WO2015198312A1

WO2015198312A1 - Compositions comprising antibodies to ceacam-1 and lag-3 for cancer therapy

Info

Publication number: WO2015198312A1
Application number: PCT/IL2015/050636
Authority: WO
Inventors: Tehila Ben-Moshe; Moti HAKIM; Yair SAPIR; Ilana MANDEL
Original assignee: Ccam Therapeutics Ltd.
Priority date: 2014-06-24
Filing date: 2015-06-23
Publication date: 2015-12-30

Abstract

The present invention provides compositions comprising antibodies against human CEACAM-1 and human LAG-3, kits comprising the same, and methods for their use in attenuating or treating cancer.

Description

COMPOSITIONS COMPRISING ANTIBODIES TO CEACAM-1 AND LAG-3 FOR

CANCER THERAPY

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions comprising antibodies to human CEACAM-1 and LAG-3 molecules, and methods for their use in treating cancer.

BACKGROUND

The transmembrane protein carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1, also known as biliary glycoprotein (BGP), CD66a and C-CAM1), is a member of the carcinoembryonic antigen family (CEA) that also belongs to the immunoglobulin superfamily. CEACAM-1 is known to interact with other known CEACAM proteins, including CD66a (CEACAM1), CD66c (CEACAM6) and CD66e (CEACAM5, CEA) proteins. It is expressed on a wide spectrum of cells, ranging from epithelial cells to those of hemopoietic origin (e.g. immune cells).

Many different functions have been attributed to the CEACAM-1 protein. It was shown that the CEACAM-1 protein is over expressed in some carcinomas of colon, prostate, as well as other types of cancer. Additional data support the central involvement of CEACAM-1 in angiogenesis and metastasis. CEACAM-1 also plays a role in the modulation of innate and adaptive immune responses. For example, CEACAM1 was shown to be an inhibitory receptor for activated T cells contained within the human intestinal epithelium (W099/52552 and Morales et al. J. Immunol. 1999, 163, 1363-1370). Additional reports have indicated that CEACAM-1 engagement either by T Cell Receptor cross-linking with Monoclonal antibodies (mAbs) or by Neisseria gonorrhoeae Opa proteins inhibits T cell activation and proliferation.

Lymphocyte-activation gene 3, also known as LAG-3 and CD223 (cluster of differentiation 223), is a protein belonging to immunoglobulin (Ig) superfamily, which in humans is encoded by the LAG3 gene. LAG-3 is a high affinity MHC class II ligand, which can associate with T cell receptors (TCRs) and down-regulate TCR signaling. As a negative regulator of proliferation and activation of T cells, LAG-3 expression in tumor-infiltrating CD4+ CD8+ lymphocytes (TILs) is associated with low effector function and T-cell anergy in-vivo. WO 2015/075725 to some of the present inventors relates to combinations of anti- CEACAM1 and anti-PD-l/PD-Ligand antibodies, and their use in treating cancer. WO 2015/075710 to some of the present inventors relates combinations of anti-CEACAMl antibodies, lymphocyte activating agent and activated lymphocytes, and their use in treating cancer. International PCT application PCT/IL2015/050007 to some of the present inventors relates to combinations of kinase inhibitors and antibodies to CEACAMl, and to their use in treating cancer. International PCT application PCT/IL2015/050433 to some of the present inventors relates to humanized antibodies, capable of specific binding to human CEACAMl molecules. There is still an unmet need in the field of cancer treatment for innovational, synergistic combinations of anti-cancer immunotherapeutic agents.

SUMMARY OF THE INVENTION

The present inventions provides novel combinations of monoclonal antibodies directed to human CEACAM-1 and human LAG-3 proteins, as well as methods for their use in treating cancer and activating human lymphocytes.

The present invention stems from the surprising finding that a combination of anti- human-CEACAM-1 antibodies and anti-human-LAG-3 antibodies has a synergistic anticancer effect, which is significantly better than the anti-cancer effect of each antibody alone.

Without being bound by any theory or mechanism, it is speculated that the antibodies of the present invention allow cells of the human immune system to affect their cytotoxic activity towards cancerous cells. More specifically, it is speculated that these antibodies prevent cytotoxic cells of the human immune system from becoming deactivated by interaction with their target cancer cells, thus allowing them to destroy these target cells.

