阅读说明：本技术 全人源的抗lag-3抗体及其应用 (Fully human anti-LAG-3 antibodies and uses thereof ) 是由 胡化静 缪小牛 张攀 刘军建 于 2018-06-19 设计创作，主要内容包括：本发明涉及特异性结合LAG-3的新型抗体和抗体片段以及含有所述抗体或抗体片段的组合物。此外,本发明涉及编码所述抗体或其抗体片段的核酸及包含其的宿主细胞,以及其治疗和诊断用途。(The present invention relates to novel antibodies and antibody fragments that specifically bind LAG-3 and compositions containing the same. Furthermore, the invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and host cells comprising the same, as well as therapeutic and diagnostic uses thereof.)

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

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

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

(iii) The CDR1, CDR2 and CDR3 sequences of the heavy chain variable region shown in SEQ ID NO 37 or 38 and the CDR1, CDR2 and CDR3 sequences of the light chain variable region shown in SEQ ID NO 42 or 43.

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

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

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

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

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

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

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

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

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

(i) SEQ ID NO: 78, HCDR1 sequence; SEQ ID NO: the HCDR2 sequence of 14; SEQ ID NO: 79, HCDR3 sequence; SEQ ID NO: the LCDR1 sequence of 27; SEQ ID NO: the LCDR2 sequence of 23; SEQ ID NO: LCDR3 sequence of 29.

3. 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;

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

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

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

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

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

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

or

(ii) (ii) a variant relative to the CDR sequence combination of (i), wherein said variant comprises at least one and not more than 15, 10 or 5, 4,3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in total over 1, 2, 3, 4,5 or preferably 6 CDR regions.

4. 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 set forth in SEQ ID NO. 33 or 34, and/or a light chain variable region comprising an amino acid sequence having at least 80%, 85% or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO. 39, 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 set forth in SEQ ID NO 35 or 36, and/or a light chain variable region comprising an amino acid sequence having at least 80%, 85% or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO 40 or 41, 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 set forth in SEQ ID NO 37 or 38, and/or a light chain variable region comprising an amino acid sequence having at least 80%, 85% or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO 42 or 43.

5. 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) a VH comprising the amino acid sequence of SEQ ID NO. 38, and a VL comprising the amino acid sequence of SEQ ID NO. 43.

6. The anti-LAG-3 antibody or antigen-binding fragment thereof of 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 IgG4Fc region having S228P, F234A, and L235A mutations.

7. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a fully human antibody, or a humanized antibody, or a chimeric antibody.

8. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: fab, Fab '-SH, Fv, single-chain antibodies such as scFv, (Fab')₂A fragment, a single domain antibody, a diabody (dAb), or a linear antibody.

9. An anti-LAG-3 antibody or antigen-binding fragment thereof, wherein the antibody has one or more of the following properties:

(i) inhibiting (e.g., competitively inhibiting) binding of any of the antibodies listed in table 3 to human LAG-3;

(ii) binds to the same or an overlapping epitope as any one of the antibodies shown in table 3;

(iii) competes for binding to human LAG-3 with any of the antibodies shown in table 3.

10. The anti-LAG-3 antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody has one or more of the following properties:

(i) binds to human LAG-3 with high affinity, e.g. with a KD value of less than 100nM, e.g. less than 50nM, e.g. 0.1-20nM, preferably 0.5-3 nM;

(ii) dissociation constant (K) for binding to human LAG-3_d) Less than 100 x 10^-4E.g. 0.5X 10^-4To 50X 10^-4Preferably 1X 10^-4To 10X 10^-4Or 1X 10^-4To 6X 10^-4s^-1；

(iii) Binds to cell surface expressed human LAG-3 with high affinity, e.g. with an EC50 value of less than 100nM, e.g. less than 50nM, e.g. 0.1-10nM, preferably less than 1nM, more preferably less than 0.5 nM;

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

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

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

(vii) inhibit the growth of human LAG-3 expressing tumor cells (preferably, skin cancer cells), particularly when used in combination with an anti-PD 1 antibody.

11. An isolated nucleic acid encoding the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of the preceding claims.

12. A vector comprising the nucleic acid of claim 12, preferably said vector is an expression vector.

13. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13, preferably the host cell is prokaryotic or eukaryotic, more preferably a yeast cell, a mammalian cell (e.g. 293 cell or CHO cell).

14. A method of making an anti-LAG-3 antibody or antigen-binding fragment thereof, the method comprising culturing the host cell of claim 14 under conditions suitable for expression of a nucleic acid encoding the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of the preceding claims, optionally isolating the antibody or antigen-binding fragment thereof, optionally the method further comprising recovering the anti-LAG-3 antibody or antigen-binding fragment thereof from the host cell.

15. An immunoconjugate comprising the anti-LAG-3 antibody, or antigen-binding fragment thereof, of any one of the preceding claims conjugated to a therapeutic or diagnostic agent.

16. A multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of the preceding claims, preferably the multispecific antibody is a bispecific antibody that binds LAG-3 and PD-1, or binds LAG-3 and PD-L1, or binds LAG-3 and PD-L2.

17. A pharmaceutical composition comprising the anti-LAG-3 antibody or antigen-binding fragment thereof of any one of the preceding claims or the immunoconjugate of claim 15 or the multispecific antibody of claim 16, and optionally a pharmaceutical adjuvant.

18. The pharmaceutical composition of claim 17, comprising a second therapeutic agent; preferably, the second therapeutic agent is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.

19. A method of preventing or treating a tumor or an infectious disease in a subject, the method comprising administering to the subject an effective amount of the anti-LAG-3 antibody or antigen-binding fragment thereof of 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 malignant melanoma.

20. A method for detecting LAG-3 in a sample, the method comprising

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

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

Technical Field

The present invention relates to novel antibodies and antibody fragments that specifically bind LAG-3 and compositions containing the same. Furthermore, the invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and host cells comprising the same, as well as related uses. Furthermore, the invention relates to 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, e.g., therapeutic modalities or agents, such as anti-PD-1 or anti-PD-L1 antibodies.

Background

Lymphocyte activation gene 3(LAG-3), also known as CD223, is a type I transmembrane protein encoded in humans by the LAG3 gene. The molecular properties and biological functions of LAG-3 have been well characterized and described, see, e.g., Sierro et al, Expert Opin Ther Targets (2011)15 (1): 91-101. LAG-3 is a CD 4-like protein expressed on the surface of T cells (particularly activated T cells), natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 has been shown to be a negative co-stimulatory receptor, i.e., 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-. The interaction between LAG3 and MHC class II is also thought to play a role in regulating dendritic cell function (Andrea et al J Immunol 168:3874-3880, 2002). Recent preclinical studies have also documented the role of LAG-3 in CD 8T-cell depletion (Blackburn et al NatImmunol 10:29-37,2009).

Studies have shown that CD8+ T cells depleted following chronic viral infection express multiple inhibitory receptors (e.g., PD-1, CD160, and 2B 4). LAG-3 is expressed at high levels following LCMV infection and blocking the PD-1/PD-L1 pathway and LAG-3 was shown to significantly reduce viral load in chronically infected mice (Blackburn et al, Nat Immunol (2009) 10: 29-37). It has also been shown that combined inhibition of the PD-1/PD-L1 pathway and LAG-3 blocker provides anti-tumor efficacy (Jing et al, Journal for immunotherapy of Cancer (2015) 3: 2).

