HK40076079B - Antibodies against human lag-3 and uses thereof - Google Patents

Description

Anti-human LAG-3 antibodies and their uses

This application is a divisional application of Chinese invention patent application No. 201910146172.2, filed on February 27, 2019, entitled "Anti-human LAG-3 monoclonal antibody and its preparation method and use".

sequence list

This application contains a sequence list, the entire contents of which are incorporated herein by reference.

Technical Field

This application generally relates to antibodies. More specifically, this application relates to fully human monoclonal antibodies that bind to human LAG-3, methods for their preparation, and their uses.

Background Technology

Lymphocyte activation gene 3 (CD223), also known as LAG-3, is a type I transmembrane protein and 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 not on resting T cells. LAG-3 shares approximately 20% amino acid sequence homology with CD4, but binds with higher affinity to MHC class II molecules and fibrinoid protein 1 (FGL1) molecules, a major functional ligand of LAG-3 independent of MHC-II, thereby providing negative regulation of T cell receptor signaling.

In vitro blockade of LAG-3 enhances T cell proliferation and cytokine production, and LAG-3-deficient mice exhibit defects in the downregulation of T cell responses induced by infection with superantigen Staphylococcus enterotoxin B, peptide, or Sendai virus. LAG-3 is expressed on activated native Treg (nTreg) and induced CD4 ⁺ FoxP3 ⁺ Treg (iTreg) cells, with expression levels higher than those observed on activated effector CD4 ⁺ T cells. Blockade of LAG-3 on Treg cells eliminates the repressor function of Treg cells, while ectopic expression of LAG-3 in non-Treg CD4 ⁺ T cells confers inhibitory activity. Based on the immunomodulatory role of LAG-3 in T cell function during chronic infection and cancer, the predicted mechanism of action of LAG-3-specific monoclonal antibodies is the inhibition of negative 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 responses to treat advanced solid tumors. These antibodies are described in patents US 20110150892 A1, US 20170101472A1, and WO2015138920A1, hereinafter referred to as BMK1, BMK7, and BMK5, respectively. As described herein, BMK8 is a humanized form of the chimeric antibody BMK5. BMK1, BMK7, and BMK8 are used as baseline antibodies in the context of this application. Therefore, there remains a need for anti-human LAG-3 antibodies with improved efficacy (e.g., high binding affinity, low cross-family reactivity, and good stability). In this application, the inventors generated a series of antibodies against LAG-3 and fully human antibodies using humanized rats. The antibodies of this application have high binding affinity, specifically bind to human LAG-3 protein without cross-family reactivity, and effectively modulate the immune response.

Invention Overview

In a broad sense, this invention relates to novel compounds, methods, compositions, and articles providing antibodies with improved efficacy. The benefits provided by this invention are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in combination with antibodies capable of reacting to a variety of targets. This invention provides antibodies that bind to human LAG-3, preferably fully human monoclonal antibodies. It also provides a method for generating hybridomas using humanized rats, nucleic acid molecules encoding anti-LAG-3 antibodies, expression vectors for expressing anti-LAG-3 antibodies, and host cells. This invention further provides a method for in vitro verification of antibody function. The antibodies of this invention provide effective agents for treating a variety of diseases by modulating human immune function.

In some aspects, the present invention includes isolated antibodies or antigen-binding portions thereof.

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

(a) Human LAG-3 is bound at 2× ^10⁻¹⁰ M or lower _KD ;

(b) Inhibits the binding of LAG-3 to major histocompatibility (MHC) class II molecules;

(c) Inhibits the binding of LAG-3 to fibrinoid protein 1 (FGL1) ligand molecules;

(d) Inhibit the binding of LAG-3 to LSECtin and/or galactolectin-3;

(e) binds to human LAG-3 without causing cross-family reactions; or

(f) It has no cross-reactivity with human CD4.

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

A) One or more heavy chain CDRs (CDRHs) selected from at least one of the following: (i) CDRH1 having at least 90% sequence identity with CDRH1 represented by one of the sequences selected from SEQ ID NO: 1 and 7; (ii) CDRH2 having at least 90% sequence identity with CDRH2 represented by one of the sequences selected from SEQ ID NO: 2 and 8; and (iii) CDRH3 having at least 90% sequence identity with CDRH3 represented by one of the sequences selected from SEQ ID NO: 3 and 9;

B) One or more light chain CDRs (CDRLs) selected from at least one of the following: (i) CDRL1 having at least 90% sequence identity with CDRL1 represented by one of the sequences selected from SEQ ID NO: 4 and 10; (ii) CDRL2 having at least 90% sequence identity with CDRL2 represented by one of the sequences selected from SEQ ID NO: 5 and 11; and (iii) CDRL3 having at least 90% sequence identity with CDRL3 represented by one of the sequences selected from SEQ ID NO: 6 and 12; or

C) One or more CDRHs of A) and B) One or more CDRLs of B).

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

A) One or more (e.g., 1, 2 or 3) heavy chain CDRs (CDRHs) selected from at least one of the following: (i) CDRH1 selected from SEQ ID NO: 1 and 7 or CDRH1 with an amino acid sequence difference of no more than 2 amino acids from the CDRH1; (ii) CDRH2 selected from SEQ ID NO: 2 and 8 or CDRH2 with an amino acid sequence difference of no more than 2 amino acids from the CDRH2; and (iii) CDRH3 selected from SEQ ID NO: 3 and 9 or CDRH3 with an amino acid sequence difference of no more than 2 amino acids from the CDRH3;

B) One or more (e.g., 1, 2, or 3) light chain CDRs (CDRLs) selected from at least one of the following: (i) CDRL1 selected from SEQ ID NO: 4 and 10 or CDRL1 differing from the amino acid sequence of CDRL1 by no more than 2 amino acid additions, deletions, or substitutions; (ii) CDRL2 selected from SEQ ID NO: 5 and 11 or CDRL2 differing from the amino acid sequence of CDRL2 by no more than 2 amino acid additions, deletions, or substitutions; and (iii) CDRL3 selected from SEQ ID NO: 6 and 12 or CDRL3 differing from the amino acid sequence of CDRL3 by no more than 2 amino acid additions, deletions, or substitutions; or

C) One or more CDRHs of A) and B) One or more CDRLs of B).

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

A) CDRH3 containing SEQ ID NO: 3 or 9; or

B) CDRH3 having at least 90% sequence identity with CDRH3 represented by one of the sequences selected from SEQ ID NO: 3 and 9; or

C) CDRH3, whose amino acid sequence differs from that of CDRH3 in A) by no more than two amino acid additions, deletions, or substitutions.

Furthermore, the isolated antibody or its antigen-binding portion binds to human LAG-3 at a _KD of 2 × ^10⁻¹⁰ M or lower.

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

(a) CDRH1 containing SEQ ID NO: 1 or consisting of therefrom;

(b) Contains SEQ ID NO: 2 or CDRH2 consisting of SEQ ID NO: 2;

(c) Contains SEQ ID NO: 3 or CDRH3 consisting of SEQ ID NO: 3;

(d) Contains SEQ ID NO: 4 or CDRL1 consisting of SEQ ID NO: 4;

(e) CDRL2 containing SEQ ID NO: 5 or consisting of therein; and

(f) Contains SEQ ID NO: 6 or CDRL3 composed of it.

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

(a) CDRH1 containing SEQ ID NO: 7 or consisting of therein;

(b) CDRH2 containing SEQ ID NO: 8 or consisting of therefrom;

(c) Contains SEQ ID NO: 9 or CDRH3 consisting of SEQ ID NO: 9;

(d) Contains SEQ ID NO: 10 or CDRL1 consisting of SEQ ID NO: 10;

(e) CDRL2 containing SEQ ID NO: 11 or consisting of therein; and

(f) Contains SEQ ID NO: 12 or CDRL3 composed of therefrom.

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

(A) Heavy chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 13; or

(iii) An amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4, or 5) amino acids added, deleted, and/or substituted compared to SEQ ID NO: 13; and/or

(B) Light chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 14; or

(iii) An amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4 or 5) amino acids added, deleted and/or substituted compared to SEQ ID NO: 14.

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

(A) Heavy chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 15; or

(iii) An amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4, or 5) amino acids added, deleted, and/or substituted compared to SEQ ID NO: 15; and/or

(B) Light chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 16; or

(iii) An amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4 or 5) amino acids added, deleted and/or substituted compared to SEQ ID NO: 16.

In some aspects, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and/or light chain variable regions of isolated antibodies as disclosed herein.

In some aspects, the present invention relates to expression vectors comprising nucleic acid molecules encoding antibodies or antigen-binding moieties as disclosed herein.

In some respects, the present invention relates to host cells comprising expression vectors as disclosed herein.

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

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

In some aspects, the present invention relates to methods for modulating antigen-specific T cell responses, including administering to a subject an antibody or antigen-binding portion thereof as disclosed herein, such that antigen-specific T cell responses in the subject are modulated.

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

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

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

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

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

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

In some aspects, the present invention relates to the use of antibodies or antigen-binding portions thereof as disclosed herein in the preparation of medicaments for the treatment or prevention of proliferative diseases such as cancer.

In some aspects, the present invention relates to the use of antibodies or antigen-binding portions thereof as disclosed herein in the preparation of diagnostic agents for diagnosing proliferative disorders such as cancer.

In some aspects, the present invention relates to antibodies or antigen-binding portions thereof as disclosed herein for the treatment or prevention of proliferative conditions such as cancer.

In some aspects, the present invention relates to kits or devices and related methods using antibodies or antigen-binding portions thereof as disclosed herein, and pharmaceutical compositions as disclosed herein that can be used to treat proliferative conditions such as cancer. To this end, the present invention preferably provides articles for treating such conditions, comprising a container containing an antibody or antigen-binding portion thereof as disclosed herein, and explanatory material for treating, improving, or preventing proliferative diseases or their progression or recurrence using the antibody or antigen-binding portion thereof as disclosed herein. In selected embodiments, the device and related methods will include the step of contacting at least one circulating tumor cell with an antibody or antigen-binding portion thereof as disclosed herein.

Specifically, the present invention relates to the following embodiments:

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

A) One or more heavy chain CDRs (CDRHs), which are selected from at least one of the following:

(i) CDRH1 having at least 90% sequence identity with CDRH1 shown in one of the sequences selected from SEQ ID NO: 1 and 7;

(ii) CDRH2 having at least 90% sequence identity with CDRH2 represented by one of the sequences selected from SEQ ID NO: 2 and 8; and

(iii) CDRH3 having at least 90% sequence identity with CDRH3 represented by one of the sequences selected from SEQ ID NO: 3 and 9;

B) One or more light chain CDRs (CDRLs), which are selected from at least one of the following:

(i) CDRL1 having at least 90% sequence identity with CDRL1 shown in one of the sequences selected from SEQ ID NO: 4 and 10;

(ii) CDRL2 having at least 90% sequence identity with CDRL2 shown in one of the sequences selected from SEQ ID NO: 5 and 11; and

(iii) A CDRL3 having at least 90% sequence identity with a CDRL3 represented by one of the sequences selected from SEQ ID NO: 6 and 12; or

C) One or more CDRHs of A) and B) One or more CDRLs of B).

