Anti-human LAG-3 antibodies and uses thereof

Abstract

Provided are novel fully human monoclonal antibodies that bind to human LAG-3. Also provided are methods for generating hybridomas using humanized rats, nucleic acid molecules encoding anti-LAG-3 antibodies, and expression vectors and host cells for expressing the anti-LAG-3 antibodies. The present invention further provides methods for validating antibody function in vitro. The antibodies of the present invention provide effective agents for treating various cancers by modulating human immune function.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201910146172.2, filed on February 27, 2019, and entitled “Anti-human LAG-3 monoclonal antibodies, preparation methods and uses thereof”.

[0002] Sequence Listing

[0003] This application contains a Sequence Listing, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present invention relates generally to antibodies. More specifically, the present invention relates to fully human monoclonal antibodies that bind to human LAG-3, methods for preparing the same, and uses thereof. Background Art

[0005] Lymphocyte activation gene 3 (CD223), also known as LAG-3, is a type I transmembrane protein that is a member of the immunoglobulin superfamily (IgSF).

[0006] 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 to the fibrillin-like protein 1 (FGL1) class, LAG-3's primary functional ligand, independent of MHC class II, thereby providing negative regulation of T cell receptor signaling.

[0007] In vitro blockade of LAG-3 enhances T cell proliferation and cytokine production, and LAG-3-deficient mice are defective in downregulating T cell responses induced by superantigen Staphylococcal enterotoxin B, peptides, or Sendai virus infection. LAG-3 plays a crucial role in the activation of natural Tregs (nTregs) and induced CD4 + FoxP3 + It is expressed on Treg (iTreg) cells, where its expression level is higher than that on activated effector CD4 + Expression levels observed on T cells. Blockade of LAG-3 on Treg cells abolishes the suppressor function of Treg cells, but not Treg CD4 + Ectopic expression of LAG-3 in T cells confers suppressive activity. Based on the immunomodulatory effects of LAG-3 on T cell function in chronic infection and cancer, the predicted mechanism of action of LAG-3-specific monoclonal antibodies is to inhibit the negative regulation of tumor-specific effector T cells.

[0008] Currently, only three potential antagonist antibodies are in early clinical development to modulate LAG-3 function and anti-tumor immune responses for the treatment of advanced solid tumors. These antibodies are described in US Patents 20110150892 A1, US Patents 20170101472 A1, and WO2015138920 A1, 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 benchmark 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-reactivity, and good stability). In this application, the inventors generated a series of antibodies and fully human antibodies against LAG-3 using humanized rats. The antibodies of the present application have high binding affinity, specifically bind to the human LAG-3 protein without cross-reactivity, and effectively modulate the immune response. SUMMARY OF THE INVENTION

[0009] Broadly speaking, the present invention relates to novel compounds, methods, compositions, and articles of manufacture that provide antibodies with improved efficacy. The benefits provided by the present invention are broadly applicable to the fields of antibody therapy and diagnosis and can be used in combination with antibodies capable of reacting with a variety of targets. The present invention provides antibodies that bind to human LAG-3, preferably fully human monoclonal antibodies. Also provided are methods for generating hybridomas using humanized rats, nucleic acid molecules encoding anti-LAG-3 antibodies, and expression vectors and host cells for expressing anti-LAG-3 antibodies. The present invention further provides methods for verifying antibody function in vitro. The antibodies of the present invention provide effective agents for treating a variety of diseases by modulating human immune function.

[0010] In some aspects, the invention includes isolated antibodies or antigen-binding portions thereof.

[0011] In some embodiments, the isolated antibody, or antigen-binding portion thereof, has one or more of the following properties:

[0012] (a) 2×10 -10 M or lower K D Binds to human LAG-3;

[0013] (b) inhibiting the binding of LAG-3 to major histocompatibility (MHC) class II molecules;

[0014] (c) inhibiting the binding of LAG-3 to the fibrillin-like protein 1 (FGL1) ligand molecule;

[0015] (d) inhibiting the binding of LAG-3 to LSECtin and / or galectin-3;

[0016] (e) binds to human LAG-3 without cross-family reactivity; or

[0017] (f) No cross-reactivity with human CD4.

[0018] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0019] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following: (i) a CDRH1 having at least 90% sequence identity to a CDRH1 set forth in one of the sequences selected from SEQ ID NOs: 1 and 7; (ii) a CDRH2 having at least 90% sequence identity to a CDRH2 set forth in one of the sequences selected from SEQ ID NOs: 2 and 8; and (iii) a CDRH3 having at least 90% sequence identity to a CDRH3 set forth in one of the sequences selected from SEQ ID NOs: 3 and 9;

[0020] B) one or more light chain CDRs (CDRLs) selected from at least one of the following: (i) a CDRL1 having at least 90% sequence identity to a CDRL1 set forth in one of the sequences selected from SEQ ID NOs: 4 and 10; (ii) a CDRL2 having at least 90% sequence identity to a CDRL2 set forth in one of the sequences selected from SEQ ID NOs: 5 and 11; and (iii) a CDRL3 having at least 90% sequence identity to a CDRL3 set forth in one of the sequences selected from SEQ ID NOs: 6 and 12; or

[0021] C) one or more CDRHs of A) and one or more CDRLs of B).

[0022] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0023] A) one or more (e.g., 1, 2, or 3) heavy chain CDRs (CDRHs) selected from at least one of the following: (i) a CDRH1 selected from SEQ ID NOs: 1 and 7, or a CDRH1 that differs from the amino acid sequence of said CDRH1 by no more than 2 amino acid additions, deletions, or substitutions; (ii) a CDRH2 selected from SEQ ID NOs: 2 and 8, or a CDRH2 that differs from the amino acid sequence of said CDRH2 by no more than 2 amino acid additions, deletions, or substitutions; and (iii) a CDRH3 selected from SEQ ID NOs: 3 and 9, or a CDRH3 that differs from the amino acid sequence of said CDRH3 by no more than 2 amino acid additions, deletions, or substitutions;

[0024] B) one or more (e.g., 1, 2, or 3) light chain CDRs (CDRLs) selected from at least one of the following: (i) a CDRL1 selected from the group consisting of SEQ ID NOs: 4 and 10, or a CDRL1 that differs from the amino acid sequence of said CDRL1 by no more than 2 amino acid additions, deletions, or substitutions; (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 5 and 11, or a CDRL2 that differs from the amino acid sequence of said CDRL2 by no more than 2 amino acid additions, deletions, or substitutions; and (iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 6 and 12, or a CDRL3 that differs from the amino acid sequence of said CDRL3 by no more than 2 amino acid additions, deletions, or substitutions; or

[0025] C) one or more CDRHs of A) and one or more CDRLs of B).

[0026] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0027] A) a CDRH3 comprising SEQ ID NO: 3 or 9; or

[0028] B) a CDRH3 having at least 90% sequence identity to a CDRH3 represented by one of the sequences selected from SEQ ID NOs: 3 and 9; or

[0029] C) a CDRH3 that differs from the amino acid sequence of the CDRH3 of A) by an amino acid addition, deletion, or substitution of no more than two amino acids,

[0030] and wherein the isolated antibody or antigen-binding portion thereof is expressed in 2×10 -10 M or lower K D Binds human LAG-3.

[0031] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0032] (a) CDRH1 comprising or consisting of SEQ ID NO: 1;

[0033] (b) CDRH2 comprising or consisting of SEQ ID NO: 2;

[0034] (c) CDRH3 comprising or consisting of SEQ ID NO: 3;

[0035] (d) CDRL1 comprising or consisting of SEQ ID NO: 4;

[0036] (e) CDRL2 comprising or consisting of SEQ ID NO: 5; and

[0037] (f) CDRL3 comprising or consisting of SEQ ID NO: 6.

[0038] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0039] (a) CDRH1 comprising or consisting of SEQ ID NO: 7;

[0040] (b) a CDRH2 comprising or consisting of SEQ ID NO: 8;

[0041] (c) a CDRH3 comprising or consisting of SEQ ID NO: 9;

[0042] (d) CDRL1 comprising or consisting of SEQ ID NO: 10;

[0043] (e) CDRL2 comprising or consisting of SEQ ID NO: 11; and

[0044] (f) CDRL3 comprising or consisting of SEQ ID NO: 12.

[0045] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0046] (A) Heavy chain variable region:

[0047] (i) an amino acid sequence comprising SEQ ID NO: 13;

[0048] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 13; or

[0049] (iii) an amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4, or 5) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 13; and / or

[0050] (B) Light chain variable region:

[0051] (i) an amino acid sequence comprising SEQ ID NO: 14;

[0052] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 14; or

[0053] (iii) an amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4 or 5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 14.

[0054] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0055] (A) Heavy chain variable region:

[0056] (i) an amino acid sequence comprising SEQ ID NO: 15;

[0057] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 15; or

[0058] (iii) an amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4, or 5) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 15; and / or

[0059] (B) Light chain variable region:

[0060] (i) an amino acid sequence comprising SEQ ID NO: 16;

[0061] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 16; or

[0062] (iii) an amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4 or 5) amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 16.

[0063] In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region and / or light chain variable region of an isolated antibody as disclosed herein.

[0064] In some aspects, the invention relates to expression vectors comprising a nucleic acid molecule encoding an antibody, or antigen-binding portion thereof, as disclosed herein.

[0065] In some aspects, the invention relates to a host cell comprising an expression vector as disclosed herein.

[0066] In some aspects, the invention relates to pharmaceutical compositions comprising at least one antibody, or antigen-binding portion thereof, as disclosed herein and a pharmaceutically acceptable carrier.

[0067] In some aspects, the invention relates to methods for preparing an anti-LAG-3 antibody, or an antigen-binding portion thereof, comprising expressing the antibody, or the antigen-binding portion thereof, in a host cell and isolating the antibody or the antigen-binding portion from the host cell.

[0068] In some aspects, the invention relates to a method of modulating an antigen-specific T cell response, comprising administering to a subject an antibody, or antigen-binding portion thereof, as disclosed herein, such that the antigen-specific T cell response in the subject is modulated.

[0069] In some aspects, the invention relates to methods of modulating an immune response in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, as disclosed herein, such that the immune response in the subject is modulated.

[0070] In some aspects, the invention relates to a method for inhibiting or blocking the binding of LAG-3 to an MHC class II or FGL1 molecule, comprising contacting the MHC class II or FGL1 molecule with an antibody or antigen-binding portion thereof as disclosed herein.

[0071] In some aspects, the present invention relates to a method for inhibiting or blocking the binding of LAG-3 to LSECtin and / or Galectin-3, comprising contacting the LSECtin and / or Galectin-3 with an antibody or antigen-binding portion thereof as disclosed herein.

[0072] In some aspects, the invention relates to methods for inhibiting the growth of tumor cells in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, as disclosed herein, such that the growth of the tumor in the subject is inhibited.

[0073] In some aspects, the invention relates to methods for treating a viral infection in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, as disclosed herein, such that the viral infection is treated in the subject.

[0074] In some aspects, the invention relates to methods for treating or preventing a proliferative disorder, such as cancer, in a subject, comprising administering to the subject an effective amount of an antibody, or antigen-binding portion thereof, as disclosed herein.

[0075] In some aspects, the invention relates to the use of an antibody, or antigen-binding portion thereof, as disclosed herein in the preparation of a medicament for treating or preventing a proliferative disorder, such as cancer.

[0076] In some aspects, the invention relates to the use of an antibody, or antigen-binding portion thereof, as disclosed herein in the preparation of a diagnostic agent for diagnosing a proliferative disorder, such as cancer.

[0077] In some aspects, the invention relates to an antibody, or antigen-binding portion thereof, as disclosed herein for use in treating or preventing a proliferative disorder, such as cancer.

[0078] In some aspects, the present invention relates to kits or devices and related methods using antibodies or antigen-binding portions thereof as disclosed herein, as well as pharmaceutical compositions as disclosed herein, which can be used to treat proliferative disorders such as cancer. To this end, the present invention preferably provides an article of manufacture that can be used to treat such disorders, comprising a container containing an antibody or antigen-binding portion thereof as disclosed herein and instructional materials for using the antibody or antigen-binding portion thereof as disclosed herein to treat, improve or prevent a proliferative disease or its progression or recurrence. In selected embodiments, the device and related methods will include a step of contacting at least one circulating tumor cell with an antibody or antigen-binding portion thereof as disclosed herein.

[0079] Specifically, the present invention relates to the following embodiments:

[0080] 1. An isolated antibody or antigen-binding portion thereof, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0081] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following:

[0082] (i) a CDRH1 having at least 90% sequence identity to a CDRH1 represented by one of the sequences selected from SEQ ID NOs: 1 and 7;

[0083] (ii) a CDRH2 having at least 90% sequence identity to a CDRH2 represented by one of the sequences selected from SEQ ID NOs: 2 and 8; and

[0084] (iii) a CDRH3 having at least 90% sequence identity to a CDRH3 represented by one of the sequences selected from SEQ ID NOs: 3 and 9;

[0085] B) one or more light chain CDRs (CDRLs) selected from at least one of the following:

[0086] (i) a CDRL1 having at least 90% sequence identity to the CDRL1 represented by one of the sequences selected from SEQ ID NOs: 4 and 10;

[0087] (ii) a CDRL2 having at least 90% sequence identity to the CDRL2 represented by one of the sequences selected from SEQ ID NOs: 5 and 11; and

[0088] (iii) a CDRL3 having at least 90% sequence identity to a CDRL3 represented by one of the sequences selected from SEQ ID NOs: 6 and 12; or

[0089] C) one or more CDRHs of A) and one or more CDRLs of B).