In view of the above-mentioned important roles of LAG-3, there is a need to develop new anti-LAG-3 antibodies, particularly fully human antibodies, that modulate their activity. Such antibodies can be better used for the treatment of tumors and other diseases such as infections. In addition, it would also be desirable to have new anti-LAG-3 antibodies that can be used in combination with other therapies (e.g., therapeutic agents such as anti-PD-1 or anti-PD-L1 antibodies) to treat tumors or infections.

Summary of The Invention

The invention provides a fully human anti-human LAG-3 antibody, and a coding gene and application thereof. Through genetic engineering means and yeast surface display technology, the inventor selects a fully human antibody of anti-human LAG-3 from a human antibody library displayed on the yeast surface, and further obtains an affinity matured high-affinity anti-human LAG-3 antibody. The fully human antibody molecule of the invention can effectively block the combination of LAG-3 and Major Histocompatibility (MHC) II molecules, combine LAG-3 expressed on activated human CD4+ T cells, and inhibit tumor growth when being applied in vivo, and particularly has obvious tumor inhibition effect when being combined with anti-PD-1 antibody. Thus, the antibodies of the invention may be used for a variety of uses, including but not limited to detecting LAG-3 protein and inhibiting tumor growth in a tumor-bearing subject.

Accordingly, in one aspect, the invention provides an antibody or antigen-binding fragment thereof that specifically binds LAG-3 (preferably 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) 3 complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO. 33 or 34, and 3 complementarity determining regions LCDR of the light chain variable region shown in SEQ ID NO. 39, or

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

(iii) 3 CDRs of the heavy chain variable region as set forth in SEQ ID NO:37 or 38, and 3 CDRs of the light chain variable region as set forth in SEQ ID NO:42 or 43.

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 properties: (i) inhibiting (e.g., competitively inhibiting) binding of any of the antibodies listed in table 3 to human LAG-3; (ii) binds to the same or an overlapping epitope as any one of the antibodies shown in table 3; (iii) competes for binding to human LAG-3 with any of the antibodies shown in table 3.

In some embodiments, the antibodies of the invention exhibit one or more of the following biological activities:

(i) binds to human LAG-3 with high affinity; (ii) to be less than 100 x 10^-4E.g. 0.5X 10^-4To 50X 10^-4Dissociation constant (K) of_d) Binds to human LAG-3; (iii) binds with high affinity to human LAG-3 expressed on the cell surface; (iv) blocking the binding of human LAG-3 to cell surface MHCII molecules; (v) (ii) binds activated CD4+ and/or CD8+ T cells expressing human LAG-3; (vi) stimulating an immune response, preferably an anti-tumor immune response; (vii) inhibiting human LAG-3 expressing tumor cells, particularly when used in combination with an anti-PD 1 antibody. Preferably, the antibody exhibits at least two, more preferably at least three, four, five, even more preferably all of the above properties.

In yet another aspect, the invention provides a nucleic acid encoding the antibody or fragment thereof of the invention, a vector comprising the nucleic acid, and a host cell comprising the vector. The invention also provides methods of making the antibodies or fragments thereof of the invention.

In still another aspect, the invention provides immunoconjugates, multispecific antibodies and pharmaceutical compositions comprising the antibodies of the invention.

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

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

Brief Description of Drawings

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

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

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

Figure 4 shows flow cytometry testing of the binding capacity of anti-LAG-3 antibodies and activated human CD4+ T cells as measured by flow cytometry.

FIG. 5 shows the tumor suppression effect of anti-LAG-3 antibody in A375-human PBMC model. ADI-31798 is an antibody of the invention. Antibody D is an anti-PD-1 Antibody ("Antibody D") disclosed in PCT/CN 2016/094122.

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

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

Detailed Description

Definition of

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 that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated 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 "comprises" is intended to mean including the stated elements, integers or steps, but not excluding any other elements, integers or steps. When the term "comprising" or "includes" is used herein, unless otherwise specified, it also encompasses the presence of stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions 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, polyclonal, single or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), fully human or chimeric antibodies or humanized antibodies, full length antibodies, and antibody fragments, so long as they exhibit the desired antigen binding activity.

As will be understood by those skilled in the art, a "whole antibody" (used interchangeably herein with "full length antibody", "whole antibody" and "whole antibody") comprises 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 3 domains, CH1, CH2, and CH 3. 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 one domain CL. The variable region is a domain in the heavy or light chain of an antibody that is involved in binding of the antibody to its antigen. The constant regions are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions. The light chains of antibodies can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. The heavy chains of antibodies can be divided into 5 major different types depending on the amino acid sequence of their heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA 2. The heavy chain constant regions corresponding to different antibody types are referred to as α, δ, ε, γ, and μ, respectively. The term "isotype" refers to the type of antibody defined by the constant region of the heavy chain of the antibody. See, e.g., Fundamental Immunology, ch.7(Paul, w. ed., 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 (used interchangeably herein with "antibody fragment" and "antigen-binding fragment"), refers to a molecule that is not an intact antibody, including those of an intact antibodyFor binding to the portion of the antigen to which the whole antibody binds. As understood by those skilled in the art, the antigen-binding portion of an antibody typically comprises amino acid residues from a "complementarity determining region" or "CDR". Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab, scFab, Fab ', F (ab')₂Fab' -SH, Fv, single-chain Fv, diabody (diabody), triabody (triabody), tetrabody (tetrabody), minibody (minibody), single domain antibody (sdAb). For a more detailed description of antibody fragments, see: basic immunology (fundamentals immunology), editors of w.e.paul, Raven Press, n.y. (1993); shorea et al (editors), antibody drug research and applications, national institutes of health press (2013); hollinger et al, PNAS USA 90: 6444-; hudson et al, nat. Med.9: 129-.

The terms "human antibody" or "fully human antibody" are used interchangeably herein and refer to an antibody comprising variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid sequences not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example, in the CDRs, particularly in CDR 3. 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 (e.g., mouse) and grafted into human framework sequences.

As used herein, the term "recombinant human antibody" includes all human antibodies that are prepared, expressed, produced, or isolated by recombinant means, e.g., (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal with human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells, e.g., transfectomas, that are transformed to express human antibodies, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, e.g., yeast display libraries, and (d) antibodies 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 and CDR regions derived from the variable regions of human germline immunoglobulin sequences. However, in certain embodiments, the recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis in the case of a human Ig sequence-transgenic animal) and the amino acid sequences of the VH and VL regions of the resulting recombinant antibodies, although derived from and related to human germline VH and VL sequences, do not naturally occur in human antibody germline repertoires in vivo.

The term "monoclonal antibody" refers herein to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., variant antibodies containing natural mutations or produced during the production of a monoclonal antibody preparation) which are typically present in small amounts.

