HK40076079A

HK40076079A - Antibodies against human lag-3 and uses thereof

Info

Publication number: HK40076079A
Application number: HK42022065387.7A
Authority: HK
Inventors: 郑勇; 吴琼; 李竞
Original assignee: 广州誉衡生物科技有限公司
Priority date: 2018-02-28
Filing date: 2022-12-08
Publication date: 2023-02-10

Description

Anti-human LAG-3 antibodies and uses thereof

The application is a divisional application of Chinese patent application No.201910146172.2 with the application date of 2019, 2, and 27, and the invention name of the monoclonal antibody against human LAG-3 and the preparation method and the application thereof.

Sequence listing

The present application contains a sequence listing and is incorporated herein by reference in its entirety.

Technical Field

The present application relates generally to antibodies. More specifically, the present application relates to fully human monoclonal antibodies that bind human LAG-3, methods of making the same, and uses thereof.

Background

Lymphocyte activation gene 3(CD223), also known as LAG-3, is a type I transmembrane protein that is a member of the immunoglobulin superfamily (IgSF).

LAG-3 is a cell surface molecule expressed on activated T cells, NK cells, B cells, and plasmacytoid dendritic cells, but which is not expressed on resting T cells. LAG-3 has about 20% amino acid sequence homology with CD4, but binds with higher affinity to MHC class II molecules and to the major functional ligand, fibrin-like protein 1(FGL1) class molecules of LAG-3, independent of MHC-II, thereby providing down-regulation of T cell receptor signaling.

In vitro blockade of LAG-3 enhances T cell proliferation and cytokine production, and LAG-3 deficient mice are deficient in down-regulation of T cell responses induced by superantigen staphylococcal enterotoxin B, peptide or sendai virus infection. LAG-3 on activated natural Treg (nTreg) and induced CD4 ⁺ FoxP3 ⁺ Treg (iTreg) cells, wherein the expression level is higher than on activated effector CD4 ⁺ Expression levels observed on T cells. Blockade of LAG-3 on Treg cells abrogates the suppressor function of Treg cells, not Treg CD4 ⁺ Ectopic expression of LAG-3 in T cells confers inhibitory activity. Based on the immunomodulatory effect of LAG-3 on T cell function in chronic infections and cancer, the predicted mechanism of action of LAG-3 specific monoclonal antibodies is to inhibit the down-regulation of tumor-specific effector T cells.

Currently, only three potential antagonist antibodies in early clinical development modulate LAG-3 function and anti-tumor immune response to treat advanced solid tumors. These antibodies are described in patents US 20110150892 a1, US 20170101472a1 and WO2015138920a1, hereinafter BMK1, BMK 7and BMK5, respectively. As described herein, BMK8 is a humanized version of the chimeric antibody BMK 5. BMK1, BMK 7and BMK8 were used as reference antibodies in the context of the present application. Thus, there remains a need for anti-human LAG-3 antibodies with improved efficacy (e.g., high binding affinity, low cross-family response, and good stability). In this application, the inventors generated a series of antibodies to LAG-3 and fully human antibodies using humanized rats. The antibodies of the present application have high binding affinity, specifically bind to human LAG-3 protein without a cross-family reaction, and are effective in modulating immune responses.

Summary of The Invention

The present invention relates broadly to novel compounds, methods, compositions and articles of manufacture that provide antibodies with improved efficacy. The benefits provided by the present invention are broadly applicable to the field of antibody therapy and diagnosis, and can be used in conjunction with antibodies capable of reacting with a variety of targets. The present invention provides antibodies, preferably fully human monoclonal antibodies, that bind to human LAG-3. Also provided are methods of producing hybridomas using humanized rats, nucleic acid molecules encoding anti-LAG-3 antibodies, expression vectors and host cells for expressing anti-LAG-3 antibodies. The invention further provides methods for verifying antibody function in vitro. The antibodies of the invention provide effective agents for the treatment of various diseases by modulating human immune function.

In some aspects, the invention includes an isolated antibody, or antigen-binding portion thereof.

In some embodiments, the isolated antibody, or antigen-binding portion thereof, has one or more of the following properties:

(a) at 2X 10 ^-10 K of M or less _D Binding human LAG-3;

(b) inhibiting binding of LAG-3 to a Major Histocompatibility (MHC) class II molecule;

(c) inhibiting the binding of LAG-3 to a fibrin-like protein 1(FGL1) ligand molecule;

(d) inhibiting the binding of LAG-3 to LSECtin and/or galectin-3;

(e) bind human LAG-3 without a cross-family response; or

(f) Has no cross-reactivity with human CD 4.

In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

A) one or more heavy chain cdrs (cdrhs) selected from at least one of: (i) and a sequence selected from SEQ ID NO:1 and 7, wherein the CDRH1 has at least 90% sequence identity to the CDRH 1; (ii) and a sequence selected from SEQ ID NO: 2 and 8 has a CDRH2 of at least 90% sequence identity to the CDRH2 shown in one of the sequences of 2 and 8; and (iii) a peptide selected from the group consisting of SEQ ID NO: 3 and 9 has a CDRH3 of at least 90% sequence identity to the CDRH3 shown in one of the sequences;

B) one or more light chain cdrs (cdrls) selected from at least one of: (i) and a sequence selected from SEQ ID NO: 4 and 10, a CDRL1 having at least 90% sequence identity to a CDRL 1; (ii) and a sequence selected from SEQ ID NO: 5 and 11, wherein the CDRL2 has at least 90% sequence identity to the CDRL 2; and (iii) a peptide selected from the group consisting of SEQ ID NO: 6 and 12, a CDRL3 having at least 90% sequence identity to a CDRL 3; or

C) One or more CDRH of a) and one or more CDRL of B).

A) one or more (e.g. 1, 2 or 3) heavy chain cdrs (cdrhs) selected from at least one of: (i) selected from the group consisting of SEQ ID NO:1 and 7 CDRH1 or CDRH1 with no more than 2 amino acid additions, deletions or substitutions difference from the amino acid sequence of CDRH 1; (ii) selected from the group consisting of SEQ ID NO: 2 and 8 or a CDRH2 that differs from the amino acid sequence of the CDRH2 by the addition, deletion or substitution of an amino acid of not more than 2 amino acids; and (iii) a sequence selected from SEQ ID NO: 3 and 9 or CDRH3 that differs from the amino acid sequence of CDRH3 by the addition, deletion or substitution of an amino acid of not more than 2 amino acids;

B) one or more (e.g., 1, 2 or 3) light chain cdrs (cdrls) selected from at least one of: (i) selected from the group consisting of SEQ ID NO: 4 and 10 CDRL1 or CDRL1 with amino acid addition, deletion or substitution differences of no more than 2 amino acids from the amino acid sequence of CDRL 1; (ii) selected from the group consisting of SEQ ID NO: 5 and 11 CDRL2 or CDRL2 with amino acid addition, deletion or substitution differences of no more than 2 amino acids from the amino acid sequence of CDRL 2; and (iii) a sequence selected from SEQ ID NO: 6 and 12 CDRL3 or CDRL3 with amino acid addition, deletion or substitution differences of no more than 2 amino acids from the amino acid sequence of CDRL 3; or

C) One or more CDRH of a) and one or more CDRL of B).

A) comprises SEQ ID NO: 3 or 9 CDRH 3; or

B) And a sequence selected from SEQ ID NO: 3 and 9, wherein the CDRH3 has a CDRH3 of at least 90% sequence identity; or

C) CDRH3 which differs from the amino acid sequence of CDRH3 of A) by no more than 2 amino acid additions, deletions or substitutions,

and wherein the isolated antibody or antigen-binding portion thereof is at 2X 10 ^-10 K of M or less _D Binding human LAG-3.

(a) comprises the amino acid sequence of SEQ ID NO:1 or CDRH1 consisting thereof;

(b) comprises the amino acid sequence of SEQ ID NO: 2 or CDRH2 consisting thereof;

(c) comprises the amino acid sequence of SEQ ID NO: 3 or a CDRH3 consisting thereof;

(d) comprises the amino acid sequence of SEQ ID NO: 4 or a CDRL1 consisting thereof;

(e) comprises the amino acid sequence of SEQ ID NO: 5 or a CDRL2 consisting thereof; and

(f) comprises SEQ ID NO: 6 or a CDRL3 consisting thereof.

(a) comprises the amino acid sequence of SEQ ID NO: 7 or a CDRH1 consisting thereof;

(b) comprises the amino acid sequence of SEQ ID NO: 8 or a CDRH2 consisting thereof;

(c) comprises the amino acid sequence of SEQ ID NO: 9 or a CDRH3 consisting thereof;

(d) comprises the amino acid sequence of SEQ ID NO: 10 or a CDRL1 consisting thereof;

(e) comprises the amino acid sequence of SEQ ID NO: 11 or a CDRL2 consisting thereof; and

(f) comprises the amino acid sequence of SEQ ID NO: 12 or a CDRL3 consisting thereof.

(A) heavy chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 13;

(ii) comprising a nucleotide sequence substantially identical to SEQ ID NO: 13, an amino acid sequence having at least 85%, at least 90%, or at least 95% identity; or

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 13 with one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3,4, or 5) amino acid additions, deletions, and/or substitutions; and/or

(B) Light chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 14;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 14 an amino acid sequence having at least 85%, at least 90%, or at least 95% identity; or

(iii) Comprising a nucleotide sequence substantially identical to SEQ ID NO: 14 with one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3,4, or 5) amino acid additions, deletions, and/or substitutions.

(A) heavy chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 15;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 15 an amino acid sequence having at least 85%, at least 90%, or at least 95% identity; or

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 15 with one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3,4, or 5) amino acid additions, deletions, and/or substitutions; and/or

(B) Light chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 16;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 16 has at least 85%, at least 90%, or at least 95% identity; or

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 16, or a sequence having one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3,4, or 5) amino acid additions, deletions, and/or substitutions.

In some aspects, the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region of an isolated antibody as disclosed herein.

In some aspects, the invention relates to expression vectors comprising a nucleic acid molecule encoding an antibody or antigen-binding portion thereof as disclosed herein.

In some aspects, the invention relates to a host cell comprising an expression vector as disclosed herein.

In some aspects, the invention relates to a pharmaceutical composition comprising at least one antibody, or antigen-binding portion thereof, as disclosed herein and a pharmaceutically acceptable carrier.

In some aspects, the invention relates to a method for making an anti-LAG-3 antibody, or antigen-binding portion thereof, comprising expressing the antibody, or antigen-binding portion thereof, in a host cell and isolating the antibody, or antigen-binding portion, from the host cell.

In some aspects, the invention relates to a method of modulating an antigen-specific T cell response, comprising administering to a subject an antibody or antigen-binding portion thereof as disclosed herein, such that the antigen-specific T cell response in the subject is modulated.

In some aspects, the invention relates to a method of modulating an immune response in a subject comprising administering to the subject an antibody, or antigen-binding portion thereof, as disclosed herein, such that the immune response in the subject is modulated.

In some aspects, the invention relates to a method for inhibiting or blocking binding of LAG-3 to a MHC class II or FGL1 molecule, comprising contacting the MHC class II or FGL1 molecule with an antibody or antigen-binding portion thereof as disclosed herein.

In some aspects, the invention relates to a method for inhibiting or blocking the binding of LAG-3 to LSECtin and/or galectin-3, comprising contacting the LSECtin and/or galectin-3 with an antibody or antigen binding portion thereof as disclosed herein.

In some aspects, the invention relates to a method for inhibiting tumor cell growth in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof as disclosed herein, such that growth of a tumor in the subject is inhibited.

In some aspects, the invention relates to a method for treating a viral infection in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof as disclosed herein, such that the viral infection is treated in the subject.

In some aspects, the invention relates to a method for treating or preventing a proliferative disorder, e.g., cancer, in a subject comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof as disclosed herein.

In some aspects, the invention relates to the use of an antibody, or antigen-binding portion thereof, as disclosed herein, in the manufacture of a medicament for the treatment or prevention of a proliferative disorder, such as cancer.

In some aspects, the invention relates to the use of an antibody, or antigen-binding portion thereof, as disclosed herein, in the preparation of a diagnostic agent for the diagnosis of a proliferative disorder, such as cancer.

In some aspects, the invention relates to an antibody or antigen binding portion thereof as disclosed herein for use in treating or preventing a proliferative disorder, such as cancer.

In some aspects, the invention relates to kits or devices and related methods using antibodies or antigen-binding portions thereof as disclosed herein, as well as pharmaceutical compositions as disclosed herein, which are useful for treating proliferative disorders, such as cancer. To this end, the invention preferably provides an article of manufacture useful for treating such disorders, comprising a container containing an antibody, or antigen-binding portion thereof, as disclosed herein and instructional material for using the antibody, or antigen-binding portion thereof, as disclosed herein to treat, ameliorate or prevent a proliferative disease, or the progression or recurrence thereof. In selected embodiments, the devices and related methods will comprise the step of contacting at least one circulating tumor cell with an antibody or antigen-binding portion thereof as disclosed herein.

In particular, the present invention relates to the following embodiments:

1. an isolated antibody or antigen-binding portion thereof, wherein the isolated antibody or antigen-binding portion thereof comprises:

A) one or more heavy chain cdrs (cdrhs) selected from at least one of:

(i) and a sequence selected from SEQ ID NO:1 and 7, wherein the CDRH1 has at least 90% sequence identity to the CDRH 1;

(ii) and a sequence selected from SEQ ID NO: 2 and 8 has a CDRH2 of at least 90% sequence identity to the CDRH2 shown in one of the sequences of 2 and 8; and

(iii) and a sequence selected from SEQ ID NO: 3 and 9 has a CDRH3 of at least 90% sequence identity to the CDRH3 shown in one of the sequences;

B) one or more light chain cdrs (cdrls) selected from at least one of:

(i) and a sequence selected from SEQ ID NO: 4 and 10, wherein the CDRL1 has a CDRL1 of at least 90% sequence identity;

(ii) and a sequence selected from SEQ ID NO: 5 and 11, wherein the CDRL2 has at least 90% sequence identity to the CDRL 2; and

(iii) and a sequence selected from SEQ ID NO: 6 and 12, a CDRL3 having at least 90% sequence identity to a CDRL 3; or

C) One or more CDRH of a) and one or more CDRL of B).

2. The isolated antibody or antigen-binding portion thereof of embodiment 1, wherein the isolated antibody or antigen-binding portion thereof comprises:

A) one or more heavy chain cdrs (cdrhs) selected from at least one of:

(i) selected from the group consisting of SEQ ID NO:1 and 7 CDRH1 or CDRH1 with no more than 2 amino acid additions, deletions or substitutions difference from the amino acid sequence of CDRH 1;

(ii) selected from the group consisting of SEQ ID NO: 2 and 8 or a CDRH2 that differs from the amino acid sequence of the CDRH2 by the addition, deletion or substitution of an amino acid of not more than 2 amino acids; and

(iii) selected from the group consisting of SEQ ID NO: 3 and 9 or CDRH3 that differs from the amino acid sequence of CDRH3 by the addition, deletion or substitution of an amino acid of not more than 2 amino acids;

B) one or more light chain cdrs (cdrls) selected from at least one of:

(i) selected from the group consisting of SEQ ID NO: 4 and 10 CDRL1 or CDRL1 with amino acid addition, deletion or substitution differences of no more than 2 amino acids from the amino acid sequence of CDRL 1;

(ii) selected from the group consisting of SEQ ID NO: 5 and 11 CDRL2 or CDRL2 with amino acid addition, deletion or substitution differences of no more than 2 amino acids from the amino acid sequence of CDRL 2; and

(iii) selected from the group consisting of SEQ ID NO: 6 and 12 CDRL3 or CDRL3 with amino acid addition, deletion or substitution differences of no more than 2 amino acids from the amino acid sequence of CDRL 3; or

C) One or more CDRH of a) and one or more CDRL of B).

