BR112016010557B1 - USE OF A MONOCLONAL ANTIBODY TO HUMAN CEACAM1, OR AN ANTIGEN-BINDING FRAGMENT THEREOF AND A MONOCLONAL ANTIBODY TO HUMAN PD-1, OR AN ANTIGEN-BINDING FRAGMENT THEREOF AND KIT - Google Patents

Abstract

COMPOSITIONS COMPRISING ANTI-CEACAM1 AND ANTI-PD ANTIBODIES FOR CANCER THERAPY. The present invention provides compositions comprising anti-CEACAM1 antibodies, compositions comprising antibodies capable of inhibiting or blocking the interaction between PD-1 and its ligands and methods for their combined use in the treatment of cancer.

Description

FIELD OF TECHNIQUE

[001] The present invention relates to cancer immunotherapy, especially to combinations of anti-CEACAM1 and anti-PD-1/PD ligand antibodies and their use in the treatment of cancer.

BASICS OF THE INVENTION

[002] The transmembrane protein carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, also known as bile glycoprotein (BGP), CD66a and C-CAM1), makes up the family of carcinoembryonic antigens (CEA) and also belongs to the immunoglobulin superfamily . Human CEACAM1 has been assigned the SwissProt accession number P13688. CEACAM1 interacts with itself and other known CEACAM proteins, including the CD66e (CEACAM6) and CD66e (CEACAM5, CEA) proteins. It is expressed in a broad spectrum of cells, ranging from epithelial cells to those of hematopoietic origin (e.g., immune cells).

[003] Many different functions have been attributed to the CEACAM1 protein. The CEACAM1 protein has been shown to be overexpressed in some colon and prostate carcinomas, as well as in other types of cancer such as melanoma. Additional data support the central involvement of CEACAM1 in angiogenesis and the occurrence of metastases. CEACAM1 also contributes to the modulation of innate and adaptive immune responses. For example, CEACAM1 has been shown to be an inhibitory receptor for activated T cells contained within the human intestinal epithelium (WO 99/52552 and Morales et al. J. Immunol. 1999, 163, 1363-1370). Additional reports have indicated that the involvement of CEACAM1 by cross-linking T cell receptors with monoclonal antibodies (mAbs) or Opa proteins from Neisseria gonorrhoeae Opa inhibits T cell activation and proliferation. Several monoclonal antibodies against the CEACAM1 protein are already known. , such as 26H7, 5F4, TEC-11, 12-140-4, 4/3/17, COL-4, F36-54, 34B1, YG-C28F2, D14HD11, b18.7.7, D11-AD11, HEA81, B1.1, CLB-gran-10, F34-187, T84.1, B6.2, B1.13, YG-C94G7, 12-140-5, TET-2 and scFv-DIATHIS1 (Watt et al. , Blood, 2001, Vol. 98, pages 1469-1479). WO 2010/12557 describes a murine monoclonal antibody against human CEACAM1. WO 2013/054331 describes a chimeric monoclonal antibody against human CEACAM1 CM10.

[004] Programmed cell death protein 1 (PD-1) is a type 1 transmembrane protein belonging to the CD28/CTLA-4 family of immunoreceptors that act as signal mediators to regulate immune responses. Human PD-1 has been assigned the SwissProt accession number Q15116. Members of the CD28/CTLA-4 family positively (CD28 and ICOS) or negatively (CTLA-4 and PD-1) regulate T cell activation. PD-1 is expressed on activated T cells, B cells, myeloid cells and in a thymocyte subset. Several anticlonal antibodies against the PD-1 protein are already known, such as MK-3475 (humanized IgG4 mAb), AMP514, BMS-936558 (fully human IgG4 mAb) and pidilizumab, also known as CT-011 (IgG1 mAb humanized) (Topalian et al., Curr. Opin. Immunol., 2012, Vol. 24(2), pages 207-212).

[005] Programmed cell death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 protein homolog 1 (B7-H1), is a protein that, in humans, is encoded by the CD274 gene. PD-L1 is a 40kDa type 1 transmembrane protein that has been speculated to play an important role in suppressing the immune system during specific events such as pregnancy, tissue allografts, and other disease states such as hepatitis. Normally, the immune system reacts to exogenous antigens in which, certain accumulation in the lymph nodes or spleen, triggers a proliferation of CD8+ T cells specific to the antigen. The formation of the PD-1/PD-L1 ligand complex transmits an inhibitory signal that reduces the proliferation of these CD8+ T cells in lymph nodes and is supplementary to that by which PD-1 is also capable of controlling the accumulation of T cells specific to exogenous antigens in lymph nodes. by apoptosis which is further mediated by a downregulation of the Bcl-2 gene (Chemnitz JM et al., 2004, Journal of Immunology, 173(2): 945-54). Engagement of PD-L1 with its receptor PD-1, found on activated T cells, B cells, and myeloid cells, releases a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. Programmed cell death (also known as PD-L2, B7-DC) is a protein that, in humans, is encoded by the PDCD1LG2 gene. PDCD1LG2 was also designated CD273 (cluster of differentiation 273).

[006]PD-1 and its ligands, PD-L1 and PD-L2, release inhibitory signals that regulate the balance between T cell activation, tolerance and immunopathology. Immune responses to exogenous and endogenous antigens require specific and balanced responses to eliminate pathogens and tumors and yet maintain tolerance. Human PD-L1 and PD-L2 have been assigned SwissProt accession numbers Q9NZQ7 and Q9BQ51, respectively. The induction and maintenance of tolerance in T cells requires that PD-1, and its ligand PD-L1 on non-hematopoietic cells, be able to limit effector T cell responses and protect tissues from immune-mediated tissue damage. The PD-1:PD-L pathway has also been usurped by microorganisms and tumors to attenuate antimicrobial or tumor immunity and facilitate chronic infection and tumor survival. Several monoclonal antibodies against the PD-L1 protein are already known, such as MEDI-4736, BMS-936559, MSB0010718C and MPDL3280A (Lu et al., J. Oncol. Pharm. Pract., 2014). Other monoclonal antibodies against the PD-L2 protein are also known, such as those disclosed in PCT Publication Nos. WO/2010/027827, WO/2010/036959 and WO/2011/066342. WO/2014/059251 relates to compositions and methods for enhancing the immune response and/or reducing individual T cell tolerance by administering inhibitors of two or more of CEACAM1, PD-1 and/or latency-associated peptide (LAP).

[007] Cancer immunotherapy is the use of the immune system to treat cancer. There are three main groups of immunotherapy: cell therapies, antibody therapies, and cytokine therapies. All exploit the fact that cancer cells often have different molecules on their surfaces that can be detected by the immune system. These molecules are known as cancer antigens. Immunotherapy is used to provoke the immune system to attack tumor cells using these cancer antigens as targets.

[008] Antibody therapies currently constitute the most successful form of immunotherapy, with many treatments approved for a wide variety of cancers. Antibodies are proteins produced by the immune system that bind to a target antigen on the surface of a cell. In normal physiology, they are used by the immune system to fight pathogens. Each antibody is specific to one or a few highly similar proteins, and those that bind to cancer antigens are used in cancer treatment. Once bound to a cancer antigen, antibodies can induce antibody-dependent cell-mediated cytotoxicity, activate the complement system, prevent the interaction of a receptor with its ligand, or release a chemotherapy or radiation payload, all of which can lead to cell death. Currently, there are antibodies approved for the treatment of cancer by the United States Food and Drug Administration (FDA): Rituximab (1997), Trastuzumab (1998), Gentuzumab ozogamicin (2000), Alentuzumab (2001), Ibritumomab tiuxetana (2002), Tositumomab (2003), Cetuximab (2004), Bevacizumab (2004), Panitumumab (2006), Ofatumumab (2009), Ipilimumab (2011) and Brentuximab vedotina (2011).

[009] Although originally approved as anticancer monotherapies, several antibodies have still been approved for use in combination with other anticancer therapies, such as chemotherapy. For example, the FDA granted approval to Rituximab (Rituxan, Genentech, Inc.) combined with fludarabine and cyclophosphamide for the treatment of previously untreated or treated patients with chronic lymphocytic leukemia (CLL). Recently, the FDA approved bevacizumab (Avastin, Genentech, Inc.) combined with paclitaxel and cisplatin or topotecan for the treatment of persistent, recurrent, or metastatic cervical cancer.

[010] There is still an unmet need for combinatorial antibody-based therapies, employing a diversity of antibodies targeting distinct or parallel mechanisms of cancer progression.

SUMMARY OF THE INVENTION

[011] The present invention provides improved combinatorial antibody-based therapies, employing a plurality of antibodies targeting distinct or parallel mechanisms of cancer progression. The present invention derives from the surprising finding that combinations of anti-CEACAM1 antibodies and antibodies directed to disrupt the binding of PD-1 to its natural ligands, PD-L1 and PD-L2, significantly and synergistically elevated lymphocyte cell cytotoxicity. , such as tumor lymphocytic infiltrate cells (TIL) and lymphokine-activated killer cells (LAK), for different types of cancer. Now, for the first time, it is revealed that stepwise pre-incubation of lymphocyte cells with these two types of antibodies, rather than simultaneous incubation, maximizes lymphocyte-mediated cytotoxicity. This unexpected finding can be advantageously exploited to improve clinical outcome, where different antibodies are administered separately to cancer patients to maximize efficacy. Another surprising finding was that blocking CEACAM1 by anti-CEACAM1 antibodies increases PD-L1 expression in cancer cells. Furthermore, for the first time, it was discovered that combinations of antibodies, anti-CEACAM1 antibodies and antibodies directed to disrupt the binding of PD-1 to its natural ligands, PD-L1 and PD-L2, synergistically attenuate the progression of established tumors in a immunocompetent murine model.

[012] The present invention thus provides, in one aspect, a pharmaceutical composition comprising a monoclonal antibody against human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), or an antigen-binding fragment thereof, and a pharmaceutical composition comprising the monoclonal antibody against at least one of human programmed death protein 1 (PD-1), human PD-L1 and PD-L2, or an antigen-binding fragment thereof, for use in the treatment of cancer by separate administration.

[013] The present invention further provides, in another aspect, a pharmaceutical composition comprising a monoclonal antibody against human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a monoclonal antibody capable of inhibiting or blocking the interaction between human programmed cell death protein 1 (PD-1) and its ligands, or an antigen-binding fragment thereof, for use in the treatment of cancer by separate administration.

[014] The present invention further provides, in another aspect, a monoclonal antibody against human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), or an antigen-binding fragment thereof, and a monoclonal antibody capable of inhibiting or blocking the interaction between human programmed cell death protein 1 (PD-1) and its ligands, or an antigen-binding fragment thereof, for use in the treatment of cancer by separate administration.

[015] The present invention further provides, in yet another, a method for treating a patient with cancer, comprising administering to the patient a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against at least one of human PD-1, PD-L1 and PD-L2, or an antigen-binding fragment thereof, by which treating cancer.

[016] The present invention provides, in another aspect, a kit comprising a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against at least one of PD- 1, human PD-L1 and PD-L2, or an antigen-binding fragment thereof.

[017] The present invention provides, in another aspect, the kit described above, for use in treating cancer.

[018] In some embodiments, the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, is capable of binding to a human CEACAM1 protein with an affinity at least close to 10-8 M. Each possibility represents a modality separate from the invention. In some embodiments, the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, is capable of binding to at least one of human CEACAM3 protein and human CEACAM5 with an affinity at least approaching 5x10-7 M. Each possibility represents a separate embodiment of the invention.