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

The term "humanized antibody" refers to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are joined to human framework sequences. Additional framework region modifications may 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 as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., 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. Epitopes can be formed of contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

The terms "lymphocyte activation gene-3" and "LAG-3" are used interchangeably and include variants, isoforms, homologs, and species homologs of LAG-3. The term "human LAG-3" refers to the complete amino acid sequence of human sequence LAG-3, e.g., human LAG-3 under Genbank accession No. NP _ 002277. The human LAG-3 sequence may differ from human LAG-3 of Genbank accession No. NP _002277, having, for example, conservative mutations or mutations in non-conserved regions in the sequence, but still having essentially the same biological function, for example, having an epitope in the extracellular domain that specifically binds to an antibody of the invention, or having a function of binding to an MHC class II molecule. In general, the human LAG-3 sequence has at least 90% identity to the human LAG-3 amino acid sequence of Genbank accession No. NP _002277 and contains amino acid residues that can be identified as a human amino acid sequence when compared to LAG-3 amino acid sequences of other species (murine). In some embodiments, the human LAG-3 sequence has at least 95%, even at least 96%, 97%, 98%, or 99% amino acid sequence identity to the human LAG-3 amino acid sequence of Genbank accession No. NP _ 002277. LAG-3 proteins may also include fragments of LAG-3, such as fragments comprising an extracellular domain as well as an extracellular domain, e.g., fragments that retain the ability to bind to any antibody of the invention, e.g., soluble LAG-3 molecules.

The term "immune response" refers to, for example, the action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines, and complement) that results in the selective damage, destruction, or elimination of invading pathogens, pathogen-infected cells or tissues, or cancer cells.

The term "specifically binds" means that the antibody binds selectively or preferentially to the antigen. If in the biological light interference measurement, the antibody is about 5x 10^-7M or less, about 1x 10^-7M or less, about 5x 10^-8M or less, about 1x 10^-8M or less, about 5x 10^-9K of M or less_DAnd binds to human LAG-3, the antibody is an antibody that "specifically binds to human LAG-3". However, antibodies that specifically bind human LAG-3 may be cross-reactive with LAG-3 proteins from other species. For example, it is specificAntibodies to human LAG-3, in some embodiments, can cross-react with LAG-3 protein of a non-human species. In other embodiments, an antibody specific for human LAG-3 may be completely specific for human LAG-3 and exhibit no species or other types of cross-reactivity, or only cross-reactivity with LAG-3 of certain species.

"affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y may be generally determined by an equilibrium dissociation constant (K)_D) Typically, the equilibrium dissociation constants are the dissociation and association rate constants (k, respectively)_disAnd k_on) The ratio of (a) to (b). Affinity can be measured by common methods known in the art. One particular method for measuring affinity is the ForteBio kinetic binding assay herein.

The term "high affinity" for an IgG antibody means that the antibody is expressed at 1x 10^-7M or less, preferably 5x 10^-8M or less, more preferably about 1x 10^-8M or less, even more preferably about 5x 10^-9K of M or less_DBinding to the target antigen. However, "high affinity" binding may vary with antibody isotype. For example, for IgM isotype, "high affinity" means that the antibody has a 1x 10^-6M or less, preferably 1x 10^-7M or less, more preferably about 1x 10^-8K of M or less_D。

An "antibody that competes for binding" with a reference antibody that binds an antigen such as LAG-3 refers to an antibody that blocks 50% or more of the binding of the reference antibody to the antigen (e.g., LAG-3) in a competition assay, and in turn, blocks 50% or more of the binding of the reference antibody to the antigen (e.g., LAG-3) in a competition assay. Exemplary competition tests are described in: "Antibodies", Harbor and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Antibodies that compete for binding may bind to the same epitope region, e.g., the same epitope, an adjacent epitope, or an overlapping epitope, as the reference antibody.

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 native sequence Fc-regions and variant Fc-regions. In one embodiment, the human IgG heavy chain Fc-region extends from Cys226 or from Pro230 of the heavy chain 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 according to the EU numbering system, also known as the EU index, as described in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest,5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

The term "variant" in relation to an antibody refers herein to an antibody comprising a region of the antibody of interest that has been altered in amino acid by at least 1, e.g., 1-30, or 1-20 or 1-10, e.g., 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions and/or insertions as compared to a reference antibody, wherein the variant substantially retains at least one biological property (e.g., antigen binding ability) of the antibody molecule prior to the alteration. The antibody region of interest may be the full length of the antibody, or a heavy chain variable region or a light chain variable region or a combination thereof, or a heavy chain CDR region(s) or a light chain CDR region(s) or a combination thereof. Herein, an antibody region having amino acid changes relative to a reference antibody region is also referred to as a "variant" of that antibody region.

As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a comparison window. The "percent sequence identity" can be calculated by: the two optimally aligned sequences are compared over a comparison window, the number of positions in the two sequences at which the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percentage of sequence identity. Optimal alignment for determining 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. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared or over a region of the target sequence.

In the context of the present invention, the percent amino acid sequence identity is determined for an antibody sequence by optimally aligning a candidate antibody sequence with a reference antibody sequence, in a preferred embodiment, optimally according to the Kabat numbering convention. After alignment, the region of the antibody of interest (e.g., the entire variable region of the heavy or light chain, or a portion thereof such as one or more CDR regions) on the reference antibody is compared to the corresponding region on the candidate antibody. The percentage 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 and reference antibody regions is divided by the total number of aligned positions for both regions (gaps not counted) and multiplied by 100 to yield a percentage. Thus, herein, a candidate antibody is considered to be an antibody having X% sequence identity to a reference antibody over a target antibody region if the candidate antibody has X% sequence identity over a region corresponding to the target antibody region of the reference antibody after alignment with the reference antibody. Herein, without specifying the target antibody region, it will be applicable to alignment over the full length of the reference antibody sequence. In some embodiments, for antibodies, the 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 regions only, while the sequences of the corresponding CDR regions remain 100% identical.

Similarly, with respect to antibody sequences, based on the alignment, candidate antibodies having amino acid changes in the target antibody region relative to a reference antibody can be determined. Thus, herein, a candidate antibody is considered to be an antibody having X amino acid changes in a region of a target antibody (e.g., a CDR region) of a reference antibody when the candidate antibody is aligned with the reference antibody over the region of the target antibody. When X is 0, the candidate antibody is considered to have the same target antibody region as the reference antibody, e.g., 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 resulting in the substitution of an 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, for example, site-directed mutagenesis and PCR-mediated mutagenesis.

Conservative substitution tables providing functionally similar amino acids are well known in the art. In a preferred aspect, the conservatively substituted residue is from conservative substitutions a, preferably the preferred conservatively substituted residues shown in table a.

TABLE A

Original residues	Exemplary substitutions	Preferred conservative substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp；Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine	Leu

Aspects of the invention are described in further detail in the following subsections.

I. anti-LAG-3 antibodies of the invention

In one aspect, the invention provides an antibody or antigen-binding fragment thereof, particularly a fully human antibody or fragment thereof, that specifically binds LAG-3, preferably human LAG-3 protein. 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 '-SH, Fv, single-chain antibodies such as scFv, (Fab')₂A fragment, a single domain antibody, a diabody (dAb), or a linear antibody.

