Therapies for the treatment of cancer

Abstract

To provide a regimen capable of strongly instructing the immune system of the body to attack cancer cells.SOLUTION: The present invention is directed to regimens for administering one or more antibody-based molecules that bind to PD-1 or PD - L1 and LAG-3 (e.g., PD-1 * LAG-3 bispecific molecules), alone or in combination with antibody-based molecules that bind to tumor antigens (TA), for the treatment of cancers. The invention particularly relates to the use of such regimens in combination with a PD-1 * LAG-3 bispecific molecule.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Patent Application No. 63 / 123,581 (filed December 10, 2020; pending), U.S. Patent Application No. 63 / 031,453 (filed May 28, 2020; pending), U.S. Patent Application No. 63 / 021,556 (filed May 7, 2020; pending), U.S. Patent Application No. 63 / 019,857 (filed May 4, 2020; pending), U.S. Patent Application No. 62 / 952,878 (filed December 23, 2019; pending), and U.S. Patent Application No. 62 / 952,859 (filed December 23, 2019; pending), each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application contains one or more sequence listings in accordance with Title 37, Code of Federal Regulations, Sections 1.821 et seq., which are disclosed in a computer-readable medium (Filename: 1301_0166PCT_Sequence_Listing_ST25.txt, created December 18, 2020, size: 70,042 bytes), which file is incorporated by reference in its entirety into this application.

[0003] The present invention provides a method for administering one or more antibody-based molecules that bind PD-1 or PD-L1 and LAG-3 (e.g., a PD-1×LAG-3 bispecific molecule), alone or in combination with an antibody-based molecule that binds a tumor antigen (TA), for the treatment of cancer. The present invention is directed to the use of such regimens in combination with PD-1 x LAG-3 bispecific molecules. The present invention is particularly directed to the use of such molecules, as well as pharmaceutical compositions and kits containing such molecules that facilitate the use of such dosing regimens in the treatment of cancer. [Background technology]

[0004] I. Cell-Mediated Immune Response The ability of T cells to optimally mediate an immune response to an antigen requires two distinct signaling interactions (1, 2). First, antigens present on the surface of antigen-presenting cells (APCs) are transferred to antigen-specific naive CD4 T cells. + Against T cells Antigen presentation is required. Such presentation delivers a signal via the T-cell receptor (TCR) that instructs T cells to mount an immune response specific to the presented antigen. Next, a series of stimulatory and inhibitory signals mediated by interactions between APCs and distinct T-cell surface molecules triggers first T-cell activation and proliferation and ultimately T-cell inhibition. Thus, the first signal confers specificity to the immune response, while the second signal determines the nature, strength, and duration of the response. Immune responses are tightly controlled by costimulatory and co-inhibitory ligands and receptors often referred to as "immune checkpoints" (Non-Patent Documents 3 and 4). These molecules provide a balanced network of positive and negative signals that provide the second signal for T-cell activation and regulate the immune response, providing protection against infection and cancer. However, some cancer cells can escape the immune system by inducing a state of T-cell depletion, in which T cells are exposed to persistent antigen and / or inflammatory signals (Non-Patent Document 5). Two immune checkpoint molecules involved in T cell exhaustion, namely, Programmed Death-1 (PD-1) and Lymphocyte Activation Gene 3 (LAG-3) (Non-Patent Document 6), are described in more detail below. will be explained.

[0005] II. Programmed Cell Death-1 ("PD-1") Programmed cell death-1 (PD-1, also known as CD279) is an immune checkpoint protein expressed on the surface of activated T cells, B cells, and monocytes. It is a roughly 31-kD type I membrane protein member of the extended CD28 / CTLA-4 family of T cell regulators that broadly downregulates immune responses (Non-Patent Document 7, Patent Documents 1-9). PD-1 mediates its inhibition of the immune system by binding to its transmembrane protein ligands: programmed cell death-ligand 1 (PD-L1, also known as B7-H1) and programmed cell death-ligand 2 (PD-L2, also known as B7-DC) (Non-Patent Document 8, Patent Documents 10-17). Under normal circumstances, immune checkpoint proteins serve as targets for inhibiting overactivation of T cells, thus preventing autoimmune damage. However, when its ligand is expressed by tumor cells, binding acts to prevent immune system cells from accessing the tumor, thus weakening the immune system's ability to recognize and destroy tumor cells (Non-Patent Document 9). Thus, overexpression of PD-L1 on tumor cells is often associated with a poor prognosis.

[0006] The role of PD-1-ligand interactions in inhibiting T cell activation and proliferation suggests that these biomolecules may serve as therapeutic targets for the treatment of inflammation and cancer. Accordingly, the use of antibodies against PD-1 and its ligands, particularly PD-L1, for the treatment of infections and tumors and for upregulating adaptive immune responses has been proposed (see Non-Patent Documents 10-12, Patent Documents 18-20, Patent Document 2, and Patent Documents 21-26). Antibodies capable of specifically binding to PD-1 and PD-L1 have been reported (see, for example, Non-Patent Documents 13 and 14, Patent Documents 27-36, Patent Document 25, Patent Documents 37-39, Patent Document 26, and Patent Documents 40-44).

[0007] III. Lymphocyte-activation gene 3 ("LAG-3") Lymphocyte activation gene 3 ("LAG-3," also known as "CD223") is a ubiquitous phospholipase A (IgG)-dependent ... + and CD8 +LAG-3 is a cell surface receptor protein expressed by T cells and NK cells, and is constitutively expressed by plasmacytoid dendritic cells. It is not expressed by B cells, monocytes, or any other cell type tested (Non-Patent Documents 16-20).

[0008] Studies have shown that LAG-3 plays an important role in downregulating T cell proliferation, function, and homeostasis, as well as in T cell exhaustion (Non-Patent Documents 16-20).

[0009] Studies have suggested that inhibition of LAG-3 function by antibody blockade can reverse LAG-3-mediated immune system inhibition and partially restore effector function (Non-patent Documents 21 and 22). Antibodies capable of specifically binding to LAG-3 have been reported (see, for example, Patent Documents 45 to 51).

[0010] IV. Bispecific molecules The provision of bispecific molecules (e.g., bispecific antibodies, bispecific diabodies, etc.) offers a key advantage over monospecific natural antibodies: the ability to co-ligate and co-localize cells expressing multiple distinct epitopes. Bispecific molecules thus have a wide range of applications, including therapeutics. Bispecificity can provide great flexibility in design and engineering for a variety of applications, providing enhanced avidity for multimeric antigens, cross-linking of different antigens, and directed targeting to specific cell types dependent on the presence of both target antigens. PD-1×LAG-3 bispecific molecules for use in the treatment of cancer and / or pathogen-associated diseases are described in U.S. Patent Nos. 5,623,149; 5,713,162; 5,713,162; and 5,713,162. In particular, a PD-1×LAG-3 bispecific diabody having novel PD-1 and LAG-3 binding domains, as well as exemplary activities, is described in U.S. Patent No. 5,623,162.

[0011] V. Tumor Antigens Tumor antigens ("TAs") include cell membrane proteins that are present only on tumor cells and not on any other cells (i.e., tumor-specific antigens), or that are characteristically present on tumor cells but also on certain normal cells (i.e., tumor-associated antigens). Tumor antigens can be targeted by antibodies and used to stimulate cells of the immune system to overcome tumor escape and play novel roles in tumor surveillance and clearance (Non-Patent Document 9; Non-Patent Documents 23-26). [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent Publication No. 2007 / 0202100 [Patent Document 2] U.S. Patent Publication No. 2008 / 0311117 [Patent Document 3] U.S. Patent Publication No. 2009 / 00110667 [Patent Document 4] U.S. Patent No. 6,808,710 [Patent Document 5] U.S. Patent No. 7,101,550 [Patent Document 6] U.S. Patent No. 7,488,802 [Patent Document 7] U.S. Patent No. 7,635,757 [Patent Document 8] U.S. Patent No. 7,722,868 [Patent Document 9] International Publication No. 01 / 14557 [Patent Document 10] U.S. Patent No. 6,803,192 [Patent Document 11] U.S. Patent No. 7,794,710 [Patent Document 12] U.S. Patent Publication No. 2005 / 0059051 [Patent Document 13] U.S. Patent Publication No. 2009 / 0055944 [Patent Document 14] U.S. Patent Publication No. 2009 / 0274666 [Patent Document 15] U.S. Patent Publication No. 2009 / 0313687 [Patent Document 16] WO 01 / 39722 [Patent Document 17] International Publication No. 02 / 086083 [Patent Document 18] U.S. Patent Publication No. 2010 / 0040614 [Patent Document 19] U.S. Patent Publication No. 2010 / 0028330 [Patent Document 20] U.S. Patent Publication No. 2004 / 0241745 [Patent Document 21] U.S. Patent Publication No. 2009 / 0217401 [Patent Document 22] U.S. Patent No. 7,521,051 [Patent Document 23] U.S. Patent No. 7,563,869 [Patent Document 24] U.S. Patent No. 7,595,048 [Patent Document 25] International Publication No. 2004 / 056875 [Patent Document 26] International Publication No. 2008 / 083174 [Patent Document 27] U.S. Patent No. 8,008,449 [Patent Document 28] U.S. Patent No. 8,552,154 [Patent Document 29] U.S. Patent Publication No. 2007 / 0166281 [Patent Document 30] U.S. Patent Publication No. 2012 / 0114648 [Patent Document 31] U.S. Patent Publication No. 2012 / 0114649 [Patent Document 32] U.S. Patent Publication No. 2013 / 0017199 [Patent Document 33] U.S. Patent Publication No. 2013 / 0230514 [Patent Document 34] U.S. Patent Publication No. 2014 / 0044738 [Patent Document 35] International Publication No. 2003 / 099196 [Patent Document 36] International Publication No. 2004 / 004771 [Patent Document 37] International Publication No. 2004 / 072286 [Patent Document 38] International Publication No. 2006 / 121168 [Patent Document 39] International Publication No. 2007 / 005874 [Patent Document 40] International Publication No. 2009 / 014708 [Patent Document 41] International Publication No. 2009 / 073533 [Patent Document 42] International Publication No. 2012 / 135408 [Patent Document 43] International Publication No. 2012 / 145549 [Patent Document 44] International Publication No. 2013 / 014668 [Patent Document 45] International Publication No. 2014 / 140180 [Patent Document 46] International Publication No. 2015 / 138920 [Patent Document 47] International Publication No. 2015 / 116539 [Patent Document 48] International Publication No. 2016 / 028672 [Patent Document 49] International Publication No. 2016 / 126858 [Patent Document 50] International Publication No. 2016 / 200782 [Patent Document 51] International Publication No. 2017 / 015560 [Patent Document 52] International Publication No. 2015 / 200119

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Non-licensed literature

[0013]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

[0014] Provided herein are regimens that can more potently instruct the body's immune system to attack cancer cells. Although the adaptive immune system can be a powerful defense against cancer and disease, it can be hindered by immunosuppressive / evasive mechanisms in the tumor microenvironment mediated by PD-1 / PD-L1 interactions or by the inhibitory activity of LAG-3. As provided herein, such immunosuppressive / evasive mechanisms can be overcome by administration of PD-1 x LAG-3 bispecific molecules. As further provided herein, dual checkpoint inhibition of the PD / PD-L1 and LAG-3 checkpoint pathways can act synergistically with the antitumor activity of TA-binding molecules, particularly those with enhanced ADCC activity. [Means for solving the problem]

[0015] The present invention is directed to regimens for administering one or more antibody-based molecules that bind PD-1 or PD-L1 and LAG-3 (e.g., PD-1 x LAG-3 bispecific molecules), alone or in combination with antibody-based molecules that bind tumor antigens (TAs), for the treatment of cancer. The present invention particularly relates to the use of such regimens in combination with PD-1 x LAG-3 bispecific molecules. The present invention is directed to the use of such molecules, as well as pharmaceutical compositions and kits containing such molecules that facilitate the use of such dosing regimens in the treatment of cancer.

[0016] In particular, the present invention relates to methods of treating cancer comprising administering a PD-1×LAG-3 bispecific molecule to a subject in need thereof, said method comprising administering to said subject a flat dose of between about 120 mg and about 800 mg of said PD-1×LAG-3 bispecific molecule.

[0017] The present invention further relates to embodiments of the above methods, wherein the cancer is characterized by expression of a tumor antigen (TA), and the method further comprises the step of administering to the subject a tumor antigen (TA)-binding molecule (TA-binding molecule).

[0018] The present invention further relates to a method of treating cancer in a subject, wherein the cancer is characterized by expression of a TA, the method comprising administering to the subject a TA-binding molecule and: (a) a bispecific molecule (PD-1 x LAG-3 bispecific molecule); or (b) a combination of a molecule that immunospecifically binds to PD-1 (a PD-1-binding molecule) and a molecule that immunospecifically binds to LAG-3 (a LAG-3-binding molecule); or (c) a bispecific molecule that immunospecifically binds to both PD-L1 and LAG-3 (a PD-L1 x LAG-3 bispecific molecule); or (d) a combination of a molecule that immunospecifically binds to PD-L1 (a PD-L1-binding molecule) and a LAG-3-binding molecule; The method includes administering

[0019] The present invention further relates to embodiments of the above-described methods, wherein the TA-binding molecule comprises an ADCC-enhanced Fc domain.

[0020] The present invention further comprises: (a) each of the above molecules is in a separate composition; or (b) each of the above molecules is in the same composition; or (c) the PD-1-binding molecule and the LAG-3-binding molecule are in the same composition, and the TA-binding molecule is in a separate composition; or (d) the PD-L1-binding molecule and the LAG-3-binding molecule are in the same composition, and the TA-binding molecule is in a separate composition; This relates to the method embodiment described above.

[0021] The present invention further relates to embodiments of the above-described methods, wherein the TA-binding molecule is an antibody.

[0022] The present invention also relates to the embodiment of the aforementioned method, wherein the PD-1-binding molecule is an antibody, the PD-L1-binding molecule is an antibody, and the LAG-3-binding molecule is an antibody.

[0023] The present invention further relates to embodiments of the aforementioned methods, wherein the method comprises administering the TA-binding molecule and the PD-1 x LAG-3 bispecific molecule.

[0024] The present invention further relates to a method for producing an ADCC-enhancing Fc domain comprising: (A) an artificial glycoform; and / or (B) Amino acid substitutions relative to the wild-type Fc region The present invention relates to an embodiment of the method described above, comprising:

[0025] The present invention further relates to a method for producing an ADCC-enhancing Fc domain comprising: (A) an artificial glycoform that is a complex N-glycosidically linked sugar chain that does not contain fucose and / or contains a bisecting O-GlcNAc; and / or (B) an amino acid substitution selected from the group consisting of: (a) one substitution selected from the group consisting of: F243L, R292P, Y300L, V305I, I332E, and P396L; (b) two substitutions selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; (3) R292P and V305I; and (4) S239D and I332E; (c) three substitutions selected from the group consisting of: (1) F243L, R292P and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P, and P396L; and (4) R292P, V305I and P396L; (d) four substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, and P396L; and (2) F243L, R292P, V305I, and P396L; or (e) five substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I, and P396L; and (2) L235V, F243L, R292P, Y300L and P396L wherein the numbering is that of the EU index as set forth in Kabat.

[0026] The invention further relates to embodiments of the aforementioned methods, wherein the ADCC-enhancing Fc domain comprises the following amino acid substitutions: L235V, F243L, R292P, Y300L, and P396L, where the numbering is that of the EU index as set forth in Kabat.

[0027] The present invention further comprises: (A) the TA is selected from Table 6A or Table 6B; and / or (B) An embodiment of the aforementioned method, wherein the TA-binding molecule comprises the VL and VH domains of an antibody selected from Table 7.

[0028] The present invention further comprises: (A) The PD-1-binding molecule is: (a) a PD-1 VL domain comprising the amino acid sequence of SEQ ID NO: 35, and a PD-1 VH domain comprising the amino acid sequence of SEQ ID NO: 39; (b) the VH and VL domains of an anti-PD-1 antibody selected from Table 1; or (c) the light chain and heavy chain of an anti-PD-1 antibody selected from Table 1 an antibody comprising: (B) The PD-L1-binding molecule is: (a) a PD-L1 VL domain comprising the amino acid sequence of SEQ ID NO: 43, and a PD-L1 VH domain comprising the amino acid sequence of SEQ ID NO: 47; (b) the VH and VL domains of an anti-PD-L1 antibody selected from Table 2; or (c) the light chain and heavy chain of an anti-PD-L1 antibody selected from Table 2 an antibody comprising: (C) The LAG-3 binding molecule is: (a) a LAG-3 VL domain comprising the amino acid sequence of SEQ ID NO: 51, and a LAG-3 VH domain comprising the amino acid sequence of SEQ ID NO: 55; (b) the VH and VL domains of an anti-LAG-3 antibody selected from Table 3; or (c) the light and heavy chains of an anti-LAG-3 antibody selected from Table 3

[0033] In one embodiment of the method described above, the antibody comprises:

[0029] The present invention further relates to a method for producing the PD-1 x LAG-3 bispecific molecule, comprising administering to a subject the PD-1 x LAG-3 bispecific molecule: (a) a PD-1 VL domain comprising the amino acid sequence of SEQ ID NO: 35, and a PD-1 VH domain comprising the amino acid sequence of SEQ ID NO: 39, or the VH and VL domains of an anti-PD-1 antibody selected from Table 1; and / or (b) a LAG-3 VL domain comprising the amino acid sequence of SEQ ID NO: 51 and a LAG-3 VH domain comprising the amino acid sequence of SEQ ID NO: 55, or the VH and VL domains of an anti-LAG-3 antibody selected from Table 3; or (c) a bispecific antibody molecule selected from Tables 4 and 5 The present invention relates to an embodiment of the method described above, comprising:

[0030] The present invention further relates to a method for producing the PD-1 x LAG-3 bispecific molecule, comprising administering to a subject the PD-1 x LAG-3 bispecific molecule: (a) two of the PD-1 binding domains described above; and (b) two of the above LAG-3 binding domains The present invention relates to an embodiment of the method described above, comprising:

[0031] The present invention further provides a method for the preparation of a PD-1 x LAG-3 bispecific molecule comprising administering to a subject a subject the PD-1 x LAG-3 bispecific molecule of SEQ ID NO: 35. In some embodiments, the method further includes a PD-1 VL domain of SEQ ID NO: 39, a PD-1 VH domain of SEQ ID NO: 51, and a LAG-3 VH domain of SEQ ID NO: 55.

[0032] The present invention further relates to embodiments of the aforementioned methods, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule comprises an Fc Region and a hinge Domain, as well as embodiments wherein the Fc Region and the hinge Domain are both of the IgG4 isotype, and the hinge Domain comprises a stabilizing mutation.

[0033] The present invention further relates to a variant Fc region comprising: (a) one or more amino acid modifications that reduce the affinity of the variant Fc region for FcγR; and / or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc region;

[0033] In one embodiment of the method described above, the variant Fc region comprises:

[0034] The present invention further comprises: (a) the modification that reduces the affinity of the variant Fc Region for an FcγR comprises the substitutions L234A; L235A; or L234A and L235A; (b) the modification that increases the serum half-life of the variant Fc region comprises the following substitutions: M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K; The numbering is that of the EU index as set forth in Kabat, relating to the above method embodiment.

[0035] The present invention further relates to embodiments of the aforementioned methods, wherein the PD-1 x LAG-3 bispecific molecule comprises two polypeptide chains set forth in SEQ ID NO: 59 and two polypeptide chains set forth in SEQ ID NO: 60.

[0036] The present invention further relates to embodiments of the aforementioned methods, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered in a flat dose of about 300 mg; and the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered in a flat dose of about 600 mg.

[0037] The invention further relates to embodiments of the above-described methods, wherein the flat dose is administered about once every two weeks, and to embodiments wherein the flat dose is administered about once every three weeks.

[0038] The present invention further relates to embodiments of the aforementioned methods, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg about once every two weeks.

[0039] The present invention further relates to embodiments of the aforementioned methods, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg about once every three weeks.

[0040] The present invention further relates to embodiments of the aforementioned methods, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered by intravenous (IV) infusion.

[0041] The present invention further relates to a method for treating cancers including adrenal gland cancer, AIDS-related cancer, alveolar soft tissue sarcoma, anal cancer (including squamous cell carcinoma of the anal canal (SCAC)), bladder cancer, bone cancer, brain and spinal cord cancer, breast cancer (HER2 + Breast cancer, also including triple-negative breast cancer (TNBC), carotid bulb tumor, cervical cancer (including HPV-associated cervical cancer), chondrosarcoma, chordoma, chromophobe clear cell renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, endometrial cancer (including unselected endometrial cancer, MSI-high endometrial cancer, dMMR endometrial cancer, and / or POLE exonuclease domain mutation-positive endometrial cancer), Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, gallbladder or bile duct cancer (including cholangiocarcinomabile duct cancer), gastric cancer, esophagogastric junction (GEJ) cancer, gestational trophoblastic Diseases, germ cell tumors, glioblastoma, head and neck cancer (including squamous cell carcinoma of the head and neck (SCCHN)), hematologic malignancies, hepatocellular carcinoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (including acute myeloid leukemia), liposarcoma / malignant lipomatous tumor, liver cancer (including hepatocellular carcinoma (HCC)), lymphoma (including diffuse large B-cell lymphoma (DLBCL) and non-Hodgkin's lymphoma (NHL)), lung cancer (including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)), medulloblastoma, melanoma (including uveal melanoma), meningioma, Merck the tumor is selected from the group consisting of: uterine cell carcinoma, mesothelioma (including mesothelial pharyngeal carcinoma), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pharyngeal cancer, pheochromocytoma, pituitary tumor, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), posterior uveal melanoma, renal metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumor of childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer, and uterine cancer.

[0042] The present invention further relates to embodiments of the above-described methods, wherein the cancer is selected from the group consisting of: anal cancer, breast cancer, bile duct cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, GEJ cancer, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, and prostate cancer.

[0043] The present invention further relates to a method for treating cancer comprising administering to a subject the method ... + In embodiments of the method described above, the cancer is selected from the group consisting of breast cancer, TNBC, cholangiocarcinoma bile duct cancer, HPV-associated cervical cancer, SCCHN, HCC, SCLC or NSCLC, NHL, prostate cancer, gastric cancer, and GEJ cancer.

[0044] The present invention further provides a method for producing a TA-binding molecule comprising administering to a mammalian animal the steps of: HER2 ) and heavy chain variable domain (VHHER2 ), a HER2 binding molecule comprising a HER2 binding domain comprising: (A) The light chain variable domain (VL HER2 ) is the CDR of SEQ ID NO: 61 L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain of margetuximab. N (VH HER2 ) is the CDR of SEQ ID NO: 66 H 1. CDR H 2, and CDR H Marge including 3 Is it the heavy chain variable domain of tuximab; (B) The light chain variable domain (VL HER2 ) is the CDR of trastuzumab L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) is the CDR of trastuzumab H 1. CDR H 2, and CDR H Contains 3; (C) the light chain variable domain (VL HER2 ) is the CDR of pertuzumab L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) is the CDR of pertuzumab H 1. CDR H 2, and CDR H Contains 3; or (D) the light chain variable domain (VL HER2 ) is the CDR of hHER2 MAB-1 L 1. C DR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) is the CDR of hHER2 MAB-1 H 1. CDR H 2, and CDR H 3, including This relates to the method embodiment described above.

[0045] The present invention further relates to embodiments of the above-described methods, wherein the HER2 binding molecule is an anti-HER2 antibody.

[0046] The present invention further relates to an embodiment of the aforementioned method, wherein the anti-HER2 antibody is margetuximab, and the method comprises administering margetuximab at a dosage of about 6 mg / kg to about 18 mg / kg about once every three weeks.

[0047] The present invention further relates to embodiments of the above-described methods, wherein said methods further comprise the step of administering a chemotherapeutic agent.

[0048] The present invention further relates to embodiments of the above-described methods, wherein the cancer is a HER2-expressing cancer, particularly wherein the HER2-expressing cancer is selected from the group consisting of: breast cancer, metastatic breast cancer, bladder cancer, gastric cancer, GEJ cancer, ovarian cancer, pancreatic cancer, and stomach cancer.

[0049] The present invention further provides a B7-H3 binding molecule, wherein the TA-binding molecule comprises a B7-H3 binding domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH): The VL is CDR of SEQ ID NO: 71 L 1. CDR L 2, and CDR L 3, and the VH is CDR of SEQ ID NO: 76 H 1. CDR H 2, and CDR H In the above-described method embodiments, including 3 Regarding.

[0050] The present invention further relates to embodiments of the aforementioned methods, wherein the TA-binding molecule is enoblituzumab, and the method comprises administering enoblituzumab at a dosage of about 6 mg / kg to about 18 mg / kg about once every three weeks.

[0051] The present invention further relates to embodiments of the above-described methods, wherein the cancer is a B7-H3-expressing cancer, particularly wherein the B7-H3-expressing cancer is selected from the group consisting of: anal cancer, SCAC, breast cancer, TNBC, head and neck cancer, SCCHN, lung cancer, NSCLC, melanoma, uveal melanoma, prostate cancer, and mCRPC.

[0052] The present invention further relates to embodiments of the above-described methods, wherein the TA-binding molecule is administered by intravenous (IV) infusion.

[0053] The invention further relates to embodiments of the above-described methods in which cells expressing LAG-3 are present in a pre-treatment cancer biopsy, and to embodiments in which cells expressing PD-1 are present in a pre-treatment cancer biopsy.

[0054] The invention further relates to embodiments of the above-described methods, wherein co-expression of LAG-3 and PD-1 in a pre-treatment cancer biopsy indicates that the patient is a candidate for the method, as well as embodiments wherein the expression is gene expression.

[0055] The invention further relates to embodiments of the aforementioned methods, wherein the cancer expresses PD-L1 on the surface of cells of less than 1% before treatment, as determined using a Combined Positive Score (CPS) or a Tumor Proportion Score (TPS). [Brief explanation of the drawings]

[0056] [Figure 1]Figure 1 is a schematic diagram of a representative covalently linked tetravalent diabody having four epitope-binding sites composed of two pairs of polypeptide chains (i.e., four total polypeptide chains). One polypeptide of each pair has an E-coil Heterodimer-Promoting Domain, and the other polypeptide of each pair has a K-coil Heterodimer-Promoting Domain. As shown, cysteine ​​residues may be present in the linker and / or Heterodimer-Promoting Domain. One polypeptide of each pair has a cysteine-containing linker (which may include all or part of the hinge region) and a CH2 and / or CH3 Domain, such that the joined chains form all or part of an Fc region. VL and VH Domains that recognize the same epitope are indicated using the same shading or fill pattern. In embodiments where the two pairs of polypeptide chains are identical (as shown) and the VL and VH Domains recognize different epitopes, the resulting molecule has four epitope-binding sites and is bispecific and bivalent for each epitope it binds. Alternatively, in embodiments where the two pairs of polypeptides are different and the VL and VH domains of each pair of polypeptides recognize different epitopes, the resulting molecule has four epitope-binding sites and is tetraspecific and monovalent for each epitope it binds. [Figure 2] Figure 2 shows the observed and model-fitted PK profiles of the PD-1 x LAG-3 bispecific molecule DART-I across the dose range of 1 mg to 1200 mg. Symbols represent observed data for individual patients, and the solid lines represent the model-fit median curves for dose groups. The horizontal dashed lines represent target threshold concentrations based on clinical experience with other PD-1-targeted agents. [Figure 3]Figures 3A-3D plot the mean (SD) percent receptor occupancy (RO) of CD4+ cells (Figures 3A, 3C) and CD8+ cells (Figures 3B, 3D) with the PD-1 x LAG-3 bispecific molecule DART-I at the end of DART-I administration and prior to the administration of the next dose in that cycle on Day 1 of Cycle 1 (Figures 3A, 3B) or Cycle 2 (Figures 3C, 3D). EOI = end of infusion after administration of the first dose in Cycle 1 or Cycle 2. PRE = pre-dose prior to administration of the next dose in Cycle 1 or Cycle 2. No error bars indicate N=1. [Figure 4] Figures 4A-4C show simulated multi-dose median PK profiles for administration of 400, 600, 800, 1000, and 1200 mg flat doses of the PD-1 x LAG-3 bispecific molecule DART-I using Q2W (Figure 4A), Q3W (Figure 4B), and Q4W (Figure 3C) regimens. The upper horizontal dashed line represents the target threshold trough concentration of 23 μg / mL, based on clinical experience with other PD-1-targeted agents; the middle horizontal dashed line represents the RO EC50 x 100; and the lower horizontal dashed line represents the RO EC50 x 10. [Figure 5] Figure 5 presents a waterfall plot of the percentage reduction in target lesions (plotted as % change from baseline) by tumor type among response-evaluable cohort expansion patients treated with the PD-1 × LAG-3 bispecific molecule DART-I. [Figure 6]Figures 6A-6E plot LAG-3 and PD-L1 scores from retrospective immunochemistry assays. Individual patient LAG-3 (Figure 6A) and PD-L1 (Figure 6B) scores from the TNBC, EOC, and NSCLC cohorts are plotted from highest to lowest. Aggregate LAG-3 scores from the TNBC, EOC, and NSCLC cohorts are plotted by clinical response (Figure 6C). Individual patient LAG-3 (Figure 6D) scores from the DLBCL cohort are plotted from highest to lowest, with PD-L1 scores provided below. Aggregate LAG-3 scores from the DLBCL cohort are plotted by clinical response (Figure 6E). PR = partial response; SD = stable disease; PD = progressive disease; CR = complete response. [Figure 7] Figure 7 plots gene expression of LAG-3 versus PD-1 (PDCD1) from a retrospective NanoString PanCancer IO 360™ assay. Cancer type is indicated as follows: circle (●) = NSCLC; diamond (◆) = P-NSCLC; triangle (△) = EOC; and square (■) = TNBC. Clinical response is indicated as follows: "R" = responding patient (partial response); "P" = progressive disease; "S" = stable disease; symbol alone indicates unknown / undetermined. [Figure 8] Figure 8 plots IFN-γ gene signature scores from the retrospective NanoString PanCancer IO 360™ assay by clinical response (PR - partial response; SD - stable disease; PD - progressive disease). Cancer types are indicated as follows: circle (●) = NSCLC; diamond (◆) = P-NSCLC; triangle (△) = EOC; and square (■) = TNBC. [Figure 9]Figure 9 shows a comparison of changes in the expression of checkpoint molecules on the surface of NK cells conditioned by exposure to TA-binding molecules with an ADCC-enhancing Fc domain or a wild-type Fc domain. Flow cytometry analysis of CD137 (top row), LAG-3 (second row), PD-1 (third row), and PD-L1 (bottom row) expression in PBMC-derived NK cells incubated with N87 HER2+ target cells in the presence of 0.005 μg / ml or 0.05 μg / ml of buffer (−), margetuximab (an anti-HER2 antibody with an ADCC-enhancing Fc domain), or r-trastuzumab (an anti-HER2 antibody with a wild-type Fc domain), respectively. The percentage of positive cells (boxed) is shown. [Figure 10] Figure 10 shows the cytotoxicity of PBMCs preconditioned by exposure to TA-binding molecules with ADCC-enhanced Fc domains or wild-type Fc domains. Plotted are cytotoxicity curves against K562 target cells mediated primarily by NK cells preconditioned with margetuximab at 0.005 μg / ml or 0.05 μg / ml (open and closed squares), r-trastuzumab at 0.005 μg / ml or 0.05 μg / ml (open and closed triangles), or buffer (closed circles). [Figure 11] Figure 11 shows a comparison of changes in the expression of checkpoint molecules on the surface of NK cells, monocytes, CD4+, and CD8+ T cells conditioned by exposure to TA-binding molecules with an ADCC-enhancing Fc domain. Flow cytometry analysis of LAG-3 (top row), PD-1 (second row), PD-L1 (third row), and CD137 (bottom row) expression in various immune cell types present in PBMCs incubated with N87 HER2+ target cells in the presence of 0.5 μg / ml of margetuximab (an anti-HER2 antibody with an ADCC-enhancing Fc domain) or a control antibody, respectively. The percentage of positive cells (boxed) is shown. [Figure 12]Figure 12 shows the cytotoxicity of PBMCs preconditioned with TA-binding molecules bearing an ADCC-enhancing Fc domain (margetuximab) or a wild-type Fc domain (r-trastuzumab) against K562 target cells (cytotoxicity is primarily mediated by NK cells) in the presence or absence of an anti-PD-1 antibody (retifanlimab) or a PD-1 x LAG3 bispecific molecule (DART-I). [Figure 13] Figure 13 shows the cytotoxicity of PBMCs preconditioned with the ADCC-enhancing TA-binding molecule (margetuximab) or control in the presence or absence of the PD-1 x LAG3 bispecific molecule (DART-I) against K562 (HER2-negative) or N87 (HER2+++) target cells (cytotoxicity is primarily mediated by NK cells). [Figure 14] Figure 14 shows a waterfall plot of preliminary clinical results for 28 evaluable patients treated with the PD-1 x LAG-3 bispecific molecule DART-I and the ADCC-enhancing TA-binding molecule margetuximab. Tumor types are indicated. Solid bars represent responses in patients receiving 600 mg DART-I + 15 mg / kg, and striped bars represent responses in patients receiving 300 mg DART-I + 15 mg / kg. [Figure 15] Figures 15A-15C plot baseline gene expression of LAG3 and PD-1 (PDCD1) from 19 baseline biopsy samples in the margetuximab and DART-I treated cohort. Dual LAG3 / PDCD1 expression at baseline is plotted in Figure 15A. Baseline expression of LAG-3 (Figure 15B) and PDCD1 (Figure 15C) is plotted against the percent change in one or more target lesions. CR = complete response; PR = partial response; SD = stable disease; PD = progressive disease. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention is directed to regimens for administering one or more antibody-based molecules that bind PD-1 or PD-L1 and LAG-3 (e.g., PD-1×LAG-3 bispecific molecules), alone or in combination with antibody-based molecules that bind a tumor antigen (TA), for the treatment of cancer. The invention particularly relates to the use of such regimens in combination with PD-1×LAG-3 bispecific molecules. The invention is directed to the use of such molecules, as well as pharmaceutical compositions and kits containing such molecules that facilitate the use of such dosing regimens in the treatment of cancer.