[019] In some embodiments, the monoclonal antibody against human CEACAM1 is selected from the group consisting of CM-24, 26H7, 5F4, TEC-11, 12-140-4, 4/3/17, COL-4, F36 -54, 34B1, YG-C28F2, D14HD11, b18.7.7, D11-AD11, HEA81, B1.1, CLB-gran-10, F34-187, T84.1, B6.2, B1.13, YG-C94G7 , 12-140-5, scFv DIATHIS1, TET-2, their antigen-binding antigens and any combination thereof. Each possibility represents a separate embodiment of the invention. In a specific embodiment, the monoclonal antibody against human CEACAM1 is CM-24, or an antigen-binding fragment thereof, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[020] In some embodiments, the monoclonal antibody or fragment thereof that recognizes CEACAM1 comprises at least one heavy chain CDR including a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO : 3, and at least one light chain CDR including a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody or fragment thereof that recognizes CEACAM1 comprises at least two heavy chain CDRs including a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and at least one light chain CDR including a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Each possibility represents a separate embodiment of the invention.

[021] In some embodiments, the monoclonal antibody or fragment thereof that recognizes CEACAM1 comprises a heavy chain CDR sequence with at least five consecutive amino acids, derived from a sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and at least one lead chain CDR sequence of at least five amino acids, derived from a sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. Each possibility represents a separate embodiment of the invention.

[022] In some embodiments, the binding site of the monoclonal antibody or fragment thereof that recognizes CEACAM1 consists of the six CDRs of SEQ ID NOs: 1, 2, 3, 4, 5 and 6. In some embodiments, the binding site of the monoclonal antibody or fragment thereof that recognizes CEACAM1 consists of the six CDRs of SEQ ID NOs: 7, 8, 9, 10, 11 and 12. In some embodiments, the binding site of the monoclonal antibody or fragment thereof that recognizes CEACAM1 consists in the six CDRs of SEQ ID NOs: 13, 14, 15, 16, 17 and 18. In some embodiments, the binding site of the monoclonal antibody or fragment thereof that recognizes CEACAM1 consists of the six CDRs of SEQ ID NOs: 19, 20 , 21, 22, 23 and 24.

[023] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a heavy chain CDR1 including a sequence shown in SEQ ID NO: 1, a heavy chain CDR2 including a sequence shown in SEQ ID NO: 2, a heavy chain CDR3 including a sequence shown in SEQ ID NO: 3, a light chain CDR1 including a sequence shown in SEQ ID NO: 4, a light chain CDR2 including a sequence shown in SEQ ID NO: 5 and a light chain CDR3 including a sequence shown in SEQ ID NO: 6, and analogues and derivatives thereof.

[024] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a heavy chain CDR1 having the sequence shown in SEQ ID NO: 7, a heavy chain CDR2 having the sequence shown in SEQ ID NO: 8 and a heavy chain CDR3 having the sequence set forth in SEQ ID NO: 9. In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a heavy chain CDR1 having the sequence shown in SEQ ID NO: 13, a heavy chain CDR2 having the sequence shown in SEQ ID NO: 14 and a heavy chain CDR3 having the sequence shown in SEQ ID NO: 15.

[025] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a light chain CDR1 having the sequence shown in SEQ ID NO: 10, a light chain CDR2 having the sequence shown in SEQ ID NO: 11 and a light chain CDR3 having the sequence set forth in SEQ ID NO: 12. In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a light chain CDR1 having the sequence shown in SEQ ID NO: 16, a light chain CDR2 having the sequence shown in SEQ ID NO: 17 and a light chain CDR3 having the sequence shown in SEQ ID NO: 18.

[026] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises CDR sequences presented in SEQ ID NOs: 13, 14, 15, 16, 17 and 18. In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises CDR sequences presented in SEQ ID NOs: 7, 8, 9, 10, 11 and 12.

[027] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a heavy chain variable domain sequence having a sequence shown in SEQ ID NO: 25, or analogous or derived therefrom having at least 97% sequence identity with said heavy chain sequence. In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a light chain variable domain sequence having a sequence set forth in SEQ ID NO: 26, or analogous or derivative thereof having at least 97 % sequence identity with said light chain sequence. In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a heavy chain variable domain having a sequence set forth in SEQ ID NO: 25 and a light chain variable domain having a sequence set forth in SEQ ID NO: 26, or analogous or derivative thereof having at least 97% sequence identity with the sequence of the antibody or fragment.

[028] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises: i. a framework sequence selected from the group consisting of: mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2 and human IgG3; and ii. six CDRs having the sequences shown in SEQ ID NOs: 13, 14, 15, 16, 17 and 18; or six CDRs having the sequences shown in SEQ ID NOs: 7, 8, 9, 10, 11 and 12; and analogues and derivatives thereof having at least 97% sequence identity with said CDR sequences, wherein the monoclonal antibody or fragment binds to at least two CEACAM subtypes with an affinity at least close to 5x10-7 M.

[029] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises human-derived constant regions selected from the group consisting of: human IgG1, human IgG2 and human IgG3. In certain embodiments, the human or humanized monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a constant region subclass of the human IgG1 subtype.

[030] In certain embodiments, the human or humanized monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises the six CDRs having the sequences shown in SEQ ID NOs: 13, 14, 15, 16, 17 and 18; or the six CDRs having the sequences shown in SEQ ID NOs: 7, 8, 9, 10, 11 and 12; and analogues and derivatives thereof having at least 95% sequence identity with said CDR sequences, wherein the monoclonal antibody binds to CEACAM1 with an affinity at least close to 10-8 M.

[031] In certain embodiments, the human or humanized monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a heavy chain sequence set forth in SEQ ID NO: 27. In certain embodiments, the human monoclonal antibody or humanized, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a set light chain sequence set forth in SEQ ID NO: 28. In certain embodiments, the human or humanized monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a heavy chain sequence presented in SEQ ID NO: 27 and a light chain sequence presented in SEQ ID NO: 28.

[032] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises at least the antigen-binding portion that is capable of binding to the same epitope on the CEACAM1 molecule to which it binds. a monoclonal antibody with the six CDR sequences shown in SEQ ID NOs: 7, 8, 9, 10, 11 and 12, or the six CDR sequences shown in SEQ ID NOs: 13, 14, 15, 16, 17 and 18 In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof, is reactive with an epitope within residues 17-29 and 68-79 of human CEACAM1 having the sequences VLLLVHNLPQQLF (SEQ ID NO:32) and YPNASLLIQNVT ( SEQ ID NO:33) respectively.

[033] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof, is reactive with an epitope comprising at least four amino acids of the sequence VLLLVHNLPQQLF (SEQ ID NO: 29). In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof, is reactive with an epitope comprising amino acid residues within the sequences VLLLVHNLPQQLF (SEQ ID NO: 29) and YPNASLLIQNVT (SEQ ID NO: 30). In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof, is reactive with an epitope within the sequences VLLLVHNLPQQLF (SEQ ID NO: 29) and PNASLLI (SEQ ID NO: 31).

[034] In certain embodiments, the antigen-binding fragment retains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least at least 80% or at least 90% of the affinity of the corresponding complete antibody for the indicated epitope or antigen. Each possibility represents a separate embodiment of the invention. In certain embodiments, the antigen-binding fragment retains at least 50% of the affinity of the corresponding full-length antibody for the indicated epitope or antigen. In certain embodiments, the antigen-binding fragment retains at least 90% of the affinity of the corresponding full-length antibody for the indicated epitope or antigen.

[035] In some embodiments, the monoclonal antibody against human PD-1 is selected from the group consisting of MK-3475, AMP514, BMS-936558, CT-011, their antigen-binding fragments and any combination thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody against human PD-L1 is selected from the group consisting of MEDI-4736, BMS-936559, MSB0010718C, MPDL3280A, antigen-binding fragments thereof, and any combination thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the anti-PD-L1 antibody is AMP-224.

[036] In some embodiments, the monoclonal antibody against human CEACAM1 is a human or humanized monoclonal antibody. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody against human PD-1 is a human or humanized monoclonal antibody. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody against human PD-L1 is a human or humanized monoclonal antibody. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody against human PD-L2 is a human or humanized monoclonal antibody. Each possibility represents a separate embodiment of the invention. In certain embodiments, a human antibody is an isolated human antibody, that is, isolated from a human donor. In certain embodiments, a human antibody is an isolated human antibody, i.e., isolated from a hybridoma cell line. In certain embodiments, a human antibody refers to a recombinant human antibody, that is, produced by recombinant DNA technology.

[037] In certain embodiments, the pharmaceutical compositions described above further comprise a lymphocyte cell or a plurality of lymphocyte cells.

[038] In some embodiments, the lymphocyte cell expresses CEACAM1, PD-1 or both. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the lymphocyte cell expresses CEACAM1 and PD-1.

[039] In some embodiments, the lymphocyte cell is selected from the group consisting of tumor lymphocytic infiltrate cell (TIL), lymphokine-activated killer cell (LAK), cytokine-induced killer cell (CIK), T cell, B cell , NK cell and any combination thereof. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the lymphocyte cell is selected from the group consisting of tumor lymphocytic infiltrate cell (TIL) and lymphokine-activated killer cell (LAK). In certain such embodiments, the lymphocyte cell is a tumor lymphocytic infiltrate (TIL) cell or a plurality of TIL cells. In certain such embodiments, the lymphocyte cell is a lymphokine-activated killer (LAK) cell or a plurality of LAK cells.

[040] In certain embodiments, the lymphocyte cell is activated. In some embodiments, the lymphocyte cell is cytotoxic to a cancer cell.

[041] In some embodiments, the cancer cell expresses CEACAM1, PD-L1, PD-L2 or any combination thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer cell expresses CEACAM1, PD-L1, or both. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer cell expresses CEACAM1, PD-L2, or both. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the cancer cell expresses CEACAM1 and PD-L1 and PD-L2.

[042] In some embodiments, the cancer is selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, colon, prostate, liver, bladder, renal, cervical, pancreatic, leukemia, myeloid, ovarian, uterine, sarcoma, bile duct and endometrial cells. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer is selected from the group consisting of melanoma and lymphoma-type cancers. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lymphoma.

[043] In some embodiments, the monoclonal antibody against at least one of human PD-1, PD-L1 and PD-L2 is capable of inhibiting or blocking the interaction between human PD-1 and its ligands. Each possibility represents a separate embodiment of the present invention.

[044] In some embodiments, the method comprises administering to said patient a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-1, or a antigen-binding fragment of this. Each possibility represents a separate embodiment of the present invention.

[045] In some embodiments, the method comprises administering to said patient a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-L1, or a antigen-binding fragment of this. Each possibility represents a separate embodiment of the present invention.

[046] In some embodiments, the method comprises administering to said patient a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-L2, or a antigen-binding fragment of this. Each possibility represents a separate embodiment of the present invention.

[047] In some embodiments of the method, the administration of two or more of the pharmaceutical compositions is carried out simultaneously. In some embodiments of the method, administration of two or more of the pharmaceutical compositions is carried out sequentially. In some embodiments of the method, the monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, is administered before the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, is administered simultaneously with the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, is administered after the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, administration of two or more of the pharmaceutical compositions is carried out simultaneously. In some embodiments of the method, administration of two or more of the pharmaceutical compositions is carried out sequentially. In some embodiments of the method, the monoclonal antibody against human PD-L1, or an antigen-binding fragment thereof, is administered before the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L1, or an antigen-binding fragment thereof, is administered simultaneously with the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L1, or an antigen-binding fragment thereof, is administered after the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L2, or an antigen-binding fragment thereof, is administered before the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L2, or an antigen-binding fragment thereof, is administered simultaneously with the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L2, or an antigen-binding fragment thereof, is administered after the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof.

[048] In some embodiments, the method described above further comprises the step of administering to said patient a pharmaceutical composition including a lymphocyte cell. In some embodiments of the method, administration of the pharmaceutical composition including a lymphocyte cell is carried out simultaneously with at least one of the pharmaceutical compositions comprising the antibodies. In some embodiments of the method, the administration of the two or more pharmaceutical compositions is carried out sequentially.

[049] In some embodiments, the lymphocyte cell is previously incubated with a monoclonal antibody against human CEACAM1, or with an antigen-binding fragment thereof, or with a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof. , before administration. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the lymphocyte cell is pre-incubated with a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, prior to administration. In certain other such embodiments, prior to administration, the lymphocyte cell is preincubated with a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, and then incubated with a monoclonal antibody against human CEACAM1, or a fragment thereof. binding to its antigen.