3. The isolated antibody or antigen-binding portion thereof of embodiment 1, wherein the isolated antibody or antigen-binding portion thereof comprises:

(f) comprises the amino acid sequence of SEQ ID NO: 6 or a CDRL3 consisting thereof.

4. The isolated antibody or antigen-binding portion thereof of embodiment 1, wherein the isolated antibody or antigen-binding portion thereof comprises:

5. The isolated antibody or antigen-binding portion thereof of embodiment 1, wherein the isolated antibody or antigen-binding portion thereof comprises:

(A) heavy chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 13;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 13, an amino acid sequence having at least 85%, 90%, or 95% identity; or

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 13 with one or more amino acid additions, deletions and/or substitutions; and/or

(B) Light chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 14;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 14 an amino acid sequence having at least 85%, 90%, or 95% identity; or

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 14 with one or more amino acid additions, deletions and/or substitutions.

6. The isolated antibody or antigen-binding portion thereof of embodiment 1, wherein the isolated antibody or antigen-binding portion thereof comprises:

(A) heavy chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 15;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 15 an amino acid sequence having at least 85%, 90% or 95% identity; or

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 15 compared to an amino acid sequence having one or more amino acid additions, deletions and/or substitutions; and/or

(B) Light chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 16;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 16 has at least 85%, 90% or 95% identity; or

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 16 compared to an amino acid sequence having one or more amino acid additions, deletions and/or substitutions.

7. The isolated antibody, or antigen-binding portion thereof, of any one of embodiments 1-6, having one or more of the following properties:

(a) at 2X 10 ^-10 K of M or less _D Binding human LAG-3;

(b) inhibiting the binding of LAG-3 to a Major Histocompatibility (MHC) class II molecule;

(d) inhibiting the binding of LAG-3 to LSECtin and/or galectin-3;

(e) bind human LAG-3 without a cross-family response; or

(f) Has no cross-reactivity with human CD 4.

8. The isolated antibody or antigen-binding portion thereof of any of embodiments 1-7, wherein the antibody is a monoclonal antibody, e.g., a fully human monoclonal antibody produced by a transgenic mammal, preferably a transgenic rat, more preferably a transgenic rat having a recombinant immunoglobulin locus.

9. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region of an isolated antibody as defined in any one of embodiments 1 to 8, e.g., the nucleic acid sequences set forth in SEQ ID NOs: 17-20.

10. An expression vector comprising the nucleic acid molecule of embodiment 9.

11. A host cell comprising the expression vector of embodiment 10.

12. A pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as defined in any one of embodiments 1-8 and a pharmaceutically acceptable carrier.

13. A method of making an antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8, comprising the steps of:

-expressing an antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 in a host cell of embodiment 11; and

-isolating the antibody or antigen-binding portion thereof from the host cell.

14. A method of modulating an antigen-specific T cell response or modulating an immune response in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof as defined in any one of embodiments 1-8, such that the antigen-specific T cell response or immune response in the subject is modulated.

15. A method of inhibiting or blocking the binding of LAG-3 to MHC class II molecules, FGL1 molecules, LSECtin and/or galectin-3, comprising contacting the MHC class II molecules, FGL1 molecules, LSECtin and/or galectin-3 with an antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8.

16. A method of inhibiting growth of a tumor cell in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof as defined in any one of embodiments 1-8, such that growth of the tumor in the subject is inhibited.

17. A method of treating or preventing a proliferative disorder, such as cancer, in a subject, comprising administering to the subject an effective amount of an antibody, or antigen-binding portion thereof, as defined in any one of embodiments 1-8.

18. Use of an antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 in the manufacture of a medicament for the treatment or prevention of a proliferative disorder, such as cancer, an autoimmune disease, an infectious disease and/or an inflammatory disease.

19. Use of an antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 in the manufacture of a diagnostic agent for the diagnosis of a proliferative disorder, such as cancer.

20. An antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 for use in the treatment or prevention of a proliferative disorder, such as cancer.

21. An antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 for use in the diagnosis of a proliferative disorder, such as cancer.

22. A kit for the treatment or diagnosis of a proliferative disorder, such as cancer, comprising a container containing at least one antibody, or antigen-binding portion thereof, as defined in any one of embodiments 1 to 8.

The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings presented herein. This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. In addition, the contents of all references, patents, and published patent applications cited throughout this application are incorporated by reference herein in their entirety.

Brief Description of Drawings

FIG. 1 shows the binding of LAG-3 antibody to human LAG-3 on the cell surface, as indicated by MFI (mean fluorescence intensity) and as measured by BD FACSCAnto II.

FIG. 2 shows the blocking of the binding of LAG-3 protein to MHC-II expressed on Raji cells.

FIG. 3 shows the blocking of the binding of LAG-3 protein to LSECtin.

FIG. 4 shows the blocking of the binding of LAG-3 protein to galectin-3.

Figure 5 shows cross-reactivity with cynomolgus monkey LAG-3 as measured by FACS.

FIG. 6 shows cross-reactivity with murine LAG-3 as measured by FACS.

Fig. 7 shows cross-reactivity with human CD4 as measured by ELISA.

Figures 8A-E show epitope binning (epitopbinding) against the benchmark antibodies BMK1, BMK7, and BMK 5.

FIGS. 9A-B show the results of epitope mapping.

FIG. 10 shows the effect of human LAG-3 antibody in a reporter gene assay.

FIG. 11 shows the effect of human LAG-3 antibody on human allogeneic mixed lymphocyte responses, as measured by ELISA and reflected by IFN- γ levels (ng/mL).

FIG. 12 shows the effect of human LAG-3 antibody on human allogenic mixed lymphocyte responses, e.g., by ³ H-thymidine incorporation was measured and proliferation was expressed by CPM (counts per minute) in triplicate wellsThe reaction is reflected.

Figures 13A-B show the results of CDC and ADCC tests performed by determining target cell lysis.

Fig. 14A-B show the results of serum stability tests, as measured by FACS and expressed by the MFI of the cells.

Detailed Description

While this invention is susceptible of embodiment in many different forms, there is disclosed herein specific illustrative embodiments thereof which are indicative of the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes mixtures of cells and the like. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms (such as "includes" and "including") is not limiting. Moreover, the ranges provided in the specification and the appended claims include all values between the endpoints and breakpoints.

Generally, the terminology associated with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art, and are subject to various general and more specific references that are cited and discussed throughout the present specificationThe procedure is described in the literature. See, e.g., Abbas et al, Cellular and Molecular Immunology,6 ^th ed.,W.B.Saunders Company(2010)；Sambrook J.&Molecular Cloning, A Laboratory Manual,3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); ausubel et al, Short Protocols in Molecular Biology A Complex of Methods from Current Protocols in Molecular Biology, Wiley, John&Sons, inc. (2002); harlow and Lane use Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al, Short Protocols in Protein Science, Wiley, John&Sons, inc. (2003). The nomenclature associated with the analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmacochemistry and the laboratory procedures and techniques described herein are those well known and commonly used in the art. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definition of

For a better understanding of the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, the term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be divided into kappa and lambda light chains. Heavy chains can be divided into μ, δ, γ, α and ε, which define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (referred to as Complementarity Determining Regions (CDRs)) separated by relatively conserved regions (referred to as Framework Regions (FRs)). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antigen binding sites, respectively. The distribution of amino acids in various regions or domains follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)) or Chothia & Lesk (1987) J.mol.biol.196: 901-917; chothia et al, (1989) Nature 342: 878-883. The antibodies may be of different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

The term "antigen-binding portion" or "antigen-binding fragment" of an antibody, which may be used interchangeably in the context of this application, refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to an antigen to which the full-length antibody specifically binds, and/or that competes for binding to the same antigen as the full-length antibody. See, generally, Fundamental Immunology, Ch.7(Paul, W., ed., second edition, Raven Press, N.Y. (1989), which is incorporated herein by reference for all purposes ₂ Fd, Fv, dAb and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capability on the polypeptide. Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., a monoclonal anti-human LAG-3 antibody provided herein) by conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as intact antibodies.

As used herein, the term "monoclonal antibody" or "mAb" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.

As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

As used herein, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity, having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.

The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.

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

As used herein, the term "LAG-3" refers to lymphocyte activation gene-3. The term "LAG-3" includes variants, isoforms, homologs, orthologs, and paralogs.

As used herein, the term "human LAG-3" refers to the complete amino acid sequence of human sequence LAG-3, e.g., human LAG-3 having Genbank accession number NP _ 002277. The human LAG-3 sequence may differ from human LAG-3 of Genbank accession No. NP _002277, e.g., having conservative mutations in non-conserved regions, and LAG-3 has substantially the same biological function as human LAG-3 of Genbank accession No. NP _ 002277. For example, the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3 specifically bound by an antibody of the present disclosure, or the biological function of human LAG-3 is to bind to an MHC class II or FGL 1-like molecule.

As used herein, the term "mouse LAG-3" refers to the complete amino acid sequence of the mouse sequence LAG-3, e.g., mouse LAG-3 having Genbank accession No. NP _ 032505.

As used herein, the term "cynomolgus monkey LAG-3" refers to the complete amino acid sequence of cynomolgus monkey LAG-3, e.g., cynomolgus monkey LAG-3 having Genbank accession number XP — 005570011.1.

As used herein, the term "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using well established methods in the art. As used herein, the term "K _D "is intended to mean the dissociation constant for a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and expressed as molar concentration (M). A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such asProvided is a system.

The term "high affinity" for an IgG antibody as used herein means having 1 × 10 affinity for the target antigen ^-7 M or less, more preferably 5X 10 ^-8 M or less, even more preferably 1X10 ^-8 M or less, even more preferably 5X 10 ^-9 M or less, and even more preferably 1X10 ^-9 K of M or less _D The antibody of (1).

The term "EC" as used herein ₅₀ ", also referred to as" half effective concentration ", refers to the concentration of drug, antibody or toxin agent that induces a response of 50% between the baseline and maximum values after a particular exposure time. In the context of the present application, EC ₅₀ In units of "nM".

The ability to "inhibit binding" or "compete for the same epitope" in this application refers to an antibody or antigen-binding fragment thereof that inhibits the binding of two molecules (e.g., human LAG-3 and human anti-LAG-3 antibody) to any detectable level. In certain embodiments, the binding of two molecules may be inhibited by at least 50% by an antibody or antigen-binding fragment thereof. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

As used herein, the term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to as an "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains and usually have a specific three-dimensional structure and specific charge characteristics. For example, an epitope typically comprises at least 3,4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique stereo-conformation, which can be a "linear" or "conformational" epitope. See, e.g., epitopic Mapping Protocols in Methods in Molecular Biology, Vol.66, G.E.Morris, Ed. (1996). In a linear epitope, all the interaction sites between a protein and an interacting molecule (e.g., an antibody) are linearly present along the primary amino acid sequence of the protein. In conformational epitopes, the interaction sites span amino acid residues that are separated from each other in the protein. Antibodies can be screened depending on the competition for binding to the same epitope as detected by conventional techniques known to those skilled in the art. For example, competition or cross-competition studies can be performed to obtain antibodies that compete or cross-compete with each other for binding to an antigen (e.g., an RSV fusion protein). In international patent application WO 03/48731, a high throughput method for obtaining antibodies binding to the same epitope is described, which is based on their cross-competition.

As used herein, the term "isolated" refers to a state that is obtained from a natural state by artificial means. An "isolated" substance or component may be one that, if it occurs in nature, is naturally occurring, is separated from the nature, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and the same high purity polynucleotide or polypeptide isolated from that natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances nor other impurities which do not affect the activity of the isolated substance.

As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds LAG-3 protein is substantially free of antibodies that specifically bind antigens other than LAG-3 protein). However, isolated antibodies that specifically bind human LAG-3 protein may be cross-reactive to other antigens, such as LAG-3 protein from other species. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector may be used to express the carried genetic material element in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda bacteriophage or M13 bacteriophage and animal viruses. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papovaviruses (e.g., SV 40). The vector may contain a number of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may comprise an origin of replication.

As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as e.coli (e.coli) or Bacillus subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus (Aspergillus), insect cells such as S2 drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

The term "identity," as used herein, refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by aligning and comparing the sequences. "percent identity" refers to the percentage of residues in a comparison molecule that are identical between amino acids or nucleotides, and is calculated based on the size of the smallest molecule being compared. For these calculations, the gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in comparative Molecular Biology, (Lesk, A.M., ed.),1988, New York: Oxford University Press; biocomputing information and Genome Projects, (Smith, D.W., ed.),1993, New York: Academic Press; computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.),1994, New Jersey: Humana Press; von Heinje, G.,1987, Sequence Analysis in Molecular Biology, New York: Academic Press; sequence Analysis Primer, (Gribskov, m.and deveux, j., eds.),1991, New York, m.stockton Press; and those described in Carillo et al, 1988, SIAMJ. applied Math.48: 1073.

As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or primed lymphocytes in an organism. It refers not only to the property of an antigen to stimulate the activation, proliferation and differentiation of specific immune cells to eventually produce immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be developed in the immune system of an organism after stimulating the organism with the antigen. Immunogenicity is the most important property of an antigen. Whether an antigen is able to successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the immunological means.

As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are known in the art and are disclosed herein. See, e.g., Graham et al, 1973, Virology 52: 456; sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, supra; davis et al, 1986, Basic Methods in Molecular Biology, Elsevier; chu et al,1981, Gene 13: 197. In a specific embodiment of the invention, the human LAG-3 gene is transfected into 293F cells.

As used herein, the term "hybridoma" and the term "hybridoma cell line" are used interchangeably. When referring to the term "hybridoma" and the term "hybridoma cell line," they also include subclones and progeny cells of the hybridoma.

As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes optical phenomena that allow analysis of real-time biospecific interactions by detecting changes in protein concentration within a Biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For a detailed description, see the examples andU.S. et al (1993) Ann.biol.Clin.51: 19-26;U.S. et al (1991) Biotechniques 11: 620-627; johnsson, B.et al (1995) J.mol.Recognit.8: 125-131; and Johnnson, B., et al (1991) anal. biochem.198: 268-.

As used herein, the term "fluorescence activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method of Sorting a heterogeneous mixture of biological cells into two or more containers, one Cell at a time, based on the specific light scattering and Fluorescence characteristics of each Cell (flowmetric. Instruments for performing FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACScat instruments from Becton Dickinson (Foster City, CA), Epics C from Coulter Epics Division (Hialeah, FL) and MoFlo from Cytomation (Colorado Springs, Colorado).

As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted Ig that binds to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely required for such killing. The major cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. FcR expression on hematopoietic cells is summarized in Table 3 at page 464 of ravech and Kinet, Annu.Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as the assay described in U.S. Pat. No. 5,500,362 or 5,821,337. Effector cells that can be used in such assays include Peripheral Blood Mononuclear Cells (PBMCs) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model as disclosed by Clynes et al PNAS (USA)95: 652-.

The term "complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind their cognate antigen. To assess complement activation, CDC assays can be performed, for example, as described in Gazzano-Santoro et al, J.Immunol.methods 202:163 (1996).

The term "subject" includes any human or non-human animal, preferably a human.

As used herein, the term "cancer" refers to solid and non-solid tumors mediated by growth, proliferation or metastasis of any tumor or malignant cell that causes a medical condition, such as leukemia.

The terms "treatment" and "treating" as used herein in the context of treating a condition generally relate to the treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, for example, inhibiting the progression of the condition, including a decrease in the rate of progression, a cessation in the rate of progression, regression of the condition, improvement of the condition, and healing of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For cancer, "treating" may refer to inhibiting or slowing tumor or malignant cell growth, proliferation or metastasis or some combination thereof. For a tumor, "treating" includes removing all or a portion of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

As used herein, the term "therapeutically effective amount" refers to the amount of active compound or material, composition or dosage form containing the active compound that, when administered in accordance with a desired treatment regimen, is effective to produce some desired therapeutic effect commensurate with a reasonable benefit/risk ratio. In particular, "therapeutically effective amount" means an amount or concentration of an antibody, or antigen-binding portion thereof, effective to treat a human LAG-3 related disease or disorder.