[0058] I. Antibodies and antibody-based molecules An antibody is an immunoglobulin molecule that binds to a target region ("epitope") of the molecule (epitope of a tumor antigen ("TA"), epitope of PD-1, epitope of PD-L, etc.) via at least one "epitope-binding domain" present in the variable region of the immunoglobulin molecule. An immunoglobulin molecule is an immunoglobulin molecule that contains an epitope-binding domain capable of immunospecifically binding to an epitope of Ig-1, Ig-2, Ig-3, or an epitope of Ig-1. Such molecules can be of any isotype class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0059] As used herein, the terms "antibody" and "antibodies" are intended to include monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, and camelized antibodies. As used herein, the term "antibody-based molecule" refers to both whole or intact antibody molecules and molecules that are not whole or intact antibodies but that contain an epitope-binding domain of an antibody (e.g., single-chain Fvs (scFvs); single-chain antibodies; Fab fragments; F(ab') fragments; disulfide-linked bispecific Fvs (sdFvs); intrabodies; diabodies, molecules comprising the VL, VH, or VL and VH domains of an antibody; and one, two, or three of the antibody light chain CDR domains, one, two, or three of the antibody heavy chain CDR domains, any one, two, three, four, or five of the antibody light and heavy chain CDR domains, or all six of the antibody light and heavy chain CDR domains). Such antibody-based molecules may be fusion proteins comprising additional components, such as peptide linkers, dimerization domains, etc.

[0060] The antibody-based molecules of the present invention are characterized by the presence of one or more epitope-binding domains as described above. , or "immunospecifically binding" to an epitope. As used herein, an antibody or epitope-binding fragment thereof is said to "immunospecifically" bind a region of another molecule (i.e., an epitope) if it reacts with or associates with the epitope more frequently, more rapidly, for longer periods of time, and / or with greater affinity or avidity than alternative epitopes (e.g., variant epitopes containing one, two, three, or more amino acid substitutions, or unrelated polypeptides with less than 50% identity). It is understood by reading this definition that, for example, an antibody-based molecule that immunospecifically binds to a first target may or may not immunospecifically or preferentially bind to a second target. Epitope-containing molecules can have immunological activity, thereby eliciting an antibody response in an animal. Such molecules are referred to as "antigens."

[0061] Natural antibodies can bind to only one epitope species (i.e., they are "monospecific"), but can also bind to multiple copies of said species (i.e., they are "bivalent"). ) or "multivalency"). In this regard, the basic structural unit of a naturally occurring whole or intact IgG antibody is a tetramer composed of four assembled polypeptide chains, i.e., two short "light chains" complexed with two long "heavy chains." Each polypeptide chain consists of an amino-terminal ("N-terminal") portion containing a "variable domain" and a carboxy-terminal ("C-terminal") portion containing at least one "constant domain." An IgG light chain consists of a single "light chain variable domain" ("VL") and a single "light chain constant domain" ("CL"). Thus, the structure of the light chain of an IgG antibody is n-VL-CL-c (where n and c represent the N- and C-termini of the polypeptide chain, respectively). An IgG heavy chain consists of a single "heavy chain variable domain" ("VH"), three "heavy chain constant domains" ("CH1", "CH2", and "CH3"), and a "hinge" region ("H") located between the CH1 and CH2 domains. Unless specifically noted otherwise, the order of domains in protein molecules described herein is from N- to C-terminus. Thus, the structure of an IgG heavy chain is n-VH-CH1-H-CH2-CH3-c (where n and c represent the N- and C-termini of the polypeptide, respectively). The ability of an intact, unmodified antibody (e.g., an IgG antibody) to bind to an epitope of an antigen depends on the presence and sequence of variable domains.

[0062] A. Constant Domains 1. Light chain constant domain One CL domain is a human IgG CLκ domain. The amino acid sequence of a representative human CLκ domain is (SEQ ID NO: 1): RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC is.

[0063] Another CL domain is the human IgG CLλ domain. The amino acid sequence of a representative human CLλ domain is (SEQ ID NO:2): QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS is.

[0064] 2. Heavy chain CH1 domain A representative CH1 domain is the human IgG1 CH1 domain. The amino acid sequence of a representative human IgG1 CH1 domain is (SEQ ID NO: 3): ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV is.

[0065] Another exemplary CH1 domain is the human IgG2 CH1 domain. The amino acid sequence of a representative human IgG2 CH1 domain is (SEQ ID NO:4): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV is.

[0066] Another exemplary CH1 domain is the human IgG3 CH1 domain. The amino acid sequence of a representative human IgG3 CH1 domain is (SEQ ID NO:5): ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRV is.

[0067] Another exemplary CH1 domain is the human IgG4 CH1 domain. The amino acid sequence of a representative human IgG4 CH1 domain is (SEQ ID NO:6): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV is.

[0068] 3. Heavy chain hinge region A representative hinge region is a human IgG1 hinge region. The amino acid sequence of a representative human IgG1 hinge region is (SEQ ID NO:7): EPKSCDKTHT CPPCP is.

[0069] Another exemplary hinge region is the human IgG2 hinge region. The amino acid sequence of a representative human IgG2 hinge region is (SEQ ID NO:8): ERKCCVECPP CP is.

[0070] Another exemplary hinge region is the human IgG3 hinge region. The amino acid sequence of a representative human IgG3 hinge region is (SEQ ID NO:9): ELKTPLGDTT HTCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CP is.

[0071] Another exemplary hinge region is the human IgG4 hinge region. The amino acid sequence of a representative human IgG4 hinge region is (SEQ ID NO: 10): ESKYGPPCPS CP is.

[0072] As described herein, an IgG4 hinge region may contain a stabilizing mutation, such as a S228P substitution (as numbered by the EU index of Kabat). The amino acid sequence of one particular stabilized IgG4 hinge region is (SEQ ID NO: 11): ESKYGPPCPP CP is.

[0073] 4. Heavy chain CH2 and CH3 domains and Fc domain The CH2 and CH3 domains of the two heavy chains interact to form the "Fc region" of an IgG antibody, which is the domain recognized by cellular Fc receptors, including but not limited to Fcγ receptors (FcγRs). As used herein, the term "Fc region" is used to define the C-terminal region of an IgG heavy chain. A portion of the Fc region (including a portion encompassing the entire Fc region) is referred to herein as an "Fc domain." An Fc domain is said to be of a particular IgG isotype, class, or subclass if its amino acid sequence is more homologous to that IgG isotype than to other IgG isotypes, although hybrid Fc domains containing portions from different isotypes are also contemplated.

[0074] The amino acid sequence of a representative human IgG1 CH2-CH3 domain is (SEQ ID NO: 12): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X and X is a lysine (K) or is absent.

[0075] The amino acid sequence of a representative human IgG2 CH2-CH3 domain is (SEQ ID NO: 13): 231 240 250 260 270 280 APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE 390 400 410 420 430 WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X and X is a lysine (K) or is absent.

[0076] The amino acid sequence of a representative human IgG3 CH2-CH3 domain is (SEQ ID NO: 14): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESSGQPENN YNTTPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE 440 447 ALHNRFTQKS LSLSPG X and X is a lysine (K) or is absent.

[0077] The amino acid sequence of a representative human IgG4 CH2-CH3 domain is (SEQ ID NO: 15): 231 240 250 260 270 280 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 340 350 360 370 380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSLG X and X is a lysine (K) or is absent.

[0078] Throughout this specification, the numbering of residues in the constant region of the IgG heavy chain is based on Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, NH1, MD (1991), which is expressly incorporated herein by reference. The term "EU index as in Kabat" refers to the numbering of human IgG1 EU antibodies.

[0079] Polymorphisms have been observed at many different positions within antibody constant regions (e.g., CH1 positions, including but not limited to, 192, 193, and 214, as numbered by the EU index of Kabat; Fc positions, including but not limited to, 270, 272, 312, 315, 356, and 358), and therefore slight differences may exist between the sequences presented herein and those of the prior art. Polymorphic forms of human immunoglobulins have been well characterized. Currently, 18 Gm allotypes are known: G1m(1, 2, 3, 17) or G1m(a, x, f, z), G2m(23) or G2m(n), G3m(5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m(b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211). In particular, it is intended that the antibodies of the present invention be used in combination with any of the immunoglobulin genes. Any allotype, isoallotype, or haplotype of the molecule may be incorporated and is not intended to be limited to the allotypes, isoallotypes, or haplotypes of the sequences presented herein. Furthermore, depending on the expression system, the C-terminal amino acid residue of the CH3 domain (bold above) can be removed post-translationally. Thus, the C-terminal residue of the CH3 domain can be any amino acid residue in the molecules of the invention. Specifically encompassed by the invention are molecules lacking the C-terminal residue of the CH3 domain. Also specifically encompassed by the invention are molecules containing a C-terminal lysine residue of the CH3 domain.

[0080] The Fc domain of the Fc domain-containing antibody-based molecules of the invention may be a complete Fc domain (e.g., a complete IgG Fc region) or only a portion of an Fc region. Optionally, the Fc domain of the Fc domain-containing antibody-based molecules of the invention does not include the C-terminal lysine amino acid residue of the wild-type IgG CH3 domain.

[0081] In classical immune function, the interaction of antibody-antigen complexes with cells of the immune system results in a variety of effects, from effector functions such as antibody-dependent cellular cytotoxicity, mast cell degranulation, and phagocytosis to lymphocyte-mediated responses. These interactions result in a wide range of responses, ranging from immunomodulatory signals, such as modulation of proliferation and antibody secretion, to immunoregulatory signals. All of these interactions are initiated by the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes is due to the structural heterogeneity of three Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A), and FcγRIII (CD16) are activating (i.e., immune system-enhancing) receptors; FcγRIIB (CD32B) is an inhibitory (i.e., immune system-decreasing) receptor. Furthermore, interaction with the neonatal Fc receptor (FcRn) mediates the recycling of IgG molecules from endosomes to the cell surface and their release into the circulation. The amino acid sequences of the CH2-CH3 domains of representative wild-type IgG1 (SEQ ID NO: 12), IgG2 (SEQ ID NO: 13), IgG3 (SEQ ID NO: 14), and IgG4 (SEQ ID NO: 15) are presented above.

[0082] Modification of the amino acid sequence of the Fc domain can provide an altered phenotype, such as an altered serum half-life, an altered stability, an altered susceptibility to cellular enzymes, an altered effector function, or a combination of such phenotypes. In particular, the present invention contemplates antibody-based molecules comprising a wild-type Fc domain or an Fc domain that has been modified to enhance its ability to mediate antibody-dependent cellular cytotoxicity (ADCC) relative to the ADCC mediated by an antibody-based molecule containing an unmodified Fc domain. Such modified Fc domains are referred to herein as "ADCC-Enhanced Fc Domains." The present invention provides Antibody-based molecules comprising an Fc domain with little or no ADCC activity are also contemplated. Thus, in certain embodiments, antibody-based molecules of the invention may be engineered to comprise an ADCC-enhanced Fc domain or an Fc domain with little or no ADCC activity. The Fc domain of an antibody-based molecule of the invention may be capable of binding to one or more Fc receptors (e.g., one or more FcγRs), although in certain embodiments, the Fc domain is a modified Fc domain with altered binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) (relative to the binding exhibited by an Fc domain without such modifications). For example, such variant Fc domains have enhanced binding to one or more activating receptors and / or reduced or no ability to bind to one or more inhibitory receptors, and exhibit enhanced ADCC activity. Alternatively, such variant Fc domains may have significantly reduced or no ability to bind to one or more activating receptors and / or have enhanced binding to one or more inhibitory receptors and exhibit little or no ADCC activity.

[0083] Modifications that reduce or eliminate FcγR binding (and ADCC activity) are known in the art and include amino acid substitutions at positions 234 and 235, at position 265, or at position 297 according to the EU index of Kabat numbering (see, e.g., U.S. Pat. No. 5,624,821). In one embodiment, an antibody-based molecule of the invention comprises an Fc domain with little or no ADCC activity comprising one, two, three, or four of the following substitutions: L234A, L235A, D265A, N297Q, and N297G. In a specific embodiment, an antibody-based molecule of the invention comprises an Fc domain with little or no ADCC activity comprising a substitution at position 234 with alanine and a substitution at position 235 with alanine (234A, 235A) according to the EU index of Kabat numbering. Alternatively, such molecules may comprise naturally occurring Fc domains with essentially reduced (or substantially no) binding to FcγRIIIA (CD16a) and / or reduced effector function (compared to the binding and effector function exhibited by wild-type IgG1 Fc domains). In a specific embodiment, the Fc-bearing molecules of the present invention comprise an IgG2 Fc domain (SEQ ID NO: 13) or an IgG4 Fc domain (SEQ ID NO: 15). When an IgG4 Fc domain is used, the present invention provides an Fc-bearing molecule comprising an IgG2 Fc domain (SEQ ID NO: 13) or an IgG4 Fc domain (SEQ ID NO: 15) with the S228P substitution in the hinge region as described above (see, e.g., SEQ ID NO: 11). The introduction of stabilizing mutations such as the above is also included.

[0084] An ADCC-enhanced Fc domain of the invention may comprise part or all of the CH2 domain and / or part or all of the CH3 domain of a complete Fc domain, or may comprise a variant CH2 and / or variant CH3 sequence (which may, for example, comprise one or more substitutions and / or insertions and / or one or more deletions relative to the CH2 or CH3 domain of the complete Fc domain). Such an Fc domain may comprise a non-Fc polypeptide portion, or may comprise a portion of a non-naturally occurring Fc domain, or may comprise a non-naturally occurring orientation of the CH2 and / or CH3 domain (e.g., two CH2 domains or two CH3 domains, or a CH3 domain joined to a CH2 domain in an N-terminal to C-terminal direction, etc.).

[0085] ADCC-enhancing Fc domains that have been identified as altering effector function (e.g., ADCC) are known in the art and include modifications that increase binding to activating Fc receptors (e.g., FcγRIIA (CD16A)) relative to inhibitory Fc receptors (e.g., FcγRIIB (CD32B)) (see, e.g., Stavenhagen, JB et al. (2007) "FcOptimization of Therapeutic Antibodies Enhances Their Ability to Kill Tumor Cells"). (See “In Vitro and In Vivo Controls Tumor Expansion via Low-Affinity Activating Fcgamma Receptors,” Cancer Res. 57(18):8882-8890). Single, double, triple, quadruple, and quintuple permutations of numbers have been described (see, e.g., U.S. Patent Nos. 6,737,056; 7,317,091; 7,355,008; 7,960,512; 8,217,147; and 8,652,466).

[0086] In one embodiment, the ADCC-enhanced Fc domain comprises an Fc domain comprising one or more amino acid substitutions (relative to a wild-type IgG Fc domain) selected from the following substitutions, as numbered according to the EU index of Kabat: S239D, F243L, D270E, R292G, R292P, Y300L, V305I, I332E, or P396L. These amino acid substitutions may be present in any combination in a human IgG Fc domain (e.g., an IgG1 Fc domain). In one embodiment, the mutant human IgG Fc domain contains S239D and I332E substitutions. In another embodiment, the mutant human IgG Fc domain contains F243L, R292P, and Y300L substitutions. In a further embodiment, the mutant human IgG Fc domain contains F243L, R292P, Y300L, V305I, and P296L substitutions. In a specific embodiment, such a human IgG ADCC-enhanced Fc domain comprises: (a) one substitution selected from the group consisting of: (1)F243L; (2)R292P; (3) Y300L; (4)V305I; (5) I332E; and (6) P396L (b) two substitutions selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; (3) R292P and V305I; and (4) S239D and I332E (c) three substitutions selected from the group consisting of: (1) F243L, R292P and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P, and P396L; and (4) R292P, V305I and P396L; (d) four substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, and P396L; and (2) F243L, R292P, V305I, and P396L; or (e) five substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I, and P396L; and (2) L235V, F243L, R292P, Y300L and P396L where the numbering is that of the EU Index as given in Kabat.

[0087] In a specific embodiment, the ADCC-enhancing Fc domain will comprise: (1) "FcMT1" ADCC-enhancing Fc domain, which contains F243L, R292P, Y300L, V305I, and P396L substitutions. Antibody-based molecules containing the FcMT1 mutant IgG1 Fc domain exhibit a 10-fold increase in binding to human CD16A (FcγRIIIA) relative to that observed with the wild-type IgG1 Fc domain, and proportionally greater enhancement in binding to CD16-158Phe compared to binding to CD16-158Val. The amino acid sequence of the "FcMT1" ADCC-enhancing Fc domain is (SEQ ID NO: 16): APELLGGPSV FL L PPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK P P EEQYNST L RVVS I LTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTP L VLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X wherein X is lysine (K) or absent; (2) "FcMT2" ADCC-enhancing Fc domain, which contains L235V, F243L, R292P, Y300L, and P396L substitutions. The FcMT2 mutant IgG1 Fc domain is a further improvement of the FcMT1 mutant IgG1 Fc domain, and has comparable CD16A-binding properties while advantageously exhibiting reduced binding to CD32B (FcγRIIB). The amino acid sequence of the "FcMT2" ADCC-enhancing Fc domain is (SEQ ID NO: 17): APEL V GGPSV FL L PPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK P P EEQYNST L RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTP L VLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X and X is lysine (K) or absent; or (3) "FcMT3" ADCC-enhancing Fc domain, which contains F243L, R292P, and Y300L substitutions. The FcMT3 mutant IgG1 Fc domain is a further improvement of the FcMT1 mutant IgG1 Fc domain, and has comparable CD16A binding properties while advantageously exhibiting reduced binding to CD32B (FcγRIIB). The amino acid sequence of the "FcMT3" ADCC-enhancing Fc domain is (SEQ ID NO: 18): APELLGGPSV FL L PPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK P P EEQYNST L RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X and X is a lysine (K) or is absent.

[0088] In an alternative embodiment, the ADCC-enhancing Fc domain does not contain fucose and / or These glycoforms include artificial glycoforms, which are complex N-glycosidic sugar chains containing isectic O-GlcNAc. Such glycoforms can be synthesized from fucosyltransferase-free cell lines (e.g., POTELLIGENT® cell lines, BioWa, Inc.; Matsushita, T. (2011) “Engineered Therapeutic Antibodies With Enhanced Effector Functions: Clinical Application Of The Potelligent® Technology,” Korean J. Hematol. 46(3):148-150) and / or cells expressing O-GlcNAc transferase. The ADCC-enhancing Fc domain can be obtained by recombinantly expressing an antibody-based molecule in a recombinant human Fc-like antibody strain (Roche GlycArt AG; Satoh, M. et al. (2006) "Non-Fucosylated Therapeutic Antibodies As Next-Generation Therapeutic Antibodies," Exp. Opin. Biol. Ther. 6(11):1161-1173). In certain embodiments, the ADCC-enhancing Fc domain comprises one or more amino acid substitutions and an artificial glycoform.

[0089] Furthermore, the serum half-life of a molecule containing an Fc domain can be increased by increasing the binding affinity of the Fc domain for FcRn. As used herein, the term "half-life" refers to the time in minutes that is a measure of the average survival time of a molecule after administration. Half-life refers to the pharmacokinetic properties of a molecule. Half-life can be expressed as the time required for 50 percent (50%) of a known amount of a molecule to be cleared from a subject's body (e.g., a human patient or other mammal) or a particular body cavity thereof, as measured in serum (i.e., circulating half-life) or other tissues. Generally, an increase in half-life leads to an increase in the mean residence time (MRT) of the administered molecule in the circulation. Modifications that can increase the half-life of Fc domain-containing molecules are known in the art and include, for example, amino acid substitutions M252Y, S254T, T256E, and combinations thereof. See, e.g., U.S. Patent Nos. 6,277,375, 7,083,784, 7,217,797, and 8,088,376; U.S. Patent Publication Nos. 2002 / 0147311 and 2007 / 0148164; and the modifications described in WO 98 / 23289, WO 2009 / 058492, and WO 2010 / 033279.

[0090] In one embodiment, an antibody-based molecule of the invention comprises a variant Fc domain comprising a substitution at position 252 with tyrosine, a substitution at position 254 with threonine, and a substitution at position 256 with glutamic acid (252Y, 254T and 256E) according to the EU index as numbered by Kabat.

[0091] The present invention also encompasses antibody-based molecules of the invention comprising an Fc domain, said Fc domain comprising: (a) one or more mutations that alter effector function and / or FcγR binding; and / or (b) one or more mutations that extend serum half-life Includes.

[0092] The present invention also encompasses antibody-based molecules of the invention comprising an Fc domain, said Fc domain comprising: (a) one or more mutations that reduce or eliminate ADCC; and / or (b) one or more mutations that extend serum half-life Includes.

[0093] A representative IgG1 sequence of the CH2 and CH3 domains of a variant Fc domain with little or no ADCC activity and extended serum half-life contains the substitutions L234A / L235A / M252Y / S254T / T256E (SEQ ID NO: 19): APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X where X is lysine (K) or absent.

[0094] A representative IgG4 sequence of the CH2 and CH3 domains of a variant Fc domain with extended serum half-life contains the substitutions M252Y / S254T / T256E (SEQ ID NO: 20): APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG X where X is lysine (K) or absent.

[0095] 5. Variable Domain The variable domain of an IgG molecule is made up of three complementarity-determining regions (CDRs), which contain the amino acid residues of the antibody that come into contact with the epitope. ), and intervening non-CDR segments called framework regions ("FR"), which generally maintain the structure of the CDR residues and position the CDRs, thereby enabling such contacts (although certain framework residues may also contact the epitope). Thus, VL and VH domains have the structure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c. The amino acid sequences of the CDRs determine whether an antibody can bind to a particular epitope. The interaction of antibody light chains and antibody heavy chains, particularly the interaction of these VL and VH domains, which are present on separate polypeptides, forms the epitope-binding domain of the antibody.

[0096] Amino acids from the mature heavy and light chain variable domains of immunoglobulins are designated by the position of the amino acid within the chain. Kabat (Sequences of Proteins of Immunological Interest, 5 thEd. Public Health Service, NH1, MD (1991)) describes numerous amino acid sequences for antibodies, identifies amino acid consensus sequences for each subgroup, assigns residue numbers to each amino acid, and identifies CDRs and FRs as defined by Kabat (Chothia, C. & Lesk, A. M. (1987) "Canonical Structures For The Hypervariable Regions Of Immunoglobulins,” J.Mol. Biol. 196:901‐917 ) defined by H 1 will be understood to start 5 residues earlier) The Kabat numbering scheme can be extended for antibodies not included in the compendium by aligning the antibody with one of the Kabat consensus sequences with reference to conserved amino acids. This method of assigning residue numbers has become standard in the art, and amino acids at identical positions in different antibodies, including chimeric or humanized variants, are readily identified. For example, amino acid 50 of a human antibody light chain occupies the same position as amino acid 50 of a murine antibody light chain. Thus, the positions within the VL and VH domains where these CDRs begin and end are well defined and can be confirmed by inspection of the sequences of the VL and VH domains (see, e.g., Martin, CR (2010) "Protein Sequence Analysis of Human Antibodies"). and Structure Analysis of Antibody Variable Domains,”In: Antibody Engineering Vol. 2 (Kontermann, R. and Dubel, S. (eds.), Springer-Verlag Berlin Heidelberg, See Chapter 3 (pages 33-51).

[0097] Polypeptides that are (or can function as) the first, second, and third CDRs of the light chain of an antibody are referred to herein as CDRs 1, 2, and 3, respectively. L 1 domain, CDR L 2 domains and CDRs L Similarly, the first, second and third domains of an antibody heavy chain are called Polypeptides which are (or can function as) the first, second, and third CDRs of are referred to herein as CDRs 1, 2, and 3, respectively. H 1 domain, CDR H 2 domains and CDRs H 3 domains and Therefore, CDR L 1 domain, CDR L 2 domains, CDR L 3 domains, CD R H 1 domain, CDR H 2 domains and CDRs H The term 3-domain refers to a The present invention is directed to polypeptides that, when incorporated into a protein, enable the protein to bind to a specific epitope, regardless of whether the protein is an antibody having a light and heavy chain, or a diabody, or a single-chain binding molecule (e.g., scFv, BiTe, etc.), or another type of protein. Thus, as used herein, the term "epitope binding domain" refers to a portion of an antibody-based molecule of the invention that is capable of immunospecifically binding to an epitope. An epitope binding domain may contain one, two, three, four, or five of the CDR domains of an antibody, or may contain all six of the CDR domains of an antibody, and may be capable of immunospecifically binding to such an epitope but may exhibit immunospecificity, affinity, or selectivity for an epitope that is different from the epitope of such an antibody. Typically, however, an epitope binding domain will contain all six of the CDR domains of such an antibody.

[0098] Epitope binding domains may comprise complete variable domains fused to constant domains or only the complementarity determining regions (CDRs) of such variable domains grafted onto appropriate framework regions. Epitope binding domains may be wild-type or modified by one or more amino acid substitutions.

[0099] Humanization of antibody-based molecules The present invention particularly encompasses antibody-based molecules comprising the VL and / or VH domains of a humanized antibody. The term "humanized" antibody refers to a chimeric molecule, generally produced using recombinant techniques. The term "chimeric" refers to a chimeric molecule prepared using a chimeric antibody, which has an epitope-binding domain of an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The polynucleotide sequence of the variable domain of such an antibody can be used for genetic engineering to generate derivatives and improve the affinity or other characteristics of the antibody. Both heavy and light chain variable domains are known to contain three complementarity-determining regions (CDRs) flanked by four framework regions (FRs), which vary in response to the antigen of interest and determine binding capacity; the framework regions are relatively conserved in a given species and are presumed to provide a scaffold for the CDRs. When preparing a non-human antibody against a specific antigen, the variable domain can be "reshaped" or "humanized." The general principle of antibody humanization involves replacing the non-human remainder of the antibody with human antibody sequences while retaining the nucleotide sequence of the epitope-binding portion of the antibody. There are four general steps to humanize a monoclonal antibody. The steps are as follows: (1) determining the nucleotide and predicted amino acid sequences of the light and heavy chain variable domains of the starting antibody; (2) designing the humanized or caninized antibody, i.e., determining the antibody framework regions to be used during the humanization or caninization process; (3) the actual humanization or caninization method / technique; and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Patent No. 4,816,567; U.S. Patent No. 5,807,715; U.S. Patent No. 5,866,692; and U.S. Patent No. 6,331,415; Lobuglio et al. (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics and Immune Response,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989). Other references describe rodent CDRs that are grafted into human supporting framework regions (FRs) before fusion with an appropriate human antibody constant domain (e.g., Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; and Jones et al. (1986) "Replacing the Complementarity-Determining Regions (See “In A Human Antibody With Those From A Mouse,” Nature 321:522-525). Other methods that may be utilized to humanize are described in Daugherty et al. (1991) "Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins," Nucl. Acids Res. 19:2471-2476 and U.S. Pat. No. 6,180,377; U.S. Pat. No. 6,180,377; Nos. 6,054,297; 5,997,867; and 5,866,692. In some embodiments, humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibody). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that have been altered and differ in sequence relative to the original antibody.

[0100] B. Bispecific molecules In some embodiments, antibody-based molecules of the invention are bispecific, such as bispecific antibodies or bispecific diabodies. Such bispecific antibody-based molecules may comprise the provided epitope-binding domains of PD-1 and LAG-3 (i.e., a PD-1 x LAG-3 bispecific molecule) or may comprise the provided epitope-binding domains of PD-L1 and LAG-3 (i.e., a PD-L1 x LAG-3 bispecific molecule). The provision of such bispecific antibody-based molecules offers important advantages over monospecific antibodies, namely, the ability to co-link PD-1 and LAG-3 on cells that co-express them and / or co-localize PD-1-expressing cells with LAG-3-expressing cells, or the ability to co-link PD-L1 and LAG-3 on cells that co-express them and / or co-localize PD-L1-expressing cells with LAG-3-expressing cells. In certain embodiments, such bispecific antibody-based molecules may bind to two different TAs.

[0101] 1. Bispecific antibodies A wide range of recombinant bispecific antibody formats have been developed (see, for example, WO 2008 / 003116, WO 2009 / 132876, WO 2008 / 003103, WO 2007 / 146968, WO 2009 / 018386, WO 2012 / 009544 and WO 2013 / 070565), most of which use linker peptides to fuse additional epitope-binding fragments (e.g. scFv, VL, VH, etc.) to or within the antibody core (IgA, IgD, IgE, IgG or IgM), or to fuse multiple epitope-binding fragments (e.g. two Fab fragments or scFvs). An alternative format uses linker peptides to fuse epitope-binding fragments (e.g., scFv, VL, VH, etc.) to dimerization domains such as CH2-CH3 domains or alternative polypeptides (WO 2005 / 070966, WO 2006 / 107786A, WO 2006 / 107617A, and WO 2007 / 046893). WO 2013 / 174873, WO 2011 / 133886, and WO 2010 / 136172 teach trispecific antibodies in which the CL and CH1 domains are switched from their natural positions and the VL and VH domains are diversified to allow binding to more than one antigen (WO 2008 / 027236; WO 2010 / 108127). WO 2013 / 163427 and WO 2013 / 119903 disclose modifying the CH2 domain to contain a fusion protein adduct containing a binding domain. WO 2010 / 028797, WO 2010028796, and WO 2010 / 028795 disclose recombinant antibodies in which the Fc region is replaced with additional VL and VH domains to form trivalent binding molecules. WO 2003 / 025018 and WO 2003012069 disclose recombinant diabodies in which each chain contains an scFv domain.WO 2013 / 006544 discloses multivalent fab molecules that are synthesized as single polypeptide chains and then subjected to proteolysis to obtain heterodimeric structures. WO 2014 / 022540, WO 201 WO 3 / 003652, WO 2012 / 162583, WO 2012 / 156430, WO 2011 / 086091, WO 2008 / 024188, WO 2007 / 024715, WO 2007 / 075270, WO 1998 / 002463, WO 1992 / 022583 and WO 1991 / 003493 disclose adding additional binding domains or functional groups to antibodies or antibody portions (for example adding a diabody to the light chain of the antibody, or adding additional VL and VH domains to the light and heavy chains of the antibody, or adding heterologous fusion proteins to each other or linking multiple Fab domains to each other). Trivalent molecules comprising covalently linked diabodies and diabody-like domains are described in WO 2015 / 184207, WO 2015 / 184203, WO 2012 / 162068, WO 2012 / 018687, WO 2010 / 080538, and WO 2006 / 113665, and are provided herein. Accordingly, it is specifically contemplated that the PD-1 x LAG-3 bispecific molecules of the invention may have the structure of any of the above-described formats and may be produced by any of the above-described methods.

