Anticancer embryonic antigen-related cell adhesion molecule 1 (CEACAM1) antibodies that inhibit neutrophil extracellular trap (NET)-mediated activity

Abstract

Pharmaceutical compositions comprising anti-CEACAM1mAb and their use in the inhibition of NET-mediated activity and in the prevention and treatment of pathologies associated with these activities are provided, and are exemplified by mAb CM24 that also exhibit effective inhibition of cancer cell migration and NET-induced platelet aggregation. Prognostic methods based on NET-biomarkers for use in patient selection and monitoring of therapeutic efficacy are also presented.

Description

Field of the Invention

[0001] The present invention belongs to the fields of immunotherapy and cell biology and relates to CEACAM1 - targeting antibodies for the prevention and treatment of processes and disorders involving neutrophil extracellular trap (NETs) activity. Background of the Invention

[0003] Neutrophils are the most abundant circulating white blood cells in humans and are an essential component of the host response to pathogens. During infection, neutrophils migrate from the peripheral blood to tissues in response to several chemotactic stimuli released within the inflamed site. They can rapidly kill pathogens after phagocytosis, but can also rapidly kill pathogens by releasing their potent antimicrobial arsenal, which includes granzyme and proteins, oxidants (reactive oxygen species: ROS), and neutrophil extracellular traps (NETs). Neutrophils also infiltrate tumors and are considered key mediators of neoplastic transformation, tumor progression, angiogenesis, and regulation of the immune response.

[0004] Cancer - associated inflammation is a driver of tumor initiation and progression, enabling cancer cells to evade immune surveillance. Infiltrating and resident immune cells in the tumor microenvironment (TME) play a fundamental role in tumor growth, metastasis formation, and response to immunotherapy. Growing evidence indicates that circulating and infiltrating neutrophils also play multiple roles in tumor initiation and progression.

[0005] NETs are extracellular decondensed chromatin networks that can include granule proteins, DNA, histones, and other substances. NETs are produced by neutrophils to entrap and kill pathogens and can form during infection, inflammation, and / or thrombosis. More specifically, when neutrophils detect a pathogen, granule proteins, DNA, and / or histones can combine within the neutrophil. The neutrophil can then eject the combined granule proteins, DNA, and / or histones through the disintegration of the nuclear and granule membranes, allowing the intracellular material to "pop out" of the cell to form NETs. NETs can then capture, bind, entrap, and / or kill pathogens in a process called NETosis. Because they are derived from neutrophils and because they play a role in combating infection, NETs are typically present extravascularly in inflamed or infected tissues.

[0006] A variety of markers have been tested to show NETosis. Methods for evaluating NETosis include detecting co-localized neutrophil-derived proteins and extracellular DNA and citrullinated histones, as well as detecting NET remnants in fluid samples and flow cytometry for detecting cell-associated NET components. NET markers include: myeloperoxidase (MPO), neutrophil elastase (NE), peptidylarginine deiminase 4 (PAD4), citrullinated histone H3 (Cit-H3), and cell-free DNA. Regarding the specificity, objectivity, and quantification of NET markers, the recognized NET markers used in clinical studies are the MPO / NE DNA complex (Mi-Hyun et al., J. Rheumatol. 2019 Dec; 46(12):1560-1569 and Sakiko et al., Clin Chim Acta. 2016 Aug 1; 459:89-93), CitH3 together with cell-free DNA (Pranav et al., Front Immunol. 2019 Jan; 24:10:28).

[0007] NETs are also involved in cancer immunoediting, progression, metastatic spread, and play a key role in the TME. NETs enhance tumor invasiveness by enhancing cancer migration and invasion capabilities and can capture circulating cancer cells to promote the formation and spread of metastases.