Advantageous biological properties of antibodies

In some embodiments, the anti-LAG-3 antibodies or fragments thereof of the invention bind human LAG-3 with high affinity, e.g., dissociation equilibrium constant (K)_D) Less than about 100nM, less than or equal to about 50nM, 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, even more preferably less than or equal to about 1nM, 0.5 nM. Preferably, K_DBy using a biological light interferometry (e.g. Fortebio affinity measurement).

In some embodiments, the dissociation constant (K) for binding of an antibody or fragment thereof of the invention to human LAG-3_dis) Less than 100 x 10^-4、60×10^-4E.g. 0.5X 10^-4To 50X 10^-4Preferably 1X 10^-4To 10X 10^-4Or 1X 10^-4To 6X 10^-4s^-1E.g. about 5X 10^-4s^-1. In some embodiments, the binding constant (K) of an anti-LAG-3 antibody molecule to human LAG-3 binding_on) Greater than 0.5 x 10⁵、1×10⁵、2×10⁵、3×10⁵、4×10⁵Or 5X 10⁵M^-1s^-1E.g. at about 5 × 10⁵M^-1s^-1K of_onBinds to human LAG-3. Preferably, K_disAnd K_onBy using a biological light interferometry (e.g. Fortebio affinity measurement).

In some embodiments, the antibodies or fragments thereof of the invention bind LAG-3 expressing cells with high affinity. In one embodiment, the cell that surface expresses human LAG-3 is a 293 cell, such as a HEK293 cell. Preferably, the antibodies bind HEK293 cells expressing human LAG-3 with an EC50 value of less than about 50nM, 30nM or 10M, e.g., 0.1-10nM, preferably less than or equal to about 8nM, 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, as determined by flow cytometry (e.g., FACS).

In some embodiments, an antibody or fragment thereof of the invention inhibits an activity associated with LAG-3, e.g., IC₅₀Values less than or equal to about 20nM, 10nM, 9nM, 8nM, 7nM, 6nM or 5nM, more preferably IC₅₀Less than or equal to about 1-7nM, 1-5nM, 6.5nM, 6nM, 5.5nM, 5nM, 4.5nM, 4nM, 3.5nM, or 3 nM. In some embodiments, the activity associated with LAG-3 is the binding of MHC class II molecules to LAG-3. In some embodiments, an antibody or fragment thereof of the invention has an IC of less than or equal to about 20nM, 10nM, e.g., 1-9nM, e.g., 1-8nM, more preferably about 1-7nM, 1-2nM, e.g., less than or equal to 6.5nM, 6nM, 5.5nM, 5nM, 4.5nM, 4nM, 3.5nM or 3nM₅₀Blocking binding of human LAG-3 to MHC class II molecules on cells expressing MHC class II molecules. 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 or fragment thereof of the invention is measured by flow cytometry (e.g., FACS).

In some embodiments, the antibodies or fragments thereof of the invention bind to activated CD4+ and/or CD8+ T cells that express human LAG-3. Preferably, the antibody is conjugated to activated human CD4 as determined by flow cytometry (e.g., FACS)⁺The T cell bound EC50 values are less than or equal to about 35pM, 30pM, 25pM, 20pM, 15pM, or 10pM, preferably about 1-20pM, 6-15pM, 6-10pM, for example less than or equal to about 12pM, 11pM, 10pM, 9pM, 8pM, 7pM, 6pM, or 5 pM. In some embodiments, flow cytometry is performed in an Accuri C6 system.

In some embodiments, an antibody or fragment thereof of the invention inhibits one or more activities of LAG-3, e.g., resulting in one or more of: increased antigen-dependent stimulation of CD4+ T lymphocytes; increased T cell proliferation; increased expression of an activating antigen (e.g., CD 25); increased expression of a cytokine (e.g., interferon- γ (IFN- γ), interleukin-2 (IL-2), or interleukin-4 (IL-4)); increased expression of chemokines (e.g., CCL3, CCL4, or CCL 5); decreased suppressive activity of Treg cells; increased T cell homeostasis; an increase in tumor-infiltrating lymphocytes; or a reduction in immune evasion by 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, growth of human skin cancer cells is inhibited in an in vivo tumor transplantation model, such as NOG mice. In some embodiments, the antibodies of the invention are used in combination with an anti-PD 1 antibody to achieve an anti-tumor effect that is significantly better than when either antibody is administered alone.

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

Antibody CDR regions

"complementarity determining regions" or "CDR regions" or "CDRs" (used interchangeably herein with hypervariable region "HVRs") are regions of amino acids in an antibody variable region that are primarily responsible for binding to an epitope of an antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR 3.

Various protocols for determining the CDR sequences of a given VH or VL amino acid sequence are known in the art. For example, Kabat Complementarity Determining Regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). And Chothia refers to the position of the structural loops (Chothia and Lesk, J.mol.biol.196:901-917 (1987)). The AbM HVR is a compromise between the Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. The "Contact" HVR is based on an analysis of the complex crystal structure available. The residues of each of these HVRs/CDR are as follows, according to different CDR determination schemes.

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

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

In one embodiment, the HVRs of an antibody of the invention are HVR sequences located at Kabat residue positions according to the numbering system of Kabat:

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

In one embodiment, the HVRs of an antibody of the invention are HVR sequences located at Kabat residue positions according to the numbering system of Kabat:

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

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

Unless otherwise indicated, in the present invention, the terms "CDR" or "CDR sequence" or "HVR sequence" encompass HVRs or CDR sequences determined in any of the ways described above.

Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), reference is made to the numbering positions according to the Kabat numbering system (Kabat et al, Sequences of proteins of Immunological Interest,5th ed. public Health Service, national institutes of Health, Bethesda, Md. (1991)).

In a preferred embodiment, the CDR sequences of the invention are shown in table 1;

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

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the region of minimum overlap 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 appreciated by those skilled in the art, the residues in the remainder of the CDR sequences can be determined by the structure and protein folding of the antibody. Thus, the present invention also contemplates variants of any of the CDRs given herein. For example, in a variant of one CDR, the amino acid residue of the smallest binding unit may remain unchanged, while the remaining CDR residues according to Kabat or Chothia definition may be replaced by conserved amino acid residues.

In some embodiments, an antibody of the invention has at least one, two, three, four, five, or six CDRs that are the same as corresponding CDRs of any of the antibodies listed in table 3, or a variant thereof. In some embodiments, an antibody of the invention has at least one, two, or three HCDRs that are the same as, or are variants of, the corresponding heavy chain CDRs of any of the antibodies listed in table 3. In some embodiments, an antibody of the invention has at least one, two, or three LCDRs that are the same as, or are variants of, the corresponding light chain CDRs of any of the antibodies listed in table 3. Herein, "corresponding CDRs" refer to CDRs that, after optimal alignment, are located in the most similar positions in the variable region amino acid sequence of the candidate antibody as the CDRs of the reference antibody. Herein, a CDR variant is a CDR that has been modified by at least one, e.g., 1 or 2 or 3 amino acid substitutions, deletions, and/or insertions, wherein the antigen binding molecule comprising the CDR variant substantially retains the biological properties of the antigen binding molecule comprising the unmodified CDR, e.g., retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., 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, in particular 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 amino acid positions corresponding to residues X of the consensus CDR sequences provided herein (e.g., SEQ ID NOs: 16,17,18,19,20,21,30,31, 32).

