WO2023105528A1 - Antibodies specific to ceacam1 - Google Patents

Abstract

The present invention provides monoclonal antibodies that recognize CEACAM1 protein with high affinity and specificity. The antibodies recognize CEACAM1 with a non-blocking activity. The present invention further provides pharmaceutical compositions comprising the antibodies and methods for their use in cancer immunotherapy, in preventing and treating viral infection and in diagnosis.

Description

ANTIBODIES SPECIFIC TO CEACAM1

FIELD OF THE INVENTION

The invention is in the field of immunotherapy and relates to antibodies and fragments thereof which bind to the protein carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a), to polynucleotide sequences encoding these antibodies and antibody fragments and to cells producing them. The invention further relates to therapeutic and diagnostic compositions comprising these antibodies and fragments and to methods of treating and diagnosing diseases, particularly cancer, using them.

BACKGROUND OF THE INVENTION

Cancer immunotherapy is one of the most promising advancements made in the past decade. Four main approaches are being used to harness immune cells to fight cancer: 1) antibodies directed against tumor antigens are injected into patients and tumors are eliminated, in part, by immune cells carrying Fc receptors that recognize the antibody-coated tumors; 2) anti-cancer immune cells are extracted from the patient, expanded in vitro (and sometimes even genetically manipulated), and then re-injected into the patient; 3) T cells engineered to express Chimeric Antigen Receptors (CAR) that recognize tumor antigens are injected into patients; and 4) activity of the immune cells is augmented by using mAbs that block inhibitory pathways (known as checkpoints) that operate to restrain the immune cells.

The transmembrane protein carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, also known as biliary glycoprotein, BGP, CD66a and C-CAM1), is a member of the carcinoembryonic antigen (CEA) family that belongs to the immunoglobulin superfamily. Human CEACAM1 has been assigned the SwissProt accession number P13688. CEACAM1 interacts with itself and with other known CEACAM proteins, including the subtypes CD66c (CEACAM6) and CD66e (CEACAM5, CEA). Human CEACAM1 is expressed on a wide spectrum of cells, ranging from epithelial cells to those of hemopoietic origin (e.g. immune cells), including many cancer cells.

Many different functions have been attributed to the CEACAM1 protein. It was shown that the CEACAM1 protein is over expressed in some carcinomas of colon, prostate, as well as other types of cancer, such as melanoma. Additional data support the central involvement of CEACAM1 in angiogenesis and metastasis. CEACAM1 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 (WO 99/52552 and Morales et al. J. Immunol. 1999, 163, 1363-1370). Additional reports have indicated that CEACAM1 engagement either by T cell receptor cross-linking with monoclonal antibodies (mAbs) or by Neisseria gonorrhea Opa proteins inhibits T cell activation and proliferation. Several monoclonal antibodies against the CEACAM1 protein are already known and some of them are tested in clinical trials. WO 2015/166484 discloses humanized antibody against CEACAM1, useful for preventing, attenuating or treating a disease associated with expression, activation or function of a CEACAM1 protein.

It is still unclear why tumors express CEACAM1, an inhibitory molecule that can potentially inhibits tumor proliferation. It is possible that tumors express CEACAM1 to inhibit the activity of CEACAM1 -positive immune cells. Since CEACAM1 also function as adhesion molecule, it is possible that tumors express it to enhance tumor motility and metastases. It is also possible that in the initial stages of tumor development inhibition of tumor growth is required for the tumor to properly establish itself in the tumor niche. Regardless, the fact that different tumors express CEACAM1 provide an opportunity to target these tumors by developing antibodies specific to protein.

Despite of the success made, there is still a need for finding additional means and approaches to fight tumors. Not all cancers respond to the mentioned immunotherapies and even within a cancer that presents susceptibility to immunological treatments, not all patients respond. In addition, tumors are likely to develop escape variants. Therefore, the use of new, more effective, specific, safe and/or stable antibodies, either alone or together with other antibodies or agents, may overcome these deficiencies.

SUMMARY OF THE INVENTION

The present invention provides according to some embodiments, antibodies and fragments thereof that recognize the protein CEACAM1 (CD66a). The anti-CEACAMl antibodies disclosed herein are capable of binding to CEACAM1 present on cancer cells and to induce their killing. The antibodies of the present invention are agonistic/activating antibodies that do not bind the N-terminal domain of CEACAM1 and therefore are not involved in interactions mediated through this domain of the protein. This unique characteristic of the antibodies of the present invention confers them with enhances safety as inhibition of homophilic interactions of CEACAM1 through its N-terminal may lead in some circumstances to tumor proliferation. These antibodies and fragment thereof are characterized by having unique sets of complementarity determining region (CDR) sequences, high affinity and high specificity to CEACAM1, and are useful in cancer immunotherapy and diagnosis, as standalone therapy and in combination with other anti-cancer agents. Furthermore, these antibodies may be used as effective anti-viral agents, alone or in combination with other agents.

It is now disclosed that the anti-CEACAMl antibodies described herein were found to reduce tumor cell viability in an immune independent manner, to induce killing of melanoma cells in vitro and reduce tumor size and weight in xenograft in vivo model in dose-dependent manner. The anti-CEACAMl antibodies of the present invention were also able to stain CEACAM1 on eosinophils of COVID-19 infected patients.

According to one aspect, the present invention provides an antibody, or an antibody fragment thereof comprising at least the antigen binding portion, which specifically binds to CEACAM1, said antibodies do not block CEACAM1 homophilic interactions.

According to some embodiments, the antibody or fragment thereof comprises a set of six CDR sequences of an antibody selected from the group consisting of CCM5.01, CCM5.18, and CCM5.19.

According to some embodiments, the antibody is selected from the group consisting of CCM5.01, CCM5.18, and CCM5.19. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the antibody or antibody fragment comprises three CDRs of a heavy-chain (HC) variable region comprising SEQ ID NO: 1 and three CDRs of a light-chain (LC) variable region comprising SEQ ID NO: 3.

There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard unequivocal method. Determination of CDR sequences from antibody heavy and light chain variable regions can be made according to any method known in the art, including but not limited to the methods known as KAB AT, Chothia and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using KABAT and some using IMGT, for example. According to some embodiments, the CDR sequences of the mAb variable regions are determined using the IMGT method.

According to some embodiments, the antibody or fragment comprises the CDR sequences of a monoclonal antibody denoted CCM5.01, namely, the three CDR sequences contained in heavy chain variable region set forth in SEQ ID NO: 1 and the three CDR sequences contained in light chain variable region set forth in SEQ ID NO: 3.

According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR1 comprising the sequence SDYAWN (SEQ ID NO: 5). According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR2 comprising the sequence YITYSGSTSYNPSLKS (SEQ ID NO: 6). According to some embodiments, the antibody or the antibody fragment comprises heavy-chain CDR3 comprising the sequence ENRYDGEMFFDV (SEQ ID NO: 7).

According to certain embodiments, the antibody or the antibody fragment comprises: (i) HC CDR1 comprising the sequence SDYAWN (SEQ ID NO: 5); (ii) HC CDR2 comprising the sequence YITYSGSTSYNPSLKS (SEQ ID NO: 6); and (iii) HC CDR3 comprising the sequence ENRYDGEMFFDV (SEQ ID NO: 7).

According to some embodiments, the antibody or the antibody fragment comprises lightchain CDR1 comprising the sequence RASQSISNDLH (SEQ ID NO: 8). According to some embodiments, the antibody or the antibody fragment comprises light-chain CDR2 comprising the sequence YAYQSIS (SEQ ID NO: 9). According to some embodiments, the antibody or the antibody fragment comprises light-chain CDR3 comprising the sequence QQSNSWPHT (SEQ ID NO: 10).

According to certain embodiments, the antibody or the antibody fragment comprises: (i) LC CDR1 comprising the sequence RASQSISNDLH (SEQ ID NO: 8); (ii) LC CDR2 comprising the sequence YAYQSIS (SEQ ID NO: 9); and (iii) LC CDR3 comprising the sequence QQSNSWPHT (SEQ ID NO: 10).

According to some specific embodiments, the antibody or fragment comprises heavy chain CDR1 sequence comprising the sequence SDYAWN (SEQ ID NO: 5), heavy chain CDR2 comprising the sequence YITYSGSTSYNPSLKS (SEQ ID NO: 6), heavy chain CDR3 comprising the sequence ENRYDGEMFFDV (SEQ ID NO: 7), light chain CDR1 comprising the sequence RASQSISNDEH (SEQ ID NO: 8), light chain CDR2 comprising the sequence YAYQSIS (SEQ ID NO: 9), and light chain CDR3 comprising the sequence QQSNSWPHT (SEQ ID NO: 10), or analogs thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the hypervariable region (HVR) sequence.

According to some specific embodiments the antibody or fragment comprises a set of six CDR sequences consisting of: i. heavy chain CDR1 having a sequence set forth in SEQ ID NO: 5; ii. heavy chain CDR2 having a sequence set forth in SEQ ID NO: 6; iii. heavy chain CDR3 having a sequence set forth in SEQ ID NO: 7; iv. light chain CDR1 having a sequence set forth in SEQ ID NO: 8; v. light chain CDR2 having a sequence set forth in SEQ ID NO: 9; and vi. light chain CDR3 having a sequence set forth in SEQ ID NO: 10

According to some embodiments, the antibody or fragment thereof comprises heavy chain variable region set forth in SEQ ID NO: 1, or an analog or derivative thereof having at least 90% sequence identity with the heavy chain variable region sequence.

According to some embodiments, the antibody or fragment thereof comprises light chain variable region set forth in SEQ ID NO: 3, or an analog thereof having at least 90% sequence identity with the light chain variable region sequence.