[0008] Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as cluster of differentiation 66a (CD66a), is a member of the carcinoembryonic antigen (CEA) gene family and belongs to the immunoglobulin (Ig) superfamily. CEACAM1 is an immune checkpoint protein that is upregulated in T cells and NK cells upon activation, and its homophilic interaction results in the inhibition of the cytotoxic effects of lymphocytes. Studies in several human tumor types have suggested that the utilization of the CEACAM1 pathway can allow for tumor immune evasion. CEACAM1 plays important roles in tumor immune evasion, metastasis, and angiogenesis, and its expression on primary cutaneous melanoma lesions strongly predicts the development of metastatic disease with poor prognosis. In addition, increased CEACAM1 expression has been observed on NK cells derived from some patients with metastatic melanoma compared to healthy donors. Preclinical animal models of tumors have shown that blockade of CEACAM1 interactions by monoclonal antibodies (mAbs) can enhance the immune response against tumors. CEACAM1 has also been suggested as a putative therapeutic target for preventing the metastatic progression of colorectal epithelial cancer (Rayes et al., Immunol. 2020 April 15; 204(8):2285-2294).

[0009] CEACAM1 is associated with angiogenesis and is an intercellular adhesion regulator of Fas-mediated apoptosis. Fas enhances the cytotoxicity of natural killer cells against tumor cells via interaction with β-catenin. High CEACAM1 expression is known to be associated with poor disease prognosis in many tumor types. CEACAM1 and the CEACAM1-CEACAM5 pathway prevent tumor cell death by inhibiting the immune activity of tumor-infiltrating lymphocytes (TILs), reducing the phosphorylation of immune receptors, and decreasing the phosphorylation levels of SHP1 / 2 in T cells and NK cells.

[0010] WO 2013 / 054331 discloses mAbs specific for human CEACAM1, which mAbs comprise a specific set of complementarity-determining regions (CDRs). Chimeric antibodies, including CM10 which is a human / mouse antibody, are also disclosed.

[0011] WO2015166484 discloses humanized anti-CEACAM1 mAbs which have a specific set of CDR sequences and several back mutations in a human framework. Among the claimed mAbs is the potent mAb designated CM24.

[0012] CM24 is a humanized IgG4 mAb which specifically and with high affinity binds to the extracellular domain of CEACAM1. CM24 is a first-in-class mAb in clinical-stage for targeting CEACAM1 and has significant potential for treating a variety of cancers. Blocking the interactions of CEACAM1-CEACAM1 and CEACAM1-CEACAM5 with CM24 is associated with anti-angiogenesis, immune entry, and checkpoint release mechanisms and enables the cytotoxic activity of lymphocytes and the killing of tumor cells by T cells and NK cells.

[0013] CM24 is currently undergoing an open-label, multi-center, multi-dose escalation and dose expansion trial (https: / / clinicaltrials.gov / ct2 / show / NCT04731467) in combination with nivolumab (anti-PD-1) in adults with selected advanced solid tumors (e.g., advanced recurrent refractory non-small cell lung cancer and metastatic pancreatic cancer).

[0014] There is an unmet need for effective agents that can inhibit NET-mediated activities for the prevention and treatment of a wide range of diseases and disorders associated with NET-mediated activities. SUMMARY OF THE INVENTION

[0016] The present invention provides a pharmaceutical composition comprising a humanized anti-CEACAM1 mAb CM24, or an mAb comprising the same set of CDR sequences, and their use in inhibiting NET-mediated activities and in the prevention and treatment of pathologies associated with these activities.

[0017] Since NET-mediated activities are involved in a wide range of pathologies, including cardiovascular, hematological, autoimmune, neoplastic and inflammatory diseases, anti-CEACAM1 mAbs and especially CM24 can be effectively used to inhibit or delay the NET-induced processes of these pathologies.

[0018] The present invention is based on the favorable results of using CM24 to block several NET-mediated activities. In the dose escalation part of a recent Phase 1 / 2 study of CM24 and nivolumab (NCT 04731467) and in exploratory studies conducted as part of this trial, it was unexpectedly found that in patients with pancreatic cancer, CM24 therapy led to a significant decrease in serum NETs levels, and this decrease was significant (persistent effect) for at least two weeks after CM24 / nivolumab treatment. Further advantageously, it was found that the pre-treatment serum levels of the NET marker myeloperoxidase (MPO) increased in patients showing stable disease (SD) or partial response (PR) and surviving longer, while they did not increase in patients showing complete non-response (progressive disease, PD), suggesting serum MPO as a biomarker for patient selection for CM24 therapy. In part C2 of the study, a significant and persistent decrease in serum MPO was detected in patients showing SD or PR, while this was not the case in patients with complete non-response (PD). Thus, for the first time, it is disclosed that MPO can be used as a prognostic biomarker for selecting patients suitable for treatment with anti-CEACAM1 antibodies and for monitoring the effectiveness of treatment with such antibodies.