[050] In some embodiments, at least two different antibodies selected from the group consisting of anti-CEACAM1, anti-PD-1, anti-PD-L1 and anti-PD-L2 are included in at least two different pharmaceutical compositions. In some embodiments, at least one antibody selected from the group consisting of anti-CEACAM1, anti-PD-1, anti-PD-L1 and anti-PD-L2 and at least one lymphocyte cell are included in the same pharmaceutical composition.

[051] In some embodiments, the kit comprises a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof. antigen of this. In some embodiments, the kit comprises a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-L1, or an antigen-binding fragment thereof. In some embodiments, the kit comprises a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-L2, or an antigen-binding fragment thereof.

[052] In certain embodiments, each pharmaceutical composition comprising an antibody, or an antigen-binding fragment thereof, is contained in a separate container within the kit.

[053] In certain embodiments, the kit further comprises a pharmaceutical composition including a lymphocyte cell. In certain such embodiments, the kit comprises a pharmaceutical composition including the lymphocyte cell and the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, or the monoclonal antibody against human PD-1, or an antigen-binding fragment thereof. this one. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the kit comprises a pharmaceutical composition including the lymphocyte cell and the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In certain such embodiments, the kit comprises a pharmaceutical composition including the lymphocyte cell and the monoclonal antibody against human PD-1, or an antigen-binding fragment thereof. In certain embodiments, the kit further comprises instructions for using the kit.

BRIEF DESCRIPTION OF THE DRAWINGS

[054] Figure 1A-B. Human melanoma cells express CEACAM1 and PD-L1 upon activation by IFN-Y. MALME 3M cells were incubated with IFN-Y for 24 hours and analyzed by FACS with a PE-conjugated anti-PD-L1 antibody (light histogram) or an isotype-matched control antibody (dark histograms) (A), or with an antibody monoclonal against human CEACAM1, conjugated to PE (light histogram), or an isotype-matched control antibody (dark histograms) (B).

[055] Figure 2A-B. Human TIL cells express PD-1 and CEACAM1. TIL cells were stained with a monoclonal antibody against human PD-1 and analyzed by FACS. Expression of PD-1 (light histogram) compared to a corresponding isotype control (dark histogram) is shown in (A), either with a monoclonal antibody against human CEACAM1, conjugated to PE (light histogram), or an antibody with isotype corresponding control (dark histograms) (B).

[056] Figure 3. Synergistic effects of anti-CEACAM1 and anti-PD-1 antibodies on the cytotoxicity of human TIL cells against human melanoma cells. Human melanoma cells were cultured in the presence of IFN-Y to induce PD-1 expression. TIL cells were incubated with various concentrations of a monoclonal antibody against human CEACAM1 (black dashed line, spherical marker), a monoclonal antibody against human PD-1 (solid gray line, rectangular marker), or a combination of both antibodies (line solid black, triangular marker). IFN-Y-treated melanoma cells were added for an overnight incubation. Results represent an average of % cytotoxicity ± SE (standard error) as determined by the classical LDH release assay from three one-well replicates per treatment. * P<0.05 paired T test compared to monoclonal antibody against human CEACAM1 only.

[057] Figure 4A-B. Synergistic effects of anti-CEACAM1 and anti-PD-1 antibodies on granzyme B levels and cytotoxicity of human TIL cells against human melanoma cells when anti-PD-1 antibodies are added before the addition of anti-CEACAM1 antibodies. Human melanoma cells were cultured in the presence of IFN-Y to induce PD-L1 expression. Human TIL cells were incubated with medium alone (black), nonspecific IgG antibody (white), various concentrations of a monoclonal antibody against human CEACAM1 (vertical lines), a monoclonal antibody against human PD-1 (horizontal lines), or with a combination of both antibodies (dots).

[058] The monoclonal antibody against human PD-1 was added first for 30 minutes at 37 °C, followed by the addition of the monoclonal antibody against human CEACAM1. MALME 3M cells treated with IFN-Y were added for overnight incubation. (A) Results represent an average of % cytotoxicity ± SE, as determined by the classical LDH release assay, from three one-well replicates per treatment. * P<0.05 Paired T test compared to anti-PD-1 only. (B) Results represent granzyme B levels ± SE, as determined by a commercial granzyme B ELISA kit, from three replicates of one well per treatment.

[059] Figure 5A-B. Synergistic effects of anti-CEACAM1 and anti-PD-L1 antibodies on granzyme B levels and cytotoxicity of human TIL cells against human melanoma cells when anti-PD-L1 antibodies are added before the addition of anti-CEACAM1 antibodies. Human melanoma cells were cultured in the presence of IFN-Y to induce PD-L1 expression. Human TIL cells were incubated with medium alone (black), nonspecific IgG antibody (white), various concentrations of a monoclonal antibody against human PD-L1 (horizontal lines), a monoclonal antibody against human CEACAM1 (vertical lines), or a combination of both. antibodies (dots). The anti-PD-L1 antibody was added first for 30 minutes at 37°C, followed by the addition of the monoclonal antibody against human CEACAM1. MALME 3M cells treated with IFN-Y were added for overnight incubation before cytotoxicity was assessed (A). Results represent an average of % cytotoxicity ± SE, as determined by the classical LDH release assay, from three one-well replicates per treatment. (B) Results represent granzyme B levels ± SE, as determined by a commercial granzyme B ELISA kit, from three one-well replicates per treatment. * P<0.05 Paired T test compared to anti-PD-L1 only.

[060] Figure 6. Synergistic effects of anti-CEACAM1 and anti-PD-1 antibodies on the cytotoxicity of human LAK cells against human melanoma cells when anti-PD-1 antibodies are added before the addition of anti-CEACAM1 antibodies. Human melanoma cells were cultured in the presence of IFN-Y to induce PD-L1 expression. Human LAK cells, generated by IL-2 activation of PBMCs from a healthy human donor, were incubated with medium alone (black), nonspecific IgG antibody (white), various concentrations of a monoclonal antibody against human CEACAM1 (vertical lines), a monoclonal antibody against human PD-1 (horizontal lines) or a combination of both antibodies (dots). The monoclonal antibody against human PD-1 was added first for 30 minutes at 37°C, followed by the addition of the monoclonal antibody against human CEACAM1. SKMEL28 cells treated with IFN-Y were added for 24 hours of incubation. Results represent an average of % cytotoxicity ± SE, as determined by the classical LDH release assay, from three one-well replicates per treatment. * P<0.05 Paired T test compared to anti-PD-1 only. The combination index was calculated as described above.

[061] Figure 7A-B. Treatment with anti-CEACAM1 antibodies increases PD-L1 expression on target cancer cells. NK cells (NK92MI) were incubated with or without a monoclonal antibody against human CEACAM1 (CM-24) (10 μg/mL), followed by the addition of human melanoma cells (SKMEL28). Cells were incubated for 24, 48, and 72 hours, and PD-L1 levels were measured at each time point by FACS analysis. A. Average ratio of anti-PD-L1 compared to an appropriate control isotype for the indicated treatments at different times. B. Representative FACS analysis of PD-L1 expression levels after 48 hours.

[062] Figure 8. Synergistic effects of anti-CEACAM1 and anti-PD-1 antibodies on tumor progression in immunocompetent mice. Murine lymphoma cells were implanted subcutaneously into the abdomen of BALB/C mice (Day 1). On Days 10, 15, and 20, mice were intravenously administered PBS (dashed black line, light circles), a murine anti-CEACAM1 antibody (solid gray line, gray rectangles), a murine anti-PD-1 antibody (gray line continuous, gray triangles) or a combination of both antibodies (solid black line, black spheres). The experiment ended on Day 22.

[063] Figure 9A-D. Individual tumor progression curves. Individual tumor progression curves of mice treated with PBS (A), a murine anti-CEACAM1 antibody (B), a murine anti-PD-1 antibody (C), or a combination of both antibodies (D).

DETAILED DESCRIPTION OF THE INVENTION

[064] The present invention derives from the surprising finding that combinations of anti-CEACAM1 antibodies and antibodies directed to disrupt the binding of PD-1 to its natural ligands, PD-L1 and PD-L2, significantly elevated the cytotoxicity of lymphocyte cells such as , for example, human lymphocytic infiltrate cells (TIL) and lymphokine-activated killer cells (LAK), for different types of cancer. It was also surprisingly found that prior staged incubation of lymphocyte cells with these antibodies, rather than simultaneous incubation, maximizes lymphocyte cytotoxicity. It was also found that the expression of CEACAM1 and PD ligands in cancer cells is interrelated, as the binding of anti-CEACAM1 antibodies to cancer cells increases the expression of PD-L1. Furthermore, such antibody combinations were found to significantly attenuate the progression of established tumors in an immunocompetent murine model, presumably exploiting the natural mouse lymphocyte milieu.

[065] Without sticking to any theory or mechanism, the hypothesis is considered, in accordance with the findings of the present invention, that lymphocytes expressing CEACAM1 and/or PD-1 could be considerably suppressed by interactions with their corresponding ligands, CEACAM1 and/or PD-L1 and/or PD-L2, which are presented, for example, by cancer cells. On the other hand, when suppressive interactions are blocked, these lymphocytes can become cytotoxic to these cancer cells upon activation and lead to the elimination of malignant cells.

[066] The present invention thus provides pharmaceutical compositions directed to obstruct two important immunosuppressive homotypic interactions, CEACAM1lymphocyte/CEACAM1cancer cell and PD-1lymphocyte/PDBand-cancer cell, and to increase the level of cytotoxic cancer cells within a patient's body. diagnosed with cancer. Without being limited by any theory or mechanism, it has been hypothesized that the compositions of the present invention generate a “double hit” combination, in which the level of anticancer cytotoxic lymphocytes within the cancer patient is elevated, while the cytotoxicity of the lymphocytes is elevated. is maintained by protective interaction with anti-CEACAM1 antibodies and/or anti-PD-1/PD-L1/PD-L2 antibodies, linked to CEACAM1 and/or PD-1 molecules presented by the lymphocytes themselves, to CEACAM1/PD molecules -L1/PD-L2 presented by cancer cells, or both.

[067] The present invention thus provides, in one aspect, a pharmaceutical composition comprising a monoclonal antibody against human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a monoclonal antibody against at least one of human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, or an antigen-binding fragment thereof, for use in treating cancer by separate administration.

[068] The present invention further provides, in another aspect, a pharmaceutical composition comprising a monoclonal antibody against human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), or an antigen-binding fragment thereof, and a pharmaceutical composition comprising a monoclonal antibody capable of inhibiting or blocking the interaction between human programmed cell death protein 1 (PD-1) and its ligands, or an antigen-binding fragment thereof, for use in the treatment of cancer by separate administration.

[069] The present invention further provides, in another aspect, a monoclonal antibody against human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), or an antigen-binding fragment thereof, and a monoclonal antibody capable of inhibiting or blocking the interaction between human programmed cell death protein 1 (PD-1) and its ligands, or an antigen-binding fragment thereof, for use in the treatment of cancer by separate administration.

[070] The present invention further provides, in yet another, a method for treating a patient with cancer, comprising administering to the patient a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against at least one of human PD-1, PD-L1 and PD-L2, or an antigen-binding fragment thereof, whereby treating cancer.

[071] Furthermore, the present invention provides, in another aspect, a kit comprising a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody at least one of Human PD-1, PD-L1 and PD-L2, or an antigen-binding fragment thereof.

[072] The present invention provides, in another aspect, the kit described above for use in treating cancer.

[073] The term “separate administration” in this specification refers to different therapeutic agents, such as antibodies, being included in separate pharmaceutical compositions. In certain embodiments, different therapeutic agents or different pharmaceutical compositions are administered to cancer patients simultaneously or one after the other. Each possibility represents a separate embodiment of the invention. For example, by the wording “a monoclonal antibody against human CEACAM1 and a monoclonal antibody against human PD-1, for use in treating cancer by separate administration” is meant that the anti-CEACAM1 antibody is contained in a composition that is different from the composition containing the anti-PD-1 antibody.