According to a specific embodiment, the antibody or fragment thereof comprises a heavy chain variable region having a sequence set forth in SEQ ID NO: 1, and a light chain variable region having a sequence set forth in SEQ ID NO: 3, or an analog thereof having at least 90% sequence identity with the light and/or heavy chain sequence.

According to some embodiments, the antibody is an isolated monoclonal antibody.

According to some embodiments, the antibody or fragment thereof recognizes human CEACAM1 with an affinity of at least 5xl0'⁸M. According to other embodiments, the antibody or antibody fragment binds with an affinity of 5xl0'⁹M, 10'⁹M, 5xl0'¹⁰M, 10'¹⁰M, or even higher to CEACAM1. According to some embodiments, the antibody or antibody fragment binds to human CEACAM1 with affinity at the range of 10'⁹M to 10'¹⁰M. Each possibility represents a separate embodiment of the invention.

According to some embodiments, antibodies of the present invention do not bind the N- domain of CEACAM1.

According to some embodiments, antibodies of the present invention bind CEACAM1, and cross-react with CEACAM5 and CEACAM8. According to other embodiments, antibodies of the present invention do not bind CEACAM3, CEACAM6 and CEACAM7.

Analogs and derivatives of the isolated antibody and the fragments described above, are also within the scope of the invention.

According to some embodiments, the antibody or antibody fragment analog have at least 95% sequence identity with the hypervariable region of the reference antibody sequence.

According to certain embodiments, the analog or derivative of the isolated antibody or fragment thereof has at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with a variable region of the reference antibody sequence. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the antibody or antibody fragment according to the invention comprises a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO: 3, or an analog having at least 95% sequence similarity with said sequence.

According to some embodiments, the analog has at least 96, 97, 98 or 99% sequence identity with an antibody light or heavy chain variable regions described above. According to some embodiments, the analog comprises no more than one amino acid substitution, deletion or addition to one or more CDR sequences of the hypervariable region, namely, any one of the CDR sequences set forth in SEQ ID NOs: 5, 6, 7, 8, 9, and 10. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the amino acid substitution is a conservative substitution.

According to some embodiments, the antibody or antibody fragment comprises a hypervariable region (HVR) having light and heavy chain regions defined above, in which 1, 2, 3, 4, or 5 amino acids were substituted, deleted and/or added. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the antibody or antibody fragment comprises a HVR having light and heavy chain regions defined above, in which one amino acid was substituted. According to specific embodiments, the antibody or antibody fragment comprises a CDR as defined above, in which one amino acid was substituted.

The present invention also provides antibodies and binding fragments thereof, comprising a heavy chain and a light chain, wherein said chains comprises heavy chain variable region sequence set forth in SEQ ID NO: 1 and light chain variable region sequence set forth in SEQ ID NO: 3.

According to some embodiments, the antibody or antibody fragment is capable of killing cancer cells.

According to a specific embodiment, the antibody is selected from the group consisting of a chimeric antibody and an antibody fragment comprising at least the antigen-binding portion of an antibody. According to specific embodiments, the antibody is a chimeric antibody. According to yet other embodiments, the chimeric antibody comprises a human constant region. According to a specific embodiment, the antibody fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fd, Fd', Fv, dAb, isolated CDR region, single chain variable region (scFV), single chain antibody (scab), "diabodies", and "linear antibodies". Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the antibody or antibody fragment comprises a heavy chain constant region selected from the group consisting of: mouse IgGl, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgGl, human IgG2, human IgG3 and human IgG4. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the antibody or antibody fragment comprises a mouse IgGl heavy chain constant region.

According to some embodiments, the monoclonal antibody is a chimeric monoclonal antibody. According to some embodiments the human constant regions of the chimeric antibody are selected from the group consisting of: human IgGl, human IgG2, human IgG3, and human IgG4.

According to some embodiments, a conjugate comprising the antibody or fragment thereof as described above is provided.

Antibodies or fragments thereof according to the present invention may be attached to a cytotoxic moiety, a radioactive moiety, or an identifiable moiety.

Polynucleotide sequences encoding antibodies, having specificity for CEACAM1 protein, as well as vectors and host cells carrying these polynucleotide sequences, are provided according to another aspect of the present invention.

According to some embodiments, polynucleotide sequences encoding the amino acid sequences of the heavy chain variable region and light chain variable region described above are provided.

According to some embodiments, the polynucleotide sequence encodes to the amino acid sequences of heavy chain variable region and light chain variable region of an antibody selected from the group consisting of CCM5.01, CCM5.18, and CCM5.19.

According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising at least one sequence set forth in SEQ ID NO: 1, and SEQ ID NO: 3.

According to yet some embodiments, the polynucleotide sequence according to the invention encodes an antibody or antibody fragment or chain comprising: a set of six CDRs wherein: heavy chain CDR1 sequence comprising the sequence SDYAWN (SEQ ID NO: 5), heavy chain CDR2 comprising the sequence YITYSGSTSYNPSLKS (SEQ ID NO: 6), heavy chain CDR3 comprising the sequence ENRYDGEMFFDV (SEQ ID NO: 7), light chain CDR1 comprising the sequence RASQSISNDLH (SEQ ID NO: 8), light chain CDR2 comprising the sequence YAYQSIS (SEQ ID NO: 9), and light chain CDR3 comprising the sequence QQSNSWPHT (SEQ ID NO: 10).

According to some embodiments, the polynucleotide sequences defined above encode a molecule selected from the group consisting of: an antibody, an antibody fragment comprising at least an antigen-binding portion, and an antibody conjugate comprising said antibody or antibody fragment. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody heavy chain variable region comprising a sequence set forth in SEQ ID NO: 1 or a variant thereof having at least 90% sequence identity.

According to some embodiments, the polynucleotide sequence encodes a monoclonal antibody light chain variable region, comprising a sequence set forth in SEQ ID NO: 3, or a variant thereof having at least 90% sequence identity.

According to some embodiments, the polynucleotide encoding for the heavy chain comprises a sequence set forth in SEQ ID NO: 2 or a variant thereof having at least 90% sequence identity. According to other embodiments, the polynucleotide encoding for the light chain comprises a sequence set forth in SEQ ID NO: 4 or a variant thereof having at least 90% sequence identity.

According to some embodiments, the polynucleotide encoding for the heavy chain comprises a sequence set forth in SEQ ID NO: 2 or a variant thereof having at least 95% sequence identity. According to other embodiments, the polynucleotide encoding for the light chain comprises a sequence set forth in SEQ ID NO: 4 or a variant thereof having at least 95% sequence identity.

According to some embodiments, the polynucleotide encoding for the heavy chain comprises a sequence set forth in SEQ ID NO: 2. According to some embodiments, the polynucleotide encoding for the light chain comprises a sequence set forth in SEQ ID NO: 4.

The present invention provides, according to some embodiments, a polypeptide comprising at least one sequence encoded by at least one polynucleotide sequence disclosed above.

In a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof according to the present invention. According to some embodiments the nucleic acid construct is a plasmid. According to some embodiments the plasmid comprises at least one polynucleotide sequence set forth in a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. Each possibility represents a separate embodiment of the present invention.

In still another aspect the present invention provides a cell capable of producing an antibody or an antibody fragment comprising the specific CDR sequences and/or specific heavy and light chain variable regions defined above.

According to some embodiments, a cell is provided comprising at least one polynucleotide sequence disclosed above.

According to some embodiments, the cell producing the monoclonal antibody in a hybridoma cell.

The present invention provides, according to another aspect, a pharmaceutical composition comprising as an active ingredient, at least one antibody, antibody fragment or conjugates thereof, that recognizes CEACAM1, and optionally at least one pharmaceutical acceptable excipient, diluent, salt or carrier, wherein said at least one antibody or antibody fragment does not block the homophilic interactions of CEACAM1.

According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or a fragment thereof which is capable of binding to an epitope within the CEACAM1 protein to which binds a monoclonal antibody selected from the group consisting of: CCM5.01, CCM5.18, and CCM5.19.

According to some embodiments, the pharmaceutical composition comprises at least one monoclonal antibody comprising a set of six CDRs wherein: HC CDR1 is SEQ ID NO: 5; HC CDR2 is SEQ ID NO: 6; HC CDR3 is SEQ ID NO: 7; LC CDR1 is SEQ ID NO: 8; LC CDR2 is SEQ ID NO: 9; and LC CDR3 is SEQ ID NO: 10.

According to some embodiments, the pharmaceutical composition comprises an antibody or fragment thereof comprising a heavy chain variable region having a sequence set forth in SEQ ID NO: 1.

According to some embodiments, the pharmaceutical composition comprises an antibody or fragment thereof comprising a light chain variable region having a sequence set forth in SEQ ID NO: 3. According to a specific embodiment, the pharmaceutical composition comprises an antibody or fragment thereof comprising a heavy chain variable region having the sequence set forth in SEQ ID NO: 1 and a light chain variable region having the sequence set forth in SEQ ID NO: 3.

According to some embodiments, the antibody, antibody fragment or antibody conjugate is capable of directly killing tumor cells independent of immune cells. According to certain embodiments, the antibody, antibody fragment or antibody conjugate does not interfere with the homophilic interactions of CEACAM1.

According to some embodiments, the pharmaceutical composition according to the present invention is for use in cancer immunotherapy. According to some embodiments, the pharmaceutical composition according to the present invention is for use in treating cancer.

According to some embodiments, the pharmaceutical composition further comprises an additional anti-cancer agent.

The cancer treatable with a composition according to the present invention may be any cancer that expresses CEACAM1. According to some embodiments, the cancer overexpresses CEACAM1.

According to some embodiments of the invention, the cancer is selected from the group consisting of a melanoma, a bladder cancer, a breast cancer, a prostate cancer, a gastric cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, a colon cancer, a cervical cancer, a kidney cancer, a lung cancer, a thyroid cancer, a brain cancer, a renal cancer, a throat cancer, a laryngeal carcinoma, a hepatic cancer, a fibrosarcoma, an endometrial cells cancer, a glioblastoma, sarcoma, a myeloid, a leukemia and a lymphoma. Each possibility represents a separate embodiment of the invention.