[0019] In cancer, blocking, inhibiting or delaying these NET-mediated activities will result in blocking, inhibiting or delaying extravasation into the vasculature, hindering the survival of cancer cells in the bloodstream, extravasation into the organ parenchyma and the formation of dormant cells or multicellular micrometastases and macrometastases.

[0020] This article first shows that CM24 effectively inhibits cancer cell migration in vivo and suppresses metastatic activity, thereby reducing the progression of the disease. The present invention provides a method for blocking the metastasis cascade promoted by NETosis using CM24, an mAb containing the same set of CDR sequences, or an antibody fragment containing at least the CM24 binding site, thus effectively blocking the metastasis cascade and preventing the seeding, spread, and subsequent exponential growth of distant metastasis colonies. The present invention is advantageous because treating and even in some cases preventing pathological conditions such as cancer and NET-related thrombotic diseases and disorders using immunological methods, rather than relying on cytotoxic therapies, greatly reduces the harmful side effects associated with such treatments.

[0021] The present invention also provides a method for selecting a subject diagnosed with cancer for treatment with an anti-CEACAM1 mAb or its fragment or conjugate, or for monitoring the effectiveness of treatment, wherein the level of a NET marker (e.g., myeloperoxidase (MPO)) is used to optimize patient selection and monitor treatment efficacy.

[0022] Thus, according to one aspect, the present invention provides a method for preventing or inhibiting NET-mediated activity, comprising using an mAb containing a set of six CDR sequences or an active fragment or conjugate thereof, wherein the heavy chain CDR1 (HC-CDR1) comprises the sequence GYAFTNNLIE (SEQ ID NO:1), the heavy chain CDR2 (HC-CDR2) comprises the sequence VINPGSGDTNYNEKFKG (SEQ ID NO:2), the heavy chain CDR3 (HC-CDR3) comprises the sequence GDYYGGFAVDY (SEQ ID NO:3), the light chain CDR1 (LC-CDR1) comprises the sequence RTSQDIGNYLN (SEQ ID NO:4), the light chain CDR2 (LC-CDR2) comprises the sequence YTSRLHS (SEQ ID NO:5), and the light chain CDR3 (LC-CDR3) comprises the sequence QQGKSLPRT (SEQ ID NO:6).

[0023] According to some embodiments, the mAb or its fragment contains a set of six CDR sequences, wherein HC-CDR1 consists of GYAFTNNLIE (SEQ ID NO:1), HC-CDR2 consists of VINPGSGDTNYNEKFKG (SEQ ID NO:2), HC-CDR3 consists of GDYYGGFAVDY (SEQ ID NO:3), LC-CDR1 consists of RTSQDIGNYLN (SEQ ID NO:4), LC-CDR2 consists of YTSRLHS (SEQ ID NO:5), and LC-CDR3 consists of QQGKSLPRT (SEQ ID NO:6).

[0024] According to another aspect, the present invention also provides a method for preventing, inhibiting or delaying a pathological process or condition involving NET-mediated activity, comprising administering to a subject in need thereof an mAb or an active fragment thereof, said mAb or active fragment thereof comprising a set of six CDR sequences, wherein HC-CDR1 consists of GYAFTNNLIE (SEQ ID NO:1), HC-CDR2 consists of VINPGSGDTNYNEKFKG (SEQ ID NO:2), HC-CDR3 consists of GDYYGGFAVDY (SEQ ID NO:3), LC-CDR1 consists of RTSQDIGNYLN (SEQ ID NO:4), LC-CDR2 consists of YTSRLHS (SEQ ID NO:5), and LC-CDR3 consists of QQGKSLPRT (SEQ ID NO:6).