[0102] 2. Bispecific diabodies The diabodies of the present invention are stable, covalently linked heterodimeric non-monospecific diabodies, and are described, for example, in Chichili, GR et al. (2015) “A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates,” Sci. Transl. Med. 7(289):289ra82; Veri, MC et al. (2010) “Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIB (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold,” Arthritis Rheum. 62(7):1933-1943; Moore, PA et al. (2011) “Application Of Dual Affinity Retargeting Molecules To AchieveOptimal Redirected T cell Killing Of B-Cell Lymphoma,”Blood 117(17):4542-4551;US Published Patent No. 2007 / 000490 See U.S. Patent Publication No. 2009 / 0060910; U.S. Patent Publication No. 2010 / 0174053; U.S. Patent Publication No. 20130295121; U.S. Patent Publication No. 2014 / 0099318; U.S. Patent Publication No. 2015 / 0175697; U.S. Patent Publication No. 2016 / 0017038; U.S. Patent Publication No. 2016 / 0194396; U.S. Patent Publication No. 2016 / 0200827; and U.S. Patent Publication No. 2017 / 0247452. Such diabodies comprise two or more covalently complexed polypeptide chains, involving the incorporation of one or more cysteine ​​residues into each of the employed polypeptide species that are capable of forming disulfide bonds, thereby covalently linking the two polypeptide chains. For example, the addition of a cysteine ​​residue to the C-terminus of such structures has been shown to allow disulfide bonding between the polypeptide chains, which stabilizes the resulting heterodimers without interfering with the binding properties of the divalent molecule. Such diabodies also contain domains that help promote heterodimerization of the polypeptide chains ("heterodimer-promoting domains").

[0103] The diabodies of the present invention are covalently complexed diabodies composed of polypeptides, which may consist of two, three, four, or five or more polypeptide chains. As used herein, the term "composed of" is intended to be non-limiting. It is intended that the diabodies of the invention comprise two polypeptide chains, and therefore, a diabody of the invention that is comprised of two polypeptide chains may comprise an additional polypeptide chain. Such a chain may have the same sequence as another polypeptide chain of the diabody, or may have a different sequence from any other polypeptide chain of the binding molecule. Diabodies of the invention may be designed to include an Fc domain.

[0104] In certain embodiments, the diabodies of the invention comprise two diabodies specific for a first epitope. The diabody is a four-chain Fc domain-containing diabody with one binding site, two binding sites specific for a second epitope, an Fc domain, and a cysteine-containing E / K-coil Heterodimer-Promoting Domain. The overall structure of such a diabody is provided in Figure 1.

[0105] Bispecific diabodies of the present invention are engineered such that the first and second polypeptides are covalently linked to one another via cysteine ​​residues along their length. Such cysteine ​​residues can be introduced into an intervening linker (Linker 1; e.g., GGGSGGGG (SEQ ID NO: 21)) separating the VL and VH domains of the polypeptides. Alternatively and more preferably, a second peptide (Linker 2) containing a cysteine ​​residue is introduced into each polypeptide chain, e.g., at a position N-terminal to the VL domain or C-terminal to the VH domain of the polypeptide chain. A preferred sequence for such Linker 2 is SEQ ID NO: 22: GGCGGG. Additionally or alternatively, cysteine ​​residues may be introduced into other domains, examples of which are provided below.

[0106] In certain embodiments, Heterodimer-Promoting Domains of the invention will comprise tandemly repeated coil domains of opposite charge. Thus, in one embodiment, one of the polypeptide chains will contain an "E-coil" domain (SEQ ID NO:23: ) with residues that form a negative charge at pH 7. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E The other of the two polypeptide chains was engineered to contain a "K-coil" domain (SEQ ID NO: 24: ) with residues that form a positive charge at pH 7. K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL KE). The presence of such a charged domain promotes association between the first and second polypeptides, thereby promoting heterodimerization. It is not important which coil is provided in the first or second polypeptide chain.

[0107] In another embodiment, a Heterodimer-Promoting Domain (e.g., SEQ ID NO: 23) is used in which one of the four tandem "E-coil" helical domains of SEQ ID NO: 23 has been modified to contain a cysteine ​​residue. E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E Similarly, in another embodiment, a Heterodimer-Promoting Domain (e.g., SEQ ID NO: 24) is utilized in which one of the four "K-coil" helical domains of SEQ ID NO: 24 has been modified to contain a cysteine ​​residue. K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO:26)) is utilized. Such embodiments advantageously combine to employ the Heterodimer-Promoting Domain of SEQ ID NO:25 and the Heterodimer-Promoting Domain of SEQ ID NO:26.

[0108] Thus, the diabody is engineered such that pairs of its polypeptide chains are covalently linked to one another via one or more cysteine ​​residues positioned along their length to form a covalent molecular complex. Such cysteine ​​residues can be introduced into the intervening linker separating the VL and VH domains of the polypeptides. Alternatively, one or more linkers (e.g., Linker 2, Linker 3, etc.) may contain a cysteine ​​residue. In specific embodiments, one or more coil domains of the coil-containing Heterodimer-Promoting Domains contain amino acid substitutions incorporating a cysteine ​​residue, such as SEQ ID NO:25 or SEQ ID NO:26. An alternative, cysteine-free Linker 2 sequence is SEQ ID NO:27:ASTKG which may be used in conjunction with the cysteine ​​residue containing Heterodimer-Promoting Domain.

[0109] Bispecific diabodies of the invention are preferably engineered to have IgG CH2-CH3 domains that can be conjugated together to form an Fc region. In a specific embodiment, bispecific diabodies of the invention comprise human IgG CH2-CH3 domains. Exemplary human IgG CH2-CH3 domains are provided above, including CH2-CH3 domains engineered to alter effector function and / or serum half-life.

[0110] In certain embodiments, bispecific diabodies of the invention are engineered with an intervening linker peptide (Linker 3) connecting the CH2 and CH3 Domains to the Heterodimer-Promoting Domain. Preferably, Linker 3 is C-terminal to the Heterodimer-Promoting Domain. Linkers that can be employed in the PD-1 x LAG-3 bispecific diabodies of the invention include: GGGS (SEQ ID NO:28), LGGGSG (SEQ ID NO:29), ASTKG (SEQ ID NO:27), LEPKSS (SEQ ID NO:30), APSSS (SEQ ID NO:31), APSSSPME (SEQ ID NO:32), GGC, and GGG. Linker 3 may comprise a portion of an IgG hinge region, alone or in addition to other linker sequences. Exemplary hinge regions are: DKTHTCPPCP (SEQ ID NO:33) or EPKSCDKTHTCPPCP (SEQ ID NO:7) from IgG1, and ERKCCVECPPCP (SEQ ID NO:8) from IgG2. ), IgG4-derived ESKYGPPCPSCP (SEQ ID NO: 10), and ESKYGPPCPPCP (SEQ ID NO: 11), an IgG4 hinge variant containing a stabilizing S228P substitution to reduce strand exchange (Lu et al., (2008) "The Effect of a Point Mutation on the Stability of IgG4 As Monitored By Analytical Ultracentrifugation,” J. Pharmaceutical Sciences 97:960-969). In certain embodiments, linker 3 may further comprise GGG, e.g., GGGDKTHTCPPCP (SEQ ID NO: 34).

[0111] II. Antibody-Based Molecules that Bind PD-1 (or PD-L1) and / or LAG-3 The present invention specifically contemplates compositions and methods comprising or employing: (1) PD-1×LAG-3 bispecific molecule; (2) monospecific PD-1 binding molecules and monospecific LAG-3 binding molecules; (3) a PD-L1 x LAG-3 bispecific molecule; or (4) Monospecific PD-L1-binding molecules and monospecific LAG-3-binding molecules wherein said monospecific binding molecule is an intact antibody and said bispecific molecule is a diabody or a bispecific antibody.

[0112] Antibody-based molecules that immunospecifically bind to human PD-1 (e.g., monospecific PD-1-binding molecules or PD-1 x LAG-3 bispecific molecules) that can be used in accordance with the present invention will comprise at least one epitope-binding domain (PD-1-binding domain) that immunospecifically binds to an epitope on PD-1.

[0113] Antibody-based molecules that immunospecifically bind to human PD-L1 (e.g., monospecific PD-L1-binding molecules or PD-L1 x LAG-3 bispecific molecules) that can be used in accordance with the present invention will comprise at least one epitope-binding domain (PD-L1-binding domain) that immunospecifically binds to an epitope on PD-L1.

[0114] Antibody-based molecules that immunospecifically bind to human LAG-3 (e.g., monospecific LAG-3 binding molecules or PD-1 x LAG-3 (or PD-L1 x LAG-3) bispecific molecules) that can be used in accordance with the present invention will comprise at least one epitope-binding domain (LAG-3 binding domain) that immunospecifically binds to an epitope on LAG-3.

[0115] In specific embodiments, the present invention contemplates antibody-based molecules comprising a PD-1-binding domain, a PD-L1-binding domain, and / or a LAG-3-binding domain, which further comprise an Fc domain. In one embodiment, the Fc domain of such molecules is a wild-type IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0116] The present invention contemplates monospecific antibody-based molecules that contain a PD-1-binding domain, a PD-L1-binding domain, or a LAG-3-binding domain, and that contain a variant Fc domain that has little or no ADCC activity. The present invention also contemplates monospecific antibody-based molecules that contain epitopes that are immunospecific for PD-1 and LAG-3, or epitopes that are immunospecific for PD-L1 and LAG-3. Bispecific antibody-based molecules (e.g., diabodies) are contemplated that comprise binding domains that contain an Fc domain that has little or no ADCC activity. In one embodiment, such molecules comprise a mutated IgG1 Fc domain comprising a substitution at position 234 with alanine and a substitution at position 235 with alanine (234A, 235A) according to the EU index of Kabat numbering. In another embodiment, such molecules comprise an IgG4 Fc domain and optionally a stabilized IgG4 hinge region (see, e.g., SEQ ID NO: 11).

[0117] In certain embodiments, antibody-based molecules comprising a PD-1-binding domain, a PD-L1-binding domain, and / or a LAG-3-binding domain comprise a variant Fc domain comprising one or more mutations that extend serum half-life. In one embodiment, such molecules comprise a variant Fc domain comprising a substitution at position 252 with tyrosine, a substitution at position 254 with threonine, and a substitution at position 256 with glutamic acid (252Y, 254T, and 256E) according to the EU index numbering of Kabat.

[0118] The present invention also encompasses antibody-based molecules comprising a PD-1-binding domain, a PD-L1-binding domain, and / or a LAG-3-binding domain, which further comprise an Fc domain, wherein the Fc domain comprises: (a) one or more mutations that reduce or eliminate ADCC; and / or (b) one or more mutations that extend serum half-life Includes.

[0119] In one embodiment, the antibody-based molecule comprises a PD-1-binding domain, a PD-L1-binding domain, and / or a LAG-3-binding domain, which comprise a variant IgG1 Fc domain comprising the substitutions L234A / L235A / M252Y / S254T / T256E (SEQ ID NO: 19), numbered according to the EU index as per Kabat.

[0120] In another embodiment, the antibody-based molecule comprises a PD-1-binding domain, a PD-L1-binding domain, and / or a LAG-3-binding domain, which comprise a mutant IgG4 Fc domain comprising the substitutions M252Y / S254T / T256E (SEQ ID NO: 20), numbered according to the EU index as per Kabat.

[0121] A. PD-1 Binding Domains and Molecules In one embodiment, the PD-1 binding domain comprises the CDRs of the VL and VH domains of SEQ ID NO: 35 and SEQ ID NO: 39. In another embodiment, the PD-1 binding domain comprises the humanized VL and VH domains of SEQ ID NO: 36 and SEQ ID NO: 39.

[0122] Such humanized VL PD‐1 The amino acid sequence of the domain is (SEQ ID NO: 35): EIVLTQSPAT LSLSPGERAT LSC RASESVD NYGMSFMN WF QQKPGQPPKL LIH AASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FC QQSKEVPY T FGGGTKVEI K is.

[0123] Such a VL PD‐1 The CDR is: CDR L 1 SEQ ID NO:36:RASESVDNYGMSFMN; CDR L 2 SEQ ID NO: 37:AASNQGS; and CDR L3 SEQ ID NO: 38: QQSKEVPYT is.

[0124] Such humanized VH PD‐1 The amino acid sequence of the domain is (SEQ ID NO: 39): QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMN WVRQA PGQGLEWIG V IHPSDSETWL DQKFKD RVTI TVDKSTSTAY MELSSLRSED TAVYYCAR EH YGTSPFAY WG QGTLVTVSS is.

[0125] Such a VH PD‐1 The CDR for the domain is: CDR H 1 SEQ ID NO:40:SYWMN; CDR H 2 SEQ ID NO: 41: VIHPSDSETWLDQKFKD; and CDR H 3 SEQ ID NO: 42: EHYGTSPFAY is.

[0126] Alternative PD-1 binding domains, and molecules containing same, have been described previously and include, but are not limited to, those presented in Table 1. These may be referred to herein by their generic names or by the INN designation.

[0127] [Table 1] JPEG2026001028000003.jpg236155 JPEG2026001028000004.jpg53153

[0128] It is specifically contemplated that the PD-1-binding molecules provided herein can be used directly in the methods of the invention, or that the sequences or polypeptide chains can be employed in the construction of alternative PD-1-binding molecules or PD-1 x LAG-3 bispecific molecules.

[0129] B. PD-L1 Binding Domains and Molecules In one embodiment, the PD-L1 binding domain comprises the CDRs of the VL and VH domains of SEQ ID NO: 43 and SEQ ID NO: 47. In another embodiment, the PD-L1 binding domain comprises the humanized VL and VH domains of SEQ ID NO: 43 and SEQ ID NO: 47.

[0130] Such humanized VL PD‐L1 The amino acid sequence of the domain is (SEQ ID NO: 43): DIQMTQSPSS LSASVGDRVT ITC KASQDVN TAVA WYQQKP GKAPKLLIY W ASTRHT GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ HYNTPLT FGQ GTKVEIK is.

[0131] Such a VL PD‐L1 The CDR is: CDR L 1 SEQ ID NO: 44: KASQDVNTAVA; CDR L 2 SEQ ID NO: 45: WASTRHT; and CDR L 3 SEQ ID NO: 46: QQHYNTPLT is.

[0132] Such a VH PD‐L1 The amino acid sequence of the humanized domain is (SEQ ID NO:47): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMS WVRQA PGKGLEWVA Y ISIGGGTTYY PDTVKGRFTI SRDNAKNTLY LQMNSLKTED TAVYYCAR QG LPYYFDY WGQ GTLVTVSS is.

[0133] Such a VH PD‐L1 The CDR is: CDR H 1 SEQ ID NO: 48: SYTM; CDR H 2 SEQ ID NO: 49: YISIGGGTTYYPDTVK; ​​and CDR H 3 SEQ ID NO: 50: QGLPYYFDY is.

[0134] Alternative PD-L1 binding domains, and molecules containing same, have been described previously and include, but are not limited to, those listed in Table 2. These may be referred to herein by their generic names or by the INN designation.

[0135] [Table 2] JPEG2026001028000006.jpg236159 JPEG2026001028000007.jpg233157

[0136] It is specifically contemplated that the PD-L1 binding molecules provided herein can be used directly in the methods of the invention, or that the sequences or polypeptide chains can be employed in the construction of alternative PD-L1 binding molecules or PD-L1 x LAG-3 bispecific molecules.

[0137] C. LAG-3 binding domains and molecules In one embodiment, the LAG-3 binding domain comprises the CDRs of the VL and VH domains of SEQ ID NO: 51 and SEQ ID NO: 55. In another embodiment, the LAG-3 binding domain comprises the humanized VL and VH domains of SEQ ID NO: 51 and SEQ ID NO: 55.

[0138] Such humanized VL LAG‐3 The amino acid sequence of the domain is (SEQ ID NO:51): DIQMTQSPSS LSASVGDRVT ITC RASQDVS SVVA WYQQKP GKAPKLLIY S ASYRYT GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ HYSTPWT FGG GTKLEIK is.

[0139] Such a VL LAG‐3 The CDR for the domain is: CDR L 1 SEQ ID NO:52: RASQDVSSVVA; CDR L 2 SEQ ID NO: 53: SASYRYT; and CDR L 3 SEQ ID NO: 54: QQHYSTPWT Includes.

[0140] Such humanized VH LAG‐3 The amino acid sequence of the domain is (SEQ ID NO: 55): QVQLVQSGAE VKKPGASVKV SCKASGYTFT DYNMD WVRQA PGQGLEWMG D INPDNGVTIY NQKFEG RVTM TTDTSTSTAY MELRSLRSDD TAVYYCAR EA DYFYFDY WGQ GTTLTVSS is.

[0141] Such a VH LAG‐3 The CDR for the domain is: CDR H 1 SEQ ID NO:56:DYNMD; CDR H2 SEQ ID NO: 57: DINPDNGVTIYNQKFEG; and CDR H 3 SEQ ID NO: 58: EADYFYFDY Includes.

[0142] Alternative LAG-3 binding domains, and molecules containing same, have been previously described and include, but are not limited to, those presented in Table 3. These may be referred to herein by their common names or by the INN designation.

[0143] [Table 3]

[0144] It is specifically contemplated that the LAG-3 binding molecules provided herein can be used directly in the methods of the invention, or that the sequences or polypeptide chains can be employed in the construction of alternative LAG-3 binding molecules or PD-1 x LAG-3 (or PD-L1 x LAG-3) bispecific molecules.

[0145] D. PD-1 x LAG-3 (or PD-L1 x LAG-3) Bispecific Molecules Antibody-based molecules that immunospecifically bind to both human PD-1 (or PD-L1) and human LAG-3 (i.e., PD-1 x LAG-3 bispecific molecules or PD-L1 x LAG-3 bispecific molecules) that can be used in accordance with the present invention will comprise at least one epitope-binding domain that immunospecifically binds to an epitope on PD-1 (or PD-L1) and at least one epitope-binding domain that immunospecifically binds to an epitope on LAG-3.

[0146] In certain embodiments, the PD-1 x LAG-3 bispecific molecules of the invention: (I) PD-1 specific CDR L 1. CDR L 2, and CDR L 3 containing the VL domain (V L PD‐1 ) and PD-1 specific CDR H1. CDR H 2, and CDR H The VH domain (VH PD‐1 a PD-1 binding domain comprising: (II) LAG-3 specific CDR L 1. CDR L 2, and CDR L VL containing 3 domains Domain (VL LAG‐3 ) and LAG-3 specific CDR H 1. CDR H 2, and CDR H 3D VH domain (VH LAG‐3 ) and a LAG-3 binding domain wherein the PD-1 binding domain and the LAG-3 binding domain are selected from those provided in Tables 1 and 3.

[0147] In another embodiment, the PD-L1 x LAG-3 bispecific molecule of the invention: (I) PD-L1-specific CDR L 1. CDR L 2, and CDR L VL domain containing 3 ( VL PD‐L1 ) and PD-L1-specific CDR H 1. CDR H 2, and CDR H Contains 3 domains VH domain (VH PD‐L1 a PD-L1 binding domain comprising: (II) LAG-3 specific CDR L 1. CDR L 2, and CDR L VL containing 3 domains Domain (VL LAG‐3 ) and LAG-3 specific CDR H 1. CDR H 2, and CDR H 3D VH domain (VH LAG‐3 ) and a LAG-3 binding domain wherein the PD-L1 binding domain and the LAG-3 binding domain are as shown in Tables 2 and 3. Select from what is offered.

[0148] One embodiment of the present invention relates to a PD-1 x LAG-3 (or PD-L1 x LAG-3) bispecific molecule comprising an Fc domain. In one embodiment, the PD-1 x LAG-3 (or PD-L1 x LAG-3) bispecific molecule comprises an Fc domain that has little or no ADCC activity. In one embodiment, the PD-1 x LAG-3 (or PD-L1 x LAG-3) bispecific molecule comprises an Fc domain that has little or no ADCC activity and comprises one or more mutations that extend serum half-life.

[0149] In certain embodiments, the PD-1 x LAG-3 bispecific molecule of the invention is a PD-1 x LAG-3 bispecific diabody, preferably a four-chain Fc domain-containing diabody, having two binding sites specific for PD-1, two binding sites specific for LAG-3, an Fc domain, and a cysteine-containing E / K-coil heterodimer-promoting domain. The overall structure of a representative PD-1 x LAG-3 bispecific diabody is provided in Figure 1. Such a molecule comprises the VL and VH domains (VL and VH, respectively) of an antibody that binds to PD-1. PD‐1 and VH PD‐1 ) and the VL and VH domains of an antibody that binds to LAG-3 (VL LAG‐3 and VH LAG‐3 ) and the PD-1 x LAG-3 bispecific diabody can thus specifically bind to an epitope on PD-1 and an epitope on LAG-3.

[0150] 1. DART-I "DART-I" (also known as "MGD013" and tebotelimab) is an exemplary PD-1 x LAG-3 bispecific molecule of the present invention. DART-I is a bispecific, four-chain, Fc domain-containing diabody containing two binding sites specific for PD-1, two binding sites specific for LAG-3, a variant IgG4 Fc domain engineered to extend half-life, and a cysteine-containing E / K-coil heterodimer-promoting domain. DART-I comprises four polypeptide chains having the amino acid sequences summarized in Table 4. These amino acid sequences are described in further detail below.

[0151] [Table 4]

[0152] The first and third polypeptide chains of DART-I are composed of, in the N-terminal to C-terminal direction: N-terminus; a VL domain (VL) of a monoclonal antibody capable of binding to LAG-3; LAG‐3 ) (SEQ ID NO: 51); intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 21)); PD-1 The VH domain (VH) of a monoclonal antibody capable of binding to PD‐1 ) (SEQ ID NO: 39); cis a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 22)); a cysteine-containing heterodimer-promoting (E-coil) domain (EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 25)); an intervening linker peptide (Linker 3) containing a stabilized IgG4 hinge region (SEQ ID NO: 11); a mutant IgG4 CH2-CH3 domain containing substitutions M252Y / S254T / T256E and lacking the C-terminal residue (SEQ ID NO: 20); and a C-terminus.

[0153] The amino acid sequences of the first and third polypeptide chains of DART-I are (SEQ ID NO: 59): DIQMTQSPSS LSASVGDRVT ITCRASQDVS SVVAWYQQKP GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYSTPWTFGG GTKLEIKGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYSFTSYWMN WVRQAPGQGL EWIGVIHPSD SETWLDQKFK DRVTITVDKS TSTAYMELSS LRSEDTAVYY CAREHYGTSP FAYWGQGTLV TVSSGGCGGG EVAACEKEVA ALEKEVAALE KEVAALEKES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG is.

[0154] The second and fourth polypeptide chains of DART-I are composed of, in the N-terminal to C-terminal direction: an N-terminus; a VL domain of a monoclonal antibody capable of binding to PD-1 (VL PD‐1 )(sequence number 35); an intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 21)); a VH domain of a monoclonal antibody capable of binding to LAG-3 (VH LAG‐3 ) (SEQ ID NO: 55); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 22)); a cysteine-containing heterodimer-promoting (K-coil) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 26)); and a C-terminus.

[0155] The amino acid sequence of the second and fourth polypeptide chains of DART-I is (SEQ ID NO: 60): EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTD YNMDWVRQAP GQGLEWMGDI NPDNGVTIYN QKFEGRVTMT TDTSTSTAYM ELRSLRSDDT AVYYCAREAD YFYFDYWGQG TTLTVSSGGC GGGKVAACKE KVAALKEKVA ALKEKVAALK E is.

[0156] Variants of DART-I can be readily generated by incorporating alternative VH / VL domains, intervening linkers, Fc domains, and / or by introducing one or more amino acid substitutions, additions, or deletions. For example, a mutant IgG1 Fc domain engineered to reduce / eliminate FcγR binding and / or ADCC activity and extend half-life is readily generated by incorporating CH2 and CH3 domains containing the substitutions L234A / L235A / M252Y / S254T / T256E (SEQ ID NO: 19) in place of SEQ ID NO: 20. Such a mutant linker 3 may comprise an IgG1 hinge (SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 34). Additional linkers and PD-1 x LAG-3 bispecific diabodies that can be used in the methods of the invention are disclosed in U.S. Patent No. 5,629,999; and U.S. Patent No. 5,629,999 (see in particular "DART-A," "DART-B," "DART-C," "DART-D," "DART-E," "DART-F," and "DART-G"; the sequences of which are set forth in Table 14).

[0157] 2. Additional PD-1 x LAG-3 (or PD-L1 x LAG-3) Bispecific Molecules Other PD-1 x LAG3 bispecific molecules that can be used in the methods of the invention have been described previously and include, but are not limited to, those presented in Table 5, and are further described below.

[0158] [Table 5]

[0159] PD-1 x LAG3 bispecific antibody-lipocalin mutein fusion proteins are described in Patent Documents 53 and 56. Examples of such antibody-lipocalin mutein fusion proteins include anti-PD-1 antibodies with a lipocalin mutein engineered to bind to LAG-3 genetically fused to the C-terminus of the heavy chain.

[0160] PD-1 x LAG-3 bispecific antibody-domain antibody (antibody-dAb) fusion proteins are described in Patent Document 54. Examples of such antibody-dAb fusion proteins include anti-LAG-3 antibodies with an anti-PD-1 dAb genetically fused to the C-terminus of the heavy chain. PD-1 x LAG-3 bispecific antibodies containing CH1 / Ck domain swaps (alone or in combination with VH / VL swaps) and / or charged amino acid substitutions at the CH1 / CL interface are described in Patent Document 55. Examples of such bispecific antibodies include: four-polypeptide chain antibodies with one PD-1-binding domain and one LAG-3-binding domain (1+1 antibody), including crossFab (with VH / VL domain swaps); and two Fab domains with mutations in CH1 / CK and the C-terminus of each heavy chain. and two crossFab domains fused together at the 2′ end, resulting in a three-polypeptide chain antibody with three distinct polypeptide chains, two PD-1-binding domains and two LAG-3-binding domains (2+2 antibodies).

[0161] PD-1 x LAG-3 bispecific antibodies having a three-polypeptide chain Fab x scFvFc structure or a two-polypeptide chain scFvFc x scFvFc structure are described in WO 2018 / 217944 and Patent Document 57. Examples of such bispecific antibodies include anti-PD1 scFvFc paired with anti-LAG3 scFvFc hole, and anti-PD1 scFvFc (heavy chain + light chain) paired with anti-LAG3 half IgG.

[0162] It is specifically contemplated that the PD-1 x LAG-3 bispecific molecules and PD-L1 x LAG-3 bispecific molecules provided herein can be used directly in the methods of the invention. Alternative PD-1 x LAG-3 bispecific molecules and PD-L1 x LAG-3 bispecific molecules can be generated that comprise all six CDRs (or VL and VH domains) of any of the PD-1, PD-L1, and LAG-3 binding molecules provided herein (see, e.g., SEQ ID NOS: 35-58, and Tables 1-5).

[0163] III. Antibody-based molecules that bind to TA Antibody-based molecules that immunospecifically bind to a tumor antigen (TA) (i.e., a TA-binding molecule) that can be used in accordance with the present invention will comprise at least one epitope-binding domain (TA-binding domain) that is capable of immunospecifically binding to an epitope of such a TA.

[0164] In certain embodiments, the present invention contemplates antibody-based molecules comprising a TA-binding domain that further comprises an Fc domain. In one embodiment, the Fc domain of the TA-binding molecule is a wild-type IgG1, IgG2, IgG3, or IgG4 Fc domain. In another embodiment, the Fc domain of the TA molecule is an ADCC-enhancing Fc domain.

[0165] The present invention also provides a TA-binding molecule comprising an Fc domain, wherein said Fc domain comprises: (a) one or more mutations and / or modifications that enhance ADCC; and / or (b) one or more mutations that extend serum half-life The present invention also encompasses TA-binding molecules comprising:

[0166] In one embodiment, the TA-binding molecule comprises an FcMT1 ADCC-enhancing Fc domain (SEQ ID NO: 16), an FcMT2 ADCC-enhancing Fc domain (SEQ ID NO: 17), or an FcMT3 ADCC-enhancing Fc domain (SEQ ID NO: 18).

[0167] A. Tumor Antigens The present invention specifically contemplates TA-binding molecules and compositions and methods comprising or employing: (1) PD-1×LAG-3 bispecific molecule; (2) monospecific PD-1 binding molecules and monospecific LAG-3 binding molecules; (3) a PD-L1 x LAG-3 bispecific molecule; or (4) Monospecific PD-L1-binding molecules and monospecific LAG-3-binding molecules wherein said monospecific binding molecule is an intact antibody and said bispecific molecule is a diabody or a bispecific antibody. In certain embodiments, said TA binding molecule comprises an ADCC-enhancing Fc domain.

[0168] Tumor antigens to which the above-described TA-binding molecules can bind include, but are not limited to, those presented in Tables 6A-6B, which may be referred to herein by their common names, abbreviations, and / or gene names.

[0169] [Table 6] JPEG2026001028000012.jpg237162 JPEG2026001028000013.jpg208160

[0170] [Table 7]

[0171] B. TA-binding domains and molecules Numerous TA-binding molecules are known or can be produced using known methods, including those described herein. TA-binding molecules can be monospecific or bispecific. Representative TA-binding molecules that contain a TA-binding domain and therefore whose sequences or polypeptide chains can be employed in constructing or used as TA-binding molecules of the invention (e.g., ADCC-enhancing TA-binding molecules) are listed in Table 7. The CDRs, VH, and VL domains of several TA-binding molecules are provided below.

[0172] [Table 8] JPEG2026001028000016.jpg236163 JPEG2026001028000017.jpg235164 JPEG2026001028000018.jpg236164 JPEG2026001028000019.jpg234164 JPEG2026001028000020.jpg85165

[0173] In one embodiment, the invention relates to a TA-binding molecule comprising the CDR domains (or VL and VH domains) of any of the TA-binding molecules listed in Table 7. In a further embodiment, the invention uses any of the TA-binding molecules listed in Table 7 or provided below. In an alternative embodiment, the invention relates to an ADCC-enhanced TA-binding molecule comprising the CDR domains (or VL and VH domains) of any of the antibodies listed in Table 7. Specific examples of ADCC-enhanced TA-binding molecules are provided below.