According to some embodiments of the invention, the cancer is selected from the group consisting of a melanoma, a bladder cancer, a breast cancer, a prostate cancer, a non-small cell lung carcinoma (NSCLC), and a gastric cancer. According to certain embodiments, the cancer is melanoma. According to some embodiments, the cancer is a solid cancer. According to some specific embodiments, the solid cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, ovarian cancer, and colon cancer.

According to some embodiments, the pharmaceutical composition according to the present invention is for use in preventing or treating viral infection.

According to some embodiments, the viral infection is caused by a virus selected from the group consisting of viruses of the corona family and in particular SARS-CoV-2, influenza of any type (including but not limited to human, avian and swine strains), cytomegalovirus (CMV), Human immune deficiency virus (HIV), herpes virus and hepatitis virus. Each possibility represents a separate embodiment of the invention.

For use in preventing (after exposure for example), or treating a viral infection, the antibodies of the present invention may be used alone or in combination with at least one additional agent.

According to some embodiments, the additional agent is an anti-viral agent.

According to yet other embodiments, the additional agent is an anti-inflammatory agent.

According to some embodiments, the additional agent is an antibody activating inhibitory receptor on immune cells, including but not limited to anti-CD300a, anti-Siglec, anti-CD48 antibody and CD48Fc. According to some embodiments, the anti CEACAM1 antibody is selected from the group consisting of CCM5.01, CCM5.18, and CCM5.19.

According to some embodiments, the anti CEACAM1 antibody or antibody fragment comprises a set of six CDRs wherein: HC CDR1 is SEQ ID NO: 5; HC CDR2 is SEQ ID NO: 6; HC CDR3 is SEQ ID NO: 7; LC CDR1 is SEQ ID NO: 8; LC CDR2 is SEQ ID NO: 9; and LC CDR3 is SEQ ID NO: 10.

According to yet another aspect, the present invention provides a method of treating cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising at least one antibody, antibody fragment or conjugate thereof, that recognizes CEACAM1, the antibody, antibody fragment or conjugate thereof does not interfere with homophilic interactions of CEACAM1. According to some embodiments of the invention, the therapeutically effective amount results in a decrease in tumor size or in the number of metastases in the subject.

According to some embodiments, the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anticancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy.

According to some embodiments, the method of treating cancer comprises administration of the antibody that recognizes CEACAM1 protein as described herein, and an additional anticancer agent. According to some embodiments, the additional anti-cancer agent is selected from the group consisting of: immune-modulator, activated lymphocyte cell, kinase inhibitor and chemotherapeutic agent.

According to other embodiments, the additional immune-modulator is an antibody, antibody fragment or antibody conjugate that binds to an antigen other than CEACAM1.

According to some embodiments of the invention, the subject is a human subject.

According to an aspect, the present invention provides a method of diagnosing or prognosing cancer in a subject, the method comprises determining the expression level of CEACAM1 in a biological sample of said subject using at least one antibody as described herein.

According to yet another aspect, the present invention provides a method of preventing or treating viral infection comprising administering to a subject in need thereof, a pharmaceutical composition comprising a therapeutically effective amount of at least one antibody, antibody fragment or conjugate thereof, that recognizes CEACAM1, the antibody, antibody fragment or conjugate thereof does not interfere with homophilic interactions of CEACAM1.

According to some embodiments, the viral infection is caused by a virus selected from the group consisting of viruses of the corona family and in particular SARS-CoV-2, influenza of any type (including but not limited to human, avian and swine strains), cytomegalovirus (CMV), Human immune deficiency virus (HIV), herpes virus and hepatitis virus. According to some embodiments, the method of preventing (after exposure for example), or treating viral infection, comprises administering of the antibodies of the present invention alone or in combination with at least one additional agent.

According to some embodiments, the additional agent is an anti-viral agent.

According to some embodiments, the method comprises administering of an anti CEACAM1 antibody selected from the group consisting of CCM5.01, CCM5.18, and CCM5.19.

According to some embodiments, the method comprises administration of an antibody or antibody fragment comprises a set of six CDRs wherein: HC CDR1 is SEQ ID NO: 5; HC CDR2 is SEQ ID NO: 6; HC CDR3 is SEQ ID NO: 7; LC CDR1 is SEQ ID NO: 8; LC CDR2 is SEQ ID NO: 9; and LC CDR3 is SEQ ID NO: 10.

The present invention further comprises, according to another aspect, a method of determining or quantifying CEACAM1 in a sample, the method comprising contacting a biological sample with an antibody or antibody fragment as described herein, and measuring the level of complex formation.

According to some embodiments, the antibody is selected from the group consisting of CCM5.01, CCM5.18, and CCM5.19.

According to some embodiments, the antibody or antibody fragment comprises a set of six CDRs wherein: HC CDR1 is SEQ ID NO: 5; HC CDR2 is SEQ ID NO: 6; HC CDR3 is SEQ ID NO: 7; LC CDR1 is SEQ ID NO: 8; LC CDR2 is SEQ ID NO: 9; and LC CDR3 is SEQ ID NO: 10.

Determining and quantifying methods may be performed in-vitro or ex-vivo according to some embodiments or may be used in diagnosing conditions associated with expression of CEACAM1. The antibodies according to the present invention may be also used to configure screening methods. For example, an enzyme-linked immunosorbent assay (ELISA), or a radioimmunoassay (RIA), as well as method such as IHC or FACS, can be constructed for measuring levels of polypeptides using the antibodies and methods known in the art.

According to some embodiments, the method for detecting or quantifying the presence of CEACAM1 expressed on cells or secreted to a biological medium, comprises the steps of: i. incubating a sample with an antibody specific to CEACAM1 or an antibody fragment thereof as described herein; and ii. detecting the bound CEACAM1 using a detectable probe.

According to some embodiments, the method further comprises the steps of: iii. comparing the amount of (ii) to a standard curve obtained from a reference sample containing a known amount of CEACAM1; and iv. calculating the amount of the CEACAM1 in the sample from the standard curve.

According to some particular embodiments the sample is body fluid.

According to some embodiments, the method is performed in-vitro or ex-vivo.

A kit for measuring the expression or presence of CEACAM1 in biological sample is also provided comprising at least one antibody or antibody fragment according to the present invention.

According to some embodiments, the kit comprises an antibody selected from the group consisting of CCM5.01, CCM5.18, and CCM5.19.

According to some embodiments, the kit comprises an antibody or antibody fragment comprising a set of six CDRs wherein: HC CDR1 is SEQ ID NO: 5; HC CDR2 is SEQ ID NO: 6; HC CDR3 is SEQ ID NO: 7; LC CDR1 is SEQ ID NO: 8; LC CDR2 is SEQ ID NO: 9; and LC CDR3 is SEQ ID NO: 10.

According to an aspect, the present invention provides a kit for detecting cancer, the diagnostic kit comprises an antibody or antibody fragment thereof as described herein.

It is to be understood that any combination of each of the aspects and the embodiments disclosed herein is explicitly encompassed within the disclosure of the present invention.

Further embodiments and the full scope of applicability of the present invention will become apparent from the drawings and detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows staining of parental 721.221 cells (left column) with the 3 anti- CEACAM1 mAbs: CCM5-1 (CCM5.01), CCM5-18 (CCM5.18) and CCM5-19 (CCM5.19). The staining of the CEACAM1 transfected 721.221 cells is shown in the right column. The staining was performed using various quantities (2pl, 5pl, lOpl, 20pl, empty histograms) of hybridomas supernatants.

Figure 2 shows staining of 721.221 cells expressing CEACAM1 with CEACAM1 -Ig incubated with the various anti- CEACAM1 mAbs: CCM5-1, CCM5-18 and CCM5-19 at various amounts as indicated (2pl, 5pl, lOpl, 20pl, empty histograms).

Figure 3 shows the binding of the mAbs to CEACAMl-IgG fusion protein. Microscale thermophoresis (MST) binding measurements of anti-CEACAMl mAbs to CEACAM1 is shown. The CEACAM1 fusion protein concentration was kept constant (7.5 nM), and the concentration of the mAbs varied from 30 mM to 0.1 nM. The binding Kd was obtained using the Hill method using the NanoTemper analysis software. Error bars represent standard error of 5 measurements.

Figure 4 shows staining of parental 1106mel cells with the 3 anti-CEACAMl mAbs: CCM5- 1, CCM5-18 and CCM5-19.

Figure 5 shows the effect of anti-CEACAMl mAbs on melanoma cell death. Death was determined using PI staining.

Figures 6A-6B show the combined effect of anti-CEACAMl and Cisplatin on Mel-14 cell viability. Figure 6A - Cell viability (MTT assay) of Mel- 14 cells treated with Cisplatin or with PBS for 72h. Data are expressed as % of viable cells. (n=l), p**=0.014. Figure 6B - Cell viability of Mel- 14 cells treated with a constant concentration of Cisplatin (2.5 ug/ml) and different concentrations of anti-CEACAMl mAbs or IgGl (as negative control) for 72h. The O.D. levels represent viable cell numbers. n=2; p**<0.009.

Figures 7A-7B show tumor growth and weight progression of human melanoma cell line (Mel- 14) transplanted as a xenograft in SCID/beige mice. Figure 7A - Tumor growth was measured using a caliper every other day until the first mice had a tumor of -15 mm³. Tumor size was calculated by the formula: width² x length/2. Figure 7B - Tumor weight after transplanting different amounts of cells. Each point indicates tumor characteristics of a single mouse.