Furthermore, it is known in the art that the CDR3 region, independently of the CDR1 and/or CDR2 regions, alone can determine the binding specificity of an antibody for an associated antigen. Also, a variety of other antibodies with the same binding specificity can be generated based on the common CDR3 sequence. See, e.g., US Patents nos.6,951,646; 6,914,128, respectively; 6,090,382; 6,818,216, respectively; 6,156,313, respectively; 6,827,925, respectively; 5,833,943, respectively; 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 region of one of the antibodies shown in table 3, wherein said antibody is capable of specifically binding to human LAG-3. In yet another embodiment, the antibody may further comprise CDR2 from the heavy and/or light chain variable region of the same antibody, or CDR2 from the heavy and/or light chain variable region of a different LAG-3 antibody. In yet another embodiment, the antibody may further comprise CDR1 from the heavy and/or light chain variable region of the same antibody, or CDR1 from the heavy and/or light chain variable region of a different LAG-3 antibody. The activity of these antibodies, including binding activity to human LAG-3, activity to block binding of LAG-3 to mhc class ii molecules, and activity to inhibit tumor growth, can be characterized by the assay methods described herein.

In yet another aspect, given that antigen binding specificity is provided primarily by the CDR1, 2, and 3 regions, in some embodiments, the VH CDR1, 2, and 3 sequences and VL CDR1, 2, and 3 sequences can be "mixed and matched" (i.e., the CDRs from different antibodies that bind the same LAG-3 antigen, although each antibody preferably contains VH CDRs 1, 2, and 3 and VLCDRs 1, 2, and 3) to produce other molecules of the invention that bind LAG-3. Such "mixed and matched" antibodies can be tested for binding to LAG-3 using binding assays known in the art (e.g., 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. Likewise, 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. The "mixing and matching" of CDRs can be performed between antibodies of the present invention as shown in table 3. In addition, it will be apparent to those skilled in the art that other antibodies of the invention can also be produced by replacing structurally similar CDR sequences of the antibodies shown herein with one or more VH and/or VL CDR region sequences from other different antibodies.

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

(i) identical to HCDR3 of the heavy chain variable region of any one of the antibodies listed in table 3; or

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

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

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

(ii) (ii) comprises a total of at least 1 and not more than 3 (preferably 1-2 or more preferably 1) amino acid changes (preferably substitutions, more preferably conservative substitutions) relative to the HCDR3 and LCDR3 of (i). Preferably, the HCDR3 and LCDR3 comprise a combination of amino acid sequences selected from: 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 or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH comprises:

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

(ii) (ii) sequences comprising at least one and no more than 10 or 5, 4,3, 2,1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in total on the three CDR regions relative to the sequence of (i).

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

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

(ii) (ii) sequences comprising at least one and no more than 10 or 5, 4,3, 2,1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in total on the three CDR regions relative to the sequence of (i).

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

(i) 6 CDR sequences identical to the 6 CDR sequences contained in the VH and VL sequences of any one of the antibodies listed in Table 3, respectively; or

(ii) (ii) sequences which collectively comprise at least one and no more than 20, 10 or 5, 4,3, 2,1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) over the 6 CDR regions relative to the sequence of (i).

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

(i) 3 complementarity determining regions HCDR of the heavy chain variable region shown in SEQ ID NO. 33 or 34, and 3 complementarity determining regions LCDR of the light chain variable region shown in SEQ ID NO. 39, or

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

(iii) 3 CDRs of the heavy chain variable region as set forth in SEQ ID NO:37 or 38, and 3 CDRs of the light chain variable region as set forth in SEQ ID NO:42 or 43.

In a preferred embodiment, the antibody or antigen binding fragment thereof of the invention comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO:33 and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences 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 comprising the CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO:34 and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences 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 comprising the CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO:35 and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences 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 comprising the CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO:36 and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences 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 comprising the CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO:37 and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences 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 comprising the CDR1, CDR2, and CDR3 sequences from the heavy chain variable region of SEQ ID NO:38 and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences from the light chain variable region of SEQ ID NO: 43.

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

(i) A heavy chain HCDR combination (HCDR1, HCDR2 and HCDR3 respectively in that order) selected from the following combinations of amino acid sequences:

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, respectively; or

(ii) A heavy chain HCDR combination (HCDR1, HCDR2 and HCDR3 respectively in that order) selected from the following combinations of amino acid sequences:

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, respectively; or

(iii) (iii) a variant of the HCDR combination of (i) or (ii) which comprises in total at least one and no more than 10 or 5, 4,3, 2,1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions.

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

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

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, respectively;

(ii) (ii) a variant of the LCDR combination of (i) which comprises in total at least one and no more than 10 or 5, 4,3, 2,1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions.

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 (HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, respectively, in that order) selected from the following combinations of amino acid sequences:

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, respectively; or

(ii) A combination of heavy and light chain CDRs (HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, respectively, in that order) selected from the following combinations of amino acid sequences:

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, respectively;

(ii) (ii) a variant of the heavy and light chain CDR combinations of (i) which comprises at least one and no more than 10 or 5, 4,3, 2,1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in total over the six CDR regions.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises 3 complementarity determining regions HCDR of a heavy chain variable region, and 3 complementarity determining regions LCDR of a light chain variable region, wherein

(i) HCDR1 comprises the amino acid sequence shown as SEQ ID No. 1 or 4 or 16 or 70 or 72, HCDR2 comprises the amino acid sequence shown as SEQ ID No. 2 or 5 or 17, HCDR3 comprises the amino acid sequence shown as SEQ ID No. 3 or 6 or 18 or 71 or 73, LCDR1 comprises the amino acid sequence shown as SEQ ID No. 22 or 31, LCDR2 comprises the amino acid sequence shown as SEQ ID No. 23, and LCDR3 comprises the amino acid sequence shown as SEQ ID No. 24; or

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Antibody variable regions

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 the light chain variable region (VL) may be further subdivided into hypervariable regions (HVRs, also known as Complementarity Determining Regions (CDRs)) with more conserved regions (i.e., Framework Regions (FRs)) interposed therebetween. Each VH and VL consists of three CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. In some cases, a single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated by screening libraries of complementary VL or VH domains using VH or VL domains from antibodies that bind the antigen (see, e.g., Portolano, S. et al, J.Immunol.150(1993) 880-.

It is known in the art that one or more residues in one or both of the two variable regions (i.e., VH and/or VL) may be modified, e.g., residue modifications, particularly conservative residue substitutions, to one or more CDR regions and/or to one or more framework regions, while the modified antibody still substantially retains at least one biological property (e.g., antigen binding ability) of the antibody molecule prior to the modification. For example, residues from a CDR region may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. The antibody binding properties or other functional properties of the mutated antibody may be assessed in vitro or in vivo assay assays. Preferably, conservative substitutions are introduced. Preferably, the residues introduced in the CDR regions are not changed by more than 1, 2, 3, 4 or 5. Furthermore, framework region residues may be mutated, for example to improve the properties of the antibody. For example, one or more framework residues may be "back-mutated" to the corresponding germline sequence residue.

