity measurement) is used.

On the other hand, competition assays can be used to identify antibodies that compete for binding to LAG-3 with any of the anti-LAG-3 antibodies disclosed herein. In certain embodiments, such a competitive antibody binds to an epitope (eg, a linear or conformational epitope) that is the same as or overlaps with the epitope bound by any of the anti-LAG-3 antibodies disclosed herein. For detailed exemplary methods for locating the epitope bound by an antibody, see Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ).

The present invention also provides an assay for identifying a biologically active anti-LAG-3 antibody. Biological activities may include, for example, binding to LAG-3 (e.g., binding to human LAG-3), blocking LAG-3 (e.g., binding to human LAG-3) binding to cell surface MHC class II molecules, binding to activated CD4 + and / or CD8 + T cells, inhibit tumor growth. For example, in a tumor suppressor model in vivo (see, e.g., Example 8), antibodies are tested for their ability to inhibit tumor growth. Antibodies having such biological activity in vivo and / or in vitro are also provided in the present invention.

It is understood that any of the above assays can be performed using an immunoconjugate or multispecific antibody of the invention in place of or in addition to an anti-LAG-3 antibody.

V. Multispecific antibodies

In yet another aspect, the invention provides a multispecific (including bispecific) antibody molecule that specifically binds LAG-3, preferably human LAG-3. In one embodiment, in a multispecific antibody, an antibody of the invention (or an antigen-binding fragment thereof) forms a first binding specificity for LAG-3. In yet another embodiment, the multispecific antibody is further directed against a second specificity of one of the following, or further comprises a second and third binding specificity directed to two molecules: PD-1, TIM-3, CEACAM (eg, CEACAM-1 or CEACAM-5), PD-L1, or PD-L2. In yet another embodiment, the multispecific antibody is a bispecific antibody that binds LAG-3 and PD-1, binds LAG-3 and PD-L1, or binds LAG-3 and PD-L2.

In one embodiment, the binding specificity is provided by the "binding site" or "antigen binding site" of the antibody (the region of the antibody molecule that actually binds to the antigen). In a preferred embodiment, the antigen binding site consists of a VH / VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). Thus, in one embodiment, a "multispecific" antibody is an antibody having at least two antigen-binding sites, each of said at least two antigen-binding sites may be a different list of the same antigen Or binding to different epitopes of different antigens.

For multispecific antibodies and their preparation, see, for example, WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010 / 145792 and WO 2010/145793.

VI. Immunoconjugates

In yet another aspect, the invention provides an immunoconjugate produced by conjugating an antibody of the invention to a heterologous molecule. In one embodiment, an antibody (or antigen-binding fragment thereof) of the invention is in a immunoconjugate with a therapeutic or diagnostic agent. In some embodiments, an antibody of the invention may be conjugated to a heterologous molecule in the form of a full-length antibody or antibody fragment. For example, conjugation can be performed in the form of Fab fragments, Fab 'fragments, F (ab)' 2 fragments, single-chain scFab antibodies, and single-chain scFv.

Linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a "cleavable linker" that facilitates release of the polypeptide upon delivery to the target site. For example, acid labile linkers, peptidase sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al. Cancer 52 (1992) 127-131; US 5,208,020) .

Therapeutic agents suitable for use in the conjugate include, but are not limited to, cytotoxins (such as cytostatic agents or cytocidal agents), drugs or radioisotopes. Examples of cytotoxic agents (eg, chemotherapeutic agents) suitable for forming immunoconjugates are known in the art, see for example WO05 / 103081. For example, cytotoxic agents include, but are not limited to: radioisotopes; growth inhibitors; enzymes and fragments thereof such as nucleases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including their Fragments and / or variants; and various known antitumor or anticancer agents.

In yet another aspect, the antibodies of the invention can be conjugated to a diagnostic or detectable agent. Such conjugates can be used as part of a clinical test (such as determining the efficacy of a particular therapy) for monitoring or predicting the onset, formation, progression, and / or severity of a disease or disorder. Such diagnosis and detection can be achieved by coupling antibodies to a detectable agent, including but not limited to a variety of enzymes, such as but not limited to horseradish peroxidase; prosthetic groups, such as but not limited to streptomyces Avidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission imaging techniques.

VII. Pharmaceutical compositions and pharmaceutical preparations

The present invention also includes a composition (including a pharmaceutical composition or a pharmaceutical preparation) comprising an anti-LAG-3 antibody or an immunoconjugate or a multispecific antibody thereof, and an anti-LAG-3 antibody or an immunoconjugate or a multispecific antibody thereof. Polynucleotide composition of sexual antibodies. These compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art. In one embodiment, the composition further comprises a second therapeutic agent, preferably, an anti-PD-1 antibody.

Pharmaceutically acceptable carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk , Glycerol, propylene, glycol, water, ethanol, etc. For the use of excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

This can be achieved by combining an anti-LAG-3 antibody, immunoconjugate or multispecific antibody of the invention with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed. (1980))) to prepare a pharmaceutical formulation comprising the invention, preferably in the form of a lyophilized formulation or an aqueous solution.

An exemplary lyophilized antibody formulation is described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine-acetate buffers.

The pharmaceutical composition or formulation of the invention may also contain one or more other active ingredients that are required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other . For example, it is desirable to also provide other anticancer active ingredients, such as chemotherapeutic agents, PD-1 axis binding antagonists (such as anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies) or anti-angiogenic agents ( (Eg bevacizumab). The active ingredients are suitably present in combination in an amount effective for the intended use.