[0090] 2. The isolated antibody or antigen-binding portion thereof of embodiment 1, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0091] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following:

[0092] (i) a CDRH1 selected from SEQ ID NOs: 1 and 7, or a CDRH1 that differs from the amino acid sequence of said CDRH1 by an amino acid addition, deletion or substitution of no more than 2 amino acids;

[0093] (ii) a CDRH2 selected from SEQ ID NOs: 2 and 8, or a CDRH2 that differs from the amino acid sequence of said CDRH2 by an amino acid addition, deletion, or substitution of no more than 2 amino acids; and

[0094] (iii) a CDRH3 selected from SEQ ID NOs: 3 and 9, or a CDRH3 that differs from the amino acid sequence of said CDRH3 by an amino acid addition, deletion, or substitution of no more than 2 amino acids;

[0095] B) one or more light chain CDRs (CDRLs) selected from at least one of the following:

[0096] (i) a CDRL1 selected from SEQ ID NOs: 4 and 10, or a CDRL1 that differs from the amino acid sequence of said CDRL1 by an amino acid addition, deletion or substitution of no more than 2 amino acids;

[0097] (ii) a CDRL2 selected from SEQ ID NOs: 5 and 11, or a CDRL2 that differs from the amino acid sequence of said CDRL2 by an amino acid addition, deletion or substitution of no more than 2 amino acids; and

[0098] (iii) a CDRL3 selected from SEQ ID NOs: 6 and 12, or a CDRL3 that differs from the amino acid sequence of said CDRL3 by an amino acid addition, deletion, or substitution of no more than two amino acids; or

[0099] C) one or more CDRHs of A) and one or more CDRLs of B).

[0100] 3. The isolated antibody, or antigen-binding portion thereof, of embodiment 1, wherein the isolated antibody, or antigen-binding portion thereof, comprises:

[0101] (a) CDRH1 comprising or consisting of SEQ ID NO: 1;

[0102] (b) CDRH2 comprising or consisting of SEQ ID NO: 2;

[0103] (c) CDRH3 comprising or consisting of SEQ ID NO: 3;

[0104] (d) CDRL1 comprising or consisting of SEQ ID NO: 4;

[0105] (e) CDRL2 comprising or consisting of SEQ ID NO: 5; and

[0106] (f) CDRL3 comprising or consisting of SEQ ID NO: 6.

[0107] 4. The isolated antibody, or antigen-binding portion thereof, of embodiment 1, wherein the isolated antibody, or antigen-binding portion thereof, comprises:

[0108] (a) CDRH1 comprising or consisting of SEQ ID NO: 7;

[0109] (b) a CDRH2 comprising or consisting of SEQ ID NO: 8;

[0110] (c) a CDRH3 comprising or consisting of SEQ ID NO: 9;

[0111] (d) CDRL1 comprising or consisting of SEQ ID NO: 10;

[0112] (e) CDRL2 comprising or consisting of SEQ ID NO: 11; and

[0113] (f) CDRL3 comprising or consisting of SEQ ID NO: 12.

[0114] 5. The isolated antibody, or antigen-binding portion thereof, of embodiment 1, wherein the isolated antibody, or antigen-binding portion thereof, comprises:

[0115] (A) Heavy chain variable region:

[0116] (i) an amino acid sequence comprising SEQ ID NO: 13;

[0117] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 13; or

[0118] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 13; and / or

[0119] (B) Light chain variable region:

[0120] (i) an amino acid sequence comprising SEQ ID NO: 14;

[0121] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 14; or

[0122] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 14.

[0123] 6. The isolated antibody, or antigen-binding portion thereof, of embodiment 1, wherein the isolated antibody, or antigen-binding portion thereof, comprises:

[0124] (A) Heavy chain variable region:

[0125] (i) an amino acid sequence comprising SEQ ID NO: 15;

[0126] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 15; or

[0127] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 15; and / or

[0128] (B) Light chain variable region:

[0129] (i) an amino acid sequence comprising SEQ ID NO: 16;

[0130] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 16; or

[0131] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 16.

[0132] 7. The isolated antibody, or antigen-binding portion thereof, of any one of embodiments 1-6, which has one or more of the following properties:

[0133] (a) 2×10 -10 M or lower K D Binds to human LAG-3;

[0134] (b) inhibiting the binding of LAG-3 to major histocompatibility (MHC) class II molecules;

[0135] (c) inhibiting the binding of LAG-3 to the fibrillin-like protein 1 (FGL1) ligand molecule;

[0136] (d) inhibiting the binding of LAG-3 to LSECtin and / or galectin-3;

[0137] (e) binds to human LAG-3 without cross-family reactivity; or

[0138] (f) No cross-reactivity with human CD4.

[0139] 8. The isolated antibody or antigen-binding portion thereof of any one of embodiments 1-7, wherein the antibody is a monoclonal antibody, e.g., a fully human monoclonal antibody, e.g., a fully human monoclonal antibody produced by a transgenic mammal, preferably a transgenic rat, more preferably a transgenic rat with a recombinant immunoglobulin locus.

[0140] 9. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region and / or light chain variable region of the isolated antibody defined in any one of embodiments 1-8, for example, the nucleic acid sequence shown in SEQ ID NOs: 17-20.

[0141] 10. An expression vector comprising the nucleic acid molecule of embodiment 9.

[0142] 11. A host cell comprising the expression vector of embodiment 10.

[0143] 12. A pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 and a pharmaceutically acceptable carrier.

[0144] 13. A method for preparing the antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8, comprising the steps of:

[0145] - expressing the antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 in the host cell of embodiment 11; and

[0146] -Isolating the antibody or antigen-binding portion thereof from the host cell.

[0147] 14. A method of modulating an antigen-specific T cell response in a subject or modulating an immune response in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof as defined in any one of embodiments 1-8, such that the antigen-specific T cell response or immune response in the subject is modulated.

[0148] 15. A method of inhibiting or blocking the binding of LAG-3 to an MHC class II molecule, an FGL1 class molecule, LSECtin and / or Galectin-3, comprising contacting the MHC class II molecule, the FGL1 class molecule, LSECtin and / or Galectin-3 with the antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8.

[0149] 16. A method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, as defined in any one of embodiments 1-8, such that the growth of the tumor in the subject is inhibited.

[0150] 17. A method of treating or preventing a proliferative disorder, such as cancer, in a subject, comprising administering to the subject an effective amount of the antibody, or antigen-binding portion thereof, as defined in any one of embodiments 1 to 8.

[0151] 18. Use of an antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 for the preparation of a medicament for treating or preventing a proliferative disorder such as cancer, an autoimmune disease, an infectious disease and / or an inflammatory disease.

[0152] 19. Use of an antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 for the preparation of a diagnostic agent for diagnosing a proliferative disorder, such as cancer.

[0153] 20. An antibody, or antigen-binding portion thereof, as defined in any one of embodiments 1 to 8 for use in treating or preventing a proliferative disorder such as cancer.

[0154] 21. An antibody or antigen-binding portion thereof as defined in any one of embodiments 1 to 8 for use in diagnosing a proliferative disorder such as cancer.

[0155] 22. A kit for treating or diagnosing a proliferative disorder, such as cancer, comprising a container containing at least one antibody, or antigen-binding portion thereof, as defined in any one of embodiments 1 to 8.

[0156] The above is an overview and therefore contains simplifications, generalizations, and omissions of details, as necessary; therefore, those skilled in the art will recognize that this overview is illustrative only and is 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. An overview is provided to simplify the introduction of some selected concepts, which will be further described in the detailed description below. This overview is not intended to identify the key features or essential features of the claimed subject matter, nor is it intended to be used as an auxiliary means for determining the scope of the claimed subject matter. In addition, the contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] Figure 1 Binding of LAG-3 antibodies to cell surface human LAG-3 is shown, expressed as MFI (mean fluorescence intensity), and measured by BD FACSCanto II.

[0158] Figure 2 Blockade of LAG-3 protein binding to MHC-II expressed on Raji cells is shown.

[0159] Figure 3 Blockade of LAG-3 protein binding to LSECtin is shown.

[0160] Figure 4 Blockade of LAG-3 protein binding to Galectin-3 is shown.

[0161] Figure 5 Cross-reactivity with cynomolgus monkey LAG-3 measured by FACS is shown.

[0162] Figure 6 Cross-reactivity with murine LAG-3 measured by FACS is shown.

[0163] Figure 7 Cross-reactivity with human CD4 measured by ELISA is shown.

[0164] Figure 8A -E shows epitope binning for benchmark antibodies BMK1, BMK7 and BMK5.

[0165] Figure 9A -B shows the results of epitope mapping.

[0166] Figure 10 The effect of human LAG-3 antibody in a reporter gene assay is shown.

[0167] Figure 11 Shown are the effects of human LAG-3 antibodies on human allogeneic mixed lymphocyte reactions as measured by ELISA and reflected by IFN-γ levels (ng / mL).

[0168] Figure 12 The effect of human LAG-3 antibodies on human allogeneic mixed lymphocyte reactions was demonstrated by 3 H-thymidine incorporation was measured and reflected by the proliferative response expressed as CPM (counts per minute) of triplicate wells.

[0169] Figure 13A -B shows the results of CDC and ADCC assays performed by determining target cell lysis.

[0170] Figure 14A -B shows the results of serum stability test as measured by FACS and expressed by MFI of cells. Detailed Description of the Invention

[0171] Although the present invention can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that proves the principle of the present invention. It should be emphasized that the present invention is not limited to the specific embodiment illustrated. In addition, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0172] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have the meanings that are commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular shall include the plural, and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a protein" includes multiple proteins; reference to "a cell" includes mixtures of cells, etc. In this application, unless otherwise indicated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include all values ​​between the endpoints and breakpoints.

[0173] Generally, terms relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and their techniques, are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6 thed., WB Saunders 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 terms, laboratory procedures, and techniques used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. In addition, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0174] definition

[0175] For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0176] As used herein, the term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be divided into kappa and lambda light chains. Heavy chains can be divided into μ, δ, γ, α and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also includes 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 three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form an antigen binding site, respectively. The distribution of amino acids in various regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or the definitions in Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342: 878-883. The antibodies can be of different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0177] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, which may be used interchangeably in the context of this application, refer to polypeptides comprising fragments of a full-length antibody that retain the ability to specifically bind to the antigen to which the full-length antibody specifically binds, and / or which compete 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 produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Under certain 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, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., the monoclonal anti-human LAG-3 antibodies provided herein) by conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and can be screened for specificity in the same manner as intact antibodies.

[0178] As used herein, the term "monoclonal antibody" or "mAb" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0179] As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies with variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present 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 CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0180] The term "human monoclonal antibody," as used herein, refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.

[0181] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0182] The term "chimeric antibody" as used herein refers to antibodies in which the variable region sequences are from one species and the constant region sequences are from another species, for example, wherein the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0183] As used herein, the term "LAG-3" refers to lymphocyte activation gene-3. The term "LAG-3" includes variants, isoforms, homologs, orthologs, and paralogs.

[0184] As used herein, the term "human LAG-3" refers to human sequence LAG-3, for example, the complete amino acid sequence of human LAG-3 having Genbank Accession No. NP_002277. The human LAG-3 sequence may differ from the human LAG-3 of Genbank Accession No. NP_002277, for example, by having conservative mutations in non-conserved regions, and the LAG-3 may have substantially the same biological function as the human LAG-3 of Genbank Accession No. 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 the present disclosure, or the biological function of human LAG-3 may be to bind to an MHC class II or FGL1 molecule.

[0185] As used herein, the term "mouse LAG-3" refers to the complete amino acid sequence of mouse sequence LAG-3, such as mouse LAG-3 having Genbank Accession No. NP_032505.

[0186] As used herein, the term "cynomolgus monkey LAG-3" refers to the complete amino acid sequence of cynomolgus monkey LAG-3, such as cynomolgus monkey LAG-3 having Genbank Accession No. XP_005570011.1.

[0187] As used herein, the term "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods well established in the art. As used herein, the term "Kd" is intended to refer to the association rate of a particular antibody-antigen interaction. D " is intended to represent the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as system.

[0188] As used herein, the term "high affinity" of an IgG antibody refers to an antibody with a high affinity 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 lower K D of antibodies.

[0189] As used herein, the term "EC 50 ”, also known as the “half-maximal effective concentration”, is the concentration of a drug, antibody, or toxicant that induces a response that is 50% between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM".

[0190] As used herein, the ability to "inhibit binding" or "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment thereof to inhibit the binding of two molecules (e.g., human LAG-3 and a human anti-LAG-3 antibody) to any detectable level. In certain embodiments, the binding of the two molecules can be inhibited by the antibody or antigen-binding fragment thereof by at least 50%. In certain embodiments, such inhibition can be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0191] As used herein, the term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to as an "antigenic determinant". An epitope or antigenic determinant is typically composed of chemically active surface groups of molecules such as amino acids, carbohydrates, or sugar side chains, and typically has a specific three-dimensional structure and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 continuous or discontinuous amino acids in a unique three-dimensional conformation, which can be a "linear" or "conformational" 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 a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span amino acid residues separated from each other in the protein. Depending on the competitiveness of the same epitope detected by conventional techniques known to those skilled in the art, antibodies can be screened. For example, competition or cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete in binding antigens (e.g., RSV fusion proteins). In International Patent Application WO 03 / 48731, a high throughput method for obtaining antibodies in conjunction with the same epitope is described, which is based on their cross-competition.