2. The isolated antibody or its antigen-binding portion according to embodiment 1, wherein the isolated antibody or its antigen-binding portion comprises:

A) One or more heavy chain CDRs (CDRHs), which are selected from at least one of the following:

(i) CDRH1 selected from SEQ ID NO: 1 and 7 or CDRH1 with an amino acid addition, deletion or substitution difference of no more than 2 amino acids from the amino acid sequence of the CDRH1;

(ii) CDRH2 selected from SEQ ID NO: 2 and 8, or CDRH2 differing from the CDRH2 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; and

(iii) CDRH3 selected from SEQ ID NO: 3 and 9 or CDRH3 with an amino acid addition, deletion or substitution difference of no more than 2 amino acids from the amino acid sequence of the CDRH3;

B) One or more light chain CDRs (CDRLs), which are selected from at least one of the following:

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

(ii) CDRL2 selected from SEQ ID NO: 5 and 11, or CDRL2 differing from the CDRL2 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; and

(iii) CDRL3 selected from SEQ ID NO: 6 and 12, or CDRL3 differing from the CDRL3 in amino acid sequence by no more than two amino acid additions, deletions, or substitutions; or

C) One or more CDRHs of A) and B) One or more CDRLs of B).

3. The isolated antibody or its antigen-binding portion according to embodiment 1, wherein the isolated antibody or its antigen-binding portion comprises:

(a) CDRH1 containing SEQ ID NO: 1 or consisting of therefrom;

(b) Contains SEQ ID NO: 2 or CDRH2 consisting of SEQ ID NO: 2;

(c) Contains SEQ ID NO: 3 or CDRH3 consisting of SEQ ID NO: 3;

(d) Contains SEQ ID NO: 4 or CDRL1 consisting of SEQ ID NO: 4;

(e) CDRL2 containing SEQ ID NO: 5 or consisting of therein; and

(f) Contains SEQ ID NO: 6 or CDRL3 composed of it.

4. The isolated antibody or its antigen-binding portion according to embodiment 1, wherein the isolated antibody or its antigen-binding portion comprises:

(a) CDRH1 containing SEQ ID NO: 7 or consisting of therein;

(b) CDRH2 containing SEQ ID NO: 8 or consisting of therefrom;

(c) Contains SEQ ID NO: 9 or CDRH3 consisting of SEQ ID NO: 9;

(d) Contains SEQ ID NO: 10 or CDRL1 consisting of SEQ ID NO: 10;

(e) CDRL2 containing SEQ ID NO: 11 or consisting of therein; and

(f) Contains SEQ ID NO: 12 or CDRL3 composed of therefrom.

5. The isolated antibody or its antigen-binding portion according to embodiment 1, wherein the isolated antibody or its antigen-binding portion comprises:

(A) Heavy chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 13; or

(iii) Contains an amino acid sequence having one or more added, deleted, and/or substituted amino acids compared to SEQ ID NO: 13; and/or

(B) Light chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 14; or

(iii) Contains an amino acid sequence having one or more added, deleted and/or substituted amino acids compared to SEQ ID NO: 14.

6. The isolated antibody or its antigen-binding portion according to embodiment 1, wherein the isolated antibody or its antigen-binding portion comprises:

(A) Heavy chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 15; or

(iii) Contains an amino acid sequence having one or more added, deleted, and/or substituted amino acids compared to SEQ ID NO: 15; and/or

(B) Light chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 16; or

(iii) Contains an amino acid sequence having one or more added, deleted and/or substituted amino acids compared to SEQ ID NO: 16.

7. The isolated antibody or its antigen-binding moiety from any one of embodiments 1-6 has one or more of the following properties:

(a) Human LAG-3 is bound at 2× ^10⁻¹⁰ M or lower _KD ;

(b) Inhibits the binding of LAG-3 to major histocompatibility (MHC) class II molecules;

(c) Inhibits the binding of LAG-3 to fibrinoid protein 1 (FGL1) ligand molecules;

(d) Inhibit the binding of LAG-3 to LSECtin and/or galactolectin-3;

(e) binds to human LAG-3 without causing cross-family reactions; or

(f) It has no cross-reactivity with human CD4.

8. An isolated antibody or its antigen-binding portion thereof from any one of embodiments 1-7, wherein the antibody is a monoclonal antibody, such as a fully human monoclonal antibody, for example, a fully human monoclonal antibody produced by a transgenic mammal, wherein the transgenic mammal is 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 1-8, for example, the nucleic acid sequences shown 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 for preparing an antibody or its antigen-binding portion as defined in any one of embodiments 1-8, comprising the following steps:

- Expressing an antibody or its antigen-binding moiety as defined in any one of embodiments 1-8 in the host cells of embodiment 11; and

- Isolate the antibody or its antigen-binding portion from the host cell.

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

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

16. A method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject an antibody or an antigen-binding portion thereof as defined in any one of embodiments 1-8, thereby inhibiting the growth of tumors in the subject.

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

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

19. Use of an antibody or antigen-binding portion thereof as defined in any one of embodiments 1-8 in the preparation of a diagnostic agent for diagnosing proliferative disorders such as cancer.

20. An antibody or its antigen-binding portion as defined in any one of embodiments 1-8, used for the treatment or prevention of proliferative diseases such as cancer.

21. An antibody or its antigen-binding portion as defined in any one of embodiments 1-8, used for the diagnosis of proliferative disorders such as cancer.

22. A kit for treating or diagnosing proliferative conditions such as cancer, comprising a container containing at least one antibody or its antigen-binding portion as defined in any one of embodiments 1-8.

The above is an overview and therefore includes simplifications, generalizations, and omissions of details where necessary; thus, those skilled in the art will recognize that this overview is merely illustrative and not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and/or devices and/or other subjects described herein will become apparent from the teachings presented herein. This overview is provided to simply introduce some alternative concepts, which will be further described in the detailed description below. This overview is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid to determining the scope of the claimed subject matter. Furthermore, the contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference in their entirety.

Brief description of the attached diagram

Figure 1 shows the binding of LAG-3 antibody to human LAG-3 on the cell surface, represented by MFI (mean fluorescence intensity) and measured by BD FACSCanto II.

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

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

Figure 4 shows the blockade of the binding of LAG-3 protein to galactolectin-3.

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

Figure 6 shows the cross-reactivity with mouse LAG-3 as measured by FACS.

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

Figures 8A-E show the epitope binning against the baseline antibodies BMK1, BMK7, and BMK5.

Figures 9A-B show the results of the epitope plotting.

Figure 10 shows the role of human LAG-3 antibody in reporter gene assay.

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

Figure 12 shows the effect of human LAG-3 antibody on the response of human allogeneic mixed lymphocytes, as reflected by ^3H -thymidine incorporation measurement and by the proliferation response expressed in CPM (counts per minute) in triplicate wells.

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

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

Invention Details

While the invention can be embodied in many different forms, what is disclosed herein are specific illustrative embodiments that demonstrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter.

Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and/or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not limiting. Moreover, the scope provided in the specification and the appended claims includes all values between endpoints and breakpoints.

Generally, the terms and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, ^6th ed., WBSaunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terminology, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well-known and commonly used in the field. Furthermore, any chapter headings used in this article are for organizational purposes only and are not to be construed as limiting the subject matter described.

definition

To better understand this invention, the definitions and explanations of relevant terms are provided below.

As used herein, the term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be divided into κ and λ light chains. The heavy chains can be divided into μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain also contains 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 three domains (CH1, CH2, and CH3). 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 (called complementarity-determining regions (CDRs)) separated by relatively conserved regions (called 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 the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen-binding sites. The distribution of amino acids in the various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883. Antibodies can have 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 containing a fragment of a full-length antibody that retains the ability to specifically bind to an antigen that specifically binds to the full-length antibody, and/or competes with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Under some conditions, antigen-binding fragments include Fab, Fab', F(ab') ₂ , Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., the monoclonal anti-human LAG-3 antibody provided in this application) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology 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 an antibody molecular formulation consisting of a single molecule. Monoclonal antibodies exhibit single-molecule binding specificity and affinity for a specific epitope.

As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies having variable regions, wherein both the frame region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of this 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 mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species, such as a mouse, has been grafted onto a human frame sequence.

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

The term "humanized antibody" refers to an antibody in which a CDR sequence derived from another mammalian species, such as a mouse, has been transplanted onto a human frame sequence. Additional frame region modifications can be performed within the human frame sequence.

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

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

As used herein, the term "human LAG-3" refers to the human sequence LAG-3, such as the complete amino acid sequence of human LAG-3 with Genbank accession number NP_002277. The human LAG-3 sequence may differ from that of human LAG-3 with Genbank accession number NP_002277, for example, by having conserved mutations in non-conserved regions, and the LAG-3 may have substantially the same biological function as that of human LAG-3 with Genbank accession number NP_002277. For example, the biological function of human LAG-3 may be to have an epitope in the extracellular domain of LAG-3 that is specifically bound by an antibody of this disclosure, or the biological function of human LAG-3 may be to bind to MHC class II or FGL1 molecules.

As used herein, the term “mouse LAG-3” refers to the mouse sequence LAG-3, such as the complete amino acid sequence of mouse LAG-3 with Genbank accession number NP_032505.

As used herein, the term "cynomolgus monkey LAG-3" refers to the cynomolgus monkey sequence LAG-3, such as the complete amino acid sequence of the cynomolgus monkey LAG-3 with Genbank accession number XP_005570011.1.

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

As used herein, “high affinity” for IgG antibodies refers to antibodies with a KD of 1× ^10⁻⁷ M or less, more preferably 5× ^10⁻⁸ M or less, even more preferably 1× ^10⁻⁸ M or less, even more preferably 5× ^10⁻⁹ M or less, and even more preferably 1× ^10⁻⁹ M or less against a _target antigen.

As used herein, the term "EC ₅₀ ," also known as "half-maximum effective concentration," refers to the concentration of a drug, antibody, or toxicant that induces a 50% response between baseline and maximum after a specific exposure time. In the context of this application, EC ₅₀ is measured in "nM."

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

As used herein, the term “epitope” refers to the antigenic portion that an immunoglobulin or antibody specifically binds to. An epitope is also known as an “antigenic determinant.” Epitopes or antigenic determinants typically consist of chemically active surface groups on the side chains of molecules such as amino acids, carbohydrates, or sugars, and usually possess a specific three-dimensional structure and specific charge characteristics. For example, an epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique stereoconformation, and can be a “linear” or “conformal” epitope. See, for example, *Epitope Mapping Protocols in Methods in Molecular Biology*, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span amino acid residues that are separated from each other within the protein. Antibodies can be screened based on the competitiveness of binding to the same epitope, as detected by conventional techniques known to those skilled in the art. For example, competitive or cross-competitive studies can be conducted to obtain antibodies that compete or cross-competitively bind to antigens (e.g., RSV fusion proteins). A high-throughput method for obtaining antibodies that bind to the same epitope, based on their cross-competition, is described in International Patent Application WO 03/48731.