The present invention provides, in one aspect, a pharmaceutical composition comprising (i) a monoclonal antibody to human carcinoembryonic antigen-related cell adhesion molecule- 1 (CEACAM-1) or an antigen-binding fragment thereof, and (ii) a monoclonal antibody to human lymphocyte-activation gene 3 (LAG-3).

The present invention further provides, in another aspect, a method for treating a cancer in a patient in need thereof, comprising administrating to the patient (i) a pharmaceutical composition comprising a monoclonal antibody to human carcinoembryonic antigen-related cell adhesion molecule- 1 (CEACAM-1) or an antigen-binding fragment thereof, and (ii) a pharmaceutical composition comprising a monoclonal antibody to human lymphocyte-activation gene 3 (LAG-3).

In certain embodiments of the method described above, the method further comprises administrating to the patient a pharmaceutical composition comprising a lymphocyte cell. The present invention further provides, in another aspect, a kit comprising (i) a monoclonal antibody to human CEAC AM- 1 or an antigen-binding fragment thereof, and (ii) a monoclonal antibody to human LAG-3 or an antigen-binding fragment thereof.

In certain embodiments, the kit described above is for use in treating cancer.

In certain embodiments, the monoclonal antibody to human CEACAM-1 or the monoclonal antibody to human LAG-3 is a human antibody, a humanized antibody, or a chimeric antibody. Each possibility represents a separate embodiment of the present invention.

In certain embodiments, the monoclonal antibody to human CEACAM-1 or the antigen-binding fragment thereof binds to a CEACAM-1 molecule on a lymphocyte cell and prevents suppression of the lymphocyte cell.

In certain embodiments, the monoclonal antibody to human CEACAM-1 or the antigen-binding fragment thereof has a heavy-chain CDR1 comprising a sequence set forth in SEQ ID NO: 1, a heavy-chain CDR2 comprising a sequence set forth in SEQ ID NO: 2, a heavy-chain CDR3 comprising a sequence set forth in SEQ ID NO: 3, a light-chain CDR1 comprising a sequence set forth 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. Analogs and derivatives of the monoclonal antibody or fragment thereof, having at least 90% sequence identity with the antigen-binding portion of the reference sequence are also within the scope of the present invention. In certain embodiments, the monoclonal antibody to human CEACAM-1 is CM-24 or the antigen-binding fragment thereof. In certain embodiments, the monoclonal antibody to human CEACAM-1 is CM-24.

In certain embodiments, the anti-CEACAM-1 antibody is selected from the group consisting of CM-24, MRG-1, 26H7, 5F4, TEC-11, 12-140-4, 4/3/17, COL-4, F36-54, 34B1, YG-C28F2, D14HD11, M8.7.7, D11-AD11, HEA81, B l. l, CLB-gran-10, F34-187, T84.1, B6.2, B1.13, YG-C94G7, 12-140-5, TET-2, variants thereof, antigen-binding fragments thereof, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

In certain embodiments, the monoclonal antibody to human LAG-3 or the antigen- binding fragment thereof binds to a LAG-3 molecule on a lymphocyte cell and prevents suppression of the lymphocyte cell.

In certain embodiments, the anti-LAG-3 antibody is selected from the group consisting of 17B4, BMS-986016, 11E3, 333210, IMP321, C9B7W, variants thereof, antigen- binding fragments thereof, and any combination thereof. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the anti-LAG3 antibody is 17B4.

In certain embodiments, the pharmaceutical composition described above further comprises a human lymphocyte cell. In certain embodiments, the pharmaceutical composition described above further comprises the human lymphocyte cell described above. In certain embodiments, the lymphocyte cell is found in the body of a cancer patient. It should be understood that, according to the present invention, anti-human-CEACAM-1 antibodies and/or anti-human-LAG-3 antibodies may bind lymphocytes both in-vivo, i.e. bind a human subject's natural lymphocytes, and/or bind lymphocytes in-vitro, i.e. bind lymphocytes outside the human subject's body, when the lymphocytes are optionally later administered back to the human subject. In certain embodiments, the lymphocyte cell expresses CEACAM-1, a LAG-3 molecule, or both. In certain embodiments, the lymphocyte cell expresses CEACAM-1 and LAG-3.