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

With regard to other components of the pharmaceutical formulation, also see those disclosed in WO2015 / 153513.

VIII. Combination products

In some embodiments, the invention also provides a combination product comprising an antibody of the invention or an antigen-binding fragment thereof, and or a fragment or immunoconjugate thereof, and one or more other therapeutic agents (e.g., a chemotherapeutic agent) , Other antibodies, cytotoxic agents, vaccines, anti-infective agents, etc.). In some embodiments, other antibodies are, for example, anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies.

In some embodiments, the combination product is used to prevent or treat a tumor. In some embodiments, the tumor is a cancer, such as a gastrointestinal cancer, such as gastric, rectal, colon, colorectal, and the like; or a skin cancer, such as a malignant melanoma. In some embodiments, the combination product is used to prevent or treat infections, such as bacterial infections, viral infections, fungal infections, protozoan infections, and the like.

IX. Therapeutic methods and uses

As used herein, the terms "individual" or "subject" are used interchangeably and refer to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits and rodents (e.g., mice and rats mouse). In particular, the subject is a human.

As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural process of a disease in an individual being treated. The desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the state of the disease, and alleviating or improving the prognosis.

In one aspect, the invention relates to a method of enhancing an immune response in a subject, said method comprising administering to said subject an effective amount of any of the anti-LAG-3 antibodies or fragments thereof described herein, or An immunoconjugate, a multispecific antibody, or a pharmaceutical composition comprising the antibody or fragment. In some embodiments, an anti-LAG-3 antibody or antigen-binding portion thereof of the invention is administered to a tumor-bearing subject to stimulate an anti-tumor immune response. In other embodiments, an antibody of the invention or an antigen-binding portion thereof is administered to a subject carrying an infection to stimulate an anti-infective immune response.

In another aspect, the invention relates to a method of treating a tumor, such as cancer, in a subject, said method comprising administering to said subject an effective amount of any of the anti-LAG-3 antibodies or fragments thereof described herein, or comprising An immunoconjugate, a multispecific antibody, or a pharmaceutical composition of the antibody or fragment.

In some embodiments, tumors, such as cancers, described herein include, but are not limited to, solid tumors, hematological cancers (eg, leukemia, lymphoma, myeloma) and their metastatic lesions. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignant tumors, such as sarcomas and cancers (e.g., adenocarcinoma) of multiple organ systems, such as invasion of the lung, breast, lymph, gastrointestinal or colorectal, genital and reproductive urinary tract (e.g., kidney cells , Bladder cells, bladder cells), pharynx, CNS (e.g., brain cells, nerve cells or glial cells), skin (e.g., melanoma), head and neck (e.g., head and neck squamous cell carcinoma (HNCC )) And those of the pancreas. For example, melanoma, colon cancer, gastric cancer, rectal cancer, renal cell carcinoma, breast cancer (eg, breast cancer that does not express one, two, or all of the estrogen receptor, progesterone receptor, or Her2 / neu, such as , Triple negative breast cancer), liver cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and / or non-squamous structure) or small cell liver cancer), prostate cancer, head or neck Cancer (eg, HPV + squamous cell carcinoma), small intestine cancer, and esophageal cancer. Examples of hematological cancers include, but are not limited to, leukemia (e.g., myeloid leukemia, lymphoid leukemia, or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma or mantle cell lymphoma (MCL)) and myeloma, for example, multiple myeloma. Cancer can be early, middle or advanced, or metastatic.

In one embodiment, the cancer is a gastrointestinal cancer such as colon cancer, or a skin cancer such as malignant melanoma and the like.

In another aspect, the invention relates to a method of treating an infectious disease, such as a chronic infection, in a subject, said method comprising administering to said subject an effective amount of any of the anti-LAG-3 antibodies or fragments thereof described herein Or an immunoconjugate, a multispecific antibody, or a pharmaceutical composition comprising the antibody or fragment. In one embodiment, the infection is a viral infection.

In some embodiments, the infectious disease is caused by a viral infection. Some examples of pathogenic viruses include (A, B, and C) hepatitis viruses, (A, B, and C) influenza viruses, HIV, herpes viruses (e.g., VZV, HSV-1, HAV-6 , HSV-II, CMV, Epstein Barr virus +), adenovirus, flavivirus, acovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, Rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papilloma, molluscum virus, poliovirus, rabies virus, JC virus and arbovirus.

For diseases that are suitable for prevention or treatment with the anti-LAG-3 antibodies or fragments thereof, immunoconjugates and multispecific antibodies of the present invention, further see WO2015 / 138920, WO2016 / 028672, WO2015 / 042246 and the like.

In some embodiments, the methods described herein further comprise administering to the subject one or more therapies (eg, a treatment modality and / or other therapeutic agent). In some embodiments, the treatment modality includes surgical treatment and / or radiation therapy.

In some embodiments, in addition to administering an antibody of the invention, the methods of the invention include administering at least one other immunostimulatory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-1 antibody These antibodies can be, for example, fully human, chimeric, or humanized antibodies.

In other embodiments, the other therapeutic agent is selected from a chemotherapeutic agent, a PD-1 axis binding antagonist (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody) or an anti-angiogenic agent (e.g., shellfish Valizumab).

In some embodiments, PD-1 axis binding antagonists include, but are not limited to, PD-1 binding antagonists, PD-L1 binding antagonists and PD-L2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand-binding partner. In a specific aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In a specific aspect, the PD-L1 binding partner is PD-1 and / or B7.1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding to its binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody).