As used herein, the term "separated" refers to a state obtained artificially from the natural state. If a "separated" substance or component exists naturally, it may be due to natural changes, separation from the natural state, or both. For example, if an unseparated polynucleotide or polypeptide exists naturally in a living organism, the same high-purity polynucleotide or polypeptide separated from that natural state is called a separated polynucleotide or polypeptide. The term "separated" does not exclude either artificially created or synthetic substances or other impurities that do not affect the activity of the separated substance.

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

As used herein, the term "vector" refers to a nucleic acid medium in which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotides inserted therein, the vector is called an expression vector. This vector can be used to express the carried genetic material elements in host cells through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain 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 or Bacillus subtilis, fungal cells such as yeast cells or 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.

As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by alignment and comparison of sequences. "Percentage identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include: Computational Molecular Biology (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Grif... The following are cited in: fin, 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 Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAMJ. Applied Math. 48:1073.

As used herein, the term "immunogenicity" refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately 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 formed in the organism's immune system after stimulation with an antigen. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the host's reactivity, and the immunization method.

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

As used herein, the terms “hybridoma” and “hybridoma cell line” are used interchangeably. When referring to the terms “hybridoma” and “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 for the 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 and U. et al. (1993) Ann. Biol. Clin. 51:19-26; U. et al. (1991) Biotechniques 11:620-627; Johnson, B. et al. (1995) J. Mol. Recognit. 8:125-131; and Johnson, B. et al. (1991) Anal. Biochem. 198:268-277.

As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments used to perform FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACSStar Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the EpicsC from Coulter Epics Division (Hialeah, FL), and the 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 binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely necessary for this killing. The main cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Optionally or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

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 (C1q) of the complement system to an antibody (appropriate subclass) that binds to its homologous antigen. To assess complement activation, a CDC assay 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 in this article, the term “cancer” refers to any tumor or malignant cell growth, proliferation, or metastasis that causes medical conditions, including solid tumors and non-solid tumors such as leukemia.

The terms "treatment" and "curing" as used in this article generally refer to treatments and therapies for humans or animals that achieve some desired therapeutic effect, such as inhibiting disease progression, including a slowdown in the rate of progression, a halt in the rate of progression, disease regression, disease improvement, and disease cure. Treatment as a preventative measure (i.e., prevention) is also included. For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

As used herein, the term "therapeutic effective amount" refers to an amount of an active compound or a material, composition, or dosage form containing the active compound that, when administered according to the desired treatment regimen, is effective in producing certain desired therapeutic effects commensurate with a reasonable benefit/risk ratio. Specifically, "therapeutic effective amount" means the amount or concentration of an antibody or its antigen-binding portion that is effective in treating LAG-3-related diseases or conditions in humans.

As used herein, the term "host cell" in this invention refers to a cell in which exogenous polynucleotides are introduced.

As used herein, the term “pharmaceuticalally acceptable” means that the carrier, diluent, excipient and/or salt thereof is chemically and/or physically compatible with the other components in the formulation and is physiologically compatible with the recipient.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or before an antigen, can enhance the organism's immune response to the antigen or alter the type of immune response. Various adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, cytokines, etc. Freund's adjuvants are currently the most commonly used adjuvants in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

Anti-LAG-3 antibody

In some aspects, the present invention includes isolated antibodies or antigen-binding portions thereof.

In the context of this application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primate-derived antibodies, CDR transplantation antibodies, human antibodies, recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, synthetic antibodies, including mutant proteins and variants thereof; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecule that exhibits preferential binding or association with the LAG-3 protein. Furthermore, unless the context otherwise specifies, 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 IgA2). 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 generated using a variety of techniques known in the art. One technique is phage display, in which a (preferably human) antibody library is synthesized on a phage, the library is screened with an antigen of interest or its antibody-binding moiety, and the antigen-bound phage is isolated from which an immunoreactive fragment 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, catalog number 27-9400-01; and Stratagene SurfZAP™ Phage Display Kit, catalog number 240612). Other methods and reagents can be used to generate and screen antibody display libraries (see, for example, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

Human antibodies can also be prepared by introducing human immunoglobulin gene loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated and human immunoglobulin genes have been introduced). Upon challenge, human antibody production is observed, which is very similar to that observed in humans in various aspects, including gene rearrangement, assembly, and antibody libraries. This method is described, for example, in U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and U.S. Patents 6,075,181 and 6,150,584 concerning XenoMouse technology; Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against the target antigen (such B lymphocytes can be obtained from individuals with neoplastic diseases or those that may have been immunized in vitro). See, for example, 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 combinations thereof. For example, monoclonal antibodies can be produced using hybridomas and well-established biochemical and genetic engineering techniques, as described in detail in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, ^1st ed. 2009; Shire et al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC, ^1st 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 Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. It should be understood that the selected binding sequence can be further modified, for example, to increase affinity for the target, humanize the target binding sequence, improve its production in cell cultures, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target binding sequences are also antibodies of the present invention. In a preferred embodiment, anti-human LAG-3 monoclonal antibodies are prepared by using hybridomas.

Generation of hybridomas that produce the human monoclonal antibodies of the present invention

To obtain hybridomas that produce the antibodies of the present invention, such as the human monoclonal antibodies of the present invention, spleen cells and/or lymph node cells from immunized mice can be isolated and fused into a suitable immortalized cell line, such as 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 transfected tumors that produce the monoclonal antibodies of the present invention

The antibodies of the present invention can also be generated in host cells transfected with tumors 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 a partial or full-length light and heavy chain, obtained by standard molecular biology techniques, is inserted into one or more expression vectors such that the gene is operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operatively linked" is intended to mean linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene.

The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (such as 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 mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and/or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses (e.g., the adenovirus major late promoter (AdMLP) and polyomavirus), or non-viral regulatory sequences such as ubiquitin promoters or β-globin promoters; and regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and long terminal repeat sequences from human T-cell leukemia virus type 1 (Takebe et al. (1988) MoI. Cell. Biol. 8:466-472). The expression vector and expression control sequence are selected to be compatible with the expression host cells used.

Antibody light chain genes and antibody heavy chain genes can be inserted into the same or different expression vectors. In some embodiments, the variable region is used to generate a full-length antibody gene of any antibody isotype by inserting it into an expression vector that already encodes the heavy chain constant region and light chain constant region of the desired isotype, such that the VH segment is operatively linked to the CH segment within the vector and the VL segment is operatively linked to the CL segment within the vector. Alternatively or additionally, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is frame-conformally linked to the N-terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the present invention may also carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells in which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216; 4,634,665 and 5,179,017). For example, selection marker genes typically confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) to host cells in which the vector has been introduced. Selection marker genes may include a dihydrofolate reductase (DHFR) gene (for DHFR-host cells with methotrexate selection/amplification) and a 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 using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells (which can assemble and secrete antibodies with appropriate folding and immunological activity).

Mammalian host cells used to express the recombinant antibodies of the present invention include Chinese hamster ovary (CHO) cells (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220) used with DHFR selection markers (e.g., as described in R. J. Kaufman and P. A. Sharp (1982) J. MoI. Biol. 159: 601-621), NSO myeloma cells, COS cells, and SP2 cells. 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 the recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to allow antibody expression in the host cells or by secreting the antibody into the culture medium in which the host cells grow. Antibodies can be recovered from the culture medium using standard protein purification methods.

Anti-LAG3 antibodies with certain properties

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

(a) Human LAG-3 is bound at 2× ^10⁻¹⁰ M or lower _KD ;

(b) Inhibits the binding of LAG-3 to major histocompatibility (MHC) class II molecules;

(c) Inhibits the binding of LAG-3 to fibrinoid protein 1 (FGL1) ligand molecules;

(d) Inhibit the binding of LAG-3 to LSECtin and/or galactolectin-3; or

(e) It binds to human LAG-3 without cross-family reactions.

The antibody of the present invention binds to human LAG-3 with high affinity. The binding of the antibody to LAG-3 can be assessed using one or more well-established techniques in the art, such as ELISA. The binding specificity of the antibody can also be determined, for example, by monitoring the binding of the antibody to cells expressing the LAG-3 protein using flow cytometry. For example, the antibody can be tested by flow cytometry in which the antibody reacts with a cell line expressing human LAG-3, such as CHO cells transfected to express LAG-3 on their cell surface. Other suitable cells for flow cytometry assays include CD4 ⁺ activated T cells expressing native LAG-3 and resisting CD3-stimulation. Alternatively or additionally, antibody binding can be tested in a BIAcore binding assay, including binding kinetics (e.g., Kd value). Other suitable binding assays include ELISA assays, such as those using recombinant LAG-3 protein. For example, the antibodies of the present invention bind to human LAG-3 protein with _{a KD} of 5× ^10⁻⁸ M _or less, 2× ^10⁻⁸ M or less, 5× ^10⁻⁹ M or less, 4× ^10⁻⁹ M or _less , 3× ^10⁻⁹ M or _less , 2× ^10⁻⁹ M or less _, 1× ^10⁻⁹ M or _less , 5× ^10⁻¹⁰ _M or less, or 1× ^10⁻¹⁰ M or less _.

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

The isolated antibodies or their antigen-binding moieties disclosed herein inhibit the binding of LAG-3 to major histocompatibility (MHC) class II molecules, FGL1 molecules, LSECtin, and/or galactohemagglutinin-3. LAG-3 negatively regulates T cell signaling and function. Ligands of LAG-3 include, for example, major histocompatibility (MHC) class II molecules, LSECtin, and galactohemagglutinin-3. LAG-3 can interact with MHC class II molecules on the cell surface (Baixeras et al. (1992) J. Exp. Med. 176:327-337; Huard et al. (1996) Eur. J. Immunol. 26:1180-1186). It has been proposed that the direct binding of LAG-3 to MHC class II molecules plays a role in downregulating antigen-dependent stimulation of CD4 ⁺ T lymphocytes (Huard et al. (1994) Eur. J. Immunol. 24: 3216-3221). Recently, Chen Lieping et al. further verified through in vitro and in vivo experiments that FGL1 is the main immunosuppressive ligand of LAG-3, and proposed a new tumor immune escape pathway FGL1-LAG-3. Blocking the interaction between FGL1 and LAG-3 can enhance anti-tumor effects (Cell. 2019 Jan 10; 176(1-2): 334-347.e12.).

Galactolectin-3 is a 31kD lectin that regulates T cell responses through several mechanisms, including apoptosis, TCR crosslinking, and TCR downregulation. Galactolectin-3 binds to LAG-3, and LAG-3 expression is essential for galactolectin-3-mediated in vitro CD8+ T cell inhibition (Kouo et al. (2015) Cancer Immunol. Res. 10. 1158: 2326-6066). Anti-LSECtin has been shown to inhibit the growth of B16 melanoma cells (Xu et al. (2014) Cancer Res. 74(13): 3418-3428).