[0025] According to another aspect, the present invention also provides a pharmaceutical composition comprising an mAb or an active fragment thereof and a pharmaceutically acceptable salt, carrier or diluent, said mAb or active fragment thereof comprising a set of six CDR sequences, wherein HC-CDR1 consists of GYAFTNNLIE (SEQ ID NO:1), HC-CDR2 consists of VINPGSGDTNYNEKFKG (SEQ ID NO:2), HC-CDR3 consists of GDYYGGFAVDY (SEQ ID NO:3), LC-CDR1 consists of RTSQDIGNYLN (SEQ ID NO:4), LC-CDR2 consists of YTSRLHS (SEQ ID NO:5), and LC-CDR3 consists of QQGKSLPRT (SEQ ID NO:6), said pharmaceutical composition being for preventing or delaying NET-mediated activity, and for preventing, inhibiting or delaying a pathological process or disorder involving NET-mediated activity.

[0026] In some embodiments, the anti-CAECAM1 antibody is a chimeric antibody. In other embodiments, the anti-CEACAM1 antibody is a humanized antibody or a partially humanized antibody.

[0027] In other embodiments, the anti-CEACAM1 antibody comprises a heavy chain variable region containing the sequence QVQLVQSGAEVKKPGASVKVSCKASGYAFTNNLIEWVRQAPGQGLEWIGVINPGSGDTNYNEKFKGRVTMTADKSISTAYMELSRLRSDDTAVYYCARGDYYGGFAVDYWGQGTTVTVSS (SEQ ID NO:7), and a light chain variable region containing the sequence DIQMTQSPSSLSASVGDRVTITCRTSQDIGNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQGKSLPRTFGGGTKVEIK (SEQ ID NO:8), or an active fragment thereof containing at least the binding site, or an antibody analogue or derivative having at least 90% identity with any one of the chain sequences.

[0028] In some embodiments, the heavy chain variable region of the antibody comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least about 95% identical to SEQ ID NO:8. In some embodiments, the heavy chain variable region of the antibody comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least about 97% identical to SEQ ID NO:8. In some embodiments, the heavy chain variable region of the antibody comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence that is at least about 99% identical to SEQ ID NO:8. Each selection represents a separate embodiment of the invention.

[0029] In some embodiments, the antibody or fragment thereof is an IgG mAb. In some embodiments, the anti-CEACAM1 mAb has a heavy chain constant region selected from IgG4, IgG1, and IgG2. In other embodiments, the antibody comprises a human IgG constant region selected from IgG1 and IgG4. In certain embodiments, the humanized antibody or fragment thereof is of the IgG4 subclass. In certain embodiments, the humanized antibody or antigen-binding fragment thereof is of the IgG1 subclass. In some embodiments, the anti-CEACAM1 antibody comprises a human κ light chain constant region. Each selection represents a separate embodiment of the invention.

[0030] In an exemplary embodiment, the anti-CEACAM1 antibody is CM24, which comprises the heavy chain sequence QVQLVQSGAEVKKPGASVKVSCKASGYAFTNNLIEWVRQAPGQGLEWIGVINPGSGDTNYNEKFKGRVTMTADKSISTAYMELSRLRSDDTAVYYCARGDYYGGFAVDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDVEVHNAKTKPREEQF N STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:9), and the light chain sequence DIQMTQSPSSLSASVGDRVTITCRTSQDIGNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQGKSLPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:10), or an active fragment thereof comprising at least the binding site, or an antibody analogue or derivative having at least 90% identity with any of the said chain sequences.

[0031] The antibodies of the methods and compositions of the present invention also include conjugates comprising the antibody or fragments thereof. According to some embodiments, the conjugate may comprise an antibody or fragment thereof attached to a cytotoxic moiety, a radioactive moiety, or an affinity or labeling tag.

[0032] Any disease or disorder in which NETs are highly involved in its pathogenesis or progression and / or in which inhibition of NETs activity will result in symptom improvement, delay, etc. is suitable for treatment with the mAbs of the present invention.