In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), and CT-011 (Pidilizumab). In some embodiments, the anti-PD-1 antibody is MDX-1106. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4). In a preferred embodiment, the anti-PD-1 antibody is an "Antibody D" described herein.

In further embodiments, an anti-LAG-3 antibody or fragment thereof, alone or in combination with a PD-1 axis binding antagonist, can also be administered in combination with one or more other therapies such as a treatment modality and / or other therapeutic agents. In some embodiments, treatment modalities include surgery (e.g., tumor resection); radiation therapy (e.g., exoparticle beam therapy, which involves three-dimensional conformal radiation therapy in which the illuminated area is designed), local irradiation (e.g., pointing at a preselected target) Or organ irradiation) or focused irradiation.

In some embodiments, an anti-LAG-3 antibody or fragment thereof of the invention can be administered in combination with a chemotherapeutic agent or a chemotherapeutic agent. In some embodiments, an anti-LAG-3 antibody or fragment thereof of the invention can be administered in combination with radiotherapy or a radiotherapy agent. In some embodiments, an anti-LAG-3 antibody or fragment thereof of the invention can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, an anti-LAG-3 antibody or fragment thereof of the invention can be administered in combination with an immunotherapy or immunotherapeutic agent, such as a monoclonal antibody.

The antibodies of the invention (and pharmaceutical compositions or immunoconjugates comprising them, and any additional therapeutic agents) can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal administration, And, if local treatment is needed, it is administered intralesionally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on whether the medication is short-term or long-term, it can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

To prevent or treat a disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease , Whether the antibody is administered for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to a patient in one treatment or after a series of treatments.

In the method of the present invention described above, instead of the antibody or antigen-binding portion of the present invention, the composition, multispecific antibody or immunoconjugate of the present invention can be administered. Alternatively, in these methods, in addition to administering an antibody or an antigen-binding portion of the present invention, a composition, a multispecific antibody, or an immunoconjugate of the present invention may be further administered.

In yet another aspect, the present invention provides the use of an anti-LAG-3 antibody, composition, immunoconjugate, multispecific antibody of the present invention in the manufacture of a medicament for use in the aforementioned method (for example, for treatment).

X. Methods and compositions for diagnosis and detection

In yet another aspect, the invention relates to a method and kit for detecting LAG-3 in a sample, wherein the method comprises: (a) contacting the sample with an antibody of the invention or an antigen-binding fragment or immunoconjugate thereof; and ( b) detecting the formation of a complex between the antibody or its antigen-binding fragment or immunoconjugate and the LAG-3 protein. In some embodiments, the sample is from a cancer patient, such as a skin cancer patient. The detection may be in vitro or in vivo.

As used herein, the term "detection" includes quantitative or qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody-molecule magnetic beads, ELISA assays Method, PCR-technology (eg, RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissue. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion. In certain embodiments, the LAG-3 to be detected is human LAG-3.

In one embodiment, an anti-LAG-3 antibody is used to select a subject suitable for treatment with an anti-LAG-3 antibody, for example where LAG-3 is a biomarker for selecting said subject. In one embodiment, a cancer or tumor can be diagnosed using an antibody of the invention, such as to evaluate (e.g., monitor) a subject for the treatment or progression of a disease described herein (e.g., a hyperproliferative or cancerous disease), its diagnosis, and / or Staging.

In certain embodiments, a labeled anti-LAG-3 antibody is provided. Labels include, but are not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radioactive labels), and portions that are detected indirectly, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and their derivatives, rhodamine and its derivatives, dansyl, umbrella Umbelliferone, luciferase, for example, firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazine dione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lyase, sugar oxidase, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as urase and xanthine oxidase, and enzymes using peroxidase dyes such as HR, lactoperoxidase, or microperoxidase, biotin / avidin , Spin labeling, phage labeling, stable free radicals, and more.

The following examples are described to assist in understanding the invention. It is not intended and should not be interpreted in any way as limiting the scope of protection of the present invention.

The following examples of the present invention relate to six exemplary antibodies (ADI-26818, ADI-26822, ADI-26836, ADI-31798, ADI-31815, and ADI-31836), the CDR regions, light chain variable regions, and heavy chains of these antibodies. The amino acid sequences of the chain variable regions, light and heavy chains, and the corresponding nucleotide sequences are listed in Tables 1-3 and the Sequence Listing of this application. In addition, the sequence numbers of the light chain constant region, heavy chain constant region, light chain variable region, and heavy chain variable region of the above exemplary antibodies of the present invention are shown in Table 5.

Examples

Example 1. Anti-human LAG-3 antibody screening to determine the parent antibody

Screening of anti-LAG-3 fully human antibodies using yeast display technology

Yeast-based antibody presentation libraries (Adimab) were amplified according to existing methods (WO2009036379; WO2010105256; WO2012009568), where the diversity of each library reached 1 × 10 ⁹ . In short, the first two rounds of screening used Miltenyi's MACS system for magnetically activated cell sorting. First, incubate the yeast cells (~ 1 × 10 ¹⁰ cells / library) in FACS washing buffer (phosphate buffer, containing 0.1% bovine serum protein) at room temperature for 15 minutes. The buffer contains 100 nM biotin Human LAG-3 antigen (Acro Biosystems, catalog number LA3-H5255-1 mg). Wash once with 50 ml of pre-chilled FACS wash buffer, resuspend the cells with 40 ml of the same wash buffer, and add 500 μl of streptavidin microbeads (Miltenyi LS) and incubate at 4 ° C for 15 minutes. Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with 5 ml FACS washing buffer, and add the cell solution to the Miltenyi LS column. After the addition was completed, the column was washed 3 times with 3 ml FACS washing buffer. The Miltenyi LS column was removed from the magnetic area and eluted with 5 ml of growth medium. The eluted yeast cells were collected and grown overnight at 37 ° C.