[0192] As used herein, the term "isolated" refers to a state obtained by artificial means from a natural state. If a substance or component is "isolated" in nature, it may be because it has undergone a natural change, or the substance has been separated from nature, or both. For example, a polynucleotide or polypeptide that is not isolated naturally exists in a living organism, and the same highly pure polynucleotide or polypeptide separated from the natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impure substances that do not affect the activity of the isolated substance.

[0193] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds a LAG-3 protein is substantially free of antibodies that specifically bind antigens other than LAG-3 protein). However, an isolated antibody that specifically binds a human LAG-3 protein may have cross-reactivity to other antigens, such as LAG-3 proteins from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0194] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can be transformed, transduced, or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). The 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. In addition, the vector may contain an origin of replication.

[0195] 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 Escherichia coli (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.

[0196] 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 as determined by alignment and comparison of the sequences. "Percent 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 molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, 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, SIAM J. Applied Math. 48:1073.

[0197] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to 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 immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the means of immunity.

[0198] As used herein, the term "transfection" refers to the process of introducing a nucleic acid into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In a specific embodiment of the present invention, the human LAG-3 gene is transfected into 293F cells.

[0199] As used herein, the term "hybridoma" and the term "hybridoma cell line" are used interchangeably. When referring to the term "hybridoma" and the term "hybridoma cell line," they also include subclones and progeny cells of the hybridoma.

[0200] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes an optical phenomenon that allows 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 Examples and U., et al. (1993) Ann. Biol. Clin. 51: 19-26; U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0201] As used herein, the term "fluorescence activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments for performing FACS are known to those skilled in the art and can be commercially available to the public. Examples of such instruments include FACSStar Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), EpicsC from Coulter Epics Division (Hialeah, FL), and MoFlo from Cytomation (Colorado Springs, Colorado).

[0202] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely required for this killing. NK cells, the main cells that mediate ADCC, 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). In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model disclosed in the people PNAS (USA) 95: 652-656 (1998) such as Clynes.

[0203] The term "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.

[0204] The term "subject" includes any human or non-human animal, preferably a human.

[0205] As used herein, the term "cancer" refers to any neoplastic or malignant cell growth, proliferation, or metastasis-mediated medical condition, both solid tumors and non-solid tumors such as leukemias.

[0206] As used herein, the terms "treat" and "treatment" in the context of treating a condition generally refer to treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, for example, inhibition of disease progression, including a decrease in the rate of progression, stagnation of the rate of progression, regression of the disease, improvement of the disease, and cure of the disease. Treatment as a preventative measure (i.e., prophylaxis) is also included. For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination thereof. For a tumor, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0207] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound, or a material, composition, or dosage form comprising an active compound, that, when administered according to a desired treatment regimen, is effective to produce some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. Specifically, "therapeutically effective amount" refers to an amount or concentration of an antibody, or antigen-binding portion thereof, that is effective to treat a human LAG-3-associated disease or disorder.

[0208] As used herein, the "host cell" of the present invention refers to a cell into which an exogenous polynucleotide is introduced.

[0209] As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0210] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to 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, for example, 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.

[0211] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiator that, when delivered to an organism together with an antigen or delivered to an organism in advance, can enhance the immune response to the antigen in the organism or change the type of immune response. There are a variety of adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0212] Anti-LAG-3 antibodies

[0213] In some aspects, the invention includes isolated antibodies or antigen-binding portions thereof.

[0214] In the context of this application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecule, as long as it exhibits preferential binding or association with LAG-3 protein. In addition, unless the context dictates otherwise, the term also includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a human monoclonal antibody.

[0215] Human antibodies can be produced using various techniques known in the art. A type of technology is phage display, in which (preferably human) antibody libraries are synthesized on phage, the library is screened with an antigen of interest or its antibody binding moiety, and the phage bound to the antigen is separated, from which immunoreactive fragments can be obtained. Methods for preparing and screening such libraries are well known in the art, and kits for producing phage display libraries are commercially available (e.g., Pharmacia recombinant phage antibody system, catalog number (Cat. No.) 27-9400-01; and Stratagene SurfZAP™ phage display kit, catalog number (Cat. No.) 240612). Other methods and reagents are also available for producing and screening antibody display libraries (see, for example, Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

[0216] Human antibodies can also be prepared by introducing human immunoglobulin 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 attack, human antibody production is observed, which is very similar to that observed in humans in all aspects, including gene rearrangement, assembly, and antibody repertoires. 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 on XenoMouse technology; Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995). Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes (such B lymphocytes can be obtained from individuals suffering from neoplastic diseases or who may have been immunized in vitro) that produce antibodies against target antigens. See, eg, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol, 147(1):86-95 (1991); and USPN 5,750,373.

[0217] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals (e.g. ) or some combination thereof. For example, monoclonal antibodies can be produced using hybridomas and art-recognized biochemical and genetic engineering techniques, as described in detail in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1 st ed.2009; Shire et.al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC,1 st ed. 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. It will be appreciated that the selected binding sequence can be further altered, for example, to increase affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its in vivo immunogenicity, generate multispecific antibodies, etc., and that antibodies comprising altered target binding sequences are also antibodies of the invention. In a preferred embodiment, anti-human LAG-3 monoclonal antibodies are prepared using hybridomas.

[0218] Generation of hybridomas producing human monoclonal antibodies of the present invention

[0219] To obtain hybridomas that produce antibodies of the invention, such as human monoclonal antibodies of the invention, splenocytes and / or lymph node cells from immunized mice can be isolated and fused to 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 production 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.

[0220] Generation of transfectomas producing monoclonal antibodies of the invention

[0221] The antibodies of the present invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody genes are linked to the vector so that the transcriptional and translational control sequences within the vector perform their intended functions of regulating transcription and translation of the antibody genes.

[0222] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the 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), adenovirus (e.g., the adenovirus major late promoter (AdMLP), and polyoma virus, or non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter can be used; also, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al. (1988) MoI. Cell. Biol. 8: 466-472). The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

[0223] In some embodiments, the antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors.In some embodiments, the variable region is used to produce the full-length antibody gene of any antibody isotype by being inserted into the expression vector of the heavy chain constant region and the light chain constant region of the required isotype encoded, so that the VH segment is operably connected to the CH segment and the VL segment in the carrier are operably connected to the CL segment in the carrier.In addition or alternatively, the recombinant expression vector can encode the signal peptide that promotes the secretion of antibody chain from the host cell.The antibody chain gene can be cloned into the vector so that the signal peptide is connected to the amino terminal of the antibody chain gene in frame.The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0224] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector of the present invention can also carry additional sequences, such as sequences (such as replication origins) and selective marker genes that regulate the replication of the vector in the host cell. Selective marker genes help select the host cells into 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, conventional selective marker genes confer resistance to drugs (such as G418, hygromycin or methotrexate) to the host cells into which the vector has been introduced. Selective marker genes can include dihydrofolate reductase (DHFR) genes (for dhfr- host cells with methotrexate selection / amplification) and neo genes (for G418 selection).

[0225] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. 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 appropriately folded and immunologically active antibodies.

[0226] Mammalian host cells for expressing 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), NSO myeloma cells, COS cells, and SP2 cells, used with a DHFR selectable marker (e.g., as described in RJ Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159: 601-621). Another expression system, particularly for use with NSO myeloma, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0227] Anti-LAG3 antibodies with certain properties

[0228] The antibodies of the present invention are characterized by specific functional characteristics or properties of the antibodies. In some embodiments, the isolated antibodies or antigen-binding portions thereof have one or more of the following properties:

[0229] (a) 2×10-10 M or lower K D Binds to human LAG-3;

[0230] (b) inhibiting the binding of LAG-3 to major histocompatibility (MHC) class II molecules;

[0231] (c) inhibiting the binding of LAG-3 to the fibrillin-like protein 1 (FGL1) ligand molecule;

[0232] (d) inhibiting the binding of LAG-3 to LSECtin and / or galectin-3; or

[0233] (e) Binds to human LAG-3 without cross-family reactivity.

[0234] The antibodies of the present invention bind to human LAG-3 with high affinity. Binding of the antibodies of the present invention to LAG-3 can be assessed using one or more techniques well established in the art, such as ELISA. The binding specificity of the antibodies of the present invention can also be determined by, for example, monitoring binding of the antibodies to cells expressing LAG-3 protein by flow cytometry. For example, the antibodies can be tested by flow cytometry assays in which the antibodies react with cell lines expressing human LAG-3, such as CHO cells that have been transfected to express LAG-3 on their cell surface. Other suitable cells for flow cytometry assays include anti-CD3-stimulated CD4 expressing native LAG-3. + Additionally or alternatively, the binding of the antibodies can be tested in a BIAcore binding assay, including binding kinetics (e.g., Kd values). Other suitable binding assays include ELISA assays, for example, using recombinant LAG-3 protein. For example, an antibody of the invention can be tested at 5×10 -8 M or lower K D Bind to human LAG-3 protein, at 2×10 -8 M or lower K D Bind to human LAG-3 protein, 5×10 -9 M or lower K D Bind to human LAG-3 protein, at 4×10 -9 M or lower K D Bind to human LAG-3 protein, at 3×10 -9 M or lower K D Bind to human LAG-3 protein, at 2×10 -9 M or lower K D Bind to human LAG-3 protein, at 1×10 -9 M or lower K D Bind to human LAG-3 protein, 5×10 -10 M or lower K DBind to human LAG-3 protein, or at 1×10 -10 M or lower K D Binds to human LAG-3 protein.

[0235] The ability of an antibody to modulate an immune response (e.g., an antigen-specific T cell response) can be indicated, for example, by the ability of the antibody to stimulate the production of interleukin-2 (IL-2) in an antigen-specific T cell response. In certain embodiments, the antibodies of the invention bind to human LAG-3 and exhibit the ability to stimulate an antigen-specific T cell response. Means for assessing the ability of an antibody to stimulate an immune response can include, for example, the ability of the antibody to inhibit tumor growth in an in vivo tumor transplant model, or the ability of the antibody to stimulate an autoimmune response.

[0236] The isolated antibodies or antigen-binding portions thereof disclosed herein inhibit the binding of LAG-3 to major histocompatibility (MHC) class II molecules, FGL1 class molecules, LSECtin and / or galectin-3. LAG-3 negatively regulates T cell signaling and function. Ligands for LAG-3 include, for example, major histocompatibility (MHC) class II molecules, LSECtin and galectin-3. LAG-3 can interact with MHC class II molecules on the cell surface (Baixeras et al. (1992) J. Exp. Med. 176: 327-337; Huard et al. (1996) Eur. J. Immunol. 26: 1180-1186). It has been proposed that direct binding of LAG-3 to MHC class II molecules plays a role in downregulating CD4 T cell signaling. + FGL1 plays a role in antigen-dependent stimulation of T lymphocytes (Huard et al. (1994) Eur. J. Immunol. 24:3216-3221). Recently, Chen Lie 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 FGL1-LAG-3 interaction can enhance anti-tumor effects (Cell. 2019 Jan 10; 176(1-2):334-347.e12.).

[0237] Galectin-3 is a 31 kD lectin that regulates T cell responses through several mechanisms, including apoptosis, TCR crosslinking, and TCR downregulation. Galectin-3 binds to LAG-3, and LAG-3 expression is essential for galectin-3-mediated inhibition of CD8+ T cells in vitro. (Kouo et al. (2015) Cancer Immunol. Res. 10.1158:2326-6066). Anti-LSECtin has been shown to inhibit B16 melanoma cell growth (Xu et al. (2014) Cancer Res. 74(13):3418-3428).

[0238] Anti-LAG3 antibodies comprising CDRs having sequence identity to a specific sequence

[0239] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0240] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following: (i) a CDRH1 having at least 90% sequence identity to a CDRH1 set forth in one of the sequences selected from SEQ ID NOs: 1 and 7; (ii) a CDRH2 having at least 90% sequence identity to a CDRH2 set forth in one of the sequences selected from SEQ ID NOs: 2 and 8; and (iii) a CDRH3 having at least 90% sequence identity to a CDRH3 set forth in one of the sequences selected from SEQ ID NOs: 3 and 9;

[0241] B) one or more light chain CDRs (CDRLs) selected from at least one of the following: (i) a CDRL1 having at least 90% sequence identity to a CDRL1 set forth in one of the sequences selected from SEQ ID NOs: 4 and 10; (ii) a CDRL2 having at least 90% sequence identity to a CDRL2 set forth in one of the sequences selected from SEQ ID NOs: 5 and 11; and (iii) a CDRL3 having at least 90% sequence identity to a CDRL3 set forth in one of the sequences selected from SEQ ID NOs: 6 and 12; or

[0242] C) one or more CDRHs of A) and one or more CDRLs of B).