[0174] In certain embodiments, the TA-binding molecule binds to a HER2 TA ("HER2-binding molecule"). In one embodiment, the HER2-binding molecule of the present invention is an anti-HER2 antibody. Antibodies that bind to human HER2 include "margetuximab," "trastuzumab," and "pertuzumab." Margetuximab (also known as mgAH22; CAS Registry Number 1350624-75-7, KEGG D10446; see, e.g., U.S. Patent No. 8,802,093) is an Fc-optimized monoclonal antibody that binds to HER2 and mediates enhanced ADCC activity. The sequence of margetuximab is provided below. Trastuzumab (also known as rhuMAB4D5, commercially available as Herceptin®; CAS Registry Number 180288-69-1; see U.S. Patent No. 5,821,337) is a humanized antibody with an IgG1 / κ constant region. The amino acid sequence of trastuzumab, for trastuzumab emtansine, can be found in WHO Drug Information, 2011, Recommended INN: List 65, 25(1):89-90. Pertuzumab (also known as rhuMAB2C4, commercially available as Perjeta™; CAS Registry Number 380610-27-5; see, e.g., WO 2001 / 000245) is another humanized antibody with an IgG1 / κ constant region. The amino acid sequence of the Fab domain of pertuzumab can be found in Protein Data Bank Accession No. 117i. Antibody "8H11" is a murine anti-HER2 monoclonal antibody that binds to an epitope on HER2 that is distinct from the epitopes recognized by margetuximab, trastuzumab, and pertuzumab (WO 2001 / 036005). A humanized variant of antibody 8H11 (referred to as "hHER2 MAB-1") has been previously described (see, e.g., WO 2018 / 156740), and representative humanized VH and VL domains are provided below.In addition to the HER2 binding molecules identified above, the present invention provides the following HER2 binding molecules: 1.44.1; 1.140; 1.43; 1.14.1; 1.100.1; 1.96; 1.18.1; 1.20; 1.39; 1.24; and 1.71.3 (disclosed in U.S. Pat. No. 8,350,011; U.S. Pat. No. 8,858,942; and WO 2008 / 019290); F5 and. C1 (disclosed in U.S. Patent Nos. 7,892,554; 8,173,424; 8,974,792; and WO 99 / 55367); and any of the HER2 binding molecules of U.S. Patent Publication Nos. 2011 / 0097323, 2013 / 017114, 2014 / 0328836, 2016 / 0130360, and 2016 / 0257761, and WO 2011 / 147986 are contemplated.

[0175] In certain embodiments, the TA-binding molecule binds to a B7-H3 TA ("B7-H3 binding molecule"). In one embodiment, the B7-H3 binding molecule of the present invention is an anti-B7-H3 antibody. Antibodies that bind to human B7-H3 include "enoblitutuzumab," "omburtamab," and "mirzotamab." Enoblituzumab (also known as mgAH22; CAS Registry Number 1350624-75-7, KEGG D11752, see, e.g., U.S. Patent No. 8,802,093) is an Fc-optimized monoclonal antibody that binds to HER2 and mediates enhanced ADCC activity. The sequence of margetuximab is provided below. Omburtamab (also known as 8H9; CAS Registry Number 1895083-75-6, see, e.g., U.S. Patent No. 7,737,258) is a murine monoclonal antibody. The amino acid sequence of omburtamab can be found in WHO Drug Information 2018, Proposed INN: List 119, 32(2):339-340. A humanized version of 8H9 is described in WO 2016 / 033 225. Mirzotamab cleztoclax (also known as ABBV-155; CAS Registry Number 2229859-12-3, see e.g., WO 2017 / 214322) is a humanized antibody with an IgG1 / κ constant region. The amino acid sequence of mirzotamab can be found in WHO Drug Information 2019, Proposed INN: List 121, 33(2): 294-6. In addition to the B7-H3 binding molecules identified above, the present invention contemplates the use of any of the following B7-H3 binding molecules: BRCA84D, BRCA69D, and PRCA157 (disclosed in WO 2011109400); L7, L8, L11, M30, and M31 (disclosed in U.S. Patent Publication No. 2013 / 0078234), hmAb-C, and the B7-H3 antibody hmAb-D (disclosed in WO 2017 / 180813).

[0176] C.ADCC-enhancing TA-binding molecule 1. Margetuximab The present invention specifically contemplates compositions and methods comprising or employing margetuximab and: (1) PD-1×LAG-3 bispecific molecule; (2) monospecific PD-1 binding molecules and monospecific LAG-3 binding molecules; (3) a PD-L1 x LAG-3 bispecific molecule; or (4) Monospecific PD-L1-binding molecules and monospecific LAG-3-binding molecules wherein said monospecific binding molecule is an intact antibody and said bispecific molecule is a diabody or a bispecific antibody.

[0177] Margetuximab contains a mutated human Fc domain that exhibits improved affinity for the CD16A receptor. The light chain of the antibody (IgGκ) has been modified to remove an N-linked glycosylation site (N65S; double underlined below).

[0178] The VL domain of margetuximab has the amino acid sequence of SEQ ID NO: 61: DIVMTQSHKF MSTSVGDRVS ITC KASQDVN TAVA WYQQKP GHSPKLLIY S ASFRYT GVPD RFTG S RSGTD FTFTISSVQA EDLAVYYC QQ HYTTPPT FGG GTKVEIK It has.

[0179] The CDR domains of the VL domain of margetuximab are: CDR L 1 SEQ ID NO: 62: KASQDVNTAVA CDR L 2 SEQ ID NO: 63: SASFRYT, and CDR L 3 SEQ ID NO: 64: QQHYTTPPT is.

[0180] The light chain of margetuximab has the amino acid sequence of SEQ ID NO: 65: DIVMTQSHKF MSTSVGDRVS ITCKASQDVN TAVAWYQQKP GHSPKLLIYS ASFRYTGVPD RFTG S RSGTD FTFTISSVQA EDLAVYYCQQ HYTTPPTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC It has.

[0181] The VH domain of margetuximab has the amino acid sequence of SEQ ID NO: 66: QVQLQQSGPE LVKPGASLKL SCTASGFNIK DTYIH WVKQR PEQGLEWIG R IYPTNGYTRY DPKFQD KATI TADTSSNTAY LQVSRLTSED TAVYYCSR WG GDGFYAMDY W GQGASVTVSS It has.

[0182] The CDR domains of the VH domain of margetuximab are: CDR H 1 SEQ ID NO: 67:DTYIH CDR H 2 SEQ ID NO: 68: RIYPTNGYTRYDPKFQD, and CDR H 3 SEQ ID NO: 69: WGGDGFYAMDY is.

[0183] The heavy chain of margetuximab comprises the FcMT2 ADCC-enhancing Fc domain (containing the L235V, F243L, R292P, Y300L, and P396L substitutions (underlined)), which has the amino acid sequence of SEQ ID NO: 70: QVQLQQSGPE LVKPGASLKL SCTASGFNIK DTYIHWVKQR PEQGLEWIGR IYPTNGYTRY DPKFQDKATI TADTSSNTAY LQVSRLTSED TAVYYCSRWG GDGFYAMDYW GQGASVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPEL V GG PSVFL L PPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKP P EEQYN ST LRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTP L V LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK It has.

[0184] A variant of the heavy chain of margetuximab comprises an FcMT1 ADCC-enhancing Fc domain (containing F243L, R292P, Y300L, V305I, and P396L substitutions (see SEQ ID NO: 16)). Another variant of the heavy chain of margetuximab comprises an FcMT3 ADCC-enhancing Fc domain (containing F243L, R292P, and Y300L substitutions (see SEQ ID NO: 18)).

[0185] 2. Enobrituzumab The present invention specifically contemplates compositions and methods comprising or employing enoblituzumab and: (1) PD-1×LAG-3 bispecific molecule; (2) monospecific PD-1 binding molecules and monospecific LAG-3 binding molecules; (3) a PD-L1 x LAG-3 bispecific molecule; or (4) Monospecific PD-L1-binding molecules and monospecific LAG-3-binding molecules wherein said monospecific binding molecule is an intact antibody and said bispecific molecule is a diabody or a bispecific antibody.

[0186] The VL domain of enoblituzumab has the amino acid sequence of SEQ ID NO: 71: DIQLTQSPSF LSASVGDRVT ITC KASQNVD TNVA WYQQKP GKAPKALIY S ASYRYS GVPS RFSG S GSGTD FTLTISSLQP EDFATYYC QQ YNNYPFT FGQ GTKLEIK It has.

[0187] The CDR domains of the VL domain of enoblituzumab are: CDR L 1 SEQ ID NO: 72: KASQNVDTNVA CDR L 2 SEQ ID NO: 73: SASYRYS, and CDR L 3 SEQ ID NO: 74: QQYNNYPFT is.

[0188] The light chain of enoblituzumab has the amino acid sequence of SEQ ID NO: 75: DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC It has.

[0189] The VH domain of enoblituzumab has the amino acid sequence of SEQ ID NO: 76: EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMH WVRQA PGKGLEWVA Y ISSDSSAIYY ADTVKG RFTI SRDNAKNSLY LQMNSLRDED TAVYYCGR GR ENIYYGSRLD Y WGQGTTVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV It has.

[0190] The CDR domains of the VH domain of enoblituzumab are: CDR H 1 SEQ ID NO: 77: SFGMH CDR H 2 SEQ ID NO: 78: YISSDSSAIYYADTVKG, and CDR H 3 SEQ ID NO: 79: GRENIYYGSRLDY is.

[0191] The heavy chain of enoblituzumab comprises the FcMT2 ADCC-enhancing Fc domain (containing the L235V, F243L, R292P, Y300L, and P396L substitutions (underlined)), which has the amino acid sequence of SEQ ID NO: 80: EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCGRGR ENIYYGSRLD YWGQGTTVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPEL V GGPSVFL L PP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKP P EEQ YNST L RVVSV LTVLHQDWLN GKEYKCKVSN KALAPIEKT ISKAKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP L VLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP Goalkeeper It has.

[0192] A variant of the heavy chain of enoblituzumab contains an FcMT1 ADCC-enhancing Fc domain (containing F243L, R292P, Y300L, V305I, and P396L substitutions (see SEQ ID NO: 16)). Another variant of the heavy chain of enoblituzumab contains an FcMT3 ADCC-enhancing Fc domain (containing F243L, R292P, and Y300L substitutions (see SEQ ID NO: 18)).

[0193] 3. Other ADCC-enhancing Fc TA-binding molecules The present invention specifically contemplates compositions and methods comprising or employing ADCC-enhancing TA-binding molecules and: (1) PD-1×LAG-3 bispecific molecule; (2) monospecific PD-1 binding molecules and monospecific LAG-3 binding molecules; (3) a PD-L1 x LAG-3 bispecific molecule; or (4) Monospecific PD-L1-binding molecules and monospecific LAG-3-binding molecules wherein said monospecific binding molecule is an intact antibody and said bispecific molecule is a diabody or a bispecific antibody.

[0194] In one embodiment, the present invention relates to an ADCC-enhancing TA-binding molecule comprising a TA-binding domain that immunospecifically binds to any of the TAs listed in Tables 6A-6B.

[0195] In one embodiment, the invention relates to an ADCC-enhancing TA-binding molecule comprising the CDR domains (or VL and VH domains) of any of the antibodies listed in Table 7. Such molecules may comprise an enhanced ADCC-enhancing Fc domain provided herein or known.

[0196] The present invention specifically contemplates compositions and methods that comprise or employ other TA-binding molecules that contain an enhanced ADCC-enhancing Fc domain, including, but not limited to, the anti-CD20 antibodies obinutuzumab (KEGG D0932; Marcus, R. et al. (2017) "Obinutuzumab for the First-Line Treatment of Follicular Lymphoma," N. Engl. J. Med. 377(14):1331-1344) and BAT4306F (Yu, J.-C. et al. (2018) "Abstract 3823:Bat4306f, An Anti-CD20 Antibody Devoid Of Fucose Modification, Demonstrates Enhanced ADCC Effect And Potent In Vivo Efficacy," Cancer Res. 78:(13 Supplement):3823); the EGFR-cMET bispecific antibody amivantamab (KEGG D11894;Yun, et al. (2020) “Antitumor Activity of Amivantamab (JNJ-61186372), an EGFR-MET Bispecific Antibody, in Diverse Models of EGFR Exon 20 Insertion-Driven NSCLC” Cancer Discovery DOI:10.1158 / 2159-8290.CD-20-0116); and anti-CD The CD19 antibodies tafasitamab (MOR208) (KEGG D11601; Kellner, C. et al. (2013) “The Fc-Engineered CD19 Antibody MOR208 (Xmab5574) Induces Natural Killer Cell-Mediated Lysis of Acute Lymphoblastic Leukemia Cells from Pediatric and Adult Patients,” Leukemia 27(7):1595-1598) and obexelimab (KEGG D11496).

[0197] IV. Manufacturing method The antibody-based molecules of the present invention can be produced recombinantly and expressed using any method known in the art for producing recombinant proteins. For example, nucleic acids encoding the polypeptide chains of such binding molecules can be constructed, introduced into an expression vector, and expressed in a suitable host cell. The binding molecules can be expressed in bacterial cells (e.g., E. coli). oli cells) or eukaryotic cells (e.g. CHO, 293E, COS, NS0 cells). Additionally, the binding molecules can be expressed in yeast cells such as Pichia or Saccharomyces.

[0198] To produce an antibody-based molecule of the invention, one or more polynucleotides encoding the molecule may be constructed, introduced into an expression vector, and expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture the host cells, and recover the molecule (see, e.g., Green, MR et al., (2012), Molecular Cloning, A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al. eds., (1998), Current (Protocols in Molecular Biology, John Wiley & Sons, NY). One or more expression vectors must have characteristics that allow the vector to replicate in a host cell. The vector must also have promoter and signal sequences necessary for expression in the host cell. Such sequences are known in the art. In addition to one or more nucleic acid sequences encoding such binding molecules, recombinant expression vectors may contain additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. Suitable methods that can be used to recombinantly express the binding molecules in plants (e.g., tobacco) or transgenic animals have been previously disclosed (see, e.g., Peeters et al. (2001) "Production of Antibodies and Antibody Fragments in Plants," Vaccine 19:2756; U.S. Patent No. 5,849,992; and Pollock et al. (1999) "Transgenic Milk As A Method For The Production of Recombinant Antibodies," J. Immunol Methods 231:147-157).

[0199] After recombinant expression of the antibody-based molecules of the present invention, they may be purified from inside or outside the host cell (e.g., from the culture medium) by any method known in the art for purifying polypeptides or polyproteins. Isolation and purification methods commonly used for antibody purification (e.g., antigen selectivity-based antibody purification schemes) may be used to isolate and purify the molecules, and are not limited to any particular method, such as column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, ion exchange chromatography, affinity chromatography (optionally after Protein A selection, where the antibody-based molecule contains an Fc region or a Protein A-binding portion thereof), particularly by affinity for a particular antigen, sizing column chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography (Marshak et al. (1996) Strategies for Protein Purification and Characterization: A Laboratory Course Manual. (Eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0200] V. Pharmaceutical Compositions The antibody-based molecules of the invention, e.g., antibodies that bind to a TA (optionally comprising an ADCC-enhancing Fc domain), antibodies that bind to PD-1, antibodies that bind to PD-L1, antibodies that bind to LAG-3, PD-1xLAG-3 bispecific molecules, or PD-L1xLAG-3 bispecific molecules, can be formulated as compositions. Compositions of the invention include bulk drug compositions (e.g., non-pure or non-sterile compositions) that can be used to prepare pharmaceutical compositions suitable for administration to a subject (e.g., a human patient or other mammal) for the treatment of cancer or other diseases and conditions. Such pharmaceutical compositions include one or more antibody-based molecules (e.g., antibodies that bind to a TA (optionally comprising an ADCC-enhancing Fc domain), antibodies that bind to PD-1, antibodies that bind to PD-L1, antibodies that bind to LAG-3, PD-1xLAG-3 bispecific molecules, or PD-L1xLAG-3 bispecific molecules) and one or more pharmaceutically acceptable carriers, and may optionally include one or more additional therapeutic agents. Such pharmaceutical compositions can be, for example, formulated in a pharmaceutically acceptable carrier. It can be supplied as an aqueous solution, a lyophilized powder, or a water-free concentrate that is specifically adapted for reconstitution with, or to be reconstituted with, such a carrier.

[0201] As used herein, the term "pharmaceutically acceptable carrier" refers to a compound that is "acceptable carrier" refers to a substance suitable for administration to animals, more particularly humans. "The term 'pharmacopoeia' refers to a diluent, solvent, dispersion medium, antibacterial and antifungal agent, excipient, or vehicle approved by a federal or state regulatory agency, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal fats, vegetable oils, or synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. The compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0202] Generally, the components of the compositions of the invention are supplied separately or mixed in dosage forms, as lyophilized powders or water-free concentrates, or as aqueous solutions in airtight containers such as vials, ampoules, or sachets labeled with the amount of active agent. When the composition is to be administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection, saline, or other diluent can be provided so that the components can be mixed prior to administration.

[0203] VI. Medical Kits The present invention also provides pharmaceutical kits comprising one or more containers containing the pharmaceutical composition of the present invention and instructional materials (e.g., warnings, package inserts, instructions, etc.). Additionally, one or more other prophylactic or therapeutic agents useful for treating a disease can also be included in the pharmaceutical kit. The containers of such pharmaceutical kits can include one or more airtight vials, ampoules, sachets, etc., labeled with the amount of active agent contained therein. If the composition is to be administered by injection, the container can be an infusion bottle or bag containing a sterile pharmaceutical-grade solution (e.g., water, saline, buffer, etc.). If the composition is to be administered by injection, the pharmaceutical kit can contain an ampoule of sterile water for injection, saline, or other diluent to facilitate mixing of the components of the pharmaceutical kit for administration to a subject (e.g., a human patient or other mammal).

[0204] In one embodiment, the pharmaceutical composition of such a kit is supplied as a lyophilized sterile powder or water-free concentrate in an airtight container, which can be reconstituted to a concentration suitable for administration to a subject, e.g., with water, saline, or other diluents. In another embodiment, the pharmaceutical composition of such a kit is supplied as an aqueous solution in an airtight container, which can be diluted to a concentration suitable for administration to a subject, e.g., with water, saline, or other diluents. The kit can further include, in one or more containers, one or more other prophylactic and / or therapeutic agents that can be used to treat cancer; and / or the kit can further include one or more cytotoxic antibodies that bind to one or more cancer antigens associated with cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biotherapeutic or hormonal therapeutic agent.

[0205] The instructional material included in the pharmaceutical kits of the present invention can be, for example, in a content and format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, and can indicate approval by such agency of the manufacture, sale, or use of the pharmaceutical composition for administration to and / or treatment of humans. The instructional material can provide, for example, information regarding the dosage contained in the pharmaceutical composition, the mode in which it can be administered, etc.

[0206] Thus, for example, the instructional material included in the pharmaceutical kits of the present invention may instruct to administer a provided pharmaceutical composition in combination with additional agents, which may be provided in the same or separate pharmaceutical kits. Such instructional material may instruct to administer a provided pharmaceutical composition about once every two weeks, about once every three weeks, or more or less frequently. Such instructional material may instruct to reconstitute / diluted to comprise or administer at a flat dose of about 120 mg, about 300 mg, about 400 mg, about 420 mg, about 600 mg, about 800 mg, or about 840 mg, or more, or to reconstitute / diluted to administer at a body weight-based dose of about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 8 mg / kg, about 10 mg / kg, about 15 mg / kg, about 18 mg / kg, or more. Such instructional material may instruct that the provided pharmaceutical composition comprises, or is to be reconstituted / diluted to comprise, a single dose, or two or more doses (e.g., 2 doses, 4 doses, 6 doses, 12 doses, 24 doses, etc.). Such instructional material included in pharmaceutical kits can combine any set of such information (e.g., the instructional material may instruct that the provided PD-1 x LAG-3 bispecific molecule-containing pharmaceutical composition comprises, or is to be reconstituted / diluted to comprise, a dose of about 400 mg or about 600 mg, and that such a dose is to be administered about once every two weeks; the instructional material may instruct that the provided pharmaceutical composition comprises, or is to be reconstituted to comprise, a dose of about 600 mg or about 800 mg, and that such a dose is to be administered about once every three weeks; and / or the instructional material may instruct that the provided HER2- or B7-H3-binding molecule-containing pharmaceutical composition comprises, or is to be reconstituted to comprise, a dose of about 15 mg / kg, and that such a dose is to be administered about once every three weeks). Such instructional material may instruct as to the mode of administration of the included pharmaceutical composition, for example, that the pharmaceutical composition be administered by intravenous (IV) infusion.The instructional materials included in the pharmaceutical kit may instruct regarding the duration or timing of administration, for example, that the included composition be administered by intravenous (IV) infusion over a period of 30 to 240 minutes, over a period of 30 to 90 minutes, etc.

[0207] The instructional material included in the pharmaceutical kits of the invention may provide instructions regarding the appropriate or desired use of the included pharmaceutical composition, for example, to administer the pharmaceutical composition (e.g., a PD-1×LAG-3 bispecific molecule) for the treatment of cancer. In certain embodiments, the instructional material included in the pharmaceutical kit may provide instructions for administering one or more pharmaceutical compositions of a PD-1 (or PD-L1)-binding molecule and a LAG-3-binding molecule, or a PD-1×LAG-3 (or PD-L1×LAG-3) bispecific molecule of the invention in combination with a TA-binding molecule (optionally having an ADCC-enhancing Fc domain) for the treatment of a cancer in which a TA (e.g., HER2 or B7-H3) is expressed. Cancers that may be treated include, but are not limited to, adrenal gland cancer, AIDS-related cancer, alveolar soft tissue sarcoma, anal cancer (including squamous cell carcinoma of the anal canal (SCAC)), bladder cancer, bone cancer, brain and spinal cord cancer, breast cancer (e.g., HER2 + breast cancer, or triple-negative breast cancer (TNBC), carotid bulb tumor, cervical cancer (including HPV-associated cervical cancer), chondrosarcoma, chordoma, chromophobe clear cell renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, endometrial cancer (including unselected endometrial cancer, MSI-high endometrial cancer, dMMR endometrial cancer, and / or POLE exonuclease domain mutation-positive endometrial cancer), Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, gallbladder cancer or bile duct cancer (cholangiocarcinomabile duct cancer), gastric cancer, esophagogastric junction (GEJ) cancer, gestational villus Cancers of the head and neck (including squamous cell carcinoma of the head and neck (SCCHN)), hematologic malignancies, hepatocellular carcinoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (including acute myeloid leukemia), liposarcoma / malignant lipomatous tumor, liver cancer (including hepatocellular carcinoma (HCC)), phospholipids Cancer (including diffuse large B-cell lymphoma (DLBCL) and non-Hodgkin's lymphoma (NHL)), lung cancer (including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)), medulloblastoma, melanoma (including uveal melanoma), meningioma, Merkel cell carcinoma, mesothelioma (including mesothelial pharyngeal carcinoma), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pharyngeal cancer, pheochromocytoma, pituitary tumor, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), posterior uveal melanoma, renal metastasis, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumor of childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma, gastric cancer cancer), synovial sarcoma, testicular cancer, thymic cancer, thymoma, thyroid cancer, and uterine cancer.

[0208] VII. Uses of the Antibody-Based Molecules of the Invention As provided herein, the PD-1×LAG-3 bispecific molecules of the invention can be used to treat or prevent a variety of disorders, including cancer. Additionally, the PD-1-binding (or PD-L1-binding), LAG-3-binding, PD-1×LAG-3 (or PD-L1×LAG-3) bispecific molecules of the invention can be used in combination with a TA-binding molecule (optionally having an ADCC-enhancing Fc domain) to treat cancers in which such TAs are expressed.

[0209] Accordingly, the present invention provides a method of treating cancer, said method comprising the step of administering to a subject in need thereof a PD-1 x LAG-3 bispecific molecule.

[0210] Furthermore, the present invention relates to a TA binding molecule and: (1) PD-1×LAG-3 bispecific molecule; (2) monospecific PD-1 binding molecules and monospecific LAG-3 binding molecules; (3) a PD-L1 x LAG-3 bispecific molecule; or (4) Monospecific PD-L1-binding molecules and monospecific LAG-3-binding molecules and (c) administering to the cancer, wherein the monospecific binding molecule is an intact antibody, the bispecific molecule is a diabody or a bispecific antibody, and the cancer expresses the TA. In certain embodiments, the TA binding molecule comprises an ADCC-enhancing Fc domain.

[0211] Provided herein are specific dosing regimens for administering the PD-1 x LAG-3 bispecific molecules, or combinations of molecules, to a subject in need thereof.

[0212] As used herein, the term "in combination" refers to the use of two or more therapeutic agents (e.g., antibody-based molecules of the invention). The use of the term "in combination" does not restrict the order in which individual therapeutic agents should be administered to a subject (e.g., a human patient or other mammal) with a disease or disorder, nor does it mean that the agents are or must be administered simultaneously; rather, it means that the agents are administered to a subject concurrently or sequentially within a time interval so as to provide an enhanced benefit over the benefit that would be provided if the agents were administered in another manner. For example, each antibody-based molecule (e.g., a TA-binding molecule, a PD-1-binding molecule (or a PD-L1-binding molecule), and a LAG-3-binding molecule; or a TA-binding molecule and a PD-1xLAG-3 (or PD-L1xLAG-3) bispecific molecule) can be administered simultaneously or at different times in any order, but, if not administered simultaneously, they should be administered sufficiently closely in time to provide the desired therapeutic or prophylactic effect. Each agent administered can be administered separately in any suitable form and by any suitable route, for example, one by the oral route and one by the parenteral route, etc. Specific dosing regimens for administering the antibody-based molecules of the invention to a subject in need thereof are provided herein.

[0213] Cancers that can be treated by administration of a PD-1 x LAG-3 bispecific molecule; or a TA-binding molecule and a PD-1 x LAG-3 (or PD-L1 x LAG-3) bispecific molecule; or a combination of a PD-1-binding molecule (or a PD-L1-binding molecule) and a LAG-3-binding molecule include, but are not limited to, adrenal gland cancer, AIDS-related cancers, alveolar soft tissue sarcoma, anal cancer (including squamous cell carcinoma of the anal canal (SCAC)), bladder cancer, bone cancer, brain and spinal cord cancer, breast cancer (HER2 + Breast cancer, including triple-negative breast cancer (TNBC), carotid tumors tumor, cervical cancer (including HPV-associated cervical cancer), chondrosarcoma, chordoma, chromophobe clear cell renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, endometrial cancer (including unselected endometrial cancer, MSI-high endometrial cancer, dMMR endometrial cancer, and / or POLE exonuclease domain mutation-positive endometrial cancer), Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, gallbladder or bile duct cancer (including cholangiocarcinomabile duct cancer), gastric cancer, gastroesophageal junction (GEJ) cancer GEJ cancer, gestational trophoblastic disease, germ cell tumors, glioblastoma, head and neck cancer (including squamous cell carcinoma of the head and neck (SCCHN)), hematologic malignancies, hepatocellular carcinoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (including acute myeloid leukemia), liposarcoma / malignant lipomatous tumor, liver cancer (including hepatocellular carcinoma (HCC)), lymphoma (including diffuse large B-cell lymphoma (DLBCL) and non-Hodgkin's lymphoma (NHL)), lung cancer (including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)), medulloblastoma, melanoma (including uveal melanoma), medulloblastoma mesothelioma, Merkel cell carcinoma, mesothelioma (including mesothelial pharyngeal carcinoma), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pharyngeal cancer, pheochromocytoma, pituitary tumor, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), posterior uveal melanoma, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumor of childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer, and uterine cancer.

[0214] In certain embodiments, the PD-1 x LAG-3 bispecific molecules of the invention are directed to: breast cancer (HER2 +It can be used to treat breast cancer and / or TNBC, bile duct cancer (including cholangiocarcinomabile duct cancer), cervical cancer (including HPV-associated cervical cancer), endometrial cancer (including unselected endometrial cancer, MSI-high endometrial cancer, dMMR endometrial cancer, and / or POLE exonuclease domain mutation-positive endometrial cancer), gastric cancer, GEJ cancer, head and neck cancer (including SCCHN), liver cancer (including HCC), lung cancer (including SCLC and / or NSCLC), lymphoma (including NHL and DLBCL), ovarian cancer, and prostate cancer.

[0215] In other embodiments, the PD-1-binding molecule (or PD-L1-binding molecule) and LAG-3-binding molecule, or PD-1×LAG-3 (or PD-L1×LAG-3) bispecific molecule of the invention, in combination with a HER2-binding molecule (e.g., margetuximab), is administered to treat HER2-, HER3-, and HER2-associated cancers, including breast cancer, metastatic breast cancer, bladder cancer, gastric cancer, GEJ cancer, ovarian cancer, pancreatic cancer, and stomach cancer. + In one such embodiment, the compound can be used to treat cancer. The PD-1 x LAG-3 bispecific molecule is used in combination with an ADCC-enhanced HER2 binding molecule. In another such embodiment, DART-I is used in combination with margetuximab.

[0216] In other embodiments, the PD-1-binding (or PD-L1-binding) and LAG-3-binding molecules, or PD-1×LAG-3 (or PD-L1×LAG-3) bispecific molecules of the invention are used in combination with a B7-H3-binding molecule (e.g., enoblituzumab) to treat: anal cancer, SCAC, breast cancer, TNBC, head and neck cancer, SCCHN, lung cancer, NSCLC, melanoma, uveal melanoma, prostate cancer, mCRPC, and other cancers. + It can be used to treat cancer. In embodiments, the PD-1 x LAG-3 bispecific molecule is an ADCC-enhancing B7-H3 binding molecule. In another such embodiment, DART-I is used in combination with enoblituzumab.

[0217] In certain embodiments, the PD-1 x LAG-3 bispecific molecule; or the TA-binding molecule and PD-1 x LAG-3 (or PD-L1 x LAG-3) bispecific molecule; or the combination of a PD-1-binding molecule (or PD-L1-binding molecule) and a LAG-3-binding molecule are administered as first-line therapy for the treatment of cancer. In other embodiments, the molecules are administered after one or more prior lines of therapy. In other embodiments, the molecules are administered in further combination with one or more additional therapies. In yet other embodiments, the molecules can be employed as adjuvant therapy at the time of or after surgical removal of a tumor to delay, inhibit, or prevent the development of metastasis. Such molecules can also be administered prior to surgery (e.g., as neoadjuvant therapy) to reduce tumor size to allow or simplify surgery, to spare tissue during surgery, and / or to reduce any resulting cosmetic damage.

[0218] In one embodiment, the PD-1×LAG-3 bispecific molecule, in combination with a TA-binding molecule (e.g., HER2 or B7-H3), is administered as a first-line therapy for the treatment of cancer. In another embodiment, the PD-1×LAG-3 bispecific molecule, in combination with a TA-binding molecule, is administered after one or more prior lines of therapy. In another embodiment, the PD-1×LAG-3 bispecific molecule, in combination with a TA-binding molecule, is also administered in combination with one or more additional therapies. In yet another embodiment, the PD-1×LAG-3 bispecific molecule of the invention, in combination with a TA-binding molecule, can be employed as adjuvant therapy at the time of or after surgical removal of a tumor. The PD-1×LAG-3 bispecific molecule of the invention can also be administered in combination with a TA-binding molecule or prior to surgery. In one such embodiment, the TA-binding molecule is a HER2-binding molecule or a B7-H3-binding molecule.

[0219] The present invention particularly encompasses the administration of a PD-1×LAG-3 bispecific molecule; or a combination of a PD-1-binding molecule (or a PD-L1-binding molecule) and a LAG-3-binding molecule, or a PD-1×LAG-3 (or PD-L1×LAG-3) bispecific molecule and a TA-binding molecule, in combination with one or more other therapies known to those of skill in the art for the treatment or prevention of cancer, including, but not limited to, current standard and experimental chemotherapy, hormonal therapy, biological therapy, immunotherapy, radiation therapy, or surgery. In some embodiments, the combination of a PD-1-binding molecule (or a PD-L1-binding molecule) and a LAG-3-binding molecule, or a PD-1×LAG-3 (or PD-L1×LAG-3) bispecific molecule, in combination with a TA-binding molecule (e.g., an ADCC-enhancing TA-binding molecule), and further to treat cancer, particularly TA-expressing cancers (e.g., HER2 + Cancer or B7-H3 + The HER2-expressing cancers are typically administered in combination with a therapeutically or prophylactically effective amount of one or more therapeutic agents known to those skilled in the art for the treatment and / or prevention of HER2-expressing cancers. Chemotherapeutic agents commonly used in the treatment of HER2-expressing cancers include, but are not limited to, anthracyclines (particularly daunorubicin, doxorubicin, and epirubicin), capecitabine, carboplatin, cyclophosphamide, leucovorin, methotrexate, oxaliplatin, taxanes (particularly docetaxel and paclitaxel), and 5-fluorouracil (5-FU).