The next round of sorting was performed using a flow cytometer: approximately 1 × 10 ⁸ of yeast cells obtained after screening by the MACS system were washed three times with FACS buffer, and were labeled with human LAG containing a low concentration of biotin (100-1nM). -3 antigen was cultured at room temperature. The culture medium was discarded, and after washing the cells twice with FACS washing buffer, the cells were mixed with LC-FITC (FITC-labeled anti-human immunoglobulin kappa light chain antibody, Southern Biotech) (diluted 1: 100) and mixed with SA -633 (Streptavidin-633, Molecular Probes) (1: 500 dilution) or SA-PE (Streptavidin-PE, Sigma) (1:50 dilution) reagents were mixed and incubated at 4 ° C for 15 minutes . Eluted twice with pre-chilled FACS wash buffer and resuspended in 0.4 ml buffer, and the cells were transferred to a filter-equipped separation tube. Cells were sorted using FACS ARIA (BD Biosciences).

The yeast cells expressing the anti-human LAG-3 antibody obtained by the screening were induced by shaking at 30 ° C for 48 hours to express the anti-human LAG-3 antibody. After induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 min, and the supernatant was harvested. The protein A was used to purify the anti-human LAG-3 antibody in the supernatant, and the pH 2.0 acetic acid solution was used to elute the anti-human LAG-3 antibody. The antibody purity was> 95%.

Antibodies ADI-26818, ADI-26822 and ADI-26836 were obtained by screening.

Example 2 Affinity optimization of anti-human LAG-3 antibodies

In order to obtain higher affinity anti-human LAG-3 antibodies, we optimized the antibodies ADI-26818, ADI-26822 and ADI-26836 by the following methods.

VHmut screening

This method uses conventional mismatch PCR to introduce mutations into the antibody heavy chain region. During PCR, the base mismatch probability was increased to about 0.01 bp by using 1uM highly mutated base analogs dPTP and 8-oxo-dGTP.

The obtained mismatched PCR products were constructed into a vector containing a heavy chain constant region by the method of homologous recombination. By this method, under the screening pressure including LAG-3 antigen titer, unlabeled antigen competition, and competition using parent antibody, we obtained a secondary library with a library capacity of 1 × 10 ⁷ . Three successful screenings were performed by FACS method.

CDRH1 / CDRH2 Screening

The CDRH3 gene of the progeny antibody obtained by the VHmut method was constructed into a 1 × 10 ⁸ diversity CDRH1 / CDRH2 gene library, and 3 rounds of screening were performed. In the first round, the MACS method was used, while in the second and third rounds, the FACS method was used to pressurize the affinity of the antibody-antigen conjugate to select the antibody with the highest affinity.

After the above affinity maturation process, we have obtained anti-human LAG-3 monoclonal antibodies with improved affinity ADI-31798, ADI-31815 and ADI-31836.

Example 3. Expression and purification in HEK293 cells

According to a conventional method in the art, a gene DNA encoding an anti-LAG-3 antibody was obtained from a yeast cell expressing the above-mentioned anti-human LAG-3 antibody, and the gene DNA was cloned into a new expression vector (pTT5) according to a conventional method.

The expression vector containing the target antibody gene and the transfection reagent PEI (Polysciences) were transiently transfected into cultured human kidney embryonic cell 293 cells (Invitrogen) according to the protocol provided by the manufacturer. After transfection, the medium was discarded and the cells were diluted to 4 x 10 ⁶ / ml with fresh medium. The cells were cultured at 37 ° C, 5% CO ₂ for 7 days, and fresh medium was added every 48 hours. After 7 days, centrifuge at 1300 rpm for 20 min. Take the supernatant and purify the supernatant with Protein A so that the purity of the antibody is> 95%. An IgG4 antibody having an IgG4-PAA Fc portion (SEQ ID No: 68) was obtained.

The following control antibodies used in the examples were expressed and purified in HEK293 cells:

对照抗体Control antibody
25F725F7

25F7 is a human LAG-3 antibody transiently expressed in HEK293 cells, and its sequence is the same as that of the antibody "25F7" in the US patent US20170137514A1. The full-length heavy and light chains of a 25F7 antibody with an IgG4-PAA Fc portion (SEQ ID No: 68) are shown in SEQ ID No: 55 and SEQ ID NO: 56.

Example 4: Affinity determination of anti-LAG-3 antibodies of the present invention

The bio-interferometry (ForteBio) assay was used to determine the equilibrium dissociation constant (K _D ) of the six exemplary antibodies of the present invention that bind to human LAG-3 (hLAG-3).

ForteBio affinity measurement was performed in accordance with existing methods (Estep, P, et al., High Throughput Solution Based Measurement, Antibody-Antigen Affinity and Epitope Binning. MAbs, 2013.5 (2): p.270-8). In brief, the sensor was equilibrated in the analysis buffer for 30 minutes below the line, and then detected online for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online to an AHQ sensor (ForteBio) for ForteBio affinity measurement. The sensor with the loaded antibody was exposed to 100 nM of LAG-3 antigen for 5 minutes, and then the sensor was transferred to the analysis buffer for 5 minutes for dissociation rate measurement. Kinetic analysis was performed using a 1: 1 binding model.

