FIELD OF THE INVENTION

This invention relates to monoclonal anti-LAG-3 antibodies, nucleic acids and expression vectors encoding the antibodies, recombinant cells containing the vectors, and compositions comprising the antibodies. Methods of making the antibodies, and methods of using the antibodies to treat diseases including cancer, inflammatory diseases, autoimmune diseases, infectious diseases, type 1 diabetes, and/or associated complications are also provided.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name 689204.4U1 Sequence Listing” and a creation date of Feb. 13, 2020, and having a size of 89 kb. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Immune suppression has long been recognized as one of the critical causes for cancer. Recently, stimulation of the immune responses to cancer cells has exhibited durable efficacy in cancer treatment. This is highlighted by the tremendous success of therapeutics that target immune checkpoint proteins such as PD-1, PD-L1 and CTLA-4. However, only a small fraction of patients responds to these immune checkpoint therapies. It raises the question whether the immune response to cancer cells is suppressed by additional mechanisms, and whether the blockage of these suppression mechanisms can treat more patients (for review, see Hahn et al., Immunotherapy 2017; 9:681-692).

In addition to PD-1, PD-L1 and CTLA-4, T effector cells are negatively modulated by multiple co-inhibitory checkpoint proteins, such as B7-H3, BTLA, LAG-3 (lymphocyte activation gene 3; CD223), TIM-3 and TIGIT. Among them, LAG-3 is expressed on the surface of activated T cells (Triebel, et al., J. Exp. Med. 1990; 171:1393-1405) and exhibits a unique co-inhibitory activity. Both LAG-3 and CD4 T cell co-receptor bind to the major histocompatibility complex (MHC) class II molecules at the same site, but the LAG-3 binding is of much higher affinity than that of CD4 (Huard, et al., Immunogenetics 1994; 39:213-217). Therefore, the expression of LAG-3 on T cells prevents the binding of CD4 to the MEW class II molecules on antigen presenting cells (APCs), inhibits the co-stimulatory activity of the MHC class II molecules, and negatively modulates the maintenance of T cell activities.

In addition to the inhibiting signal into pre-activated LAG-3+ cells triggered by the binding of MHC class II molecules to LAG-3, it has been reported that LAG-3 on the surface of T regulatory (Treg) cells also suppresses the activity of dendritic cells (DCs) through binding to MEW class II molecules (Huang, et al., Immunity 2004; 21:503-513; Liang, et al., J Immunol 2008; 180:5916-5926). Such backward signaling has the potential to further suppress immune response by downregulating the activity of DCs, and consequently downregulating the activation of naïve T cells and other LAG-3 negative immune cells whose activation and/or activity depend on DCs.

LAG-3 is also expressed on the surface of natural killer cells (Baixeras, et al., J. Exp. Med 1992; 176:327-337), B cells (Kisielow, et al., Eur. J. Immunol. 2005; 35:2081-2088) and plasmacytoid dendritic cells (Workman, et al., J Immunol 2009; 182:1885-1891). Compared with the inhibitory activity on T cells, its function in other immune cells is poorly understood. In addition to binding to MEW class II molecules, LAG-3 is also reported to bind to LSECtin (Xu, et al., Cancer Res 2014; 74:3418-3428), a dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) family lectin on the surface of dendritic cells, and the binding is suggested to negatively regulate T cell proliferation. A therapeutic monoclonal antibody (mAb) that blocks these functions of LAG-3 may provide additional therapeutic values.

The binding of MHC class II molecules to T cell receptor (TCR) and the subsequent T cell activation require an antigen. Therefore, LAG-3 specifically inhibits antigen-activated T cells, which is unique from other inhibitory checkpoint mechanisms. Taken together, the LAG-3/MHC class II axis plays an important role in bridging the innate and adaptive immunities. A therapeutic monoclonal antibody that blocks the LAG-3/MHC class II binding has the potential to reactivate an immune response to cancer by itself as a monotherapy or in combination with other immuno-oncology therapeutics such as anti-PD1, anti-PD-L1, anti-CTLA-4, anti-TIM-3, and anti-CD47 antibodies.

LAG-3 limits Treg proliferation and function and may contribute to Treg insufficiency in autoimmune diseases such as type 1 diabetes (Zhang et al., Sci Immunol. 2017; 31:2(9).) An anti-LAG-3 mAb that improves Treg activity has the therapeutic potential to treat type 1 diabetes.

BRIEF SUMMARY OF THE INVENTION

In one general aspect, the invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that bind LAG-3.