[074] The term “CEACAM1” is used to refer to the protein product of the CEACAM1 gene, for example, NP_001020083.1, NP_001703.2. In humans, to date, 11 different CEACAM1 variants resulting from splicing have been detected. Individual CEACAM1 isoforms differ with respect to the number of extracellular immunoglobulin-like domains (e.g., CEACAM1 with four extracellular immunoglobulin-like domains is known as CEACAM1-4), membrane anchorage, and/or the length of their tails. cytoplasmic (e.g., CEACAM1-4 with a long cytoplasmic tail is known as CEACAM1-4L and CEACAM1-4 with a short cytoplasmic tail is known as CEACAM1-4S). The N-terminal domain of CEACAM1 starts immediately after the signal peptide and its structure is considered to be IgV-like. For example, in CEACAM1 annotation P13688, the IgV-like N-terminal domain is formed by 108 amino acids, from the 35th to the 142nd amino acid. This domain has been identified as responsible for homophilic binding activity (Watt et al., 2001, Blood. 98, 1469-79). All variants including these splice variants are included within the term “CEACAM1”.

[075] The term “pharmaceutical composition”, in this specification, refers to a composition comprising at least one biologically active ingredient. Antibodies, their antigen-binding fragments, and lymphocyte cells constitute non-limiting examples of biologically active ingredients.

[076] The term "antibody" is used in the broadest sense, including monoclonal antibodies (including complete or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), immunomodulatory agents and antibody fragments with sufficient size to retain and exhibit the desired full biological activity of the antibody.

[077] The term “immunomodulatory agent” or “immunomodulatory protein” or “antibody fragment”, used interchangeably, includes synthetic or genetically engineered proteins that act like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the variable regions, such as "Fv" fragments consisting of the heavy and light chain variable regions, and single-chain recombinant polypeptide molecules, in which the variable regions, light and heavy. , are connected by a spacer peptide (linker) ("scFv proteins"). Fragments can be constructed in different ways to produce multivalent and/or multispecific binding forms. The antibody or antibodies according to the invention include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof such as Fc, Fab or F(ab')2 fragments. Included within the scope of the invention are chimeric antibodies; human and humanized antibodies; recombinant antibodies produced by genetic engineering and fragments thereof.

[078] “A monoclonal antibody against human CEACAM1”, “an antibody that recognizes CEACAM1”, “an antibody against CEACAM1” or “an antibody to CEACAM1” is an antibody that binds to the CEACAM1 protein with sufficient affinity and specificity. Typically, a monoclonal antibody against human CEACAM1 is capable of binding to CEACAM1 with a minimum affinity close to 10-8 or 10-9 M. Some anti-CEACAM1 monoclonal antibodies are capable of binding to CEACAM3, 5 and/or 8 with a minimum affinity close to 5x10-7 M. In certain embodiments, the monoclonal antibody against human CEACAM1 is able to avoid interfering with or dissociating an interaction between CEACAM1, presented by lymphocytes, and CEACAM1 presented by cancer cells.

[079] A “neutralizing antibody”, in this specification, refers to a molecule having an antigen binding site to a specific receptor or target ligand capable of reducing or inhibiting (blocking) activity or signaling through a receptor , as determined in in vivo or in vitro assays, in accordance with the specification.

[080] The term "monoclonal antibody", in this specification, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population are identical, except for possible natural mutations that may be present in minimal quantities. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, unlike polyclonal antibody preparations that typically include different antibodies directed against distinct determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be interpreted to mean that production of the antibody requires any particular method. Monoclonal Abs can be obtained by methods known to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention can be created by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or can be created by recombinant DNA methods (see , e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage-displayed antibody libraries using the techniques described in Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222 :581-597, for example.

[081] The term “plurality”, in this specification, refers to two or more of the specified object.

[082] mAbs can be from any class of immunoglobulin including IgG, IgM, IgE, IgA. A hybridoma producing a mAb can be cultured in vitro or in vivo. High titers of mAbs can be obtained by in vivo production when individual hybridoma cells are injected intraperitoneally into pristin-primed BALB/c mice to produce ascitic fluid containing high concentrations of the desired mAbs. Monoclonal Abs of the IgM or IgG isotype can be purified from such ascitic fluid or culture supernatants using column chromatography methods well known to those skilled in the art.

[083] The term “antigenic determinant” or “epitope” according to the invention refers to the region of an antigen molecule that specifically reacts with a certain antibody. An "antigen" is a molecule or part of a molecule capable of eliciting the formation of antibodies and of being bound by an antibody. An antigen can have one or more than one epitope. An antigen according to the present invention is a CEACAM1 protein or a fragment thereof.

[084] Antibodies, or immunoglobulins, comprise two heavy chains linked by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds with a "Y"-shaped configuration. Proteolytic digestion of an antibody produces Fv (variable fragment) and Fc (crystallizable fragment) domains. The antigen-binding domains, Fab, include regions where the polypeptide sequence varies. The term F(ab')2 represents two Fab' arms joined by disulfide bonds. The central axis of the antibody is called the Fc fragment. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end, with the variable domain of the light chain aligned with the variable domain of the heavy chain and the constant domain of the light chain aligned with the first. heavy chain constant domain (CH1). The variable domains of each pair of light and heavy chains form the antigen-binding site. The domains in the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, joined by three hypervariable domains known as complementarity determining regions (CDR1-3). These domains contribute to the specificity and affinity of the antigen-binding site. The heavy chain isotype (gamma, alpha, delta, epsilon or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa, K or lambda, À) found in all classes of antibodies.

[085] The terms “monoclonal antibody against human CEACAM1”, “monoclonal antibody against human PD-1”, “anti-PD-L1 antibody” and “anti-PD-L2 antibody”, in this specification, refer to molecules complete immunoglobulin molecules, e.g., IgMs, IgDs, IgEs, IgAs or IgGs, antigen-binding domains of such immunoglobulin molecules, e.g., Fab fragments, Fab' fragments, F(ab)2 fragments, F(ab) fragments 2 chimeric or chimeric Fab', chimeric Fab fragments or isolated VH or CDR regions and known isoforms and modifications of immunoglobulins, e.g., single-chain antibodies or single-chain Fv fragments (scAB/scFv) or bispecific antibody constructs, capable of bind to their indicated targets. The terms “anti-human CEACAM1 antibody”, “anti-human PD-1 antibody”, “monoclonal antibody against human PD-L1” and “monoclonal antibody against human PD-L2”, in this specification, refer to antibodies capable of to bind to its indicated targets, where these targets are of human origin.

[086] The terms “antigen-binding fragment of an antibody” and “antigen-binding fragment”, as used interchangeably in this specification, refer to one or more fragments of an antibody that retain the ability to specifically bind to the revealed antigen. For example, the antigen-binding fragment may include, among others, Fab fragment, F(ab')2 fragment, scFv fragment, dAb fragment, CDR-containing fragment, or isolated CDR. Therefore, an antigen-binding fragment of a monoclonal antibody against human CEACAM1 may be, for example, a Fab fragment of a monoclonal antibody against human CEACAM1, or any molecule that mimics the sequences and structure of such a Fab fragment, without having been directly obtained from a monoclonal antibody against human CEACAM1, for example, by chemical or enzymatic cleavage.

[087] "Antibody fragments" comprise only a part of an intact antibody, generally including an antigen-binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments covered by the present definition include: (i) the Fab fragment, with VL, CL, VH and CH1 domains; (ii) the Fab' fragment, which is a Fab fragment with one or more cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd fragment with VH and CH1 domains; (iv) the Fd' fragment with VH and CHI domains and one or more cysteine residues at the C-terminus of the CH1 domain; (v) the Fv fragment with the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) consisting of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulfide bridge in the hinge region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Huston et al., PNAS (USA) 1988, 85.5879 -5883); (x) "diabodies" with two antigen-binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (see, for example, EP 404,097; WO 93 /11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) "linear antibodies" comprising a pair of tandem FD segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al. Protein Eng ., 1995, 8, 1057-1062; and U.S. Patent No. 5,641,870).

[088] By the term "single-chain variable fragment (scFv)", we mean a fusion of the variable regions of the immunoglobulin heavy and light chains, joined by a short spacer (usually serine, glycine). Single-chain antibodies can be polypeptides composed of a single chain with antigen-binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of the light and heavy chain of an immunoglobulin (VH-VL linked or single-chain Fv (scFv)). . Either VH or VL may reproduce natural monoclonal antibody sequences or one or both chains may comprise a CDR-FR construct of the type described in U.S. Patent 5,091,513, the contents of which are incorporated herein in their entirety by reference in this patent application. . Separate polypeptides analogous to the light and heavy chain variable regions are held together by a spacer polypeptide (linker). Methods of producing such single-chain antibodies, especially when the DNA encoding the polypeptide structures of the VH and VL chains are known, can be carried out in accordance with the methods described, for example, in U.S. Patents 4,946,778, 5,091,513 and 5,096,815, the contents of which are incorporated herein in their entirety by reference in this patent application.

[089] Single-chain antibodies can be polypeptides composed of a single chain with antigen-binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of the light and heavy chain of an immunoglobulin, that is, VH-VL linked or Fv single chain (scFv).

[090] The term "molecule with the antigen-binding portion of an antibody", in this specification, is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also its antigen-binding reactive moiety, including, among others, the Fab fragment, the Fab' fragment, the F(ab')2 fragment, the variable portion of the heavy and/or light chain thereof, Fab mini antibodies (see WO 93/15210, WO 96/13583, WO 96/37621, the contents of which are incorporated herein in their entirety by reference in this patent application), dimeric bispecific mini antibodies (see Muller et al., 1998) and chimeric or single chain incorporating such reactive moiety, as well as any other type of molecule or cell into which such reactive moiety of the antibody has been physically inserted, such as a chimeric T cell receptor or a T cell having such a receptor, or molecules designed to release parts therapeutics through a portion of the molecule containing such a reactive moiety. Such molecules can be generated by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.

[091] The term "antigen" in this specification refers to a molecule or part of a molecule capable of eliciting the formation of antibodies and/or of being bound by an antibody. An antigen can have one or more than one epitope. For example, the CEACAM1, PD-1, PD-L1 and/or PD-L2 proteins are each considered an antigen by the present invention. In preferred embodiments, the antigens are human antigens.

[092] The term “antibody capable of inhibiting or blocking the interaction between PD-1 and its ligands”, in this specification, refers to any antibody or its antigen-binding fragment that interferes, inhibits, decreases, eliminates or prevents an interaction between a PD-1 molecule, for example, presented by a lymphocyte cell, and a PD-L1 and/or PD-L2 molecule, for example, presented by a cancer cell, optionally interacting chemically and/or physically with PD-1 and/or PD-L1 and/or PD-L2.

[093] The term “treating cancer”, in this specification, refers to administering effective therapeutic amounts of agents, such as antibodies and/or lymphocyte cells, to a patient diagnosed with cancer to inhibit the further growth of malignant cells in the patient, inhibit the spread of malignant cells in the patient and/or cause the death of malignant cells in the patient. Thus, in certain embodiments, treating cancer means attenuating tumor progression, inhibiting the spread of malignant cells in the patient, causing the death of malignant cells in the patient, and any combination thereof. Each possibility represents a separate embodiment of the invention. In certain embodiments, treating cancer means attenuating tumor progression, causing the death of malignant cells in the patient, or both.

[094] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to some extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., slow down and preferably stop) tumor metastases; inhibit tumor growth to some extent; and/or alleviate to some extent one or more of the symptoms associated with the disorder. To the extent that it can prevent the growth of and/or eliminate existing cancer cells, the drug may be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can, for example, be gauged by evaluating duration of survival, time to disease progression (TTP), response rates (RR), duration of response, and/or the quality of life.

[095] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include melanoma, lung, thyroid, breast, colon, prostate, liver, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterine, sarcoma, biliary, or endometrial cancer.