Anti-LAG3 antibodies containing CDRs with sequence identity to a specific sequence

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

A) One or more heavy chain CDRs (CDRHs) selected from at least one of the following: (i) CDRH1 having at least 90% sequence identity with CDRH1 represented by one of the sequences selected from SEQ ID NO: 1 and 7; (ii) CDRH2 having at least 90% sequence identity with CDRH2 represented by one of the sequences selected from SEQ ID NO: 2 and 8; and (iii) CDRH3 having at least 90% sequence identity with CDRH3 represented by one of the sequences selected from SEQ ID NO: 3 and 9;

B) One or more light chain CDRs (CDRLs) selected from at least one of the following: (i) CDRL1 having at least 90% sequence identity with CDRL1 represented by one of the sequences selected from SEQ ID NO: 4 and 10; (ii) CDRL2 having at least 90% sequence identity with CDRL2 represented by one of the sequences selected from SEQ ID NO: 5 and 11; and (iii) CDRL3 having at least 90% sequence identity with CDRL3 represented by one of the sequences selected from SEQ ID NO: 6 and 12; or

C) One or more CDRHs of A) and B) One or more CDRLs of B).

Unless otherwise stated, amino acids may be assigned to each CDR according to one of the following numbering schemes: Kabat et al. (1991) Sequences of Proteins of Immunological Interest ( ^5th Ed.), USDept. 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, ^3rd Ed., Wily-VCHVerlag GmbH and Co.

Variable regions and CDRs in antibody sequences can be identified according to general rules already developed in the art (as described above, such as 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 “Abysis” website at www.bioinf.org.uk/abs (maintained by A.C. Martin of the Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005). The Abysis database is preferred for sequence analysis. It integrates sequence data from Kabat, IMGT, and the Protein Database (PDB) with structural data from the PDB. See *Protein Sequence and Structure Analysis of Antibody Variable Domains*, in *Antibody Engineering Lab Manual* (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13:978-3540413547, also available at bioinforg.uk/abs). The Abysis database website also includes general rules developed for identifying CDRs that can be used in accordance with the teachings herein. Unless otherwise stated, all CDRs described herein are derived from Kabat's Abysis database website.

The percentage 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 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using 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 at http://www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

Alternatively or additionally, the protein sequence of the present invention can be further used as a “query sequence” to perform a search against a public database to, for example, identify relevant sequences. Such a search can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J.MoI.Biol. 215:403-10. A BLAST protein search with a score of 50 and a word length of 3 can be performed using the XBLAST program to obtain amino acid sequences homologous to the antibody molecule of the present invention. For vacancy alignment for comparative purposes, vacancy BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402, can be used. When using the BLAST and vacancy BLAST programs, the default parameters of the respective programs (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 the sequences described above. As an illustrative example, the antibody may comprise CDRH1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with CDRH1 represented by one of the sequences selected from SEQ ID NO: 1 and 7.

In some embodiments, the isolated antibody or its antigen-binding moiety comprises: anti-LAG3 antibodies with added, deleted, or substituted amino acids.

A) One or more heavy chain CDRs (CDRHs) selected from at least one of the following: (i) CDRH1 selected from SEQ ID NO: 1 and 7 or CDRH1 with an amino acid sequence difference of no more than 2 amino acids from the CDRH1; (ii) CDRH2 selected from SEQ ID NO: 2 and 8 or CDRH2 with an amino acid sequence difference of no more than 2 amino acids from the CDRH2; and (iii) CDRH3 selected from SEQ ID NO: 3 and 9 or CDRH3 with an amino acid sequence difference of no more than 2 amino acids from the CDRH3;

B) One or more light chain CDRs (CDRLs) selected from at least one of the following: (i) CDRL1 selected from SEQ ID NO: 4 and 10 or CDRL1 differing from the amino acid sequence of CDRL1 by no more than two amino acid additions, deletions, or substitutions; (ii) CDRL2 selected from SEQ ID NO: 5 and 11 or CDRL2 differing from the amino acid sequence of CDRL2 by no more than two amino acid additions, deletions, or substitutions; and (iii) CDRL3 selected from SEQ ID NO: 6 and 12 or CDRL3 differing from the amino acid sequence of CDRL3 by no more than two amino acid additions, deletions, or substitutions; or

C) One or more CDRHs of A) and B) One or more CDRLs of B).

Preferably, the CDR of the isolated antibody or its antigen-binding moiety contains no more than two amino acids or no more than one amino acid conserved substitution. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein/peptide comprising an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such as substitutions of physically or functionally similar residues (e.g., having 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), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, 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-LAG3 antibody containing CDR

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

(a) CDRH1 containing SEQ ID NO: 1;

(b) CDRH2 containing SEQ ID NO: 2;

(c) CDRH3 containing SEQ ID NO: 3;

(d) CDRL1 containing SEQ ID NO: 4;

(e) CDRL2 containing SEQ ID NO: 5; and

(f) CDRL3 containing SEQ ID NO: 6.

In a specific implementation scheme, the isolated antibody or its antigen-binding portion comprises:

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

(b) CDRH2 consisting of SEQ ID NO: 2;

(c) CDRH3 consisting of SEQ ID NO: 3;

(d) CDRL1 consisting of SEQ ID NO: 4;

(e) CDRL2 consisting of SEQ ID NO: 5; and

(f) CDRL3 consisting of SEQ ID NO: 6.

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

(a) CDRH1 containing SEQ ID NO: 7;

(b) CDRH2 containing SEQ ID NO: 8;

(c) CDRH3 containing SEQ ID NO: 9;

(d) CDRL1 containing SEQ ID NO: 10;

(e) CDRL2 containing SEQ ID NO: 11; and

(f) CDRL3 containing SEQ ID NO: 12.

In a specific implementation scheme, the isolated antibody or its antigen-binding portion comprises:

(a) CDRH1 consisting of SEQ ID NO: 7;

(b) CDRH2 consisting of SEQ ID NO: 8;

(c) CDRH3 consisting of SEQ ID NO: 9;

(d) CDRL1 consisting of SEQ ID NO: 10;

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

(f) CDRL3 consisting of SEQ ID NO: 12.

Anti-LAG3 antibodies containing heavy chain variable regions and light chain variable regions

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

(A) Heavy chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 13; or

(iii) Contains an amino acid sequence having one or more added, deleted, and/or substituted amino acids compared to SEQ ID NO: 13; and/or

(B) Light chain variable region:

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

(ii) Contains an amino acid sequence that has at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 14;

(iii) Contains an amino acid sequence having one or more added, deleted and/or substituted amino acids compared to SEQ ID NO: 14.

In a specific implementation scheme, the isolated antibody or its antigen-binding portion comprises:

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

(b) Light chain variable region containing the amino acid sequence of SEQ ID NO: 14.

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

(A) Heavy chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 15; or

(iii) Contains an amino acid sequence having one or more added, deleted, and/or substituted amino acids compared to SEQ ID NO: 15; and/or

(B) Light chain variable region:

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

(ii) Contains an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NO: 16; or

(iii) Contains an amino acid sequence having one or more added, deleted and/or substituted amino acids compared to SEQ ID NO: 16.

In a specific implementation scheme, the isolated antibody or its antigen-binding portion comprises:

(a) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15; and/or

(b) Light chain variable region containing the amino acid sequence of SEQ ID NO: 16.

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

In some further embodiments, the isolated antibody or its antigen-binding moiety may contain conserved substitutions or modifications of amino acids in the variable regions of the heavy and/or light chains. It is understood in the art that certain conserved sequence modifications that do not eliminate antigen binding can be made. See, for example, 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.

As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein/peptide comprising an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such as substitutions of physically or functionally similar residues (e.g., having 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 with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, 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).

Sub-warehouse and table-top plotting

It will be further understood that the disclosed antibody will associate or bind to discrete epitopes or immunogenic determinants presented by a selected target or fragment thereof. In some embodiments, the epitope or immunogenic determinant comprises chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments may have specific three-dimensional structural features and/or specific charge properties. Thus, as used herein, the term "epitaxy" includes any protein determinant capable of specifically binding to or otherwise interacting with a molecule with an immunoglobulin or T-cell receptor. In some embodiments, an antibody is considered to specifically bind (or immune-specifically 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, an antibody is considered to specifically bind to an antigen when the equilibrium dissociation constant (K_{_D}) is less than or equal to 10 ^{^-6} M or less than or equal to 10 ^{^-7} M, more preferably when K_{_D} is less than or equal to 10 ^{^-7} M, and even more preferably when K_{_D} is less than or equal to 10 ^{^-9} M.

Epitopes formed by consecutive amino acids (sometimes called “linear” or “continuous” epitopes) are generally retained during protein denaturation, while epitopes formed by ternary folding are usually lost after protein denaturation. In any case, antibody epitopes typically contain at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation.

In this regard, it should be understood that, in some embodiments, epitopes may bind to or reside in one or more regions, domains, or motifs of, for example, the 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 typically consisting of ten to twenty consecutive amino acid residues.

In any case, once the desired epitope on the antigen is identified, it becomes possible to generate an antibody against that epitope, for example, by immunizing with a peptide containing the epitope using the techniques described in this invention. Alternatively, during the discovery process, antibody generation and characterization can elucidate information about the desired epitope located in a specific domain or motif. From this information, antibodies that bind to the same epitope can be competitively screened. This is achieved by conducting competitive studies to discover antibodies that competitively bind to each other, i.e., antibodies competitively bind to antigens. A high-throughput method for compartmentalizing antibodies based on their cross-competition is described in WO 03/48731. Other methods involving compartmentalization or domain-level or epitope mapping, including antibody competition or expression of antigen fragments in yeast, are well known in the art.

As used herein, the term "bin" refers to a method 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 present invention, these bins do not always bind directly to epitopes, and such initial determination of epitope binding can be further refined and confirmed by other recognized methods in the art as described herein. However, it is understood that the verifiable allocation of antibodies to individual bins provides information that can indicate the therapeutic potential of the disclosed antibodies.

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

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

Nucleic acid molecules encoding the antibodies of this invention

In some aspects, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and/or light chain variable regions of isolated antibodies as disclosed herein.

The nucleic acids of this 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 further described below), the light and heavy chains of the antibody prepared via hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acid encoding such antibody can be recovered from the gene library.

By operatively linking the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), the isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably an IgG1 or IgG4 constant region, and most preferably an IgG4 constant region.

By operatively linking DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ or λ constant region.

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

Preferred nucleic acid molecules of the present invention are nucleic acid molecules encoding the VH and VL sequences of monoclonal antibodies 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L. The DNA sequences encoding the VH sequences of 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are shown in SEQ ID NO: 17 and 19, respectively. The DNA sequences encoding the VL sequences of 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are shown in SEQ ID NO: 18 and 20, respectively. In some embodiments, the nucleic acids 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 with SEQ ID NO: 17-20, respectively. In some implementations, the percentage of identity is derived from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

Pharmaceutical Composition

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

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 compositions of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-LAG-3 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, combinations or more of various components known in the art.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, 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, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and/or propyl arsenate. As disclosed in this invention, antibodies or antigen-binding fragments containing compositions disclosed herein can be oxidized in a solvent containing one or more antioxidants such as methionine that reduce the antibody or its antigen-binding fragment. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this invention provides compositions comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. This invention further provides various methods in which antibodies or their antigen-binding fragments are mixed with one or more antioxidants such as methionine. Thus, antibodies or their antigen-binding fragments can be prevented from oxidation, thereby extending their shelf life and/or increasing their activity.

Application, formulation and dosage

The pharmaceutical compositions of the present invention can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, or implantation or inhalation. The compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on 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 inhalers and their controlled-release formulations.