[0243] Unless otherwise indicated, the assignment of amino acids to each CDR may be based on one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5 thEd.),USDept.of Health and Human Services,PHS,NIH,NIH Publication no.91-3242The Chothia Society,1987,PMID:3681981The Chothia Society,1989,PMID:2 687698;MacCallum Publications,1996,PMID:8876650;MacDubel,Ed.(2007)Handbook of Therapeutic Antibodies,3 rd Ed.,Wily-VCH Publishing House.

[0244] The variable region and CDR in the antibody sequence can be identified according to the general rules (as mentioned above, such as Kabat numbering system) that have been developed in the art or by comparing the sequence with the database of known variable regions. 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, the method for identifying these regions has been described in 2000. The exemplary database of antibody sequence is described in and can be obtained from " Abysis " website (maintained by AC Martin of Department of Biochemistry&Molecular Biology University College London, London, England) and VBASE2 website www.vbase2.org on www.bioinf.org.uk / abs, such as Retter et al., Nucl.Acids Res., 33 (Database issue): described in D671-D674 (2005). Preferably, the sequence is analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB, see "Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual" by Dr. Andrew CR Martin (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 to identify CDRs that can be used according to the teachings of this article. Unless otherwise noted, all CDRs described herein are obtained according to the Abysis database website of Kabat.

[0245] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0246] Additionally or alternatively, the protein sequences of the present invention can be further used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecules of the present invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0247] In other embodiments, the CDR amino acid sequence can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-described sequences. As an illustrative example, the antibody can comprise a CDRH1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a CDRH1 set forth in one of the sequences selected from SEQ ID NOs: 1 and 7.

[0248] Anti-LAG3 Antibodies Comprising CDRs with Amino Acid Additions, Deletions, or Substitutions In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0249] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following: (i) a CDRH1 selected from SEQ ID NOs: 1 and 7, or a CDRH1 that differs from the amino acid sequence of said CDRH1 by no more than two amino acid additions, deletions or substitutions; (ii) a CDRH2 selected from SEQ ID NOs: 2 and 8, or a CDRH2 that differs from the amino acid sequence of said CDRH2 by no more than two amino acid additions, deletions or substitutions; and (iii) a CDRH3 selected from SEQ ID NOs: 3 and 9, or a CDRH3 that differs from the amino acid sequence of said CDRH3 by no more than two amino acid additions, deletions or substitutions;

[0250] B) one or more light chain CDRs (CDRLs) selected from at least one of the following: (i) a CDRL1 selected from the group consisting of SEQ ID NOs: 4 and 10, or a CDRL1 that differs from the amino acid sequence of said CDRL1 by no more than 2 amino acid additions, deletions or substitutions; (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 5 and 11, or a CDRL2 that differs from the amino acid sequence of said CDRL2 by no more than 2 amino acid additions, deletions or substitutions; and (iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 6 and 12, or a CDRL3 that differs from the amino acid sequence of said CDRL3 by no more than 2 amino acid additions, deletions or substitutions; or

[0251] C) one or more CDRHs of A) and one or more CDRLs of B).

[0252] Preferably, the CDR of the isolated antibody or its antigen-binding portion thereof contains no more than 2 amino acids or no more than 1 amino acid conservative substitution. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted by another amino acid residue with a similar side chain, such as physical or functionally similar residues (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent bonds or hydrogen bonds, etc.) to corresponding amino acid residues. 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, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0253] Anti-LAG3 antibodies containing CDRs

[0254] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0255] (a) CDRH1 comprising SEQ ID NO: 1;

[0256] (b) CDRH2 comprising SEQ ID NO: 2;

[0257] (c) CDRH3 comprising SEQ ID NO: 3;

[0258] (d) CDRL1 comprising SEQ ID NO: 4;

[0259] (e) CDRL2 comprising SEQ ID NO: 5; and

[0260] (f) CDRL3 comprising SEQ ID NO:6.

[0261] In specific embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0262] (a) CDRH1 consisting of SEQ ID NO: 1;

[0263] (b) CDRH2 consisting of SEQ ID NO: 2;

[0264] (c) CDRH3 consisting of SEQ ID NO: 3;

[0265] (d) CDRL1 consisting of SEQ ID NO: 4;

[0266] (e) CDRL2 consisting of SEQ ID NO: 5; and

[0267] (f) CDRL3 consisting of SEQ ID NO:6.

[0268] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0269] (a) CDRH1 comprising SEQ ID NO: 7;

[0270] (b) CDRH2 comprising SEQ ID NO: 8;

[0271] (c) CDRH3 comprising SEQ ID NO: 9;

[0272] (d) CDRL1 comprising SEQ ID NO: 10;

[0273] (e) CDRL2 comprising SEQ ID NO: 11; and

[0274] (f) CDRL3 comprising SEQ ID NO:12.

[0275] In specific embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0276] (a) CDRH1 consisting of SEQ ID NO: 7;

[0277] (b) CDRH2 consisting of SEQ ID NO: 8;

[0278] (c) CDRH3 consisting of SEQ ID NO: 9;

[0279] (d) CDRL1 consisting of SEQ ID NO: 10;

[0280] (e) CDRL2 consisting of SEQ ID NO: 11; and

[0281] (f) CDRL3 consisting of SEQ ID NO:12.

[0282] Anti-LAG3 antibody comprising a heavy chain variable region and a light chain variable region

[0283] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0284] (A) Heavy chain variable region:

[0285] (i) an amino acid sequence comprising SEQ ID NO: 13;

[0286] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 13; or

[0287] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 13; and / or

[0288] (B) Light chain variable region:

[0289] (i) an amino acid sequence comprising SEQ ID NO: 14;

[0290] (ii) comprising an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 14;

[0291] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 14.

[0292] In specific embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0293] (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 13; and / or

[0294] (b) A light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

[0295] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0296] (A) Heavy chain variable region:

[0297] (i) an amino acid sequence comprising SEQ ID NO: 15;

[0298] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 15; or

[0299] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 15; and / or

[0300] (B) Light chain variable region:

[0301] (i) an amino acid sequence comprising SEQ ID NO: 16;

[0302] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 16; or

[0303] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to SEQ ID NO: 16.

[0304] In specific embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0305] (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15; and / or

[0306] (b) A light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.

[0307] In other embodiments, the amino acid sequences of the heavy chain variable region and / or light chain variable region can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned respective sequences. As an illustrative example, the antibody can 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 to the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15.

[0308] In some further embodiments, the isolated antibody or antigen-binding portion thereof may comprise conservative substitutions or modifications of amino acids in the variable region of the heavy and / or light chain. It is understood in the art that certain conservative sequence modifications can be made that do not eliminate antigen binding. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0309] The term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted with 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 bonds or hydrogen bonds, etc.) to corresponding amino acid residues. 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, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0310] Binning and epitope mapping

[0311] It will be further understood that the disclosed antibodies will associate or bind to discrete epitopes or immunogenic determinants presented by the selected target or fragment thereof. In certain embodiments, epitopes or immunogenic determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge characteristics. Thus, as used herein, the term "epitope" includes any protein determinant that is capable of specifically binding to an immunoglobulin or T cell receptor or otherwise interacting with a molecule. In certain embodiments, an antibody is considered to specifically bind (or immunospecifically bind or react) to an antigen when the antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, when the equilibrium dissociation constant (K D ) is less than or equal to 10 -6M or less than or equal to 10 -7 M, more preferably when K D Less than or equal to 10 -7 When M, the specific binding of antibody to antigen is equal to 10 -8 M, or even more preferably when K D Less than or equal to 10 -9 When M, the antibody is said to bind specifically to the antigen.

[0312] Epitopes formed by consecutive amino acids (sometimes referred to as "linear" or "continuous" epitopes) are generally retained upon protein denaturation, whereas epitopes formed by tertiary folding are generally lost upon protein denaturation. In any case, an antibody epitope generally comprises at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation.

[0313] In this regard, it will be understood that, in certain embodiments, an epitope can be associated with or located within one or more regions, domains, or motifs of, for example, a LAG-3 protein. Similarly, the art-recognized term "motif" will be used according to its ordinary meaning and will generally refer to a short conserved region of a protein, typically ten to twenty consecutive amino acid residues.

[0314] In any case, once the desired epitope on the antigen has been determined, it is possible to produce antibodies against the epitope, for example by immunizing with a peptide comprising the epitope using the technology described in the present invention. Alternatively, in the discovery process, the generation and characterization of antibodies can illustrate information about the desired epitope located in a specific domain or motif. From this information, competitive screening can be used to identify antibodies that are combined with the same epitope. The method for achieving this is to conduct competitive studies to find antibodies that competitively bind to each other, i.e., antibodies compete for binding to the antigen. A high-throughput method for binning antibodies based on their cross-competition is described in WO 03 / 48731. Other methods of binning or domain level or epitope mapping including antibody competition or antigen fragment expression on yeast are well known in the art.

[0315] As used herein, the term "binning" refers to a method for grouping or classifying antibodies based on antigen binding characteristics and competition. Although these techniques are useful for defining and classifying the antibodies of the present invention, these bins are not always directly bound to epitopes, and this initial determination of epitope binding can be further improved and confirmed by other recognized methods in the art and as described herein. However, it will be understood that empirically assigning antibodies to individual bins provides information that can indicate the therapeutic potential of the disclosed antibodies.

[0316] More specifically, whether a selected reference antibody (or fragment thereof) binds to the same epitope or cross-competes for binding (i.e., is in the same bin) as a second test antibody can be determined by using methods known in the art and shown in the Examples herein.

[0317] Other compatible epitope mapping techniques include alanine scanning mutagenesis, peptide blotting (Reineke (2004) Methods Mol Biol 248: 443-63) (specifically incorporated herein by reference in its entirety) or peptide cleavage analysis. In addition, epitope excision, epitope extraction and chemical modification of antigens can be used (Tomer (2000) Protein Science 9: 487-496) (specifically incorporated herein by reference in its entirety).

[0318] Nucleic acid molecules encoding the antibodies of the present invention

[0319] In some aspects, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region and / or light chain variable region of an isolated antibody as disclosed herein.

[0320] Nucleic acids of the present invention can be obtained using standard molecular biology techniques. For hybridoma-expressed antibodies (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as further described below), cDNA encoding the light and heavy chains of the antibodies prepared by the 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), nucleic acids encoding such antibodies can be recovered from the gene library.

[0321] The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding the VH region to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), supra), 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.

[0322] By operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, the isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequence of human light chain constant region genes is known in the art (see, for example, Kabat et al., supra), and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0323] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to mean that the two DNA fragments are connected so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0324] Preferred nucleic acid molecules of the present invention are nucleic acid molecules encoding the VH and VL sequences of the 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L monoclonal antibodies. The DNA sequences encoding the VH sequences of 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are shown in SEQ ID NOs: 17 and 19, 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 NOs: 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 to SEQ ID NOs: 17-20, respectively. In some embodiments, the percentage of identity arises from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0325] Pharmaceutical composition

[0326] In some aspects, the invention relates to pharmaceutical compositions comprising at least one antibody, or antigen-binding portion thereof, as disclosed herein and a pharmaceutically acceptable carrier.

[0327] Components of the composition

[0328] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition 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 auxiliary substances, combinations of various components known in the art, or more.

[0329] 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, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propyl arsenate. As disclosed herein, in a solvent containing an antibody or antigen-binding fragment of the composition disclosed herein that contains one or more antioxidants such as methionine that reduce the antibody or antigen-binding fragment thereof, it may be oxidized. Redox can prevent or reduce the reduction in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present invention provides a composition comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. The present invention further provides various methods in which an antibody or antigen-binding fragment thereof is mixed with one or more antioxidants such as methionine. Thus, the antibody or antigen-binding fragment thereof can be protected from oxidation to extend its shelf life and / or increase its activity.

[0330] Administration, formulation and dosage

[0331] The pharmaceutical compositions of the present invention can be administered to a subject in need thereof in vivo via various routes, including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardial, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into solid, semisolid, liquid, or gaseous formulations, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. Suitable formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0332] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0333] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic vehicles suitable for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosage regimen (i.e., dosage, timing, and repetition) will depend on the specific individual and individual's medical history as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0334] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such tumor cells, reducing tumor cell proliferation or delaying the development of metastases. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the present invention may be suitable.

[0335] Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit with any risks or adverse side effects. The dosage level selected will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, time of administration, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, sex, age, weight, condition, general health and previous medical history of the patient. The amount of the compound and route of administration are ultimately determined by the physician, veterinarian or clinician, but the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0336] Generally, the antibodies of the present invention, or their antigen-binding portions thereof, can be administered in various 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; 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 certain embodiments, the dosage 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, at least about 10 mg / kg body weight.

[0337] In any case, the antibodies or antigen-binding portions thereof of the present invention are preferably administered to subjects in need thereof as needed. The frequency of administration can be determined by one skilled in the art, for example, by the attending physician based on considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, and the like.

[0338] In certain preferred embodiments, a course of treatment involving an antibody or antigen-binding portion thereof of the invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibody or antigen-binding portion thereof of the invention 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 will be appreciated that the dosage may be varied or the interval adjusted based on patient response and clinical practice.