In certain embodiments, the lymphocyte cell is activated. In certain embodiments, the lymphocyte cell is cytotoxic to a cancer cell. In certain embodiments, the lymphocyte cell is directly cytotoxic to a cancer cell, for example by interacting with the cancer cell and causing lysis or apoptosis. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the lymphocyte cell is indirectly cytotoxic to a cancer cell, for example by releasing cytokine(s).

In certain embodiments, the cancer cell expresses CEACAM-1, MHC class II, or both. In certain embodiments, the cancer cell expresses CEACAM-1 and MHC class II. In certain embodiments, the cancer is selected from the group consisting of a melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, and endometrial cells cancers. Each possibility represents a separate embodiment of the invention. In certain embodiments, the cancer is melanoma.

In certain embodiments, the lymphocyte cell is a natural killer (NK) cell or a T cell. In certain embodiments, the lymphocyte cell is a T cell. In certain embodiments, the T cell is a tumor infiltrating lymphocyte (TIL) cell.

In certain embodiments, the pharmaceutical compositions described above are for use in treating cancer.

In certain embodiments of the method described above, the lymphocyte cell is incubated with the monoclonal antibody to human CEACAM-1 or with the monoclonal antibody to human LAG-3, or with at least one antigen-binding fragment thereof, prior to the administration. In certain embodiments of the method described above, the lymphocyte cell is incubated with the monoclonal antibody to human CEACAM-1 or with an antigen-binding fragment thereof, and with the monoclonal antibody to human LAG-3 or with an antigen- binding fragment thereof, prior to the administration. Each possibility represents a separate embodiment of the invention.

The present invention further provides the use of the pharmaceutical compositions and kits described above in preparing a medicament for treating cancer.

In certain embodiments, the target cell is selected from the group consisting of a cancer cell and an antigen-presenting cell. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the antigen-presenting cell is selected from the group consisting of a dendritic cell, a macrophage, a B cell, an epithelial cell, a fibroblast, a thymic epithelial cell, a thyroid epithelial cell, a glial cell, a pancreatic beta cell, a vascular endothelial cell and any combination thereof. Each possibility represents a separate embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Human melanoma cells express CEACAM-1, and HLA-DR upon IFN-γ activation. Human melanoma cells (MEL526) were incubated with IFN-γ for 24 hours followed by FACS analysis (Figure 1A) with a APC-conjugated anti-HLA-DR antibody (clone L243, full histogram) or isotype control matched antibody (empty histograms), or (Figure IB) with a PE-conjugated anti-CEACAM-1 antibody (CM-24-S36, full histogram) or isotype control matched antibody (empty histograms).

Figure 2. Human tumor-infiltrating lymphocytes (TILs) express lymphocyte- activation gene 3 (LAG- 3). Human TILs were stained with an antibody against LAG-3, followed by FACS analysis. The expression of LAG-3 (empty histogram) compared to a matching isotype control (full histogram) is presented.

Figure 3. Synergistic effects of anti-CEACAM-1 and anti-LAG-3 antibodies on the cytotoxicity of TILs against human melanoma cells at low effector to target ratio. Human melanoma cells were grown in the presence of IFN-γ to induce HLA-DR expression. TIL cells were incubated with a control antibody (IgG) or with various concentrations of an anti-CEACAM-1 antibody (CM-24), an anti-LAG-3 antibody (clone 17B4) or with a combination of both antibodies. The IFN-y-treated melanoma cells were then added for an overnight incubation in a low effector to target ratio of 2.5: 1. Results represent an average of % cytotoxicity +SE. * P<0.05 paired T-test compared to each antibody alone.

Figure 4. Synergistic effects of anti-CEACAM-1 and anti-LAG-3 antibodies on the cytotoxicity of TILs against human melanoma cells at high effector to target ratio.

Human melanoma cells were grown in the presence of IFN-γ to induce HLA-DR expression. TIL cells were incubated with a control antibody (IgG) or with various concentrations of an anti-CEACAM-1 antibody (CM-24), an anti-LAG-3 antibody or a combination of both antibodies. The IFN-y-treated melanoma cells were then added for an overnight incubation in a high effector to target ratio of 7.5:1. Results represent an average of % cytotoxicity +SE. * P<0.05 paired T-test compared to each antibody alone. ** P<0.005 paired T-test compared to each antibody alone. DETAILED DESCRIPTION OF THE INVENTION

The present invention generally provides novel combinations of immunotherapeutic agents having a surprisingly prominent anti-cancer effect, which is significantly stronger than the anti-cancer effect of each agent alone, and methods for the use of these agents and combinations in treating cancer. More specifically, the present invention provides combinations of antibodies against the human transmembrane proteins carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) and lymphocyte-activation gene 3 (LAG-3), expressed by human lymphocytes, which surprisingly together act in synergy to promote the cytotoxic activity of the lymphocytes towards cancerous cells.