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 components, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Similarly, a specific dosing regimen (i.e., dose, time, and repetition) will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetic parameters (e.g., half-life, clearance, etc.).

The frequency of administration can be determined and adjusted during treatment, and is based on reducing the number of proliferating or tumorigenic cells, maintaining this reduction in tumor cells, reducing tumor cell proliferation, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, a continuously releasing formulation of the therapeutic composition of the present invention may be suitable.

Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or adverse side effects. The chosen dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, timing of administration, compound clearance rate, duration of treatment, other drugs, compounds and/or materials used in combination, severity of the condition, and species, the patient's sex, age, weight, condition, general health status, and previous medical history. The amount of compound and route of administration are ultimately determined by a physician, veterinarian, or clinician, but a dosage is typically chosen to achieve a local concentration at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

Typically, the antibodies or antigen-binding portions of the present invention can be administered in a variety of ranges. These include about 5 μg/kg body weight to about 100 mg/kg body weight per dose; about 50 μg/kg body weight to about 5 mg/kg body weight per dose; and about 100 μg/kg body weight to about 10 mg/kg body weight per dose. Other ranges include about 100 μg/kg body weight to about 20 mg/kg body weight per dose and about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the dose is at least about 100 μg/kg body weight, at least about 250 μg/kg body weight, at least about 750 μg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, and at least about 10 mg/kg body weight.

In any case, the antibody or its antigen-binding portion of the present invention is preferably administered to subjects in need as required. Those skilled in the art can determine the frequency of administration, for example, based on considerations by the attending physician regarding the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health condition of the subject being treated.

In some preferred embodiments, the treatment process involving the antibody or antigen-binding portion thereof of the present invention will comprise multiple doses of the selected pharmaceutical product administered over several weeks or months. More specifically, the antibody or antigen-binding portion thereof of the present invention may 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 or interval may be varied or adjusted based on patient response and clinical practice.

The dosage and regimen of the disclosed therapeutic composition may also be determined empirically in individuals receiving one or more administrations. For example, an incremental dose of the therapeutic composition as described herein may be administered to an individual. In selected embodiments, the dosage may be gradually increased, decreased, or mitigated based on empirical determination or observed side effects or toxicities. To assess the efficacy of the selected composition, biomarkers of a specific disease, condition, or disease 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 microscopic examination of tumor samples; measurement of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein; reduction of pain or paralysis; improvement of tumor-related speech, vision, breathing, or other disabilities; increased appetite; or improvement in quality of life or prolonged survival as measured by administered tests. Those skilled in the art will understand that the dosage will vary depending on the individual, the type of tumor, the stage of the tumor, whether the tumor has begun to metastasize to other sites within the individual, and past and concurrent treatments.

Compatible formulations intended for parenteral administration (e.g., intravenous injection) will contain an antibody or its antigen-binding moiety at a concentration of about 10 μg/mL to about 100 mg/mL. In some selected embodiments, the concentration of the antibody or its antigen-binding moiety will include 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, 100 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL, 500 μg/mL, 600 μg/mL, 700 μg/mL, 800 μg/mL, 900 μg/mL, or 1 mg/mL. In other preferred embodiments, the ADC concentration will include 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 8 mg/mL, 10 mg/mL, 12 mg/mL, 14 mg/mL, 16 mg/mL, 18 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or 100 mg/mL.

Application of the present invention

The antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo uses, including, for example, the detection of LAG-3 or an enhanced immune response by blocking LAG-3. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects, to enhance immunity under various conditions. Immune responses can be modulated, for example, enhanced, stimulated, or upregulated.

Preferred subjects include patients who require enhanced immune responses. The method is particularly suitable for treating patients with conditions that can be treated by enhancing immune responses, such as T-cell-mediated immune responses. In one specific embodiment, the method is particularly suitable for in vivo treatment of cancer. To achieve antigen-specific enhancement of the immune response, the anti-LAG-3 antibody can be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject carrying a tumor or virus). When the LAG-3 antibody is administered together with another agent, the two can be administered in any order or simultaneously.

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

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

Cancer treatment

Non-limiting examples of such suitable indicators include increased T cell proliferation and/or increased cytokine production in the presence of an antibody. In a preferred embodiment, interleukin-2 production is stimulated in antigen-specific T cells. The invention also provides a method for stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering to the subject an antibody of the invention or its antigen-binding portion thereof, thereby stimulating an immune response (e.g., an antigen-specific T cell response). In a preferred embodiment, the subject is a cancer-carrying subject and an immune response against the tumor is stimulated. Cancer blockade of LAG-3 by an antibody can enhance a patient's immune response against cancer cells. Anti-LAG-3 antibodies can be used alone or in combination with other immunogenic agents, standard cancer therapeutic agents, or other antibodies.

Examples of cancers that can be treated using the methods of the present invention include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, and chronic or acute leukemia, including acute bone cancer. Myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) cancer, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environment-induced cancers including asbestos-induced cancers, and combinations of the aforementioned cancers.

Antibodies or their antigen-binding portions can be used in combination with chemotherapy or radiotherapy.

Used in combination with chemotherapy

Antibodies or their antigen-binding portions can be used in combination with anticancer agents, cytotoxic agents, or chemotherapy agents.

The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat cell-proliferating conditions such as cancer, and include, but are not limited to, cytotoxic agents, cell inhibitors, anti-angiogenic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents, and immunotherapy agents. It should be understood that, in selected embodiments as described above, such anticancer agents may comprise conjugates and may be bound to a disclosed site-specific antibody prior to administration. More specifically, in some embodiments, a selected anticancer agent is linked to an unpaired cysteine residue of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are explicitly contemplated within the scope of this invention. In other embodiments, the disclosed anticancer agent is administered in combination with a site-specific conjugate comprising the various therapeutic agents 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 damage. In some embodiments, the substance 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 from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-acinthin, localized aspergillin), plants (abrusatilin, ricin, saccharitoxin, quercetin, American pokeweed antiviral protein, saponins, white tree toxin, momoridin, trichosanthes pollen protein, barley toxin, tung oil (Aleurites fordii) protein, caryophyllin protein, Phytolacca mericana protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, salsa inhibitor, white tree toxin, mitegellin, localized aspergillin, phenolmycin, neomycin and trichothecene compounds), or animals (e.g., small molecule toxins or enzymatically active toxins from cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including its fragments and/or variants).

For the purposes of this invention, "chemotherapy agents" include chemical compounds (e.g., cytotoxic agents or cell inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and/or survival of cancer cells. These chemical agents typically target intracellular processes required for cell growth or division, and are therefore particularly effective for cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally, chemotherapy agents can include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become sexual or produce tumorigenic progeny (e.g., TIC). These agents are often used in combination and are often the most effective, for example, in regimens such as CHOP or FOLFIRI.

Examples of anticancer agents (as components of site-specific conjugates or in their unconjugated state) that can be used in combination with the site-specific constructs of the present invention include, but are not limited to, alkylating agents, alkyl sulfonates, aziridine, ethyleneimine and methylmelamine, acetogenins, camptothecin, lichenin, callystatin, CC-1065, cryptophycins, dolastidine, leutherobin, sildenafil, and salsa. Sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esporamycin, chromophores of enediyne antibiotics, aclacinomysins, actinomycins, atrazomycin, azoserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinomamycin, chromomycinis, styromycin, styromycin Erythromycin, Detoxin, 6-diazo-5-oxo-L-leucine, Doxorubicin, Epirubicin, Isorubicin, Idarubicin, Ephedrine, Mitomycin, Mycophenolic acid, Nogamycin, Oligomycin, Pepromycin, Potfiromycin, Purulin, Triamcinolone Acetonide, Rodoxine, Streptomycin, Streptozotocin, Tuberculin, Ubenimex, Netostatin, Zorobacterium; Anti-metabolites, Erlotinib, Vemurafenib, Crizotinib, Sorafenib, Ibrutinib, Enzalutamide, Folic acid analogues, Purines Analogs, androgens, anti-adrenergics, folic acid supplements such as frolinic acid, glucuronolactone, aldehyde phosphoramide glycoside, aminolevulinic acid, emuramicin, acridine, bestrabucil, bisulfite, edarax, defofamine, colchicine, diazinon, elfornithine, eletine, epoxigerin, etogluconate, gallium nitrate, hydroxyurea, lentinan, chlordamine, maytansine compounds Inoids, mitoxantrone, mitoxantrone, mopidanmol, nitraerine, pentostatin, methamidoxacin, pirarubicin, loxoantrone, podophyllic acid, 2-ethylhydrazine, procarbazine, polysaccharide complex (JHS Natural Products, Eugene, OR), razorcinol; crizolam; germanospiramine; tinuzonic acid; triaminoquinone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrulin A (ve rracurin A), baculosporin A and serpentin); urethane; vincristine; dacarbazine; mannomustine; dibromomannitol; dibromoeutherol; piperobromethane; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes; chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine Vinorelbine; Norfloxacin; Teniposide; Idatroxa; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; Difluoromethylornithine; Retinoids; Capecitabine; Cobbutatin; Leucovorin; Oxaliplatin; Inhibitors of PKC-α, Raf, H-Ras, EGFR, and VEGF-A (which reduce cell proliferation), and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes... Antihormonal agents used to regulate or inhibit the hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as trisatabine (a 1,3-dioxane cyclopentane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; topoisomerase 1 inhibitors; rmRH; vinorelbine and esporomycin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

Used in combination with radiotherapy

This invention also provides combinations of antibodies or their antigen-binding portions with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combined therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeted agents. Typically, radiotherapy is administered in a pulsed manner over a period of about 1 to 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

diagnosis

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

In some implementations, the binding of an antibody to specific cells in a sample may indicate that the sample may contain tumorigenic cells, thereby suggesting that an individual with cancer can be effectively treated with the antibodies described herein.

Samples can be analyzed using a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluorescence immunoassay, immunoblotting, Western blot analysis, and flow cytometry. Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, etc., as known to those skilled in the art.

Drug packaging and reagent kits

Pharmaceutical packages and kits containing one or more containers comprising one or more doses of an antibody or its antigen-binding moiety are also provided. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or its antigen-binding moiety, with or without one or more other reagents. For other embodiments, such a unit dose is supplied in a single-use, pre-filled syringe. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; buffers, such as phosphates; and/or formulated within a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In some preferred embodiments, the composition comprises 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 packaged conjugate composition is intended for the treatment of selected oncological conditions.

The present invention also provides a kit for generating single- or multiple-dose administration units of site-specific conjugates and optionally one or more anticancer agents. The kit includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials, such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed conjugate or non-conjugate form of the conjugate. In other preferred embodiments, the container includes a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container and optionally contain one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the antibody or its antigen-binding portion of the present invention, such a kit may contain any one or more anticancer agents, such as chemotherapeutic or radiotherapy agents; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and/or other anticancer agents.