[0220] Another aspect of the present invention involves an improved method for determining a subject's suitability for such treatment by measuring the level of PD-L1 expression in the subject's tumor cells prior to the initiation of such treatment. PD-L1 expression on greater than 10% of tumor cells has been established as a clinically relevant cutoff point for treatment with certain PD-1-binding (or PD-L1-binding) molecules. Methods for measuring the level of PD-L1 expression are known in the art (de Vicente, JC et al. (2018) "PD-L1 Expression in Tumor Cells"). Is an Independent Unfavorable Prognostic Factor in Oral Squamous Cell Carcinoma ,” Cancer Epidemiol. Biomarkers Prev. 28(3):546-554;Davis, AA et al. (2019) “The Role OfPD-L1 Expression As A Predictive Biomarker: An Analysis Of All US Food And DrugAdministration (FDA) Approvals Of Immune Checkpoint Inhibitors,” J. ImmunoTher. Canc.7:278:1-8; Khozin, For example, such a measurement is performed by Dako S. et al. EnVision Flex+Visualization System (Dako This can be achieved using a mouse monoclonal PD-L1 antibody (clone 22C3, 1:200 dilution; PD-L1 IHC 22C3 pharmDx; Dako SK006) by using a fluorochrome autostainer. In such an assay, formalin-fixed, paraffin-embedded tumor biopsies are incubated in the presence of a monoclonal mouse anti-PD-L1 antibody (clone 22C3). PD-L1 protein expression is determined using the tumor proportion score (TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity; or by the total positive score (CPS), which is the number of PD-L1-stained cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells multiplied by 100.

[0221] Finding that a subject's tumor exhibits less than 1% PD-L1 expression (as determined using the total positive score (CPS) or tumor proportion score (TPS) in IHC analysis) prior to treatment is indicative of the patient's suitability for the treatment methods of the invention, particularly methods involving administering a PD-1-binding (or PD-L1-binding) and LAG-3-binding molecule, or a PD-1×LAG-3 (or PD-L1×LAG-3) bispecific molecule in combination with an ADCC-enhancing TA-binding molecule. Such suitability is also enhanced in subjects who have previously failed or responded inadequately to at least one prior treatment, including prior treatment with a PD-1-binding molecule or a PD-L1-binding molecule without treatment with an ADCC-enhancing TA-binding molecule. The present invention encompasses methods of treating cancer by administering to a subject a TA-binding molecule and: a PD-1 x LAG-3 (or PD-L1 x LAG-3) bispecific molecule; or a combination of a PD-1-binding molecule (or a PD-L1-binding molecule) and a LAG-3-binding molecule, wherein expression of PD-L1 on the surface of cells of the cancer prior to treatment is less than 1% as determined using a total positive score (CPS) or a tumor proportion score (TPS).

[0222] VIII. Administration and Dosage The antibody-based molecules of the invention (e.g., PD-1 x LAG-3 bispecific molecules) can be administered to a subject, e.g., a subject in need thereof, e.g., a human patient, in a variety of ways. For many applications, the route of administration will be one of: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), or intramuscular injection. Intra-articular delivery may also be used. Other modes of parenteral administration may also be used. Examples of such modes include: intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injection.

[0223] The antibody-based molecules of the present invention can be administered as a flat dose or as a weight-based dose (e.g., a dose in mg / kg of patient body weight). The dose can also be selected to reduce or avoid the production of antibodies against the administered antibody-based molecule. The dosing regimen is adjusted to provide a desired response, e.g., a therapeutic response or a combined therapeutic effect. Generally, multiple doses of the antibody-based molecule (and optionally additional agents) can be used to provide a subject with a bioavailable amount of the agent. As used herein, the term "dose" refers to a specified amount of drug treatment administered at one time. The term "dosage" refers to the administration of a dose in a specific amount, number, and frequency over a specified period of time; thus, the term "dosage" refers to With respect to the timing of administration of a dose (i.e., dosing), the term "about" is intended to refer to a range of ±3 days of the stated administration. Illustrated.

[0224] As used herein, the term "flat dose" refers to a dose that is administered based on the patient's weight. "Weight-based dose" refers to a dose that does not contain a therapeutically effective amount and includes physically discrete units of the antibody-based molecule to be administered (e.g., an antibody that binds to a TA, an antibody that binds to PD-1, an antibody that binds to PD-L1, an antibody that binds to LAG-3, or a PD-1xLAG-3 (or PD-L1xLAG-3) bispecific molecule) suitable for use as a single dose to a subject to be treated, each such unit containing a predetermined quantity (calculated to produce a desired therapeutic effect) of the antibody-based molecule, in association with a pharmaceutical carrier and, optionally, an additional agent. Single or multiple flat doses may be administered. As used herein, the term "weight-based dose" refers to a dose that does not contain a therapeutically effective amount and includes physically discrete units of the antibody-based molecule to be administered (e.g., an antibody that binds to a TA, an antibody that binds to PD-1, an antibody that binds to PD-L1, an antibody that binds to LAG-3, or a PD-1xLAG-3 (or PD-L1xLAG-3) bispecific molecule) suitable for use as a single dose to a subject to be treated, each such unit containing a predetermined quantity (calculated to produce a desired therapeutic effect) of the antibody-based molecule, in association with a pharmaceutical carrier, and optionally, an additional agent. Single or multiple flat doses may be administered. As used herein, the term "weight-based dose" refers to a dose that does not contain a therapeutically effective amount. "Dose" refers to the individual amount of a molecule of the invention administered per unit of patient weight, e.g., It refers to milligrams of drug per kilogram of subject's body weight (mg / kg body weight; abbreviated herein as "mg / kg"). The calculated dose is administered based on the subject's body weight at baseline. Typically, a significant change in body weight (10% or more) from baseline or established plateau body weight will prompt a recalculation of the dose. Single or multiple doses can be administered in a dosing regimen. The composition comprising the antibody-based molecule can be administered to a subject in need thereof by injection.

[0225] Thus, in some embodiments, antibody-based molecules that bind to TAs (particularly ADCC-enhancing TA-binding molecules), PD-1 or PD-L1, and / or LAG-3 are administered to a subject in need thereof according to an approved prescription dosing regimen, which may incorporate a flat dose or weight-based dose. Approved prescription dosing regimens for the above molecules have been previously described (e.g., package inserts for trastuzumab, pertuzumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, tafasitamab, etc. are available on the US National Library of Medicine website: dailymed.nlm.nih.gov / dailymed / ). In certain embodiments, antibody-based molecules that bind to PD-1, or PD-L1, and / or LAG-3 are administered to a subject in need thereof at a flat dose of about 120 mg to about 800 mg. In certain embodiments, antibody-based molecules that bind to TA (e.g., antibody-based molecules that bind to HER2 or B7-H3) are administered to a subject in need thereof at a body weight-based dose of about 2 mg / kg to about 18 mg / kg.

[0226] In certain embodiments, the PD-1 x LAG-3 bispecific molecule (e.g., DART-I) is administered to a subject in need thereof in a flat dose of about 120 mg to about 800 mg. In certain embodiments, the PD-1 x LAG-3 bispecific molecule is administered to a subject in need thereof in a flat dose of about 120 mg, about 300 mg, about 400 mg, about 600 mg, or about 800 mg. In a specific embodiment, the PD-1 x LAG-3 bispecific molecule is administered to a subject in need thereof in a flat dose of about 400 mg. In another specific embodiment, the PD-1 x LAG-3 bispecific molecule is administered to a subject in need thereof in a flat dose of about 600 mg. In another specific embodiment, the PD-1 x LAG-3 bispecific molecule is administered to a subject in need thereof in a flat dose of about 800 mg. In certain embodiments, an anti-PD-1 antibody (e.g., retifanlimab) is administered to a subject in need thereof at a flat dose of about 120 mg to about 750 mg. In certain embodiments, an anti-PD-1 antibody is administered to a subject in need thereof at a flat dose of about 375 mg, about 500 mg, or about 750 mg. In a specific embodiment, an anti-PD-1 antibody is administered to a subject in need thereof at a flat dose of about 375 mg. In another specific embodiment, an anti-PD-1 antibody is administered to a subject in need thereof at a flat dose of about 500 mg. In certain embodiments, an anti-LAG-3 antibody (e.g., leratolimab) is administered to a subject in need thereof at a flat dose of about 80 mg to about 200 mg. In certain embodiments, an anti-LAG-3 antibody is administered to a subject in need thereof at a flat dose of about 80 mg to about 200 mg. A subject in need thereof is administered a flat dose of about 80 mg, about 100 mg, or about 160 mg. In a specific embodiment, an anti-LAG-3 antibody is administered to a subject in need thereof at a flat dose of about 160 mg. With respect to flat doses or flat dosing, the term "about" is intended to refer to a range of ±10% of the stated dose, so that, for example, a dose of about 600 mg would be 540 mg to 660 mg. With respect to dosing, the term "about" is intended to refer to a range of ±3 days of the stated dose.

[0227] In certain embodiments, a HER2 or B7-H3 binding molecule (e.g., an anti-HER2 antibody, an anti-B7-H3 antibody) is administered to a subject in need thereof at a body weight-based dose of about 2 mg / kg to about 18 mg / kg. In certain embodiments, a HER2 or B7-H3 binding molecule is administered to a subject in need thereof at a dose of about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 8 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 18 mg / kg. In a specific embodiment, a HER2 or B7-H3 binding molecule is administered to a subject in need thereof at a dose of about 15 mg / kg. In another specific embodiment, a first dose of a HER2 binding molecule is administered to a subject in need thereof at a dose of about 8 mg / kg, followed by one or more additional doses of the HER2 binding molecule at a dose of about 6 mg / kg. In another specific embodiment, a first dose of a HER2-binding molecule is administered to a subject in need thereof at a dose of about 4 mg / kg, followed by one or more additional doses of the HER2-binding molecule at doses of about 2 mg / kg. With respect to weight-based doses, the term "about" is intended to refer to a range of ±10% of the stated dose, so that, for example, a dose of about 15 mg / kg would be 13.6 mg / kg to 16.5 mg / kg.

[0228] In certain embodiments, the HER2-binding molecule is administered to a subject in need thereof in a flat dose of about 420 mg to about 1650 mg. In a specific embodiment, the HER2-binding molecule is administered to a subject in need thereof in a flat dose of about 420 mg. In another specific embodiment, the HER2-binding molecule is administered to a subject in need thereof in a flat dose of about 600 mg. In another specific embodiment, the HER2-binding molecule is administered to a subject in need thereof in a flat dose of about 840 mg. In another specific embodiment, the HER2-binding molecule is administered to a subject in need thereof in a flat dose of about 1650 mg. In another specific embodiment, a first dose of the HER2-binding molecule is administered to a subject in need thereof in a flat dose of about 840 mg, followed by one or more additional doses of the HER2-binding molecule in flat doses of about 420 mg.

[0229] A dosage of an antibody-based molecule (e.g., a dose of an antibody that binds a TA, an antibody that binds PD-1, an antibody that binds PD-L1, an antibody that binds LAG-3, or a PD-1xLAG-3 (or PD-L1xLAG-3) bispecific molecule) can be administered at periodic intervals over a period of time (a course of treatment) sufficient to encompass at least two doses, at least four doses, at least six doses, at least 12 doses, or at least 24 doses. For example, a dosage may be administered, for example, once or twice daily, or about 1 to 4 times per week. In certain embodiments, a dosage may be administered once per week ("Q1W"), once every two weeks ("Q2W"), once every three weeks ("Q3W"), once every four weeks ("Q4W"), etc. Such periodic administration may continue for a period of time, for example, from about 1 to 52 weeks or for more than 52 weeks. Such a course of treatment may be divided into multiple increments, e.g., 2 to 24 weeks, about 3 to 7 weeks, about 4 weeks, or about 6 weeks, or about 8 weeks, or about 12 weeks, or about 24 weeks, each referred to herein as a "cycle," during which a set number of doses are administered. is administered. The dosage and / or frequency of administration may be the same or different during each cycle. Factors that may affect the dosing and timing required to effectively treat a subject include, for example, the severity of the subject's disease or disorder, the formulation, route of delivery, previous treatments, overall health, and / or age, and the presence of other diseases in the subject's body. Additionally, treatment of a subject with a therapeutically effective amount of a compound may involve a single treatment or a series of multiple treatments. It can include.

[0230] It is contemplated that a subject may be provided with multiple doses of an antibody-based molecule (e.g., an antibody that binds to a TA, an antibody that binds to PD-1, an antibody that binds to PD-L1, an antibody that binds to LAG-3, a PD-1xLAG-3 (or PD-L1xLAG-3) bispecific molecule). The amount of each antibody-based molecule in each such dose may be the same or different from previously administered doses. Thus, for example, therapy may be provided by administering the "first" (or "last") dose of the antibody-based molecule. administration of a "loading" dose, followed by a small "first" dose of the antibody-based molecule. For example, if the first dose of the antibody-based molecule is approximately 8 mg / kg, the second dose will be less than 8 mg / kg (e.g., about 6 mg / kg). In some embodiments, subsequent doses are administered at the same concentration as the second smaller dose. In some embodiments, the same dose of the antibody-based molecule is administered throughout the course of treatment. In some embodiments, the TA-binding molecule that binds HER2 is administered at a first dose that is a flat dose of about 4 mg / kg, about 8 mg / kg, or about 840 mg, followed by a second smaller dose, where the second dose is administered about three weeks after administration of the first dose. In some embodiments, a further subsequent dose of the HER2-binding molecule is administered, where the subsequent dose is administered about three weeks after administration of the second dose or after the previous one of the subsequent doses.

[0231] A "dosing regimen" is the administration of a medication to a patient at one or more predetermined periodicities, at a predetermined frequency (or set of multiple such frequencies), and at a predetermined dose (or set of multiple such doses).

[0232] An exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule (e.g., DART-I) at a flat dose of about 120 mg Q2W. Another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 300 mg Q2W. Yet another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 300 mg Q3W. Another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 400 mg Q2W. Another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 400 mg Q3W. Another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 600 mg Q2W. Yet another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 600 mg Q3W. Other exemplary dosing regimens comprise administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 800 mg Q2W, or administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 800 mg Q3W. As provided herein, such dosing regimens may further comprise administration of a TA-binding molecule. In one embodiment, the PD-1 x LAG-3 bispecific molecule is administered according to a dosing regimen provided herein in combination with an approved TA-binding molecule (e.g., trastuzumab, pertuzumab, etc.) administered according to an approved prescribed dosing regimen. In one embodiment, the PD-1 x LAG-3 bispecific molecule is administered according to a dosing regimen provided herein in combination with an approved ADCC-enhancing TA-binding molecule (e.g., tafasitamab, etc.) administered according to an approved prescribed dosing regimen. In certain embodiments of the above-described dosing regimens, the PD-1 x LAG-3 bispecific molecule is DART-I. In one such embodiment, DART-I is administered at a flat dose of about 600 mg Q3W. In another such embodiment, DART-I is administered at a flat dose of about 600 mg Q3W in combination with an approved TA-binding molecule (e.g., trastuzumab, pertuzumab, etc.) administered according to an approved prescription dosing regimen.In another such embodiment, DART-I is administered at a flat dose of about 600 mg Q3W in combination with an approved ADCC-enhancing TA binding molecule (e.g., tafasitamab) administered according to an approved prescribed dosing regimen.

[0233] Another exemplary dosing regimen comprises administering an anti-PD-1 antibody (e.g., retifanlimab) Q3W at a flat dose of about 375 mg, and an anti-LAG-3 antibody (e.g., leratolimab) Q4W at a flat dose of about 160 mg. Another exemplary dosing regimen comprises administering an anti-PD-1 antibody Q4W at a flat dose of about 500 mg, and an anti-LAG-3 antibody Q4W at a flat dose of about 160 mg. Yet another exemplary dosing regimen comprises administering an anti-PD-1 antibody Q4W at a flat dose of about 750 mg, and an anti-LAG-3 antibody Q4W at a flat dose of about 160 mg. As provided herein, such dosing regimens may further comprise administration of a TA-binding molecule. In one embodiment, the anti-PD-1 antibody and anti-LAG-3 antibody are administered according to the dosing regimen provided herein in combination with an approved TA-binding molecule (e.g., trastuzumab, pertuzumab, etc.), administered according to an approved prescription dosing regimen. In one embodiment, the anti-PD-1 antibody and anti-LAG-3 antibody are administered according to the dosing regimen provided herein in combination with an approved ADCC-enhancing TA-binding molecule (e.g., tafasitamab, etc.), administered according to an approved prescription dosing regimen. In a specific embodiment of the above-described dosing regimen, the anti-PD-1 antibody is retifanlimab and the anti-LAG-3 antibody is leratolimab. In one such embodiment, retifanlimab is administered at a flat dose of about 375 mg Q3W, leratolimab is administered at a flat dose of about 160 mg Q4W, and the approved TA-binding molecule (e.g., trastuzumab, pertuzumab, etc.) is administered according to an approved prescription dosing regimen. In another such embodiment, retifanlimab is administered at a flat dose of about 500 mg Q4W, leratolimab is administered at a flat dose of about 160 mg Q4W, and the approved TA-binding molecule (e.g., trastuzumab, pertuzumab, etc.) is administered according to an approved prescribed dosing regimen. In another such embodiment, retifanlimab is administered at a flat dose of about 375 mg Q3W, leratolimab is administered at a flat dose of about 160 mg Q4W, and the approved ADCC-enhancing TA-binding molecule (e.g., tafasitamab, etc.) is administered according to an approved prescribed dosing regimen.In yet another such embodiment, retifanlimab is administered at a flat dose of about 500 mg Q4W, leratolimab is administered at a flat dose of about 160 mg Q4W, and the approved ADCC-enhancing TA binding molecule (e.g., tafasitamab) is administered according to an approved prescribed dosing regimen.

[0234] In one embodiment, the PD-1 x LAG-3 bispecific molecule is administered in combination with an ADCC-enhanced TA-binding molecule according to the dosing regimens provided herein. An exemplary combination dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule (e.g., DART-I) Q2W at a flat dose of about 120 mg, and administration of the ADCC-enhanced HER2 or B7-H3 binding molecule (e.g., margetuximab or enoblituzumab) Q3W at a dose of about 2 mg / kg to about 18 mg / kg. Another exemplary combination dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule (e.g., DART-I) Q3W at a flat dose of about 120 mg, and administration of the ADCC-enhanced HER2 or B7-H3 binding molecule (e.g., margetuximab or enoblituzumab) Q3W at a dose of about 2 mg / kg to about 18 mg / kg. Another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule Q2W at a flat dose of about 300 mg and administration of the ADCC-enhanced HER2 or B7-H3 Q3W at a dose of about 2 mg / kg to about 18 mg / kg. Yet another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule Q3W at a flat dose of about 300 mg and administration of the ADCC-enhanced HER2 or B7-H3 binding molecule Q3W at a dose of about 2 mg / kg to about 18 mg / kg. Another exemplary dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule Q2W at a flat dose of about 400 mg and administration of the ADCC-enhanced HER2 or B7-H3 binding molecule Q3W at a dose of about 2 mg / kg to about 18 mg / kg. Another exemplary dosing regimen involves administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of 400 mg Q3W and administration of the ADCC-enhanced HER2 or B7-H3 binding molecule at a dose of about 2 mg / kg to about 18 mg / kg Q3W. and the ADCC-enhanced HER2 or B7-H3 binding molecule Q3W at a dose of about 2 mg / kg to about 18 mg / kg. Another specific dosing regimen includes administering the PD-1 x LAG-3 bispecific molecule Q3W at a flat dose of about 600 mg and administering the ADCC-enhanced HER2 or B7-H3 binding molecule Q3W at a dose of about 2 mg / kg to about 18 mg / kg. Another specific dosing regimen includes administering the PD-1 x LAG-3 bispecific molecule Q2W at a flat dose of about 800 mg and administering the ADCC-enhanced HER2 or B7-H3 binding molecule (e.g., margetuximab or enoblituzumab) Q3W at a dose of about 2 mg / kg to about 18 mg / kg. Another specific dosing regimen comprises administration of the PD-1 x LAG-3 bispecific molecule at a flat dose of about 800 mg Q3W and administration of the ADCC-enhanced HER2 or B7-H3 binding molecule at a dose of about 2 mg / kg to about 18 mg / kg Q3W. In certain embodiments of the above dosing regimen, the PD-1 x LAG-3 bispecific molecule is DART-I. In some embodiments of the above dosing regimen, the ADCC-enhanced HER2 binding molecule is margetuximab. In some embodiments of the above dosing regimen, the ADCC-enhanced B7-H3 binding molecule is enoblituzumab.

[0235] Preferably, in the above-described embodiments, administration is at a predetermined frequency or periodicity, such that administration occurs 1-3 days before, 1-3 days after, or on the day of the scheduled dose, or within 1-3 days of the scheduled interval, e.g., once every 3 weeks (±3 days). Typically, in the above-described embodiments, the PD-1×LAG-3 bispecific molecule and the ADCC-enhanced HER2 or B7-H3 binding molecule are administered by IV infusion within a 24-hour period. In specific embodiments, the PD-1×LAG-3 bispecific molecule and the ADCC-enhanced HER2 or B7-H3 binding molecule are administered by IV infusion according to any of the above-described dosing regimens for a duration (i.e., a course of treatment) of at least 1 month or more, at least 3 months or more, at least 6 months or more, or at least 12 months or more. Treatment durations of at least 6 months or more, or at least 12 months or more, or until remission of the disease or unmanageable toxicity is observed are specifically contemplated. In certain embodiments, treatment is continued for a period following remission of the disease.

[0236] In certain embodiments, antibody-based molecules are administered via IV infusion. Therefore, antibody-based molecules are typically diluted (separately or together) in an infusion bag containing a suitable diluent, such as 0.9% sodium chloride. Because infusion or allergic reactions are possible, pre-medication to prevent such reactions is recommended, and anaphylaxis precautions must be observed during antibody administration. Such IV infusions can be administered to subjects over a period of 30 minutes to 24 hours. In certain embodiments, IV infusions are delivered over a period of about 30 to 240 minutes, about 30 to 180 minutes, about 30 to 120 minutes, or about 30 to 90 minutes, or over a period of about 60 to 90 minutes, or over a period of about 60 to 75 minutes, or over a shorter period, provided the subject does not exhibit signs or symptoms of an adverse infusion reaction.

[0237] As noted above, while a variety of dosages and routes of administration may be employed to provide antibody-based molecules to a subject in need thereof in accordance with the present invention, certain combinations, dosages, and routes of administration are particularly provided for use in such treatments. The use of the PD-1 x LAG-3 bispecific diabodies of the present invention in combination with anti-HER2 or anti-B7-H3 antibodies (e.g., margetuximab, trastuzumab, pertuzumab, and / or enoblituzumab) in such dosages and administrations is particularly described herein.

[0238] Thus, the dosing regimen described above may involve the administration of a flat dose of about 300 mg to about 800 mg of the PD-1 x LAG-3 bispecific diabody in combination with a dose of about 2 mg / kg to about 15 mg / kg, and / or a flat dose of about 420 to 840 mg of an anti-HER2 or anti-B7-H3 antibody. wherein the molecules are administered Q3W (±3 days). In certain embodiments, the PD-1 x LAG-3 bispecific diabody is administered at a flat dose of about 300 mg, about 400 mg, about 600 mg, or about 800 mg, and the anti-HER2 or anti-B7-H3 antibody is administered at a dose of about 2 mg / kg, about 4 mg / kg, about 6 mg / kg, about 8 mg / kg, or about 15 mg / kg. In other embodiments, the PD-1 x LAG-3 bispecific diabody is administered at a flat dose of about 300 mg, about 400 mg, about 600 mg, or about 800 mg, and the anti-HER2 antibody is administered at a flat dose of about 420 mg or about 840 mg.

[0239] (A) In certain embodiments, the PD-1 x LAG-3 bispecific diabody is administered at a flat dose of about 300 mg. In such embodiments, when the anti-HER2 or anti-B7-H3 antibody to be administered is margetuximab or enoblituzumab, respectively, such margetuximab or enoblituzumab is administered at a dose of about 15 mg / kg body weight. Alternatively, in such embodiments, when the anti-HER2 antibody to be administered is trastuzumab, the first dose of trastuzumab is administered at a dose of about 8 mg / kg, followed by one or more additional doses of trastuzumab, each at a dose of about 6 mg / kg, or the first dose of trastuzumab is administered at a dose of about 4 mg / kg, followed by one or more additional doses of trastuzumab, each at a dose of about 2 mg / kg. Alternatively, in such embodiments, where the anti-HER2 antibody to be administered is pertuzumab, the first dose of pertuzumab is administered at a dose of about 840 mg, followed by one or more additional doses of pertuzumab each at a dose of about 420 mg.

[0240] (B) In certain embodiments, the PD-1 x LAG-3 bispecific diabody is administered with an anti-HER2 or anti-B7-H3 antibody at a flat dose of about 400 mg. In such embodiments, when the anti-HER2 or anti-B7-H3 antibody to be administered is margetuximab or enoblituzumab, respectively, such margetuximab or enoblituzumab is administered at a dose of about 15 mg / kg body weight. Alternatively, in such embodiments, when the anti-HER2 antibody to be administered is trastuzumab, the first dose of trastuzumab is administered at a dose of about 8 mg / kg, followed by one or more additional doses of trastuzumab, each at a dose of about 6 mg / kg, or the first dose of trastuzumab is administered at a dose of about 4 mg / kg, followed by one or more additional doses of trastuzumab, each at a dose of about 2 mg / kg. Alternatively, in such embodiments, where the anti-HER2 antibody to be administered is pertuzumab, the first dose of pertuzumab is administered at a dose of about 840 mg, followed by one or more additional doses of pertuzumab each at a dose of about 420 mg.

[0241] (C) In certain embodiments, the PD-1 x LAG-3 bispecific diabody is administered at a flat dose of about 600 mg. In such embodiments, when the anti-HER2 or anti-B7-H3 antibody to be administered is margetuximab or enoblituzumab, respectively, such margetuximab or enoblituzumab is administered at a dose of about 15 mg / kg body weight. In such embodiments, when the anti-HER2 antibody to be administered is trastuzumab, the first dose of trastuzumab is administered at a dose of about 8 mg / kg, followed by one or more additional doses of trastuzumab, each at a dose of about 6 mg / kg, or the first dose of trastuzumab is administered at a dose of about 4 mg / kg, followed by one or more additional doses of trastuzumab, each at a dose of about 2 mg / kg. Alternatively, in such embodiments, where the anti-HER2 antibody to be administered is pertuzumab, the first dose of pertuzumab is administered at a dose of about 840 mg, followed by one or more additional doses of pertuzumab each at a dose of about 420 mg.

[0242] (D) In ​​certain embodiments, the PD-1 x LAG-3 bispecific diabody is In such embodiments, when the anti-HER2 or anti-B7-H3 antibody to be administered is margetuximab or enoblituzumab, respectively, such margetuximab or enoblituzumab is administered at a dose of about 15 mg / kg body weight. Alternatively, when the anti-HER2 antibody to be administered is trastuzumab, the first dose of trastuzumab is administered at a dose of about 8 mg / kg, followed by one or more additional doses of trastuzumab, each at a dose of about 6 mg / kg, or the first dose of trastuzumab is administered at a dose of about 4 mg / kg, followed by one or more additional doses of trastuzumab, each at a dose of about 2 mg / kg. Alternatively, where the anti-HER2 antibody to be administered is pertuzumab, the first such dose of pertuzumab is administered at a dose of about 840 mg, followed by one or more additional doses of pertuzumab each at a dose of about 420 mg.

[0243] In any of the above embodiments, the PD-1 x LAG-3 bispecific diabody and the anti-HER2 or anti-B7-H3 antibody are administered by IV infusion in parallel, sequentially, or alternatingly, or at different times within a 24-hour period. In any of the above embodiments, the PD-1 x LAG-3 bispecific diabody is DART-I.

[0244] The present invention also provides dosing regimens in which a PD-1 x LAG-3 bispecific diabody is administered in combination with two different anti-HER2 antibodies (e.g., trastuzumab and pertuzumab), where the administration of each molecule is according to any of the embodiments described above or according to an approved prescribed dosing regimen.

[0245] IX. EMBODIMENTS OF THE INVENTION Having now generally described the present invention, it will be more readily understood by reference to the following numbered embodiments ("EA" and "EB"), which are provided by way of example and are not intended to limit the invention unless otherwise specified.

[0246] EA1. A method of treating cancer comprising administering a PD-1×LAG-3 bispecific molecule to a subject in need thereof, said method comprising administering to the subject a flat dose of between about 120 mg and about 800 mg of the PD-1×LAG-3 bispecific molecule.

[0247] EA2. The method of EA1, wherein the cancer is characterized by expression of a tumor antigen (TA), and the method further comprises administering to the subject a tumor antigen (TA)-binding molecule (TA-binding molecule).

[0248] EA3. A method of treating cancer in a subject, said cancer being characterized by expression of a TA, said method comprising administering to said subject a TA binding molecule, and further administering to said subject: (a) a bispecific molecule (PD-1 x LAG-3 bispecific molecule); or (b) a combination of a molecule that immunospecifically binds to PD-1 (a PD-1-binding molecule) and a molecule that immunospecifically binds to LAG-3 (a LAG-3-binding molecule); or (c) a bispecific molecule that immunospecifically binds to both PD-L1 and LAG-3 (a PD-L1 x LAG-3 bispecific molecule); or (d) a combination of a molecule that immunospecifically binds to PD-L1 (a PD-L1-binding molecule) and a LAG-3-binding molecule; The method of claim 1, further comprising administering

[0249] EA4. The method of EA2 or EA3, wherein said TA-binding molecule comprises an ADCC-enhancing Fc domain.

[0250] EA5.(a) each of the above molecules is in a separate composition; or (b) each of the above molecules is in the same composition; or (c) the PD-1-binding molecule and the LAG-3-binding molecule are in the same composition, and the TA-binding molecule is in a separate composition; or (d) any one of the methods of EA2-EA4, wherein the PD-L1-binding molecule and the LAG-3-binding molecule are in the same composition, and the TA-binding molecule is in a separate composition.

[0251] EA6. The method of any one of EA2-EA5, wherein said TA-binding molecule is an antibody.

[0252] EA7. The method of any one of EA2-EA6, wherein the PD-1 binding molecule is an antibody.

[0253] EA8. The method of any one of EA2 to EA6, wherein the PD-L1 binding molecule is an antibody.

[0254] EA9. The method of any one of EA2-EA8, wherein said LAG-3 binding molecule is an antibody.

[0255] EA10. Any one of methods EA3-EA6, wherein the method comprises administering the TA-binding molecule and the PD-1×LAG-3 bispecific molecule.

[0256] EA11. Any one of EA3-EA9, wherein the method comprises administering the TA-binding molecule and the PD-1-binding molecule in combination with a LAG-3-binding molecule.

[0257] EA12. Any one of methods EA3-EA6, wherein the method comprises administering the TA-binding molecule and the PD-L1×LAG-3 bispecific molecule.

[0258] EA13. Any one of EA3-EA9, wherein the method comprises administering the TA-binding molecule and the PD-L1-binding molecule in combination with a LAG-3-binding molecule.

[0259] EA14. The ADCC-enhancing Fc domain is: (a) artificial glycoforms; and / or (b) Amino acid substitutions relative to the wild-type Fc region Any one of methods EA4 to EA13, including:

[0260] EA15. The method of EA14, wherein the ADCC-enhancing Fc domain comprises an artificial glycoform that is a complex N-glycosidically linked glycan that does not contain fucose and / or contains a bisecting O-GlcNAc.