[096] The term "antineoplastic composition" refers to a composition useful in the treatment of cancer, comprising at least one active therapeutic agent capable of inhibiting or preventing tumor growth or function and/or causing the destruction of tumor cells. Suitable therapeutic agents in an antineoplastic composition for treating cancer include, among others, chemotherapeutic agents, radioactive isotopes, toxins, cytokines such as interferons and antagonistic agents directed against cytokines, cytokine receptors or antigens associated with tumor cells.

[097] The term "kit", in this specification, refers to a combination of reagents and other materials. It is contemplated that the kit may include reagents such as antibodies, antibody mixtures, buffers, diluents and other aqueous solutions and/or one or more vials or other storage containers. The term "kit" is not intended to be restricted to a particular combination of reagents and/or other materials.

[098] The present invention also provides pharmaceutical compositions aimed at simultaneously obstructing two important immunosuppressive interactions: CEACAM1 lymphocyte/CEACAM1 cancer cell and PD-1 lymphocyte/PD Ligand cancer cell. Thus, in some embodiments, the pharmaceutical compositions comprise a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a monoclonal antibody against human PD-1 or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the pharmaceutical compositions comprise a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a monoclonal antibody against human PD-L1 or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the pharmaceutical compositions comprise a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a monoclonal antibody against human PD-L2 or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the pharmaceutical compositions comprise a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, and a monoclonal antibody against PD-L1. human or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the pharmaceutical compositions comprise a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, and a monoclonal antibody against PD-L2. human or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention.

[099] In one aspect, the present invention provides a pharmaceutical composition comprising an anti-human CEACAM1 monoclonal antibody, or an antigen-binding fragment thereof, and a monoclonal antibody selected from the group consisting of an anti-PD-1 antibody. human, or an antigen-binding fragment thereof, an anti-human PD-L1 antibody, or an antigen-binding fragment thereof, and an anti-human PD-L2 antibody or an antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition comprises a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a monoclonal antibody against human PD-1 or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the pharmaceutical composition comprises a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a monoclonal antibody against human PD-L1 or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the pharmaceutical composition comprises a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a monoclonal antibody against human PD-L2 or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the pharmaceutical composition comprises a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, and a monoclonal antibody against PD-L1. human or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the pharmaceutical composition comprises a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, and a monoclonal antibody against PD-L2. human or an antigen-binding fragment thereof. Each possibility represents a separate embodiment of the invention.

[0100] The expression “the pharmaceutical compositions comprise antibody A and antibody B” refers to at least two separate pharmaceutical compositions, each comprising at least one antibody that is different from the antibody comprised in the other pharmaceutical composition. The expression “the pharmaceutical composition comprises” refers to a single pharmaceutical composition comprising at least two different antibodies.

[0101] Many monoclonal antibodies against the CEACAM1 protein are already known, all of which are considered suitable for use in the compositions and methods of the present invention. Nevertheless, in some embodiments, the monoclonal antibody against human CEACAM1 is selected from the group consisting of CM-24, 26H7, 5F4, TEC-11, 12-140-4, 4/3/17, COL-4, F36 -54, 34B1, YG-C28F2, D14HD11, b18.7.7, D11-AD11, HEA81, B1.1, CLB-gran-10, F34-187, T84.1, B6.2, B1.13, YG-C94G7 , 12-140-5, scFv DIATHIS1 and TET-2, their antigen-binding fragments and any combination thereof. Each possibility represents a separate embodiment of the invention.

[0102] In a specific embodiment, the monoclonal antibody against human CEACAM1 is CM-24, its antigen-binding fragment and any combination thereof. Each possibility represents a separate embodiment of the invention.

[0103] CM-24 CDR segments were identified using two methods with different algorithms: 1. IMGT algorithm (Lefranc et al., 1999, Nucleic Acids Research, 27, 209212); 2. KABAT algorithm (Wu T.T. and Kabat E.A., 1970, J. Exp. Med. 132, 211250).

[0104] Table 1 summarizes the CDR sequences determined using both methods, as well as the minimum consensus sequence and the combined sequence of sequences identified using both methods. Table 1. CDR sequences where X1 is absent or is Thr (T) and X2 is absent or is Ile (I).

[0105] In some embodiments, the monoclonal antibody or fragment thereof that recognizes CEACAM1 comprises at least one heavy chain CDR including a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO : 3, and at least one light chain CDR including a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Each possibility represents a separate embodiment of the invention.

[0106] In some embodiments, the monoclonal antibody or fragment thereof that recognizes CEACAM1 comprises at least two heavy chain CDRs including a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO : 3, and at least one light chain CDR including a sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Each possibility represents a separate embodiment of the invention.

[0107] In some embodiments, the monoclonal antibody or fragment thereof that recognizes CEACAM1 comprises at least one heavy chain CDR sequence with at least five consecutive amino acids derived from a sequence selected from the group consisting of: SEQ ID NO: 19 , SEQ ID NO: 20 and SEQ ID NO: 21, and at least one light chain CDR sequence with at least five amino acids derived from a sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. Each possibility represents a separate embodiment of the invention.

[0108] In some embodiments, the binding site of the monoclonal antibody or fragment thereof that recognizes CEACAM1 consists of the six CDRs of SEQ ID NOs: 1, 2, 3, 4, 5 and 6. In some embodiments, the binding site of the monoclonal antibody or fragment thereof that recognizes CEACAM1 consists of the six CDRs of SEQ ID NOs: 7, 8, 9, 10, 11 and 12. In some embodiments, the binding site of the monoclonal antibody or fragment thereof that recognizes CEACAM1 consists of the six CDRs of SEQ ID NOs: 13, 14, 15, 16, 17 and 18. In some embodiments, the monoclonal antibody binding site or fragment thereof that recognizes CEACAM1 consists of the six CDRs of SEQ ID NOs: 19, 20, 21, 22 , 23 and 24.

[0109] In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises a heavy chain CDR1 including a sequence set forth in SEQ ID NO: 1, a heavy chain CDR2 including a sequence set forth in SEQ ID NO: 2, a heavy chain CDR3 including a sequence shown in SEQ ID NO: 3, a light chain CDR1 including a sequence shown in SEQ ID NO: 4, a light chain CDR2 including a sequence shown in SEQ ID NO : 5 and a light chain CDR3 including a sequence shown in SEQ ID NO: 6 and analogues and derivatives thereof.

[0110] In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises a heavy chain CDR1 having the sequence set forth in SEQ ID NO: 7, a heavy chain CDR2 having the sequence set forth in SEQ ID NO: 8 and a heavy chain CDR3 having the sequence shown in SEQ ID NO: 9. In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises a heavy chain CDR1 having the sequence shown in SEQ ID NO: 13, a heavy chain CDR2 having the sequence shown in SEQ ID NO: 14 and a heavy chain CDR3 having the sequence shown in SEQ ID NO: 15.

[0111] In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises a light chain CDR1 having the sequence set forth in SEQ ID NO: 10, a light chain CDR2 having the sequence set forth in SEQ ID NO: 11 and a light chain CDR3 having the sequence shown in SEQ ID NO: 12. In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises a light chain CDR1 having the sequence shown in SEQ ID NO: 16, a light chain CDR2 having the sequence shown in SEQ ID NO: 17 and a light chain CDR3 having the sequence shown in SEQ ID NO: 18.

[0112] In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises CDR sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17 and 18. In certain embodiments, the antibody monoclonal or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises CDR sequences presented in SEQ ID NOs: 7, 8, 9, 10, 11 and 12.

[0113] In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises a heavy chain variable domain sequence having a sequence set forth in SEQ ID NO: 25, or an analogue or derivative thereof having at least at least 97% sequence identity with said heavy chain sequence. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof that recognizes human CEACAM1 comprises a light chain variable domain sequence having a sequence set forth in SEQ ID NO: 26, or an analogue or derivative thereof having at least 97% of sequence identity with said light chain sequence. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof that recognizes human CEACAM1 comprises a heavy chain variable domain having a sequence set forth in SEQ ID NO: 25 and a light chain variable domain having a sequence set forth in SEQ ID NO: 26, or an analogue or derivative thereof having at least 97% sequence identity with the sequence of the antibody or fragment.

[0114] In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises: i. a framework sequence selected from the group consisting of: mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, human IgG3; and ii. six CDRs having the sequences shown in SEQ ID NOs: 13, 14, 15, 16, 17 and 18; or six CDRs having the sequences shown in SEQ ID NOs: 7, 8, 9, 10, 11 and 12; and analogues and derivatives thereof having at least 97% sequence identity with said CDR sequences, wherein the monoclonal antibody or fragment binds to at least two CEACAM subtypes with an affinity at least close to 5x10-7 M.

[0115] In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises human-derived constant regions selected from the group consisting of: human IgG1, human IgG2 and human IgG3. In certain embodiments, the human or humanized monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises a constant region subclass of the human IgG1 subtype.

[0116] In certain embodiments, the human or humanized monoclonal antibody or an antigen-binding fragment thereof that recognizes human CEACAM1 comprises the six CDRs having the sequences set forth in SEQ ID NOs: 13, 14, 15, 16, 17 and 18; or the six CDRs having the sequences shown in SEQ ID NOs: 7, 8, 9, 10, 11 and 12; and analogues and derivatives thereof having at least 95% sequence identity with said CDR sequences, wherein the monoclonal antibody binds to CEACAM1 with an affinity at least close to 10-8 M.

[0117] In certain embodiments, the human or humanized monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a heavy chain sequence set forth in SEQ ID NO: 27. In certain embodiments, the human monoclonal antibody or humanized, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises a light chain sequence set forth in SEQ ID NO: 28. In certain embodiments, the human or humanized monoclonal antibody, or an antigen-binding fragment thereof that recognizes Human CEACAM1 comprises a heavy chain sequence presented in SEQ ID NO: 27 and a light chain sequence presented in SEQ ID NO: 28.

[0118] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof that recognizes human CEACAM1, comprises at least the antigen-binding portion, which is capable of binding to the same epitope on the CEACAM1 molecule to which it binds. binds a monoclonal antibody having the six CDR sequences shown in SEQ ID NOs: 7, 8, 9, 10, 11 and 12, or the six CDR sequences shown in SEQ ID NOs: 13, 14, 15, 16, 17 and 18. In certain embodiments, the monoclonal antibody or an antigen-binding fragment thereof is reactive with an epitope within residues 17-29 and 68-79 of human CEACAM1 having the sequences VLLLVHNLPQQLF (SEQ ID NO:32) and YPNASLLIQNVT (SEQ ID NO:33) respectively.

[0119] In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof, is reactive with an epitope comprising at least four amino acids of the sequence VLLLVHNLPQQLF (SEQ ID NO: 29). In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof, is reactive with an epitope comprising amino acid residues within the sequences VLLLVHNLPQQLF (SEQ ID NO: 29) and YPNASLLIQNVT (SEQ ID NO: 30). In certain embodiments, the monoclonal antibody, or an antigen-binding fragment thereof, is reactive with an epitope within the sequences VLLLVHNLPQQLF (SEQ ID NO: 29) and PNASLLI (SEQ ID NO: 31).

[0120] In some embodiments, the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, is capable of binding to a human CEACAM1 protein with an affinity at least close to 10-8 M. Each possibility represents an embodiment separate from the invention. In some embodiments, the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, is capable of binding to a human CEACAM3 and human CEACAM5 protein with an affinity at least approaching 5x10-7 M. Each possibility represents an embodiment separate from the invention.

[0121] Several monoclonal antibodies against the PD-1 protein are already known, all of which are considered suitable for use in the compositions and methods of the present invention. However, in some embodiments, the monoclonal antibody against human PD-1 is selected from the group consisting of MK-3475, AMP514, BMS-936558, CT-011, antigen-binding fragments thereof, and any combination thereof. Each possibility represents a separate embodiment of the invention.