More specifically, the kits may have a single container containing the disclosed antibody or its antigen-binding moiety, with or without additional components, or they may have different containers for each desired reagent. In cases where a combination therapeutic agent for conjugation is provided, a single solution may be premixed in molar equivalents or in a manner where one component is more than another. Alternatively, the conjugates and any optional anticancer agents in the kit may be stored separately in different containers prior to administration to the patient. The kits may also include a second/third container for containing sterile, pharmaceutically acceptable buffers or other diluents such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

As briefly described above, the kit may also contain tools for administering antibodies or their antigen-binding portions and any optional components to a patient, such as one or more needles, IV bags, or syringes, or even eye drops, pipettes, or other similar devices through which the preparation can be injected or introduced into an animal or applied to a diseased area of the body. The kits of the present invention typically also include devices for containing vials or the like, and other tightly sealed components for commercial sale, such as injection or blow-molded plastic containers, in which the desired vials and other devices are placed and held.

Sequence List Overview

This application is accompanied by a sequence listing containing numerous nucleic acid and amino acid sequences. The table below provides an overview of the included sequences.

Example

The invention generally described herein will be more readily understood by referring to the following embodiments, which are provided by way of example and are not intended to limit the invention. These embodiments are also intended to represent all or only of the experiments conducted below.

Example 1

Material preparation

1. Production of immunogens

Nucleic acids encoding human LAG-3 ECD (extracellular domain, ECD) with SEQ ID NO: 22 or encoding full-length human LAG-3 with SEQ ID NO: 24 were synthesized by Sangon Biotech. The amino acid sequence of LAG-3 ECD and the DNA sequence encoding it are shown in SEQ ID NO: 21 and 22, respectively, and the amino acid sequence of full-length LAG-3 and the DNA sequence encoding it are shown in SEQ ID NO: 23 and 24, respectively. 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-3 ECD and various tags (including human Fc, mouse Fc, and His tags) were obtained by transfecting the human LAG-3 gene into 293F cells (ThermoFisher). Cells were cultured in FreeStyle 293 expression medium (ThermoFisher) at 37°C and 5% _CO2 . Five days after culture, the supernatant harvested from the transiently transfected cell culture was used for protein purification. The fusion protein was purified using nickel, protein A, and/or SEC columns. Untagged LAG-3ECD protein was generated by cleaving the ECD-hFc fusion protein at its cleavage site using factor Xa protease (New England Biolabs). The purified protein was used for immunization, screening, and characterization.

2. Generation of benchmark antibodies

Based on the information disclosed in patent applications US 20110150892 A1 and US 20170101472 A1, anti-human LAG-3 baseline antibodies (BMK1 and BMK7, where BMK1 is referred to as “25F7” in US 20110150892 A1 and BMK7 as “H4sH15482P” in US 20170101472 A1) were synthesized. The baseline antibody BMK8 is a humanized form of the chimeric antibody BMK5, described in WO2015138920A1 and referred to as “BAP050-chi”. In WO2015138920A1, BMK8 is referred to as “BAP050-hum01”. As described in Section 1 above, the synthesized gene sequence was integrated into plasmid pcDNA3.3. 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 transiently transfected cell culture was used for protein purification. The reference antibody was purified from the supernatant.

3. Establish stable cell lines

Human, mouse, and cynomolgus monkey LAG-3 transfected cell lines were generated. Briefly, using the Lipofectamine 2000 transfection kit, 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, according to the manufacturer's protocol. 48–72 hours after transfection, the transfected cells were cultured in medium containing blastcinon for selection and testing of LAG-3 expression. Cell lines expressing human LAG-3, cynomolgus monkey LAG-3, and mouse LAG-3 were obtained through limiting dilutions.

Example 2

Antibody hybridoma generation

1. Immunity and cell fusion

Twenty-four-week-old OMT rats (transgenic rats with recombinant immunoglobulin loci, as described in US8,907,157B2) were alternately immunized with 12.5 μg hFc-labeled human LAG-3 ECD protein and 12.5 μg His-labeled mouse LAG-3 from an adjuvant to generate antibodies with both frame and CDR regions derived from human germline immunoglobulin sequences. Serum was collected from immunized rats every two weeks, and serum titers against human LAG-3 were measured by ELISA. Plates coated with human LAG-3.ECD.hFc were incubated with diluted rat serum (first 1:100, then 3-fold diluted in 2% BSA) for 2 hours. Goat anti-rat IgG-Fc-HRP was used as the secondary antibody. Color development was initiated by dispensing 100 μL of TMB substrate, followed by termination with 100 μL of 2N HCl. Absorbance was read at 450 nM using a microplate reader. Once the antibody titer was sufficiently high, rats were given a final booster dose of 40 μg of human LAG-3 ECD protein in adjuvant-free DPBS. On the day of fusion, lymph nodes and spleens were aseptically removed from the immunized rats and prepared into single-cell suspensions. The isolated cells were then mixed with myeloma cells SP2/0 at a 1:1 ratio. Electrocellular fusion was performed using a BTX 2000 Electrocell manipulator. Cells were then seeded at a density of 1 × ^10⁴ cells/well in 96-well plates and cultured at 37°C, 5% _CO₂ until ready for selection.

2. Preliminary and confirmatory screening of hybridoma supernatant

ELISA was used as the first screening method to test the binding of hybridoma supernatant to LAG-3 protein. The plates from Section 1 of this embodiment were coated overnight at 4°C with 1 μg/mL human LAG-3 ECD.hFc. After blocking and washing, the hybridoma supernatant was transferred to the coated plates and incubated at room temperature for 1 hour. The plates were then washed and incubated with the secondary antibody goat anti-rat IgG HRP for 1 hour. After washing, TMB substrate was added, and the colorimetric reaction was terminated with 2M HCl. The absorbance was read at 450 nm using a microplate reader.

To confirm the natural binding of the LAG-3 antibody to the conformational LAG-3 molecule expressed on the cell membrane, flow cytometry analysis was performed on the LAG-3-transfected CHO-K1 cell line. CHO-K1 cells expressing human LAG-3 were transferred to 96-well U-bottom plates at a density of 1 x ^10⁵ cells/well. Hybridoma supernatant was then transferred to the plates and incubated at 4°C for 1 h. After washing with 1×PBS/1% BSA, the secondary antibody (goat anti-rat IgG Alexa647) was added and the cells were incubated at 4°C in the dark for 0.5 h. The cells were then washed and resuspended in 1×PBS/1% BSA, followed by flow cytometry analysis. Antibody binding 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 also performed as a control.

The blocking activity of antibodies was used as a confirmatory screening to select potential antibody hits. The ability of the selected antibodies to block the binding of LAG-3 protein to the human MHC-II-expressing Raji cell line was tested by FACS analysis. Raji cells were transferred to 96-well U-bottom plates at a density of 1 x ^10⁵ cells/well. The supernatant was incubated with mFc-labeled LAG-3 protein at 4°C for 30 min. The mixture was then transferred to 96-well plates seeded with Raji cells. The cells were incubated with the secondary antibody PE-labeled goat anti-mouse IgG antibody (non-cross-reactive to rat IgG Fc, Jackson Immunoresearch Lab) at 4°C in the dark for 0.5 h. The cells were then washed and resuspended in 1×PBS/1% BSA and analyzed by flow cytometry.

3. Hybridoma subcloning:

Once specific binding was verified through preliminary and confirmatory screening, monoclonal anti-hLAG-3 antibodies were obtained by subcloning positive hybridoma cell lines using a semi-solid medium method. In this method, for each hybridoma cell line, cells were diluted in semi-solid cloning medium (STEMCELL Technologies) and seeded into 6-well plates. Cells were cultured in an incubator (37°C, 5% _CO2 ) for 8–10 days until monoclonal antibodies 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 against human LAG-3, as described above.

Example 3

Hybridoma sequencing and construction of fully human antibody molecules

1. Hybridoma sequencing

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, the antibody VH and VL genes were amplified from the cDNA using a set of 3'-constant region degenerate primers and 5'-degenerate primers (complementary to the upstream signal sequence coding region of the Ig variable sequence). Table 5 shows reagent information, including the manufacturer.

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

Table 1. cDNA amplification reaction (20 μL)

Table 2. cDNA amplification reaction conditions

Step 1 Step 2 Step 3 Step 4 Temperature (°C) 25 50 85 4 time 10min 50min 5min ∞

Table 3. PCR reaction system (50 μL)

Components quantity cDNA 2.0μL Premix Ex Taq 25μL 5’-Degenerate primer set (10pM) 2.5μL 3’-Degenerate primers for the constant region (10 pM) 1μL <![CDATA[ddH₂O]]> 19.5μL

Table 4. PCR Reaction Conditions

Table 5. Reagent Information

The two lead antibodies were named “1.53.3-uAb-IgG4k” and “3.40.19-uAb-IgG4L”, respectively.

The CDR sequence of 1.53.3-uAb-IgG4k is as follows:

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

The sequences of the heavy and light chain variable regions of 1.53.3-uAb-IgG4k are as follows:

The CDR sequence of 3.40.19-uAb-IgG4L is as follows:

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

The sequences of the heavy and light chain variable regions of 3.40.19-uAb-IgG4L are as follows:

2. Construction of fully human antibody molecules

The VH and VL genes were re-amplified using cloning primers containing appropriate restriction sites, and then cloned into expression vectors to generate corresponding chimeric antibody clones.

Example 4

LAG-3 antibody binds to human LAG-3 on the cell surface.

Various concentrations of test antibodies, 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 Figure 1, and _EC50 is shown in Table 6.

Table 6

Ab <![CDATA[EC₅₀(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 Figure 1 and Table 6, the _EC50 (0.13) of 3.40.19-uAb-IgG4L binding to cell surface LAG-3 was significantly lower than that of all three benchmark antibodies, BMK1 (0.32), BMK7 (0.61), and BMK8 (0.90). Furthermore, the _EC50 (0.43) of 1.53.3-uAb-IgG4k binding to cell surface LAG-3 was much lower than _that of BMK7 (0.61) and BMK8 (0.90). These results indicate that 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L can effectively bind to human LAG-3, and their binding efficacy is superior to or equivalent to that of the benchmark antibodies.

Example 5

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

The antibody was serially diluted in 1% BSA-PBS and incubated with mFc-labeled LAG-3 protein at 4°C for 30 min. The mixture was transferred to 96-well plates seeded with Raji cells. The 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 using FlowJo software (version 7.6.1). Data are shown in Figure 2, and _EC50 is shown in Table 7.

Table 7

Ab <![CDATA[EC₅₀(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 Figure 2 and Table 7, surprisingly, the _EC50 (0.67) for the binding of 3.40.19-uAb-IgG4L to MHC-II expressed on Raji cells was significantly lower than that of all three benchmark antibodies, BMK1 (0.76), BMK7 (1.25), and BMK8 (0.88). Furthermore, the _EC50 (0.80) for the binding of 1.53.3-uAb- _IgG4k to MHC-II expressed on Raji cells was lower than _that of BMK7 (1.25) and BMK8 (0.88). These results indicate that 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are effective in blocking the binding of MHC-II expressed on Raji cells, and their blocking effect is superior to or equivalent to that of the benchmark antibodies.

Example 6

Blocking the binding of LAG-3 protein to LSECtin and galactoglobulin-3

96-well plates were coated overnight at 4°C with 0.5 μg/mL human LSECtin or galactolectin-3, respectively. 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 plates and incubated at room temperature for 1 hour. The plates were then washed and incubated with the corresponding secondary antibody for 60 minutes. After washing, TMB substrate was added, and the colorimetric reaction was terminated with 2M HCl. The absorbance was read at 450 nm using a microplate reader. The data are shown in Figures 3 and 4. _EC50 is shown in Table 8.