[0033] In some embodiments, the subject in need of treatment has been diagnosed with a neoplastic disease, i.e., has cancer. In some embodiments, the neoplastic disease is a solid tumor. According to some embodiments, the cancer is metastatic cancer or a tumor. In some embodiments, the neoplastic disease is selected from the group consisting of: epithelial cancer, lymphoma, blastoma, sarcoma, melanoma, cancer of unknown primary, skin cancer, lung cancer, thyroid cancer, parathyroid cancer, breast cancer, heart cancer, thymic cancer, bone cancer, soft tissue cancer, brain cancer, retinal cancer, ophthalmic cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, pancreatic cancer, biliary cancer, liver cancer, bladder cancer, adrenal cancer, kidney cancer, urogenital cancer, testicular cancer, cervical cancer, fallopian tube cancer, ovarian cancer, uterine cancer, vulvar cancer or endometrial cancer. In some embodiments, the neoplastic disease is a hematological cancer. In some embodiments, the hematological cancer is selected from lymphoma, leukemia, myelodysplastic syndrome, myeloproliferative disorder and myeloma. Each selection represents a separate embodiment of the invention.

[0034] According to some embodiments, the cancer is selected from the group consisting of: pancreatic cancer, lung cancer and melanoma.

[0035] According to some embodiments, treatment with the anti-CEACAM1 antibody or antibody fragment of the invention results in the prevention, inhibition or delay of at least one of the following: the formation of metastases, the migration or spread of metastases, the adhesion of metastases, the intravasation of cancerous cells into the vasculature, the survival of cancer cells in the bloodstream, the extravasation of cancer cells into the organ parenchyma, and the formation of dormant cell or multicellular metastases.

[0036] In a further aspect, the invention provides a method of preventing, delaying or inhibiting the formation, migration, spread, adhesion or progression of metastases, comprising administering an mAb against CEACAM1 as defined above.

[0037] In some embodiments, the formation, migration or spread of metastases is prevented or inhibited after tumor resection surgery.

[0038] In some embodiments, the patient undergoing surgery has been treated with an additional anti-cancer therapy selected from the group consisting of chemotherapy, radiation and immunotherapy.

[0039] The invention also provides an anti-CEACAM1 mAb for use in treating NET-mediated disorders or complications in a subject in need of such treatment, the method comprising:

[0040] (i) determining the level of at least one NET biomarker in a biological sample obtained from the subject;

[0041] (ii) Compare the level of said at least one NET biomarker with a reference value or a control sample value; and

[0042] (iii) If the level of the NET biomarker in the sample is significantly higher than the reference value or the control sample value, administer an anti-CEACAM1 antibody to the subject.

[0043] According to some embodiments, the subject is a patient diagnosed with or suspected of having cancer.

[0044] According to some embodiments, the subject is diagnosed with or suspected of having a non-malignant NET-related disease, disorder or complication, including treatment-induced complications.

[0045] According to some embodiments, the levels of at least two NET biomarkers are measured.

[0046] Any biomarker known in the art for detecting NETosis can be used in the methods of the invention. According to some embodiments, detecting NETosis includes detecting co-localized neutrophil-derived proteins and extracellular DNA and citrullinated histones, detecting NET remnants in fluid samples, and flow cytometry for detecting cell-attached NET components. NET biomarkers include, but are not limited to: myeloperoxidase (MPO), neutrophil elastase (NE), peptidylarginine deiminase 4 (PAD4), citrullinated histone H3 (Cit-H3), and cell-free DNA. According to some embodiments, at least one biomarker is selected from MPO, NE, and DNA complexes.

[0047] According to some embodiments, at least one NET biomarker is selected from the group consisting of myeloperoxidase (MPO), neutrophil elastase (NE), peptidylarginine deiminase 4 (PAD4), citrullinated histone H3 (Cit-H3), and cell-free DNA. According to some embodiments, at least one NET biomarker is selected from MPO, NE, and DNA complexes. According to certain embodiments, the NET biomarker is MPO. Each selection represents a separate embodiment of the invention.

[0048] According to some specific embodiments, the NET biomarker is MPO.

[0049] Thus, according to some embodiments, the invention provides an anti-CEACAM1 mAb for use in treating cancer in a subject in need of such treatment, the method comprising:

[0050] (i) Determining the level of myeloperoxidase (MPO) in a biological sample obtained from a subject diagnosed with cancer;

[0051] (ii) Compare the MPO level with a reference value or a control sample value; and

[0052] (iii) If the MPO level in the sample is significantly higher than the reference value or the control sample value, administer an anti-CEACAM1 antibody to the subject.

[0053] In some embodiments, a significantly higher MPO level corresponds to an increase of at least about 100%, at least about 200%, or at least about 300% relative to a reference value or a control sample value.