Provided are isolated monoclonal antibodies or antigen-binding fragments thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the polypeptide sequences of:a. SEQ ID NOs:35, 36, 37, 86, 87, and 88, respectively;b. SEQ ID NOs:38, 39, 40, 89, 90, and 91, respectively;c. SEQ ID NOs:41, 42, 137, 138, 93, and 94, respectively;d. SEQ ID NOs:139, 140, 141, 142, 99, and 143, respectively;e. SEQ ID NOs:144, 145, 146, 147, 148, and 149, respectively;f. SEQ ID NOs:65, 66, 67, 116, 117, and 118, respectively;g. SEQ ID NOs:68, 69, 70, 119, 120, and 121, respectively;h. SEQ ID NOs:71, 72, 73, 122, 123, and 124, respectively;i. SEQ ID NOs:74, 75, 76, 125, 126, and 127, respectively;j. SEQ ID NOs:77, 78, 79, 128, 129, and 130, respectively; ork. SEQ ID NOs:80, 150, 151, 131, 132, and 133, respectively;
wherein the antibody or antigen-binding fragment thereof specifically binds LAG-3, preferably human LAG-3.

SEQ ID NO:137 is represented by the amino acid sequence ARGGYX1DYVWFPY, wherein X1is an amino acid selected from the group consisting of Y and F.

SEQ ID NO:138 is represented by the amino acid sequence QSIX1YSNGYTY, wherein X1is an amino acid selected from the group consisting of L and V.

SEQ ID NO:139 is represented by the amino acid sequence GYTX1X2X3YY, wherein X1is an amino acid selected from the group consisting of F and L; X2is an amino acid selected from the group consisting of T and S; X3is an amino acid selected from the group consisting of G and N.

SEQ ID NO:140 is represented by the amino acid sequence INPYNGDX1, wherein X1is an amino acid selected from the group consisting of T and I.

SEQ ID NO:141 is represented by the amino acid sequence X1RDDGYX2VX3X4FDX5, wherein X1is an amino acid selected from the group consisting of V and A; X2is an amino acid selected from the group consisting of H and Y; X3is an amino acid selected from the group consisting of R and Y; X4is an amino acid selected from the group consisting of F and Y; and X5is an amino acid selected from the group consisting of V, Y, and C.

SEQ ID NO:142 is represented by the amino acid sequence QDIX1X2X3, wherein X1is an amino acid selected from the group consisting of S and G; X2is an amino acid selected from the group consisting of D and G; and X3is an amino acid selected from the group consisting of Y, S, and R.

SEQ ID NO:143 is represented by the amino acid sequence LQX1X2X3SPPT, wherein X1is an amino acid selected from the group consisting of Y, C, and N; X2is an amino acid selected from the group consisting of V and A; and X3is an amino acid selected from the group consisting of S and N.

SEQ ID NO:144 is represented by the amino acid sequence GYSFX1DYN, wherein X1is an amino acid selected from the group consisting of S and T.

SEQ ID NO:145 is represented by the amino acid sequence IX1LDX2X3X4T, wherein X1is an amino acid selected from the group consisting of N and T; X2is an amino acid selected from the group consisting of S and Y; X3is an amino acid selected from the group consisting of A and G; and X4is an amino acid selected from the group consisting of A and T.

SEQ ID NO:146 is represented by the amino acid sequence AX1YDY, wherein X1is an amino acid selected from the group consisting of S and C.

SEQ ID NO:147 is represented by the amino acid sequence QDISX1Y, wherein X1is an amino acid selected from the group consisting of H and N.

SEQ ID NO:148 is represented by the amino acid sequence X1TS, wherein X1is an amino acid selected from the group consisting of E and A.

SEQ ID NO:149 is represented by the amino acid sequence LQYAX1YPLT, wherein X1is an amino acid selected from the group consisting of T and S.

SEQ ID NO:150 is represented by the amino acid sequence of IYPGRGX1P, wherein X1is an amino acid selected from the group consisting of D and N.

SEQ ID NO:151 is represented by the amino acid sequence of EIYYGNYX1DY, wherein X1is an amino acid selected from the group consisting of I and L.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises:(a) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1, and a light chain variable region having the polypeptide sequence of SEQ ID NO:2;(b) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:3, and a light chain variable region having the polypeptide sequence of SEQ ID NO:4;(c) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:5, and a light chain variable region having the polypeptide sequence of SEQ ID NO:6;(d) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:7, and a light chain variable region having the polypeptide sequence of SEQ ID NO:8;(e) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:9, and a light chain variable region having the polypeptide sequence of SEQ ID NO:10;(f) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:11, and a light chain variable region having the polypeptide sequence of SEQ ID NO:12;(g) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:13, and a light chain variable region having the polypeptide sequence of SEQ ID NO:14;(h) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15, and a light chain variable region having the polypeptide sequence of SEQ ID NO:16;(i) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:17, and a light chain variable region having the polypeptide sequence of SEQ ID NO:18;(j) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:19, and a light chain variable region having the polypeptide sequence of SEQ ID NO:20;(k) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:21, and a light chain variable region having the polypeptide sequence of SEQ ID NO:22;(l) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:23, and a light chain variable region having the polypeptide sequence of SEQ ID NO:24;(m) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:25, and a light chain variable region having the polypeptide sequence of SEQ ID NO:26;(n) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:27, and a light chain variable region having the polypeptide sequence of SEQ ID NO:28;(o) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:29, and a light chain variable region having the polypeptide sequence of SEQ ID NO:30;(p) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:31, and a light chain variable region having the polypeptide sequence of SEQ ID NO:32; or(q) a heavy chain variable region having the polypeptide sequence of SEQ ID NO:33, and a light chain variable region having the polypeptide sequence of SEQ ID NO:34.