[0261] EA16. The method of EA14 or EA15, wherein the ADCC-enhancing Fc domain comprises one or more amino acid substitutions selected from F243L, R292P, Y300L, V305I, I332E, and P396L.

[0262] EA17. The ADCC-enhancing Fc domain comprises an amino acid substitution selected from the group consisting of: (a) one substitution selected from the group consisting of: F243L, R292P, Y300L, V305I, I332E, and P396L; (b) two substitutions selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; (3) R292P and V305I; and (4) S239D and I332E; (c) three substitutions selected from the group consisting of: (1) F243L, R292P and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P, and P396L; and (4) R292P, V305I and P396L; (d) four substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, and P396L; and (2) F243L, R292P, V305I, and P396L; or (e) five substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I, and P396L; and (2) L235V, F243L, R292P, Y300L and P396L and the numbering is that of the EU index as set out in Kabat, any one of EA14 to EA16.

[0263] EA18. The method of any one of EA14 to EA16, wherein the ADCC-enhancing Fc domain comprises the amino acid substitutions: L235V, F243L, R292P, Y300L, and P396L, wherein the numbering is that of the EU index as set forth in Kabat.

[0264] EA19. The method of any one of EA14 to EA16, wherein said ADCC-enhancing Fc domain comprises the amino acid substitutions S239D and I332E, wherein the numbering is that of the EU index as set forth in Kabat.

[0265] EA20. The method of any one of EA2 to EA19, wherein said TA is selected from Table 6A or Table 6B.

[0266] EA21. The method of any one of EA2 to EA19, wherein said TA-binding molecule comprises the VL and VH domains of an antibody selected from Table 7.

[0267] EA22. The PD-1 binding molecule is: (a) a PD-1 VL domain comprising the amino acid sequence of SEQ ID NO: 35, and a PD-1 VH domain comprising the amino acid sequence of SEQ ID NO: 39; (b) the VH and VL domains of an anti-PD-1 antibody selected from Table 1; or (c) the light chain and heavy chain of an anti-PD-1 antibody selected from Table 1 The method according to any one of EA3 to EA7, EA9, EA11, and EA14 to EA21, wherein the antibody comprises

[0268] EA23. The PD-L1 binding molecule is: (a) a PD-L1 VL domain comprising the amino acid sequence of SEQ ID NO: 43, and a PD-L1 VH domain comprising the amino acid sequence of SEQ ID NO: 47; (b) the VH and VL domains of an anti-PD-L1 antibody selected from Table 2; or (c) the light chain and heavy chain of an anti-PD-L1 antibody selected from Table 2 The method according to any one of EA3 to EA6, EA8 to EA9, and EA13 to EA21, wherein the antibody comprises

[0269] EA24. The LAG-3 binding molecule is: (a) a LAG-3 VL domain comprising the amino acid sequence of SEQ ID NO: 51, and a LAG-3 VH domain comprising the amino acid sequence of SEQ ID NO: 55; (b) the VH and VL domains of an anti-LAG-3 antibody selected from Table 3; or (c) the light and heavy chains of an anti-LAG-3 antibody selected from Table 3 The method according to any one of EA3 to EA9, EA11, and EA13 to EA23, wherein the antibody comprises

[0270] EA25. The PD-1 x LAG-3 bispecific molecule described above: (a) a PD-1 VL domain comprising the amino acid sequence of SEQ ID NO: 35, and a PD-1 VH domain comprising the amino acid sequence of SEQ ID NO: 39, or the VH and VL domains of an anti-PD-1 antibody selected from Table 1; and / or (b) a LAG-3 VL domain comprising the amino acid sequence of SEQ ID NO: 51 and a LAG-3 VH domain comprising the amino acid sequence of SEQ ID NO: 55, or the VH and VL domains of an anti-LAG-3 antibody selected from Table 3; or (c) a bispecific antibody molecule selected from Tables 4 and 5 Any one of methods EA1 to EA6, EA10, and EA14 to EA21, including:

[0271] EA26. The PD-1 x LAG-3 bispecific molecule described above: (a) CDR of SEQ ID NO: 35 L 1. CDR L 2, and CDR L Light chain variable domain containing 3 (VL PD‐1 ) and the PD-1-specific CDR of SEQ ID NO: 39 H 1. CDR H 2, and CDR H 3, including the heavy chain variable domain (VH PD‐1 a PD-1 binding domain comprising: (b) CDR of SEQ ID NO: 51 L 1. CDR L 2, and CDR L Light chain variable domain containing 3 (VL LAG‐3 ) and the LAG-3 specific CDR of SEQ ID NO: 55 H 1. CDR H 2, and CDR H 3, including the heavy chain variable domain (VH LAG‐3 ) and a LAG-3 binding domain Any one of methods EA1 to EA6, EA10, and EA14 to EA21, including:

[0272] EA27. The PD-1 x LAG-3 bispecific molecule described above: (a) two of the PD-1 binding domains described above; and (b) two of the above LAG-3 binding domains Any one of methods EA1 to EA6, EA10, EA14 to EA21, and EA25 to EA26, including:

[0273] EA28. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA27, wherein the PD-1 x LAG-3 bispecific molecule comprises a VL domain of SEQ ID NO: 35 and a VH domain of SEQ ID NO: 39.

[0274] EA29. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA28, wherein the PD-1 x LAG-3 bispecific molecule comprises a VL domain of SEQ ID NO: 51 and a VH domain of SEQ ID NO: 55.

[0275] EA30. The method of any one of EA1-EA6, EA10, EA12, EA14-EA21, and EA25-EA29, wherein the PD-1×LAG-3 bispecific molecule or the PD-L1×LAG-3 bispecific molecule comprises an Fc region.

[0276] EA31. The method of EA30, wherein said Fc region is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0277] EA32. The method of EA30 or EA31, wherein the PD-1 x LAG-3 bispecific molecule, or the PD-L1 x LAG-3 bispecific molecule, further comprises a hinge domain.

[0278] EA33. The method of EA32, wherein the Fc region and the hinge domain are both of the IgG4 isotype, and the hinge domain comprises a stabilizing mutation.

[0279] EA34. The Fc region is a variant Fc region comprising: (a) one or more amino acid modifications that reduce the affinity of the variant Fc region for FcγR; and / or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc region; The method of any one of EA30 to EA33, wherein the mutant Fc region comprises:

[0280] EA35. The method of EA34, wherein the modification that reduces the affinity of the variant Fc region for an FcγR comprises the substitutions L234A; L235A; or L234A and L235A, and the numbering is that of the EU index as set forth in Kabat.

[0281] EA36. The method of EA34 or EA35, wherein the modification that increases the serum half-life of the variant Fc region comprises the following substitutions: M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, wherein the numbering is that of the EU index as set forth in Kabat.

[0282] EA37. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA36, wherein the PD-1 x LAG-3 bispecific molecule comprises two polypeptide chains of SEQ ID NO: 59 and two polypeptide chains of SEQ ID NO: 60.

[0283] EA38. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 120 mg.

[0284] EA39. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 300 mg.

[0285] EA40. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 400 mg.

[0286] EA41. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg.

[0287] EA42. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 800 mg.

[0288] EA43. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA42, wherein the flat dose is administered approximately once every two weeks.

[0289] EA44. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA42, wherein the flat dose is administered approximately once every three weeks.

[0290] EA45. Any one of the methods EA1-EA6, EA10, EA14-EA21, EA25-EA37, EA40, and EA43, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 400 mg about once every two weeks.

[0291] EA46. Any one of the methods EA1-EA6, EA10, EA14-EA21, EA25-EA37, EA41, and EA43, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg approximately once every two weeks.

[0292] EA47. Any one of the methods EA1-EA6, EA10, EA14-EA21, EA25-EA37, EA41, and EA44, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg about once every three weeks.

[0293] EA48. Any one of the methods EA1-EA6, EA10, EA14-EA21, EA25-EA37, EA42, and EA44, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 800 mg about once every three weeks.

[0294] EA49. The method of any one of EA1-EA6, EA10, EA14-EA21, and EA25-EA48, wherein the PD-1×LAG-3 bispecific molecule or the PD-L1×LAG-3 bispecific molecule is administered by intravenous (IV) infusion.

[0295] EA50. The method of EA49, wherein the intravenous (IV) infusion is over a period of 30 to 240 minutes.

[0296] EA51. The method of EA49, wherein the intravenous (IV) infusion is over a period of about 30 to 90 minutes.

[0297] EA52. The above cancers are: adrenal gland cancer, AIDS-related cancer, alveolar soft tissue sarcoma, anal cancer (including squamous cell carcinoma of the anal canal (SCAC)), bladder cancer, bone cancer, brain and spinal cord cancer, breast cancer (HER2 + Breast Cancer, Younger including triple-negative breast cancer (TNBC), carotid bulb tumor, cervical cancer (including HPV-associated cervical cancer), chondrosarcoma, chordoma, chromophobe clear cell renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, endometrial cancer (including unselected endometrial cancer, MSI-high endometrial cancer, dMMR endometrial cancer, and / or POLE exonuclease domain mutation-positive endometrial cancer), Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, gallbladder or bile duct cancer (including cholangiocarcinomabile duct cancer), gastric cancer, esophagogastric junction (GEJ) cancer, pregnancy trophoblastic disease, germ cell tumors, glioblastoma, head and neck cancer (including squamous cell carcinoma of the head and neck (SCCHN)), hematologic malignancies, hepatocellular carcinoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (including acute myeloid leukemia), liposarcoma / malignant lipomatous tumor, liver cancer (including hepatocellular carcinoma (HCC)), lymphoma (including diffuse large B-cell lymphoma (DLBCL) and non-Hodgkin's lymphoma (NHL)), lung cancer (including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)), medulloblastoma, melanoma (including uveal melanoma), meningioma, any one of methods EA1 to EA51, wherein the cancer is selected from the group consisting of: leukemia, leukemia (including mesothelioma, pharyngeal carcinoma), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pharyngeal cancer, pheochromocytoma, pituitary tumor, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), posterior uveal melanoma, renal metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumor of childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer, and uterine cancer.

[0298] EA53. The above cancers are: anal cancer, breast cancer, bile duct cancer, cervical cancer, colorectal cancer. The method of EA52, wherein the cancer is intestinal cancer, endometrial cancer, gastric cancer, GEJ cancer, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, or prostate cancer.

[0299] EA54. The above cancer is HER2 + The method of EA52 or EA53, wherein the cancer is breast cancer or TNBC.

[0300] EA55. The above cancer is bile duct cancer. 2 or EA53 method.

[0301] EA56. The method of EA52 or EA53, wherein said cancer is HPV-associated cervical cancer.

[0302] EA57. The method of EA52 or EA53, wherein said cancer is SCCHN.

[0303] EA58. The method of EA52 or EA53, wherein said cancer is HCC.

[0304] EA59. The method of EA52 or EA53, wherein said cancer is SCLC or NSCLC.

[0305] EA60. The method of EA52 or EA53, wherein said cancer is NHL.

[0306] EA61. The method of EA52 or EA53, wherein said cancer is prostate cancer.

[0307] EA62. The method of EA52 or EA53, wherein said cancer is gastric cancer.

[0308] EA63. The TA-binding molecule comprises a light chain variable domain (VL HER2 ) and heavy chain variable domain (VH HER2 ), a HER2 binding molecule comprising a HER2 binding domain comprising: (A) The light chain variable domain (VL HER2 ) is the CDR of SEQ ID NO: 61L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain of margetuximab. N (VH HER2 ) is the CDR of SEQ ID NO: 66 H 1. CDR H 2, and CDR H Marge including 3 Is it the heavy chain variable domain of tuximab; (B) The light chain variable domain (VL HER2 ) is the CDR of trastuzumab L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) is the CDR of trastuzumab H 1. CDR H 2, and CDR H Contains 3; (C) the light chain variable domain (VL HER2 ) is the CDR of pertuzumab L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) is the CDR of pertuzumab H 1. CDR H 2, and CDR H Contains 3; or (D) the light chain variable domain (VL HER2 ) are the CDRs of hHER2 MAB-1 L 1. C DR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) are the CDRs of hHER2 MAB-1 H 1. CDR H 2, and CDR H Any one of EA2 to EA62, including 3 Two ways.

[0309] EA64. The method of any one of EA2 to EA63, wherein said HER2 binding molecule is an anti-HER2 antibody.

[0310] EA65. The method of EA64, wherein the anti-HER2 antibody is margetuximab, and the method comprises administering margetuximab at a dosage of about 6 mg / kg to about 18 mg / kg about once every three weeks.

[0311] EA66. The method of EA65, wherein margetuximab is administered about once every three weeks at a dose selected from the group consisting of: about 6 mg / kg, about 10 mg / kg, about 15 mg / kg, and about 18 mg / kg.

[0312] EA67. The method of EA65 or EA66, wherein the PD-1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg about once every three weeks, and margetuximab is administered at a dose of about 15 mg / kg about once every three weeks.

[0313] EA68. The method of any one of EA63 to EA67, wherein the method further comprises administering a chemotherapeutic agent.

[0314] EA69. The method of any one of EA63-EA68, wherein said cancer is a HER2-expressing cancer.

[0315] EA70. The method of EA69, wherein said HER2-expressing cancer is breast cancer, metastatic breast cancer, bladder cancer, gastric cancer, GEJ cancer, ovarian cancer, pancreatic cancer, or stomach cancer.

[0316] EA71. The TA-binding molecule is a B7-H3-binding molecule comprising a B7-H3-binding domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH): The VL is the CDR of SEQ ID NO: 71 L 1. CDR L 2, and CDR L 3, including The VH is the CDR of SEQ ID NO: 76 H 1. CDR H 2, and CDR H EA2~E, including 3 Either one of the A62 methods.

[0317] EA72. The method of any one of EA2 to EA62, and EA71, wherein said TA binding molecule is enoblituzumab.

[0318] EA73. The method of EA72, wherein said enoblituzumab is administered at a dosage of about 6 mg / kg to about 18 mg / kg about once every three weeks.

[0319] EA74. The method of EA73, wherein enoblituzumab is administered about once every three weeks at a dose selected from the group consisting of about 6 mg / kg, about 10 mg / kg, about 15 mg / kg, and about 18 mg / kg.

[0320] EA75. The method of EA73 or EA74, wherein the PD-1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg about once every three weeks and enoblituzumab is administered at a dose of about 15 mg / kg about once every three weeks.

[0321] EA76. The method of any one of EA71-EA75, wherein said cancer is a B7-H3-expressing cancer.

[0322] EA77. The method of EA76, wherein the B7-H3 expressing cancer is: anal cancer, SCAC, breast cancer, TNBC, head and neck cancer, SCCHN, lung cancer, NSCLC, melanoma, uveal melanoma, prostate cancer, mCRPC.

[0323] EA78. The method of any one of EA2-EA77, wherein said TA binding molecule is administered by intravenous (IV) infusion.

[0324] EA79. The method of EA78, wherein the IV infusion is over a period of about 30 to 240 minutes.

[0325] EA80. The method of EA78, wherein the IV infusion is over a period of about 30 to 90 minutes.

[0326] EA81. The PD-1 x LAG-3 bispecific molecule and the TA-binding molecule are distinct The method of any one of EA1 to EA6, EA10, EA14 to EA21, and EA25 to EA80, wherein the separate compositions are administered to the subject in parallel in pharmaceutical compositions, the separate compositions being administered within a 24 hour period.

[0327] EA82. Any one of the methods EA1-EA6, EA10, EA14-EA21, and EA25-EA80, wherein the PD-1 x LAG-3 bispecific molecule and the TA-binding molecule are administered to the subject sequentially in separate pharmaceutical compositions, the second composition administered being administered at least 24 hours after the first composition administered.

[0328] EA83. Any one of methods EA1-EA82, wherein the subject has been previously treated with CAR T cell therapy.

[0329] EA84. Any one of the methods EA1-EA6, EA10, EA14-EA21, and EA25-EA82, wherein the PD-1×LAG-3 bispecific molecule or the PD-L1×LAG-3 bispecific molecule is administered concurrently with or following CAR T-cell therapy treatment.

[0330] EA85. The method of any one of EA1 to EA84, wherein cells expressing LAG-3 are present in a biopsy of said cancer prior to treatment.

[0331] EA86. The method of any one of EA1 to EA85, wherein cells expressing PD-1 are present in a biopsy of said cancer prior to treatment.

[0332] EA87. Any one of the methods of EA1-EA86, wherein co-expression of PD-1 and LAG-3 in a pre-treatment biopsy of said cancer indicates that said patient is a candidate for said method.

[0333] EA88. The method of EA87, wherein the expression is gene expression.

[0334] EA89. Any one of the methods EA1-EA88, wherein said cancer expresses PD-L1 on the surface of cells of less than 1% prior to said treatment as determined using a total positive score (CPS) or tumor proportion score (TPS).

[0335] EA90. Any one of methods EA1-EA89, wherein said subject has previously failed to respond or had an inadequate response to at least one prior treatment.

[0336] EA91. The method of EA90, wherein at least one of the prior treatments was treatment with a PD-1 binding molecule or a PD-L1 binding molecule.

[0337] EB1. A PD-1 x LAG-3 bispecific molecule for use in treating cancer in a subject, wherein the PD-1 x LAG-3 bispecific molecule is for administration at a flat dose of about 120 mg to about 800 mg.

[0338] EB2. The PD-1 x LAG-3 bispecific molecule of EB1, wherein the cancer is characterized by expression of a TA, and the PD-1 x LAG-3 bispecific molecule is used in combination with a TA-binding molecule.

[0339] EB3. For treating cancer characterized by expression of the above TAs: (I) TA-binding molecules; (II)(a) a PD-1 x LAG-3 bispecific molecule; or (b) a combination of a PD-1-binding molecule and a LAG-3-binding molecule; or (c) a PD-L1 x LAG-3 bispecific molecule; or (d) a combination of a PD-L1-binding molecule and a LAG-3-binding molecule; A combination of.

[0340] EB4. The PD-1 x LAG-3 bispecific molecule of EB2, or the combination of EB3, or the combination of EB7, wherein the TA-binding molecule comprises an ADCC-enhancing Fc domain.

[0341] EB5.(a) each of the above molecules is in a separate composition; or (b) each of the above molecules is in the same composition; or (c) the PD-1-binding molecule and the LAG-3-binding molecule are in the same composition, and the TA-binding molecule is in a separate composition; or (d) A PD-1×LAG-3 bispecific molecule of EB2 or EB4, or a combination of any one of EB2-4, or a combination of EB7 or 8, wherein the PD-L1-binding molecule and the LAG-3-binding molecule are in the same composition and the TA-binding molecule is in a separate composition.

[0342] EB6. The PD-1 x LAG-3 bispecific molecule of any one of EB2 and EB4-5, or a combination of any one of EB3-5, or a combination of any one of EB7-EB9, wherein the TA-binding molecule is an antibody.

[0343] EB7. The combination of any one of EB3 to EB6, wherein the PD-1 binding molecule is an antibody.

[0344] EB8. The combination of any one of EB3 to EB6, wherein the PD-L1 binding molecule is an antibody.

[0345] EB9. Any one of the combinations of EB3 to EB8, wherein the LAG-3 binding molecule is an antibody.

[0346] EB10. Any one of the combinations of EB3-EB6, using the TA-binding molecule and the PD-1 x LAG-3 bispecific molecule.

[0347] EB11. Any one of the combinations EB3-EB9, wherein the TA-binding molecule and the PD-1-binding molecule are used in combination with a LAG-3-binding molecule.

[0348] EB12. Any one of the combinations of EB3 to EB6, using the TA-binding molecule and the PD-L1xLAG-3 bispecific molecule.

[0349] EB13. Any one of the combinations EB3-EB9, wherein the TA-binding molecule and the PD-L1-binding molecule are used in combination with a LAG-3-binding molecule.

[0350] EB14. The ADCC-enhancing Fc domain is: (a) artificial glycoforms; and / or (b) Amino acid substitutions relative to the wild-type Fc region any one of PD-1 x LAG-3 bispecific molecules EB4 to EB6, or any one combination of EB4 to EB9.

[0351] EB15. The ADCC-enhancing Fc domain is an artificial glycoform that is a complex N-glycosidically linked glycan that does not contain fucose and / or contains a bisecting O-GlcNAc. a PD-1 x LAG-3 bispecific molecule of EB14, or a combination of EB14, comprising:

[0352] EB16. The PD-1 x LAG-3 bispecific molecule of EB14 or EB15, or the combination of EB14 or EB15, wherein the ADCC-enhancing Fc domain comprises one or more amino acid substitutions selected from F243L, R292P, Y300L, V305I, I332E, and P396L.

[0353] EB17. The ADCC-enhancing Fc domain comprises an amino acid substitution selected from the group consisting of: (a) one substitution selected from the group consisting of: F243L, R292P, Y300L, V305I, I332E, and P396L; (b) two substitutions selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; (3) R292P and V305I; and (4) S239D and I332E; (c) three substitutions selected from the group consisting of: (1) F243L, R292P and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P, and P396L; and (4) R292P, V305I and P396L; (d) four substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, and P396L; and (2) F243L, R292P, V305I, and P396L; or (e) five substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I, and P396L; and (2) L235V, F243L, R292P, Y300L and P396L any one of the PD-1 x LAG-3 bispecific molecules EB14 to EB16, or any one of the combinations EB14 to EB16, wherein the numbering is that of the EU index as set forth in Kabat.

[0354] EB18. The PD-1 x LAG-3 bispecific molecule of any one of EB14 to EB16, or any one of the combinations EB14 to EB16, wherein the ADCC-enhancing Fc domain comprises the following amino acid substitutions: L235V, F243L, R292P, Y300L, and P396L, where the numbering is that of the EU index as set forth in Kabat.

[0355] EB19. The PD-1 x LAG-3 bispecific molecule of any one of EB14 to EB16, or any one of the combinations of EB14 to EB16, wherein the ADCC-enhancing Fc domain comprises the amino acid substitutions S239D and I332E, and the numbering is that of the EU index as set forth in Kabat.

[0356] EB20. The TA is selected from Table 6A or Table 6B: any one of PD-1 x LAG-3 bispecific molecules: EB2, EB4-EB6, and EB14-EB19, or any one combination of EB3-EB19.

[0357] EB21. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4-EB6, and EB14-EB19, or any one of the combinations EB3-EB19, wherein the TA-binding molecule comprises the VL and VH domains of an antibody selected from Table 7.

[0358] EB22. The PD-1 binding molecule is: (a) a PD-1 VL domain comprising the amino acid sequence of SEQ ID NO: 35, and a PD-1 VH domain comprising the amino acid sequence of SEQ ID NO: 39; (b) the VH and VL domains of an anti-PD-1 antibody selected from Table 1; or (c) the light chain and heavy chain of an anti-PD-1 antibody selected from Table 1 any one of EB3 to EB7, EB9, EB11, and EB14 to EB21, which are antibodies comprising the above.

[0359] EB23. The PD-L1 binding molecule is: (a) a PD-L1 VL domain comprising the amino acid sequence of SEQ ID NO: 43, and a PD-L1 VH domain comprising the amino acid sequence of SEQ ID NO: 49; (b) the VH and VL domains of an anti-PD-L1 antibody selected from Table 2; or (c) the light chain and heavy chain of an anti-PD-L1 antibody selected from Table 2 any one combination of EB3 to EB6, EB8 to EB9, and EB13 to EB21, which are antibodies comprising the above.

[0360] EB24. The LAG-3 binding molecule is: (a) a LAG-3 VL domain comprising the amino acid sequence of SEQ ID NO: 51, and a LAG-3 VH domain comprising the amino acid sequence of SEQ ID NO: 55; (b) the VH and VL domains of an anti-LAG-3 antibody selected from Table 3; or (c) the light and heavy chains of an anti-LAG-3 antibody selected from Table 3 any one of EB3 to EB9, EB11, and EB13 to EB23, which are antibodies comprising the above.

[0361] EB25. The PD-1 x LAG-3 bispecific molecule described above: (a) a PD-1 VL domain comprising the amino acid sequence of SEQ ID NO: 35, and a PD-1 VH domain comprising the amino acid sequence of SEQ ID NO: 39, or the VH and VL domains of an anti-PD-1 antibody selected from Table 7; and / or (b) a LAG-3 VL domain comprising the amino acid sequence of SEQ ID NO: 51 and a LAG-3 VH domain comprising the amino acid sequence of SEQ ID NO: 55, or the VH and VL domains of an anti-LAG-3 antibody selected from Table 9; or (c) a bispecific antibody molecule selected from Tables 4 and 5 any one of EB2, EB4 to EB6, and EB14 to EB21, or any one combination of EB3 to EB6, EB10, and EB14 to EB21, comprising the PD-1 x LAG-3 bispecific molecule.

[0362] EB26. The PD-1 x LAG-3 bispecific molecule described above: (a) CDR of SEQ ID NO: 35 L 1. CDR L 2, and CDR L Light chain variable domain containing 3 (VL PD‐1 ) and the PD-1-specific CDR of SEQ ID NO: 39 H 1. CDR H 2, and CDR H 3, including the heavy chain variable domain (VH PD‐1 a PD-1 binding domain comprising: (b) CDR of SEQ ID NO: 51 L 1. CDR L 2, and CDR L Light chain variable domain containing 3 (VL LAG‐3 ) and the LAG-3 specific CDR of SEQ ID NO: 55 H 1. CDR H2, and CDR H 3, including the heavy chain variable domain (VH LAG‐3 ) and a LAG-3 binding domain any one of EB2, EB4 to EB6, and EB14 to EB21, or any one combination of EB3 to EB6, EB10, and EB14 to EB21, comprising the PD-1 x LAG-3 bispecific molecule.

[0363] EB27. The PD-1 x LAG-3 bispecific molecule: (a) two of the PD-1 binding domains described above; and (b) two of the above LAG-3 binding domains any one of EB2, EB4 to EB6, EB14 to EB21, and EB25 to EB26, or any one combination of EB3 to EB6, EB10, EB14 to EB21, and EB25 to EB26, comprising the PD-1 x LAG-3 bispecific molecule.

[0364] EB28. The PD-1 x LAG-3 bispecific molecule comprises a VL domain of SEQ ID NO: 35 and a VH domain of SEQ ID NO: 39; any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4 to EB6, EB14 to EB21, and EB25 to EB27; or any one combination of EB3 to EB6, EB10, EB14 to EB21, and EB25 to EB27.

[0365] EB29. The PD-1 x LAG-3 bispecific molecule comprises a VL domain of SEQ ID NO: 51 and a VH domain of SEQ ID NO: 39; any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4 to EB6, EB14 to EB21, and EB25 to EB28, or any one combination of EB3 to EB6, EB10, EB14 to EB21, and EB25 to EB28.

[0366] EB30. The PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule comprises an Fc Region; any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4 to EB6, EB14 to EB21, and EB25 to EB29, or any one combination of EB2 to EB6, EB10, EB12, EB14 to EB21, and EB25 to EB29.

[0367] EB31. The PD-1 x LAG-3 bispecific molecule of EB30, or a combination of EB30, wherein the Fc region is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0368] EB32. The PD-1 x LAG-3 bispecific molecule of EB30 or EB31, or a combination of EB30 or EB31, wherein the PD-1 x LAG-3 bispecific molecule, or the PD-L1 x LAG-3 bispecific molecule, further comprises a hinge domain.

[0369] EB33. The PD-1 x LAG-3 bispecific molecule of EB32, or the combination of EB32, wherein the Fc region and the hinge domain are both of the IgG4 isotype, and the hinge domain comprises a stabilizing mutation.

[0370] EB34. The Fc region is a variant Fc region comprising: (a) one or more amino acid modifications that reduce the affinity of the variant Fc region for FcγR; and / or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc region; a PD-1 x LAG-3 bispecific molecule of any one of EB30 to EB33, or a combination of any one of EB30 to EB33, wherein the variant Fc Region comprises:

[0371] EB35. A PD-1 x LAG-3 bispecific molecule of EB34, or a combination of EB34, wherein the modification that reduces the affinity of the variant Fc Region for FcγR comprises the substitutions L234A; L235A; or L234A and L235A, where numbering is that of the EU index as set forth in Kabat.

[0372] EB36. The PD-1 x LAG-3 bispecific antibody of EB34 or EB35, wherein the modification that extends the serum half-life of the variant Fc Region comprises substitutions of M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, where the numbering is that of the EU index as set forth in Kabat. A combination of EB34 or EB35.

[0373] EB37. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 through EB6, EB14 through EB21, and EB25 through EB36, or any one combination of EB3 through EB6, EB10, EB14 through EB21, and EB25 through EB36, wherein the PD-1 x LAG-3 bispecific molecule comprises two polypeptide chains of SEQ ID NO: 59 and two polypeptide chains of SEQ ID NO: 60.

[0374] EB38. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 through EB6, EB14 through EB21, and EB25 through EB37, or a combination of any one of EB3 through EB6, EB10, EB14 through EB21, and EB25 through EB37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration in a flat dose of about 120 mg.

[0375] EB39. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 through EB6, EB14 through EB21, and EB25 through EB37, or a combination of any one of EB3 through EB6, EB10, EB14 through EB21, and EB25 through EB37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration in a flat dose of about 300 mg.

[0376] EB40. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4-EB6, EB14-EB21, and EB25-EB37, or a combination of any one of EB3-EB6, EB10, EB14-EB21, and EB25-EB37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration in a flat dose of about 400 mg.

[0377] EB41. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 through EB6, EB14 through EB21, and EB25 through EB37, or a combination of any one of EB3 through EB6, EB10, EB14 through EB21, and EB25 through EB37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration in a flat dose of about 600 mg.

[0378] EB42. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4-EB6, EB14-EB21, and EB25-EB37, or a combination of any one of EB3-EB6, EB10, EB14-EB21, and EB25-EB37, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration in a flat dose of about 800 mg.

[0379] EB43. The above flat dose is for administration approximately once every two weeks of any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4 through EB6, EB14 through EB21, and EB25 through EB42, or any one of the combinations EB3 through EB6, EB10, EB14 through EB21, and EB25 through EB42.

[0380] EB44. The above flat dose is for administration approximately once every three weeks of any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4 through EB6, EB14 through EB21, and EB25 through EB42, or any one of the combinations EB3 through EB6, EB10, EB14 through EB21, and EB25 through EB42.

[0381] EB45. The PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration at a flat dose of about 400 mg approximately once every two weeks. , EB2, EB4 to EB6, EB14 to EB21, EB25 to EB37, EB40, and EB43, or a combination of any one of EB3 to EB6, EB10, EB14 to EB21, EB25 to EB37, EB40, and EB43.

[0382] EB46. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 through EB6, EB14 through EB21, EB25 through EB37, EB41, and EB43, or a combination of any one of EB3 through EB6, EB10, EB14 through EB21, EB25 through EB37, EB41, and EB43, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration about once every two weeks at a flat dose of about 600 mg.

[0383] EB47. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 through EB6, EB14 through EB21, EB25 through EB37, EB41, and EB44, or a combination of any one of EB3 through EB6, EB10, EB14 through EB21, EB25 through EB37, EB41, and EB44, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration about once every three weeks at a flat dose of about 600 mg.

[0384] EB48. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 through EB6, EB14 through EB21, EB25 through EB37, EB42, and EB44, or a combination of any one of EB3 through EB6, EB10, EB14 through EB21, EB25 through EB37, EB42, and EB44, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration about once every three weeks at a flat dose of about 800 mg.

[0385] EB49. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 through EB6, EB14 through EB21, and EB25 through EB48, or a combination of any one of EB3 through EB6, EB10, EB14 through EB21, and EB25 through EB48, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration by intravenous (IV) infusion.

[0386] EB50. The PD-1 x LAG-3 bispecific molecule of EB49, or a combination of EB49, administered intravenously (IV) over a period of 30 to 240 minutes.