As used herein, a "host cell" of the invention refers to a cell into which an exogenous polynucleotide is introduced.

The term "pharmaceutically acceptable" as used herein means that the carrier, diluent, excipient, and/or salt thereof is chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active agent, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19th ed. pennsylvania: machine Publishing Company,1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, and ionic strength enhancing agents. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered to an organism with an antigen or delivered to an organism in advance, can enhance the immune response to the antigen in the organism or alter the type of immune response. There are a variety of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., freund's complete adjuvant and freund's incomplete adjuvant), corynebacterium pumilus, lipopolysaccharides, cytokines, and the like. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

anti-LAG-3 antibodies

In the context of the present application, "antibody" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecules, so long as they exhibit preferential binding or association with LAG-3 protein. Furthermore, unless the context dictates otherwise, the term also includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a human monoclonal antibody.

Human antibodies can be produced using various techniques known in the art. One technique is phage display, in which a (preferably human) antibody library is synthesized on phage, the library is screened with the antigen of interest or an antibody-binding portion thereof, and the antigen-binding phage is isolated, from which immunoreactive fragments can be obtained. Methods for preparing and screening such libraries are well known in the art, and kits for generating phage display libraries are commercially available (e.g., Pharmacia recombinant phage antibody System, Cat. No. 27-9400-01; and Stratagene SurfZAP phage display kit, Cat. No. 240612). Still other methods and reagents are useful for generating and screening antibody display libraries (see, e.g., Barbas et al, Proc. Natl. Acad. Sci. USA 88: 7978-.

Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated and into which human immunoglobulin genes have been introduced). Upon challenge, human antibody production was observed, which is very similar to that observed in humans in every aspect, including gene rearrangement, assembly, and antibody repertoire. Such a process is described, for example, in us patent 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,661,016 and U.S. Pat. Nos. 6,075,181 and 6,150,584 to XenoMouse; lonberg and Huszar, Intern.Rev.Immunol.13:65-93 (1995). Alternatively, human antibodies can be prepared by immortalization of human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes can be obtained from individuals with neoplastic disease or may have been immunized in vitro). See, e.g., Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); boerner et al, J.Immunol,147(l):86-95 (1991); and U.S. p.n.5,750, 373.

Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals (e.g.) Or some combination thereof. For example, Monoclonal Antibodies can be produced using hybridomas and art-recognized biochemical and genetic engineering techniques, as described in detail in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies, From Bench to clinical, John Wiley and Sons,1 ^st ed.2009；Shire et.al.(eds.)Current Trends in Monoclonal Antibody Development and Manufacturing,Springer Science+Business Media LLC,1 ^st ed.2010; harlow et al, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed.1988; hammerling, et al, in: Monoclonal Antibodies and T-Cell hybrids 563-681(Elsevier, N.Y.,1981), each of which is incorporated herein by reference in its entirety. It will be appreciated that the selected binding sequence may be further altered, for example, to increase affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce a multispecific antibodyAntibodies and the like, and antibodies comprising altered target binding sequences are also antibodies of the invention. In a preferred embodiment, anti-human LAG-3 monoclonal antibodies are prepared by using hybridomas.

Generation of hybridomas producing the human monoclonal antibodies of the invention

To obtain hybridomas that produce antibodies of the invention, e.g., human monoclonal antibodies of the invention, spleen cells and/or lymph node cells from an immunized mouse can be isolated and fused to a suitable immortalized cell line, e.g., a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The generation of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

Generation of transfectomas producing monoclonal antibodies of the invention

Antibodies of the invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked into a vector such that transcriptional and translational control sequences within the vector perform their intended functions of regulating transcription and translation of the antibody gene.

The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. These regulatory sequences are described, for example, in Goeddel (Gene Expression technology. methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Exemplary regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers from Cytomegalovirus (CMV), Simian Virus 40(SV40), adenoviruses (e.g., adenovirus major late promoter (AdMLP) and polyoma virus, or non-viral regulatory sequences such as ubiquitin promoter or beta-globin promoter may be used, as well as regulatory elements consisting of sequences of different origins, such as SRa promoter system, comprising sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al (1988) MoI.cell. biol.8: 466-472.) expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene may be inserted into the same or different expression vectors. In some embodiments, the variable regions are used to generate full length antibody genes of any antibody isotype by inserting them into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype, such that the VH segments are operably linked to the CH segments and the VL segments within the vector are operably linked to the CL segments within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may also carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically a selectable marker gene confers resistance to a drug (e.g., G418, hygromycin or methotrexate) on a host cell into which the vector has been introduced. Selectable marker genes may include the dihydrofolate reductase (DHFR) gene (for DHFR-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The term "transfection" of various forms is intended to cover the usually used to introduce exogenous DNA into prokaryotic or eukaryotic host cells in various techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection. The antibodies of the invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete appropriately folded and immunologically active antibodies.

Mammalian host cells for expression of recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) (including DHFR CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 4277: 4216-one 621), NSO myeloma cells, COS cells and SP2 cells, used with a DHFR selection marker (e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. MoI.biol.159: 601-one 621). In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When a recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secreting the antibody into the medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

anti-LAG 3 antibodies with certain properties

The antibodies of the invention are characterized by specific functional characteristics or properties of the antibodies. In some embodiments, the isolated antibody, or antigen-binding portion thereof, has one or more of the following properties:

(a) at 2X 10 ^-10 K of M or less _D Binding human LAG-3;

(d) inhibiting the binding of LAG-3 to LSECtin and/or galectin-3; or

(e) Human LAG-3 was bound without a cross-family response.

The antibodies of the invention bind human LAG-3 with high affinity. Antibodies of the invention andbinding of LAG-3 can be assessed using one or more techniques well established in the art, such as ELISA. The binding specificity of an antibody of the invention can also be determined by monitoring the binding of the antibody to cells expressing LAG-3 protein, e.g., by flow cytometry. For example, antibodies can be tested by flow cytometry assays, in which the antibody reacts with a cell line expressing human LAG-3, such as CHO cells that have been transfected to express LAG-3 on their cell surface. Other suitable cells for flow cytometry assays include anti-CD 3-stimulated CD4 expressing native LAG-3 ⁺ Activated T cells. Additionally or alternatively, the binding of the antibody can be tested in a BIAcore binding assay, including binding kinetics (e.g., Kd values). Other suitable binding assays include ELISA assays, for example using recombinant LAG-3 protein. For example, the antibodies of the invention are present at 5X 10 ^-8 K of M or less _D Binding to human LAG-3 protein at 2X 10 ^-8 K of M or less _D Binding to human LAG-3 protein at 5X 10 ^-9 K of M or less _D Binding to human LAG-3 protein at 4X 10 ^-9 K of M or less _D Binding to human LAG-3 protein at 3X 10 ^-9 K of M or less _D Binding to human LAG-3 protein at 2X 10 ^-9 K of M or less _D Binding to human LAG-3 protein at 1X10 ^-9 K of M or less _D Binding to human LAG-3 protein at 5X 10 ^-10 K of M or less _D Binding to human LAG-3 protein, or at 1X10 ^-10 K of M or less _D Binds human LAG-3 protein.

The ability of an antibody to modulate an immune response (e.g., an antigen-specific T cell response) can be indicated, for example, by the ability of the antibody to stimulate the production of interleukin-2 (IL-2) in an antigen-specific T cell response. In certain embodiments, the antibodies of the invention bind human LAG-3 and exhibit the ability to stimulate an antigen-specific T cell response. Means for assessing the ability of an antibody to stimulate an immune response may include, for example, the ability of an antibody to inhibit tumor growth in an in vivo tumor transplantation model, or the ability of an antibody to stimulate an autoimmune response.

The isolated antibody or antigen-binding portion thereof as disclosed herein inhibits LAG-3 and Major Histocompatibility (MHC) class II molecule, FGL1Binding of a peptoid, LSECtin and/or galectin-3. LAG-3 negatively regulates T cell signaling and function. Ligands for LAG-3 include, for example, Major Histocompatibility (MHC) class II molecules, LSECtin, and galectin-3. LAG-3 can interact with MHC class II molecules on the cell surface (Baixeras et al (1992) J.exp.Med.176: 327-. Direct binding of LAG-3 to MHC class II molecules has been proposed to down-regulate CD4 ⁺ Plays a role in antigen-dependent stimulation of T lymphocytes (Huard et al (1994) Eur. J. Immunol.24: 3216-3221). Recently, it was further verified by demonstrate-in vivo and in vitro experiments that FGL1 is a major immunosuppressive ligand of LAG-3, a novel tumor immune escape pathway FGL1-LAG-3 was proposed, and blocking FGL1-LAG-3 interaction could enhance antitumor effect (cell.2019Jan 10; 176(1-2):334-347.e 12.).

Galectin-3 is a 31kD lectin that regulates T cell responses by several mechanisms, including apoptosis, TCR cross-linking, and TCR down-regulation. Galectin-3 binds to LAG-3, and LAG-3 expression is essential for galectin-3 mediated inhibition of CD8+ T cells in vitro. (Kouo et al (2015) Cancer Immunol. Res.10.1158: 2326-6066). anti-LSECtin has been shown to inhibit B16 melanoma cell growth (Xu et al (2014) Cancer Res.74(13): 3418-3428).

anti-LAG 3 antibodies comprising CDRs having sequence identity to a particular sequence

C) One or more CDRH of a) and one or more CDRL of B).

Unless otherwise indicated, the assignment of amino acids to each CDR can be according to one of the numbering schemes provided below: kabat et al (1991) Sequences of Proteins of Immunological Interest (5) ^th Ed.), US depth.of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242; chothia et al, 1987, PMID 3681981; chothia et al, 1989, PMID 2687698; MacCallum et al, 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies,3 ^rd Ed.,Wily-VCH Verlag GmbH and Co.。

The variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as described above, e.g., the Kabat numbering system) or by aligning the sequence to a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY,2001 and Dinarello et al, Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in and available from the "Absysis" website (maintained by Department of Biochemistry & Molecular Biology University College London, London, A.C. Martin of England) and the VBASE2 website www.vbase2.org on www.bioinf.org.uk/abs, as described in Retter et al, Nucl. acids Res.,33(Database issue): D671-D674 (2005). The sequences are preferably analyzed using the Abysis database, which integrates Sequence data from the Kabat, IMGT, and Protein Database (PDB) with structural data from the PDB, see Protein Sequence and Structure Analysis of Antibody Variable Domains in the book by Dr.Andrew C.R.Martin (Ed.: Duebel, S.and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13:978-3540413547, also available on the website bio for g.uk/abs). The Abysis database website also includes general rules that have been developed for identifying CDRs that can be used in accordance with the teachings herein. Unless otherwise indicated, all CDRs described herein were obtained from the Abysis database website of Kabat.

The percent identity between two amino acid sequences can be determined using the algorithm of e.meyers and w.miller (comput.appl.biosci.,4:11-17(1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J.mol. biol.48:444-453(1970)), which has been incorporated into the GAP program in the GCG software package (available from http:// www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, with a GAP weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6.

Additionally or alternatively, the protein sequences of the invention may further be used as "query sequences" to perform searches against public databases, for example to identify related sequences. Such a search may be performed using the XBLAST program (version 2.0) of Altschul, et al (1990) J.MoI.biol.215: 403-10. BLAST protein searches using the XBLAST program can be performed with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of the present invention. To obtain gap alignments for comparison purposes, gap BLAST can be used, as described in Altschul et al, (1997) Nucleic Acids Res.25(17): 3389-3402. When BLAST and gapped BLAST programs are used, the default parameters for each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

In other embodiments, the CDR amino acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each of the sequences described above. As an illustrative example, the antibody may comprise an amino acid sequence identical to a sequence selected from SEQ ID NOs:1 and 7 has a CDRH1 of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

anti-LAG 3 antibodies comprising CDRs with amino acid additions, deletions, or substitutions in some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

A) one or more heavy chain cdrs (cdrhs) selected from at least one of: (i) selected from the group consisting of SEQ ID NO:1 and 7 CDRH1 or CDRH1 with no more than 2 amino acid additions, deletions or substitutions difference from the amino acid sequence of CDRH 1; (ii) selected from the group consisting of SEQ ID NO: 2 and 8 or a CDRH2 that differs from the amino acid sequence of the CDRH2 by the addition, deletion or substitution of an amino acid of not more than 2 amino acids; and (iii) a sequence selected from SEQ ID NO: 3 and 9 or CDRH3 that differs from the amino acid sequence of CDRH3 by the addition, deletion or substitution of an amino acid of not more than 2 amino acids;

B) one or more light chain cdrs (cdrls) selected from at least one of: (i) selected from the group consisting of SEQ ID NO: 4 and 10 CDRL1 or CDRL1 with amino acid addition, deletion or substitution differences of no more than 2 amino acids from the amino acid sequence of CDRL 1; (ii) selected from the group consisting of SEQ ID NO: 5 and 11 CDRL2 or CDRL2 with amino acid addition, deletion or substitution differences of no more than 2 amino acids from the amino acid sequence of CDRL 2; and (iii) a sequence selected from SEQ ID NO: 6 and 12 CDRL3 or CDRL3 with no more than 2 amino acid additions, deletions or substitutions difference from the amino acid sequence of the CDRL 3; or

C) One or more CDRH of a) and one or more CDRL of B).

Preferably, the CDRs of the isolated antibody or antigen binding portion thereof contain conservative substitutions of no more than 2 amino acids or no more than 1 amino acid. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the basic properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include those in which an amino acid residue is substituted with another amino acid residue having a similar side chain, e.g., a substitution of a physically or functionally similar residue (e.g., of similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al, Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94:412-417(1997), which are incorporated herein by reference).

anti-LAG 3 antibodies comprising CDRs

(a) comprises SEQ ID NO: CDRH1 of 1;

(b) comprises the amino acid sequence of SEQ ID NO: 2 CDRH 2;

(c) comprises the amino acid sequence of SEQ ID NO: 3 CDRH 3;

(d) comprises the amino acid sequence of SEQ ID NO: CDRL1 of 4;

(e) comprises the amino acid sequence of SEQ ID NO: CDRL2 of 5; and

(f) comprises the amino acid sequence of SEQ ID NO: CDRL3 of 6.

In particular embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

(a) consisting of SEQ ID NO:1 CDRH 1;

(b) consisting of SEQ ID NO: 2 CDRH 2;

(c) consisting of SEQ ID NO: 3, CDRH 3;

(d) consisting of SEQ ID NO: 4 CDRL 1;

(e) consisting of SEQ ID NO: 5 into CDRL 2; and

(f) consisting of SEQ ID NO: 6 in the form of CDRL 3.

(a) comprises the amino acid sequence of SEQ ID NO: CDRH1 of 7;

(b) comprises the amino acid sequence of SEQ ID NO: CDRH2 of 8;

(c) comprises the amino acid sequence of SEQ ID NO: CDRH3 of 9;

(d) comprises the amino acid sequence of SEQ ID NO: CDRL1 of 10;

(e) comprises SEQ ID NO: CDRL2 of 11; and

(f) comprises the amino acid sequence of SEQ ID NO: CDRL3 of 12.

(a) consisting of SEQ ID NO: 7 CDRH 1;

(b) consisting of SEQ ID NO: 8 into CDRH 2;

(c) consisting of SEQ ID NO: 9, CDRH 3;

(d) consisting of SEQ ID NO: 10 CDRL 1;

(e) consisting of SEQ ID NO: 11 CDRL 2; and

(f) consisting of SEQ ID NO: 12 in CDRL 3.

anti-LAG 3 antibody comprising heavy chain variable region and light chain variable region

(A) heavy chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 13;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 13, an amino acid sequence having at least 85%, at least 90%, or at least 95% identity; or

(B) Light chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 14;

(ii) comprises a nucleotide sequence substantially identical to SEQ ID NO: 14 an amino acid sequence having at least 85%, at least 90%, or at least 95% identity;

(a) consisting of SEQ ID NO: 13, or a heavy chain variable region consisting of the amino acid sequence of seq id no; and/or

(b) Comprises the amino acid sequence of SEQ ID NO: 14, or a light chain variable region of the amino acid sequence of seq id No. 14.