The present invention stems from the surprising finding that combinations of antibodies against human CEACAM-1 and human LAG-3 have a synergistic effect on promoting anti-cancer cytotoxic activity of human lymphocytes.

Without being bound to any theory or mechanism, it is speculated that while antibodies to human CEACAM-1 hinder the suppressive interactions between lymphocytes and their target cancer cells, antibodies to human LAG-3 hinder similar suppressive interactions between the lymphocytes and the cancer cells and/or initiate or promote lymphocyte cytotoxicity toward these target cells.

The present invention provides, in one aspect, a pharmaceutical composition comprising (i) a monoclonal antibody to human carcinoembryonic antigen-related cell adhesion molecule- 1 (CEACAM-1) or an antigen-binding fragment thereof, and (ii) a monoclonal antibody to human lymphocyte-activation gene 3 (LAG-3). The present invention further provides, in another aspect, a method for treating a cancer in a patient in need thereof, comprising administrating to the patient (i) a pharmaceutical composition comprising a monoclonal antibody to human carcinoembryonic antigen-related cell adhesion molecule- 1 (CEACAM-1) or an antigen-binding fragment thereof, and (ii) a pharmaceutical composition comprising a monoclonal antibody to human lymphocyte-activation gene 3 (LAG-3).

The present invention further provides, in another aspect, a kit comprising (i) a monoclonal antibody to human CEAC AM- 1 or an antigen-binding fragment thereof, and (ii) a monoclonal antibody to human LAG-3 or an antigen-binding fragment thereof.

The term "pharmaceutical composition" as used herein refers to any composition comprising at least one chemical or biological agent and a pharmaceutically acceptable carrier. Non-limiting examples of agents according to the present invention are antibodies to human CEACAM-1 proteins, and/or antibodies to human KIR proteins.

The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multi-specific antibodies (e.g., bi-specific antibodies), and antibody fragments long enough to exhibit the desired biological activity. Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds in a "Y" shaped configuration. Proteolytic digestion of an antibody yields Fv (Fragment variable) and Fc (Fragment crystalline) domains. The antigen binding domains, Fab, include regions where the polypeptide sequence varies. The term F(ab')2 represents two Fab' arms linked together by disulfide bonds. The central axis of the antibody is termed 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, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CHI). The variable domains of each pair of light and heavy chains form the antigen-binding site. The domains on 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 hyper-variable domains known as complementarity determining regions (CDRs 1-3). These domains contribute specificity and affinity of the antigen-binding site. The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu) determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa, κ or lambda, λ) found in all antibody classes.

The antibody according to the present invention is a molecule comprising at least the antigen-binding portion of an antibody. 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 the Fab or F(ab')2 fragments. Single chain antibodies also fall within the scope of the present invention.

"Antibody fragments" comprise only a portion 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 encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at 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) in the same polypeptide chain (see, e.g., 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-CHl-VH-CHl) which, 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. Pat. No. 5,641,870).

Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain i.e. linked VH-VL or single chain Fv (scFv).

The term "neutralizing antibody" as used herein refers to a molecule having an antigen-binding site to a specific receptor or ligand target capable of reducing or inhibiting (blocking) activity or signaling through a receptor, as determined by in-vivo or in-vitro assays, as per the specification.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. mAbs may 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 may be made by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage 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. The mAbs of the present invention may be of any immunoglobulin class including IgG, IgM, IgE, IgA. A hybridoma producing a mAb may be cultivated in-vitro or in-vivo. High titers of mAbs can be obtained by in-vivo production where cells from the individual hybridomas are injected intra-peritoneally into pristine -primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. mAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

The term "human antibody" as used herein refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. 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 in the art.

The term "humanized antibody" as used herein refers to an antibody that has its CDRs (complementarily determining regions) derived from a non-human species immunoglobulin and the remainder of the antibody molecule derived mainly from a human immunoglobulin.