Figures 8A-8C show the effect of anti- CEACAM1 on tumor growth of Mel-14 cell xenograft in SCID/Beige mice. Figure 8A - A schematic representation of the treatment schedule. Mel- 14 cells (5xl0⁶ / 200 pl) were injected subcutaneously (sc) into SCID/beige mice at day 0. Intraperitoneal injections (ip) of anti-CEACAMl or IgGl (as negative control), were performed 30 min later and then every other day until tumors reached 15 mm³. At this time, mice were euthanized, tumor excised and weighted. Figures 8B-8C - Tumor weight (mg) of anti-CEACAMl or IgGl treated mice with dose of 50 pg/kg (B) and 500 pg/kg (C).

Figure 9 shows flow cytometry staining of A549 human lung carcinoma cells using anti- CEACAMl (empty histogram) or the negative isotype control IgGl (filled histogram). One representative experiment of at least 3 is presented.

Figures 10A-10D show the effect of anti-CEACAMl mAb on immune cells of transgenic mice expressing CEACAM1 following injection of B16-F10 melanoma cells. Figure 10A - Schematic representation of the treatment schedule of B16-F10 cells grafted mice. B16-F10 cells (5X10⁵ /100 pl) were injected subcutaneously (sc) into hCeacaml^+l+ x in.sCeacam I '' mice at day 0. Intraperitoneal injections (ip) of anti-hCeacaml, anti-mCeacaml or mlgGl (lOOpg/mice) were performed after day 8 (palpable tumor) and then every other day until tumors reached 15mm³ and mice were euthanized. Figures 10B and 10C - Tumor size (mm³, B) and weight of treated mice (C). Figure 10D - number of CD3 T cells in spleen of euthanized mice (3 mice/group).

Figure 11 shows the binding of anti-CEACAMl mAbs (EC-Sab3, B3-17, and CCM5.01) to CEACAM1 molecule that includes (CCl-Fc) or lacks (CCldN-Fc) the N-terminal domain. Binding was assessed by absorbance using spectrophotometer at 450nm.

Figures 12A and 12B show CEACAM1 expression of eosinophils from healthy donor and 4 Covid-19 patients, as detected by anti hCEACAMl mAb CCM5.01. Figure 12A - Representative histogram of CEACAM1 expression. Figure 12B - CEACAM1 expression as staining index.

Figure 13 depicts the binding of mAbs CCM5.01, CCM5.19, and F1-C5 to CEACAMl-Fc:

CEACAM1 fusion protein and CEACAMl-dN-Fc: CEACAM1 fusion protein that does not contain the N-terminal domain, compared to CCl/3/5-Sab positive control and isotype matched negative control mAh. Data are representative of three independent experiments.

Figure 14 demonstrates the binding of mAbs CCM5.01 and CCM5.19 and respective positive control antibodies, to CHO, CHO-mouse CEACAM1, CHO-human CEACAM1, CHO-human CEACAM3, CHO-human CEACAM5, CHO-human CEACAM6, CHO-human CEACAM7, and CHO-human CEACAM8 transfectants. Data shown represent the median values of one out of three different, representative experiments.

Figures 15A-15B show the effect of CCM5.01 mAb on colon cells death. Colon cancer cell lines HT-29 (Figure 15b) and LoVo cells (Figure 15A) transfected with long (L) or short (S) CEACAM1 isoforms were incubated with CCM5.01 for 72hr. Cell death was then determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium reduction (MTT) assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibodies specific to the human protein CEACAM1 that are effective in inhibition of tumor growth. The invention also provides production and use of the antibodies as therapeutic agents. In particular, the mAbs of the present invention may be used for augmenting anti-tumor killing activity, and as diagnostic reagents. In some embodiments, the invention provides antibodies specific to CEACAM1 for efficient induction of apoptosis of cancer cells.

Advantageously, the antibodies of the invention induce an inhibitory effect directly on the tumor cells, without dependency on immune components. The inhibitory signal induced by the antibodies is mediated by the phosphorylation of the SH2 domain-containing protein tyrosine phosphatase- SHP1, inducing apoptosis of the tumor cells by increase and activation of the tumor suppressor protein p53 (Zaffran et al., Cancer Gene Therapy, 29; 1676-1685 (2022)). This is in contrast to other known anti-CEACAMl antibodies that inhibit the inhibitory signal induced by the tumor cells on immune cells.

The new respiratory coronavirus from Wuhan in China is causing intense research activity in the world. Viruses are evidently associated with respiratory infections ranging in severity from the common cold to pneumonia and death. More than 200 viral types have been associated with the common cold, of which 50% of infections are rhinovirus, but also respiratory syncitial virus, influenza - and coronaviruses, particularly human coronavirus- 229E (HCoV-229E), which is ‘relatively benign’. Although monocytes are much more resistant to HCoV-229E, dendritic cells are rapidly killed by the virus. This is performed by invasion of HCoV-229E to the dendritic cells, followed by a rapid replication of the virus, which eventually resulted with cell death within a few hours of infection (Mesel-Lemoine et al., 2012). Dendritic cells are the sentinel cells in the respiratory tract, and plasmacytoid dendritic cells are a crucial antiviral defense via interferon production. Thus, these viruses can impair control of viral dissemination and the formation of long-lasting immune memory.

The cellular receptor for HcoV-229E is CD- 13 (aminopeptidase-N). The cellular receptors for coronaviruses are critical for cell entry. Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and HCoV-NL63, bind to angiotensin converting enzyme 2 (ACE2) on ciliated cells in the respiratory tract, the crystal structure is known (Song et al., 2018). The SARS-CoV spike (S) glycoprotein (with S 1 and S2 sub-units) on the outer envelope binds to ACE2, fusing viral and cellular membranes, triggering conformational transformations. Cleavage of the S1/S2 subunits by proteases is critical. Indeed, Simmons et al (2005) showed that SARS-CoV infection involved receptor binding, conformational changes receptor binding and subsequent cathepsin L (and type II transmembrane serine proteases) proteolysis within endosomes - with inhibitors of cathepsin L preventing viral entry. SARS- CoV may lead to markedly elevated cytokine levels, leading to tissue damage, pneumonitis, and acute respiratory distress syndrome (ARDS). The spike proteins in SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) are crucial for host specificity and jumping between species, e.g. from bats to humans (Lu et al., 2015), and also the recent crossover to swine acute diarrhea syndrome coronavirus (SADS-CoV) to pigs (Zhou et al., 2018). There is intense interest in the complicated ways whereby coronaviruses engage with their target receptors, are activated and replicated. The rapid evolution of coronaviruses is critical as humans have little or no pre-existing immunity to SARS- or MERS-CoV.

ACE2 has been identified as a receptor for the novel coronavirus that was identified as the cause of the respiratory disease outbreak in Wuhan in late 2019 (referred to as 2019 - nCoV by the WHO, or Wuhan coronavirus). 2019-nCoV is a beta coronavirus, and, in common with the SARS-C0V8, the viral spike protein engages ACE2 for viral entry. The data presented by Letko and Munzter (BiorXiv7) is preliminary and has not been peer-reviewed, but a rapid pace of research is needed in the present circumstances. For example, in another recent, preliminary report, ACE2 receptor expression is highly expressed in type II alveolar cells (AT2) with much individual variation and the authors reported that the cells also expressed many genes involved in viral reproduction and transmission. While these findings are preliminary, the rapid publications in bioRxiv and similar archives are crucial in what is now officially a global health emergency.

Various viruses, including influenza virus, human cytomegalovirus and murine corona virus induce CEACAM1 expression on epithelia and endothelial cells and on cells of the immune system. Viruses use CEACAM1 to enter and infect these cells. However, upon induction, CEACAM1 suppresses the viruses in an SHP2-dependent process and achieves this also by suppressing mTOR-mediated protein biosynthesis.

The antibodies of the present invention activate CEACAM1 receptor ITIMs to dephosphorylate various signal transduction proteins via SHP-1 and SHP-2 phosphatases and therefore have an anti-viral activity and protect the cells from killing by viral infection.

Without wishing to be bound to any mechanism of action, the anti-CEACAMl activating antibodies of the present invention are effectors in a broad antiviral activity by at least one of the following mechanisms: i. binding to cell associated membrane CEACAM1, expressed for example on lung epithelial cells, lung dendritic cells and other immune cells, and therefore stimulating anti-viral activity. ii. competing with virus CEACAM-1 binding to the cell surface CEACAM1. iii. binding to virus CEACAM-1 and therefore antagonize its binding to the cell surface CEACAM1.

The antibodies described herein are according to some embodiments for use in treating viral infection.

According to some embodiments, the viral infection is caused by a virus selected from the group consisting of viruses of the corona family and in particular SARS-CoV-2, influenza of any type (including but not limited to human, avian and swine strains), cytomegalovirus (CMV), Human immune deficiency virus (HIV), herpes virus and hepatitis virus.

For use in preventing (after exposure for example), or treating anti-viral infection, the antibodies of the present invention may be used alone or in combination with at least one additional agent.

According to some embodiments, the additional agent is an anti-viral agent. According to other embodiments, the additional agent is an anti-inflammatory agent.

The term "antigen" as used herein refers to a molecule or a portion of a molecule capable of eliciting antibody formation and being specifically bound by an antibody. An antigen may have one or more than one epitope. The specific binding 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 some embodiments of the present invention is a CEACAM protein. According to specific embodiments, the antigen is CEACAM 1.

The term “CEACAM1” is used to refer to the protein product of the CEACAM1 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 P13688, 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 antibodies or a fragment thereof according to the invention bind to an epitope in CEACAM 1. Specifically, the antibodies bind to an epitope within the ectodomain (extracellular part) of the CEACAM 1 protein. According to some embodiments, antibodies of the present invention do not bind the N-domain of CEACAM 1.