(A) heavy chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 15;

(iii) Comprising a nucleotide sequence substantially identical to SEQ ID NO: 15 compared to an amino acid sequence having one or more amino acid additions, deletions and/or substitutions; and/or

(B) Light chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 16;

(a) consisting of SEQ ID NO: 15, or a light chain variable region consisting of the amino acid sequence of seq id no; and/or

(b) Comprises the amino acid sequence of SEQ ID NO: 16, or a light chain variable region of the amino acid sequence of seq id No. 16.

In other embodiments, the amino acid sequence of the heavy chain variable region and/or the light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each of the sequences described above. As an illustrative example, an antibody may comprise an amino acid sequence identical to SEQ ID NO: 15 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In some further embodiments, the isolated antibody, or antigen-binding portion thereof, may comprise conservative substitutions or modifications of amino acids in the variable region of the heavy and/or light chain. It is understood in the art that certain conservative sequence modifications may be made that do not eliminate antigen binding. See, e.g., Brummell et al (1993) Biochem 32: 1180-8; de Wildt et al (1997) prot.Eng.10: 835-41; komissarov et al (1997) J.biol.chem.272: 26864-26870; hall et al (1992) J.Immunol.149: 1605-12; kelley and O' Connell (1993) biochem.32: 6862-35; Adib-Conquy et al (1998) int. Immunol.10:341-6 and Beers et al (2000) Clin. Can. Res.6: 2835-43.

The term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or alter the basic properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include those in which an amino acid residue is substituted with another amino acid residue having a similar side chain, e.g., a physically or functionally similar residue (e.g., of similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al, Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94:412-417(1997), which are incorporated herein by reference).

Binning and epitope mapping

It will be further understood that the disclosed antibodies will associate or bind to discrete epitopes or immunogenic determinants presented by the selected target or fragment thereof. In certain embodiments, an epitope or immunogenic determinant includes a chemically active surface grouping of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have particular three-dimensional structural characteristics and/or specific charge characteristics. Thus, as used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T cell receptor or otherwise interacting with a molecule. In certain embodiments, an antibody is considered to specifically bind (or immunospecifically bind or react) to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, the dissociation constant (K) is balanced when _D ) Less than or equal to 10 ^-6 M is less than or equal to 10 ^-7 When M, it is more preferable when K _D Less than or equal to 10 ^-7 M, antibody is said to bind antigen specifically to equal 10 ^-8 M, even more preferably when K _D Less than or equal to 10 ^-9 M, the antibody is said to specifically bind to the antigen.

Epitopes formed by contiguous amino acids (sometimes referred to as "linear" or "contiguous" epitopes) are typically retained when the protein is denatured, while epitopes formed by tertiary folding are typically lost after the protein is denatured. In any case, an antibody epitope typically comprises at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation.

In this regard, it is to be understood that, in certain embodiments, an epitope may be bound to or located within one or more regions, domains or motifs of, for example, a LAG-3 protein. Similarly, the art-recognized term "motif will be used in accordance with its general meaning and should generally refer to a short conserved region of a protein of typically ten to twenty contiguous amino acid residues.

In any event, once the desired epitope on the antigen is determined, it is possible to generate antibodies against that epitope, for example by immunizing with an epitope-containing peptide using the techniques described in the present invention. Alternatively, in the discovery process, the generation and characterization of antibodies can elucidate information about the desired epitope located in a particular domain or motif. From this information, antibodies that bind to the same epitope can be competitively screened. One way to achieve this is to conduct competition studies to find antibodies that compete for binding to each other, i.e., antibodies compete for binding to antigen. A high throughput method for binning antibodies based on their cross-competition is described in WO 03/48731. Binning or domain level or epitope mapping including antibody competition or other methods of antigen fragment expression on yeast are well known in the art.

As used herein, the term "binning" refers to methods for grouping or classifying antibodies based on antigen binding characteristics and competition. While these techniques are useful for defining and classifying the antibodies of the invention, these bins (bins) do not always bind directly to the epitope, and this initial determination of epitope binding can be further improved and confirmed by other recognized methods in the art and as described herein. However, it will be appreciated that assigning antibodies to the various bins empirically provides information that can be indicative of the therapeutic potential of the disclosed antibodies.

More specifically, it can be determined whether a selected reference antibody (or fragment thereof) binds to the same epitope or cross-competes for binding (i.e., within the same compartment) with a second test antibody by using methods known in the art and set forth in the examples herein.

Other compatible epitope mapping techniques include alanine scanning mutants, peptide blotting (Reineke (2004) Methods Mol Biol 248: 443-63) (specifically incorporated herein by reference in its entirety) or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of an antigen (Tomer (2000) Protein Science 9: 487-496) can be used (specifically, incorporated herein by reference in its entirety).

Nucleic acid molecules encoding the antibodies of the invention

The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cdnas encoding the light and heavy chains of antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

Isolated nucleic acids encoding a VH region can be converted to full-length heavy chain genes by operably linking the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH 3). The sequence of the human heavy chain constant region gene is known in the art (see, e.g., Kabat et al (1991), supra), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is more preferably an IgG1 or IgG4 constant region, most preferably an IgG4 constant region.

An isolated nucleic acid encoding a VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding a light chain constant region CL. The sequence of the human light chain constant region gene is known in the art (see, e.g., Kabat et al, supra) and DNA fragments comprising these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda constant region.

Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding either VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to mean that two DNA segments are linked such that the amino acid sequences encoded by the two DNA segments are maintained in frame.

Preferred nucleic acid molecules of the invention are those encoding the VH and VL sequences of the 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L monoclonal antibodies. The DNA sequences encoding the VH sequences of 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are shown in SEQ ID NOs:17 and 19. The DNA sequences encoding the VL sequences of 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are shown in SEQ ID NOs: 18 and 20. In some embodiments, the nucleic acids are identical to SEQ ID NOs:17-20 have at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the percentage of identity results from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

Pharmaceutical composition

Components of the composition

The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the invention may also be administered in combination with, for example, another immunostimulant, an anti-cancer agent, an anti-viral agent, or a vaccine, such that the anti-LAG-3 antibody enhances the immune response to the vaccine. The pharmaceutically acceptable carrier may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antimicrobial agent, isotonic agent, buffer, antioxidant, anesthetic, suspending/dispersing agent, chelating agent, diluent, adjuvant, excipient or nontoxic auxiliary substance, a combination of various components known in the art or more.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorants, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and/or propyl arsenate. As disclosed herein, the antibody or antigen-binding fragment of the disclosed compositions may be oxidized in a solvent containing the antibody or antigen-binding fragment of the disclosed composition comprising one or more antioxidants, such as methionine, that reduce the antibody or antigen-binding fragment thereof. Redox can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Thus, in some embodiments, the present invention provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants, such as methionine. The invention further provides methods wherein the antibody or antigen-binding fragment thereof is mixed with one or more antioxidants, such as methionine. Thus, the antibody or antigen-binding fragment thereof may be prevented from oxidation, to extend its shelf-life and/or increase activity.

Administration, formulation and dosage

The pharmaceutical compositions of the present invention may be administered in vivo to a subject in need thereof by a variety of routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention may be formulated in solid, semi-solid, liquid or gaseous form; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected according to the intended application and treatment regimen.

Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and controlled release dosage forms thereof.

Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, ringer's solution or lactated ringer's injection. Similarly, the particular dosage regimen (i.e., dose, time and repetition) will depend on the particular individual and the individual's medical history as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining such a reduction in tumor cells, reducing proliferation of tumor cells or delaying the development of metastases. In some embodiments, the dose administered may be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of the therapeutic compositions of the present invention may be suitable.

One skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risk or deleterious side effects. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the particular compound, the administration, the time of administration, the rate of clearance of the compound, the duration of the treatment, other drugs, compounds and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health and prior medical history of the patient. The amount of the compound and the route of administration are ultimately at the discretion of the physician, veterinarian, or clinician, but the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect, without causing substantial deleterious or adverse side effects.

In general, the antibodies of the invention, or antigen binding portions thereof, can be administered in a variety of ranges. These include from about 5 μ g/kg body weight to about 100mg/kg body weight per dose; about 50 μ g/kg body weight to about 5mg/kg body weight per dose; about 100. mu.g/kg body weight to about 10mg/kg body weight per dose. Other ranges include from about 100 μ g/kg body weight to about 20mg/kg body weight per dose and from about 0.5mg/kg body weight to about 20mg/kg body weight per dose. In certain embodiments, the dose is at least about 100 μ g/kg body weight, at least about 250 μ g/kg body weight, at least about 750 μ g/kg body weight, at least about 3mg/kg body weight, at least about 5mg/kg body weight, at least about 10mg/kg body weight.

In any event, the antibodies of the invention or antigen-binding portions thereof are preferably administered to a subject in need thereof as needed. The frequency of administration can be determined by one skilled in the art, e.g., by the attending physician based on considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like.

In certain preferred embodiments, the course of treatment involving the antibodies or antigen-binding portions thereof of the present invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody or antigen-binding portion thereof of the invention can be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage may be varied or the interval adjusted based on patient response and clinical practice.

The dosage and regimen of the disclosed therapeutic compositions can also be determined empirically in individuals given one or more administrations. For example, an individual may be administered a incremental dose of a therapeutic composition produced as described herein. In selected embodiments, the dosage may be gradually increased or decreased or lessened based on empirically determined or observed side effects or toxicity, respectively. To assess the efficacy of the selected composition, markers of a particular disease, disorder, or condition can be tracked as previously described. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopy of tumor samples; measuring the reduction in pain or paralysis of an indirect tumor marker (e.g., PSA for prostate cancer) or tumorigenic antigen identified according to the methods described herein; improvement in speech, vision, respiration or other disability associated with the tumor; appetite increase; or an increase in quality of life or an increase in survival as measured by accepted tests. Those skilled in the art will appreciate that the dosage will vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other locations in the individual, and the treatment used in the past and the treatment used concurrently.

A compatible formulation for parenteral administration (e.g., intravenous injection) will comprise the antibody, or antigen-binding portion thereof, at a concentration of about 10 μ g/mL to about 100 mg/mL. In certain selected embodiments, the concentration of the antibody, or antigen-binding portion thereof, will comprise 20. mu.g/mL, 40. mu.g/mL, 60. mu.g/mL, 80. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 300. mu.g/μ g/mL, 400. mu.g/mL, 500. mu.g/mL, 600. mu.g/mL, 700. mu.g/mL, 800. mu.g/mL, 900. mu.g/mL, or 1 mg/mL. In other preferred embodiments, the ADC concentration will comprise 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 8mg/mL, 10mg/mL, 12mg/mL, 14mg mL, 16mg/mL, 18mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL or 100 mg/mL.

Application of the invention

The antibodies, antibody compositions, and methods of the invention have a number of in vitro and in vivo uses, including, for example, the detection of LAG-3 or the enhanced immune response by blocking LAG-3. These molecules can be administered to cultured cells, for example, in vitro or ex vivo, or to human subjects, for example, in vivo, to enhance immunity in various situations. The immune response may be modulated, e.g., enhanced, stimulated or up-regulated.

Preferred subjects include human patients in need of an enhanced immune response. The methods are particularly useful for treating human patients having a condition that can be treated by enhancing an immune response (e.g., a T cell-mediated immune response). In a particular embodiment, the method is particularly suitable for the in vivo treatment of cancer. To achieve antigen-specific enhancement of immunity, an anti-LAG-3 antibody may be administered with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a tumor-or virus-bearing subject). When the antibody to LAG-3 is administered with another agent, the two may be administered in any order or simultaneously.

The present invention further provides a method for detecting the presence of human LAG-3 antigen or measuring the amount of human LAG-3 antigen in a sample, comprising contacting the sample and a control sample with a human monoclonal antibody, or antigen-binding portion thereof, that specifically binds human LAG-3 under conditions that allow for the formation of a complex between the antibody, or portion thereof, and human LAG-3. And detecting the formation of complexes, wherein differential complex formation between the samples as compared to the control sample indicates the presence of human LAG-3 antigen in the sample. Furthermore, the anti-LAG-3 antibodies of the invention can be used to purify human LAG-3 by immunoaffinity purification.

In view of the ability of the anti-LAG-3 antibodies of the invention to inhibit the binding of LAG-3 to MHC class II or FGL1 molecules and to stimulate antigen-specific T cell responses, the invention also provides in vitro and in vivo methods of stimulating, enhancing or up-regulating antigen-specific T cell responses using the antibodies of the invention. For example, the invention provides a method of stimulating an antigen-specific T cell response comprising administering to a subject an antibody, or antigen-binding portion thereof, of the invention, thereby stimulating an antigen-specific T cell response. Any suitable indicator of antigen-specific T cell response may be used to measure the antigen-specific T cell response.

Treatment of cancer

Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of antibodies and/or increased cytokine production in the presence of antibodies. In a preferred embodiment, the interleukin-2 production of antigen-specific T cells is stimulated. The invention also provides a method of stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, of the invention, such that the immune response (e.g., an antigen-specific T cell response) is stimulated. In a preferred embodiment, the subject is a cancer-bearing subject and stimulates an immune response against the tumor. Cancer blockade of LAG-3 by antibodies can enhance the immune response of a patient to cancer cells. anti-LAG-3 antibodies can be used alone or in combination with other immunogenic agents, standard cancer therapeutics, or other antibodies.

Examples of cancers that can be treated using the methods of the invention include bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, non-hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myelogenous leukemia, chronic granulocytic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, solid tumors of children, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, renal pelvis, cancer of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

The antibody or antigen binding portion thereof can be used in combination with chemotherapy or radiotherapy.

Used in combination with chemotherapy

The antibody or antigen binding portion thereof can be used in combination with an anti-cancer agent, cytotoxic agent, or chemotherapeutic agent.

The term "anti-cancer agent" or "anti-proliferative agent" means any agent useful in the treatment of cell proliferative disorders such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiation therapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapy, radiation therapy and anti-metastatic agents, and immunotherapeutic agents. It is to be understood that in selected embodiments as described above, such anti-cancer agents may comprise conjugates and may be conjugated to the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, a selected anticancer agent is linked to an unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Accordingly, such engineered conjugates are expressly contemplated within the scope of the present invention. In other embodiments, the disclosed anti-cancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and/or causes cell destruction. In certain embodiments, the agent is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins of bacteria (e.g., diphtheria toxin, pseudomonas endotoxin and exotoxin, staphylococcal enterotoxin a), fungi (e.g., alpha-sarcin, restrictocin), plants (abrin, ricin, gelonin, mistletoe, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii protein, dianthin protein, phytacca merica protein (PAPI, PAPII and PAP-S), momordica charantia inhibitors, leprosy toxin, croton toxin, alkannin inhibitors, gelonin, mitegellin, restrictocin, phenomycin, neomycin and trichothecene family compounds) or animals (e.g., cytotoxic rnases, such as extracellular pancreatic rnases; dnase I, including fragments and/or variants thereof).

For purposes of the present invention, "chemotherapeutic agents" include chemical compounds (e.g., cytotoxic or cytostatic agents) that nonspecifically reduce or inhibit the growth, proliferation, and/or survival of cancer cells. These chemical agents are generally directed to intracellular processes required for cell growth or division and are therefore particularly effective on cancer cells which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting the cells from entering mitosis. In general, a chemotherapeutic agent may include any chemical agent that inhibits or is designed to inhibit a cancer cell or a cell that may become sexually or produce tumorigenic progeny (e.g., TIC). These agents are often used in combination and are often most effective, for example, in regimens such as CHOP or FOLFIRI.