As used herein, the term "chimeric antibody" refers to an antibody in which at least one of the antibody chains (heavy or light) comprises variable region sequences from one species (e.g., mouse) and constant region sequences from another species (e.g., human). The term "chimeric antibody" is intended to encompass antibodies in which: (i) the heavy chain is chimeric but the light chain comprises variable and constant regions from only one species; (ii) the light chain is chimeric but the heavy chain comprises variable and constant regions from only one species; and (iii) both the heavy chain and the light chain are chimeric.

The terms "molecule having the antigen-binding portion of an antibody" and "antigen- binding-fragments" as used herein is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab' fragment, the F(ab')2 fragment, the variable portion of the heavy and/or light chains thereof, Fab mini-antibodies (see WO 93/15210, US patent application 08/256,790, WO 96/13583, US patent application 08/817,788, WO 96/37621, US patent application 08/999,554, the entire contents of which are incorporated herein by reference), dimeric bispecific mini-antibodies (see Muller et al., 1998) and single-chain antibodies incorporating such reactive fraction, as well as any other type of molecule in which such antibody reactive fraction has been physically inserted. Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques. The invention also provides conservative amino acid variants of the antibody molecules according to the invention. Variants according to the invention also may be made that conserve the overall molecular structure of the encoded proteins. Given the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e. "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. The term "antibody analog" as used herein refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

The term "antibody variant" as used herein refers to any molecule comprising the antibody of the present invention. For example, fusion proteins in which the antibody or an antigen-binding-fragment thereof is linked to another chemical entity is considered an antibody variant.

The term "antigen" as used herein refers to a molecule or a portion of a molecule capable of eliciting antibody formation and being bound by an antibody. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens. An antigen according to the present invention is a CEACAM-1 protein or a fragment thereof.

The term "antigenic determinant" or "epitope" as used herein refers to the region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from an epitope can be used, alone or in conjunction with a carrier moiety, applying methods known in the art, to immunize animals and to produce additional polyclonal or monoclonal antibodies. Isolated peptides derived from an epitope may be used in diagnostic methods to detect antibodies and as therapeutic agents when inhibition of said antibodies is required. The term "CEACAM-1" is used to refer to the protein product of the CEACAM-1 gene e.g., NP_001020083.1, NP_001703.2. In humans, 11 different CEACAM-1 splice variants have been detected so far. Individual CEACAM-1 isoforms differ with respect to the number of extracellular immunoglobulin-like domains (for example, CEACAM-1 with four extracellular immunoglobulin-like domains is known as CEACAM-1 -4), membrane anchorage and/or the length of their cytoplasmic tail (for example, CEACAM-1 -4 with a long cytoplasmic tail is known as CEACAM-1 -4L and CEACAM-1 -4 with a short cytoplasmic tail is known as CEACAM-1 -4S). The N-terminal domain of CEACAM-1 starts immediately after the signal peptide and its structure is regarded as IgV-type. For example, in CEACAM-1 annotation PI 3688, the N-terminal IgV-type domain is comprised of 108 amino acids, from amino acid 35 to 142. This domain was identified as responsible for the homophilic binding activity (Watt et al., 2001, Blood. 98, 1469-79). All variants, including these splice variants are included within the term "CEACAM- 1".

The terms "anti-CEACAM-1 antibody", "an antibody which recognizes CEACAM-1", "an antibody against CEACAM-1" and "an antibody to CEACAM-1" are interchangeable, known in the field of immunotherapy, and are generally used herein to refer to an antibody or to an antibody fragment that binds or is capable of binding to a CEACAM-1 protein with sufficient affinity and specificity to affect a biological activity or outcome.

The term "anti-LAG-3 antibody", "an antibody which recognizes LAG-3", "an antibody against LAG-3", "an antibody to LAG-3" and "anti-CD223 antibody" are interchangeable, known in the field of immunotherapy, and are generally used herein to refer to an antibody or to an antibody fragment that binds or is capable of binding to a lymphocyte - activation gene 3 protein with sufficient affinity and specificity to affect a biological activity or outcome.