[0339] The dosage and regimen of the disclosed therapeutic compositions can also be determined empirically in individuals given one or more administrations. For example, an individual can be given incremental doses of a therapeutic composition produced as described herein. In selected embodiments, the dosage can be gradually increased or decreased or reduced, respectively, based on empirically determined or observed side effects or toxicity. To assess the efficacy of the selected composition, markers of a particular disease, disorder, or condition can be tracked as described above. 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 specimens; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein, reduction of pain or paralysis; improvement in speech, vision, breathing, or other disabilities associated with the tumor; increased appetite; or improved quality of life or prolonged survival as measured by accepted tests. One skilled in the art will appreciate that the dosage will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and previous and concurrent treatments.

[0340] Compatible formulations for parenteral administration (e.g., intravenous injection) will comprise the antibody, or its antigen-binding portion thereof, at a concentration of about 10 μg / mL to about 100 mg / mL. In certain selected embodiments, the concentration of the antibody, or its antigen-binding portion thereof, will include 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / 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.

[0341] Application of the present invention

[0342] The antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo uses, including, for example, detection of LAG-3 or enhancing immune responses by blocking LAG-3. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or to human subjects in vivo, for example, to enhance immunity in various situations. The immune response can be modulated, for example, enhanced, stimulated, or upregulated.

[0343] Preferred subjects include human patients in need of an enhanced immune response. The methods are particularly suitable for treating human patients with conditions treatable by enhancing an immune response (e.g., a T cell-mediated immune response). In a specific embodiment, the methods are particularly suitable for treating cancer in vivo. To achieve antigen-specific enhancement of immunity, anti-LAG-3 antibodies 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 an antibody against LAG-3 is administered together with another agent, the two agents can be administered in any order or simultaneously.

[0344] The present invention further provides a method for detecting the presence of a human LAG-3 antigen in a sample or measuring the amount of a human LAG-3 antigen, comprising contacting the sample and a control sample with a human monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to human LAG-3 under conditions that allow for the formation of a complex between the antibody, or portion thereof, and human LAG-3. Complex formation is then detected, wherein differential complex formation between the sample and the control sample indicates the presence of the human LAG-3 antigen in the sample. Furthermore, the anti-LAG-3 antibodies of the present invention can be used to purify human LAG-3 by immunoaffinity purification.

[0345] Given the ability of the anti-LAG-3 antibodies of the present invention to inhibit the binding of LAG-3 to MHC class II or FGL1 molecules and 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 antibodies of the present invention. For example, the present invention provides methods for stimulating antigen-specific T cell responses, comprising administering an antibody of the present invention, or an antigen-binding portion thereof, to a subject, thereby stimulating an antigen-specific T cell response. Antigen-specific T cell responses can be measured using any suitable antigen-specific T cell response indicator.

[0346] Cancer treatment

[0347] Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of the antibody and / or increased cytokine production in the presence of the antibody. In a preferred embodiment, interleukin-2 production by antigen-specific T cells is stimulated. The present invention also provides a method of stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering an antibody of the present invention, or an antigen-binding portion thereof, to the subject, thereby stimulating an immune response (e.g., an antigen-specific T cell response). In a preferred embodiment, the subject is a cancer-bearing subject and an immune response against the tumor is stimulated. Cancer blocking of LAG-3 by antibodies can enhance the patient's immune response to cancer cells. Anti-LAG-3 antibodies can be used alone or in combination with other immunogenic agents, standard cancer therapeutics, or other antibodies.

[0348] 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, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including acute bone marrow cancer, Myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) cancer, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including cancers induced by asbestos, and combinations of said cancers.

[0349] The antibodies, or antigen-binding portions thereof, can be used in combination with chemotherapy or radiation therapy.

[0350] Used in combination with chemotherapy

[0351] The antibodies, or antigen-binding portions thereof, can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0352] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat a cell proliferative disorder, such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatics, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, and anti-metastatic and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise a conjugate and may be combined with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer agent is linked to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are explicitly contemplated within the scope of the present invention. In other embodiments, the disclosed anticancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.

[0353] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell destruction. In certain embodiments, the 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 endotoxins and exotoxins, Staphylococcal enterotoxin A), fungi (e.g., α-sarcin, restrictocin), plants (abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, hordeotoxin, Aleurites fordii proteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, phytoncidin inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin, and trichothecenes), or animals (e.g., cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including fragments and / or variants thereof).

[0354] For purposes of the present invention, "chemotherapeutic agents" include chemical compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. These chemicals are typically directed against the 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. Typically, chemotherapeutic agents can include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become resistant or produce tumorigenic offspring (e.g., TICs). These agents are typically used in combination and are typically the most effective, for example, in regimens such as CHOP or FOLFIRI.

[0355] Examples of anticancer agents that can be used in combination with the site-specific constructs of the present invention (either as a component of a site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimine and methylmelamine, acetogenins, camptothecins, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, sarcodipine, saccharin ... yin), spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicins, chromophores of enediyne antibiotics, aclacinomycins, actinomycins, anthramycins, azoserine, bleomycins, actinomycin C, carabicin, carminomycin, carmomycin, chromomycins, dactinomycin, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, epirubicin, esorubicin, idarubicin, mexilomycin, mitomycin, mycophenolic acid, nogamycin, olivomycin, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, zoloft, daunorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenergics, folic acid supplements such as furinic acid acid), aceglucuronolactone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, colcemid, diacridone, elfornithine, elliptonium acetate, apocillon, etoglucagon, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids, mitoguanidine, mitoxantrone, mopidanmol, nitraerine, pentostatin, methamidine, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazine, procarbazine, Polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizolan; spirogermanamine; tenuzolic acid; triazinon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculocin A, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chloranbucil; Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine, Vinorelbine; Novolin; Teniposide; Edatrexate; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); the topoisomerase inhibitor RFS 2000; Difluoromethylornithine; Retinoids; Capecitabine; Combretastatin; 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 foregoing. Also included within this definition are antihormonal agents used to modulate or inhibit hormonal effects on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; Topoisomerase 1 inhibitors; rmRH; vinorelbine and esperamicin, and pharmaceutically acceptable salts, acids or derivatives of any one of the foregoing.

[0356] Used in combination with radiation therapy

[0357] The present invention also provides a combination of an antibody or an antigen-binding portion thereof with radiotherapy (i.e., any mechanism for inducing DNA damage locally in tumor cells, such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electron emission, etc.). Combination therapies using directed 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 means. Typically, radiotherapy is administered in a pulsed manner over a period of about 1 week to about 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.

[0358] diagnosis

[0359] The present invention provides methods for detecting, diagnosing or monitoring proliferative disorders in vitro and in vivo and screening cells from patients to identify tumor cells including methods of tumorigenic cells. Such methods include identifying individuals with cancer for treatment or monitoring the progression of cancer, 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 or level of binding of the antibody to a bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or reporter molecule as described herein.

[0360] In some embodiments, binding of an antibody to specific cells in a sample can indicate that the sample is likely to contain tumorigenic cells, thereby indicating that an individual with cancer can be effectively treated with the antibodies described herein.

[0361] Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostics or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, and the like, known to those skilled in the art.

[0362] Drug packaging and kits

[0363] Also provided are pharmaceutical packages and kits comprising one or more containers containing one or more doses of an antibody or antigen-binding portion thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or antigen-binding portion thereof, with or without one or more other agents. For other embodiments, such a unit dose is supplied in a disposable prefilled syringe for injection. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; a buffer such as phosphate, etc.; and / or be formulated within a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder that can be reconstituted after adding a suitable liquid (e.g., sterile water or saline solution). In certain 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 encapsulated conjugate composition is used to treat a selected tumor disease condition.

[0364] The present invention also provides a kit for producing a single-dose or multi-dose administration unit of a site-specific conjugate and, optionally, one or more anticancer agents. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from a variety of materials, such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed conjugate in conjugated or non-conjugated form. In other preferred embodiments, the container includes a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). Such a kit typically contains a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container and, optionally, one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnosis or combination therapy. For example, in addition to the antibodies or antigen-binding portions thereof of the present invention, such a kit may contain any one or more anticancer agents, such as chemotherapeutic agents or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0365] More specifically, the kit can have a single container containing the disclosed antibody or antigen-binding portion thereof, with or without additional components, or they can have different containers for each required reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution can be premixed in a molar equivalent combination or in a manner where one component is more than another. Alternatively, the conjugate and any optional anticancer agent of the kit can be stored separately in different containers before being administered to a patient. The kit can also include a second / third container device for holding a sterile pharmaceutically acceptable buffer or other diluent such as antibacterial water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0366] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as a dry powder. When the reagents or components are provided in dry powder form, the powder may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in a separate container.

[0367] As briefly described above, the kit may also contain a means for administering the antibody or antigen-binding portion thereof 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 formulation can be injected or introduced into an animal or applied to an affected area of ​​the body. The kits of the present invention typically also include a device for containing vials or the like and other tightly closed components for commercial sale, such as injection or blow-molded plastic containers, in which the required vials and other devices are placed and retained.

[0368] Sequence Listing Overview

[0369] This application is accompanied by a sequence listing comprising a number of nucleic acid and amino acid sequences. The following table provides an overview of the sequences included.

[0370]

[0371] Example

[0372] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention. These examples are not intended to represent that the experiments below are all or the only experiments performed.

[0373] Example 1

[0374] Material preparation

[0375] 1. Generation of Immunogens

[0376] Nucleic acids encoding the human LAG-3 ECD (extracellular domain, ECD) with SEQ ID NO: 22 or full-length human LAG-3 with SEQ ID NO: 24 were synthesized by Sangon Biotech. The amino acid sequence of the LAG-3 ECD and the DNA sequence encoding it are shown in SEQ ID NOs: 21 and 22, respectively, and the amino acid sequence of the full-length LAG-3 and the DNA sequence encoding it are shown in SEQ ID NOs: 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 the 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). The cells were cultured at 37°C, 5% CO₂ in FreeStyle 293 Expression Medium (ThermoFisher). After 5 days of culture, supernatants harvested from transiently transfected cell cultures were used for protein purification. The fusion protein was purified by nickel, protein A, and / or SEC columns. Untagged LAG-3 ECD protein was generated by cleavage of the ECD-hFc fusion protein at the cleavage site using Factor Xa protease (New England Biolabs). The purified protein was used for immunization, screening, and characterization.

[0377] 2. Generation of benchmark antibodies

[0378] Reference anti-human LAG-3 antibodies (BMK1 and BMK7, BMK1 referred to as "25F7" in US 20110150892 A1 and BMK7 referred to as "H4sH15482P" in US 20170101472 A1) were synthesized based on information disclosed in patent applications US 20110150892 A1 and US 20170101472 A1. Reference antibody BMK8 is a humanized version of the chimeric antibody BMK5, described in WO2015138920A1 and referred to as "BAP050-chi." BMK8 is referred to as "BAP050-hum01" in WO2015138920A1. As described in Section 1 above, the synthesized gene sequence was integrated into the plasmid pcDNA3.3. The plasmid was transiently transfected into 293F cells. The cells were cultured in the same manner as described in Section 1. After 5 days of culture, supernatants from transiently transfected cell cultures were harvested and used for protein purification. Benchmark antibodies were purified from the supernatants.

[0379] 3. Establishment of Stable Cell Lines

[0380] Generation of human, mouse, and cynomolgus monkey LAG-3 transfected cell lines. Briefly, Flp-In-293, Flp-In-CHO, or 293F cells were transfected with pcDNA3.3 expression vectors containing full-length human, mouse, and cynomolgus monkey LAG-3, respectively, using the Lipofectamine 2000 transfection kit according to the manufacturer's protocol. 48-72 hours after transfection, transfected cells were cultured in blasticidin-containing medium for selection and testing of LAG-3 expression. Human, cynomolgus monkey, and mouse LAG-3-expressing cell lines were obtained by limiting dilution.

[0381] Example 2

[0382] Antibody hybridoma generation

[0383] 1. Immunization and Cell Fusion

[0384] 24-week-old OMT rats (transgenic rats with recombinant immunoglobulin loci, as described in US Pat. No. 8,907,157 B2) were alternately immunized with 12.5 μg of hFc-tagged human LAG-3 ECD protein and 12.5 μg of His-tagged mouse LAG-3 in adjuvant to generate antibodies in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Blood was collected from immunized rats every two weeks for serum collection, and the anti-human LAG-3 titer in the serum was measured by ELISA. Plates coated with human LAG-3.ECD.hFc were incubated with diluted rat serum (first 1:100, then diluted 3-fold in 2% BSA) for 2 hours. Goat anti-rat IgG-Fc-HRP was used as the secondary antibody. Color was developed 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 antibody titers reached a sufficiently high level, rats were given a final boost with 40 μg of human LAG-3 ECD protein in DPBS without adjuvant. On the day of fusion, lymph nodes and spleens were removed from the immunized rats under sterile conditions and prepared into single-cell suspensions. The isolated cells were then mixed with myeloma SP2 / 0 cells at a 1:1 ratio. Electrocellular fusion was performed using a BTX 2000 Electrocell manipulator. The cells were then plated at 1 × 10 4 Cells were seeded at a density of 10 cells / well in 96-well plates and incubated at 37°C, 5% CO2 until ready for screening.