Examples of anti-cancer agents that may be used in combination with the site-specific constructs of the invention (as components or in an unconjugated state of the site-specific conjugates) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimine and methyl melamine, polyacetyls (acetogenins), camptothecins, bryostatins, calicheastatins (callystatins), CC-1065, croutoxins (cryptophycins), dolastatins, duocarmycins, eleutherobin (eleutherobin), coprostanin, saxodidin (sarcodictyins), spongin (spongistatin), mechlorethamine, antibiotics, enediynes, dynemics, bisphosphonates, epothilones, chromogens of chromogenes, clarithromycins (acacins), actinomycins, atramycins, azatrinins, bleomycin, actinomycins (carbapenems), caracinomycins (mycins), carcinomycins (mycins), mycins (mycins), Dactinomycin, daunorubicin, ditetracycline, 6-diazo-5-oxo-L-norleucine,Doxorubicin, epirubicin, esorubicin, idarubicin, sisomicin, mitomycin, mycophenolic acid, nogomycin, olivomycin, pelomycin, bodhimycin (potfiromycin), puromycin, triiron doxorubicin, roxobicin, streptonigrin, streptozotocin, tubercidin, ubenimex, setastatin, zorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folatesAnalogs, purine analogs, androgens, anti-adrenaline, folic acid supplements such as furinic acid (frolicic acid), acetoglucuronolactone, aldphosphoramide glycosides, aminolevulinic acid, eniluracil, amsacrine, besmeacil (besmeabil), bisantrene, edatrexate, deflazamine (deffamine), colchicine, disazoquinone, efonicine (elfornitine), etiracetam, epothilones, etogrel, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids (maytansinoids), mitoguanhydrazone, mitoxantrone, motdan (mopidantramol), nitrene (nitrene), pentostatin, mechlorethamine, pirarubicin, loxinoquinone, podophyllinic acid, 2-ethylhydrazine, procarbazine,Polysaccharide complexes (JHS Natural Products, Eugene, OR), Razoxan; rhizomycin; a texaphyrin; a germanium spiroamine; tenuronic acid; a tri-imine quinone; 2,2' -trichlorotriethylamine; trichothecenes (especially T-2 toxin, Verlucurin A (verracurin A), bacillocin A and snakeheaded; uratan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; cassitoxin (gacytosine); arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chlorambucil (chlorenbucil);gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; a platinum analog; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; the concentration of the vincristine is controlled by the concentration of the vincristine,vinorelbine; noscapine; (ii) teniposide; edatrexae; daunorubicin; aminopterin; (ii) Hirodad; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine; a retinoid; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A(which reduces cell proliferation), and a pharmaceutically acceptable salt, acid or derivative of any of the above. Also included in this definition are anti-hormonal agents used to modulate or inhibit hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase that modulates estrogen production in the adrenal glands, and anti-androgens; and troxacitabine (1, 3-dioxolane nucleoside cytosine analogues); antisense oligonucleotides, ribozymes such as inhibitors of VEGF expression and inhibitors of HER2 expression; a vaccine is provided which comprises a vaccine,rIL-2；a topoisomerase 1 inhibitor;rmRH; vinorelbine and epsipromycin, and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing.

Used in combination with radiotherapy

The invention also provides the combination of an antibody or antigen-binding portion thereof with radiotherapy (i.e., any mechanism used to induce DNA damage locally within tumor cells, such as gamma-irradiation, X-ray, UV-irradiation, microwaves, electron emission, etc.). Combination therapies using targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates may be used in conjunction with targeted anti-cancer agents or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 to about 2 weeks. Radiation therapy may be administered to a subject with head and neck cancer for about 6 to 7 weeks. Optionally, the radiation therapy may be administered as a single dose or as multiple sequential doses.

Diagnosis of

The invention provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods of screening cells from patients to identify tumor cells, including tumorigenic cells. Such methods include identifying an individual having cancer for treatment or monitoring the progression of cancer, comprising contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an antibody described herein, and detecting the presence or absence or level of binding of the bound antibody to bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or reporter as described herein.

In some embodiments, binding of an antibody to a particular cell in a sample can indicate that the sample is likely to contain a tumorigenic cell, thereby indicating that an individual having cancer can be effectively treated with an antibody described herein.

Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblot assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, and the like, as known to those skilled in the art.

Pharmaceutical pack and kit

Pharmaceutical packages and kits comprising one or more containers comprising one or more doses of an antibody, or antigen-binding portion thereof, are also provided. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or antigen-binding portion thereof, with or without one or more additional agents. For other embodiments, such unit doses are supplied in single use prefilled syringe injection syringes. In other embodiments, the composition contained in a unit dose may comprise saline, sucrose, or the like; buffers such as phosphate and the like; and/or formulated at a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder, which may be reconstituted after addition of a suitable liquid (e.g., sterile water or saline solution). In certain preferred embodiments, the compositions comprise one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is used to treat the selected neoplastic disease condition.

The invention also provides kits for producing single-dose or multi-dose administration units of the site-specific conjugate and optionally one or more anti-cancer agents. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed of a variety of materials, such as glass or plastic, and contain a pharmaceutically effective amount of the disclosed conjugates in conjugated or unconjugated form. In other preferred embodiments, the container includes a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits typically comprise a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally one or more anti-cancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable preparations for use in diagnosis or combination therapy. For example, such kits may contain, in addition to an antibody or antigen-binding portion thereof of the invention, any one or more anti-cancer agents, such as chemotherapeutic agents or radiotherapeutic agents; an anti-angiogenic agent; an anti-transfer agent; a targeted anti-cancer agent; a cytotoxic agent; and/or other anti-cancer agents.

More specifically, kits can have a single container containing the disclosed antibodies or antigen-binding portions thereof, with or without additional components, or they can have different containers for each desired reagent. Where a combination therapeutic agent is provided for conjugation, the single solutions may be premixed in molar equivalent combinations or in a manner such that one component is more than the other. Alternatively, the conjugate and any optional anti-cancer agent of the kit may be stored separately in separate containers prior to administration to a patient. The kit may also comprise a second/third container means for holding sterile pharmaceutically acceptable buffers or other diluents such as bacteriostatic water for injection (BWFI), Phosphate Buffered Saline (PBS), ringer's solution and dextrose solution.

When the components of the kit are provided as one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous or saline solution. However, the components of the kit may be provided as a dry powder. When the agent or component is provided in dry powder form, the powder may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container.

As briefly mentioned above, the kit may also contain means for administering the antibody, or antigen-binding portion thereof, and any optional components to the patient, such as one or more needles, i.v. bags or syringes, or even eye droppers, pipettes, or other similar devices, through which the formulation may be injected or introduced into the animal or administered to the affected area of the body. The kits of the invention will also typically include a means for holding vials or the like, as well as other tightly closed components for commercial sale, such as injection or blow molded plastic containers, in which the desired vials and other devices are placed and held.

Overview of sequence listing

The present application is accompanied by a sequence listing comprising a number of nucleic acid and amino acid sequences. The following table provides a summary of the sequences involved.

Examples

The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention. These examples are not intended to be representative of the experiments below being all or only experiments performed.

Example 1

Preparation of the Material

1. Production of immunogens

Synthesis of a peptide having SEQ ID NO: 22 encodes human LAG-3ECD (extracellular domain, ECD) or a polypeptide having the amino acid sequence of SEQ ID NO: 24 of a nucleic acid encoding full length human LAG-3. The amino acid sequence of the LAG-3ECD and the DNA sequence encoding it are shown in SEQ ID NO: 21 and 22, the amino acid sequence of full-length LAG-3 and the DNA sequence encoding it are shown in SEQ ID NO: 23 and 24. The LAG-3 gene fragment was amplified from the synthesized nucleic acid and inserted into the expression vector pcDNA3.3 (ThermoFisher). The inserted LAG-3 gene fragment was further confirmed by DNA sequencing. Fusion proteins containing human LAG-3ECD and various tags, including human Fc, mouse Fc and His tag, were obtained by transfecting human LAG-3 gene into 293F cells (ThermoFisher). Cells were incubated at 37 ℃ with 5% CO ₂ Next, the cells were cultured in FreeStyle 293 expression medium (ThermoFisher). After 5 days of culture, the supernatant harvested from the transient transfected cell culture was used for protein purification. The fusion protein is purified by nickel, protein a and/or SEC columns. Untagged LAG-3ECD proteins were generated by cleavage of the ECD-hFc fusion protein at the cleavage site using factor Xa protease (New England Biolabs). The purified proteins were used for immunization, screening and characterization.

2. Generation of reference antibody

Anti-human LAG-3 baseline antibodies were synthesized based on the information disclosed in patent applications US 20110150892 a1 and US 20170101472a1 (BMK1 and BMK7, where BMK1 is referred to as "25F 7" in US 20110150892 a1 and BMK7 is referred to as "H4 sH 15482P" in US 20170101472a 1). The benchmark antibody BMK8 is a humanized version of the chimeric antibody BMK5, BMK5 is described in WO2015138920a1 and is referred to as "BAP 050-chi". In WO2015138920A1 BMK8 is referred to as "BAP 050-hum 01". The synthetic gene sequence was integrated into plasmid pcDNA3.3 as described in section 1 above. The plasmid was transiently transfected into 293F cells. Cells were cultured in the same manner as described in section 1. After 5 days of culture, the supernatant harvested from the transient transfected cell culture was used for protein purification. The reference antibody was purified from the supernatant.

3. Establishment of Stable cell lines

Human, mouse and cynomolgus monkey LAG-3 transfected cell lines were generated. Briefly, Flp-In-293, Flp-In-CHO or 293F cells were transfected with pcDNA3.3 expression vectors containing full-length human, mouse and cynomolgus monkey LAG-3, respectively, using the Lipofectamine 2000 transfection kit according to the manufacturer's protocol. 48-72 hours after transfection, transfected cells were cultured in blasticidin-containing medium for selection and tested for LAG-3 expression. Cell lines expressing human LAG-3, cell lines expressing cynomolgus monkey LAG-3 and cell lines expressing mouse LAG-3 were obtained by limiting dilution.

Example 2

Antibody hybridoma production

1. Immunization and cell fusion

24-week-old OMT rats (transgenic rats with recombinant immunoglobulin loci, as described in US8,907,157B2) were alternatively immunized with 12.5 μ g hFc-tagged human LAG-3ECD protein and 12.5 μ g His-tagged mouse LAG-3 in adjuvant to generate antibodies with framework and CDR regions derived from human germline immunoglobulin sequences. The immunized rats were bled every two weeks for serum collection, and the titer against human LAG-3 in the serum was measured by ELISA. Plates coated with human LAG-3.ecd. hfc were incubated with diluted rat serum (first 1:100, then diluted 3-fold in 2% BSA) for 2 hours. Goat anti-rat-IgG-Fc-HRP was used as secondary antibody. Color was developed by dispensing 100. mu.L of TMB substrate and then stopped with 100. mu.L of 2N HCl. Absorbance was read at 450nM using a microplate reader. Once the antibody titer reached a sufficiently high value, rats were finally boosted with 40 μ g of human LAG-3ECD protein in DPBS without adjuvant. On the day of fusion, lymph nodes and spleens were removed from immunized rats under sterile conditions and prepared as single cell suspensions. The isolated cells were then compared with myeloma cells SP2/0 at a ratio of 1:1, and mixing. The electric cell fusion was performed using a BTX 2000 Electro cell manipulator. The cells were then plated at 1X10 ⁴ Individual cells/well were seeded in 96-well plates at 37 ℃ with 5% CO ₂ Incubate until ready for screening.

2. Preliminary screening and confirmation screening of hybridoma supernatants

Hybridoma supernatants were tested for binding to LAG-3 protein using an ELISA assay as the first screening method. The plates of section 1 of this example were coated with 1. mu.g/mL human LAG-3 ECD. hFc overnight at 4 ℃. After blocking and washing, hybridoma supernatants were transferred to coated plates and incubated at room temperature for 1 hour. The plates were then washed and subsequently incubated with a secondary goat anti-rat IgG HRP for 1 hour. After washing, TMB substrate was added and the chromogenic reaction was stopped with 2M HCl. Absorbance at 450nm was read using a microplate reader.

To confirm the natural binding of LAG-3 antibodies to conformational LAG-3 molecules expressed on cell membranes, flow cytometry analysis was performed on LAG-3 transfected CHO-K1 cell lines. CHO-K1 cells expressing human LAG-3 at 1X10 ⁵ The density of individual cells/well was transferred to a 96-well U-bottom plate. Hybridoma supernatants were then transferred to plates and incubated at 4 ℃ for 1 hour. After washing with 1 XPBS/1% BSA, a secondary goat anti-rat IgG Alexa647 was added and the cells incubated for 0.5 h at 4 ℃ in the dark. Cells were then washed and resuspended in 1 XPBS/1% BSA and then analyzed by flow cytometry. Binding of the antibody to the parental CHO-K1 cell line was performed in parallel as a negative control. Antibody binding to the parental CHO-K1 cell line was performed as a control.

Blocking activity of the antibodies was used as a confirmatory screen to select potential antibody hits. The selected antibodies were tested for their ability to block the binding of LAG-3 protein to the human MHC-II expressing cell line Raji by FACS analysis. Raji cells were cultured at 1X10 ⁵ The density of individual cells/well was transferred to a 96-well U-bottom plate. The supernatant was incubated with the mFc-labeled LAG-3 protein at 4 ℃ for 30 minutes. The mixture was transferred to a 96-well plate seeded with Raji cells. A secondary PE-labeled goat anti-mouse IgG antibody (no cross-reactivity to rat IgG Fc, Jackson Immunoresearch Lab) was incubated with the cells at 4 ℃ for 0.5 hour in the dark. Cells were then washed and resuspended in 1 × PBS/1% BSA and analyzed by flow cytometry.

3. Hybridoma subcloning:

once specific binding was verified by preliminary and confirmatory screening, the positive hybridoma cell line was subcloned by using the semi-solid medium method to obtain monoclonal anti-hLAG-3 antibody. In the semi-solid medium method, for each hybridoma cell line, the cells are in a semi-solid stateCloning media (STEMCELL Technologies) were diluted and plated in 6-well plates. In an incubator (37 ℃, 5% CO) ₂ ) The cells were cultured for 8-10 days until monoclonals were visible in the semi-solid medium. Clones were picked and transferred to 96-well plates in HAT medium (hypoxanthine-aminopterin-thymidine medium) containing 10% FBS. Positive clones were confirmed by binding ELISA and FACS for human LAG-3 as described above.

Example 3

Hybridoma sequencing and fully human antibody molecule construction

1. Sequencing of hybridomas

Total RNA was isolated from hybridoma cells using the RNeasy Plus Mini kit (Qiagen) and first strand cDNA was prepared as shown in tables 1 and 2. As shown in tables 3 and 4, antibody VH and VL genes were amplified from cDNA by using 3 '-constant region degenerate primers and 5' -degenerate primer sets (which are complementary to the upstream signal sequence coding region of the Ig variable sequences). Table 5 shows reagent information including the manufacturer.

The PCR product (10. mu.L) was ligated into the pMD18-T vector, and 10. mu.L of the ligation product was transformed into Top10 competent cells. Transformed cells were plated on 2-YT + Cab plates and incubated overnight at 37 ℃. Positive clones were randomly selected for sequencing at Shanghai Biosune Biotech co.

TABLE 1 cDNA amplification reaction (20. mu.L)

TABLE 2 cDNA amplification reaction conditions

	Step 1	Step 2	Step 3	Step 4
					Temperature (. degree.C.)	25	50	85	4
Time	10min	50min	5min	∞

TABLE 3 PCR reaction system (50. mu.L)

Components	Measurement of
		cDNA	2.0μL
Premix Ex Taq	25μL
		5' -degenerate primer set (10pM)	2.5μL
3' -constant regionAnd primer (10pM)	1μL
		ddH ₂ O	19.5μL

TABLE 4 PCR reaction conditions

TABLE 5 reagent information

The two lead antibodies were named "1.53.3-uAb-IgG 4 k" and "3.40.19-uAb-IgG 4L", respectively.