The terms "capable of preventing suppression of a lymphocyte cell", "prevents suppression of a lymphocyte cell" and "capable of inhibiting or blocking lymphocyte cell suppression" as used herein are interchangeable, and generally refer to the capability of an antibody to interact with a CEACAM-1 or a LAG-3 molecule presented by a lymphocyte cell in such a way that the cell becomes less responsive, preferably insensitive, to signals otherwise preventing the cell from becoming cytotoxic toward a target cell, for example by causing lysis or apoptosis. Such signals may be, for example, interactions with CEACAM-1 molecules and/or MHC class I complexes, which are known to suppress the cytotoxic activity of lymphocyte cells by interacting with the lymphocytes' CEACAM-1 and/or LAG-3 molecules, respectively.

The phrase "capable of inhibiting or blocking the interaction" as used herein refers to the capability of an agent, for example an antibody, to interact with a at least one target, for example a cell's membrane-bound receptor, in such a way that the target becomes less accessible, preferably inaccessible, to binding by at least one other agent, for example the receptor's natural antigen.

The term "activated lymphocyte cell" as used herein refers to any cell of the immune system, having a cytotoxic effect towards a target cell, for example by releasing cytokine(s) or granzyme B, causing lysis or apoptosis. The term "cytotoxic lymphocyte cell" as used herein refers to any cell of the immune system which causes lysis or apoptosis of cancer cells by any mechanism.

The term "target cell" as used herein refers to any cell exhibiting unregulated or abnormal cell growth. The term "target cell" as used herein further refers to any cell exhibiting an MHC class II molecule. The term "antigen-presenting cell" or "accessory cell" as interchangeably used herein refers to any cell that displays foreign antigens complexed with major histocompatibility complexes (MHC's) on their surfaces. The term "cancer cell" as used herein refers to any cell exhibiting unregulated or abnormal cell growth. The term "cytotoxicity" means the quality of being toxic to cells. The term

"cytotoxicity" as used herein refers to the degree to which a lymphocyte cell is able to induce the death, lysis or otherwise destruction of cancer cells.

The term "treating cancer" as used herein, refers to administering therapeutic effective 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, to inhibit the spread of the malignant cells in the patient, and/or to cause the death of malignant cells in the patient.

As used herein the term "treating" further means to ameliorate one or more symptoms associated with the referenced disorder. 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 may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life. As used herein, the term "preventing" means to mitigate a symptom of the referenced disorder.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. The terms "pre-incubated" and "incubated" as used herein refers to the incubation of at least two chemical or biological agents, for example a cell and an antibody directed against an antigen presented by the cell, under conditions to allow and/or promote an interaction between the agents. In some embodiments, the interaction is a physical interaction. In some embodiments, the interaction is a high affinity physical interaction. As used herein, the term "kit" is used in reference to a combination of reagents and other materials. It is contemplated that the kit may include reagents such antibodies and/or storage vials or other containers. It is not intended that the term "kit" be limited to a particular combination of reagents and/or other materials.

The terms "pre-incubated" and "incubated" as used herein refers to the incubation of at least two chemical or biological agents, for example a cell and an antibody directed against an antigen presented by the cell, under conditions to allow and/or promote an interaction between the agents. In some embodiments, the interaction is a physical interaction. In some embodiments, the interaction is a high affinity physical interaction.

The following examples are intended to illustrate how to make and use the compounds and methods of this invention and are in no way to be construed as a limitation. Although the invention will now be described in conjunction with specific embodiments thereof, it is evident that many modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the appended claims. EXAMPLES

Example 1 - Human melanoma cells express CEACAM-1, and further express HLA-DR upon IFN-γ activation.

Human melanoma cells (MEL526) were incubated with IFN-γ for 24 hours followed by FACS analysis (Figure 1A) with a APC-conjugated anti-HLA-DR antibody (clone L243, full histogram) or isotype control matched antibody (empty histograms), or (Figure IB) with a PE-conjugated anti-CEACAM-1 antibody (CM-24-S36, full histogram) or isotype control matched antibody (empty histograms). CM-24, developed by the present inventors, is a humanized antibody against CEACAM1, which has a heavy-chain CDR1 comprising a sequence set forth in SEQ ID NO: 1, a heavy-chain CDR2 comprising a sequence set forth in SEQ ID NO: 2, a heavy-chain CDR3 comprising a sequence set forth in SEQ ID NO: 3, a light-chain CDR1 comprising a sequence set forth 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.

Figures 1A and IB demonstrate that the human melanoma cells used in the experiments express both CEACAM-1 and HLA-DR.

Example 2 - Human TILs express LAG-3.