[0054] In another aspect, the present invention provides a method of treating cancer in a subject in need of such treatment, comprising:

[0055] (i) Determining the level of MPO in a biological sample obtained from a subject diagnosed with cancer;

[0056] (ii) Comparing the MPO level with a reference value or a control sample value; and

[0057] (iii) If the MPO level in the sample is significantly higher than the reference value or the control sample value, administer anti-CEACAM1 to the subject.

[0058] In some embodiments, the biological sample is a blood sample. In still other embodiments, the blood sample is selected from whole blood, serum, and plasma. In other embodiments, the biological sample obtained from the subject is a biopsy, such as a tissue or a liquid biopsy or particularly a tumor biopsy.

[0059] The present invention also provides a method of selecting a subject suitable for treatment with an anti-CEACAM1 antibody, comprising the steps of: (i) providing a biological sample from the subject; (ii) determining the level of at least one NET biomarker in the sample of step (i), and (iii) comparing the level of the at least one NET biomarker with a reference value or a control sample value, wherein a significant increase in the level of the NET biomarker relative to the reference value or the control sample value indicates that the subject is likely to have a therapeutic response to the anti-CEACAM1 antibody.

[0060] According to some embodiments, the subject is diagnosed with cancer or suspected of having cancer.

[0061] According to some embodiments, the subject is diagnosed with or suspected of having a non-malignant NET-related disease, disorder, or complication, including treatment-induced complications.

[0062] According to some embodiments, at least one NET marker is selected from the group consisting of myeloperoxidase (MPO), neutrophil elastase (NE), peptidylarginine deiminase 4 (PAD4), citrullinated histone H3 (Cit-H3), and cell-free DNA. According to some embodiments, at least one NET marker is selected from MPO, NE, and DNA complexes. According to certain embodiments, the NET marker is MPO. Each of these selections represents a separate embodiment of the invention.

[0063] According to some embodiments, the levels of at least two NET markers are measured.

[0064] According to some specific embodiments, the NET marker is MPO, and the invention provides a method of selecting a cancer subject suitable for treatment with an anti-CEACAM1 antibody, comprising the steps of: (i) providing a biological sample from the subject; (ii) determining the level of MPO in the sample of step (i), and (iii) comparing the MPO level with a reference value or a control sample value, wherein a significant increase in the MPO level relative to the reference value or the control sample value indicates that the subject may be therapeutically responsive to the anti-CEACAM1 antibody.

[0065] In some embodiments, an increase in the MPO level relative to a reference value or a control sample value corresponds to an increase of at least about 100%. In other embodiments, the increase corresponds to at least about 200%. In still other embodiments, the increase corresponds to at least about 300%. In still more embodiments, an increase in the MPO level identified in a patient characterizes the patient as expected to develop a severe form of cancer.

[0066] In some embodiments, the anti-CEACAM1 mAb or antibody fragment comprises a set of CDR sequences consisting of: SEQ ID No. 1-6. In other embodiments, the anti-CEACAM1 mAb is CM24.

[0067] In some embodiments, the subject selected for treatment has been diagnosed with solid tumor cancer. In other embodiments, the solid tumor cancer is selected from pancreatic cancer, lung cancer, and melanoma cancer. In other embodiments, the patient receives a combination of anti-CEACAM1 mAb therapy and at least one other anti-cancer treatment (such as chemotherapy). In certain embodiments, the patient receives a combination of anti-CEACAM1 mAb therapy and anti-PD-1 antibody therapy.

[0068] In another aspect, a method of inhibiting therapy-induced thrombosis is provided, comprising administering an anti-CEACAM1 mAb or a fragment thereof, the anti-CEACAM1 mAb or fragment thereof comprising a set of six CDR sequences, wherein heavy chain CDR1 (HC-CDR1) comprises the sequence GYAFTNNLIE (SEQ ID NO:1), heavy chain CDR2 (HC-CDR2) comprises the sequence VINPGSGDTNYNEKFKG (SEQ ID NO:2), heavy chain CDR3 (HC-CDR3) comprises the sequence GDYYGGFAVDY (SEQ ID NO:3), light chain CDR1 (LC-CDR1) comprises the sequence RTSQDIGNYLN (SEQ ID NO:4), light chain CDR2 (LC-CDR2) comprises the sequence YTSRLHS (SEQ ID NO:5), and light chain CDR3 (LC-CDR3) comprises the sequence QQGKSLPRT (SEQ ID NO:6). According to some embodiments, the mAb is CM24. According to some embodiments, the therapy that induces thrombosis is selected from immunotherapy, surgery, radiation, hormone therapy, and chemotherapy.