[0385] 2. Preliminary Screening and Confirmatory Screening of Hybridoma Supernatants

[0386] An ELISA assay was used as the first screening method to test hybridoma supernatants for binding to LAG-3 protein. The plates from Section 1 of this example were coated overnight at 4°C with 1 μg / mL of human LAG-3 ECD.hFc. After blocking and washing, the hybridoma supernatants were transferred to the coated plates and incubated at room temperature for 1 hour. The plates were then washed and incubated with a secondary antibody, goat anti-rat IgG HRP, for 1 hour. After washing, TMB substrate was added, and the color reaction was stopped with 2M HCl. The absorbance was read at 450 nm using a microplate reader.

[0387] To confirm the natural binding of LAG-3 antibodies to conformational LAG-3 molecules expressed on the cell membrane, flow cytometry analysis was performed on LAG-3 transfected CHO-K1 cells. CHO-K1 cells expressing human LAG-3 were cultured at 1x10 5 Cells were transferred to a 96-well U-bottom plate at a density of 10 cells / well. Hybridoma supernatants were then transferred to the plates and incubated at 4°C for 1 hour. After washing with 1× PBS / 1% BSA, secondary antibody goat anti-rat IgG Alexa647 was added and incubated with the cells at 4°C in the dark for 0.5 hours. The cells were then washed and resuspended in 1× PBS / 1% BSA before analysis by flow cytometry. Antibody binding to the parental CHO-K1 cell line was performed in parallel as a negative control.

[0388] The blocking activity of the antibodies was used as a confirmatory screen 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 cell line Raji was tested by FACS analysis. Raji cells were plated at 1x10 5 Cells were transferred to a 96-well U-bottom plate at a density of 10 cells / well. The supernatant was incubated with mFc-tagged LAG-3 protein at 4°C for 30 minutes. The mixture was transferred to a 96-well plate seeded with Raji cells. The secondary antibody, PE-labeled goat anti-mouse IgG (non-cross-reactive with rat IgG Fc, Jackson Immunoresearch Lab), was incubated with the cells at 4°C in the dark for 0.5 hours. The cells were then washed, resuspended in 1×PBS / 1% BSA, and analyzed by flow cytometry.

[0389] 3. Hybridoma subcloning:

[0390] Once specific binding was verified by primary and confirmatory screening, positive hybridoma cell lines were subcloned using a semi-solid medium method to obtain monoclonal anti-hLAG-3 antibodies. In the semi-solid medium 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 single colonies were visible in the semi-solid medium. Colonies 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.

[0391] Example 3

[0392] Hybridoma sequencing and construction of fully human antibody molecules

[0393] 1. Hybridoma Sequencing

[0394] 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. Antibody VH and VL genes were amplified from cDNA using a 3'-constant region degenerate primer and a 5'-degenerate primer set complementary to the upstream signal sequence coding region of the Ig variable sequence as shown in Tables 3 and 4. Table 5 shows reagent information, including manufacturer information.

[0395] 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.

[0396] Table 1. cDNA amplification reaction (20 μL)

[0397]

[0398] Table 2. cDNA amplification reaction conditions

[0399] Step 1 Step 2 Step 3 Step 4 Temperature (℃) 25 50 85 4 time 10min 50min 5min ∞

[0400] Table 3. PCR reaction system (50 μL)

[0401] Components quantity cDNA 2.0 μL Premix Ex Taq 25 μL 5'-degenerate primer set (10pM) 2.5 μL 3'-Constant region degenerate primer (10 pM) 1 μL <![CDATA[ddH2O]]> 19.5μL

[0402] Table 4. PCR reaction conditions

[0403]

[0404] Table 5. Reagent information

[0405]

[0406] The two lead antibodies were named "1.53.3-uAb-IgG4k" and "3.40.19-uAb-IgG4L", respectively.

[0407] The CDR sequence of 1.53.3-uAb-IgG4k was determined as follows:

[0408] describe SEQ ID NO. Sequence information CDRH1 1 GGSFSGYYWS CDRH2 2 EINHRGNTNYNPSLKS CDRH3 3 GEDYSDYDYYGDF CDRL1 4 RASQSISSYLA CDRL2 5 AASNRAT CDRL3 6 QQRSNWPLT

[0409] The sequences of the heavy and light chain variable regions of 1.53.3-uAb-IgG4k are as follows:

[0410]

[0411] The CDR sequence of 3.40.19-uAb-IgG4L is as follows:

[0412] describe SEQ ID NO. Sequence information CDRH1 7 GDSISSTSYYWG CDRH2 8 SFYYSGSTYYNPSLKS CDRH3 9 MQLWSYDVDV CDRL1 10 TGTSSDVGGYDYVA CDRL2 11 DVSERPS CDRL3 12 SSYTSTTTLVV

[0413] The sequences of the heavy and light chain variable regions of 3.40.19-uAb-IgG4L are as follows:

[0414]

[0415]

[0416] 2. Construction of fully human antibody molecules

[0417] The VH and VL genes were re-amplified using cloning primers containing appropriate restriction sites and cloned into expression vectors to generate the corresponding chimeric antibody clones.

[0418] Example 4

[0419] Binding of LAG-3 antibodies to cell surface human LAG-3

[0420] Various concentrations of test antibodies, positive and negative controls were added to human LAG-3 transfected cells, and then antibody binding on the cell surface was detected by corresponding PE-labeled secondary antibodies. Figure 1 In, EC 50 Shown in Table 6.

[0421] Table 6

[0422] Ab <![CDATA[EC 50 (nM)]]> 1.53.3-uAb-IgG4k 0.43 3.40.19-uAb-IgG4L 0.13 BMK1 0.32 BMK7 0.61 BMK8 0.90

[0423] Surprisingly, if Figure 1 As shown in Table 6, the EC of 3.40.19-uAb-IgG4L binding to cell surface LAG-3 50 (0.13) was significantly lower than all three benchmark antibodies BMK1 (0.32), BMK7 (0.61) and BMK8 (0.90). In addition, the EC of 1.53.3-uAb-IgG4k binding to cell surface LAG-3 50 (0.43) is much lower than the EC of BMK7 (0.61) and BMK8 (0.90). 50 These results indicate that 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L can effectively bind to human LAG-3, and the binding effect is better than or equivalent to the benchmark antibody.

[0424] Example 5

[0425] Blockade of LAG-3 protein binding to MHC-II expressed on Raji cells

[0426] Antibodies were serially diluted in 1% BSA-PBS and incubated with mFc-labeled LAG-3 protein at 4°C for 30 minutes. The mixture was transferred to a 96-well plate seeded with Raji cells. Goat anti-mouse IgG Fc-PE antibody was used to detect the binding of LAG-3 protein to Raji cells. MFI was assessed by flow cytometry and analyzed by FlowJo software (version 7.6.1). Data are shown in Figure 2 In, EC 50 Shown in Table 7.

[0427] Table 7

[0428] Ab <![CDATA[EC 50 (nM)]]> 1.53.3-uAb-IgG4k 0.80 3.40.19-uAb-IgG4L 0.67 BMK1 0.76 BMK7 1.25 BMK8 0.88

[0429] like Figure 2 As shown in Table 7, surprisingly, the EC of 3.40.19-uAb-IgG4L binding to MHC-II expressed on Raji cells was 50 (0.67) was significantly lower than the EC values ​​of all three benchmark antibodies BMK1 (0.76), BMK7 (1.25), and BMK8 (0.88). 50 Furthermore, the EC binding of 1.53.3-uAb-IgG4k to MHC-II expressed on Raji cells 50 (0.80) is lower than the EC of BMK7 (1.25) and BMK8 (0.88). 50 These results indicate that 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L are effective in blocking binding to MHC-II expressed on Raji cells, and the blocking effect is better than or equivalent to the benchmark antibody.

[0430] Example 6

[0431] Blockade of LAG-3 protein binding to LSECtin and galectin-3

[0432] 96-well plates were coated with 0.5 μg / mL of human LSECtin or galectin-3 at 4°C overnight. The antibodies were serially diluted in 1% BSA-PBS and mixed with mFc-tagged LAG-3 protein. After blocking and washing, the mixture was transferred to the plate and incubated at room temperature for 1 hour. The plate was then washed and subsequently incubated with the corresponding secondary antibody for 60 minutes. After washing, TMB substrate was added and the color reaction was stopped with 2M HCl. The absorbance at 450 nm was read using a microplate reader. The data are shown in Figure 3 and 4 In. EC 50 Shown in Table 8.

[0433] Table 8

[0434]

[0435] like Figure 3 and 4 As shown in 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 galectin-3, and the blocking effect is better than or equivalent to that of the benchmark antibody.

[0436] Example 7

[0437] Complete kinetic binding affinity testing

[0438] Full kinetic binding affinity as measured by surface plasmon resonance (SPR):

[0439] The affinity and binding kinetics of the antibodies for human LAG-3 were characterized using a Biacore 8K SPR assay. Goat anti-human Fc was pre-immobilized onto a sensor chip (CM5), and the anti-LAG-3 antibodies were captured upon injection into the chip. Various concentrations of human LAG-3 protein and running buffer were flowed through the sensor chip at a flow rate of 30 μL / min, with a 300-second association phase followed by a 3600-second dissociation phase. Association and dissociation curves were fitted using a 1:1 Langmuir binding model using the Biacore 8K evaluation software. The data are shown in Table 9.

[0440] Table 9

[0441] 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

[0442] Binding affinity of LAG-3 antibodies to cell surface LAG-3 molecules as tested by fluorescence-activated cell sorting (FACS)

[0443] The binding affinity of the antibodies to cell surface LAG-3 was measured by FACS analysis. Flp-In-293 cells expressing human LAG-3 were cultured at 5 × 10 5 The cells were washed once and resuspended in 1 × PBS / 1% BSA and analyzed by flow cytometry. Fluorescence intensity was converted into bound molecules / cells based on quantitative beads (QuantumTM MESF kit, Bangs Laboratories, Inc.). Affinity was calculated using Graphpad Prism 5. The data are shown in Table 10.

[0444] Table 10

[0445] 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

[0446] As tested by SPR and FACS, the antibodies of the invention represented by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L effectively bound to human LAG-3, and the binding was better than or equivalent to the benchmark antibody.

[0447] Example 8

[0448] Orthologs (across species) and homologs (across families) binding

[0449] Cross-reactivity with cynomolgus monkey LAG-3 and mouse LAG-3

[0450] Cross-reactivity with cynomolgus monkey and mouse LAG-3 was measured by FACS. Flp-In-CHO cells expressing mouse LAG-3 or cynomolgus monkey LAG-3 expressing 293F cells were cultured at 1×10 5 The density of cells / well was transferred to a 96-well U-bottom plate. The test antibodies were serially diluted in wash buffer (1×PBS / 1% BSA) and incubated with cells at 4°C for 1 hour. After washing with 1×PBS / 1% BSA, the corresponding secondary antibody was applied and incubated with cells at 4°C in the dark for 1 hour. The cells were then washed and resuspended in 1×PBS / 1% BSA and analyzed by flow cytometry. The data are shown in Figure 5 and Figure 6 In. EC 50Shown in Table 11.

[0451] Table 11

[0452] Ab <![CDATA[EC 50 (nM)]]> 1.53.3-uAb-IgG4k 4.01 3.40.19-uAb-IgG4L 3.92 BMK1 86.0 BMK7 2.65 BMK8 3.05

[0453] like Figure 5 As shown in FIG, the LAG-3 antibodies "1.53.3-uAb-IgG4k" and "3.40.19-uAb-IgG4L" of the present invention bind to cell surface cynomolgus monkey LAG-3. Figure 6 As shown, the LAG-3 antibodies "1.53.3-uAb-IgG4k" and "3.40.19-uAb-IgG4L" of the present invention do not bind to cell surface mouse LAG-3.

[0454] Cross-reactivity with human CD4

[0455] Cross-reactivity with human CD4 was measured by ELISA. Plates were coated with 1 μg / mL of human CD4 overnight at 4°C. After blocking and washing, 1 μg / mL of LAG-3 antibody was added to the plates and incubated at room temperature for 1 hour. The plates were then washed and subsequently incubated with the corresponding secondary antibody for 45 minutes. After washing, TMB substrate was added and the color reaction was stopped with 2M HCl. The absorbance at 450 nm was read using a microplate reader. The data are shown in Figure 2. Figure 7 shown.

[0456] These results indicate that the LAG-3 antibodies "1.53.3-uAb-IgG4k" and "3.40.19-uAb-IgG4L" of the present invention do not bind to human CD4 protein.

[0457] Example 9

[0458] Epitope binning for BMK1, BMK7, and BMK5

[0459] The binding epitopes of the LAG-3 antibody were binned against the benchmark antibodies BMK1, BMK7, and BMK5 by FACS analysis. Flp-In-293 cells expressing human LAG-3 on the cell surface were incubated with biotinylated benchmark antibodies at a concentration of 1 μg / mL for 1 hour, and then serially diluted LAG-3 antibodies were added. Streptavidin-PE antibody (Jackson ImmunoresearchLab) was used to detect the binding of the benchmark antibodies to the cells. MFI was assessed by flow cytometry and analyzed by FlowJo. Data are shown in Figure 5. Figure 8A -E as shown.

[0460] It was found that 1.53.3-uAb-IgG4k of the present invention competes with BMK, but 3.40.19-uAb-IgG4L does not. 1.53.3-uAb-IgG4k shares a similar epitope with BMK1 and BMK7, but not with BMK5. Surprisingly, 3.40.19-uAb-IgG4L has a distinct epitope from all BMK1, BMK7, and BMK5.