[0122] Several monoclonal antibodies against the PD-L1 protein are already known, all of which are considered suitable for use in the compositions and methods of the present invention. However, in some embodiments, the monoclonal antibody against human PD-L1 is selected from the group consisting of MEDI-4736, BMS-936559, MSB0010718C, MPDL3280A, antigen-binding fragments thereof, and any combination thereof. Each possibility represents a separate embodiment of the invention. A non-limiting example of an antibody according to the present invention is AMP-224, also known as B7-DCIg, which is a fusion protein of the soluble PD-L1 receptor and the Fc domain of an antibody, described in PCT Publications of Patent Application Nos. WO/2010/027827 and WO/2011/066342.

[0123] Several monoclonal antibodies against the PD-L2 protein are already known, such as those described in the PCT Patent Application Publication in WO/2010/036959, which are all considered suitable for use in the compositions and methods of the present invention .

[0124] When the process of developing a specific antibody involves generation in a non-human immune system (such as that in mice), the protein sequences of the antibodies produced are partially distinct from those of homologous antibodies occurring naturally in humans and are therefore , potentially immunogenic when administered to human patients.

[0125] Thus, to avoid immunogenicity when administered to a human patient, in some embodiments, the monoclonal antibody against human CEACAM1 is a human or humanized monoclonal antibody. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody against human PD-1 is a human or humanized monoclonal antibody. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody against human PD-L1 is a human or humanized monoclonal antibody. Each possibility represents a separate embodiment of the invention. In some embodiments, the monoclonal antibody against human PD-L2 is a human or humanized monoclonal antibody. Each possibility represents a separate embodiment of the invention.

[0126] The term "human antibody" in this specification is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in CDRs. However, the term "human antibody" in this specification is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. In certain embodiments, the term "human antibody" refers to an isolated human antibody, that is, isolated from a human donor. In certain embodiments, the term "human antibody" refers to a human antibody isolated from a hybridoma cell line. In certain embodiments, the term "human antibody" refers to a recombinant human antibody, that is, produced by recombinant DNA technology.

[0127] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and/or has been created using any of the techniques described here for creating human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known on the subject. In one embodiment, the human antibody is selected from a library of phage-displayed antibodies, wherein the phage-displayed library expresses human antibodies (Vaughan et al. Nature Biotechnology 1996 14,309-314; Sheets et al. PNAS (USA) , 1998, 95, 61576162; Hoogenboom and Winter, J. Mol. Biol., 1991, 227, 381; Marks et al., J. Mol. Biol., 1991, 222, 581). Human antibodies can also be created by introducing human immunoglobulin loci into transgenic animals, for example, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon exposure, production of human antibodies is observed, which closely resembles that seen in humans in all aspects, including gene rearrangement, assembly and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5 545 806; 5,569,825; 5 625 126; 5 633 425; 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, human antibody can be prepared via immortalization of human B lymphocytes, producing an antibody directed against a target antigen (such B lymphocytes can be recovered from an individual or can be immunized in vitro). See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1):86-95 (1991); and U.S. Patent No. 5,750,373.

[0128] The term "recombinant human antibody" in this specification is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a combinatorial library of recombinant human antibodies, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the Recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human germline antibody repertoire in vivo.

[0129] The term "humanized antibody" in this specification is intended to include antibodies whose CDRs (complementarity determining regions) derive from an immunoglobulin from a non-human species, and the remainder of the antibody molecule is derived primarily from an human immunoglobulin.

[0130] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. Most often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the receptor are replaced by residues from a hypervariable region from a non-human species (donor antibody), such as mouse, rat, rabbit or primate. non-human having the desired specificity, affinity and capacity. In some cases, residues from the framework region (FR) of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody comprises substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of an immunoglobulin sequence. human. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see Jones et al., Nature 1986, 321, 522-525; Riechmann et al., Nature 1988, 332, 323329; and Presta, Curr. Op. Struct. Biol., 1992 2, 593-596.

[0131] The term "recombinant human antibody" in this specification is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a combinatorial library of recombinant human antibodies, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the Recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human germline antibody repertoire in vivo.

[0132] Without being bound by any mechanism or theory, it is suggested that the substantial anticancer cytotoxic effect demonstrated by the combination of antibodies of the present invention, in vitro and in vivo, can be reinforced by exogenous lymphocyte cells. Thus, in certain embodiments, the pharmaceutical compositions described above further comprise a lymphocyte cell or a plurality of lymphocyte cells.

[0133] The term “lymphocyte cell” or “lymphocyte” in this specification refers to any of natural killer (NK) cells (usually involved in cell-mediated cytotoxic innate immunity), T cell (usually involved in cytotoxic adaptive immunity cell-mediated), B cell (usually involved in antibody-driven adaptive humoral immunity), a plurality and any combination of such cells. Peripheral blood mononucleated cells (PBMC), tumor lymphocytic infiltrate cells (TIL) and lymphokine-activated killer (LAK) cells (usually involved in eliminating tumor cells) are also considered lymphocytic cells. Non-limiting examples of lymphocytes include cytotoxic lymphocytes (CTLs, CD8+ or CD4+), NK cells (CD2+), and T helper cells (CD4+).

[0134] The terms “lymphokine-activated killer (LAK) cell”, “lymphokine-activated killer cell” and “LAK cell” are used interchangeably and refer to a lymphocyte cell that has been stimulated or activated to eliminate tumor cells, i.e. , to become more cytotoxic against tumor cells. LAK cells can be produced from homogeneous or heterogeneous cell populations, therefore the above terms further refer to homogeneous or heterogeneous cell populations, stimulated or activated to become more cytotoxic against tumor cells. In specific embodiments, LAK cells are produced from PBMC cells by IL-2.

[0135] It has been formally well established that known lymphocyte activating agents, such as IL-2, Interferon-Y (IFN-Y) and/or anti-CD3 monoclonal antibodies, are capable of transforming lymphocytes, such as PBMC cells, so that they become more cytotoxic, thus producing a population of cells called “activated cytotoxic lymphocyte cells”, “activated lymphocyte cells”, “cytotoxic lymphocyte cells” or “activated lymphocytes”, in vitro and in vivo. The present invention, and others, provide guidance to one of ordinary skill in the art on how to activate PBMCs to produce diverse populations of activated cytotoxic lymphocyte cells without adverse side effects. For example, Stephen E. Ettinghausen and colleagues established that systemic administration of recombinant IL-2 stimulates in vivo proliferation of lymphoid cells in tissues (Stephen E. Ettinghausen et al., The Journal of Immunology, 1985, Vol. 135, No. 2, pages 1488-1497), and that recombinant IL-2 stimulates the in vivo proliferation of LAK cells received by adoptive transfer (Stephen E. Ettinghausen et al., The Journal of Immunology, 1985, Vol. 135, No 5, pages 3623-3635). Joseph H. Phillips and colleagues have established that continuous administration of IL-2 at 100,000 U IL-2/kg every 8 weeks is sufficient to prime PBMC cells to become LAK cells in patients with advanced colon carcinoma, malignant melanoma, or breast cancer. kidney cells (J. Clin. Oncol., 1987, Vol. 5, p. 1933-1941). Cytokine-induced killer cells (CIK) are human CD8 T cells activated in vitro that have acquired nonspecific antitumor cytotoxicity and thus represent a cell population with a dual T cell and NK cell phenotype. Thanks to their in vitro intratumoral housing and lack of graft-versus-host (GVH) reactivity, CIK cells have been extensively used in cancer patients in autologous or allogeneic settings. M. Introna et al. (Immunology Letters, 2013, Vol. 155, pages 27-30) provide a summary of the main biological characteristics of CIK cells, as well as their most prominent results.

[0136] In some embodiments, the lymphocyte cell expresses CEACAM1, PD-1 or both. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the lymphocyte cell expresses CEACAM1 and PD-1.

[0137] In some embodiments, the lymphocyte cell is selected from the group consisting of tumor lymphocytic infiltrate cell (TIL), lymphokine-activated killer cell (LAK), cytokine-induced killer cell (CIK), T cell, B cell , NK cell and any combination thereof. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the lymphocyte cell is selected from the group consisting of tumor lymphocytic infiltrate cell (TIL) and lymphokine-activated killer cell (LAK). In certain such embodiments, the lymphocyte cell is a tumor lymphocytic infiltrate (TIL) cell or a plurality of TIL cells. In certain such embodiments, the lymphocyte cell is a lymphokine-activated killer (LAK) cell or a plurality of LAK cells.

[0138] Without being bound by any mechanism or theory, it is suggested that the desired anticancer cytotoxic effect demonstrated by the lymphocytes of the present invention in vitro can be maximized using activated lymphocyte cells. Thus, in certain embodiments, the lymphocyte cell is activated.

[0139] The term “activated” in this specification refers to any lymphocyte cell exhibiting cytotoxic activity, for example, against a cancer cell line, such as human melanoma cells (MALME 3M or SKMEL28), or exhibiting high levels of granzyme B production.

[0140] In some embodiments, the lymphocyte cell is cytotoxic to a cancer cell.

[0141] The term “cytotoxic”, in this specification, refers to an agent (such as a lymphocyte cell) that is harmful to the structure and/or function of a cell (such as a cancer cell). For example, when exposed to dysfunctional somatic cells, cytotoxic T lymphocytes (CTLs) release the cytotoxins perforin, granzymes, and granulysin. Through the action of perforin, granzymes penetrate the cytoplasm of the target cell and their serine protease function triggers the caspase cascade, which is a series of cysteine proteases that ultimately lead to apoptosis (programmed cell death).

[0142] The compositions and methods provided by the present invention demonstrate to be effective against several types of cancer unrelated by origin. In some embodiments, the cancer cell expresses CEACAM1, PD-L1, PD-L2, or any combination thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer cell expresses CEACAM1, PD-L1, or both. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer cell expresses CEACAM1, PD-L2, or both. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the cancer cell expresses CEACAM1 and PD-L1 and PD-L2.

[0143] In some embodiments, the cancer is selected from the group consisting of melanoma, lymphoma, lung cancer, thyroid, breast, colon, prostate, liver, bladder, renal, cervical, pancreatic, leukemia, myeloid, ovarian, of uterus, sarcoma, biliary and endometrial cells. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer is selected from the group consisting of melanoma and lymphoma-type cancers. Each possibility represents a separate embodiment of the present invention. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lymphoma.

[0144] In some embodiments, the monoclonal antibody against at least one of human PD-1, PD-L1 and PD-L2 is capable of inhibiting or blocking the interaction between human PD-1 and its ligands. Each possibility represents a separate embodiment of the present invention.

[0145] As described above, the present invention provides a method that uses targeted pharmaceutical compositions to simultaneously obstruct two important immunosuppressive interactions: CEACAM1 lymphocyte/CEACAM1 cancer cell and PD-1 lymphocyte/PD Ligand cancer cell. Accordingly, in some embodiments, the method comprises administering to said patient a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-1, or a antigen-binding fragment of this.

[0146] In some embodiments, the method comprises administering to said patient a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-L1, or a antigen-binding fragment of this.

[0147] In some embodiments, the method comprises administering to said patient a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-L2, or a antigen-binding fragment of this.

[0148] In some embodiments of the method, the monoclonal antibody against human PD-1 or an antigen-binding fragment thereof is administered before the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-1 or an antigen-binding fragment thereof is administered simultaneously with the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-1 or an antigen-binding fragment thereof is administered after the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof. In some embodiments of the method, administration of two or more of the pharmaceutical compositions is carried out simultaneously. In some embodiments of the method, administration of two or more of the pharmaceutical compositions is carried out sequentially. In some embodiments of the method, the monoclonal antibody against human PD-L1 or an antigen-binding fragment thereof is administered before the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L1 or an antigen-binding fragment thereof is administered simultaneously with the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L1 or an antigen-binding fragment thereof is administered after the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L2 or an antigen-binding fragment thereof is administered before the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L2 or an antigen-binding fragment thereof is administered simultaneously with the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof. In some embodiments of the method, the monoclonal antibody against human PD-L2 or an antigen-binding fragment thereof is administered after the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof.