[0387] EB51. The intravenous (IV) infusion of the PD-1 x LAG-3 bispecific molecule of EB49, or the combination of EB49, over a period of approximately 30 to 90 minutes.

[0388] EB52. The cancers listed above are: adrenal gland cancer, AIDS-related cancer, alveolar soft tissue sarcoma, anal cancer (including squamous cell carcinoma of the anal canal (SCAC)), bladder cancer, bone cancer, brain and spinal cord cancer, breast cancer (HER2+ Breast Cancer, Younger including triple-negative breast cancer (TNBC), carotid bulb tumor, cervical cancer (including HPV-associated cervical cancer), chondrosarcoma, chordoma, chromophobe clear cell renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, endometrial cancer (including unselected endometrial cancer, MSI-high endometrial cancer, dMMR endometrial cancer, and / or POLE exonuclease domain mutation-positive endometrial cancer), Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, gallbladder or bile duct cancer (including cholangiocarcinomabile duct cancer), gastric cancer, esophagogastric junction (GEJ) cancer, pregnancy Germ cell tumors, glioblastoma, head and neck cancer (including squamous cell carcinoma of the head and neck (SCCHN)), hematologic malignancies, hepatocellular carcinoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (including acute myeloid leukemia), liposarcoma / malignant lipomatous tumor, liver cancer (including hepatocellular carcinoma (HCC)) , lymphoma (including diffuse large B-cell lymphoma (DLBCL) and non-Hodgkin's lymphoma (NHL)), lung cancer (including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)), medulloblastoma, melanoma (including uveal melanoma), meningioma, Merkel cell carcinoma, mesothelioma (including mesothelial pharyngeal carcinoma), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, childhood cancer, peripheral nerve sheath tumor, pharyngeal cancer, pheochromocytoma, pituitary tumor, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), posterior uveal melanoma, renal metastasis, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumor of childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma, gastric cancer the PD-1×LAG-3 bispecific molecules of any one of EB2, EB4-EB6, EB14-EB21, and EB25-EB51, or any one combination of EB3-EB51, are selected from the group consisting of uterine cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer, and uterine cancer.

[0389] EB53. The PD-1 x LAG-3 bispecific molecule of EB52, or a combination of EB52, wherein the cancer is: anal cancer, breast cancer, bile duct cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, GEJ cancer, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, or prostate cancer.

[0390] EB54. The above cancers are HER2 + EB52 or EB53 PD-1 x LAG-3 bispecific molecules, or a combination of EB52 or EB53, for breast cancer or TNBC.

[0391] EB55. The above cancer is bile duct cancer (cholangiocarcinoma bile duct cancer), EB5 PD-1 x LAG-3 bispecific molecules of EB52 or EB53, or a combination of EB52 or EB53.

[0392] EB56. The PD-1 x LAG-3 bispecific molecule of EB52 or EB53, or a combination of EB52 or EB53, wherein the cancer is HPV-associated cervical cancer.

[0393] EB57. The PD-1 x LAG-3 bispecific molecule of EB52 or EB53, or a combination of EB52 or EB53, wherein the cancer is SCCHN.

[0394] EB58. The PD-1 x LAG-3 bispecific molecule of EB52 or EB53, or a combination of EB52 or EB53, wherein the cancer is HCC.

[0395] EB59. The PD-1 x LAG-3 bispecific molecule of EB52 or EB53, or a combination of EB52 or EB53, wherein the cancer is SCLC or NSCLC.

[0396] EB60. The PD-1 x LAG-3 bispecific molecule of EB52 or EB53, or the EB52 or EB53 combination, wherein the cancer is NHL.

[0397] EB61. The PD-1 x LAG-3 bispecific molecule of EB52 or EB53, or a combination of EB52 or EB53, wherein the cancer is prostate cancer.

[0398] EB62. The PD-1 x LAG-3 bispecific molecule of EB52 or EB53, or a combination of EB52 or EB53, wherein the cancer is gastric cancer.

[0399] EB63. The TA-binding molecule comprises a light chain variable domain (VL HER2 ) and heavy chain variable domain (VH HER2 ), a HER2 binding molecule comprising a HER2 binding domain comprising: (A) The light chain variable domain (VL HER2 ) is the CDR of SEQ ID NO: 61 L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain of margetuximab. N (VH HER2 ) is the CDR of SEQ ID NO: 66 H 1. CDR H 2, and CDR H Marge including 3 Is it the heavy chain variable domain of tuximab; (B) The light chain variable domain (VL HER2 ) is the CDR of trastuzumab L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) is the CDR of trastuzumab H 1. CDR H 2, and CDR H Contains 3; (C) the light chain variable domain (VL HER2 ) is the CDR of pertuzumab L 1. CDR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) is the CDR of pertuzumab H 1. CDRH 2, and CDR H Contains 3; or (D) the light chain variable domain (VL HER2 ) are the CDRs of hHER2 MAB-1 L 1. C DR L 2, and CDR L 3, and the heavy chain variable domain (VH HER2 ) are the CDRs of hHER2 MAB-1 H 1. CDR H 2, and CDR H 3, including EB2, EB4-EB6, EB 14 to EB21, and any one of PD-1 x LAG-3 bispecific molecules EB25 to EB62, or any one combination of EB3 to EB62.

[0400] EB64. The HER2 binding molecule is an anti-HER2 antibody. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4 to EB6, EB14 to EB21, and EB25 to EB63, or a combination of any one of EB3 to EB63.

[0401] EB65. The PD-1 x LAG-3 bispecific molecule of EB64, or a combination of EB64, wherein the anti-HER2 antibody is margetuximab, and the margetuximab is for administration about once every three weeks at a dosage of about 6 mg / kg to about 18 mg / kg.

[0402] EB66. Margetuximab: The PD-1 x LAG-3 bispecific molecule of EB65, or the combination of EB65, for administration about once every three weeks at a dose selected from the group consisting of about 6 mg / kg, about 10 mg / kg, about 15 mg / kg, and about 18 mg / kg.

[0403] EB67. The PD-1 x LAG-3 bispecific molecule of EB65 or EB66, or a combination of EB65 or EB66, wherein the PD-1 x LAG-3 bispecific molecule is for administration about once every three weeks at a flat dose of about 600 mg and the margetuximab is for administration about once every three weeks at a dose of about 15 mg / kg.

[0404] EB68. Any one of the PD-1 x LAG-3 bispecific molecules of EB63 to EB67, or any one of the combinations EB63 to EB67, wherein the PD-1 x LAG-3 bispecific molecule or combination is for administration with a chemotherapeutic agent.

[0405] EB69. The PD-1 x LAG-3 bispecific molecule of any one of EB63 to EB68, or a combination of any one of EB63 to EB68, wherein the cancer is a HER2-expressing cancer.

[0406] EB70. The PD-1 x LAG-3 bispecific molecule of EB69, or a combination of EB69, wherein the HER2-expressing cancer is breast cancer, metastatic breast cancer, bladder cancer, gastric cancer, GEJ cancer, ovarian cancer, pancreatic cancer, or stomach cancer.

[0407] EB71. The TA-binding molecule is a B7-H3-binding molecule comprising a B7-H3-binding domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH): The VL is the CDR of SEQ ID NO: 71 L 1. CDR L 2, and CDR L 3, including The VH is the CDR of SEQ ID NO: 76 H 1. CDR H 2, and CDR H 3, including EB2, E any one of PD-1 x LAG-3 bispecific molecules B4 to EB6, EB14 to EB21, or EB25 to EB62, or any one combination of EB3 to EB62.

[0408] EB72. The TA-binding molecule is enoblituzumab. Any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4 to EB6, EB14 to EB21, EB25 to EB62, and EB71, or any one combination of EB3 to EB62, and EB71.

[0409] EB73. The PD-1 x LAG-3 bispecific molecule of EB72, or the combination of EB72, wherein the enoblituzumab is for administration at a dosage of about 6 mg / kg to about 18 mg / kg about once every three weeks.

[0410] EB74. The PD-1 x LAG-3 bispecific molecule of EB73, or the combination of EB73, wherein enoblituzumab is for administration about once every three weeks at a dose selected from the group consisting of about 6 mg / kg, about 10 mg / kg, about 15 mg / kg, and about 18 mg / kg.

[0411] EB75. The combination of EB73 or EB74 PD-1 x LAG-3 bispecific molecule, or EB73 or EB74, wherein the PD-1 x LAG-3 bispecific molecule is for administration about once every three weeks at a flat dose of about 600 mg and enoblituzumab is for administration about once every three weeks at a dose of about 15 mg / kg.

[0412] EB76. Any one of the PD-1 x LAG-3 bispecific molecules EB71 to EB75, or any one combination of EB71 to EB75, wherein the cancer is a B7-H3-expressing cancer.

[0413] EB77. The B7-H3 expressing cancers are: anal cancer, SCAC, breast cancer, TNBC, head and neck cancer, SCCHN, lung cancer, NSCLC, melanoma, uveal melanoma, prostate cancer, mCRPC; PD-1 x LAG-3 bispecific molecule of EB76, or combination of EB76.

[0414] EB78. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4-EB6, EB14-EB21, and EB25-EB77, or a combination of any one of EB3-EB77, wherein the TA-binding molecule is for administration by intravenous (IV) infusion.

[0415] EB79. The PD-1 x LAG-3 bispecific molecule of EB78, or a combination of EB78, administered by IV infusion over a period of approximately 30 to 240 minutes.

[0416] EB80. EB78, a PD-1 x LAG-3 bispecific molecule, or a combination of EB78, administered by IV infusion over a period of approximately 30 to 90 minutes.

[0417] EB81. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4-EB6, EB14-EB21, and EB25-EB80, or a combination of any one of EB3-EB6, EB10, EB14-EB21, and EB25-EB80, wherein the PD-1 x LAG-3 bispecific molecule and the TA-binding molecule are for concurrent administration to the subject in separate pharmaceutical compositions, wherein the separate compositions are for administration within a 24 hour period.

[0418] EB82. Any of EB2, EB4-EB6, EB14-EB21, and EB25-EB80, wherein the PD-1 x LAG-3 bispecific molecule and the TA-binding molecule are for sequential administration to the subject in separate pharmaceutical compositions, the second composition being for administration at least 24 hours after the first composition. Any one of the PD-1 x LAG-3 bispecific molecules, or any one of the combinations of EB3 to EB6, EB10, EB14 to EB21, and EB25 to EB80.

[0419] EB83. The subject has been previously treated with CAR T-cell therapy, any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4-EB6, EB14-EB21, and EB25-EB82, or any one of the combinations EB3-EB6, EB10, EB14-EB21, and EB25-EB82.

[0420] EB84. The PD-1 x LAG-3 bispecific molecule of any one of EB2, EB4-EB6, EB14-EB21, and EB25-EB83, or a combination of any one of EB3-EB6, EB10, EB14-EB21, and EB25-EB82, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is for administration in conjunction with or following CAR T-cell therapy treatment.

[0421] EB85. Any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4-EB6, EB14-EB21, and EB25-EB84, or any one combination of EB3-EB84, where LAG-3-expressing cells are present in a biopsy from the above cancer prior to treatment.

[0422] EB86. Any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4 to EB6, EB14 to EB21, and EB25 to EB85, or any one combination of EB3 to EB85, where PD-1-expressing cells are present in a biopsy from the above cancer prior to treatment.

[0423] EB87. Any one of the PD-1 x LAG-3 bispecific molecules EB1 to EB86, or any one of the combinations EB3 to EB86, wherein co-expression of PD-1 and LAG-3 in a pre-treatment biopsy of the cancer indicates that the patient is a candidate for the method.

[0424] EB88. Expression is gene expression, PD-1 x LAG-3 bispecific molecule of EB87, or combination of EB87.

[0425] EB89. Any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4-EB6, EB14-EB21, and EB25-EB88, or any one combination of EB3-EB88, wherein said cancer prior to said treatment expresses PD-L1 on the surface of cells of less than 1% as determined by the combined positive score (CPS) or tumor proportion score (TPS).

[0426] EB90. The subject has previously failed to respond or had an inadequate response to at least one prior treatment with any one of the PD-1 x LAG-3 bispecific molecules EB2, EB4-EB6, EB14-EB21, and EB25-EB89, or any one of the combinations EB3-EB89.

[0427] EB91. The PD-1 x LAG-3 bispecific molecule of EB90, or the combination of EB88, where at least one of the previous treatments was treatment with a PD-1 or PD-L1 binding molecule. [Example]

[0428] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Where specific materials are mentioned, this is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art will be able to produce equivalents without the exercise of inventive faculty and without departing from the scope of the invention. A means or a reaction can be developed.

[0429] Example 1 Phase I trials A Phase I clinical study is underway to determine patient tolerability of DART-I (a bispecific molecule that binds to PD-1 and LAG-3, also known as MGD013 and tebotelimab). The study includes a dose escalation phase and a cohort expansion phase. The study has been approved by the Institutional Review Boards at each clinical site, and all patients have signed written informed consent.

[0430] For the initial dose escalation and dose expansion cohorts, DART-I was administered once every 2 weeks (Q2W). For the purposes of this study, an 8-week (56-day) cycle was used, in which DART-I was administered Q2W beginning on day 1 of each 2-week period of the first cycle (i.e., on days 1, 15±1, 29±1, and 43±1), and Q2W beginning on day 1±1 of each subsequent cycle. Patients may receive multiple 8-week Q2W treatment cycles depending on their tolerability and response to treatment in this study.

[0431] In further expansion cohorts, DART-I will be administered once every three weeks (Q3W). For the purposes of this study, three-week cycles (21 days each) will be used. DART-I will be administered on day 1 of the first cycle and days 1 ± 3 of each subsequent cycle. Patients may receive multiple three-week (Q3W) treatment cycles depending on their tolerability and response to treatment in this study.

[0432] In the combination expansion cohort, DART-I and the anti-HER2 antibody margetuximab (a TA-binding molecule with an ADCC-enhancing Fc domain) will both be administered once every three weeks (Q3W). For the purposes of this study, 3-week cycles (21 days each) will be used, in which DART-I and margetuximab will be administered on day 1 of the first cycle and days 1 ± 3 of each subsequent cycle. Patients may receive multiple 3-week Q3W treatment cycles depending on their tolerability and response to treatment in this study.

[0433] In these studies, doses of DART-I are diluted in saline to concentrations ranging from 0.12 mg / mL to 6.4 mg / mL and administered via an IV line using a commercially available syringe or infusion pump over approximately 60 to 75 minutes.

[0434] In these studies, margetuximab doses are diluted in saline to concentrations ranging from 2.4 to 7.2 mg / mL and administered by IV infusion over approximately 30 to 120 minutes using a commercially available syringe or infusion pump.

[0435] Antitumor activity was assessed using the conventional Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 (Eisenhauer, EA, et al. (2009) “New Response Evaluation Criteria in Solid Tumors: Revised RECIST Guideline (Version 1.1)” Eur. J. Cancer. 45(2):228-247); Immune-related Response Evaluation Criteria in Solid Tumors (irRECIST)" (Wolchok, JD, et al., (2009) "Guidelines For The Evaluation Of Immune Therapy Activity In Solid Tumors: Immune-Related Response Criteria." Clin. Cancer Res, 15:7412-7420); or Revised International Working Group criteria for response assessment, if applicable (i.e., Lugano classification; Cheson, BD, et al., (2014) "Recommendations For Initial Evaluation, Staging, And Response Assessment Of Hodgkin And Non-Hodgkin Lymphoma: The The stenosis is evaluated using the Lugano Classification. J. Clin. Oncol, 32:3059-3068.

[0436] In the dose-escalation phase, successively escalating flat doses from 1 mg up to 1200 mg were administered Q2W to consecutive cohorts of 1 to 6 patients, each evaluated (Table 8). Patients who were deemed ineligible for dose escalation at various dose levels were replaced. Additional patients were also enrolled at multiple dose levels of interest to gain additional clinical experience. The dose-escalation phase enrolled patients with unresectable, locally advanced, or metastatic solid tumors, regardless of histology. Forty-seven patients (49% experienced checkpoints) were treated with dose escalation, and the maximum tolerated dose was not defined.

[0437] [Table 9]

[0438] Based on the overall clinical data from the dose escalation phase, including but not limited to observed clinical activity, peripheral receptor occupancy, and pharmacokinetics (PK), a dose of 600 mg administered Q2W was initially selected as the dosing regimen to be evaluated in the cohort expansion phase.

[0439] Patients with distinct malignancies (including NSCLC (pre-checkpoint therapy and checkpoint-naive cohorts); SCCHN (pre-checkpoint therapy and checkpoint-naive cohorts); SCLC; cholangiocarcinoma bile duct cancer; HCC; cervical cancer; TNBC; epithelial ovarian cancer (EOC); DLBCL; and gastric cancer) will be treated with DART-I administered at a flat dose of 600 mg Q2W in the initial cohort of the cohort expansion phase. Based in part on pharmacokinetic (PK) and receptor occupancy (RO) data detailed below, additional cohorts in the cohort expansion phase (initially patients with gastric cancer or EOC) will be treated with DART-I administered at a flat dose of 600 mg Q3W.

[0440] Another cohort enrolling patients with advanced or metastatic solid tumors expressing the HER2 tumor antigen (i.e., HER2+ solid tumors, particularly HER2+ gastric or breast cancer) will receive DART-I and margetuximab sequentially on the same day. DART-I (300 mg or 600 mg) will be administered followed by margetuximab (15 mg / kg) Q3W. This cohort followed a conventional 3+3 approach, beginning with enrolling three patients at the 300 mg dose level of DART-I, followed by patients treated with DART-I at the 600 mg dose level.

[0441] Pharmacokinetics (PK) Ongoing studies have demonstrated that DA in the dosage regimen range of 1 to 1200 mg Q2W. The pharmacokinetic profile of RT-I was evaluated. Serum PK samples were collected (i) pre-dose, (ii) end of infusion (EO) for the first dose on Day 1 of Cycles 1-2. (i), and (iii) 2, 4, 24, 72, and 168 hours after the start of infusion. Additional serum PK samples were collected pre-dose and EOI for each additional dose administered during cycles 1-2, and concentrations of DART-I in human serum were measured using ELISA. Briefly, assay plates were coated overnight with 2 μg / mL of capture antibody (anti-idiotypic antibody, "anti-ID," recognizing the LAG-3 domain of DART-I). Nonspecific sites were blocked with 1X phosphate buffered saline containing 0.1% Tween-20. 0.5% bovine serum albumin (BSA) in PBS (PBS) After blocking with , the plate is incubated with DART-I standard calibrators, quality controls, and test samples. DART-I present in standard calibrators, quality controls, and test samples is captured. Captured DART-I is detected by sequential addition of 0.25 μg / mL 2A5-biotin (biotinylated anti-EK coil antibody) followed by a 1:10,000 dilution of streptavidin-HRP. Bound HRP activity is quantified by luminescence light generation with ELISA PICO substrate. Luminescence light intensity is measured as relative light units (RLU) using a Victor X4 plate reader. DA A standard curve is generated by fitting the RLU signals from the DART-I standards to a four-parameter logistic model: 1 / y, which relates the light intensity to the concentration of DART-I. 2 Determined by interpolation from the standard curve using a four-parameter curve fit with weighting.

[0442] A preliminary PK compartmental modeling approach was used to analyze the data using the WinNonlin PK analysis program (Phoenix® 64 WinNonlin®, Version 8.0, Certara Inc., Princeton, NJ). The model used was one or two open compartments, with a weighting factor of the reciprocal of the predicted concentration squared. This model was fitted to the first dose data from cycle 1, day 1 (C1D1), and initial estimates were generated by WinNonlin.

[0443] Forty-five subjects (all dosed Q2W) were evaluable for preliminary PK analysis (one patient each at 1 and 3 mg Q2W, four patients at 10 mg Q2W, five patients at 30 mg Q2W, six patients at 120 mg Q2W, nine patients at 400 mg Q2W, eight patients at 600 mg Q2W, seven patients at 800 mg Q2W, and four patients at 1200 mg Q2W). The PK profiles are presented in Figure 2.

[0444] PK parameters are summarized by treatment in Table 9. These results demonstrate a dose-related increase in exposure to the first dose of DART-I. Over the dose range of 1 to 1200 mg, max increased proportionally with dose (slope: 0.9 85 [90% confidence interval (CI): 0.949~1.022]), AUC of the first dose (INF) increased more than proportionally with dose (slope: 1.3 45 [90% CI: 1.294-1.397]). Over the dose range of 1-1200 mg, total body clearance (CL) values ​​decreased with increasing dose, and the steady-state volume of distribution (V ss ), and elimination half-life (t 1 / 2 ) values ​​increased with increasing dose. However, over the dose range of 400 to 1200 mg, CL, V ss , and t 1 / 2 The effect did not appear to be dose-dependent, although there was a slight trend with increasing dose. The mean half-life of DART-I was approximately 11 days, and the volume of distribution indicated that DART-I distribution was blood volume limited.

[0445] [Table 10]

[0446] Abbreviations: AUC(INF) = area under the serum drug concentration-time curve (time point zero to infinity); C1D1 = day 1 of cycle 1; Cmax = maximum observed serum concentration; CL = total body clearance; CV = coefficient of variation; GeoMean = geometric mean; N = number of patients; NR = not reported; Q2W = once every 2 weeks; SD = standard deviation; t1 / 2 = elimination half-life; Vss = steady-state volume of distribution

[0447] Pharmacodynamics (PD) The receptor occupancy (RO) profile of DART-I was evaluated over a dose range of 1 to 1200 mg Q2W. The RO of DART-I in each sample was determined by fluorescence-activated cell sorting (FACS). Briefly, five aliquots of whole blood per sample were distributed into five 12 x 75 mm tubes. Two of these aliquots were spiked with DART-I, and one spiked sample was used for each RO panel. After a 30-minute incubation at room temperature (RT), all aliquots were treated with red blood cell lysis buffer (BD Biosciences) for 15 minutes at room temperature in the dark, followed by centrifugation at 1200 rpm for 5 minutes. The supernatant was removed, and the leukocyte-containing cell pellet was washed with 2 ml of FBS staining buffer (BD Biosciences). Two aliquots (one spiked) were stained with antibody panel 1, two aliquots (one spiked) with antibody panel 2 (see Table 10), and one aliquot was stained with the appropriate isotype control in a total volume of 100 μL for 30 minutes at room temperature in the dark. Samples were washed twice with 2 mL of FACS buffer. 0.2 μg of streptavidin, R-phycoerythrin conjugate (SAPE, Life Technologies) was added to the antibody panel 2 aliquot, and the mixture was mixed and incubated for 30 minutes at room temperature in the dark, followed by a single wash with 2 mL of FACS buffer. Cells were resuspended in 200 μL of staining buffer with or without DAPI (0.1 μg / mL) (panel 1 and 2 samples) or without DAPI (isotype samples) and acquired on a FACS Canto II after 10 minutes. Geometric Mean Fluorescent Intensity (gMFI) is recorded for the entire CD4+ or CD8+ population in the IgG4 or EK channel at all time points. The cycle 1 day 1 (C1D1) pre-dose sample is considered background and subtracted from all samples (if C1D1 pre-dose sample data is unavailable, then add (Isotype samples are used.) Receptor occupancy (RO), expressed as a percentage (%), is calculated using the following formula:

[0448]

number

[0449] [Table 11]

[0450] Fifty-six subjects (all dosed Q2W) were evaluable for preliminary PD analysis (one patient each at 1 and 3 mg Q2W, three at 10 mg Q2W, five at 30 mg Q2W, seven at 120 mg Q2W, nine at 400 mg Q2W, sixteen at 600 mg Q2W, eight at 800 mg Q2W, and six at 1200 mg Q2W). Percent receptor occupancy (RO) of CD4+ and CD8+ cells at EOI (end of infusion after administration of the first dose of cycle 1 or cycle 2) and PRE (before administration of the next dose) are presented in Figures 3A-3D. The relationship between DART-I concentration and binding to CD4+ and CD8+ cells was investigated using the Emax model: E = (Emax * C) / (EC50 + C), where E = % binding, Emax = maximal % binding, EC50 = half maximal effective concentration, and C = DART-I concentration. DART-I had an EC50 of 0.045 and 0.011 μg / mL for CD4+ and CD8+ cells, respectively. 50 Maximum RO was observed at doses of 120 mg and above throughout the Q2W dosing interval, with 90% of maximum RO achieved at 0.6 and 0.1 μg / mL for CD4+ and CD8+ cells, respectively.

[0451] PK and target concentration modeling Further PK simulations (see above for details regarding data analysis and modeling) based on serum concentration data from patients (n=28) receiving 400 mg to 1200 mg Q2W dosing regimens were performed for the 1-compartment: V and CL, and the 2-compartment: V1, V2, CL and CLD regimens. Simulated multi-dose median PK profiles for 400, 600, 800, 1000, and 1200 mg DART-I using the Q2W regimen are shown in Figures 4A, 4B, and 4C, respectively. As shown in Figures 4A-4C, the median PK profiles demonstrated that administration of 400 mg or more of DART-I using the Q2W regimen and 600 mg or more of DART-I using the Q3W regimen resulted in DART-I target trough concentrations (C) of 23 μg / mL or greater. trough Furthermore, all simulated DART-I doses and regimens achieved a 100×RO EC50 of 4.5 μg / mL. 50 That's all for DART-IC trough is obtained.

[0452] These studies support the efficacy of many doses and regimens that achieve the target threshold trough concentration (23 μg / mL). These studies support the efficacy of dosing regimens comprising administration of about 400 mg or more of the PD-1 x LAG-3 bispecific molecules of the invention Q2W, particularly about 600 mg of the PD-1 x LAG-3 bispecific molecules of the invention Q2W. These studies also support the efficacy of dosing regimens comprising administration of about 600 mg or more of the PD-1 x LAG-3 bispecific molecules of the invention Q3W, particularly about 600 mg or more of the PD-1 x LAG-3 bispecific molecules of the invention Q3W. Furthermore, as noted above, the maximum RO was observed at doses of 120 mg or more throughout the Q2W dosing regimen. Thus, these studies support the efficacy of dosing regimens comprising administration of about 120 mg or more and Q2W to provide target trough concentrations of the PD-1 x LAG-3 bispecific molecules of the invention sufficient to achieve the maximum RO.

[0453] Summary of early clinical findings Post-treatment findings are provided for the first 188 patients (47 patients (49%) in the Q2W dose escalation group and 141 patients (33%) in the subsequent Q2W cohort expansion group). Treatment-related adverse events (TRAEs) occurred in 117 / 188 (62.2%) patients, most commonly Common adverse events were fatigue (n=33) and nausea (n=20). The rate of grade 3 or higher TRAEs was 19.7%. Immune-related adverse events were consistent with those observed with anti-PD-1 antibodies. The mean half-life was approximately 11 days; peripheral blood flow cytometry confirmed complete and sustained on-target binding during treatment at doses of 120 mg and above.

[0454] Of the first 39 response-evaluable dose-escalation patients treated with DART-I monotherapy at doses ranging from 1 to 1200 mg Q2W, three partial responses (PRs) were confirmed and 19 patients had stable disease per RECIST 1.1 in patients with triple-negative breast cancer (TNBC), mesothelioma, or gastric cancer. While the study is ongoing and data are being finalized, Figure 5 presents a waterfall plot showing the percent reduction in target lesions for 120 response-evaluable cohort expansion patients who received DART-I monotherapy at 600 mg Q2W. In the monotherapy solid tumor expansion cohort (i.e., excluding diffuse large B-cell lymphoma [DLBCL]), seven responses by RECIST 1.1 have been observed to date (3 confirmed / 4 unconfirmed), including six PRs (ovarian, NSCLC, and TNBC [n=2 each]) and one complete response [CR] (NSCLC). Fifty-one patients had stable disease. Additional results from 75 response-evaluable patients in the TNBC, EOC, and NSCLC (checkpoint inhibitor (CPI)-naive and prior PD-1 checkpoint therapy) expansion cohorts are summarized in Table 11.

[0455] [Table 12]

[0456] In the DLBCL expansion cohort, one CR and one PR were observed by Lugano classification among two evaluable patients. Notably, a DLBCL patient, status: CD19-targeted CAR T cells, relapsed, demonstrated a CR after one DART-I infusion (600 mg). A checkpoint inhibitor-naive NSCLC patient (following lobectomy and carboplatin plus pemetrexed treatment) demonstrated a CR after four 8-week cycles (600 mg Q2W DART-I infusions). Additional results from the 13 response-evaluable patients in the DLBCL expansion cohort are summarized in Table 12. In this larger group, seven patients responded, encompassing activated B-cell (ABC), germinal center B-cell (GCB), and double-hit (MYC / BCL2) molecular subtypes. Response duration ranged from 1 (second scan data pending) to 168 days, with six of the seven responding patients remaining active. The monotherapy was generally well tolerated in heavily pretreated patients with R / R DLBCL. Infusion-related reactions were manageable, and there was no evidence of tumor lysis syndrome. These results demonstrate antitumor activity in CAR T cell-treated and CAR-naive patients with R / R DLBCL, representing a variety of molecular subtypes, with a preliminary ORR of 53.8%.

[0457] [Table 13]

[0458] In a cohort of patients with HER-2+ tumors treated with DART-I in combination with an anti-HER-2 antibody (margetuximab), two HER2+ breast cancer patients demonstrated partial responses (PRs) (one confirmed and one unconfirmed) among the first five evaluable patients treated. In particular, a heavily pretreated breast cancer patient with extensive chest wall disease and liver and lung metastases demonstrated PRs at the first on-treatment disease assessment and subsequently demonstrated a partial response (PR) with the combination. Two weeks after a single dose of the combination, patients showed dramatic disease regression. Furthermore, responses were observed in several patients after prior anti-PD-1 therapy. Further results for the combination cohort are provided below.

[0459] Pretreatment tumor biopsies were evaluated for both LAG-3 and PD-L1 expression. Briefly, LAG-3 expression was examined using the LAG-3 Ab clone EPR4392(2) (Abcam) IHC assay on the Ventana Discovery Ultra platform. Positivity was defined as at least one LAG-3+ tumor-infiltrating lymphocyte (TIL) per hot spot field (HSF) at 40x magnification. PD-L1 TPS / CPS expression was determined according to the instructions for the Agilent PD-L1 (22C3) pharmDx kit. As used herein, "-ve" indicates "negative" and "+ve" indicates "positive."

[0460] Retrospective immunohistochemistry (IHC) was performed. Briefly, archival biopsies from TNBC, EOC, and NSCLC expansion cohorts were analyzed by IHC for LAG-3 (N=46) or PD-L1 (N=45). Ab clone EPR4392(2) (Abcam) IHC assay was performed by Ventana The study was performed on the Discovery Ultra platform. The LAG-3 score was determined by calculating the mean number of LAG-3+ cells per 40x magnification field across five LAG-3+ hotspots. PD-L1 expression was measured using Agilent The PD-L1 (22C3) pharmDx kit was used to determine TPS (NSCLC), which was calculated according to the interpretation manual. CPS (EOC, TNBC) was calculated as follows: number of PD-L1+ cells (tumor and immune) / total number of viable tumor cells × 100. A CPS < 1 or TPS < 1% was considered negative. The LAG-3 and PD-L1 scores of individual patients who demonstrated a clinical response are plotted in Figures 6A and 6B, respectively. The LAG-3 scores plotted by clinical response are plotted in Figure 6C.

[0461] Additionally, IHC analysis was performed on biopsies obtained from DLBCL patients (who relapsed after CD19-targeted CAR T-cell therapy) who showed a complete response after a single dose of DART-I. Lymph node biopsies before and after CAR T-cell therapy (pre-DART-I treatment) were evaluated by multiplex IF (fluorescence) staining using the HALO® image analysis platform for the expression of CD3 (a T-cell marker), CD79a (a B-cell marker), and PD-1 and LAG-3. DAPI staining was used to determine the total number of cells and the number of cells positive for each marker. The number of single-, double-, and triple-positive cells as a percentage of DAPI-stained cells is presented in Table 13 and shows that the number of cells positive for PD-1 and / or LAG-3 and / or CD3 was significantly higher after CAR T-cell therapy. LAG-3 expression was the most commonly observed in the biopsies examined in this analysis.