[0461] Example 10

[0462] Domain mapping and epitope mapping

[0463] 1. Domain Mapping

[0464] LAG-3 has an extracellular domain of 421 aa (P30-L450), including four extracellular immunoglobulin superfamily (IgSF)-like domains, namely domain 1 ("D1," aa. 37-167), domain 2 ("D2," aa 168-252.), domain 3 ("D3," aa. 265-343), and domain 4 ("D4," aa. 348-419). Ten variants were constructed by replacing the following residues of the human LAG-3 extracellular domain with the corresponding mouse LAG-3 amino acids (also referred to as "aa" in the context of this disclosure).

[0465] (1) Variant 1: xPro1.FL-x1: Human LAG-3 aa 168 to 419 replaced by the mouse counterpart

[0466] (2) Variant 2: xPro1.FL-x2: human LAG-3 aa 37 to 167 and aa 265 to 419 replaced by their mouse counterparts

[0467] (3) Variant 3: xPro1.FL-x3: human LAG-3 aa 37 to 252 and aa 348-419 replaced by their mouse counterparts

[0468] (4) Variant 4: xPro1.FL-x4: human LAG-3 aa 37 to 343 replaced by the mouse counterpart

[0469] (5) Variant 5: xPro1.FL-x5: human LAG-3 aa 265 to 419 replaced by the mouse counterpart

[0470] (6) Variant 6: xPro1.FL-x6: human LAG-3 aa 37 to 167 and aa 348 to 419 replaced by their mouse counterparts

[0471] (7) Variant 7: xPro1.FL-x7: human LAG-3 aa 37 to 252 replaced by the mouse counterpart

[0472] (8) Variant 8: xPro1.FL-x8: human LAG-3 aa 168 to 343 replaced by the mouse counterpart

[0473] (9) Variant 9: xPro1.FL-x9: human LAG-3 aa 348 to 419 replaced by the mouse counterpart

[0474] (10) Variant 10: xPro1.FL-x10: human LAG-3 aa 37 to 167 replaced by the mouse counterpart

[0475] These 10 variants were cloned into pcDNA3 vector and used for 293F cell transfection. Briefly, 293F cells were diluted to a density of 1 × 10 6 48 hours after transfection, the cells were transfected by flow cytometry.

[0476] Antibody binding to chimeric LAG-3 variants or full-length human / mouse LAG-3 was analyzed by flow cytometry. Briefly, 1 μg / mL of antibody was incubated with transfected 293F cells expressing chimeric LAG-3 for 1 hour at 4°C, followed by incubation with 3 μg / mL goat anti-human IgG Fc R-PE (Jackson) at 4°C for 40 minutes. Cells were analyzed by flow cytometry.

[0477] Antibodies 1.53.3-uAb-IgG4k and 3.40.19-uAb-hIgG4L were tested for their ability to bind to these 10 variants, and the results are shown in Table 12 below.

[0478] Table 12. Binding MFI values ​​of LAG-3 antibodies to 10 variants

[0479]

[0480]

[0481] According to the FACS binding activity of the antibodies, both lead antibodies, i.e., "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, 131 aa) was performed by alanine scanning experiments.

[0482] 2. Epitope Mapping

[0483] Human LAG-3 was subjected to an alanine scanning experiment for epitope mapping. Alanine residues on human LAG-3 were mutated to glycine codons, and all other residues (except cysteine ​​residues) were mutated to alanine codons. For each residue in the human LAG-3 extracellular domain (ECD), site-specific amino acid substitutions were performed using two sequential PCR steps. The pcDNA3.3-LAG-3-D12.mFc plasmid 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 in the first PCR step using the QuikChange lightning multi-site-directed mutagenesis kit (Agilent technologies, Palo Alto, CA). After the mutant strand synthesis reaction, the parent template was digested with Dpn I endonuclease. In the second PCR step, a linear DNA expression cassette containing the CMV promoter, extracellular domains 1 and 2 (D1 and D2) of LAG-3, an mFc-tag, and herpes simplex virus thymidine kinase (TK) polyadenylation was amplified and transiently expressed in Expi293 cells (Life Technologies, Gaithersburg, MD) at 37°C and quantified by protein A-HPLC and an mFc-ELISA quantification kit (Bethyl, USA).

[0484] For ELISA binding assays, antibodies 1.53.3-uAb-IgG4k or 3.40.19-uAb-hIgG4L (2 μg / mL) were coated onto plates. Following interaction with supernatants containing a quantified amount of LAG-3 mutants or human LAG-3-ECD.D12.mFc protein, an HRP-conjugated anti-mFc antibody (1:5000; Bethyl, USA) was added as a detection antibody. Absorbance was normalized to the mean absorbance of the control mutants. After setting an additional cutoff (<0.75) for the fold change in binding, the final epitope residues were identified. Hotspots for antibodies 1.53.3-uAb-IgG4k and 3.40.19-uAb-hIgG4L are shown in Tables 13 and 14.

[0485] Table 13.1.53.3-uAb-IgG4k Antibody Hotspots

[0486]

[0487]

[0488] Table 14.3.40.19-uAb-IgG4L Antibody Hotspots

[0489]

[0490]

[0491] Since there is no existing LAG-3 structure, the structure of LAG-3 (aa: 31-431) was modeled based on the known myelin-linked glycoprotein structure (PDB: 5FLU, sequence identity 18%). Based on the alanine scanning results, hot spots of the two antibodies were identified and shown in Figure 9A and Figure 9B middle.

[0492] Based on the results, it can be seen that the 1.53.5-uAb-IgG4k antibody binds to the W92 site belonging to the outer loop (G70-Y99), while the 3.40.19-uAb-IgG4L antibody binds to the L134-P138 region.

[0493] Example 11

[0494] In vitro functionality of LAG-3 antibodies tested by cell-based assays

[0495] The role of human LAG-3 antibodies in reporter gene assays

[0496] Jurkat cells expressing human LAG-3 and a stably integrated IL-2 luciferase reporter gene were seeded in 96-well plates along with Raji cells in the presence of SEE. Test antibodies were added to the cells. The plates were incubated overnight at 37°C, 5% CO2. Following incubation, reconstituted luciferase substrate was added and luciferase intensity was measured by a microplate spectrophotometer. Data are shown in Figure 10 In, EC 50 Shown in Table 15.

[0497] Table 15

[0498] Ab <![CDATA[EC 50 (nM)]]> 1.53.3-uAb-IgG4k 1.07 3.40.19-uAb-IgG4L 0.21 BMK1 0.59 BMK7 2.65 BMK8 65.3

[0499] like Figure 10 As shown in Table 15, the LAG-3 antibody enhanced the IL-2 pathway in Jurkat cells in the reporter gene assay.50 Significantly lower than all three benchmark antibodies.

[0500] Effects of human LAG-3 antibody on human allogeneic mixed lymphocyte reaction

[0501] Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from healthy donors using Ficoll-Paque PLUS gradient centrifugation. Monocytes were isolated using a human monocyte enrichment kit according to the manufacturer's instructions. Cells were cultured for 5 to 7 days in a medium containing GM-CSF and IL-4 to generate dendritic cells (DCs). Human CD4 + T cell enrichment kit to isolate human CD4 + T cells. Purified CD4 + T cells were co-cultured with allogeneic immature DCs (iDCs) and various concentrations of LAG-3 antibodies in 96-well plates. On day 5, culture supernatants were collected for IFN-γ assay and T cell proliferation assay. Human IFN-γ was measured by ELISA using matched antibody pairs. Plates were pre-coated with a capture antibody specific for human IFN-γ (Pierce-M700A). Biotin-conjugated anti-IFN-γ antibody (Pierce-M701B) was used as the detection antibody. In the last 16 hours, 1 μCi / well of IFN-γ was added. 3 H-thymidine. Measured by scintillation counting 3 H-thymidine incorporation was performed and the proliferation response was expressed as CPM (counts per minute) of triplicate wells. Data are shown in Figure 11 and 12 middle.

[0502] like Figure 11 As shown in FIG, the LAG-3 antibodies "1.53.3-uAb-IgG4k" and "3.40.19-uAb-IgG4L" of the present invention enhance IFN-γ secretion in a mixed lymphocyte reaction. Figure 12 As shown, the LAG-3 antibodies "1.53.3-uAb-IgG4k" and "3.40.19-uAb-IgG4L" of the present invention enhanced T cell proliferation in a mixed lymphocyte reaction.

[0503] Example 12

[0504] ADCC and CDC testing

[0505] To assess the ability to trigger Fc effector functions, it was evaluated whether anti-LAG-3 antibodies could mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities.

[0506] ADCC testing

[0507] Flp-In-293 cells expressing human LAG-3 were preincubated with various concentrations of LAG-3 antibodies in 96-well round-bottom plates for 30 minutes. PBMCs were then added as effectors at an effector / target ratio of 50:1. The plates were maintained at 37°C, 5% CO₂ for 4 hours. Target cell lysis was determined using an LDH-based cytotoxicity assay kit. Absorbance was read at 492 nm using a microplate reader. Herceptin and the HER2-expressing cell line SK-Br-3 were used as positive controls.

[0508] CDC testing

[0509] The target, Flp-In-293 expressing human LAG-3, and various concentrations of LAG-3 antibodies were mixed in a 96-well round-bottom plate. Human complement was added at a final dilution of 1:50. The plates were incubated at 37°C, 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 Raji cell line were used as positive controls.

[0510] Figure 13A and 13B Data from ADCC and CDC assays are shown. It was demonstrated that the LAG-3 antibodies of the present invention represented by 1.53.3-uAb-IgG4k and 3.40.19-uAb-IgG4L did not mediate ADCC ( Figure 13A ) and CDC( Figure 13B )Effect.

[0511] Example 13

[0512] Serum stability test

[0513] Lead Ab was incubated in freshly isolated human serum (serum content >95%) at 37°C. At the indicated time points, aliquots of serum-treated samples were removed from the incubator and snap-frozen in liquid nitrogen and then stored at 80°C until ready for testing. Samples were quickly thawed immediately before stability testing. Human LAG-3 transfected cells were incubated with various concentrations of lead antibody at 4°C for 1 hour. PE-labeled goat anti-human IgG was used to detect binding of the lead antibody to the cells. The MFI of the cells was measured by flow cytometry (BD FACSCanto II) and analyzed by FlowJo. The data are shown in Figure 14A and 14B middle.

[0514] 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.