1.53.3-uAb-IgG4k CDR sequences were determined as follows:

description of the invention	SEQ ID NO.	Sequence information
			CDRH1	1	GGSFSGYYWS
CDRH2	2	EINHRGNTNYNPSLKS
			CDRH3	3	GEDYSDYDYYGDF
CDRL1	4	RASQSISSYLA
			CDRL2	5	AASNRAT
CDRL3	6	QQRSNWPLT

1.53.3-uAb-IgG4k has the following sequence for the heavy and light chain variable regions:

the CDR sequences of 3.40.19-uAb-IgG4L are as follows:

description of the preferred embodiment	SEQ ID NO.	Sequence information
			CDRH1	7	GDSISSTSYYWG
CDRH2	8	SFYYSGSTYYNPSLKS
			CDRH3	9	MQLWSYDVDV
CDRL1	10	TGTSSDVGGYDYVA
			CDRL2	11	DVSERPS
CDRL3	12	SSYTSTTTLVV

3.40.19-uAb-IgG4L variable heavy and light chain regions have the following sequences:

2. construction of fully human antibody molecules

VH and VL genes were reamplified using cloning primers containing appropriate restriction sites and cloned into expression vectors to generate corresponding chimeric antibody clones.

Example 4

Binding of LAG-3 antibodies to human LAG-3 on cell surface

Various concentrations of test antibody, positive and negative controls were added to human LAG-3 transfected cells, and antibody binding on the cell surface was then detected by the corresponding PE-labeled secondary antibody. Data are shown in FIG. 1, EC ₅₀ Shown in table 6.

TABLE 6

Ab	EC ₅₀ (nM)
		1.53.3-uAb-IgG4k	0.43
3.40.19-uAb-IgG4L	0.13
		BMK1	0.32
BMK7	0.61
		BMK8	0.90

Surprisingly, as shown in FIG. 1 and Table 6, EC binding of 3.40.19-uAb-IgG4L to cell surface LAG-3 ₅₀ (0.13) was significantly lower than all three baseline antibodies BMK1(0.32), BMK7(0.61) and BMK8 (0.90). In addition, 1.53.3-uAb-IgG4k binds to cell surface LAG-3EC ₅₀ (0.43) much lower EC than BMK7(0.61) and BMK8(0.90) ₅₀ . These results indicate that 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are effective in binding human LAG-3 and have a binding effect superior to or equivalent to that of human LAG-3A reference antibody.

Example 5

Blockade of binding of LAG-3 protein to MHC-II expressed on Raji cells

The antibodies were serially diluted in 1% BSA-PBS and incubated with mFc labeled LAG-3 protein at 4 ℃ for 30 minutes. The mixture was transferred to a 96-well plate seeded with Raji cells. Binding of LAG-3 protein to Raji cells was detected using goat anti-mouse IgG Fc-PE antibody. MFI was assessed by flow cytometry and analyzed by FlowJo software (version 7.6.1). Data are shown in FIG. 2, EC ₅₀ Shown in table 7.

TABLE 7

Ab	EC ₅₀ (nM)
		1.53.3-uAb-IgG4k	0.80
3.40.19-uAb-IgG4L	0.67
		BMK1	0.76
BMK7	1.25
		BMK8	0.88

As shown in FIG. 2 and Table 7, unexpectedly, the EC of 3.40.19-uAb-IgG4L binding to MHC-II expressed on Raji cells ₅₀ (0.67) significantly lower EC than all three baseline antibodies BMK1(0.76), BMK7(1.25) and BMK8(0.88) ₅₀ . Furthermore, 1.53.3-uAb-IgG4k binds to MHC-II expressed on Raji cells ₅₀ (0.80) EC lower than BMK7(1.25) and BMK8(0.88) ₅₀ . These results indicate that 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are effective in blocking binding to MHC-II expressed on Raji cells, and that the blocking effect is superior to or equivalent to that of the baseline antibody.

Example 6

Blockade of LAG-3 protein binding to LSECtin and galectin-3

96-well plates were coated with 0.5. mu.g/mL human LSECtin or galectin-3, respectively, overnight at 4 ℃. The antibodies were serially diluted in 1% BSA-PBS and mixed with mFc labeled LAG-3 protein. After blocking and washing, the mixture was transferred to a plate and incubated at room temperature for 1 hour. The plates were then washed and subsequently incubated with the corresponding secondary antibodies for 60 minutes. After washing, TMB substrate was added and the chromogenic reaction was stopped with 2M HCl. Absorbance at 450nm was read using a microplate reader. The data are shown in figures 3 and 4. EC (EC) ₅₀ Shown in table 8.

TABLE 8

As shown in fig. 3 and 4 and table 7, both 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L were effective in blocking the binding of LAG-3 to LSECtin or galectin-3, and the blocking effect was superior or comparable to that of the baseline antibody.

Example 7

Complete kinetic binding affinity assay

Full kinetic binding affinity by Surface Plasmon Resonance (SPR) test:

the affinity and binding kinetics of the antibodies to human LAG-3 were characterized by SPR assay using Biacore 8K. Goat anti-human Fc was pre-immobilized onto a sensor chip (CM5) and anti-LAG-3 antibodies were captured when the chip was injected. Various concentrations of human LAG-3 protein and running buffer were flowed over the sensor chip at a flow rate of 30. mu.L/min for 300s of association phase followed by 3600s of dissociation. Association and dissociation curves 1: 1Langmuir binding model fitting. The data are shown in table 9.

TABLE 9

Ab	ka(1/Ms)	kd(1/s)	KD(M)
				1.53.3-uAb-IgG4k	6.60E+05	3.33E-05	5.05E-11
3.40.19-uAb-IgG4L	1.05E+06	1.11E-05	1.06E-11
				BMK1	4.87E+05	3.34E-04	6.85E-10
BMK7	2.13E+05	1.06E-04	4.97E-10
				BMK8	8.46E+04	6.74E-06	7.97E-11

Binding affinity of LAG-3 antibodies to cell surface LAG-3 molecules as tested by Fluorescence Activated Cell Sorting (FACS)

The binding affinity of the antibody to cell surface LAG-3 was measured by FACS analysis. Flp-In-293 cells expressing human LAG-3 at 5X 10 ⁵ The density of individual cells/mL was transferred to a 96-well U-shaped bottom plate. The antibodies tested were serially diluted in wash buffer (1 XPBS/1% BSA) and incubated with the cells for 1 hour at 4 ℃. A secondary goat anti-human IgG Fc FITC (3.5 moles FITC per mole IgG) was added and incubated at 4 ℃ for 0.5 hours in the dark. Cells were then washed once and resuspended in 1 × PBS/1% BSA and analyzed by flow cytometry. The fluorescence intensity was converted to bound molecules/cells based on quantitative beads (QuantumTM MESF kit, Bangs Laboratories, Inc.). Affinity was calculated using Graphpad Prism 5. The data are shown in table 10.

Watch 10

Ab	KD(M)
		1.53.3-uAb-IgG4k	1.60E-10
3.40.19-uAb-IgG4L	5.30E-11
		BMK1	2.70E-10
BMK7	5.80E-10
		BMK8	9.40E-10

The antibodies of the invention represented by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L bound human LAG-3 efficiently and with binding better than or equal to the baseline antibody as tested by SPR and FACS.

Example 8

Orthologues (across species) and homologs (across families) binding

Cross-reactivity with cynomolgus monkey LAG-3 and murine LAG-3

Cross-reactivity to cynomolgus and murine LAG-3 was measured by FACS. Flp-In-CHO cells expressing murine LAG-3 or cynomolgus monkey LAG-3 expressing 293F cells at 1x10 ⁵ The density of individual cells/well was transferred to a 96-well U-bottom plate. The test antibodies were serially diluted in wash buffer (1 XPBS/1% BSA) and incubated with the cells for 1 hour at 4 ℃. After washing with 1 XPBS/1% BSA, the corresponding secondary antibody was applied and the cells were incubated for 1 hour at 4 ℃ in the dark. Cells were then washed and resuspended in 1 XPBS/1% BSA and then analyzed by flow cytometry. The data are shown in fig. 5 and 6. EC (EC) ₅₀ Shown in table 11.

TABLE 11

Ab	EC ₅₀ (nM)
		1.53.3-uAb-IgG4k	4.01
3.40.19-uAb-IgG4L	3.92
		BMK1	86.0
BMK7	2.65
		BMK8	3.05

As shown in FIG. 5, the LAG-3 antibodies "1.53.3-uAb-IgG 4 k" and "3.40.19-uAb-IgG 4L" of the present invention bind to cynomolgus monkey LAG-3 on the cell surface. As shown in fig. 6, the LAG-3 antibodies "1.53.3-uAb-IgG 4 k" and "3.40.19-uAb-IgG 4L" of the present invention did not bind to cell surface mouse LAG-3.

Cross-reactivity with human CD4

Cross-reactivity with human CD4 was measured by ELISA. Plates were coated with 1. mu.g/mL human CD4 overnight at 4 ℃. After blocking and washing, 1. mu.g/mL of LAG-3 antibody was added to the plate and incubated at room temperature for 1 hour. The plates were then washed and subsequently incubated with the corresponding secondary antibodies for 45 minutes. After washing, TMB substrate was added and the chromogenic reaction was stopped with 2M HCl. Absorbance at 450nm was read using a microplate reader. The data are shown in FIG. 7.

These results indicate that the LAG-3 antibodies "1.53.3-uAb-IgG 4 k" and "3.40.19-uAb-IgG 4L" of the present invention do not bind to human CD4 protein.

Example 9

Epitope binning for BMK1, BMK7, and BMK5

The binding epitopes of the LAG-3 antibody were binned by FACS analysis against the reference antibodies BMK1, BMK 7and BMK 5. Flp-In-293 cells expressing human LAG-3 on the cell surface were incubated with biotinylated reference antibody at a concentration of 1ug/mL for 1 hour, followed by addition of serial dilutions of LAG-3 antibody. The binding of the reference antibody to the cells was detected using a streptavidin-PE antibody (Jackson Immunoresearch Lab). MFI was assessed by flow cytometry and analyzed by FlowJo. The data are shown in FIGS. 8A-E.

1.53.3-uAb-IgG4k of the invention was found to compete with BMK, but 3.40.19-uAb-IgG4L did not compete with BMK. 1.53.3-uAb-IgG4k shares a similar epitope with BMK1 and BMK7, but not with BMK 5. Surprisingly, 3.40.19-uAb-IgG4L has a different epitope than all of BMK1, BMK7, and BMK 5.

Example 10

Domain mapping and epitope mapping

1. Domain mapping

LAG-3 has an extracellular domain of 421aa (P30-L450) and includes four extracellular immunoglobulin superfamily (IgSF) -like domains, namely domain 1 ("D1," aa.37-167), domain 2 ("D2," aa 168-252 "), domain 3 (" D3, "aa.265-343), and domain 4 (" D4, "aa.348-419). 10 variants were constructed by replacing the following residues of the human LAG-3 extracellular domain with the corresponding mouse LAG-3 amino acid (also referred to as "aa" in the context of this disclosure).

(1) Variant 1: xPro1.FL-x1 human LAG-3 aa 168 to 419 replacement by the mouse counterpart

(2) Variant 2: xPro1.FL-x2 replacement of human LAG-3 aa 37 to 167 and aa 265 to 419 by the mouse counterpart

(3) Variant 3 xPro1.FL-x3 replacement of human LAG-3 aa 37 to 252 and aa 348-419 by the corresponding part of the mouse

(4) Variant 4: xPro1.FL-x4 human LAG-3 aa 37 to 343 replaced by the mouse counterpart

(5) Variant 5 xPro1.FL-x5 replacement of human LAG-3 aa 265 to 419 by the mouse counterpart

(6) Variant 6 xPro1.FL-x6 replacement of human LAG-3 aa 37 to 167 and aa 348 to 419 by the mouse counterpart

(7) Variant 7 xPro1.FL-x7 replacement of human LAG-3 aa 37 to 252 by the mouse counterpart

(8) Variant 8 xPro1.FL-x8 replacement of human LAG-3 aa 168 to 343 by the mouse counterpart

(9) Variant 9: xPro1.FL-x9 human LAG-3 aa 348 to 419 replacement by the mouse counterpart

(10) Variant 10: xPro1.FL-x10 human LAG-3 aa 37 to 167 replacement by the mouse counterpart

These 10 variants were cloned into pcDNA3 vector and used for 293F cell transfection. Briefly, 293F cells were diluted to a density of 1X10 with FreeStyle 293F medium ⁶ Individual cells/mL, and 3 mL/well aliquots were added to 24-well plates. Transfection was performed using 293fectin reagent (Life Technologies). For each transfection, 3 μ g of DNA was diluted in 150 μ L of Opti-MEM I serum-reduced medium (Life Technologies) and then combined with 6 μ L of 293fectin reagent pre-diluted in 150 μ L of Opti-MEM I serum-reduced medium. The DNA/Lipofectamine mixture was allowed to stand at 25 ℃ for 20 minutes before addition to the culture. Transfected cells were analyzed by flow cytometry 48 hours after transfection.

Antibody binding to chimeric LAG-3 variants or full-length human/mouse LAG-3 was analyzed by flow cytometry. Briefly, 1. mu.g/mL of antibody was incubated with transfected 293F cells expressing chimeric LAG-3 for 1 hour at 4 ℃ followed by 3. mu.g/mL of goat anti-human IgG Fc R-PE (Jackson) for 40 minutes at 4 ℃. Cells were analyzed by flow cytometry.

The binding ability of antibodies 1.53.3-uAb-IgG4k and 3.40.19-uAb-hIgG4L to these 10 variants was examined and the results are shown in table 12 below.

TABLE 12 bound MFI values of LAG-3 antibodies to 10 variants

Both lead antibodies, i.e., "1.53.3-uAb-IgG 4 k" and "3.40.19-uAb-hIgG 4L", bind to domain 1 (i.e., aa.37-167) according to the FACS binding activity of the antibody. Thus, further epitope mapping of domain 1(G37-Q167,131aa) was performed by alanine scanning experiments.

2. Epitope mapping

Alanine scanning experiments were performed on human LAG-3 for epitope mapping. Alanine residues on human LAG-3 were mutated to glycine codons and all other residues (except cysteine residues) were mutated to alanine codons. For each residue of the extracellular domain (ECD) of human LAG-3, site amino acid substitutions were made using two sequential PCR steps. The pcDNA3.3-LAG-3-D12.mFc plasmid encoding ECD domain 1 and domain 2 of human LAG-3 and the C-terminal mFc-tag was used as template and a set of mutagenesis primers was used for the first step PCR using QuikChange lightning multiple site directed mutagenesis kit (Agilent technologies, Palo Alto, Calif.). After the mutant strand synthesis reaction, the parental template is digested with the Dpn I endonuclease. In a second PCR, a linear DNA expression cassette comprising the CMV promoter, extracellular domains 1 and 2 of LAG-3 (D1 and D2), the mFc-tag and herpes simplex virus Thymidine Kinase (TK) polyadenylation was amplified and transiently expressed in Expi293 cells at 37 ℃ (Life Technologies, Gaithersburg, Md) and quantified by protein A-HPLC and mFc-ELISA quantification kit (Bethyyl, USA).

For ELISA binding assays, antibodies 1.53.3-uAb-IgG4k or 3.40.19-uAb-hIgG4L (2 μ g/mL) were coated on plates. HRP-conjugated anti-mFc antibody (1: 5000; Bethyl, USA) was added as detection antibody after interaction with supernatant containing quantified LAG-3 mutant or human LAG-3-ECD.D12.mFc protein. The absorbance was normalized to the average absorbance of the control mutants. After setting an additional cut-off (<0.75) for the fold change in binding, the finally determined epitope residues were identified. The hot spots of antibodies 1.53.3-uAb-IgG4k and 3.40.19-uAb-hIgG4L are shown in tables 13 and 14.