CDR grafting is another form of antibody variable region modification known in the art. Since the 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 framework regions of different antibodies with different properties. Thus, in one embodiment, the invention relates to an anti-LAG-3 antibody, or antigen-binding fragment thereof, comprising heavy and light chain CDR sequences from one of the antibodies of table 3, but having different framework region sequences. Framework region sequences for substitution can be obtained from public DNA databases, including germline antibody gene sequences, or from published LAG-3 antibody sequences. Germline DNA encoding human heavy and light chain variable region genes can be obtained, for example, from GenBank databases. Antibody protein sequences can be compared to protein sequences in databases using sequence similarity search tools, such as Gapped BLAST. Preferably, the framework sequence used in the substitution has structural similarity to the framework sequence of the antibody of the invention selected for alteration, e.g., a framework sequence having at least 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more sequence identity.

In yet another embodiment, VH and VL sequences from an exemplary antibody of the invention (one of the antibodies shown in table 3) can be "mixed and matched" with other, different anti-LAG-3 antibodies (preferably, another antibody shown in table 3) to produce additional antibodies of the invention that bind LAG-3. In mixing and matching these chains, it is preferred to replace VH sequences from a particular VH/VL pairing with VH sequences that are structurally similar. Likewise, VL sequences from a particular VH/VL pairing 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 (e.g., ELISA, and other assays described in the examples section).

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

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

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

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

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

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

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

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

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

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

In one embodiment, a variant of a VH sequence has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity in amino acid sequence to a reference VH sequence (preferably, over the full length or over the three regions of CDR1, 2 and 3). In one embodiment, a variant of a VH sequence comprises 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) in amino acid sequence compared to a reference VH sequence (preferably, over the full length or over the three regions of CDR1, 2 and 3). Preferably the sequence differences do not occur in the CDR regions.

In a preferred embodiment, a variant of a VL sequence has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity in amino acid sequence to the reference VL sequence (preferably, over the full length or over the three CDR1, 2 and 3 regions). In a preferred embodiment, a variant of a VL sequence comprises 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) 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). Preferably the sequence differences do not occur in the CDR regions.

In a preferred embodiment, the antibody of the invention comprises a heavy chain variable region and light chain variable region 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 consists of said amino acid sequence pairs. The invention also provides variants of the antibody, e.g., variants having at least 95-99% identity in 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 comprises no more than 10, preferably no more than 5 (e.g., 3, 2,1 or 0) amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) over 1 or more CDR (preferably all 3 CDR) regions relative to the reference antibody.

In any of the above embodiments, preferably, the light chain variable region VL of the antibody variant comprises no more than 10, preferably no more than 5 (e.g., 3, 2,1 or 0) amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) over 1 or more CDR (preferably all 3 CDR) regions relative to the reference antibody.

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

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, the antibodies of the invention comprise a heavy chain Fc region, such as an Fc region of the IgG1, IgG2, or IgG4 isotype. In one embodiment, the antibody of the invention comprises an IgG4-Fc region having a serine to proline mutation at amino acid residue 228 (EU numbering) (S228P). In yet another preferred embodiment, the antibody of the invention comprises an IgG4-PAA Fc moiety. The IgG4-PAAFc moiety has a serine to proline mutation at position 228 (S228P), and a phenylalanine to alanine mutation at position 234(EU numbering) and a leucine to alanine mutation at position 235(EU numbering). The S228P mutation is a mutation in the hinge region of the tumor constant region that can reduce or eliminate inter-heavy chain disulfide bridge heterogeneity. 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, e.g., a human kappa light chain constant region.

In yet another preferred embodiment, the Fc region comprises the amino acid sequence of SEQ ID NO. 68, or an amino acid sequence which comprises at least one, two or three, but not more than 20, 10 or 5 amino acid changes relative to the amino acid sequence of SEQ ID NO. 68, or a sequence which has at least 95-99% identity to the amino acid sequence of SEQ ID NO. 68.

In a preferred embodiment, the 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 yet another preferred embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO:69, or an amino acid sequence comprising at least one, two or three, but not more than 20, 10 or 5 amino acid changes relative to the amino acid sequence of SEQ ID NO:69, or a sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO: 68.

In some preferred embodiments, the antibody of the invention comprises a heavy chain, and said heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 44, 45, 46, 47, 48 and 49, or an amino acid sequence comprising at least one, two or three, but NO more than 20, 10 or 5 amino acid changes relative thereto, or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto. Preferably, the amino acid changes do not occur in the CDR regions, more preferably, 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 NOs 63, 64, 65, 66 and 67, or an amino acid sequence comprising at least one, two or three, but NO more than 20, 10 or 5 amino acid changes relative thereto, or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto. Preferably, the amino acid changes do not occur in the CDR regions, more preferably, do not occur in the variable regions.

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

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

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

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

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

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

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

wherein the variant comprises an amino acid sequence that is at least one, two or three, but not more than 20, 10 or 5 amino acid changes, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to a corresponding reference sequence. Preferably, the amino acid changes do not occur in the CDR regions, more preferably not in the variable regions.

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

Exemplary antibody sequences

The present invention provides fully human antibodies that specifically bind LAG-3 (e.g., human LAG-3) isolated and characterized as in the examples. The antibody variable region VH and VL sequences 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. Tables 4 and 5 show the heavy and light chain amino acid sequences of exemplary antibodies of the invention. Tables 6 and 7 show the coding nucleotide sequences of the variable regions VH and VL of exemplary antibodies of the invention.

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, an antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding capacity) of the antibody prior to alteration. In some embodiments, the alteration does not result in the antibody variant losing binding to the antigen, but optionally may confer properties such as increased antigen affinity and different effector functions.

It will be appreciated that the variable heavy or light chain regions, or the respective CDR regions, of the antibody may be altered individually or in combination. In some embodiments, the amino acid change in one or more or all three heavy chain CDRs is no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. Preferably, the amino acid change is an amino acid substitution, preferably a conservative substitution. In some embodiments, the amino acid change in one or more or all three light chain CDRs is no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. In some embodiments, the amino acid change in one or more or all 6 CDRs is no more than 1, 2, 3, 4,5, 6, 7, 8, 9 or 10. Preferably, the amino acid change is an amino acid substitution, preferably a conservative substitution. In some embodiments, an antibody variant has at least 80%, 85%, 90%, or 95%, or 99% or more amino acid identity to a reference antibody over a region of the target antibody sequence. For example, in one embodiment, an antibody of the invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over the 3 heavy chain CDR regions as compared to a reference antibody (e.g., one of the antibodies listed in table 3). In one embodiment, an antibody of the invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over the 3 light chain CDR regions as compared to a reference antibody (e.g., one of the antibodies listed in table 3). In another embodiment, an antibody of the invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over the 6 CDR regions as compared to a reference antibody (e.g., one of the antibodies listed in table 3). 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 more sequence identity in the heavy chain variable region as compared to a reference antibody (e.g., one of the antibodies listed in table 3). 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 more sequence identity in the light chain variable region as compared to a reference antibody (e.g., one of the antibodies listed in table 3). 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 more sequence identity over the heavy chain variable region and/or the light chain variable region as compared to a reference antibody (e.g., one of the antibodies listed in table 3).