[0149] In some embodiments, the method described above further comprises the step of administering to said patient a pharmaceutical composition comprising a lymphocyte cell. In some embodiments of the method, administration of the pharmaceutical composition comprising a lymphocyte cell is carried out simultaneously with at least one of the pharmaceutical compositions comprising the antibodies. In some embodiments of the method, the administration of the two or more pharmaceutical compositions is carried out sequentially.

[0150] Surprisingly, it was found that the binding of anti-CEACAM1 antibodies and anti-PD-1 antibodies to their respective targets on lymphocyte cells is somewhat interrelated. Thus, in some embodiments, the lymphocyte cell is previously incubated with a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, or with a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof prior to administration. Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the lymphocyte cell is previously incubated with a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof prior to administration. . In certain other such embodiments, prior to administration, the lymphocyte cell is preincubated with a monoclonal antibody against human PD-1, or an antigen-binding fragment thereof, and then incubated with a monoclonal antibody against human CEACAM1 or a fragment thereof. binding to its antigen.

[0151] The expression “previously incubated with”, in this specification, refers to the fact that the lymphocyte cell, the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof and/or the monoclonal antibody against PD-1 human antibody or an antigen-binding fragment thereof are contained in the same pharmaceutical compositions under such conditions as to allow one or both antibodies, or antigen-binding fragments thereof, to bind to their targets presented by the lymphocyte cell in a specific manner.

[0152] The human leukocyte antigen (HLA) system is the locus of genes that encode proteins on the surface of cells that are responsible for regulating the immune system in humans. HLA genes represent the human versions of the major histocompatibility complex (MHC) genes that are found in most vertebrates. HLAs are routinely used to match a patient suffering from a disease with a healthy donor for cell or organ transplantation. The best result in transplantation occurs when the patient's HLAs and the donor's HLAs are closely compatible, preferably identical. The term “autologous lymphocyte cell” in this specification refers to a lymphocyte cell obtained or derived from a cancer patient, optionally expanded or incubated with anti-CEACAM1 antibodies, or antigen-binding fragments thereof, and/or antibodies anti-PD-1 or its antigen-binding fragments, and administered to the same cancer patient. The term “autologous lymphocyte cell” in this specification further refers to a lymphocyte cell obtained or derived from a compatible human leukocyte antigen (HLA) donor.

[0153] In some embodiments, at least two different antibodies selected from the group consisting of anti-CEACAM1, anti-PD-1, anti-PD-L1 and anti-PD-L2 are comprised in at least two different pharmaceutical compositions. In some embodiments, at least one antibody selected from the group consisting of anti-CEACAM1, anti-PD-1, anti-PD-L1 and anti-PD-L2 and at least one lymphocyte cell are comprised in the same pharmaceutical composition.

[0154] In some embodiments, the kit comprises a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-1 or an antigen-binding fragment thereof. this one. In some embodiments, the kit comprises a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-L1 or an antigen-binding fragment thereof. In some embodiments, the kit comprises a pharmaceutical composition including a monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, and a pharmaceutical composition including a monoclonal antibody against human PD-L2 or an antigen-binding fragment thereof.

[0155] In certain embodiments, each pharmaceutical composition including an antibody, or an antigen-binding fragment thereof, is contained in a separate container within the kit.

[0156] In certain embodiments, the kit further comprises a pharmaceutical composition including a lymphocyte cell. In certain such embodiments, the kit comprises a pharmaceutical composition including the lymphocyte cell and the monoclonal antibody against human CEACAM1, or an antigen-binding fragment thereof, or the monoclonal antibody against human PD-1 or an antigen-binding fragment thereof. . Each possibility represents a separate embodiment of the present invention. In certain such embodiments, the kit comprises a pharmaceutical composition including the lymphocyte cell and the monoclonal antibody against human CEACAM1 or an antigen-binding fragment thereof. In certain such embodiments, the kit comprises a pharmaceutical composition including the lymphocyte cell and the monoclonal antibody against human PD-1 or an antigen-binding fragment thereof. In certain embodiments, the kit further comprises instructions for using the kit.

[0157] The following examples are intended to illustrate how to create and use the compounds and methods of this invention and should not be interpreted in any way as a limitation. Although the invention will now be described together with its specific embodiments, it is clear that many modifications and variations will be clear to those skilled in the art. Therefore, the intention is that all modifications and variations fall within the spirit and broad scope of the attached claims.

EXAMPLES Example 1 - Human melanoma cells express CEACAM1 and PD-L1 upon activation by IFN-Y

[0158] Human melanoma cells (MALME 3M) were incubated with IFN-γ (2000 units/mL) for 24 hours, followed by FACS analysis with a PE-conjugated anti-PD-L1 antibody (clone 29E.2A3) or with a monoclonal antibody against human CEACAM1 conjugated to PE (CM-24, developed by cCAM Biotherapeutics). A similar assay was conducted for isotype control.

[0159] Figure 1 demonstrates that the human melanoma cells used in the experiments express CEACAM1 and PD-L1.

Example 2 - Human TIL cells express PD-1 and CEACAM1

[0160] Human TIL cells (expanded tumor-infiltrating lymphocytes from patients #14 at the Ella institute, Lot 14, TIL14) were stained with a monoclonal antibody against human PD-1 (clone E12.2H7) or with a monoclonal antibody against human CEACAM1 conjugated to PE (CM-24), followed by FACS analysis. Expression of PD-1 (A) and CEACAM1 (B) is shown in comparison to an isotype-matched control.

[0161] Figure 2 demonstrates that the human TIL cells used in the experiments express PD-1 and CEACAM1.

Example 3 - Synergistic effects of anti-CEACAM1 and anti-PD-1 antibodies on the cytotoxicity of human TIL cells against human melanoma cells

[0162] Human melanoma cancer cells (MALME 3M) were cultured in the presence of IFN-γ to induce PD-L1 expression. Human TIL cells (TIL14) were incubated with a monoclonal antibody against human CEACAM1 (CM-24) (0.01 μg/mL, 0.05 μg/mL, 0.1 μg/mL, 0.5 μg/mL), a monoclonal antibody against human PD-1 (clone E12.2H7) or with a combination of both antibodies (0.005; 0.025; 0.05 and 0.25 μg/mL of each antibody) for 30 minutes at 37 °C.

[0163] Human melanoma cancer cells, treated with IFN-Y, were added for overnight incubation, before cytotoxicity assessment (Figure 3). Results represent an average of % cytotoxicity ± SE as determined by the classical LDH release assay from three one-well replicates per treatment. * P<0.05 Paired T test compared to CM-24 only. The combination index (CI) calculated was < 0.2, according to the following equation:

[0164] Figure 3 demonstrates that both antibodies, anti-CEACAM1 and anti-PD-1, were able to bind to their respective targets on human lymphocytes, such as TIL cells, and that this binding significantly increased the toxicity of TIL cells against human cancer cells during each monotherapy alone. Therefore, the data presented in Figure 3 indicate that protecting lymphocytes from immunosuppressive signaling from target cancer cells results in considerable cytotoxicity against these cancer cells.

Example 4 - Synergistic effects of anti-CEACAM1 anti-PD-1 antibodies on granzyme B levels and on the cytotoxicity of human TIL cells against human melanoma cells when anti-PD-1 antibodies are added before the addition of anti-CEACAM1 antibodies

[0165] Human melanoma cancer cells (MALME 3M) were cultured in the presence of IFN-Y to induce PD-L1 expression. Human TIL cells (TIL14) were incubated with medium only (black), nonspecific IgG antibody (0.8 μg/mL, white), various concentrations (0.05 μg/mL, 0.1 μg/mL, 0.2 μg /mL, 0.4 μg/mL, 0.8 μg/mL) of a monoclonal antibody against human CEACAM1 (CM-24), a monoclonal antibody against human PD-1 (clone E12.2H7), or a combination of both antibodies (0.05 μg/mL each, 0.1 μg/mL each, 0.2 μg/mL each, 0.4 μg/mL each, 0.8 μg/mL each).

[0166] The monoclonal antibody against human PD-1 was added first for 30 minutes at 37 ° C, followed by the addition of the monoclonal antibody against human CEACAM1. Human melanoma cancer cells, treated with IFN-Y, were added for overnight incubation before cytotoxicity assessment (Figure 4A). The calculated combination index (CI) was 0.15. Results represent an average of % cytotoxicity ± SE as determined by the classical LDH release assay from three one-well replicates per treatment. * P<0.05 Paired T test compared to anti-PD-1 only. In the same assay, the level of the cytotoxic granzyme B protein, which is secreted upon activation of cytotoxic cells, was assessed by a commercial ELISA kit for granzyme B (Figure 4B). The results represent the average level of granzyme B, calculated from three replicates of one well per treatment.

[0167] Figure 4 demonstrates that anti-CEACAM1 antibodies and anti-PD-1 antibodies are capable of binding to their respective targets on human lymphocytes, such as TIL cells, and that this binding increases granzyme B secretion and the toxicity of human TIL cells against human cancer cells. Figure 4 further demonstrates that the binding of these antibodies to human TIL cells is interrelated to some extent, justifying future study of their binding mechanism. Similar to the data presented in Figure 3, Figure 4 again indicates that protecting lymphocytes from immunosuppressive signaling from target cancer cells results in considerable cytotoxicity against cancer cells and suggests that timing could be a critical factor in combination therapy.

Example 5 - Synergistic effects of anti-CEACAM1 and anti-PD-L1 antibodies on granzyme B levels and on the cytotoxicity of human TIL cells against human melanoma cells when anti-PD-L1 antibodies are added prior to the addition of anti-CEACAM1 antibodies. CEACAM1

[0168] Human melanoma cells (MALME 3M) were cultured in the presence of IFN-Y to induce PD-L1 expression. Human TIL cells (TIL14) were incubated with medium only (black), nonspecific IgG antibody (0.8 μg/mL, white), various concentrations (0.05 μg/mL, 0.1 μg/mL, 0.2 μg /mL, 0.4 μg/mL, 0.8 μg/mL) of a monoclonal antibody against human CEACAM1 (CM-24), a monoclonal antibody against human PD-L1 (clone 29E.2A3), or a combination of both antibodies (0.05 μg/mL each, 0.1 μg/mL each, 0.2 μg/mL each, 0.4 μg/mL each, 0.8 μg/mL each). The anti-PD-L1 antibody was added first for 30 minutes at 37°C, followed by the addition of the monoclonal antibody against human CEACAM1.

[0169] Human melanoma cancer cells, treated with IFN-γ, were added for overnight incubation before cytotoxicity assessment (Figure 5A). The calculated combination index (CI) was 0.67. Results represent an average of % cytotoxicity ± SE as determined by the classical LDH release assay from three one-well replicates per treatment. * P<0.05 Paired T test compared to anti-PD-L1 alone. In the same assay, levels of the cytotoxic granzyme B protein, which is secreted upon activation of cytotoxic cells, were assessed by a commercial granzyme B ELISA kit (Figure 5B). The results represent the average level of granzyme B, calculated from three replicates of one well per treatment.

[0170] Figure 5 demonstrates that anti-CEACAM1 antibodies and anti-PD-L1 antibodies are capable of binding to their respective targets on human lymphocytes (such as TIL cells) and human cancer cells (such as melanoma cells), and that this binding increases granzyme B secretion and toxicity of human TIL cells against human cancer cells. Figure 5 further demonstrates that blocking PD-1/PD-L1 and CEACAM1/CEACAM1 interactions can result in a synergistic effect. Therefore, the data presented in Figure 5 indicate that protecting lymphocytes from the immunosuppressive signal of PD-1 lymphocyte/PD ligand cancer cell results in considerable cytotoxicity against these cancer cells, regardless of the antigen targeted, be it PD-1, PD-L1 or PD-L2. .