Human TILs (TIL14) were stained with an antibody against LAG-3 (clone 17B4), followed by FACS analysis. The expression of LAG-3 (empty histogram) compared to a matching isotype control (full histogram) is presented. TIL14 are tumor infiltrating lymphocytes expanded form patients # 14 in Ella institute, Israel.

Figure 2 demonstrates that the human TIL cells used in the experiments expressed LAG-3.

Example 3 - Synergistic effects of an anti-CEACAM-1 antibody and anti-LAG3 antibody on the cytotoxicity of human TILs against human melanoma cells when using a 2.5: 1 effector to target ratio.

Human melanoma cells (MEL526, CEACAM-1 positive) were grown in the presence of IFN-γ to induce HLA-DR expression. TIL cells (TIL14) were incubated with a control antibody (IgG) or with various concentrations (0.625μg/ml, 1.25μg/ml, 2^g/ml, 5μg/ml, l(^g/ml) of an anti-CEACAM-1 antibody (CM-24), an anti-LAG-3 antibody (clone 17B4) or a combination of both antibodies (0.625μg/ml each, 1.25μg/ml each, 2^g/ml each, 5μg/ml and lC^g/ml each) for 30 minutes at 37°C.

The IFN-y-treated melanoma cells were then added for an overnight incubation in a low effector to target ratio of 2.5:1. Results represent an average of % cytotoxicity +SE as determined by classical LDH release assay from triplicate wells per treatment. * P<0.05 paired T-test compared to each antibody alone. Combination index was calculated to be < 0.43. Figure 3 demonstrates that anti-CEACAM-1 antibodies and anti-LAG3 antibodies are able to bind their respective targets on human lymphocytes such as TIL cells, and that this binding increases the toxicity of the TILs against human cancer cells in a synergistic manner, with a combination index lower than 0.43. Example 4 - Synergistic effects of an anti-CEACAM-1 antibody and anti-LAG3 on the cytotoxicity of TILs against melanoma cells when using a 7.5:1 effector to target ratio.

Human melanoma cells (MEL526, CEACAM-1 positive) were grown in the presence of IFN-γ to induce HLA-DR expression. TIL cells (TIL14) were incubated with a control antibody (IgG) or with various concentrations (0^g/ml, lμg/mί, 2μg/ml, 4μg/ml, 8μg/ml) of an anti-CEACAM-1 antibody (CM-24), an anti-LAG-3 antibody (clone 17B4) or a combination of both antibodies (0^g/ml each, ^g/ml each, 2μg/ml each, 4μg/ml each and 8μg/ml each) for 30 minutes at 37°C.

The IFN-y-treated melanoma cells were then added for an overnight incubation in a high effector to target ratio of 7.5: 1. Results represent an average of % cytotoxicity +SE as determined by classical LDH release assay from triplicate wells per treatment. * P<0.05 paired T-test compared to each antibody alone. ** P<0.005 paired T-test compared to each antibody alone. Combination index was calculated to be <0.45.

Figure 4 demonstrates that anti-CEACAM-1 antibodies and anti-LAG3 antibodies are able to bind their respective targets on human lymphocytes such as TIL cells, and that this binding increases the toxicity of the TILs against human cancer cells in a synergistic manner, with a combination index lower than 0.45.

Claims

1. A pharmaceutical composition comprising:

(i) a monoclonal antibody to human carcinoembryonic antigen-related cell adhesion molecule- 1 (CEACAM-1) or an antigen-binding fragment thereof, and (ii) a monoclonal antibody to human lymphocyte-activation gene 3 (LAG-3).

2. The pharmaceutical composition of claim 1, wherein the monoclonal antibody to human CEACAM-1 or the monoclonal antibody to human LAG-3 is a human antibody, a humanized antibody, or a chimeric antibody.

3. The pharmaceutical composition of claim 1, wherein the monoclonal antibody to human CEACAM-1 or the antigen-binding fragment thereof binds to a CEACAM-1 molecule on a lymphocyte cell and prevents suppression of the lymphocyte cell.

4. The pharmaceutical composition of claim 1, wherein the monoclonal antibody to human CEACAM-1 or the antigen-binding fragment thereof has a heavy-chain CDR1 comprising a sequence set forth in SEQ ID NO: 1, a heavy-chain CDR2 comprising a sequence set forth in SEQ ID NO: 2, a heavy-chain CDR3 comprising a sequence set forth in SEQ ID NO: 3, a light-chain CDR1 comprising a sequence set forth 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.