[0515] Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from its spirit or central features. Since the foregoing description of the present invention discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims for an indication of the scope and content of the present invention. Sequence Listing <110> Guangzhou Yuheng Biotechnology Co., Ltd. <120> Anti-human LAG-3 antibodies and uses thereof <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 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 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 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 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> CDRL3 of 3.40.19 - uAb - IgG4L <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> VH of 1.53.3 - uAb - IgG4k <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> VL of 1.53.3 - uAb - IgG4k <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> VH of 3.40.19-uAb-IgG4L <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> VL of 3.40.19 - uAb - IgG4L <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> VH of 1.53.3 - uAb - IgG4k <400> 17 60. caggtgcagc tacagcagtg gggcgcagga cttttgaagc cttcggagac cctgtccctc acctgcggtg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120 ccagggatgg ggctggagtg gattgggga atcaatcatc gtggaacac caactacaac ccgtccctca agagtcgcgt caccatatca gaagacacgt ccaagaacca gttctccctg aggctgagct ctgtgaccgc cgcggacacg gctgtgtatt tctgtacgag aggagaggac 300 tatagtgact acgattacta tggggacttc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 18 <211> 321 <212> DNA <213> The snowstorm <220> <223> 1.53.3-uAb-IgG4kVL <400> 18 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctcaagggga aagagccacc ctctcctgca gggccagtca gagtattagc agctcttag cctggtacca acagaaacct 180. ggccaggctc ccaggctcct catctatgct gcatccaaca gggccactgg catcccagcc aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct gaagattttg caatttatta ctgtcagcag cgtagcaact ggcctctcac tttcggcgga 300 gggaccaagg tggagatcaa a 321 <210> 19 <211> 360 <212> DNA <213> Synthetic sequence <220> <223> VH of 3.40.19-uAb-IgG4L <400> 19 cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtga ctccatcagc agtactagtt actactgggg ctggatccgc 120 cagcccccag ggaaggggct ggagtggatt gggagtttct attatagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagtcacc atttccgtag acacgtccaa gaaccagttc 240 tccctgaagc tgaactctgt gaccgccgca gacacggctg tgtattactg tgcgaggatg 300 cagctatggt cgtacgatgt ggacgtctgg ggccaaggga ccacggtcac cgtctcctca 360 <210> 20 <211> 333 <212> DNA <213> Synthetic sequence <220> <223> VL of 3.40.19-uAb-IgG4L <4op> 20 cagtctgccc tgactcaacc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 tcctgcactg gaaccagcag tgacgttggt gggtatgact atgtcgcctg gtaccaacaa 120 cacccaggca aagtccccaa actcatgatt tatgatgtca gtgagcggcc ctcaggggtt 180 tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240 caggctgagg acgaggctga ttattactgc agctcatata caagcaccac cactctcgtt 300 gtgttcggcg gagggaccaa gctgtccgtc ctg 333 <210> 21 <211> 421 <212> PRT <213> Artificial sequence <220> <223> Human LAG-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 Human LAG-3 <400> 23 Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp 1 5 10 15 Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro Val Val 20 25 30 Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45 Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60 His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu 65 70 75 80 Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro 85 90 95 Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100 105 110 Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln 115 120 125 Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala 130 135 140 Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser Cys 145 150 155 160 Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165 170 175 Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser 180 185 190 Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln 195 200 205 Gly Arg Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser 210 215 220 Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly 225 230 235 240 Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn 245 250 255 Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val Tyr Ala 260 265 270 Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala Gly Val 275 280 285 Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly 290 295 300 Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu 305 310 315 320 Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His 325 330 335 Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr 340 345 350 Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu 355 360 365 Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser 370 375 380 Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala 385 390 395 400 Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln 405 410 415 Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser 420 425 430 Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly 435 440 445 His Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu 450 455 460 Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro 465 470 475 480 Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln 485 490 495 Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro Glu Pro 500 505 510 Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu 515 520 525 <210> 24 <211> 1575 <212> DNA <213> Artificial Sequence <220> <223> Full-length human LAG-3 <400> 24 atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccagtg 60 aagcctctcc agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc 120 cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg 180 gtcacttggc agcatcagcc agacagtggc ccgcccgctg ccgcccccgg ccatcccctg 240 gcccccggcc ctcacccggc ggcgccctcc tcctgggggc ccaggccccg ccgctacacg 300 gtgctgagcg tgggtcccgg aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc 360 cagctggatg agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg 420 cgcgcggacg ccggcgagta ccgcgccgcg gtgcacctca gggaccgcgc cctctcctgc 480 cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca gccccccagg atctctcaga 540 gcctccgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg 600 cattggttcc ggaaccgggg ccagggccga gtccctgtcc gggagtcccc ccatcaccac 660 ttagcggaaa gcttcctctt cctgccccaa gtcagcccca tggactctgg gccctggggc 720 tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct cactgttctg 780 ggtctggagc ccccaactcc cttgacagtg tacgctggag caggttccag ggtggggctg 840 ccctgccgcc tgcctgctgg tgtgggggacc cggtctttcc tcactgccaa gtggactcct 900 cctggggag gccctgacct cctggtgact ggagacaatg gcgactttac ccttgacta 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> Synthetic 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 murine LAG-3 <400> 26 atgagggagg acctgctcct tggctttttg cttctgggac tgctttggga agctccagtt 60 gtgtcttcag ggcctgggaa agagctcccc gtggtgtggg cccaggaggg agctcccgtc 120 catcttccct gcagcctcaa atcccccaac ctggatccta actttctacg aagaggaggg 180 gttatctggc aacatcaacc agacagtggc caacccactc ccatcccggc ccttgacctt 240 caccagggga tgccctcgcc tagacaaccc gcacccggtc gctacacggt gctgagcgtg 300 gctccaggag gcctgcgcag cgggaggcag cccctgcatc cccacgtgca gctggaggag 360 cgcggcctcc agcgcgggga cttctctctg tggttgcgcc cagctctgcg caccgatgcg 420 ggcgagtacc acgccaccgt gcgcctcccg aaccgcgccc tctcctgcag tctccgcctg 480 cgcgtcggcc aggcctcgat gattgctagt ccctcaggag tcctcaagct gtctgattgg 540 gtccttttga actgctcctt cagccgtcct gaccgcccag tctctgtgca ctggttccag 600 ggccagaacc gagtgcctgt ctacaactca ccgcgtcatt ttttagctga aactttcctg 660 ttactgcccc aagtcagccc cctggactct gggacctggg gctgtgtcct cacctacaga 720 gatggcttca atgtctccat cacgtacaac ctcaaggttc tgggtctgga gcccgtagcc 780 cctctgacag tgtacgctgc tgaaggttct agggtggagc tgccctgtca tttgccccca 840 ggagtgggga ccccttcttt gctcattgcc aagtggactc ctcctggagg aggtcctgag 900 ctccccgtgg ctggaaagag tggcaatttt acccttcacc ttgaggctgt gggtctggca 960 caggctggga cctacacctg tagcatccat ctgcagggac agcagctcaa tgccactgtc acgttggcgg tcatcacagt gactcccaaa tccttcgggt tacctggctc ccggggggag 1080 ctgttgtgtg aggtaacccc ggcatctgga aaggaagat ttgtgtggcg tcccctgaac 1140 aatctgtcca ggagttgccc gggccctgtg ctggagattc aggaggccag gctccttgct gagcgatggc agtgtcagct gtacgagggc cagaggcttc ttggagcgac agtgtacgcc 1260 gcagagtcta gctcaggcgc ccacagtgct aggagaatct caggtgacct taaaggaggc 1320 catctcgttc tcgttctcat ccttggtgcc ctctccctgt tccttttggt ggccggggcc 1380 tttggctttc actggtggag aaaacagttg ctactgaga gattttctgc ctttagaacat gggattcagc catttccggc tcagaggag ataggagc tggagcgaga actggagacg gagatgggac aggagccgga gcccgagccg gagccacagc tggagccaga gcccaggcag 1560 ctc 1563 <210> 27 <211> 533 <212> PRT <213> The snowstorm <220> <223> High-pressure LAG-3 <220> <221> misc_feature <222> (74)..(74) <223> Xaa can be any naturally occurring amino acid <400> 27 Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp 1 5 10 15 Val Ala Pro Val Lys Pro Pro Gln Pro Gly Ala Glu Ile Ser Val Val 20 25 30 Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile 35 40 45 Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50 55 60 His Gln Pro Asp Ser Gly Pro Pro Ala Xaa Ala Pro Gly His Pro Pro 65 70 75 80 Val Pro Gly His Arg Pro Ala Ala Pro Tyr Ser Trp Gly Pro Arg Pro 85 90 95 Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100 105 110 Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln 115 120 125 Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala 130 135 140 Gly Glu Tyr Arg Ala Thr Val His Leu Arg Asp Arg Ala Leu Ser Cys 145 150 155 160 Arg Leu Arg Leu Arg Val Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165 170 175 Gly Ser Leu Arg Thr Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser 180 185 190 Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Ser Arg Gly Gln 195 200 205 Gly Arg Val Pro Val Gln Gly Ser Pro His His His Leu Ala Glu Ser 210 215 220 Phe Leu Phe Leu Pro His Val Gly Pro Met Asp Ser Gly Leu Trp Gly 225 230 235 240 Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn 245 250 255 Leu Thr Val Leu Gly Leu Glu Pro Ala Thr Pro Leu Thr Val Tyr Ala 260 265 270 Gly Ala Gly Ser Arg Val Glu Leu Pro Cys Arg Leu Pro Pro Ala Val 275 280 285 Gly Thr Gln Ser Phe Leu Thr Ala Lys Trp Ala Pro Pro Gly Gly Gly 290 295 300 Pro Asp Leu Leu Val Ala Gly Asp Asn Gly Asp Phe Thr Leu Arg Leu 305 310 315 320 Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Ile Cys His Ile Arg 325 330 335 Leu Gln Gly Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr 340 345 350 Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu 355 360 365 Cys Glu Val Thr Pro Ala Ser Gly Gln Glu His Phe Val Trp Ser Pro 370 375 380 Leu Asn Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala 385 390 395 400 Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu His Gln 405 410 415 Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser 420 425 430 Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Arg Ala Gly 435 440 445 His Leu Pro Leu Phe Leu Ile Leu Gly Val Leu Phe Leu Leu Leu Leu 450 455 460 Val Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro 465 470 475 480 Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala Gln 485 490 495 Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Leu Glu Pro Glu Pro 500 505 510 Glu Leu Glu Arg Glu Leu Gly Pro Glu Pro Glu Pro Gly Pro Glu Pro 515 520 525 Glu Pro Glu Gln Leu 530 <210> 28 <211> 1599 <212> DNA <213> Artificial Sequence <220> <223> Full-length Cynomolgus 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 gggaccgcgc cctctcctgc 480 cgccttcgtc tgcgcgtggg ccaggcctcg atgactgcca gccccccagg gtctctcagg 540 acctctgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg 600 cattggttcc ggagccgtgg ccagggccga gtccctgtcc aggggtcccc ccatcaccac 660 ttagcggaaa gcttcctctt cctgccccat gtcggcccca tggactctgg gctctggggc 720 tgcatcctca cctacagaga tggcttcaat gtctccatca tgtataacct cactgttctg 780 ggtctggagc ccgcaactcc cttgacagtg tacgctggag caggttccag ggtggagctg 840 ccctgccgcc tgcctcctgc tgtggggacc cagtctttcc ttactgccaa gtgggctcct 900 cctgggggag gccctgacct cctggtggct ggagacaatg gcgactttac ccttcgacta 960 gaggatgtaa gccaggccca ggctgggacc tacatctgcc atatccgtct acagggacag 1020 cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc ctttgggtca 1080 cctggctccc tggggaagct gctttgtgag gtgactccag catctggaca agaacacttt 1140 gtgtggagcc ccctgaacac cccatcccag aggagtttct caggaccatg gctggaggcc 1200 caggaagccc agctcctttc ccagccttgg caatgccagc tgcaccaggg ggagaggctt 1260 cttggagcag cagtatactt cacagaactg tctagcccag gtgcacaacg ctctgggaga 1320 gccccagggg ccctccgagc aggccacctc ccgctgtttc tcatccttgg tgtccttttt 1380 ctgctccttt tggtgactgg agcctttggc tttcaccttt ggagaagaca gtggcgacca 1440 agaagatttt ctgccttaga gcaagggatt caccctccgc aggctcagag caagatagag 1500 gagctcgagc aagaaccgga gctggaacca gagccggagc tggagcgcga gctggggccg 1560 gagcccgagc cggggcctga gcccgagccg gagcagctc 1599

Claims

1. An isolated antibody, or antigen-binding portion thereof, that binds to LAG-3, wherein the isolated antibody, or antigen-binding portion thereof, 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 antigen-binding portion thereof of claim 1, wherein the isolated antibody or antigen-binding portion thereof comprises: (A) Heavy chain variable region: (i) comprising the amino acid sequence of SEQ ID NO: 15; (ii) comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 15; or (iii) comprising an amino acid sequence having one or more amino acid additions, deletions and / or substitutions in the framework regions compared to SEQ ID NO: 15; and (B) Light chain variable region: (i) comprising the amino acid sequence of SEQ ID NO: 16; (ii) comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 16; or (iii) comprising an amino acid sequence having one or more amino acid additions, deletions and / or substitutions in the framework regions compared to SEQ ID NO:

16.

3. The isolated antibody or antigen-binding portion thereof of claim 1 , which has one or more of the following properties: (a) 2×10 -10 M or lower K D Binds to human LAG-3; (b) inhibiting the binding of LAG-3 to major histocompatibility (MHC) class II molecules; (c) inhibiting the binding of LAG-3 to the fibrillin-like protein 1 (FGL1) ligand molecule; (d) inhibiting the binding of LAG-3 to LSECtin and / or galectin-3; (e) binds to human LAG-3 without cross-family reactivity; or (f) No cross-reactivity with human CD4.

4. The isolated antibody, or antigen-binding portion thereof, of any one of claims 1 to 3, wherein the antibody is a monoclonal antibody.

5. The isolated antibody or antigen-binding portion thereof of claim 4, wherein the monoclonal antibody is a fully human monoclonal antibody.

6. The isolated antibody or antigen-binding portion thereof 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 antigen-binding portion thereof 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 nucleic acid sequences encoding the heavy chain variable region and the light chain variable region of the isolated antibody defined in any one of claims 1 to 8.

10. The nucleic acid molecule of claim 9, comprising the nucleic acid sequence 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 to 8 and a pharmaceutically acceptable carrier.

14. A method for preparing the antibody or antigen-binding portion thereof as defined in any one of claims 1 to 8, comprising the steps of: - expressing the antibody or antigen-binding portion thereof in a host cell of claim 12; and - isolating the antibody or antigen-binding portion thereof from the host cell.

15. Use of the antibody or antigen-binding portion thereof as defined in any one of claims 1 to 8 in the preparation of an agent for inhibiting or blocking the binding of LAG-3 to an MHC class II molecule, an FGL1 class molecule, LSECtin and / or Galectin-3 in vitro, said inhibition or blocking comprising contacting the MHC class II molecule, the FGL1 class molecule, LSECtin and / or Galectin-3 with the antibody or antigen-binding portion thereof as defined in any one of claims 1 to 8.

16. Use of an antibody or antigen binding portion thereof as defined in any one of claims 1 to 8 for the preparation of a medicament for inhibiting the growth of tumor cells in a subject, wherein the tumor is cutaneous or intraocular malignant melanoma or renal cancer.

17. Use of an antibody, or antigen-binding portion thereof, as defined in any one of claims 1 to 8 for the preparation of a medicament for treating cancer in a subject, wherein the cancer is cutaneous or intraocular malignant melanoma or renal cancer.

18. Use of an antibody or antigen-binding portion thereof as defined in any one of claims 1 to 8 in the preparation of a diagnostic agent for diagnosing a cancer expressing LAG-3.

19. A kit comprising a container comprising an antibody, or antigen-binding portion thereof, as defined in any one of claims 1 to 8.

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