In the experiments performed as described in the above assay, the affinity of ADI-26818, ADI-26822, ADI-26836, ADI-31798, ADI-31815, ADI-31836 and control antibody 25F7 is shown in Table 6.

Table 6: Measurement of binding kinetics of antibodies of the invention by biological light interference

It can be seen that the above six exemplary antibodies of the present invention all show extremely high affinity, among which ADI-31798, ADI-31815 and ADI-31836 have higher affinity than 25F7.

Example 5: Binding of the Anti-LAG-3 Antibody of the Invention to Human LAG-3

The binding of the above-mentioned 6 exemplary antibodies of the present invention to human LAG-3 was measured in a flow cytometry-based assay.

293 cells (293-hLAG-3 cells) overexpressing human LAG-3 were generated by transfecting the pCHO1.0 vector (Invitrogen) carrying human LAG-3 cDNA (Sino Biological) cloned to the multicloning site MCS.

293-hLAG-3 cells (0.2 × 10 ⁶ cells) with different concentrations of experimental antibodies (ADI-26818, ADI-26822, ADI-26836, ADI-31798, ADI-31815, ADI-31836 and control antibody 25F7) Mix (antibody dilution method: the highest antibody concentration is 500 nM, three-fold dilution in PBS containing 0.1% bovine serum albumin (BSA), a total of 8 concentrations were tested). Incubate on ice for 30 minutes. The cells were then washed at least twice, a 1: 100 diluted secondary antibody (PE-labeled goat anti-human IgG antibody, Southern Biotech, final concentration 5 μg / ml) was added, and incubated on ice (protected from light) for 30 minutes. Cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed on an Accuri C6 system (BD Biosciences) and a concentration-dependent curve was fitted according to its MFI.

ADI-26818, ADI-26822, and ADI-26836 bind hLAG-3 overexpressed on HEK293 cells with EC50 values of 1.181nM, 1.500nM, and 1.437nM, respectively. Binding capacity is comparable (EC50 value of control antibody 25F7 is 3.339 nM) (see Figure 1).

In experiments performed as described above, affinity-optimized anti-hLAG-3 antibodies ADI-31798, ADI-31815, and ADI-31836 bind hLAG-3 overexpressed on HEK293 cells with EC50 values of 0.201 nM and 1.293, respectively. nM and 0.562nM are superior to the binding ability of the control antibody 25F7 to hLAG-3 overexpressed on HEK293 cells (EC50 value 3.339nM). (See Figure 2)

Example 6. Blocking of the interaction between human LAG-3 ligand MHCII and LAG-3 by the anti-LAG-3 antibody of the present invention

The ability of ADI-31798, ADI-31815, and ADI-31836 to block the binding of human LAG-3 to MHCII (HLA) on the cell surface was measured by flow cytometry.

CHO cells (CHO-DR cells) overexpressing human HLA-DR were generated by transfecting the pCHO1.0 vector (Invitrogen) carrying human HLA-DR (Sino Biological) cloned into MCS.

Antigen rhLAG3 protein (huFc) (Sino Biological) was diluted to 40 nM, 50 μl / well. Add different concentrations of antibodies (ADI-31798, ADI-31815 and ADI-31836 and the control antibody 25F7, starting from the highest concentration of 80nM, three-fold gradient dilution, a total of 8 dilution gradients), 50μl / well, incubate on ice in PBS for 30min The final concentration of the antigen is 20nM, and the maximum final concentration of the antibody is 40nM. CHO-DR cells were adjusted to 3 × 10 ⁵ cells / well, 100 μl / well. The cells were centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in the antigen-antibody mixture. Incubate on ice for 30 min, add 100 μl / well PBS, centrifuge at 300 g for 5 min, wash once in PBS, add 100 μl of goat anti-human IgG-PE (Southern Biotech) / well diluted 1: 100, bath in ice for 20 min, add PBS 100 μl / well, Centrifuge at 300g for 5 min and wash once in PBS. Resuspend in 100 μl PBS and measure the fluorescence signal value of the cells with a flow cytometer.

The experimental results show that ADI-31798, ADI-31815 and ADI-31836 can effectively block the binding of LAG-3 and its ligand MHCII (HLA-DR), and its blocking ability is equivalent to that of control antibody 25F7. Specifically, the IC50s of the capabilities of ADI-31798, ADI-31815, and ADI-31836 to block the binding of human LAG-3 and MHCII (HLA-DR) are 4.701 nM, 3.575 nM, and 6.657 nM, respectively. The IC50 of the ability of the control antibody 25F7 to block the binding of human LAG-3 to MHCII (HLA-DR) was 5.141 nM. (See Figure 3)

Example 7. Binding Experiment of Antibodies to Activated T Cells

After activation of human CD4 + T cells, LAG-3 protein is expressed on the surface. In this study, the binding capacity of ADI-31798, ADI-31815, ADI-31836 and activated human CD4 ⁺ T cells was measured by flow cytometry.

Anti-CD3 / CD28 magnetic beads (Gibco) were added to CD4 ⁺ T cells for 3 days at a ratio of magnetic beads / CD4 ⁺ T cells = 1: 1, and the cell density was adjusted to 1 × 10 ⁶ cells / ml. Wells are 150μl / well, other wells are 100μl / well, antibody is added to the first row of wells, the final concentration is 10nM, mix well, 50μl is sucked into the next row of wells, and so on. Make 3 replicates per sample. Ice bath for 30 min, centrifuge at 400 g for 5 min, wash twice in PBS, add 50 μl of 1: 100 diluted PE-anti-human Fc antibody, ice bath for 30 min, centrifuge at 400 g for 5 min, wash twice in PBS, resuspend in 60 μl PBS, and then flow through the cells Analysis. Flow cytometry was performed on the Accuri C6 system (BD Biosciences) and a concentration-dependent curve was fitted according to its MFI.