Example 6 - Synergistic effects of anti-CEACAM1 and anti-PD-1 antibodies on the cytotoxicity of human LAK cells against human melanoma cells when anti-PD-1 antibodies are added before the addition of anti-CEACAM1 antibodies

[0171] Human melanoma cells (SKMEL28, CEACAM1 positive, PD-L1 positive) were cultured in the presence of IFN-Y to induce PD-L1 expression. Human LAK (lymphokine-activated killer) cells generated by activation of PBMCs, from a healthy human donor, with IL-2 (500 units/mL) for 7 days were incubated with medium only (black), nonspecific IgG antibody (0, 8 μg/mL, blank), various concentrations (0.05 μg/mL, 0.1 μg/mL, 0.2 μg/mL, 0.4 μg/mL, 0.8 μg/mL) of a monoclonal antibody against human CEACAM1 (CM-24), a monoclonal antibody against human PD-1 (clone E12.2H7) or a combination of both antibodies (0.05 μg/mL each, 0.1 μg/mL each, 0. 2 μg/mL each, 0.4 μg/mL each, 0.8 μg/mL each). The monoclonal antibody against human PD-1 was added first for 30 minutes at 37°C, followed by the addition of the monoclonal antibody against human CEACAM1.

[0172] Human melanoma cells, treated with IFN-y, were added for 24 hours of incubation, before cytotoxicity assessment (Figure 6). The calculated combination index (CI) was < 0.2. Results represent an average of % cytotoxicity ± SE as determined by the classical LDH release assay from three one-well replicates per treatment. * P<0.05 Paired T test compared to anti-PD-1 alone.

[0173] Figure 6 demonstrates that anti-CEACAM1 antibodies and anti-PD-1 antibodies are capable of binding to their respective targets on activated human lymphocytes, such as LAK cells, and that this binding increases the toxicity of human LAK cells against cells of human cancer. Figure 6 further demonstrates that the binding of these antibodies to LAK cells is somewhat interrelated, justifying future study of their mechanism, and that this mechanism is present in a variety of activated lymphocytes.

Example 7 - Treatment with anti-CEACAM1 antibodies increases PD-L1 expression in target cancer cells

[0174] Human NK cells (NK92MI) were incubated with or without a monoclonal antibody against human CEACAM1 (10 μg/mL CM-24), followed by the addition of human melanoma cancer cells (SKMEL28). Cells were incubated for 24, 48, and 72 hours, and PD-L1 levels were measured at each time point by FACS analysis. Figure 7A illustrates the mean anti-PD-L1 ratio levels compared to an appropriate isotype control for the indicated treatments at the different time points. B. Representative FACS analysis of PD-L1 levels after 48 hours.

[0175] Figure 7 demonstrates that the expression of CEACAM1 and PD-L1 in cancer cells is indeed interrelated. The addition of anti-CEACAM1 antibodies results in lower expression of PD-L1 in surviving cancer cells, thus providing additional support for combined treatment with both agents. It may therefore be beneficial to treat cancer by first administering anti-CEACAM1 antibodies and then additionally administering anti-PD-L1 and/or anti-PD-L2 antibodies, as the number of PD-L1 proteins on cancer cells remains relatively low. high, making cells more sensitive to treatment with anti-PD-1/PD-L1 antibodies, implying that combinatorial therapy may improve clinical outcome.

[0176] The data presented in Examples 4 to 6 demonstrate a surprising finding, according to which the administration of different antibodies at separate times, rather than simultaneously, maximizes the cytotoxic effect of lymphocytes against cancer cells. Without being bound by any theory or mechanism, this finding can be linked to another surprising finding of the present invention, according to which treatment with anti-CEACAM1 antibodies increases the expression of PD-L1 in target cancer cells. Hypothetically, this would support the need for a plurality of antibodies to improve the efficacy of cytotoxic lymphocytes. It can be predicted that administration of anti-PD-1 will first block PD-1 molecules on lymphocytes, subsequent administration of anti-CEACAM1 antibodies will block CEACAM1 molecules on lymphocytes and/or target cancer cells and increase expression of ligands of PD-1 on target cancer cells. However, considering that PD-1 molecules on lymphocytes are already blocked, high expression levels of PD-1 ligands on target cancer cells do not prevent lymphocytes from exerting their full cytotoxic potential efficiently.

Example 8 - Synergistic effects of anti-CEACAM1 and anti-PD-1 antibodies on tumor progression in immunocompetent mice

[0177] Murine lymphoma cells (5*106, A20) were grafted into the abdomen of Balb/C mice by subcutaneous injection on Day 1. By Day 10, tumors reached an average volume of 45 mm3, and the mice were randomized to 4 separate groups (11-12 mice per group) and intravenously received PBS (Figure 8, dashed black line, light circles), CC-1 (murine anti-CEACAM1 antibody, 6 mg/kg, Figure 8, solid gray line , gray rectangles), PRM-1 (murine anti-PD-1 antibody, 6 mg/kg, Figure 8, solid gray line, gray triangles) or a combination of CC-1 and PRM-1 (6 mg/kg each, Figure 8, solid black line, black spheres). Treatments were repeated on Days 15 and 20. The experiment was terminated on Day 22. The effect of a monoclonal antibody against human CEACAM1 alone, a monoclonal antibody against human PD-1 alone, and a combination of both antibodies in inhibiting the tumor growth was followed.

[0178] Immunocompetent Balb/C mice were selected for this experiment to allow evaluation of the biological activity of anti-murine CEACAM1 and anti-murine PD-1 antibodies in mice with an intact immune system and to evaluate the entire immune system reaction against murine cancer cells. As a whole, this model simulates therapies in which cancer patients would receive combinations of anti-human CEACAM1 and anti-human PD-1/PD-L1/PD-L2 antibodies. Without sticking to any theory or mechanism, the hypothesis is considered that a combination of anti-CEACAM1 and anti-PD-1/PD-L1/PD-L2 antibodies would prohibit cancer cells from avoiding the activation and cytotoxicity of the system. patient's immune system, thus producing a significant anticancer response.

[0179] Figure 8 demonstrates that anti-CEACAM1 antibodies and anti-PD-1/PD-L1/PD-L2 antibodies are capable of binding to their respective targets on tumor cells and/or immune cells in vivo, and that this Combined binding significantly attenuates tumor progression when compared to each monotherapy. This result is highly important, as it attests to the efficacy and potential of using anti-CEACAM1 and anti-PD-1/PD-L1/PD-L2 combined, even in established tumors of considerable volume, which simulate the clinical scenario in which patients with established tumors are being treated.

[0180] Figure 9 provides tumor progression curves for each mouse individually, grouped by their respective therapies (control, anti-CEACAM1 antibodies, anti-PD-1 antibodies or a combination of anti-CEACAM1 and anti-PD-1 antibodies) .

Example 9 - Treatment in a cancer xenograft model with a combination of an anti-CEACAM-1 antibody and an anti-PD-1 or anti-PD-L1 antibody

[0181] Tumors were xenografted into SCID NOD mice by subcutaneous (SC) injection of a cancer cell line (e.g., melanoma cell line such as SKMEL5). The effect of an anti-CEACAM-1 antibody (e.g., CM-24) alone, that of a monoclonal antibody against human PD-1 or a monoclonal antibody against human PD-L1 alone, and the effect of a combination of both antibodies, with or without TIL cells is examined for tumor eradication, inhibition of tumor growth and/or mortality rate of mice.

[0182] For example, SCID-NOD mice were randomized to the groups described in Table 2. Table 2. Groups of SCID-NOD mice *The administration of TIL cells by adoptive transfer aims to compensate for the absence of the immune system in this line of mice.

[0183] Tumor initiation - Cancer cells in PBS (e.g., melanoma) are administered to each mouse by SC injection into its right flank. Treatment - Treatment begins on the day of tumor inoculation. Tumor growth measurements - Measurements occur twice a week using a caliper and carried out by a person who is unaware of the experimental procedure. Measurements start from the day of tumor initiation. Outcome - ~60-80 days from the day of tumor initiation. On the closing day of the trial, all mice are sacrificed after total body bleeding and serum separation. Tumors are transferred for fixation in a separate tube per mouse.Example 10 - Monotherapy of Anti-PD-1/anti-PD-L1//anti-PD-L2/anti-CEACAM1 versus a combination therapy with anti-CEACAM1 and anti- PD-1/anti-PD-L1/anti-PD-L2 in Human Patients With Untreated, Unresectable, or Metastatic Cancer *The Experiment continues until documented disease progression, discontinuation due to toxicity, withdrawal of patient consent or the end of the study.

Claims (17)

1. Use of: a monoclonal antibody to human CEACAM1 or an antigen-binding fragment thereof; and a monoclonal antibody to human PD-1, or an antigen-binding fragment thereof, CHARACTERIZED by the fact that it is for the manufacture of a pharmaceutical composition for treating cancer by separate administration, wherein said monoclonal antibody to PD-1 1 is capable of inhibiting or blocking the interaction between human PD-1 and its ligand, and wherein said monoclonal antibody to human CEACAM1 has: a heavy chain CDR1 comprising a sequence shown in SEQ ID NO: 1, a CDR2 of heavy chain comprising a sequence set out in SEQ ID NO: 2, a heavy chain CDR3 comprising a sequence set out in SEQ ID NO: 3, a light chain CDR1 comprising a sequence set out in SEQ ID NO: 4, a light chain CDR2 comprising a sequence set forth in SEQ ID NO: 5, and a light chain CDR3 comprising a sequence set forth in SEQ ID NO: 6. 2. Use, according to claim 1, CHARACTERIZED by the fact that it comprises a monoclonal antibody to human CEACAM1 and a monoclonal antibody to human PD-1. 3. Use according to claim 1, CHARACTERIZED by the fact that the monoclonal antibody to human CEACAM1 or an antigen-binding fragment thereof is capable of binding with an affinity of at least about 10-8 M to CEACAM1 human. 4. Use according to claim 3, CHARACTERIZED by the fact that said monoclonal antibody to human CEACAM1 or an antigen-binding fragment thereof is capable of binding with an affinity of at least about 5x10-7 M to at least one of human CEACAM3 and human CEACAM5. 5. Use, according to claim 1, CHARACTERIZED by the fact that said monoclonal antibody to human CEACAM1 is CM-24, or an antigen-binding fragment thereof. 6. Use according to claim 1, CHARACTERIZED by the fact that said monoclonal antibody to human PD-1 is selected from the group consisting of MK-3475, AMP514, BMS-936558, CT-011 and any combination thereof. 7. Use, according to claim 1, CHARACTERIZED by the fact that said monoclonal antibodies are human or humanized monoclonal antibodies. 8. Use, according to any one of claims 1 to 7, CHARACTERIZED by the fact that it further comprises a human lymphocyte cell. 9. Use, according to claim 8, CHARACTERIZED by the fact that said human lymphocyte cell expresses CEACAM1, PD-1, or both. 10. Use, according to claim 9, CHARACTERIZED by the fact that said human lymphocyte cell expresses CEACAM1 and PD-1. 11. Use according to claim 8, CHARACTERIZED by the fact that said human lymphocyte cell is selected from the group consisting of a tumor lymphocytic infiltrate cell (TIL), a lymphokine-activated killer cell (LAK), a killer cell induced cytokine (CIK), a T cell, a B cell, an NK cell, and any combination thereof. 12. Use, according to claim 11, CHARACTERIZED by the fact that said human lymphocyte cell is selected from the group consisting of a tumor lymphocytic infiltrate cell and a lymphokine-activated killer cell. 13. Use, according to claim 8, CHARACTERIZED by the fact that said human lymphocyte cell is activated. 14. Use, according to claim 8, CHARACTERIZED by the fact that said human lymphocyte cell is cytotoxic to a cancer cell. 15. Use, according to claim 14, CHARACTERIZED by the fact that said cancer cell expresses CEACAM1. 16. Use according to claim 1, CHARACTERIZED by the fact that said cancer is selected from the group consisting of melanoma, lymphoma, lung, thyroid, breast, colon, prostate, liver, bladder, renal, cervical, pancreatic cancer , leukemia, myeloid, ovarian, uterine, sarcoma, biliary and endometrial cells. 17. Use, according to claim 1, CHARACTERIZED by the fact that said cancer is selected from the group consisting of melanoma cancer and lymphoma cancer.