Also provided are isolated monoclonal antibodies or antigen-binding fragments thereof to LAG-3 that specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO:192. Also provided are isolated monoclonal antibodies or antigen-binding fragments thereof to LAG-3 that specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO:193. Also provided are isolated monoclonal antibodies or antigen-binding fragments thereof to LAG-3 that specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO:158. Also provided are isolated monoclonal antibodies or antigen-binding fragments thereof to LAG-3 that specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO:180. Also provided are isolated monoclonal antibodies or antigen-binding fragments thereof to LAG-3 that specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO:194. The isolated monoclonal antibodies or antigen-binding fragments thereof can, for example, inhibit LAG-3 activity.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is capable of blocking binding of LAG-3 to WIC class II molecules.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is capable of blocking binding of LAG-3 to LSECtin.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric.

In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is human or humanized. In certain embodiments, the humanized monoclonal antibody or antigen-binding fragment thereof comprises:a. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:195, and a light chain variable region having the polypeptide sequence of SEQ ID NO:208b. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:196, and a light chain variable region having the polypeptide sequence of SEQ ID NO:206;c. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:197, and a light chain variable region having the polypeptide sequence of SEQ ID NO:205;d. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:197, and a light chain variable region having the polypeptide sequence of SEQ ID NO:206;e. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:197, and a light chain variable region having the polypeptide sequence of SEQ ID NO:207;f. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:198, and a light chain variable region having the polypeptide sequence of SEQ ID NO:205;g. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:199, and a light chain variable region having the polypeptide sequence of SEQ ID NO:209;h. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:200, and a light chain variable region having the polypeptide sequence of SEQ ID NO:209;i. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:201, and a light chain variable region having the polypeptide sequence of SEQ ID NO:209;j. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:202, and a light chain variable region having the polypeptide sequence of SEQ ID NO:209;k. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:203, and a light chain variable region having the polypeptide sequence of SEQ ID NO:209;l. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:204, and a light chain variable region having the polypeptide sequence of SEQ ID NO:209;m. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:199, and a light chain variable region having the polypeptide sequence of SEQ ID NO:210;n. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:200, and a light chain variable region having the polypeptide sequence of SEQ ID NO:210;o. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:201, and a light chain variable region having the polypeptide sequence of SEQ ID NO:210;p. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:202, and a light chain variable region having the polypeptide sequence of SEQ ID NO:210;q. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:203, and a light chain variable region having the polypeptide sequence of SEQ ID NO:210; orr. a heavy chain variable region having the polypeptide sequence of SEQ ID NO:204, and a light chain variable region having the polypeptide sequence of SEQ ID NO:210.

Also provided are isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention disclosed herein.

Also provided are vectors comprising the isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention.

Also provided are host cells comprising the vectors comprising the isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention.

In certain embodiments, provided is a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.

Also provided are methods of blocking binding of LAG-3 to MHC class II molecules in a subject in need thereof, comprising administering to the subject the pharmaceutical compositions of the invention.

Also provided are methods of blocking binding of LAG-3 to LSECtin in a subject in need thereof, comprising administering to the subject the pharmaceutical compositions of the invention.

Also provided are methods of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical compositions of the invention. The cancer can be any liquid or solid cancer, for example, it can be selected from, but not limited to, a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, a glioma, a glioblastoma, and other solid tumors, and a non-Hodgkin's lymphoma (NHL), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.

Also provided are methods of treating an infectious disease in a subject in need thereof, comprising administering to the subject the pharmaceutical compositions of the invention.

Also provided are methods of treating an inflammatory disease in a subject in need thereof, comprising administering to the subject the pharmaceutical compositions of the invention.

Also provided are methods of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject the pharmaceutical compositions of the invention.

Also provided are methods of treating a type 1 diabetes in a subject in need thereof, comprising administering to the subject the pharmaceutical compositions of the invention.

Also provided are methods of producing the monoclonal antibody or antigen-binding fragment thereof of the invention, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment under conditions to produce the monoclonal antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the cell or culture.

Also provided are methods of producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of the invention, comprising combining the monoclonal antibody or antigen-binding fragment with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

Also provided are methods of determining a level of LAG-3 in a subject. The methods comprise (a) obtaining a sample from the subject; (b) contacting the sample with an antibody or antigen-binding fragment thereof of the invention; and (c) determining the level of LAG-3 in the subject. In certain embodiments, the sample is a tissue sample. The tissue sample can, for example, be a cancer tissue sample. In certain embodiments, the sample is a blood sample.