Table 8

As shown in Figures 3 and 4 and Table 7, both 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L can effectively block the binding of LAG-3 to LSECtin or galactohemagglutinin-3, and their blocking effect is superior to or equivalent to that of the baseline antibody.

Example 7

Complete kinetic binding affinity test

Full kinetic binding affinity tested via surface plasmon resonance (SPR):

The affinity and binding kinetics of the antibody against human LAG-3 were characterized using a SPR assay with Biacore 8K. Goat anti-human Fc was pre-immobilized onto a sensor chip (CM5), and anti-LAG-3 antibody was captured upon chip injection. Various concentrations of human LAG-3 protein and running buffer were flowed through the sensor chip at a flow rate of 30 μL/min for a 300 s association phase, followed by a 3600 s dissociation phase. Association and dissociation curves were fitted using a 1:1 Langmuir binding model in the Biacore 8K evaluation software. 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

The binding affinity of LAG-3 antibody to LAG-3 molecules on the cell surface was measured by fluorescence-activated cell sorting (FACS).

The binding affinity of the antibody to LAG-3 on the cell surface was measured by FACS analysis. Flp-In-293 cells expressing human LAG-3 were transferred to 96-well U-bottom plates at a density of 5 × ^10⁵ cells/mL. The antibody to be tested was serially diluted in wash buffer (1 × PBS/1% BSA) and incubated with the cells at 4°C for 1 h. Secondary antibody, goat anti-human IgG FcFITC (3.5 mol FITC per mol IgG), was added, and the cells were incubated at 4°C in the dark for 0.5 h. The cells were then washed once and resuspended in 1 × PBS/1% BSA and analyzed by flow cytometry. Fluorescence intensity was converted to bound molecules/cells based on quantitative beads (Quantum™ MESF kit, Bangs Laboratories, Inc.). Affinity was calculated using a Graphpad Prism 5. The data are shown in Table 10.

Table 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

As tested by SPR and FACS, the antibodies of the present invention, represented by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L, effectively bind human LAG-3, and the binding effect is superior to or equivalent to that of the benchmark antibody.

Example 8

Combination of orthologs (across species) and homologs (across families)

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

Cross-reactivity with cynomolgus monkey and mouse LAG-3 was measured using FACS. Mouse LAG-3-expressing Flp-In-CHO cells or cynomolgus monkey LAG-3-expressing 293F cells were transferred to 96-well U-bottom plates at a density of 1 x ^10⁵ cells/well. The test antibody was serially diluted in wash buffer (1×PBS/1% BSA) and incubated with cells at 4°C for 1 h. After washing with 1×PBS/1% BSA, the appropriate secondary antibody was applied, and the cells were incubated with the antibody at 4°C in the dark for 1 h. Cells were then washed and resuspended in 1×PBS/1% BSA and analyzed by flow cytometry. Data are shown in Figures 5 and 6. EC ₅₀ is shown in Table 11.

Table 11

Ab <![CDATA[EC₅₀(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 Figure 5, the LAG-3 antibodies “1.53.3-uAb-IgG4k” and “3.40.19-uAb-IgG4L” of this invention bind to cynomolgus monkey LAG-3 on the cell surface. As shown in Figure 6, the LAG-3 antibodies “1.53.3-uAb-IgG4k” and “3.40.19-uAb-IgG4L” of this invention do not bind to mouse LAG-3 on the cell surface.

Cross-reactivity with human CD4

Cross-reactivity with human CD4 was measured by ELISA. Plates were coated with 1 μg/mL human CD4 overnight at 4°C. After blocking and washing, 1 μg/mL LAG-3 antibody was added to the plates and incubated at room temperature for 1 hour. The plates were then washed and incubated with the corresponding secondary antibody for 45 minutes. After washing, TMB substrate was added, and the colorimetric reaction was terminated with 2M HCl. The absorbance at 450 nm was read using a microplate reader. The data are shown in Figure 7.

These results indicate that the LAG-3 antibodies “1.53.3-uAb-IgG4k” and “3.40.19-uAb-IgG4L” of this invention do not bind to human CD4 protein.

Example 9

Tabletop compartments for BMK1, BMK7 and BMK5

LAG-3 antibody binding epitopes were fragmented against the baseline antibodies BMK1, BMK7, and BMK5 using FACS analysis. Flp-In-293 cells expressing human LAG-3 were incubated with 1 μg/mL biotinylated baseline antibody for 1 hour, followed by the addition of serially diluted LAG-3 antibody. The binding of the baseline antibody to cells was detected using streptavidin-PE antibody (Jackson Immunoresearch Lab). MFI was assessed by flow cytometry and analyzed using FlowJo. Data are shown in Figures 8A-E.

It was found that 1.53.3-uAb-IgG4k of the present invention competes with BMK, but 3.40.19-uAb-IgG4L does not compete with BMK. 1.53.3-uAb-IgG4k shares close epitopes with BMK1 and BMK7, but not with BMK5. Surprisingly, 3.40.19-uAb-IgG4L has epitopes that are different from all of BMK1, BMK7, and BMK5.

Example 10

Domain plotting and epitope plotting

1. Domain Plotting

LAG-3 has 421 amino acids (P30-L450) of extracellular domains, including four extracellular immunoglobulin superfamily (IgSF)-like domains: 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). Ten variants were constructed by replacing the following residues in the extracellular domains of human LAG-3 with the corresponding mouse LAG-3 amino acids (also referred to as “aa” in the context of this disclosure).

(1) Variant 1: xPro1.FL-x1: human LAG-3 aa 168 to 419 were replaced by the corresponding mouse portion.

(2) Variant 2: xPro1.FL-x2: human LAG-3 aa 37 to 167 and aa 265 to 419 were replaced by the corresponding mouse portions.

(3) Variant 3: xPro1.FL-x3: human LAG-3 aa 37 to 252 and aa 348 to 419 were replaced by the corresponding mouse portions.

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

(5) Variant 5: xPro1.FL-x5: human LAG-3 aa 265 to 419 was replaced by the corresponding mouse portion.

(6) Variant 6: xPro1.FL-x6: human LAG-3 aa 37 to 167 and aa 348 to 419 were replaced by the corresponding mouse portions.

(7) Variant 7: xPro1.FL-x7: human LAG-3 aa 37 to 252 was replaced by the corresponding mouse portion.

(8) Variant 8: xPro1.FL-x8: human LAG-3 aa 168 to 343 was replaced by the corresponding mouse portion.

(9) Variant 9: xPro1.FL-x9: human LAG-3 aa 348 to 419 were replaced by the corresponding mouse portion.

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

Ten variants were cloned into the pcDNA3 vector and used for 293F cell transfection. Briefly, 293F cells were diluted to a density of 1 × ^10⁶ cells/mL using FreeStyle293F medium, and aliquots of 3 mL/well were added to 24-well plates. Transfection was performed using 293fectin reagent (Life Technologies). For each transfection, 3 μg of DNA was diluted in 150 μL of Opti-MEM I serum-depleted medium (Life Technologies), and then combined with 6 μL of 293fectin reagent pre-diluted in 150 μL of Opti-MEM I serum-depleted medium. The DNA/Lipofectamine mixture was allowed to stand at 25°C for 20 min before being added to the culture. Transfected cells were analyzed by flow cytometry 48 hours post-transfection.

The binding of the antibody to either the chimeric LAG-3 variant or the full-length human/mouse LAG-3 was analyzed by flow cytometry. In short, 1 μg/mL of antibody was incubated with transfected 293F cells expressing chimeric LAG-3 at 4°C for 1 hour, followed by incubation with 3 μg/mL goat anti-human IgG Fc R-PE (Jackson) at 4°C for 40 minutes. Cell analysis was then performed using flow cytometry.

The binding affinity of antibodies 1.53.3-uAb-IgG4k and 3.40.19-uAb-hIgG4L to these 10 variants was tested, and the results are shown in Table 12 below.

Table 12. MFI values of LAG-3 antibody binding to 10 variants

Based on the FACS binding activity of the antibodies, both lead antibodies, namely "1.53.3-uAb-IgG4k" and "3.40.19-uAb-hIgG4L", bound to domain 1 (i.e., aa.37-167). Therefore, further epitope mapping of domain 1 (G37-Q167, 131aa) was performed using an alanine scanning assay.

2. Tabletop plotting

Alanine scanning assays were performed on human LAG-3 for epitope mapping. Alanine residues in 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-specific amino acid substitutions were performed using two sequential PCR steps. The plasmid pcDNA3.3-LAG-3-D12.mFc, encoding ECD domains 1 and 2 of human LAG-3 with a C-terminal mFc-tag, was used as a template, and a set of mutagenic primers was used for the first step of PCR using the QuikChange Lightning Multisite Directed Mutagenesis Kit (Agilent Technologies, Palo Alto, CA). After mutant strand synthesis, the parental template was digested with Dpn I endonuclease. In the second step of PCR, a linear DNA expression cassette containing the CMV promoter, extracellular domains 1 and 2 (D1 and D2) of LAG-3, the mFc tag, and polyadenylated herpes simplex virus thymidine kinase (TK) was amplified and transiently expressed in Expi293 cells at 37°C (Life Technologies, Gaithersburg, MD). Quantification was performed by protein A-HPLC and mFc-ELISA kits (Bethyl, USA).

For ELISA binding assays, antibodies 1.53.3-uAb-IgG4k or 3.40.19-uAb-hIgG4L (2 μg/mL) were coated onto the plate. After interaction with the supernatant containing a quantified amount of LAG-3 mutant or human LAG-3-ECD.D12.mFc protein, HRP-conjugated anti-mFc antibody (1:5000; Bethyl, USA) was added as the detection antibody. Absorbance was normalized to the mean absorbance of the control mutant. The definitively identified epitope residues were identified after setting an additional cutoff point (<0.75) for the binding fold change. Hot spots for antibodies 1.53.3-uAb-IgG4k and 3.40.19-uAb-hIgG4L are shown in Tables 13 and 14.

Table 13.1.53.3-uAb-IgG4k antibody hotspots

Table 14.3.40.19-uAb-IgG4L antibody hotspots

Since the LAG-3 structure was not present, the structure of LAG-3 (aa:31-431) was modeled based on the known myelin-linked glycoprotein structure (PDB:5FLU, sequence identity 18%). Hotspots of the two antibodies were identified based on alanine scanning results and are shown in Figures 9A and 9B.

Based on the results, it can be seen that the 1.53.5-uAb-IgG4k antibody binds to the W92 site of 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 antibody tested by cell-based assay

The role of human LAG-3 antibody in reporter gene assay

Jurkat cells expressing human LAG-3 and a stably integrated IL-2 luciferase reporter gene were seeded together with Raji cells in 96-well plates in the presence of SEE. Test antibodies were added to the cells. The plates were incubated overnight at 37°C and 5% _CO2 . After incubation, reconstructed luciferase substrate was added, and luciferase intensity was measured using a microplate spectrophotometer. Data are shown in Figure 10, and _EC50 is shown in Table 15.