5. The pharmaceutical composition of claim 4, wherein the monoclonal antibody to human CEACAM-1 is CM-24 or the antigen-binding fragment thereof.

6. The pharmaceutical composition of claim 5, wherein the monoclonal antibody to human CEACAM-1 is CM-24.

7. The pharmaceutical composition of claim 1, wherein the monoclonal antibody to human LAG-3 or the antigen-binding fragment thereof binds to a LAG-3 molecule on a lymphocyte cell and prevents suppression of the lymphocyte cell.

8. The pharmaceutical composition of claim 3 or claim 7, wherein the pharmaceutical composition further comprises the human lymphocyte cell.

9. The pharmaceutical composition of claim 3 or claim 7, wherein the lymphocyte cell is found in the body of a cancer patient.

10. The pharmaceutical composition of claim 8 or claim 9, wherein the lymphocyte cell expresses CEACAM-1, a LAG-3 molecule, or both.

11. The pharmaceutical composition of claim 10, wherein the lymphocyte cell expresses CEACAM-1 and LAG-3.

12. The pharmaceutical composition of claim 8 or claim 9, wherein the lymphocyte cell is activated.

13. The pharmaceutical composition of claim 8 or claim 9, wherein the lymphocyte cell is cytotoxic to a cancer cell.

14. The pharmaceutical composition of claim 13, wherein the cancer cell expresses CEACAM- 1 , MHC class II, or both.

15. The pharmaceutical composition of claim 14, wherein the cancer cell expresses CEACAM-1 and MHC class II.

16. The pharmaceutical composition of claim 13, wherein the cancer is selected from the group consisting of a melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, and endometrial cells cancers.

17. The pharmaceutical composition of claim 16, wherein the cancer is melanoma.

18. The pharmaceutical composition of any one of the claims 1 to 17, for use in treating cancer.

19. The pharmaceutical composition of any one of the claims 1 to 18, wherein the lymphocyte cell is a natural killer (NK) cell or a T cell.

20. The pharmaceutical composition of claim 19, wherein the lymphocyte cell is a T cell.

21. The pharmaceutical composition of claim 20, wherein the T cell is a tumor infiltrating lymphocyte (TIL) cell.

22. A method for treating a cancer in a patient in need thereof, comprising administrating to the patient: (i) a pharmaceutical composition comprising a monoclonal antibody to human carcinoembryonic antigen-related cell adhesion molecule- 1 (CEACAM-1) or an antigen-binding fragment thereof, and

(ii) a pharmaceutical composition comprising a monoclonal antibody to human lymphocyte-activation gene 3 (LAG-3).

23. The method of claim 22, further comprising administrating to the patient a pharmaceutical composition comprising a lymphocyte cell.

24. The method of claim 23, wherein the lymphocyte cell expresses CEACAM-1, LAG-3, or both.

25. The method of claim 24, wherein the lymphocyte cell expresses CEACAM-1 and LAG-3.

26. The method of claim 22, wherein the cancer's cells express CEACAM-1, an MHC class II complex, or both.

27. The method of claim 26, wherein the cancer's cells express CEACAM-1 and an MHC class II complex.

28. The method of claim 22, wherein the cancer is selected from the group consisting of a melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, and endometrial cells cancers.

29. The method of claim 28, wherein the cancer is melanoma.

30. The method of claim 23, wherein the lymphocyte cell is incubated with the monoclonal antibody to human CEAC AM- 1 or with the monoclonal antibody to human LAG- 3, or with at least one antigen-binding fragment thereof, prior to the administration.

31. The method of claim 30, wherein the lymphocyte cell is incubated with the monoclonal antibody to human CEACAM-1 or with an antigen-binding fragment thereof, and with the monoclonal antibody to human LAG-3 or with an antigen-binding fragment thereof, prior to the administration.

32. A kit comprising: (i) a monoclonal antibody to human CEACAM-1 or an antigen-binding fragment thereof, and

(ii) a monoclonal antibody to human LAG-3 or an antigen-binding fragment thereof.

33. The kit of claim 32, for use in treating cancer.

34. Use of a pharmaceutical composition according to any one of claims 1 to 21, in preparing a medicament for treating cancer.

35. Use of a kit according to claim 32 in preparing a medicament for treating cancer.