According to some embodiments, antibodies of the present invention bind CEACAM 1, and cross-react with CEACAM5 and CEACAM8. According to other embodiments, antibodies of the present invention do not bind CEACAM3, CEACAM6 and CEACAM7.

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.

It should be noted that the affinity can be quantified using known methods such as, Surface Plasmon Resonance (SPR), and can be calculated using, e.g., a dissociation constant, Kd, such that a lower Kd reflects higher affinity.

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 crystallizable) 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. 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.

CDR identification or determination from a given heavy or light chain variable sequence, is typically made using one of few methods known in the art. For example, such determination is made according to the Kabat (Wu T.T and Kabat E.A., J Exp Med, 1970; 132:211-50) and IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77).

When the term “CDR having a sequence”, or a similar term is used, it includes options wherein the CDR comprises the specified sequences and also options wherein the CDR consists of the specified sequence.

The antigen specificity of an antibody is based on the hyper variable region (HVR), namely the unique CDR sequences of both light and heavy chains that together form the antigen-binding domain (ABD). 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, K or lambda, ). Both isotopes are found in all antibody classes.

The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments long enough to exhibit the desired biological activity, namely binding to CEACAM1.

Antibody or antibodies according to the invention includes 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

"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 VE, 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., Proc. Natl. Acad. Sci. (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-CH1-VH-CH1) 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). Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-1 17 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 70:163- 167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv).

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). Techniques for the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to CEACAM1.

The term "monoclonal antibody" (mAb) 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, 495-7, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597. The design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy (VH) and variable light (VL) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria are described in Fields et at. 2013, 8(6): 1125-48.

The mAbs of the present invention may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD. 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 may be purified from such ascites fluids, or from culture supernatants, using methods well known to those of skill in the art.

Anti-idiotype antibodies specifically immunoreactive with the hypervariable regions of an antibody of the invention are also comprehended.

The invention provides a monoclonal antibody or an antibody fragment comprising an antigen binding domain (ABD) which comprises three CDRs of a light chain and three CDRs of a heavy chain, wherein said ABD has at least 90% sequence identity or similarity with an ABD of a monoclonal mouse antibody selected from the group consisting of CCM5.01, CCM5.18, and CCM5.19.

According to some embodiments, the ABD has at least 90% sequence identity or similarity with an ABD of a monoclonal mouse antibody comprising a heavy variable chain comprising the amino acid sequence SEQ ID NO: 1 and a light variable chain comprising the amino acid sequence SEQ ID NO: 3 (herein identified as CCM5.01). Such antibody may have an ABD domain having at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, at least 99% sequence identity or similarity or 100% sequence identity with corresponding ABD of antibodies CCM5.01, CCM5.18, or CCM5.19.

Sequence identity is the amount of amino acids or nucleotides which match exactly between two different sequences. Sequence similarity permits conservative substitution of amino acids to be determined as identical amino acids.

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,

"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.

Analogs and variants of the antibody sequences are also within the scope of the present application. These include, but are not limited to, conservative and non-conservative substitution, insertion and deletion of amino acids within the sequence. Such modification and the resultant antibody analog or variant are within the scope of the present invention as long as they confer, or even improve the binding of the antibody to the CEACAM1.

Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration, and targeting to specific cell populations, immunogenicity, and the like. One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, according to one table known in the art, the following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

It should be emphasized that the variant chain sequences are determined by sequencing methods using specific primers. Different sequencing methods employed on the same sequence may result in slightly different sequences due to technical issues and different primers, particularly in the sequence terminals.

The terms "molecule having the antigen-binding portion of an antibody" and “antigen- binding-fragments” as used herein are 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 e.g., 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), 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 antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species, or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851- 6855 (1984)). In addition, complementarity determining region (CDR) grafting may be performed to alter certain properties of the antibody molecule including affinity or specificity. A non-limiting example of CDR grafting is disclosed in US patent No. 5,225,539.

Chimeric antibodies are molecules of which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Antibodies that have variable region framework residues substantially from human antibody (termed an acceptor antibody) and CDRs substantially from a mouse antibody (termed a donor antibody) are also referred to as humanized antibodies. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (for example PCT patent applications Nos. WO 86/01533, WO 97/02671, WO 90/07861, WO 92/22653 and US patents Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539).

According to some embodiments, the antibody is a monoclonal antibody.

According to some specific embodiments, the monoclonal antibody is a chimeric monoclonal antibody.

According to some embodiments, the chimeric antibody comprises human-derived constant regions.

According to some embodiments the human constant regions of the chimeric antibody are selected from the group consisting of: human IgGl, human IgG2, human IgG3, and human IgG4.

According to a particular embodiment, a chimeric monoclonal antibody which recognizes CEACAM1 is provided comprising a set of six CDRs wherein: HC CDR1 is (SEQ ID NO: 5); HC CDR2 is (SEQ ID NO: 6); HC CDR3 is (SEQ ID NO: 7); LC CDR1 is (SEQ ID NO: 8); LC CDR2 is (SEQ ID NO: 9); and LC CDR3 is (SEQ ID NO: 10).

Pharmacology

In pharmaceutical and medicament formulations, the active agent is preferably utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired exposure. Typically, the antibodies and fragments and conjugates thereof of the present invention comprising the antigen binding portion of an antibody or comprising another polypeptide including a peptide-mimetic will be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control release of active ingredient (molecule comprising the antigen binding portion of an antibody) or to prolong its presence in a patient's system. Numerous suitable drug delivery systems are known and include, e.g., implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. The rate of release of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles.

The pharmaceutical composition of this invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intraarticulary, intralesionally, intratumorally or parenterally. Ordinarily, intravenous (i.v.) administration is used for delivering antibodies.

It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered, its persistence in the blood circulation, and the judgment of the treating physician.

As used herein 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.

The cancer amendable for treatment by the present invention includes, but is not limited to: melanoma, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high-grade immunoblastic NHL; highgrade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. The cancerous conditions amendable for treatment of the invention include metastatic cancers.

According to other embodiments, the pharmaceutical composition according to the invention is for use in treating cancer characterized by overexpression of CEACAM1.

The molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

The pharmaceutical composition according to the present invention may be administered together with an anti-neoplastic composition.

The term "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative measures.

The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, melanoma, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include melanoma, lung, thyroid, breast, colon, prostate, hepatic, bladder, renal, gastric, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, or endometrial cancer.

According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen comprising administration of at least one additional anti-cancer agent.

According to some embodiments, the anti-cancer agent is selected from the group consisting of an antimetabolite, a mitotic inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, an asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof. Each possibility represents a separate embodiment of the invention.

Monoclonal antibodies according to the present invention may be used as part of combined therapy with at least one anti-cancer agent. According to some embodiments, the additional anti-cancer agent is an immuno-modulator, an activated lymphocyte cell, a kinase inhibitor or a chemotherapeutic agent.

According to some embodiments, the anti-cancer agent is an immuno-modulator, whether agonist or antagonist, such as antibody against an immune checkpoint molecule.

Checkpoint immunotherapy blockade has proven to be an exciting new venue of cancer treatment. Immune checkpoint pathways consist of a range of co-stimulatory and inhibitory molecules which work in concert in order to maintain self-tolerance and protect tissues from damage by the immune system under physiological conditions. Tumors take advantage of certain checkpoint pathways in order to evade the immune system. Therefore, the inhibition of such pathways has emerged as a promising anti-cancer treatment strategy.

According to other embodiments the additional anti-cancer agent is a chemotherapeutic agent.

In some embodiments, the pharmaceutical composition according to the present invention is for use in treating cancer or for use in enhancing the immune response.

The term "enhancing immune response" refers to increasing the responsiveness of the immune system and inducing or prolonging its memory. The pharmaceutical composition according to the present invention may be used to stimulate immune system upon vaccination. Thus, in one embodiment the pharmaceutical composition can be used for improving vaccination.

In certain embodiments, the cancer is selected from lung, thyroid, breast, colon, melanoma, prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, and endometrial cells cancer. Each possibility represents a separate embodiment of the invention.

According to some embodiments, a pharmaceutical composition, comprising at least one antibody or fragment thereof according to the present invention, and a pharmaceutical composition, comprising an additional immuno-modulator or a kinase inhibitor, are used in treatment of cancer by separate administration.

According to still another aspect the present invention provides a method of treating cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a monoclonal antibody or antibody fragment according to the present invention.

The term “effective amount” as used herein refers to a sufficient amount of the monoclonal antibody or the antibody fragment that, when administered to a subject will have the intended therapeutic effect. The effective amount required to achieve the therapeutic end result may depend on a number of factors including, for example, the specific type of the tumor and the severity of the patient's condition, and whether the combination is further coadministered with radiation. The effective amount (dose) of the active agents, in the context of the present invention should be sufficient to affect a beneficial therapeutic response in the subject over time, including but not limited to inhibition of tumor growth, reduction in the rate of tumor growth, prevention of tumor and metastasis growth and enhanced survival.

The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered enterally or parenterally. Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

Antibodies are generally administered in the range of about 0.1 to about 20 mg/kg of patient weight, commonly about 0.5 to about 10 mg/kg, and often about 1 to about 5 mg/kg. In this regard, it is preferred to use antibodies having a circulating half-life of at least 12 hours, preferably at least 4 days, more preferably up to 21 days. Chimeric antibodies are expected to have circulatory half-lives of up to 14-21 days. In some cases, it may be advantageous to administer a large loading dose followed by periodic (e.g., weekly) maintenance doses over the treatment period. Antibodies can also be delivered by slow-release delivery systems, pumps, and other known delivery systems for continuous infusion.

The present invention also provides a method of preventing or treating a viral infection comprising administering to a subject at least one antibody specific to CEACAM1 as described herein, or a fragment thereof comprising at least the antigen binding domain.

The term "about" means that an acceptable error range, e.g., up to 5% or 10%, for the particular value should be assumed.