Table 15

Ab <![CDATA[EC₅₀(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 Figure 10, the LAG-3 antibody enhanced the IL-2 pathway of Jurkat in the reporter gene assay. Furthermore, as shown in Table 15, the _EC50 of 3.40.19-uAb-IgG4L was significantly lower than that of all three benchmark antibodies in this assay.

Effect of human LAG-3 antibody on human allogeneic mixed lymphocyte response

Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from healthy donors using Ficoll-Paque PLUS gradient centrifugation. PBMCs were isolated using a human mononuclear cell enrichment kit according to the manufacturer's instructions. Cells were cultured for 5 to 7 days in medium containing GM-CSF and IL-4 to generate dendritic cells (DCs). Human CD4 ⁺ T cells were isolated using a human CD4 ⁺ T cell enrichment kit according to the manufacturer's protocol. The purified CD4 ⁺ T cells were co-cultured with allogeneic immature DCs (iDCs) and various concentrations of LAG-3 antibody in 96-well plates. On day 5, the culture supernatant was collected for IFN-γ and T cell proliferation assays. Human IFN-γ was measured by ELISA using matched antibody pairs. Plates were pre-coated with a human IFN-γ-specific capture antibody (Pierce-M700A). A biotin-conjugated anti-IFN-γ antibody (Pierce-M701B) was used as the detection antibody. ³ H-thymidine was added at 1 μC/well during the last 16 hours. ^3H -thymidine incorporation was measured by scintillation counting, and the proliferation reaction was expressed as CPM (counts per minute) of triplicate wells. The data are shown in Figures 11 and 12.

As shown in Figure 11, the LAG-3 antibodies “1.53.3-uAb-IgG4k” and “3.40.19-uAb-IgG4L” of the present invention enhance IFN-γ secretion in mixed lymphocyte reactions. Furthermore, as shown in Figure 12, the LAG-3 antibodies “1.53.3-uAb-IgG4k” and “3.40.19-uAb-IgG4L” of the present invention enhance T cell proliferation in mixed lymphocyte reactions.

Example 12

ADCC and CDC tests

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

ADCC test

Flp-In-293 cells expressing human LAG-3 and various concentrations of LAG-3 antibody were pre-incubated in 96-well round-bottom plates for 30 minutes; then PBMCs were added as effector at an effector/target ratio of 50:1. The plates were maintained at 37°C and 5% _CO2 for 4 hours. Target cell lysis was determined using an LDH-based cytotoxicity assay kit. Absorbance at 492 nm was read using a microplate reader. Herceptin and the HER2-expressing cell line SK-Br-3 were used as positive controls.

CDC test

Flp-In-293 expressing human LAG-3 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 incubated at 37°C and 5% CO2 for ₂ hours. Target cell lysis was determined using CellTiter-Glo. Absorbance was read using a microplate reader. Rituximab and the CD20-expressing cell line Raji were used as positive controls.

Figures 13A and 13B show the data from the ADCC and CDC tests. This demonstrates that the LAG-3 antibodies of the present invention, represented by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L, do not mediate ADCC (Figure 13A) and CDC (Figure 13B) effects.

Example 13

Serum stability test

The leader antibody was incubated in freshly isolated human serum (serum purity >95%) at 37°C. At specified time points, aliquots of the serum-treated samples were removed from the incubator and rapidly frozen in liquid nitrogen, then stored at 80°C until ready for testing. Samples were rapidly thawed immediately prior to stability testing. Human LAG-3 transfected cells were incubated with various concentrations of leader antibody at 4°C for 1 hour. PE-labeled goat anti-human IgG was used to detect the binding of the leader antibody to cells. The molecular weight fraction (MFI) of cells was measured by flow cytometry (BD FACSCanto II) and analyzed via FlowJo. Data are shown in Figures 14A and 14B.

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

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

sequence list

<110> Guangzhou Yuhang Biotechnology Co., Ltd.

<120> Anti-human LAG-3 antibodies and their uses

<130> IDC226033

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> 1.53.3-uAb-IgG4k CDRH1

<400> 1

Gly Gly Ser Phe Ser Gly Tyr Tyr Trp Ser

1 5 10

<210> 2

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> 1.53.3-uAb-IgG4k CDRH2

<400> 2

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

1 5 10 15

<210> 3

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> 1.53.3-uAb-IgG4k CDRH3

<400> 3

Gly Glu Asp Tyr Ser Asp Tyr Asp Tyr Tyr Gly Asp Phe

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> 1.53.3-uAb-IgG4k CDRL1

<400> 4

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> 1.53.3-uAb-IgG4k CDRL2

<400> 5

Ala Ala Ser Asn Arg Ala Thr

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> 1.53.3-uAb-IgG4k CDRL3

<400> 6

Gln Gln Arg Ser Asn Trp Pro Leu Thr

1 5

<210> 7

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> 3.40.19-uAb-IgG4L of CDRH1

<400> 7

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

1 5 10

<210> 8

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> 3.40.19-uAb-IgG4L CDRH2

<400> 8

Ser Phe Tyr Tyr Ser Gly Ser Thr Tyr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 9

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> 3.40.19-uAb-IgG4L of CDRH3

<400> 9

Met Gln Leu Trp Ser Tyr Asp Val Asp Val

1 5 10

<210> 10

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> 3.40.19-uAb-IgG4L of CDRL1

<400> 10

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

1 5 10

<210> 11

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> 3.40.19-uAb-IgG4L CDRL2

<400> 11

Asp Val Ser Glu Arg Pro Ser

1 5

<210> 12

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> 3.40.19-uAb-IgG4L CDRL3

<400> 12

Ser Ser Tyr Thr Ser Thr Thr Thr Leu Val Val

1 5 10

<210> 13

<211> 121

<212> PRT

<213> Artificial Sequence

<220>

<223> 1.53.3-uAb-IgG4k VH

<400> 13

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

1 5 10 15

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> 1.53.3-uAb-IgG4k VL

<400> 14

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

35 40 45

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 16

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> 3.40.19-uAb-IgG4L of 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 ggttatact 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 agagaaacct 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 attccgtag 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 of 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 ttatactgc agctcatata caagcaccac cactctcgtt 300

gtgttcggcg gagggaccaa gctgtccgtc ctg 333

<210> 21

<211> 421

<212> PRT

<213> Artificial Sequence

<220>

<223> Human LAG-3 ECD

<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-3 ECD

<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 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 Glu Glu Pro Glu Pro Glu Pro Glu Pro

500 505 510

Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Glu Glu

515 520 525

<210> 24

<211> 1575

<212> DNA

<213> Artificial Sequence

<220>

<223> Full-length LAG-3

<400> 24

atgtggggagg 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 gggacgcgc cctctcctgc 480

cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca gcccccccagg 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

cctggggggag 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 accccttcacc 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 crab-eating macaque 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 Glu 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 crab-eating macaque 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 gggacgcgc 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

cctggggggag 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 (19)

1. A separated antibody or antigen-binding moiety thereof capable of binding to LAG-3, wherein the separated antibody or antigen-binding moiety comprises: CDRH1 as shown in SEQ ID NO: 7, CDRH2 as shown in SEQ ID NO: 8, CDRH3 as shown in SEQ ID NO: 9, CDRL1 as shown in SEQ ID NO: 10, CDRL2 as shown in SEQ ID NO: 11, and CDRL3 as shown in SEQ ID NO: 12. 2. The isolated antibody or its antigen-binding portion according to claim 1, wherein the isolated antibody or its antigen-binding portion comprises: (A) Heavy chain variable region: (i) Contains the amino acid sequence of SEQ ID NO: 15; (ii) Contains an amino acid sequence having at least 85% identity with SEQ ID NO: 15; or (iii) Contains an amino acid sequence having one or more added, deleted, and/or substituted amino acids in the frame region compared to SEQ ID NO: 15; and (B) Light chain variable region: (i) Contains the amino acid sequence of SEQ ID NO: 16; (ii) Contains an amino acid sequence having at least 85% identity with SEQ ID NO: 16; or (iii) Contains an amino acid sequence having one or more amino acid additions, deletions and/or substitutions in the frame region compared to SEQ ID NO: 16. 3. The isolated antibody or its antigen-binding moiety of claim 1, having one or more of the following properties: (a) Human LAG-3 is bound to _KD at 2 × ^10⁻¹⁰ M or lower; (b) Inhibits the binding of LAG-3 to major histocompatibility (MHC) class II molecules; (c) Inhibits the binding of LAG-3 to fibrinoid protein 1 (FGL1) ligand molecules; (d) Inhibit the binding of LAG-3 to LSECtin and/or galactolectin-3; (e) Binds to human LAG-3 without causing cross-family reactions; or (f) It has no cross-reactivity with human CD4. 4. The isolated antibody or its antigen-binding portion according to any one of claims 1-3, wherein the antibody is a monoclonal antibody. 5. The isolated antibody or its antigen-binding portion of claim 4, wherein the monoclonal antibody is a fully human monoclonal antibody. 6. The isolated antibody or its antigen-binding portion of claim 5, wherein the fully human monoclonal antibody is a fully human monoclonal antibody produced by a transgenic mammal. 7. The isolated antibody or its antigen-binding portion of claim 6, wherein the transgenic mammal is a transgenic rat. 8. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the transgenic rat is 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 a light chain variable region of an isolated antibody as defined in any one of claims 1-8. 10. The nucleic acid molecule of claim 9, comprising the nucleic acid sequences shown in SEQ ID NO: 19-20. 11. An expression vector comprising the nucleic acid molecule of claim 9 or 10. 12. A host cell comprising the expression vector of claim 11. 13. A pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as defined in any one of claims 1-8 and a pharmaceutically acceptable carrier. 14. A method for preparing an antibody or its antigen-binding portion as defined in any one of claims 1-8, comprising the following steps: - Express the antibody or its antigen-binding moiety in the host cells of claim 12; and - Isolate the antibody or its antigen-binding portion from the host cell. 15. Use of the antibody or antigen-binding portion thereof as defined in any one of claims 1-8 in the preparation of a reagent for inhibiting or blocking the binding of LAG-3 to MHC class II molecules, FGL1 molecules, LSECtin and/or galactohemagglutinin-3 in vitro, wherein the inhibition or blocking comprises contacting the MHC class II molecules, FGL1 molecules, LSECtin and/or galactohemagglutinin-3 with the antibody or antigen-binding portion thereof as defined in any one of claims 1-8. 16. Use of an antibody or antigen-binding portion thereof as defined in any one of claims 1-8 in the preparation of a medicament for inhibiting the growth of tumor cells in a subject, wherein the tumor is a skin or intraocular malignant melanoma or renal cell carcinoma. 17. Use of an antibody or antigen-binding portion thereof as defined in any one of claims 1-8 in the preparation of a medicament for treating cancer in a subject, wherein said cancer is malignant melanoma of the skin or eye or kidney cancer. 18. Use of the antibody or antigen-binding portion thereof as defined in any one of claims 1-8 in the preparation of a diagnostic agent for diagnosing cancers expressing LAG-3. 19. A kit comprising a container containing an antibody or an antigen-binding portion thereof as defined in any one of claims 1-8.