The experimental results show that ADI-31798, ADI-31815, and ADI-31836 can bind to activated human CD4 ⁺ T cells, with EC50 values of 0.00595nM, 0.0128nM, and 0.0127nM, respectively. The binding ability is better than the control antibody 25F7 (the control antibody's EC50 value is 0.0356nM) (see Figure 4).

Example 8. Antitumor activity of anti-LAG-3 antibodies of the present invention

In this study, A375 (ATCC) human malignant melanoma skin cancer cells were used to determine the antitumor effect of anti-LAG-3 antibodies on NOG mice. Human PBMCs (AllCells) were injected intravenously in advance, and then A375 tumor-bearing mouse models were established by subcutaneous inoculation. After tumor formation, they were divided into groups and treated with different antibodies. The tumor volume and body weight of mice in each group were monitored during the administration period. The drug frequency is 2 times / week, and the drug is administered for 2 weeks for a total of 5 times. The monitoring frequency is 2 times / week, and the monitoring is continued for 4 weeks. The dosage and method of administration are as follows. After administration, the relative tumor suppression rate (TGI%) was calculated, and the calculation formula was as follows: TGI% = 100% * (h-IgG control group tumor volume-treatment group tumor volume) / (h-IgG control group tumor volume-h- Tumor volume before administration in the IgG control group), where the mean tumor volume before administration in the h-IgG control group was 71 mm ³ .

Mice: NOG mice, female, 7-8 weeks (weeks of age of mice at the time of tumor cell inoculation), weighing 17.6-24.2 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Mice were acclimated for 7 days after arrival, and then began research.

Cells: Human skin cancer cells A375 (ATCC # CRL-1619) were purchased from ATCC and routinely subcultured in strict accordance with ATCC requirements for subsequent in vivo experiments. Collect the cells by centrifugation, resuspend the cells in sterile PBS and adjust the cell density to 30 × 10 ⁶ cells / ml. After NOG mice have been intravenously injected with human PBMC, the right back is shaved, and A375 cells are injected subcutaneously at 0.2 ml / head. The tumor volume of each mouse was measured 7 days after tumor cell inoculation, and mice with an average tumor volume in the range of 70-71 mm ³ were selected and randomly grouped according to tumor volume. The antitumor activity of the anti-LAG-3 antibody used alone or in combination with the anti-PD-1 antibody was measured as follows.

Administration: The mice were divided into four groups (6 mice in each group), and each group was injected subcutaneously with the following doses of antibody:

(1) Human IgG, 20mg / kg;

(2) Anti-PD-1 antibody (Antibody D, PCT / CN2016 / 094122), 10 mg / kg;

(3) LAG-3 (ADI-31798), 10mg / kg;

(4) LAG-3 (ADI-31798), 10 mg / kg + anti-PD-1 antibody (Antibody D), 10 mg / kg.

The anti-PD-1 antibody "Antibody D" is an anti-human anti-PD-1 antibody disclosed in PCT / CN2016 / 094122. Human IgG is a human IgG preparation obtained from Equitech-Bio.

On the 7th, 10th, 14th, 17th, and 21st days after tumor cell inoculation, the above four groups of antibodies were administered to the mice in each group at the same dose as above.

Analysis: Tumors and body weights were measured twice a week throughout the study period, and the mice were euthanized when the tumors reached the endpoint (tumor volume> 3000 mm ³ ) or when the mice had a> 20% weight loss. Determination of the maximum major axis of tumor (L) and the maximum axial width (W) using a vernier caliper and tumor volume was calculated by the following ^{formula: V = L × W 2/} 2. Tumor size and time were plotted from mice from each group. Analysis of variance (ANOVA) was used to determine statistical significance. P values of <0.05 were considered statistically significant in all analyses.

The experimental results are shown in Table 7 and Figure 5. It can be seen that when the anti-LAG-3 monoclonal antibody ADI-31798 and the anti-PD-1 monoclonal antibody "Antibody D" of the present application are used in combination with the IgG control (equitech-Bio) and these two Compared with the respective antibodies, they can significantly inhibit tumor growth.

Table 7. Tumor suppression rates on day 24

Claims

An antibody or antigen-binding fragment thereof that specifically binds human LAG-3, comprising

(i) CDR1 sequence, CDR2 sequence and CDR3 sequence of the heavy chain variable region as shown in SEQ ID NO: 33 or 34, and CDR1 sequence and CDR2 sequence of the light chain variable region as shown in SEQ ID NO: 39 And CDR3 sequences, or

(ii) the CDR1 sequence, CDR2 sequence and CDR3 sequence of the heavy chain variable region as shown in SEQ ID NO: 35 or 36, and the CDR1 sequence of the light chain variable region as shown in SEQ ID NO: 40 or 41, CDR2 and CDR3 sequences; or

(iii) the CDR1 sequence, CDR2 sequence, and CDR3 sequence of the heavy chain variable region as shown in SEQ ID NO: 37 or 38, and the CDR1 sequence of the light chain variable region as shown in SEQ ID NO: 42 or 43, CDR2 and CDR3 sequences.
An antibody or antigen-binding fragment thereof that specifically binds human LAG-3, comprising