Diagnosis

The present invention further discloses methods for diagnosing and prognosing cancer. According to an aspect, the present invention provides a diagnostic and/or prognostic method of cancer or infectious disease in a subject, the method comprises the step of determining the expression level of CEACAM1 in a biological sample of said subject using at least one antibody as described herein.

The term "biological sample" encompasses a variety of sample types obtained from an organism that may be used in a diagnostic or monitoring assay. The term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen, or tissue cultures or cells derived therefrom and the progeny thereof. Additionally, the term may encompass circulating tumor or other cells. The term specifically encompasses a clinical sample, and further includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids including aqueous humour and vitreous for eyes samples, and tissue samples. The term also encompasses samples that have been manipulated in any way after procurement, such as treatment with reagents, solubilisation, or enrichment for certain components.

Determining the expression level of CEACAM1 can be performed by a labeled anti- CEACAM1 antibody as described herein. Determining the expression can be performed, for example, by ELISA or by flow cytometry.

The method of the invention can further comprise the step of comparing said level of expression to a control level.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed as limiting the scope of the invention.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, immunological and recombinant DNA techniques. Such techniques are well known in the art. Other general references referring to well-known procedures are provided throughout this document for the convenience of the reader.

Materials & Methods

Cell line cultures - Human melanoma cell lines (Mel-14, Mel-620, 1106Mel, 1610Mel, provided by Prof. Ofer Mandelboim from the Lautenberg Center for Immunology and Cancer Research, Faculty of Medicine, The Hebrew University of Jerusalem) and the murine melanoma cell line B16-F10 (provided by Prof. Michal Lotem, Hadassah Medical Center, Department of Oncology, Jerusalem, Israel) were cultured at 37°C in 5% CO₂ in Dulbecco's Modified Eagle Medium (DMEM, Gibco-ThermoFisher, Waltham, MA, USA) supplemented with 10% fetal calf serum (FCS, Sigma, USA), penicillin (lOOIU/ml) + streptomycin (lOOpg/ml) (Biological Industries, Beit Haemek, Israel) and 1% (vol/vol) sodium pyruvate (Biological Industries, Beit Haemek, Israel).

HC4 hepatocellular carcinoma, LS174 colon cancer, PANC-1 pancreatic cancer, ES-2 ovarian cancer and LOVO colorectal adenocarcinoma cell lines (provided by Dr. Joseph Tam and Prof. Rachel Bar-Shavit, Hebrew University of Jerusalem and Hadassah Medical Center, Israel) were cultured in DMEM (Gibco-ThermoFisher) supplemented with 10% FBS (Sigma), L-glutamine (2 mM), penicillin (100 lU/ml) + streptomycin (100 pg/ml) (Biological Industries). All cell lines were periodically checked for the CEACAM1 membrane expression by flow cytometry (FC).

Cell viability - The viability of melanoma (Mel) and LOVO cells was determined by MTT assay (Sigma, MO, USA). Melanoma cells (5xl0³/100pl) were seeded in a flat bottom 96 well cell culture plate (Thermo Scientific Nunc, Qiryat Shemona, Israel) in their respective culture medium and incubated over night at 37°C in 5% CO₂. The medium was then replaced with fresh DMEM 10% FCS containing different concentrations (0.1, 1, 5, and lOpg/ml) of anti-CEACAMl mAbs (clones 01 & 19), or IgGl isotype control (Mouse IgGl K Isotype Control Purified, without azide, eBioscience, ThermoFisher). Cell viability was determined after 72hr (a time point that was found to be the optimal one) by adding MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, lOpl/well) and incubating (37°C, 5% CO₂) them for 3 hours (until the purple-gray colored salt, is detectable). Plates were centrifuged (150g, 5min, 4°c), the supernatants discarded, and the pellet consisting of the purple-gray MTT formazan dissolved in DMSO (lOOpl, Sigma, MO, USA). Optical density (OD) absorption was measured at 570nm in ELISA reader (BIO-TEK, Winooski, USA). The viability of B16-F10 cells was determined by using anti-mouse CEACAM1 mAb (mCEACAMl) (provided by Dr. Bernhard Singer from the Institute of Anatomy, Essen, Germany) following the same protocol.

For combinational study, Cisplatin was first assessed (provided by Prof Dan Gibson, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem) IC50 on Mel- 14 cells. Then Mel- 14 were incubated for 72hr with a constant concentration of Cisplatin (2.5pg/ml) and increasing doses of anti-CEACAMl (0.1, 0.5, 1, 2.5, 5, lOpg/ml) and cell viability was determined by MTT as described above.

Flow Cytometry - Detection of CEACAM1 on the different cancer cell lines was performed by FC. Cells (IxlO⁵ cell/lOOpl/well) were resuspended in FC buffer containing PBS/0.1% bovine serum albumin (BSA) and seeded in 96-well plates, washed in the same medium, and resuspended in blocking buffer (PBS 5% Goat serum, Biological Industries, Beit Haemek, Israel) for 15min on ice. Then, cells were washed and incubated with anti-CEACAMl mAb or IgGl (2.5pg/ml) or FC buffer only for 40min on ice. Cells were washed again followed by incubation with the secondary Ab FITC goat anti-mouse (1:200, Jackson ImmunoResearch, USA) for 30min on ice. Afterward, the cells were washed three times, resuspended in 200pl FC buffer and analyzed. Data were acquired by FC (FACSCalibur; BD Biosciences) and analyzed using FlowJo software (Tree Star Inc, Ashland, OR, USA).

Binding assay - Micro-Scale Thermophoresis (MST) was used to establish the binding affinity between anti-CEACAMl mAb (clones 01 and 19) and CEACAM1. The CEACAM1 fusion protein (provided by Prof. Ofer Mandelboim) was labeled using the monolith NT protein labeling kit RED-NHS (NanoTemper Technologies GmbH, Munich, Germany) according to the company instructions.

MST experiments were performed on a NanoTemper Monolith NT.647 with blue/red filters (NanoTemper Technologies GmbH, Munich, Germany). Samples in PBS were loaded into standard/premium treated capillaries. Labelled CEACAM1 fusion protein was loaded at constant concentration of 175nM and anti-CEACAMl was added at concentrations from 30mM to O.lnM (serial dilution of 1:1). Measurements were performed at 22 °C using 20% MST power with laser off/on times of 5 s and 30 s, respectively. All experiments were repeated five times for each measurement. Data analyses were performed using the NanoTemper analysis software. The Kd constants between a protein and its ligand can be calculated using the saturation binding curve at equilibrium.

Apoptosis assay - Melanoma cell apoptosis was quantified by FC using AnnexinV/PI staining (BD, San Jose, CA, USA). Mels-14 cells seeded in 6 wells plate in their medium (5xl0⁵ cells/2ml/wells) were incubated overnight (ON). Anti-CEACAMl or IgGl (lOpg/ml) were then added and cells incubated for an additional 24hr. The cells were then washed with binding buffer (Thermo Fisher), stained with AnnexinV-FITC and acquired by FC in FACS calibur.

Mouse melanoma xenograft model - SCID/beige mice (males, 6-7 weeks old, Harlan, ENVIGO) were maintained in SPF conditions. After three acclimatization days, mice were shaved and injected subcutaneously (sc) in the right flank with Mel- 14 cells (5xl0⁶ /200pl cold PBS). Anti-CEACAMl or IgGl (50 or 500pg/kg in lOOpl ice cold PBS were injected intraperitoneally (ip) 30 minutes after tumor inoculation and thereafter every other day until tumors reached ~15mm³ (according to HUJI ethical animal guidelines) and mice were consequently euthanized by isoflurane. Every other day, tumor growth was measured using a caliper and mice were weighted to detect a decrease in weight or signs of sickness. Upon mice euthanasia, tumors were excised, weighed and fixed in 4% formaldehyde and paraffin- embedded for histopathologic or immunohistochemically studies.

Statistical analysis - Data were expressed as the mean +/- standard error (SEM). Unpaired two-tailed Student’s t-test (Prizm) was used to examine statistical significance. P values less than 0.05 were considered statistically significant.

Anti CD66a and anti CEACAM1 are used herein interchangeably.

Example 1. Preparation and analysis of anti CEACAM1 antibodies

A recombinant protein, for use as an immunogen, was produced and purified. The recombinant immunogen combines extracellular part of the human protein and human Fc region of an immunoglobulin G carrier. BALB/c mice were injected with 50 pg of the immunogen in complete Freund's adjuvant and 2 weeks later in incomplete Freund's adjuvant. After 2 weeks, the sera were screened for the antibody titer. The best responders (the serum was monitored by ELISA assay for the titer of the antibodies) were boosted with the immunogen in PBS. Three days later, spleen cells were collected, and after lysis of red blood cells, fused with SP2/0 cells. The cells were seeded in 20 % RPMI 1640 medium containing hypoxanthine, aminopterine, and thymidine for hybridoma selection and screened for mAbs using ELISA. Stable hybridoma cell lines were generated by fusing SP2/0 myeloma cells with spleen cells of an immunized mouse. Positive outcomes (cell lines secreting antibodies that recognize the immunogen) were further selected to develop a product that will have several differentiating characteristics: a) high yield to reduce the antibody-production costs and enable the testing of antibodies in a number of different techniques that require purified antibodies; b) the lack of cross-reactivity with other ligands of the immune cell receptors with emphasis on other members of the CEACAM family (transfectants expressing on their plasma membrane different ligands and different protein domains were used for this purpose); c) a strong binding capacity to the native, mature human CEACAM 1 molecules expressed on the surface of live cells. Indeed, the human and mouse CEACAM1 have high level of homology and it is not easy to generate a mouse monoclonal antibody that recognizes a human homologue. More importantly, human CEACAM 1 is extensively glycosylated on its extracellular region. Finally, there are a number of CEA members who share structural units. For these reasons, it is not easy to generate an antibody that recognizes a native protein using common antigens (such as E. coli derived ones). Antibodies that recognize a native human CEACAM 1 form on live cells were identified, which is a prerequisite to develop an agent that would bind and affect the human cells during the treatment.