In addition, changes may be made to the Fc region of the antibody. Changes to 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 cellular cytotoxicity. In addition, the antibodies of the invention may be chemically modified (e.g., attached to PEG) or have their glycosylation pattern altered.

In certain embodiments, the Fc region may comprise an Fc-region having one or more amino acid substitutions that enhance ADCC activity, e.g., substitutions at positions 298, 333, and/or 334 of the Fc-region (EU numbering of residues). In some embodiments, the Fc-region can also be altered to result in altered (i.e., increased or decreased) C1q binding and/or Complement Dependent Cytotoxicity (CDC) (see, e.g., U.S. Pat. No. 6,194,551, WO99/51642 and Idusogene, E.E., et al, J.Immunol.164(2000) 4178-.

In other embodiments, the Fc may be altered to increase or decrease its degree of glycosylation and/or alter its glycosylation pattern. Addition or deletion of glycosylation sites to the Fc can be conveniently achieved by altering the amino acid sequence so as to create or remove one or more glycosylation sites. For example, one or more amino acid substitutions may be made to eliminate one or more glycosylation sites, thereby eliminating glycosylation at that site. Antibodies with altered types of glycosylation can be prepared, for example, low or afucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of the antibody. The present invention also contemplates antibody variants having at least one galactose residue in an oligosaccharide linked to an Fc region. These antibody variants may have increased CDC function.

In certain embodiments, the invention also contemplates antibody variants having some, but not all, effector functions, which make them ideal candidates for 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 deleterious. For example, the Fc region may comprise a mutation that eliminates or reduces effector function, such as a human IgG1Fc region with mutations P329G and/or L234A and L235A, or a human IgG4Fc region with mutations P329G and/or S228P and L235E.

In certain embodiments, it may be desirable to generate cysteine engineered antibodies, such as "thio mabs," in which one or more residues of the antibody are replaced with a cysteine residue. For example, the number of cysteine residues in the hinge region of an antibody may be altered, e.g., to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. Residues such as U.S. Pat. No. 5,677,425.

In certain embodiments, the antibodies provided herein can be further modified to contain a non-protein 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), for example, to increase the (e.g., serum) half-life of the antibody. Methods for protein pegylation are known in the art and can be applied to the antibodies of the invention. See, for example, EP 0154316 and EP 0401384.

Polynucleotides, vectors and hosts

The invention provides a nucleic acid encoding any of the above anti-LAG-3 antibodies or fragments thereof. Also provided are vectors comprising the nucleic acids. In one embodiment, the vector is an expression vector. Also provided are host cells comprising the nucleic acids or the vectors. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or 293 cell). 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 variable region and/or a heavy chain variable region of an antibody, or a nucleic acid encoding an amino acid sequence of a light chain and/or a heavy chain of an antibody. Exemplary nucleic acid sequences encoding the heavy chain variable region of an antibody comprise a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence selected from SEQ ID NOs 57,58,59,60,61 or 62, or a nucleic acid sequence selected from 57,58,59,60,61 or 62. Exemplary nucleic acid sequences encoding an antibody light chain variable region include nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence selected from SEQ ID NOs 50,51,52,53, or 54, or a nucleic acid sequence selected from SEQ ID NOs 50,51,52,53, or 54. The polypeptides encoded by these polynucleotides are capable of exhibiting LAG-3 antigen binding capacity when expressed from a suitable expression vector.

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

As will be appreciated by those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence may 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, the 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.

These polynucleotide sequences may be generated by de novo solid phase DNA synthesis or by PCR mutagenesis of existing sequences (e.g., the sequences shown in tables 6-7) encoding LAG-3 binding antibodies or binding fragments thereof using methods well known in the art.

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 (YACs). 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 antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199 and 5,840,523, and also Charlton, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, NJ, 2003), page 245-. After expression, the antibody 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 a yeast cell, a mammalian cell, or other cell suitable for use in the production of an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains in which the glycosylation pathway has been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. biotech.22: 1409-: 210-215(2006). Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for growth in suspension may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293HEK or 293 cells, as described, e.g., in Graham et al, J.Gen Virol.36: 59 (1977)), and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA77: 216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki and Wu, Methods in molecular biology, Vol.248 (B.K.C.Lo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003).

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 expression of the antibody, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium). For recombinant production of anti-LAG-3 antibodies, nucleic acids encoding the antibodies (e.g., the antibodies described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Determination of

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, antibodies of the invention are tested for antigen binding activity. For example, the methods can be performed by methods known in the art, such as ELISA, Western blotting, and the like, or by the exemplary methods disclosed in the examples hereinTo determine binding to human LAG-3. For example, the assay can be performed using flow cytometry, wherein the antibody is reacted with a cell line expressing human LAG-3, such as HEK293 cells transfected to express human LAG-3 on the cell surface. Other cells are also suitable for flow cytometry, including activated CD4+ T cells expressing native LAG-3. Alternatively, binding of the antibody, including binding kinetics (e.g., K)_DValue), can be determined in a biophotonic interferometry using recombinant LAG-3 protein. In some embodiments, a biophotonic interferometry (e.g., Fortebio affinity measurement) is used.

In another aspect, a competition assay 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 competitive antibodies bind to an epitope (e.g., 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. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "Epitope Mapping Protocols", Methods in molecular biology vol.66(Humana Press, Totowa, N.J.).

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

It will be appreciated that any of the above assays can be performed using the immunoconjugates or multispecific antibodies of the invention in place of or in addition to the anti-LAG-3 antibody.

Multispecific antibodies

In a further aspect, the present invention provides multispecific (including bispecific) antibody molecules that specifically bind LAG-3 (preferably human LAG-3). In one embodiment, in a multispecific antibody, an antibody (or antigen-binding fragment thereof) of the invention forms a first binding specificity for LAG-3. In yet another embodiment, the multispecific antibody further comprises a second specificity for one of the following, or further comprises a second and third binding specificity for both of the following molecules: PD-1, TIM-3, CEACAM (e.g., 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 an antibody (the region of the antibody molecule that actually binds to an antigen). In a preferred embodiment, the antigen binding site is comprised 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 which may bind to a different epitope of the same antigen or to a different epitope of a different antigen.

For multispecific antibodies and their preparation, reference may be made to, for example, the descriptions in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO2010/145792 and WO 2010/145793.

Immunoconjugates

In yet another aspect, the invention provides immunoconjugates produced by conjugating an antibody of the invention to a heterologous molecule. In one embodiment, in an immunoconjugate, an antibody of the invention (or antigen-binding fragment thereof) is combined 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, the conjugation may be carried out in the form of fragments such as Fab fragments, Fab 'fragments, F (ab)' 2 fragments, single chain scFab antibodies, single chain scFv, etc.

Linkers may be used to covalently link the 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 (Chari et al, Cancer Research 52(1992)127- > 131; U.S. Pat. No. 5,208,020) can be used.