(a) SEQ ID NO: 1 or 4 or 16 HCDR1 sequence, SEQ ID NO: 2 or 5 or 17 HCDR2 sequence, SEQ ID NO: 3 or 6 or 18 HCDR3 sequence, SEQ ID ID NO: 22 or 31 LCDR1 sequence, LCDR2 sequence of SEQ ID NO: 23, and LCDR3 sequence of SEQ IDNO: 24; or

(b) HCDR1 sequence of SEQ ID NO: 7, HCDR2 sequence of SEQ ID NO: 8, HCDR3 sequence of SEQ ID NO: 9, LCDR1 sequence of SEQ ID NO: 22 or 31, LCDR2 sequence of SEQ ID NO: 23, And SEQ ID NO: 25 or 26 or 30 LCDR3 sequence; or

(c) SEQ ID NO: 10 or 13 or 19 HCDR1 sequence, SEQ ID NO: 11 or 14 or 20 HCDR2 sequence, SEQ ID NO: 12 or 15 or 21 HCDR3 sequence, SEQ ID NO: 27 or 31 LCDR1 sequence, LCDR2 sequence of SEQ ID NO: 23, and LCDR3 sequence of SEQ ID NO: 28 or 29 or 32; or

(d) SEQ ID NO: 70 HCDR1 sequence; SEQ ID NO: 2 HCDR2 sequence; SEQ ID NO: 71 HCDR3 sequence; SEQ ID NO: 22 LCDR1 sequence; SEQ ID ID NO: 23 LCDR2 sequence; SEQ ID NO: 24 LCDR3 sequence; or

(e) HCDR1 sequence of SEQ ID NO: 72; HCDR2 sequence of SEQ ID NO: 5; HCDR3 sequence of SEQ ID NO: 73; LCDR1 sequence of SEQ ID NO: 22; LCDR2 sequence of SEQ ID NO: 23; SEQ ID NO: 24 LCDR3 sequence; or

(f) HCDR1 sequence of SEQ ID NO: 74; HCDR2 sequence of SEQ ID NO: 8; HCDR3 sequence of SEQ ID NO: 75; LCDR1 sequence of SEQ ID NO: 22; LCDR2 sequence of SEQ ID NO: 23; SEQ ID LCDR3 sequence of NO: 25; or

(g) HCDR1 sequence of SEQ ID NO: 74; HCDR2 sequence of SEQ ID NO: 8; HCDR3 sequence of SEQ ID NO: 75; LCDR1 sequence of SEQ ID NO: 22; LCDR2 sequence of SEQ ID NO: 23; SEQ ID LCDR3 sequence of NO: 26; or

(h) HCDR1 sequence of SEQ ID NO: 76; HCDR2 sequence of SEQ ID NO: 11; HCDR3 sequence of SEQ ID NO: 77; LCDR1 sequence of SEQ ID NO: 27; LCDR2 sequence of SEQ ID NO: 23; SEQ ID LCDR3 sequence of NO: 28; or

(i) HCDR1 sequence of SEQ ID NO: 78; HCDR2 sequence of SEQ ID NO: 14; HCDR3 sequence of SEQ ID NO: 79; LCDR1 sequence of SEQ ID NO: 27; LCDR2 sequence of SEQ ID NO: 23; SEQ ID LCDR3 sequence of NO: 29.
An antibody or antigen-binding fragment thereof that specifically binds human LAG-3, comprising a heavy chain variable region and a light chain variable region, wherein the antibody comprises:

(i) a combination of CDR sequences selected from:

-CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO: 33, and CDR1, CDR2, and CDR3 sequences from the light chain variable region of SEQ ID NO: 39;

-CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO: 34, and CDR1, CDR2, and CDR3 sequences from the light chain variable region of SEQ ID NO: 39;

-CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO: 35, and CDR1, CDR2, and CDR3 sequences from the light chain variable region of SEQ ID NO: 40;

-CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO: 36, and CDR1, CDR2, and CDR3 sequences from the light chain variable region of SEQ ID NO: 41;

-CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO: 37, and CDR1, CDR2, and CDR3 sequences from the light chain variable region of SEQ ID NO: 42; or

-CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO: 38, and CDR1, CDR2, and CDR3 sequences from the light chain variable region of SEQ ID NO: 43;

or

(ii) a variant of a combination of CDR sequences relative to (i), wherein said variant comprises at least one and not more than 15, 10 or more of 1, 2, 3, 4, 5 or preferably 6 CDR regions 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
An antibody or antigen-binding fragment thereof that specifically binds human LAG-3, comprising

(i) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 33 or 34, and / or a SEQ ID NO: 39 The light chain variable region of an amino acid sequence having an amino acid sequence having at least 80%, 85%, or 90% sequence identity, or

(ii) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 35 or 36, and / or a SEQ ID NO: 40 Or a light chain variable region of an amino acid sequence having an amino acid sequence of at least 80%, 85%, or 90% sequence identity, or

(iii) a heavy chain variable region comprising an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 37 or 38, and / or a SEQ ID NO: 42 Or the light chain variable region of the amino acid sequence shown in 43 having an amino acid sequence having at least 80%, 85%, or 90% sequence identity.
The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:

(a) a VH comprising the amino acid sequence of SEQ ID NO: 33, and a VL comprising the amino acid sequence of SEQ ID NO: 39;