Three anti-CEACAMl mAbs denoted CCM5.01, CCM5.18 and CCM5.19, were generated. All clones were found to be stable, having an IgGl isotype and produce large amount of antibodies (more than 1 pg/ml). As can be seen in Figure 1, the three antibodies specifically recognize CEACAM1 transfected 721.221 cells and do not recognize the parental, non-transfected cells. The three antibodies are non-blocking and do not block the binding of CEACAM 1-Ig to CEACAM 1 expressed on the cells (Figure 2).

The affinity of two of the anti-CEACAMl mAbs CCM5.01 (also denoted clone 01), and CCM5.19 (also denoted clone 19), was evaluated using the MST method. CEACAM1 fusion protein, labeled with NT-647 red dye, was mixed with different concentrations of anti- CEACAMl mAbs. Data analysis by NanoTemper confirmed that both mAbs from clone 01 and clone 19, bind to CEACAM 1 (Kd of 44 nM and 31 nM, respectively) with a stronger affinity for clone 19 (Figure 3).

Example 2. Anti CEACAM1 antibodies reduce viability of melanoma cells in vitro To assay the effect of the 3 anti- CEACAM1 mAbs on tumor cell growth in vitro, 1106mel cell line, derived from a melanoma patient were used. Human melanoma 1106mel cells were incubated with the three anti- CEACAM1 mAbs (CCM5.01, CCM5.18 and CCM5.19) or with the control mouse IgG mAb, and the effect of the antibodies on cell death in vitro was determined. As can be seen in Figure 4, all three anti- CEACAM1 mAbs recognized the cells expressing CEACAM1 efficiently. Around 90% CEACAM1 -mediated death is observed (Figure 5). Similar results of significant death induced by CCM5.01 were obtained with two human colon cell lines HT-29 and LoVo cells (Figure 15)..

Example 3. Combined in vitro effects of anti- CEACAM1 mAb with Cisplatin

The effect of anti-CEACAMl mAb combined with Cisplatin, a commonly chemotherapy used in melanoma, was tested in vitro. First, the IC50 of Cisplatin alone on Mel- 14 cells was determined. Human melanoma Mel- 14 cells were incubated with different concentrations of Cisplatin for 72hr and cell viability was evaluated by MTT (Figure 6A). A dose-dependent response was observed with an IC50 of 2.05pg/ml. For combination assay, the same cells were incubated with a 2.5μg/ml of Cisplatin and increasing concentrations of anti- CEACAMl mAbs or IgGl (0.1, 0.5, 1, 2.5, 5, and lOpg/ml) for 72hr. In the presence of both Cisplatin and anti-CEACAMl, an improved effect was demonstrated with a reduction of -65% in cell viability (Figure 6B).

Example 4. Anti-CEACAMl mAbs reduce the development of Mel-14 tumor growth in SCID/Beige mice

The effect of anti-CEACAMl was studied in vivo using a Mel-14 xenograft model in immunosuppressed SCID/beige mice. The tumor progression in mice injected s.c. with different amounts (2, 4, 8 and 10xl0⁶) Mel- 14 cells was first evaluated (calibration experiment). Tumor size was followed until it reached - 15mm³, and excised tumors were weighted (Figures 7A and 7B). The amount of 5xl0⁶ cells /mouse for a xenograft was chosen for further experiments.

To test anti-CEACAMl mAbs effects, mice were injected i.p. with either anti- CEACAMl, IgGl (50 and 500pg/kg), or PBS 30 minutes after tumor cells graft (s.c., day 0), and then injected every other day. The results shown in Figures 8A-8C and in Table 1 indicate that the difference between tumor weight of treated and untreated mice increases in dose- dependent manner.

Table 1. Data analysis of tumor weights. The % of decrease was obtained by dividing the tumor weight of anti-CEACAMl treated mice by the tumor weight of the control IgGl group from the same experiment and dose.

Example 5. CEACAM1 is expressed on human hepatocellular carcinoma (HC4), colorectal adenocarcinoma (LOVO) and epithelial colon cancer (LS174) cell lines and its activation decreases their survival

To evaluate whether CEACAM1 is expressed on tumors other than melanoma several tumor cell lines for CEACAM1 were stained and analyzed using the flow cytometry (FC) method. Positive staining was evident on human hepatocellular carcinoma (HC4), colorectal adenocarcinoma (LOVO) and human epithelial colon (LS174) cell lines. To test whether CEACAM1 activation can also inhibit the growth of these cell lines, LOVO cell lines were exposed to anti-CEACAMl mAbs (CCM5.01 CCM5.18 and CCM5.19 at lOpg/ml), or IgGl for 72hr, and cell viability was checked by MTT. Preliminary results indicate a reduction of up to 40% in cell viability in response to the anti-CEACAMl mAbs.

Example 6. CEACAM1 is expressed in lung cells

A549 human lung carcinoma cells (IxlO⁵ cell/lOOpl/well) were resuspended in FACS buffer (PBS/0.1% bovine serum albumin (BSA)) and seeded in 96-well plates, washed in the same medium, and resuspended in blocking buffer (PBS 5% Goat serum, Biological Industries, Beit Haemek, Israel) for 15min on ice. Then, cells were washed and incubated with anti- CEACAMl mAb CCM5.01 or IgGl (2.5pg/ml in FACS buffer) for 40min on ice. Cells were washed again followed by incubation with the secondary Ab FITC goat anti-mouse (1:200, Jackson ImmunoResearch, USA) in the dark for 30min on ice. Afterward, the cells were washed three times, resuspended in 200pl FACS buffer, meshed and analyzed. Data were acquired by Cytoflex and analyzed using FlowJo software (Tree Star Inc, Ashland, OR, USA).

The results shown in Figure 9 indicate the ability of the anti-CEACAMl monoclonal antibody of the present invention to stain human CEACAM1 expressed on lung cells, that is relevant not only to lung cancer but also to viral infections, such as S ARS -Co V-2inf ection.

Example 7. Additional in vivo evaluation of anti- CEACAM1 mAbs activity

To analyze the in vivo effect of the antibodies on tumors, SCID/beige mice (that are deficient for T, B and NK cells and therefore accept human tumor xenografts), are injected with various CEACAM1 -positive tumors such as melanoma, lung, colorectal, and bladder cancer. After tumor establishment, the mice are injected with the anti- CEACAM1 mAbs of the invention and with a negative control antibody.

Example 8. Anti-CEACAMl mAbs support the anti-cancer activity of immune cells expressing CEACAM1 in vivo

To determine the effect of anti-CEACAMl mAbs on immune cells expressing CEACAM1 in vivo, B16-F10 murine melanoma cells were injected subcutaneously in the right flank of 6-7 weeks male transgenic mice expressing human but not murine CEACAM1 (hCEACAMl+/+ x msCEACAMl-/-). The mice were treated immediately after cell inoculation and then 3 times a week with intraperitoneal injections of either anti-human CEACAM1 or anti-murine CEACAM1 mAbs or with their isotype control IgGl (lOOpg/mice). The tumor growth was monitored by measuring its size using a caliper for 16 days until reaching the size of 15mm³. Mice were then sacrificed, and tumor weight was measured. Spleen from mice bearing tumors were extracted, washed in PBS and meshed through 70pm filter. Then, total cells number were counted and stained using standard flow cytometry staining protocol. T cells were stained using FITC conjugated anti-CD3 monoclonal antibody.

The results shown in Figures 10B and 10C represent a decrease in tumor growth and tumor weight, respectively, by anti-human CEACAM1 mAbs, indicating that these antibodies may support the immune cells to fight against cancer. Moreover, spleenocytes analysis by flow cytometry (Figure 10D) showed an increase in CD3⁺ cells, demonstrating that anti-human CEACAM1 mAb increase T cells number.

Therefore, besides its direct anti-tumor effect (by activating CEACAM1 on melanoma cells), anti-human CEACAM1 mAb also act on the immune level by increasing the immune cell number and contributing to a stronger anti-tumor immunity

Example 9. Determining the binding site of anti-CEACAMl mAb

CEACAM1 extracellular domain contains immunoglobulin variable-region-like (IgV- like) domain at the N terminus of the protein and 1 to 3 immunoglobulin constant-region-type- 2-like (IgC2-like) domains. To determine whether the anti-CEACAMl mAb of the present invention bind the N-terminal domain or another binding site of the CEACAM1 molecule, the mAbs was compared to 2 different known anti-CEACAMl mAbs, LC-SAB3 that binds to the N-terminal domain of CEACAM1, and F1-C5 that binds another site of the molecule. For this purpose, a standard ELISA protocol was performed. The mAbs were incubated on ELISA plate coated with CEACAM1 bearing (CCl-Fc) or lacking (CCldN-Fc) the N-terminal domain, followed by incubation with HRP. Binding was detected by plate reader at OD450nm.

The results shown in Figure 11 indicate that the anti-CECAMl CCM5.01, similarly to F1-C5 antibody, do not bind the N-terminal domain of the CEACAM1 molecule.

Example 10. Staining of CEACAM1 on human eosinophils

To evaluate whether CEACAM1 is upregulated in viral infection, its expression on eosinophils from healthy donor and 4 Covid- 19 patients was determined by using standard flowcytometry staining protocol. Eosinophils were isolated using anti-CCR3-APC antibody and stained by the anti-CEACAMl mAb CCM5.01 followed by FITC-anti-mouse IgG.