Therapeutic agents suitable for use in the conjugates include, but are not limited to, cytotoxins (e.g., cytostatic or cytocidal agents), drugs, or radioisotopes. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art, see, e.g., WO 05/103081. For example, cytotoxic agents include, but are not limited to: a radioactive isotope; a growth inhibitor; enzymes and fragments thereof such as nucleic acid hydrolases; (ii) an antibiotic; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; 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 testing procedure (e.g., to determine 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 may be achieved by coupling the antibody to a detectable agent, including but not limited to various enzymes, such as but not limited to horseradish peroxidase; prosthetic groups such as, but not limited to, streptavidin/biotin and avidin/biotin; a fluorescent substance; a luminescent substance; a radioactive substance; and positron emitting metal and non-radioactive paramagnetic metal ions for use in various positron emission tomography procedures.

Pharmaceutical compositions and pharmaceutical formulations

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

Pharmaceutical carriers suitable for use in the present invention may 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 may also be employed as liquid carriers, particularly 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 and the like. For the use of excipients and their use, see also "Handbook of pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, pharmaceutical press, London, Chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may 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.

Pharmaceutical formulations comprising the invention may be prepared by mixing an anti-LAG-3 antibody, immunoconjugate or multispecific antibody of the invention of the desired purity with one or more optional Pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer.

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

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

For further components of pharmaceutical formulations, see also those disclosed in WO 2015/153513.

VI. combination product

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

In some embodiments, the combination product is for use in the prevention or treatment of a tumor. In some embodiments, the tumor is a cancer, e.g., a gastrointestinal cancer, e.g., gastric, rectal, colon, colorectal, etc.; or skin cancer, such as malignant melanoma. In some embodiments, the combination product is used to prevent or treat an infection, such as a bacterial infection, a viral infection, a fungal infection, a protozoan infection, and the like.

Methods of treatment and uses

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). In particular, the subject is a human.

As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual undergoing treatment. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

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

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

In some embodiments, a tumor, e.g., a cancer, described herein includes, but is not limited to, a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, myeloma), and metastatic lesions thereof. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignancies, e.g., sarcomas and carcinomas (e.g., adenocarcinomas) of multiple organ systems, such as those that affect the lung, breast, lymph, gastrointestinal or colorectal tract, genitalia and genitourinary tract (e.g., kidney 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 pancreas. For example, melanoma, colon cancer, gastric cancer, rectal cancer, renal cell carcinoma, breast cancer (e.g., breast cancer that does not express one, two, or all of the estrogen receptors, progesterone receptors, or Her2/neu, e.g., 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 (e.g., HPV + squamous cell carcinoma), small intestine cancer, and esophageal cancer. Examples of hematological cancers include, but are not limited to, leukemias (e.g., myeloid leukemia, lymphoid leukemia, or Chronic Lymphocytic Leukemia (CLL)), lymphomas (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 myelomas, e.g., multiple myeloma. The cancer may be in an early, intermediate or advanced stage or a metastatic cancer.

In one embodiment, the cancer is a cancer of the gastrointestinal tract, 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, e.g., a chronic infection, in a subject, the method comprising administering to the subject an effective amount of any of the anti-LAG-3 antibodies or fragments thereof described herein, or an immunoconjugate, multispecific antibody, or pharmaceutical composition comprising the antibody or fragment. In one embodiment, the infection is a viral infection.

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

Diseases suitable for prevention or treatment with the anti-LAG-3 antibodies or fragments thereof, immunoconjugates and multispecific antibodies of the present invention can be further seen in WO2015/138920, WO2016/028672, WO2015/042246 and the like.

In some embodiments, the methods described herein further comprise co-administering to the subject one or more therapies (e.g., treatment modalities and/or other therapeutic agents). 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, a method of the invention further comprises administering at least one other immunostimulatory antibody, e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, and/or an anti-CTLA-1 antibody, which antibodies can be, e.g., fully human, chimeric, or humanized antibodies.

In other embodiments, the additional 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., bevacizumab).

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 SLEB 2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD 273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding its binding partner. In a particular 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 its binding partner. In a particular aspect, the PD-L2 binding partner is PD-1. The antagonist may be an antibody, antigen binding fragment thereof, immunoadhesin, fusion protein or oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., 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), Merck3475(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 registry number 946414-94-4). In a preferred embodiment, the anti-PD-1 Antibody is "Antibody D" as described herein.

In further embodiments, the 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, e.g., treatment modalities and/or other therapeutic agents. In some embodiments, the treatment modality includes surgery (e.g., tumor resection); radiation therapy (e.g., external particle beam therapy, which involves three-dimensional conformal radiation therapy in which an irradiation region is designed), localized irradiation (e.g., irradiation directed at a preselected target or organ), focused irradiation, or the like.

In some embodiments, the anti-LAG-3 antibodies or fragments thereof of the present invention may be administered in combination with a chemotherapeutic or chemotherapeutic agent. In some embodiments, the anti-LAG-3 antibody or fragment thereof of the present invention may be administered in combination with a radiation or radiotherapy agent. In some embodiments, the anti-LAG-3 antibodies or fragments thereof of the present invention may be administered in combination with a targeted therapy or targeted therapeutic. In some embodiments, the anti-LAG-3 antibodies or fragments thereof of the present invention can be administered in conjunction with an immunotherapy or immunotherapeutic agent, e.g., a monoclonal antibody.

The antibodies of the invention (and pharmaceutical compositions or immunoconjugates comprising the same, and any additional therapeutic agent) can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal administration, and, if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, for example intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (either 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 course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

In the above-described methods of the invention, the compositions, multispecific antibodies, or immunoconjugates of the invention can be administered in place of the antibodies or antigen-binding portions of the invention. Alternatively, in these methods, in addition to administering an antibody or antigen-binding portion of the invention, a composition, multispecific antibody, or immunoconjugate of the invention may be further administered.

In a further aspect, the invention provides the use of an anti-LAG-3 antibody, composition, immunoconjugate, multispecific antibody, of the invention in the manufacture of a medicament for use in the aforementioned methods (e.g. for treatment).

IX. methods and compositions for diagnosis and detection

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

The term "detection" as used herein includes quantitative or qualitative detection, exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises a cell 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, the anti-LAG-3 antibody is used to select a subject suitable for treatment with the anti-LAG-3 antibody, e.g., where LAG-3 is a biomarker for selecting the subject. In one embodiment, an antibody of the invention can be used to diagnose cancer or tumor, e.g., to assess (e.g., monitor) the treatment or progression of, diagnosis and/or staging of a disease (e.g., hyperproliferative or cancerous disease) described herein in a subject.

In certain embodiments, labeled anti-LAG-3 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, by 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 luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl (dansyl), umbelliferone (umbelliferone), luciferase (luceriferase), e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lytic enzymes, carbohydrate oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes utilizing a hydrogen peroxide oxidation dye precursor such as HR, lactoperoxidase, or microperoxidase, biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like.

The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed as, limiting the scope of the invention in any way.

The following examples of the invention relate to 6 exemplary antibodies (ADI-26818, ADI-26822, ADI-26836, ADI-31798, ADI-31815 and ADI-31836) whose CDR regions, light and heavy chain variable regions, light and heavy chain amino acid sequences, and corresponding nucleotide sequences are set forth in tables 1-8 of the present application. In addition, the light chain constant region, heavy chain constant region, light chain variable region and heavy chain variable region of the above-described exemplary antibodies of the invention are numbered as shown in table 9.

Examples