(b) a VH comprising the amino acid sequence of SEQ ID NO: 34, and a VL comprising the amino acid sequence of SEQ ID NO: 39;

(c) a VH comprising the amino acid sequence of SEQ ID NO: 35, and a VL comprising the amino acid sequence of SEQ ID NO: 40;

(d) a VH comprising the amino acid sequence of SEQ ID NO: 36, and a VL comprising the amino acid sequence of SEQ ID NO: 41;

(e) a VH comprising the amino acid sequence of SEQ ID NO: 37, and a VL comprising the amino acid sequence of SEQ ID NO: 42;

(f) VH comprising the amino acid sequence of SEQ ID NO: 38, and VL comprising the amino acid sequence of SEQ ID NO: 43.
The anti-LAG-3 antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody is an antibody or antigen-binding fragment thereof in the form of IgG1, IgG2, or IgG4, preferably an IgG4 Fc region having S228P, F234A and L235A mutations .
The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody is a fully human antibody, or a humanized antibody, or a chimeric antibody.
The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab ', Fab'-SH, Fv, a single-chain antibody such as scFv, (Fab') 2 fragment, single domain antibody, diabody (dAb) or linear antibody.
An anti-LAG-3 antibody or antigen-binding fragment thereof, wherein the antibody has one or more of the following characteristics:

(i) inhibiting (eg, competitively inhibiting) the binding of any of the antibodies listed in Table 3 to human LAG-3;

(ii) binding to the same or overlapping epitope as any of the antibodies shown in Table 3;

(iii) Competitive binding to human LAG-3 with any of the antibodies shown in Table 3.
The anti-LAG-3 antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody has one or more of the following characteristics:

(i) binding to human LAG-3 with a high affinity, for example with a KD value of less than 100 nM, such as less than 50 nM, such as 0.1-20 nM, preferably 0.5-3 nM;

(ii) the dissociation constant (K d ) bound to human LAG-3 is less than 100 × 10 -4 , such as 0.5 × 10 -4 to 50 × 10 -4 , preferably 1 × 10 -4 to 10 × 10 -4 , Or 1 × 10 -4 to 6 × 10 -4 s -1 ;

(iii) binding to human LAG-3 expressed on the cell surface with a high affinity, for example with an EC50 value of less than 100 nM, such as less than 50 nM, such as 0.1-10 nM, preferably less than 1 nM, more preferably less than 0.5 nM;

(iv) blocking the binding of human LAG-3 to cell surface MHCII molecules, preferably with an IC50 value of less than 100 nM, for example, less than 50 nM, 20 nM, preferably 1-10 nM, more preferably less than 5 nM;

(v) Binding of activated CD4 + and / or CD8 + T cells expressing human LAG-3, preferably the EC50 value of the antibody binding to activated human CD4 + T cells is less than or equal to about 35 pM, preferably about 1-20 pM, 6- 15pM;

(vi) stimulating an immune response, preferably an anti-tumor immune response;

(vii) Inhibiting the growth of tumor cells (preferably, skin cancer cells) expressing human LAG-3, especially when used in combination with anti-PD1 antibodies.
An isolated nucleic acid encoding the anti-LAG-3 antibody or antigen-binding fragment thereof according to any one of the preceding claims.
A vector comprising the nucleic acid of claim 11, preferably the vector is an expression vector.
A host cell comprising the nucleic acid of claim 11 or the vector of claim 12, preferably, the host cell is prokaryotic or eukaryotic, more preferably a yeast cell, a mammalian cell (such as a 293 cell or a CHO cell) ).
A method of preparing an anti-LAG-3 antibody or antigen-binding fragment thereof, the method comprising culturing claim 13 under conditions suitable for expressing a nucleic acid encoding the anti-LAG-3 antibody or antigen-binding fragment of any one of the preceding claims. A host cell, optionally isolating the antibody or antigen-binding fragment thereof, and optionally the method further comprises recovering the anti-LAG-3 antibody or antigen-binding fragment thereof from the host cell.
An immunoconjugate comprising an anti-LAG-3 antibody or an antigen-binding fragment thereof according to any one of the preceding claims conjugated to a therapeutic or diagnostic agent.
A multispecific antibody comprising the antibody of any one of the preceding claims or an antigen-binding fragment thereof, preferably the multispecific antibody is that which binds LAG-3 and PD-1, or that binds LAG-3 and PD-L1, or A bispecific antibody that binds LAG-3 and PD-L2.
A pharmaceutical composition comprising an anti-LAG-3 antibody according to any one of the preceding claims or an antigen-binding fragment thereof or an immunoconjugate according to claim 15 or a multispecific antibody according to claim 16 and optionally a pharmaceutical excipient.
The pharmaceutical composition of claim 17, comprising a second therapeutic agent; preferably, said second therapeutic agent is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.
A method for preventing or treating a tumor or infectious disease in a subject, said method comprising administering to said subject an effective amount of an anti-LAG-3 antibody or antigen-binding fragment thereof according to any one of the preceding claims, or The immunoconjugate of claim 15, or the multispecific antibody of claim 16, or the pharmaceutical composition of claims 17-18. Preferably, the tumor is a skin cancer, such as a malignant melanoma.
Method for detecting LAG-3 in a sample, said method comprising

(a) contacting the sample with an anti-LAG-3 antibody or an antigen-binding fragment thereof according to any one of the preceding claims; and

(b) detecting the formation of a complex between the anti-LAG-3 antibody or its antigen-binding fragment and LAG-3; optionally, the anti-LAG-3 antibody is detectably labeled.