TABLE 13.1.53.3 Hot Point of uAb-IgG4k antibody

TABLE 14.3.40.19 Hot Point of uAb-IgG4L antibody

The structure of LAG-3(aa:31-431) was modeled based on the known myelin-linked glycoprotein structure (PDB:5FLU, 18% sequence identity) due to the absence of the LAG-3 structure. Based on the alanine scan results, hot spots for both antibodies were identified and are shown in fig. 9A and 9B.

Based on the results, it can be seen that the 1.53.5-uAb-IgG4k antibody binds to the W92 site belonging to the outer loop (G70-Y99), while the 3.40.19-uAb-IgG4L antibody binds to the L134-P138 region.

Example 11

In vitro function of LAG-3 antibodies tested by cell-based assays

Role of human LAG-3 antibody in reporter gene assay

Jurkat cells expressing human LAG-3 and having a stably integrated IL-2 luciferase reporter gene were seeded in 96-well plates with Raji cells in the presence of SEE. Test antibodies were added to the cells. Plates were incubated at 37 ℃ with 5% CO ₂ Incubate overnight. After incubation, reconstituted luciferase substrate was added and luciferase intensity was measured by microplate spectrophotometer. Data are shown in FIG. 10, EC ₅₀ Shown in table 15.

Watch 15

Ab	EC ₅₀ (nM)
		1.53.3-uAb-IgG4k	1.07
3.40.19-uAb-IgG4L	0.21
		BMK1	0.59
BMK7	2.65
		BMK8	65.3

As shown in FIG. 10, LAG-3 antibody enhanced the IL-2 pathway of Jurkat in a reporter gene assay. Furthermore, as shown in Table 15, EC of 3.40.19-uAb-IgG4L in this assay ₅₀ Significantly lower than all three baseline antibodies.

Effect of human LAG-3 antibody on human allogenic Mixed lymphocyte reaction

Human Peripheral Blood Mononuclear Cells (PBMC) were freshly isolated from healthy donors using Ficoll-Paque PLUS gradient centrifugation. Monocytes were isolated using a human monocyte enrichment kit according to the manufacturer's instructions. The cells were cultured in a medium containing GM-CSF and IL-4 for 5 to 7 days to generate Dendritic Cells (DCs). Using human CD4 according to the manufacturer's protocol ⁺ T cell enrichment kit for separating human CD4 ⁺ T cells. Purifying CD4 ⁺ T cells were co-cultured with allogeneic immature dc (idc) and various concentrations of LAG-3 antibody in 96-well plates. On day 5, culture supernatants were collected for IFN- γ and T cell proliferation assays. Human IFN- γ was measured by ELISA using matched antibody pairs. The plates were pre-coated with a capture antibody specific for human IFN-. gamma. (Pierce-M700A). Use of biotin-conjugated anti-IFN-gamma antibodies (Pierc)e-M701B) as a detection antibody. At the last 16 hours, add at 1. mu. Ci/well ³ H-thymidine. Measured by scintillation counting ³ H-thymidine incorporation and proliferation response is expressed as CPM (counts per minute) of triplicate wells. The data are shown in figures 11 and 12.

As shown in FIG. 11, the LAG-3 antibodies "1.53.3-uAb-IgG 4 k" and "3.40.19-uAb-IgG 4L" of the present invention enhanced IFN- γ secretion in mixed lymphocyte reactions. Furthermore, as shown in fig. 12, the LAG-3 antibodies "1.53.3-uAb-IgG 4 k" and "3.40.19-uAb-IgG 4L" of the present invention enhanced T cell proliferation in mixed lymphocyte reactions.

Example 12

ADCC and CDC assays

To assess the ability to trigger Fc effector function, it was assessed whether anti-LAG-3 antibodies can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities.

ADCC assay

Flp-In-293 cells expressing human LAG-3 and various concentrations of LAG-3 antibody were preincubated In 96-well round bottom plates for 30 min; PBMC were then added as effectors at an effector/target ratio of 50: 1. the plates were maintained at 37 ℃ and 5% CO ₂ The next 4 hours. Target cell lysis was determined by LDH-based cytotoxicity detection kit. The absorbance at 492nm was read using a microplate reader. Herceptin and HER2 expressing cell line SK-Br-3 were used as positive controls.

CDC test

Flp-In-293 expressing human LAG-3 as a target and various concentrations of LAG-3 antibody were mixed In 96-well round bottom plates. Human complement was added at a final dilution of 1: 50. The plates were maintained at 37 ℃ and 5% CO ₂ And then for 2 hours. Target cell lysis was determined by CellTiter-Glo. The absorbance was read using a microplate reader. Rituximab and the cell line Raji expressing CD20 were used as positive controls.

Figures 13A and 13B show data for ADCC and CDC assays. It was demonstrated that the LAG-3 antibody of the present invention, represented by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L, did not mediate ADCC (FIG. 13A) and CDC (FIG. 13B) effects.

Example 13

Serum stability test

The lead Ab was incubated in freshly isolated human serum (serum content > 95%) at 37 ℃. At the indicated time points, aliquots of the serum-treated samples were removed from the incubator and snap frozen in liquid nitrogen, then stored at 80 ℃ until ready for testing. The samples were quickly thawed immediately prior to stability testing. Human LAG-3 transfected cells were incubated with various concentrations of leader antibody for 1 hour at 4 ℃. PE-labeled goat anti-human IgG was used to detect binding of the lead antibody to the cells. The MFI of the cells was measured by flow cytometry (BD facscan II) and analyzed by FlowJo. The data are shown in fig. 14A and 14B.

The LAG-3 antibody of the present invention, represented by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L, was demonstrated to be stable in fresh human serum for 14 days.

Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from the spirit or central characteristics thereof. Since the foregoing description of the invention discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the following claims as indicating the scope and content of the invention.

Sequence listing

<110> Guangzhou Yuheng Biotechnology Limited

<120> anti-human LAG-3 antibody and use thereof

<130> IDC226033

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<170> PatentIn version 3.5

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Gly Asp Ser Ile Ser Ser Thr Ser Tyr Tyr Trp Gly

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Met Gln Leu Trp Ser Tyr Asp Val Asp Val

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Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asp Tyr Val Ala

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Asp Val Ser Glu Arg Pro Ser

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Ser Ser Tyr Thr Ser Thr Thr Thr Leu Val Val

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<223> 1.53.3-uAb-IgG4k VH

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Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

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Thr Leu Ser Leu Thr Cys Gly Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Met Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys

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Ser Arg Val Thr Ile Ser Glu Asp Thr Ser Lys Asn Gln Phe Ser Leu

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Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys Thr

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Arg Gly Glu Asp Tyr Ser Asp Tyr Asp Tyr Tyr Gly Asp Phe Trp Gly

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Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 107

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<223> 1.53.3-uAb-IgG4k VL

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Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Gln Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Ile Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> 3.40.19-uAb-IgG4L VH

<400> 15

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Ser Thr

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

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Trp Ile Gly Ser Phe Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

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Cys Ala Arg Met Gln Leu Trp Ser Tyr Asp Val Asp Val Trp Gly Gln

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Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 16

<211> 111

<212> PRT

<213> Artificial sequence

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<223> 3.40.19-uAb-IgG4L VL

<400> 16

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asp Tyr Val Ala Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Glu Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Thr

85 90 95

Thr Thr Leu Val Val Phe Gly Gly Gly Thr Lys Leu Ser Val Leu

100 105 110

<210> 17

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> 1.53.3-uAb-IgG4k VH

<400> 17

caggtgcagc tacagcagtg gggcgcagga cttttgaagc cttcggagac cctgtccctc 60

acctgcggtg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggatgg ggctggagtg gattggggaa atcaatcatc gtggaaacac caactacaac 180

ccgtccctca agagtcgcgt caccatatca gaagacacgt ccaagaacca gttctccctg 240

aggctgagct ctgtgaccgc cgcggacacg gctgtgtatt tctgtacgag aggagaggac 300

tatagtgact acgattacta tggggacttc tggggccagg gaaccctggt caccgtctcc 360

tca 363

<210> 18

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> 1.53.3-uAb-IgG4k VL

<400> 18

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctcaagggga aagagccacc 60

ctctcctgca gggccagtca gagtattagc agctacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgct gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg caatttatta ctgtcagcag cgtagcaact ggcctctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 19

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> 3.40.19-uAb-IgG4L VH

<400> 19

cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtga ctccatcagc agtactagtt actactgggg ctggatccgc 120

cagcccccag ggaaggggct ggagtggatt gggagtttct attatagtgg gagcacctac 180

tacaacccgt ccctcaagag tcgagtcacc atttccgtag acacgtccaa gaaccagttc 240

tccctgaagc tgaactctgt gaccgccgca gacacggctg tgtattactg tgcgaggatg 300

cagctatggt cgtacgatgt ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 20

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> 3.40.19-uAb-IgG4L VL

<400> 20

cagtctgccc tgactcaacc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgacgttggt gggtatgact atgtcgcctg gtaccaacaa 120

cacccaggca aagtccccaa actcatgatt tatgatgtca gtgagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc agctcatata caagcaccac cactctcgtt 300

gtgttcggcg gagggaccaa gctgtccgtc ctg 333

<210> 21

<211> 421

<212> PRT

<213> Artificial sequence

<220>

<223> human LAG-3ECD

<400> 21

Pro Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser

1 5 10 15

Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val

20 25 30

Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly

35 40 45

His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly

50 55 60

Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu

65 70 75 80

Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg

85 90 95

Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg

100 105 110

Ala Asp Ala Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala

115 120 125

Leu Ser Cys Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala

130 135 140

Ser Pro Pro Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn Cys

145 150 155 160

Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn

165 170 175

Arg Gly Gln Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu

180 185 190

Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly

195 200 205

Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile

210 215 220

Met Tyr Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr

225 230 235 240

Val Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro

245 250 255

Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro

260 265 270

Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe Thr

275 280 285

Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys

290 295 300

His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala

305 310 315 320

Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly

325 330 335

Lys Leu Leu Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val

340 345 350

Trp Ser Ser Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp

355 360 365

Leu Glu Ala Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln

370 375 380

Leu Tyr Gln Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu

385 390 395 400

Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu

405 410 415

Pro Ala Gly His Leu

420

<210> 22

<211> 1263

<212> DNA

<213> Artificial sequence

<220>

<223> human LAG-3ECD

<400> 22

ccggtggtgt gggcccagga gggggctcct gcccagctcc cctgcagccc cacaatcccc 60

ctccaggatc tcagccttct gcgaagagca ggggtcactt ggcagcatca gccagacagt 120

ggcccgcccg ctgccgcccc cggccatccc ctggcccccg gccctcaccc ggcggcgccc 180

tcctcctggg ggcccaggcc ccgccgctac acggtgctga gcgtgggtcc cggaggcctg 240

cgcagcggga ggctgcccct gcagccccgc gtccagctgg atgagcgcgg ccggcagcgc 300

ggggacttct cgctatggct gcgcccagcc cggcgcgcgg acgccggcga gtaccgcgcc 360

gcggtgcacc tcagggaccg cgccctctcc tgccgcctcc gtctgcgcct gggccaggcc 420

tcgatgactg ccagcccccc aggatctctc agagcctccg actgggtcat tttgaactgc 480

tccttcagcc gccctgaccg cccagcctct gtgcattggt tccggaaccg gggccagggc 540

cgagtccctg tccgggagtc cccccatcac cacttagcgg aaagcttcct cttcctgccc 600

caagtcagcc ccatggactc tgggccctgg ggctgcatcc tcacctacag agatggcttc 660

aacgtctcca tcatgtataa cctcactgtt ctgggtctgg agcccccaac tcccttgaca 720

gtgtacgctg gagcaggttc cagggtgggg ctgccctgcc gcctgcctgc tggtgtgggg 780

acccggtctt tcctcactgc caagtggact cctcctgggg gaggccctga cctcctggtg 840

actggagaca atggcgactt tacccttcga ctagaggatg tgagccaggc ccaggctggg 900

acctacacct gccatatcca tctgcaggaa cagcagctca atgccactgt cacattggca 960

atcatcacag tgactcccaa atcctttggg tcacctggat ccctggggaa gctgctttgt 1020

gaggtgactc cagtatctgg acaagaacgc tttgtgtgga gctctctgga caccccatcc 1080

cagaggagtt tctcaggacc ttggctggag gcacaggagg cccagctcct ttcccagcct 1140

tggcaatgcc agctgtacca gggggagagg cttcttggag cagcagtgta cttcacagag 1200

ctgtctagcc caggtgccca acgctctggg agagccccag gtgccctccc agcaggccac 1260

ctc 1263

<210> 23

<211> 525

<212> PRT

<213> Artificial sequence

<220>

<223> full-Length human LAG-3

<400> 23

Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp

1 5 10 15

Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val

20 25 30

Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile

35 40 45

Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu

65 70 75 80

Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro

85 90 95

Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly

100 105 110

Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln

115 120 125

Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala

130 135 140

Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser Cys

145 150 155 160

Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro

165 170 175

Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser

180 185 190

Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln

195 200 205

Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser

210 215 220

Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly

225 230 235 240

Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn

245 250 255

Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala

260 265 270

Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala Gly Val

275 280 285

Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly

290 295 300

Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu

305 310 315 320

Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His

325 330 335

Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr

340 345 350

Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu

355 360 365

Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser

370 375 380

Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala

385 390 395 400

Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln

405 410 415

Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser

420 425 430

Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly

435 440 445

His Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu

450 455 460

Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro

465 470 475 480

Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln

485 490 495

Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro Glu Pro

500 505 510

Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu

515 520 525

<210> 24

<211> 1575

<212> DNA

<213> Artificial sequence

<220>

<223> full-Length human LAG-3

<400> 24

atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccagtg 60

aagcctctcc agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc 120

cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg 180

gtcacttggc agcatcagcc agacagtggc ccgcccgctg ccgcccccgg ccatcccctg 240

gcccccggcc ctcacccggc ggcgccctcc tcctgggggc ccaggccccg ccgctacacg 300

gtgctgagcg tgggtcccgg aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc 360

cagctggatg agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg 420

cgcgcggacg ccggcgagta ccgcgccgcg gtgcacctca gggaccgcgc cctctcctgc 480

cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca gccccccagg atctctcaga 540

gcctccgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg 600

cattggttcc ggaaccgggg ccagggccga gtccctgtcc gggagtcccc ccatcaccac 660

ttagcggaaa gcttcctctt cctgccccaa gtcagcccca tggactctgg gccctggggc 720

tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct cactgttctg 780

ggtctggagc ccccaactcc cttgacagtg tacgctggag caggttccag ggtggggctg 840

ccctgccgcc tgcctgctgg tgtggggacc cggtctttcc tcactgccaa gtggactcct 900

cctgggggag gccctgacct cctggtgact ggagacaatg gcgactttac ccttcgacta 960

gaggatgtga gccaggccca ggctgggacc tacacctgcc atatccatct gcaggaacag 1020

cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc ctttgggtca 1080

cctggatccc tggggaagct gctttgtgag gtgactccag tatctggaca agaacgcttt 1140

gtgtggagct ctctggacac cccatcccag aggagtttct caggaccttg gctggaggca 1200

caggaggccc agctcctttc ccagccttgg caatgccagc tgtaccaggg ggagaggctt 1260

cttggagcag cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga 1320

gccccaggtg ccctcccagc aggccacctc ctgctgtttc tcatccttgg tgtcctttct 1380

ctgctccttt tggtgactgg agcctttggc tttcaccttt ggagaagaca gtggcgacca 1440

agacgatttt ctgccttaga gcaagggatt caccctccgc aggctcagag caagatagag 1500

gagctggagc aagaaccgga gccggagccg gagccggaac cggagcccga gcccgagccc 1560

gagccggagc agctc 1575

<210> 25

<211> 521

<212> PRT

<213> Artificial sequence

<220>

<223> full Length mouse LAG-3

<400> 25

Met Arg Glu Asp Leu Leu Leu Gly Phe Leu Leu Leu Gly Leu Leu Trp

1 5 10 15

Glu Ala Pro Val Val Ser Ser Gly Pro Gly Lys Glu Leu Pro Val Val

20 25 30

Trp Ala Gln Glu Gly Ala Pro Val His Leu Pro Cys Ser Leu Lys Ser

35 40 45

Pro Asn Leu Asp Pro Asn Phe Leu Arg Arg Gly Gly Val Ile Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Gln Pro Thr Pro Ile Pro Ala Leu Asp Leu