The results shown in Figure 12A and 12B display shift in the curve to the right with the CCM5.01 mAb indicating higher CEACAM1 cell surface expression on eosinophils in the 4 Covid-19 patients tested in comparison to healthy donor. It is concluded that CEACAM1 expression is upregulation followed the SARS-CoV-2 viral infection and that the mAbs of the present invention are capable of bind to this protein expressed in human eosinophils.

Example 11. Sequencing of CCM5.01 antibody

Total RNA was isolated from the hybridoma cells following the technical manual of TRIzol® Reagent. Total RNA was then reverse-transcribed into cDNA using either isotypespecific anti-sense primers or universal primers following the technical manual of PrimeScriptTM 1^st Strand cDNA Synthesis Kit. Antibody fragments of heavy chain and light chain were amplified according to the standard operating procedure (SOP) of rapid amplification of cDNA ends (RACE) of GenScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. The obtained sequences of CCM5.01 monoclonal antibody are:

Heavy chain (SEQ ID NO: 1) - amino acids

DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYITYSGS

TSYNPSLKSRISISRDTSKNQFFLQLNSVTSEDTATYYCARENRYDGEMFFDVWGAG TTVTVSS

Heavy chain (SEQ ID NO: 2) - nucleic acids

GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGT

CCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTG

GATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAACCTACAG

TGGTAGCACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCAGTCGAGAC

ACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTAGTGAAGACACAG

CCACATATTACTGTGCAAGAGAAAATAGGTACGACGGAGAAATGTTCTTCGATG

TCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

Light chain (SEQ ID NO: 3) - amino acids

DIVLTQSPVTLSVTPGDSVSLSCRASQSISNDLHWYHQKSHESPRLLIKYAYQSISGIPS

RFSGTGSGTDFTLSINSVETEDFGMYFCQQSNSWPHTFGGGTKLEIK

Light chain (SEQ ID NO: 4) - nucleic acids

GATATTGTGCTAACTCAGTCTCCAGTCACCCTGTCTGTGACTCCAGGAGATAGCG

TCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACGACCTACACTGGTATCA

CCAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTACCAGTCCATC

TCTGGGATCCCCTCCAGGTTCAGTGGCACTGGATCAGGGACAGATTTCACTCTCA

GTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAA

CAGCTGGCCTCACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

HC CDR1 (SEQ ID NO: 5) - SDYAWN

HC CDR2 (SEQ ID NO: 6) - YITYSGSTSYNPSLKS

HC CDR3 (SEQ ID NO: 7) - ENRYDGEMFFDV

LC CDR1 (SEQ ID NO: 8) - RASQSISNDLH LC CDR2 (SEQ ID NO: 9) YAYQSIS

LC CDR3 (SEQ ID NO: 10) - QQSNSWPHT

Example 12. CCM5.01 and CCM5.19 mAbs do not bind to the N domain of human CEACAM1.

Human CEACAMl-Fc and CEACAMldN-Fc lacking the N domain, respectively, were coated to an ELISA plate (NUNC maxisorp). After blocking remaining binding sites with BSA, indicated CCl/3/5-Sab (as positive control), CCM5.01, CCM5.19, F1-C5 and isotype matched control mAh were added and incubated for 4 h at RT. For detection, the HRP-coupled goat anti mouse pAb was utilized. Subsequently, TMB substrate was added, and the enzymatic reaction was stopped by sulfuric acid and measured in a Tecan Sunrise ELISA plate reader at 450 nm. Data were run in triplicates and are representative of three independent experiments. As demonstrated in Figure 13, the antibodies of the present invention, CCM5.01, CCM5.19, and F1-C5 do not bind the extracellular N-terminal domain of CEACAM1.

Example 13. CCM5.01 and CCM5.19 mAbs bind to human CEACAM1, CEACAM5 and CEACAM8.

Antibodies CCM5.01, CCM5.19 and respective positive control antibodies were incubated with CHO, CHO-mouse CEACAM1, CHO-human CEACAM1, CHO-human CEACAM3, CHO-human CEACAM5, CHO-human CEACAM6, CHO-human CEACAM7, CHO-human CEACAM8 transfectants. After washing, FITC-labeled secondary goat anti mouse antibody was applied. Cell surface binding was analyzed utilizing a FACScalibur flow cytometer (Becton Dickinson) and analyzed by CellQuestPro (Becton Dickinson). Data shown represent the median values of one out of three different, representative experiments. As shown in Figure 14, CCM5.01, CCM5.19 that bind CEACAM1, cross-react with CEACAM5 and CEACAM8 but do not bind CEACAM3, CEACAM6 and CEACAM7. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

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Claims

1. An antibody, or an antibody fragment thereof comprising at least the antigen binding portion, which specifically binds to CEACAM1, said antibody or fragment thereof comprising three complementarity determining regions (CDRs) of a heavy-chain (HC) variable region comprising SEQ ID NO: 1 and three CDRs of a light-chain (LC) variable region comprising SEQ ID NO: 3.

2. The antibody or the antibody fragment according to claim 1, comprising a set of six CDRs wherein heavy chain CDR1 sequence comprising the sequence SDYAWN (SEQ ID NO: 5), heavy chain CDR2 comprising the sequence YITYSGSTSYNPSLKS (SEQ ID NO: 6), heavy chain CDR3 comprising the sequence ENRYDGEMFFDV (SEQ ID NO: 7), light chain CDR1 comprising the sequence RASQSISNDLH (SEQ ID NO: 8), light chain CDR2 comprising the sequence YAYQSIS (SEQ ID NO: 9), and light chain CDR3 comprising the sequence QQSNSWPHT (SEQ ID NO: 10), or analogs thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the hypervariable region (HVR) sequence.

3. The antibody or the antibody fragment according to any one of claims 1 to 2, comprising a heavy chain variable sequence set forth in SEQ ID NO: 1, or an analog having at least 95% sequence similarity with said heavy chain variable region sequence.

4. The antibody or the antibody fragment according to any one of claims 1 to 3, comprising a light chain variable sequence set forth in SEQ ID NO: 3, or an analog having at least 95% sequence similarity with said light chain variable region sequence.

5. The antibody or the antibody fragment according to any one of claims 1 to 4, comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO: 1 and the light chain comprises SEQ ID NO: 3.

6. A variant of an antibody or antibody fragment according to claim 5, having at least 95% identity with said antibody light or heavy chain.

7. The antibody or antibody fragment according to any one of claims 1 to 6, wherein the antibody binds to CEACAM1 with an affinity of 10^-8M to 10^-10M.

8. The antibody or antibody fragment according to any one of the preceding claims, wherein the antibody or fragment thereof does not block CEACAM1 homophilic interactions.

9. A polynucleotide sequence encoding at least one region of a HC or a LC sequence of an antibody or antibody fragment according to any one of claims 1 to 8.

10. The polynucleotide sequence of claim 9, encoding an antibody heavy chain variable region, wherein the polynucleotide sequence comprises a sequence set forth in SEQ ID NO: 2, or a variant thereof having at least 90% identity to said sequences.

11. The polynucleotide sequence of claim 9, encoding an antibody light chain variable region, wherein the polynucleotide sequence comprises a sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 90% identity to said sequences.

12. A plasmid comprising at least one polynucleotide sequence according to any one of claims 9 to 11.

13. A cell comprising a polynucleotide sequence according to any one of claims 9 to 11.

14. A cell capable of producing an antibody according to any one of claims 1 to 7.

15. A pharmaceutical composition comprising as an active ingredient, at least one antibody or fragment thereof, according to any one of claims 1 to 7, and a pharmaceutical acceptable excipient, diluent, salt or carrier.

16. The pharmaceutical composition of claim 15 for use in treating cancer.

17. The pharmaceutical composition of claim 15 for use in preventing or treating a viral infection.

18. The pharmaceutical composition for use of claim 17, wherein the viral infection is caused by a virus selected from the group consisting of viruses of the corona family, influenza, cytomegalovirus (CMV), human immune deficiency virus (HIV), herpes virus and hepatitis virus.

19. The pharmaceutical composition for use of claim 18, wherein the virus is SARS-CoV-2. 0. A method of treating cancer, comprising administering to a subject in need thereof, a pharmaceutical composition according to claim 15. 1. The method of claim 20, further comprising an additional anti-cancer therapy selected from surgery, chemotherapy, radiotherapy, and immunotherapy. 2. The method of claim 20, further comprising administering to said subject an additional immuno-modulator, activated lymphocyte cell, kinase inhibitor, chemotherapeutic agent or any other anti-cancer agent. 3. The method of claim 22, wherein the additional immune-modulator is an antibody against an immune checkpoint molecule. 4. The method of claim 20 wherein the cancer is selected from the group consisting of a melanoma, a bladder cancer, a breast cancer, a prostate cancer, a non-small cell lung carcinoma (NSCLC), a gastric cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, a colon cancer, a cervical cancer, a kidney cancer, a lung cancer, a thyroid cancer, a brain cancer, a renal cancer, a throat cancer, a laryngeal carcinoma, a hepatic cancer, a fibrosarcoma, an endometrial cells cancer, a glioblastoma, sarcoma, a myeloid, a leukemia and a lymphoma.

25. The method of claim 20, wherein the cancer is melanoma. 26. The method of claim 20, wherein the cancer is a solid cancer.

27. The method of claim 20, wherein treating results in preventing or reducing metastases formation, growth or spread in a subject.

28. A method of preventing or treating a viral infection, comprising administering to a subject in need thereof, a pharmaceutical composition according to claim 15. 29. The method of claim 28, wherein the viral infection is caused by a virus selected from the group consisting of: viruses of the corona family and in particular SARS-CoV-2, influenza, cytomegalovirus (CMV), human immune deficiency virus (HIV), herpes virus and hepatitis virus.

30. A kit for diagnosing a cancer in a subject comprising at least one antibody or antibody fragment according to any one of claims 1 to 7.