[0462] [Table 14]

[0463] Additional pretreatment biopsies from the DLBCL expansion cohort (N=11) were analyzed for LAG-3 and PD-L1 expression by IHC essentially as described above. The results are shown in Figures 6D and 6E. Figure 6D plots individual patients by LAG-3 expression from highest to lowest, with the number of responders per LAG-3 expression range shown on the right. Additionally, the PD-L1 score (CPS) is shown in the box below the plot. Figure 6E plots LAG-3 expression by response. These results indicate that DLBCL patients with higher baseline levels of LAG-3 are more likely to have improved responses.

[0464] Using the NanoString PanCancer IO 360™ assay, we investigated gene expression, including enriched 14 immune cell types and 32 immuno-oncological signatures, from archival biopsies from EOC (N = 14), NSCLC (N = 25, including pre-primary checkpoint-treated post-NSCLC (P-NSCLC)), and TNBC (N = 13) expansion cohorts. LAG-3 versus PD-1 (PDCD1) expression is plotted in Figure 7, demonstrating that responding patients exhibit high levels of expression of both LAG-3 and PD-1 (indicated by dotted circles). IFN-γ gene signature (CXCL9, CXCL10, CXC11, STAT1) scores are plotted by clinical response in Figure 8, demonstrating that patients with partial responses have high IFN-γ gene signature scores. These studies demonstrate that response is associated with high baseline LAG-3 / PD-1 expression and IFN-γ gene signature scores.

[0465] These data demonstrate that the PD-1 x LAG-3 bispecific molecules of the invention (e.g., DART-I) exhibited an acceptable safety profile and also showed promising evidence of anti-tumor activity, particularly in patients with tumors exhibiting relatively high levels of LAG-3 expression and relatively high IFN-γ gene signature scores. These data demonstrate that multiple dosing regimens for the molecules (especially DART-I) including administration of about 400 mg or more of the molecules (especially DART-I) Q2W (especially about 400 mg and Q2W, or about 600 mg and Q2W), and about 600 mg or more of the molecules (especially DART-I) Q3W (especially about 600 mg and Q3W, or about 800 mg and Q3W) resulted in target C levels of 23 μg / mL or greater. trough An alternative dosing regimen is 100 × ROEC 50 Target C above trough To achieve this, approximately 120 mg These studies include administering the above molecules Q2W. These studies further support the administration of the PD-1 x LAG-3 bispecific molecules of the invention according to any of the doses and regimens described above in combination with a TA-binding molecule, particularly a HER2-binding molecule (e.g., an anti-HER2 antibody), for the treatment of HER2-expressing (HER2+) cancers. In particular, the administration of about 600 mg or more of the above molecules (particularly DART-I) using a Q3W regimen in combination with a TA-binding molecule, such as a HER2-binding molecule (e.g., margetuximab at 15 mg / kg administered Q3W), which may also be administered using a Q3W regimen.

[0466] Example 2 TA-binding molecules mediated changes in checkpoint expression and NK cell activity The ability of TA-binding molecules containing an ADCC-enhanced Fc domain and a wild-type Fc domain to mediate changes in the expression of checkpoint molecules on the surface of immune effector cells, particularly NK cells, was evaluated in vitro. Furthermore, the effect on in vitro cytotoxic activity, particularly NK cell cytotoxicity, was assessed. Briefly, PBMC effector cells (0.5 × 10 ) were cultured in the presence of margetuximab (a TA-binding molecule that binds to an epitope on HER2 and contains an ADCC-enhanced Fc domain, i.e., an ADCC-enhanced TA-binding molecule), a replica of trastuzumab (which binds to the same epitope on HER2 but contains a wild-type Fc domain, "r-trastuzumab"), or PBS (phosphate-buffered saline) alone. 6 / ml) against TA HER2 N87 target cells (HER2 positive) +++ gastric cancer cell line) (0. 05×10 6 The antibody was co-incubated with 10:1 IgG1 / ml (E:T ratio was 10:1). The antibody was used at 0.005 μg / ml and 0.05 μg / ml, and 20 μg / ml of IL-2 was added to the cultures. RPMI 1640 medium containing L-glutamine, supplemented with 10% FBS, 10 mM HEPE buffer, and penicillin-streptomycin, was used as the culture medium.

[0467] On day 3, a portion of each sample was removed and the cell surface expression of checkpoint proteins CD137, LAG-3, PD-1, and PD-L1 on NK cells was examined by fluorescence-activated cell sorting (FACS). To define immune cell subsets and cell surface expression of checkpoint proteins, the following antibodies were used: CD3-V500, CD4-PerCP Cy5.5, CD8-FITC, CD56-PE, Lag-3-PE-Cy7, PDL-1-APC, CD137-BV421, and PD-1-BV650. Cell surface staining was performed by incubating cells with the Ab cocktail in FACS buffer for 30 minutes at 4°C, followed by washing with PBS. The labeled cells were then resuspended in FACS buffer. FACS samples were acquired using an LSRFortessa flow cytometer and analyzed using FlowJo software. Representative FACS plots are shown in Figure 11, with checkpoint-positive NK cells boxed and percentages indicated. As seen in Figure 11, margetuximab significantly upregulated the expression of CD137, LAG-3, and PD-L1 compared to r-trastuzumab.

[0468] On day 6, a portion of the remaining sample was used to provide effector cells for a cytotoxicity assay using PKH26 red-labeled K562 cells (a HER2 myeloid leukemia cell line) as target cells at E:T ratios of 0.3:1, 1:1, 3:1, and 10:1. After a 4-hour incubation, cells were harvested, and cytotoxicity was determined by FACS analysis of 7-AAD and Annexin V as markers to distinguish live, apoptotic, and dead cells, according to the manufacturer's instructions. The percent cytotoxicity observed at each E:T ratio is plotted in Figure 12. Because K562 target cells do not express HER2, killing in this assay is not directly mediated by binding of the anti-HER2 antibody to the K562 target cells, but rather reflects enhanced cytotoxic activity (primarily of NK cells) mediated by prior exposure to the anti-HER2 antibody in the presence of TA-positive target cells. (Figure 12) As shown in Figure 1, margetuximab mediates a more potent enhancement of NK cell cytotoxic activity than r-trastuzumab. These results indicate that TA-binding molecules containing ADCC-enhancing Fc domains are more potent mediators of PD-L1 and LAG-3 expression and cytotoxic activity (mainly of NK cells).

[0469] The ability of ADCC-enhancing TA-binding molecules to mediate changes in expression of checkpoint molecules on the surface of additional immune cell types was investigated. Briefly, PBMC effector cells (1.5 x 10 6 cells) were cultured in the presence of margetuximab (0.5 μg / ml) or a control antibody (MGAWN1, 0.5 μg / ml). 6 / ml) to N87 target cells (HER2 +++ The cells were co-incubated with NK cells (day 3), monocytes (day 2), and CD4+ cells (day 3) at an E:T ratio of 15:1. RPMI 1640 medium containing L-glutamine, supplemented with 10% FBS, 10 mM HEPE buffer, and penicillin-streptomycin, was used as the culture medium. On days 2 and 3, NK cells (day 3), monocytes (day 2), and CD4+ cells were cultured. + (Day 3), and CD8 +The cell surface expression of checkpoint proteins CD137, LAG-3, PD-1, and PD-L1 on T cells (day 3) was examined by FACS. To define immune cell subsets and cell surface expression of checkpoint proteins, the following antibodies (Abs) were used: CD3-V500, CD4-PerCP Cy5.5, CD8-FITC, CD14-FITC, CD56-PE, Lag-3-PE-Cy7, PDL-1-APC, CD137-BV421, and PD-1-BV650. Cell surface staining was performed by incubating cells with the Ab cocktail in FACS buffer for 30 minutes at 4°C, followed by washing with PBS. The labeled cells were then resuspended in FACS buffer. FACS samples were acquired using an LSRFortessa flow cytometer and analyzed using FlowJo software. A representative FACS plot is shown in Figure 13, with checkpoint-positive immune cells boxed and the percentage indicated. As seen in Figure 13, margetuximab, an ADCC-enhancing TA-binding molecule, mediated upregulation of LAG-3 and PD-L1 expression in all cells examined, with the most potent upregulation observed in monocytes, NK cells, and CD8 T cells. CD137 was upregulated in NK cells, and PD-1 in CD4 T cells. + and CD8 + It was upregulated in both T cells.

[0470] Example 3 In vitro combination studies As described above, TA-binding molecules in general, and those with an ADCC-enhancing Fc domain in particular, have been found to synergistically mediate the upregulation of the checkpoint molecules PD-L1 and LAG-3. The activity of TA-binding molecules in combination with checkpoint inhibitors that block LAG-3 and / or the PD-1 / PD-L1 inhibitory checkpoint pathway was investigated in vitro. Briefly, PBMC effector cells (0.5 × 10 ) were cultured in the presence of the TA-binding molecules margetuximab (containing an ADCC-enhancing Fc domain) or r-trastuzumab (containing a wild-type Fc domain), a control antibody (MGAWN1, an anti-WNV mAb containing a wild-type human IgG1 Fc domain), or PBS alone, or in combination with retifanlimab (a PD-1 binding molecule), DART-I (a bispecific molecule that binds both PD-1 and LAG-3). 6 / ml) to N87 target cells (HER2 +++ The cells were co-incubated with exogenous IL-2 (20 μg / ml) at a 20:1 ratio. Assays were performed with or without exogenous IL-2 (20 μg / ml), representing optimal and suboptimal conditions. The anti-HER2 antibody was used at 0.005 μg / ml and / or 0.05 μg / ml, the anti-PD-1 antibody retifanlimab was used at 5 μg / ml, and the PD-1 x LAG-3 bispecific molecule DART-I was used at 5 μg / ml. On day 6, effector cells were harvested, and cytotoxicity against K562 target cells (E:T = 10:1) was determined by FACS using 7-AAD and Annexin V as markers to distinguish live, apoptotic, and dead cells, essentially as described above. The percent cytotoxicity observed for suboptimal conditions from a representative donor was: It is plotted in Figure 14.

[0471] As shown in Figure 14, in this assay, minimal enhancement of cytotoxicity was observed by combining r-trastuzumab with the PD-1 checkpoint inhibitor retifanlimab or the PD-1 × LAG-3 dual checkpoint inhibitor DART-I. In contrast, the PD-1 × LAG-3 dual checkpoint inhibitor DART-I enhanced cytotoxicity in combination with margetuximab, a TA-binding molecule with an ADCC-enhancing Fc domain. In some donors, the PD-1 checkpoint inhibitor retifanlimab was also observed to enhance the cytotoxicity of margetuximab.

[0472] In another study, the activity of margetuximab, an ADCC-enhancing TA-binding molecule, alone or in combination with DART-I, a PD-1 × LAG-3 dual checkpoint inhibitor, was further investigated. Briefly, PBMC effector cells (1 × 10 ) were cultured in the presence of the TA-binding molecule margetuximab (0.005 μg / ml) or a control antibody (MGAWN1, 0.005 μg / ml) alone or in combination with DART-I (5 μg / ml). 6 / ml) N87 target cells (HER2 +++ gastric cancer cell line) at a ratio of 15:1 The cells were co-incubated with exogenous IL-2 (20 μg / ml). Assays were performed with or without exogenous IL-2 (20 μg / ml), representing optimal and suboptimal conditions. On day 7, effector cells were harvested, and cytotoxicity against PKH26 red-labeled K562 target cells (E:T = 10:1) was determined by FACS using 7-AAD and Annexin V as markers to distinguish live, apoptotic, and dead cells, according to the manufacturer's instructions. Cytotoxicity against luciferase-expressing N87 cells (E:T = 3:1) was determined by assessing residual cellular luciferase activity using the Steady-Glo Luciferase Assay System (Promega). The percent cytotoxicity observed for suboptimal conditions from a representative donor is plotted in Figure 15. As shown in Figure 15, PBMCs conditioned with margetuximab, an ADCC-enhancing TA binding molecule, significantly increased the binding of margetuximab-opsonized K562 cells and HER2 compared to PBMCs conditioned with control Ab. +++ N87 cells These molecules demonstrated high cytotoxic activity (mainly NK cells) against tumors. As mentioned above, the PD-1 × LAG-3 bispecific molecule, DART-I, enhanced cytotoxicity in combination with the ADCC-enhancing TA-binding molecule, margetuximab. Together, these studies demonstrate that dual checkpoint inhibition of the PD / PD-L1 and LAG-3 checkpoint pathways can synergize with the antitumor activity of TA-binding molecules, particularly those with enhanced ADCC activity.

[0473] Example 4 Phase I clinical study - HER2+Arm As mentioned above, in an ongoing Phase I clinical study, a cohort of patients with advanced or metastatic HER2+ solid tumors (specifically HER2+ gastric or breast cancer) are receiving DART-I (a bispecific molecule that binds to PD-1 and LAG-3) and margetuximab (a TA-binding molecule that binds to HER2 and has an ADCC-enhancing Fc domain).

[0474] The clinical results for 28 evaluable patients with HER2+ solid tumors (including the first 5 patients mentioned above) are summarized in Figure 16. Objective response rate (ORR) (unpublished data) The ORR (including patients with confirmed responses) was 28.6% (8 / 28), and the disease control rate was 50% (14 / 28). Table 14 summarizes the response rates of these patients by cancer type. The ORR of 28.65% favorably indicated the efficacy of pemafibrate in HER2+ mBC. A single-arm, multicenter Phase 1b / 2 trial of brolizumab plus trastuzumab reported an ORR of 11.5% (n=52) in HER2+ mBC (15% ORR (n=6 / 40) in PD-L1 positive patients; and 0% ORR (n=0 / 12) in PD-L1 negative patients), comparable to the Panacea study (Loi, et al. 2019 Lancet Oncol.Mar;20(3):371-382. doi: 10.1016 / S1470-2045(18)30812-X.). Treatment was well tolerated, Responding patients continue treatment, and further enrollment into HER2+ tumor-specific cohorts is ongoing.

[0475] [Table 15]

[0476] Available pretreatment tumor biopsies were evaluated for both LAG-3 and PD-L1 expression. Briefly, LAG-3 expression was examined using the LAG-3 Ab clone EPR4392(2) (Abcam) IHC assay on the Ventana Discovery Ultra platform. Positivity was defined as at least one LAG-3+ tumor-infiltrating lymphocyte (TIL) per hot spot field (HSF) at ×40 magnification. PD-L1 TPS / CPS expression was determined according to the instructions for the Agilent PD-L1 (22C3) pharmDx kit. LAG-3 expression by IHC varied between patients and did not correlate with response. The majority of responding patients were observed to have PD-L1-negative tumors (PD-L1 expression ≤1) by IHC. The high response rate in PD-L1-negative patients in this combination study, which utilized PD-1 and LAG-3 dual checkpoint blockade in combination with a TA-binding molecule with an ADCC-enhancing Fc domain, is in contrast to published data (e.g., Loi, S. et al. (2019) “Pembrolizumab Plus Trastuzumab InTrastuzumab-Resistant, Advanced, HER2-Positive Breast Cancer (PANACEA): aSingle-Arm, Multicentre, Phase 1b-2 Trial,” LancetOncol. 20(3):371-382. The published data show that the response rate in HER2+ breast cancer patients treated with trastuzumab plus anti-PD-1 or anti-PD-L1 antibodies was 0% in PD-L1-negative patients and only 15% in PD-L1-positive patients. The high response rate may reflect the synergistic effect of combining an ADCC-enhancing TA-binding molecule with dual checkpoint blockade of the PD / PD-L1 and LAG-3 checkpoint pathways.

[0477] The NanoString PanCancer IO 360™ assay was used to examine gene expression, including enriched 14 immune cell types and 32 immuno-oncological signatures, from archival biopsies of a cohort of 19 HER2+ advanced solid tumors treated with margetuximab and DART-I. Normalized expression scores (normalized from 0 to 100) of LAG-3 were plotted against PDCD1 (Figure 17A). Normalized LAG-3 and PDCD1 expression levels were correlated with the best percent change from baseline in target lesions. The correlations are plotted in Figures 17B and 17C, respectively. This gene expression analysis shows that responding patients exhibit higher expression of both LAG-3 and PDCD1 mRNA in their baseline biopsy samples.

[0478] In this clinical trial cohort, the dual checkpoint inhibitor DART-I combined with the ADCC-enhancing TA-binding molecule margetuximab was generally well tolerated, with a safety profile consistent with DART-I monotherapy. Evidence of antitumor activity was observed in refractory patients with a variety of tumor types expressing the HER2 tumor antigen (i.e., HER2+ tumors). Baseline LAG-3 and PD-1 mRNA expression appear to be associated with clinical response, although the majority of responding patients had baseline PD-L1 expression ≤1 (by IHC).

[0479] In summary, dual checkpoint blockade of the PD-1 / PD-L1 and LAG-3 checkpoint pathways with molecules such as DART-I can synergize with the antitumor activity of TA-binding molecules (especially those with enhanced ADCC activity, such as margetuximab). Such a combination may be more effective than treatment with TA-binding molecules alone or in combination with checkpoint blockade of the PD-1 / PD-L1 pathway alone, and may be useful for treating PD-L1-negative patients.

[0480] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in terms of specific embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of this invention which generally follow the principles of the invention, including departures from the present disclosure as come within known methods or customary practices in the art to which this invention pertains, and as applicable to the essential features hereinbefore described.

Claims

1. 1. A method of treating cancer, comprising administering to a subject in need thereof a PD-1 x LAG-3 bispecific molecule, said method comprising administering to the subject a flat dose of about 120 mg to about 800 mg of the PD-1 x LAG-3 bispecific molecule.

2. 10. The method of claim 1, wherein the cancer is characterized by expression of a tumor antigen (TA), and the method further comprises administering to the subject a tumor antigen (TA)-binding molecule (TA-binding molecule).

3. 1. A method of treating cancer in a subject, wherein the cancer is characterized by expression of a TA, the method comprising administering to the subject a TA-binding molecule and: (a) a bispecific molecule (PD-1 x LAG-3 bispecific molecule); or (b) a combination of a molecule that immunospecifically binds to PD-1 (a PD-1-binding molecule) and a molecule that immunospecifically binds to LAG-3 (a LAG-3-binding molecule); or (c) a bispecific molecule that immunospecifically binds to both PD-L1 and LAG-3 (PD-L1 x LAG-3 bispecific molecule); or (d) a combination of a molecule that immunospecifically binds to PD-L1 (a PD-L1-binding molecule) and a LAG-3-binding molecule; The method of claim 1, further comprising administering

4. The method of claim 2 or 3, wherein the TA-binding molecule comprises an ADCC-enhancing Fc domain.

5. (a) each said molecule is in a separate composition; or (b) each of said molecules is in the same composition; or (c) the PD-1-binding molecule and the LAG-3-binding molecule are in the same composition, and the TA-binding molecule is in a separate composition; or (d) the PD-L1-binding molecule and the LAG-3-binding molecule are in the same composition, and the TA-binding molecule is in a separate composition.

6. The method of any one of claims 2 to 5, wherein the TA-binding molecule is an antibody.

7. 7. The method of any one of claims 2-6, wherein the PD-1-binding molecule is an antibody, the PD-L1-binding molecule is an antibody, and the LAG-3-binding molecule is an antibody.

8. 7. The method of any one of Claims 3-6, wherein the method comprises administering the TA-binding molecule and the PD-1 x LAG-3 bispecific molecule.

9. The ADCC-enhancing Fc domain comprises: (A) an artificial glycoform; and / or (B) Amino acid substitutions relative to the wild-type Fc region The method according to any one of claims 4 to 8, comprising:

10. The ADCC-enhancing Fc domain comprises: (A) an artificial glycoform that is a complex N-glycosidic sugar chain that does not contain fucose and / or contains a bisected O-GlcNAc; and / or (B) an amino acid substitution selected from the group consisting of: (a) one substitution selected from the group consisting of: F243L, R292P, Y300L, V305I, I332E, and P396L; (b) two substitutions selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; (3) R292P and V305I; and (4) S239D and I332E; (c) three substitutions selected from the group consisting of: (1) F243L, R292P and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P, and P396L; and (4) R292P, V305I and P396L; (d) four substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, and P396L; and (2) F243L, R292P, V305I, and P396L; or (e) five substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I, and P396L; and (2) L235V, F243L, R292P, Y300L and P396L and the numbering is that of the EU index as set forth in Kabat.

11. 11. The method of claim 9 or 10, wherein the ADCC-enhancing Fc domain comprises the amino acid substitutions: L235V, F243L, R292P, Y300L and P396L, numbering being that of the EU index as set forth in Kabat.

12. (A) the TA is selected from Table 6A or Table 6B; and / or (B) The method of any one of claims 2 to 11, wherein the TA-binding molecule comprises the VL and VH domains of an antibody selected from Table 7.

13. (A) the PD-1-binding molecule comprises: (a) a PD-1 VL Domain comprising the amino acid sequence of SEQ ID NO: 35, and a PD-1 VH Domain comprising the amino acid sequence of SEQ ID NO: 39; (b) the VH and VL domains of an anti-PD-1 antibody selected from Table 1; or (c) the light chain and heavy chain of an anti-PD-1 antibody selected from Table 1 an antibody comprising: (B) the PD-L1-binding molecule is: (a) a PD-L1 VL domain comprising the amino acid sequence of SEQ ID NO: 43, and a PD-L1 VH domain comprising the amino acid sequence of SEQ ID NO: 47; (b) the VH and VL domains of an anti-PD-L1 antibody selected from Table 2; or (c) the light chain and heavy chain of an anti-PD-L1 antibody selected from Table 2 an antibody comprising: (C) the LAG-3 binding molecule is: (a) a LAG-3 VL domain comprising the amino acid sequence of SEQ ID NO: 51, and a LAG-3 VH domain comprising the amino acid sequence of SEQ ID NO: 55; (b) the VH and VL domains of an anti-LAG-3 antibody selected from Table 3; or (c) the light and heavy chains of an anti-LAG-3 antibody selected from Table 3 The method according to any one of claims 3 to 7 and 9 to 12, wherein the antibody comprises:

14. The PD-1 x LAG-3 bispecific molecule comprises: (a) a PD-1 VL Domain comprising the amino acid sequence of SEQ ID NO: 35, and a PD-1 VH Domain comprising the amino acid sequence of SEQ ID NO: 39, or the VH and VL Domains of an anti-PD-1 antibody selected from Table 1; and / or (b) a LAG-3 VL domain comprising the amino acid sequence of SEQ ID NO: 51, and a LAG-3 VH domain comprising the amino acid sequence of SEQ ID NO: 55, or the VH and VL domains of an anti-LAG-3 antibody selected from Table 3; or (c) a bispecific antibody-based molecule selected from Tables 4 and 5 The method of any one of claims 1 to 6, 8, and 9 to 12, comprising:

15. The PD-1 x LAG-3 bispecific molecule comprises: (a) two of the PD-1-binding domains; and (b) two of the LAG-3 binding domains The method of any one of claims 1 to 6, 8, 9 to 12, and 14, comprising:

16. 16. The method of any one of Claims 1-6, 8, 9-12, and 14-15, wherein the PD-1 x LAG-3 bispecific molecule comprises a PD-1 VL Domain of SEQ ID NO: 35, a PD-1 VH Domain of SEQ ID NO: 39, a LAG-3 VL Domain of SEQ ID NO: 51, and a LAG-3 VH Domain of SEQ ID NO:

55.

17. 17. The method of any one of Claims 1-6, 8, 9-12, and 14-16, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule comprises an Fc Region and a hinge Domain.

18. 18. The method of claim 17, wherein the Fc region and the hinge domain are both of the IgG4 isotype, and the hinge domain comprises a stabilizing mutation.

19. The Fc region is a variant Fc region comprising: (a) one or more amino acid modifications that reduce the affinity of said variant Fc Region for an FcγR; and / or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc region. The method of claim 17 or 18, wherein the variant Fc region comprises:

20. (a) the modification that reduces the affinity of the variant Fc Region for an FcγR comprises the substitution of L234A; L235A; or L234A and L235A; (b) the modifications that increase the serum half-life of the variant Fc Region comprise the following substitutions: M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K; 20. The method of claim 19, wherein the numbering is that of the EU Index as set forth in Kabat.

21. 21. The method of any one of Claims 1-6, 9-12, and 14-20, wherein the PD-1 x LAG-3 bispecific molecule comprises two polypeptide chains set forth in SEQ ID NO:59 and two polypeptide chains set forth in SEQ ID NO:

60.

22. 22. The method of any one of claims 1-6, 9-12, and 14-21, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 300 mg.

23. 22. The method of any one of claims 1-6, 9-12, and 14-21, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg.

24. Claims 1 to 6, 9 to 12, and 14 to 23, wherein the flat dose is administered approximately once every two weeks.

10. The method according to any one of claims 1 to 9.

25. 24. The method of any one of claims 1-6, 9-12, and 14-23, wherein the flat dose is administered about once every three weeks.

26. 25. The method of any one of claims 1-6, 9-12, 14-21, 23, and 24, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg about once every two weeks.

27. 26. The method of any one of claims 1-6, 9-12, 14-21, 23, and 25, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered at a flat dose of about 600 mg about once every three weeks.

28. 28. The method of any one of claims 1-6, 9-12, and 14-27, wherein the PD-1 x LAG-3 bispecific molecule or the PD-L1 x LAG-3 bispecific molecule is administered by intravenous (IV) infusion.

29. Such cancers include: adrenal gland cancer, AIDS-related cancer, alveolar soft tissue sarcoma, anal cancer (including squamous cell carcinoma of the anal canal (SCAC)), bladder cancer, bone cancer, brain and spinal cord cancer, breast cancer (HER2 + Breast cancer or triple negative tumors of the breast (including transverse nephropathy), carotid artery tumor, cervical cancer (including HPV-associated cervical cancer), chondrosarcoma, chordoma, chromophobe clear cell renal carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic small round cell tumor, ependymoma, endometrial cancer (including unselected endometrial cancer, MSI-high endometrial cancer, dMMR endometrial cancer, and / or POLE exonuclease domain mutation-positive endometrial cancer), Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, gallbladder cancer or bile duct cancer (including cholangiocarcinomabile duct cancer) ), gastric cancer, esophagogastric junction (GEJ) cancer, gestational trophoblastic disease, germ cell tumors, glioblastoma, head and neck cancer (including squamous cell carcinoma of the head and neck (SCCHN)), hematologic malignancies, hepatocellular carcinoma, pancreatic islet cell tumors, Kaposi's sarcoma, kidney cancer, leukemia (including acute myeloid leukemia), liposarcoma / malignant lipomatous tumors, liver cancer (including hepatocellular carcinoma (HCC)), lymphoma (including diffuse large B-cell lymphoma (DLBCL) and non-Hodgkin's lymphoma (NHL)), lung cancer (including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)), medulloblastoma, melanoma (uveal melanoma) 29. The method of any one of claims 1 to 28, wherein the cancer is selected from the group consisting of: mesothelioma, meningioma, Merkel cell carcinoma, mesothelioma (including mesothelial pharyngeal carcinoma), multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pharyngeal cancer, pheochromocytoma, pituitary tumor, prostate cancer (including metastatic castration-resistant prostate cancer (mCRPC)), posterior uveal melanoma, renal metastatic carcinoma, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, small round blue cell tumor of childhood (including neuroblastoma and rhabdomyosarcoma), soft tissue sarcoma, squamous cell carcinoma, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroid cancer, and uterine cancer.

30. 30. The method of claim 29, wherein the cancer is selected from the group consisting of: anal cancer, breast cancer, bile duct cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, GEJ cancer, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, and prostate cancer.

31. The cancer is: HER2 + 30. The method of claim 28 or 29, wherein the cancer is selected from the group consisting of breast cancer, TNBC, cholangiocarcinoma bile duct cancer, HPV-associated cervical cancer, SCCHN, HCC, SCLC or NSCLC, NHL, prostate cancer, gastric cancer, and GEJ cancer.

32. The TA-binding molecule comprises a light chain variable domain (VL HER2 ) and a heavy chain variable domain (VH HER2 ), a HER2 binding molecule comprising a HER2 binding domain comprising: (A) the light chain variable domain (VL HER2 ) is the CDR of SEQ ID NO: 61 L 1. CDR L 2, and CDR L 3, wherein the heavy chain variable domain is a light chain variable domain of margetuximab comprising N (VH HER2 ) is the CDR of SEQ ID NO:66 H 1. CDR H 2, and CDR H Marge including 3 the heavy chain variable domain of tuximab; (B) the light chain variable domain (VL HER2 ) is the CDR of trastuzumab L 1. CDR L 2, and CDR L 3, wherein the heavy chain variable domain (VH HER2 ) is the CDR of trastuzumab H 1. CDR H 2, and CDR H Contains 3; (C) the light chain variable domain (VL HER2 ) is the CDR of pertuzumab L 1. CDR L 2, and CDR L 3, wherein the heavy chain variable domain (VH HER2 ) is the CDR of pertuzumab H 1. CDR H 2, and CDR H Contains 3; or (D) the light chain variable domain (VL HER2 ) are the CDRs of hHER2 MAB-1 L 1. C Dr. L 2, and CDR L 3, wherein the heavy chain variable domain (VH HER2 ) are the CDRs of hHER2 MAB-1 H 1. CDR H 2, and CDR H Including 3, The method according to any one of claims 2 to 31.

33. The method of any one of claims 2 to 32, wherein the HER2 binding molecule is an anti-HER2 antibody.

34. 34. The method of claim 33, wherein the anti-HER2 antibody is margetuximab, and the method comprises administering margetuximab at a dosage of about 6 mg / kg to about 18 mg / kg about once every three weeks.

35. The method of any one of claims 32 to 34, wherein the method further comprises administering a chemotherapeutic agent.

36. The method of any one of claims 2 to 35, wherein the cancer is a HER2-expressing cancer.

37. 37. The method of claim 36, wherein the HER2-expressing cancer is selected from the group consisting of: breast cancer, metastatic breast cancer, bladder cancer, gastric cancer, GEJ cancer, ovarian cancer, pancreatic cancer, and stomach cancer.

38. The TA-binding molecule is a B7-H3-binding molecule comprising a B7-H3-binding domain comprising a light chain variable domain (VL) and a heavy chain variable domain (VH): The VL is the CDR of SEQ ID NO: 71 L 1. CDR L 2, and CDR L 3, wherein said VH comprises CDR of SEQ ID NO: 76 H 1. CDR H 2, and CDR H Any of claims 2 to 31, including claim 3 1. The method according to claim 1.

39. 39. The method of any one of claims 2-31 and 38, wherein the TA-binding molecule is enoblituzumab, and the method comprises administering enoblituzumab at a dosage of about 6 mg / kg to about 18 mg / kg about once every three weeks.

40. The method of any one of claims 2 to 31 and 38 to 39, wherein the cancer is a B7-H3-expressing cancer.

41. 41. The method of claim 40, wherein the B7-H3 expressing cancer is selected from the group consisting of: anal cancer, SCAC, breast cancer, TNBC, head and neck cancer, SCCHN, lung cancer, NSCLC, melanoma, uveal melanoma, prostate cancer, and mCRPC.

42. 42. The method of claim 2, wherein the TA-binding molecule is administered by intravenous (IV) infusion.

1. The method according to claim 1.

43. 43. The method of any one of claims 1 to 42, wherein cells expressing LAG-3 are present in a biopsy of said cancer prior to said treatment.

44. 44. The method of any one of claims 1-43, wherein cells expressing PD-1 are present in a biopsy of the cancer prior to treatment.

45. 45. The method of any one of claims 2-44, wherein expression of PD-L1 on the surface of cells of the cancer prior to treatment is less than 1% as determined using a Combined Positive Score (CPS) or a Tumor Proportion Score (TPS).

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