65 70 75 80

His Gln Gly Met Pro Ser Pro Arg Gln Pro Ala Pro Gly Arg Tyr Thr

85 90 95

Val Leu Ser Val Ala Pro Gly Gly Leu Arg Ser Gly Arg Gln Pro Leu

100 105 110

His Pro His Val Gln Leu Glu Glu Arg Gly Leu Gln Arg Gly Asp Phe

115 120 125

Ser Leu Trp Leu Arg Pro Ala Leu Arg Thr Asp Ala Gly Glu Tyr His

130 135 140

Ala Thr Val Arg Leu Pro Asn Arg Ala Leu Ser Cys Ser Leu Arg Leu

145 150 155 160

Arg Val Gly Gln Ala Ser Met Ile Ala Ser Pro Ser Gly Val Leu Lys

165 170 175

Leu Ser Asp Trp Val Leu Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg

180 185 190

Pro Val Ser Val His Trp Phe Gln Gly Gln Asn Arg Val Pro Val Tyr

195 200 205

Asn Ser Pro Arg His Phe Leu Ala Glu Thr Phe Leu Leu Leu Pro Gln

210 215 220

Val Ser Pro Leu Asp Ser Gly Thr Trp Gly Cys Val Leu Thr Tyr Arg

225 230 235 240

Asp Gly Phe Asn Val Ser Ile Thr Tyr Asn Leu Lys Val Leu Gly Leu

245 250 255

Glu Pro Val Ala Pro Leu Thr Val Tyr Ala Ala Glu Gly Ser Arg Val

260 265 270

Glu Leu Pro Cys His Leu Pro Pro Gly Val Gly Thr Pro Ser Leu Leu

275 280 285

Ile Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Glu Leu Pro Val Ala

290 295 300

Gly Lys Ser Gly Asn Phe Thr Leu His Leu Glu Ala Val Gly Leu Ala

305 310 315 320

Gln Ala Gly Thr Tyr Thr Cys Ser Ile His Leu Gln Gly Gln Gln Leu

325 330 335

Asn Ala Thr Val Thr Leu Ala Val Ile Thr Val Thr Pro Lys Ser Phe

340 345 350

Gly Leu Pro Gly Ser Arg Gly Lys Leu Leu Cys Glu Val Thr Pro Ala

355 360 365

Ser Gly Lys Glu Arg Phe Val Trp Arg Pro Leu Asn Asn Leu Ser Arg

370 375 380

Ser Cys Pro Gly Pro Val Leu Glu Ile Gln Glu Ala Arg Leu Leu Ala

385 390 395 400

Glu Arg Trp Gln Cys Gln Leu Tyr Glu Gly Gln Arg Leu Leu Gly Ala

405 410 415

Thr Val Tyr Ala Ala Glu Ser Ser Ser Gly Ala His Ser Ala Arg Arg

420 425 430

Ile Ser Gly Asp Leu Lys Gly Gly His Leu Val Leu Val Leu Ile Leu

435 440 445

Gly Ala Leu Ser Leu Phe Leu Leu Val Ala Gly Ala Phe Gly Phe His

450 455 460

Trp Trp Arg Lys Gln Leu Leu Leu Arg Arg Phe Ser Ala Leu Glu His

465 470 475 480

Gly Ile Gln Pro Phe Pro Ala Gln Arg Lys Ile Glu Glu Leu Glu Arg

485 490 495

Glu Leu Glu Thr Glu Met Gly Gln Glu Pro Glu Pro Glu Pro Glu Pro

500 505 510

Gln Leu Glu Pro Glu Pro Arg Gln Leu

515 520

<210> 26

<211> 1563

<212> DNA

<213> Artificial sequence

<220>

<223> full Length mouse LAG-3

<400> 26

atgagggagg acctgctcct tggctttttg cttctgggac tgctttggga agctccagtt 60

gtgtcttcag ggcctgggaa agagctcccc gtggtgtggg cccaggaggg agctcccgtc 120

catcttccct gcagcctcaa atcccccaac ctggatccta actttctacg aagaggaggg 180

gttatctggc aacatcaacc agacagtggc caacccactc ccatcccggc ccttgacctt 240

caccagggga tgccctcgcc tagacaaccc gcacccggtc gctacacggt gctgagcgtg 300

gctccaggag gcctgcgcag cgggaggcag cccctgcatc cccacgtgca gctggaggag 360

cgcggcctcc agcgcgggga cttctctctg tggttgcgcc cagctctgcg caccgatgcg 420

ggcgagtacc acgccaccgt gcgcctcccg aaccgcgccc tctcctgcag tctccgcctg 480

cgcgtcggcc aggcctcgat gattgctagt ccctcaggag tcctcaagct gtctgattgg 540

gtccttttga actgctcctt cagccgtcct gaccgcccag tctctgtgca ctggttccag 600

ggccagaacc gagtgcctgt ctacaactca ccgcgtcatt ttttagctga aactttcctg 660

ttactgcccc aagtcagccc cctggactct gggacctggg gctgtgtcct cacctacaga 720

gatggcttca atgtctccat cacgtacaac ctcaaggttc tgggtctgga gcccgtagcc 780

cctctgacag tgtacgctgc tgaaggttct agggtggagc tgccctgtca tttgccccca 840

ggagtgggga ccccttcttt gctcattgcc aagtggactc ctcctggagg aggtcctgag 900

ctccccgtgg ctggaaagag tggcaatttt acccttcacc ttgaggctgt gggtctggca 960

caggctggga cctacacctg tagcatccat ctgcagggac agcagctcaa tgccactgtc 1020

acgttggcgg tcatcacagt gactcccaaa tccttcgggt tacctggctc ccgggggaag 1080

ctgttgtgtg aggtaacccc ggcatctgga aaggaaagat ttgtgtggcg tcccctgaac 1140

aatctgtcca ggagttgccc gggccctgtg ctggagattc aggaggccag gctccttgct 1200

gagcgatggc agtgtcagct gtacgagggc cagaggcttc ttggagcgac agtgtacgcc 1260

gcagagtcta gctcaggcgc ccacagtgct aggagaatct caggtgacct taaaggaggc 1320

catctcgttc tcgttctcat ccttggtgcc ctctccctgt tccttttggt ggccggggcc 1380

tttggctttc actggtggag aaaacagttg ctactgagaa gattttctgc cttagaacat 1440

gggattcagc catttccggc tcagaggaag atagaggagc tggagcgaga actggagacg 1500

gagatgggac aggagccgga gcccgagccg gagccacagc tggagccaga gcccaggcag 1560

ctc 1563

<210> 27

<211> 533

<212> PRT

<213> Artificial sequence

<220>

<223> full-length cynomolgus monkey LAG-3

<220>

<221> misc_feature

<222> (74)..(74)

<223> Xaa can be any naturally occurring amino acid

<400> 27

Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp

1 5 10 15

Val Ala Pro Val Lys Pro Pro Gln Pro Gly Ala Glu Ile Ser Val Val

20 25 30

Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile

35 40 45

Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln

50 55 60

His Gln Pro Asp Ser Gly Pro Pro Ala Xaa Ala Pro Gly His Pro Pro

65 70 75 80

Val Pro Gly His Arg Pro Ala Ala Pro Tyr Ser Trp Gly Pro Arg Pro

85 90 95

Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly

100 105 110

Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln

115 120 125

Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala

130 135 140

Gly Glu Tyr Arg Ala Thr Val His Leu Arg Asp Arg Ala Leu Ser Cys

145 150 155 160

Arg Leu Arg Leu Arg Val Gly Gln Ala Ser Met Thr Ala Ser Pro Pro

165 170 175

Gly Ser Leu Arg Thr Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser

180 185 190

Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Ser Arg Gly Gln

195 200 205

Gly Arg Val Pro Val Gln Gly Ser Pro His His His Leu Ala Glu Ser

210 215 220

Phe Leu Phe Leu Pro His Val Gly Pro Met Asp Ser Gly Leu Trp Gly

225 230 235 240

Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn

245 250 255

Leu Thr Val Leu Gly Leu Glu Pro Ala Thr Pro Leu Thr Val Tyr Ala

260 265 270

Gly Ala Gly Ser Arg Val Glu Leu Pro Cys Arg Leu Pro Pro Ala Val

275 280 285

Gly Thr Gln Ser Phe Leu Thr Ala Lys Trp Ala Pro Pro Gly Gly Gly

290 295 300

Pro Asp Leu Leu Val Ala Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu

305 310 315 320

Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Ile Cys His Ile Arg

325 330 335

Leu Gln Gly Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr

340 345 350

Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu

355 360 365

Cys Glu Val Thr Pro Ala Ser Gly Gln Glu His Phe Val Trp Ser Pro

370 375 380

Leu Asn Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala

385 390 395 400

Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu His Gln

405 410 415

Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser

420 425 430

Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Arg Ala Gly

435 440 445

His Leu Pro Leu Phe Leu Ile Leu Gly Val Leu Phe Leu Leu Leu Leu

450 455 460

Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro

465 470 475 480

Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln

485 490 495

Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Leu Glu Pro Glu Pro

500 505 510

Glu Leu Glu Arg Glu Leu Gly Pro Glu Pro Glu Pro Gly Pro Glu Pro

515 520 525

Glu Pro Glu Gln Leu

530

<210> 28

<211> 1599

<212> DNA

<213> Artificial sequence

<220>

<223> full-length cynomolgus monkey LAG-3

<220>

<221> misc_feature

<222> (219)..(220)

<223> n is a, c, g, or t

<400> 28

atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctctgggt ggctccagtg 60

aagcctcccc agccaggggc tgagatctcg gtggtgtggg cccaggaggg ggctcctgcc 120

cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg 180

gtcacttggc agcatcaacc agacagtggc ccgcccgcnn ccgcccccgg ccaccccccg 240

gtccccggcc atcgcccggc ggcgccctac tcttgggggc ccaggccccg ccgctacacg 300

gtgctgagcg tgggtcctgg aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc 360

cagctggatg agcgcggccg gcagcgcggg gacttctcgc tgtggctgcg cccagcccgg 420

cgcgcggacg ccggcgagta ccgcgccacg gtgcacctca gggaccgcgc cctctcctgc 480

cgccttcgtc tgcgcgtggg ccaggcctcg atgactgcca gccccccagg gtctctcagg 540

acctctgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg 600

cattggttcc ggagccgtgg ccagggccga gtccctgtcc aggggtcccc ccatcaccac 660

ttagcggaaa gcttcctctt cctgccccat gtcggcccca tggactctgg gctctggggc 720

tgcatcctca cctacagaga tggcttcaat gtctccatca tgtataacct cactgttctg 780

ggtctggagc ccgcaactcc cttgacagtg tacgctggag caggttccag ggtggagctg 840

ccctgccgcc tgcctcctgc tgtggggacc cagtctttcc ttactgccaa gtgggctcct 900

cctgggggag gccctgacct cctggtggct ggagacaatg gcgactttac ccttcgacta 960

gaggatgtaa gccaggccca ggctgggacc tacatctgcc atatccgtct acagggacag 1020

cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc ctttgggtca 1080

cctggctccc tggggaagct gctttgtgag gtgactccag catctggaca agaacacttt 1140

gtgtggagcc ccctgaacac cccatcccag aggagtttct caggaccatg gctggaggcc 1200

caggaagccc agctcctttc ccagccttgg caatgccagc tgcaccaggg ggagaggctt 1260

cttggagcag cagtatactt cacagaactg tctagcccag gtgcacaacg ctctgggaga 1320

gccccagggg ccctccgagc aggccacctc ccgctgtttc tcatccttgg tgtccttttt 1380

ctgctccttt tggtgactgg agcctttggc tttcaccttt ggagaagaca gtggcgacca 1440

agaagatttt ctgccttaga gcaagggatt caccctccgc aggctcagag caagatagag 1500

gagctcgagc aagaaccgga gctggaacca gagccggagc tggagcgcga gctggggccg 1560

gagcccgagc cggggcctga gcccgagccg gagcagctc 1599

Claims

as shown in SEQ ID NO: 7, as shown in SEQ ID NO: 8, CDRH2 as shown in SEQ ID NO: 9, CDRH3 as shown in SEQ ID NO: 10, CDRL1 as shown in SEQ ID NO: 11, and CDRL2 as shown in SEQ ID NO: CDRL3 shown in fig. 12.

2. The isolated antibody, or antigen-binding portion thereof, of claim 1, wherein the isolated antibody, or antigen-binding portion thereof, comprises:

(A) heavy chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 15;

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 15 compared to an amino acid sequence having one or more amino acid additions, deletions and/or substitutions in the framework region; and/or

(B) Light chain variable region:

(i) comprises the amino acid sequence of SEQ ID NO: 16;

(iii) Comprises a nucleotide sequence substantially identical to SEQ ID NO: 16 compared to an amino acid sequence having one or more amino acid additions, deletions and/or substitutions in the framework region.

3. The isolated antibody, or antigen-binding portion thereof, of claim 1, which has one or more of the following properties:

(a) at 2X 10 ^-10 K of M or less _D Binding human LAG-3;

(d) inhibiting the binding of LAG-3 to LSECtin and/or galectin-3;

(e) bind human LAG-3 without a cross-family response; or

(f) Has no cross-reactivity with human CD 4.

4. The isolated antibody or antigen-binding portion thereof of any of claims 1-3, wherein the antibody is a monoclonal antibody, such as a fully human monoclonal antibody produced by a transgenic mammal, preferably a transgenic rat, more preferably a transgenic rat having a recombinant immunoglobulin locus.

5. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region of an isolated antibody as defined in any one of claims 1 to 4, e.g., the nucleic acid sequences set forth in SEQ ID NOS 19-20.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the expression vector of claim 6.

8. A pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as defined in any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

9. A method of preparing an antibody or antigen-binding portion thereof as defined in any one of claims 1 to 4, comprising the steps of:

-expressing the antibody or antigen-binding portion thereof in the host cell of claim 7; and

-isolating the antibody or antigen-binding portion thereof from the host cell.

10. Use of an antibody or antigen-binding portion thereof as defined in any one of claims 1 to 4 in the manufacture of a medicament for modulating an antigen-specific T cell response in a subject or modulating an immune response in a subject.

11. A method of inhibiting or blocking the in vitro binding of LAG-3 to MHC class II molecules, FGL1 molecules, LSECtin and/or galectin-3, comprising contacting the MHC class II molecules, FGL1 molecules, LSECtin and/or galectin-3 with an antibody or antigen binding portion thereof as defined in any one of claims 1 to 4 in vitro.

12. Use of an antibody or antigen-binding portion thereof as defined in any one of claims 1 to 4 in the manufacture of a medicament for inhibiting the growth of tumor cells in a subject.

13. Use of an antibody or antigen-binding portion thereof as defined in any one of claims 1 to 4 in the manufacture of a medicament for the treatment or prevention of cancer, an autoimmune disease, an infectious disease and/or an inflammatory disease in a subject.

14. The use of claim 13, wherein the infectious disease is a viral infection.

15. Use of an antibody or antigen-binding portion thereof as defined in any one of claims 1 to 4 in the manufacture of a diagnostic agent for the diagnosis of a proliferative disorder, such as cancer.

16. A kit comprising a container containing an antibody or antigen-binding portion thereof as defined in any one of claims 1-4.