[0069] In a further aspect, a method for administering an anti-CEACAM1 antibody to inhibit cancer cell invasion into the vasculature is provided.

[0070] In a further aspect, a method for administering an anti-CEACAM1 antibody treatment to inhibit cancer cell invasion into the surrounding extracellular matrix is provided.

[0071] In a further aspect, a method for administering an anti-CEACAM1 antibody treatment to inhibit cancer cell extravasation into the organ parenchyma is provided.

[0072] In a further aspect, a method for administering an anti-CEACAM1 antibody treatment to prevent seeding and exponential growth of distant metastasis colonies is provided.

[0073] Any route of administration suitable for delivering a protein or antibody can be used with the compositions and methods of the present invention, and the administered compositions are formulated according to the mode of administration. According to some embodiments, the mAb is administered parenterally. According to some embodiments, the mAb is administered via a route selected from: intravenous, intramuscular, subcutaneous, intratumoral, intradermal, intraarterial, intraarticular, intralesional, or submucosal, intranasal, oral, and topical.

[0074] Generally, intravenous (i.v.) administration by infusion or injection is used. In other embodiments, the anti-CEACAM1 composition is administered via the intratumoral route. In other embodiments, the composition is administered during or after surgery.

[0075] A method of treating cancer or non-cancerous NET-related diseases or disorders according to the present invention includes, according to some embodiments, administering to a subject in need thereof at least one dose of the above-described mAb against CEACAM1, in a range of 0.01 mg / kg to 50 mg / kg body weight.

[0076] According to some embodiments, the at least one dose is selected from the group consisting of: 0.01 - 0.1 mg / kg; 0.1 - 1 mg / kg; 1 - 10 mg / kg; and 10 - 50 mg / kg.

[0077] According to some embodiments, the method includes administering multiple doses of the mAb, wherein the multiple doses are the same or different. According to some embodiments, the method includes administering multiple escalating doses. According to some embodiments, the method includes administering at least one cycle for at least 12 weeks.

[0078] According to some embodiments, the treatment duration is 2 - 60 weeks. According to other embodiments, the treatment duration is 12 - 50 weeks. According to some specific embodiments, the treatment duration is selected from the group consisting of: 12 - 20 weeks, 20 - 30 weeks, and 30 - 50 weeks. According to still other embodiments, the treatment regimen includes several administration cycles each lasting at least 12 weeks.

[0079] According to some embodiments, the treatment regimen includes 1 - 8 cycles, each cycle including 2 - 6 infusions of the anti-CEACAM mAb for a duration of at least 4 weeks. According to some embodiments, the treatment regimen includes 2 - 6 cycles, each cycle including 4 infusions of the anti-CEACAM mAb for a duration of at least 4 weeks.

[0080] According to some embodiments, the administration is once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once every 5 weeks. Each possibility represents a separate embodiment of the present invention.

[0081] According to some embodiments, the treatment regimen includes 1 - 10 cycles, each cycle including 2 - 5 infusions of the above-described mAb every 1 - 4 weeks, followed by 2 - 8 weeks between each cycle.

[0082] According to some embodiments, a dose escalation regimen is provided, which includes starting the administration at 0.01 mg / kg and continuing to 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg. According to still other embodiments, the treatment regimen includes 6 cycles of 4 infusions, each administered every 2 weeks.

[0083] According to some embodiments, the subject is human.

[0084] According to some embodiments, a human subject is diagnosed with a NET-related condition. According to some embodiments, a human subject is diagnosed with cancer. According to some embodiments, the human subject is a cancer patient who has undergone tumor resection surgery.

[0085] The methods and uses provided by the present invention may include treatment with an anti-CEACAM1 antibody or fragment alone or as part of a treatment regimen that includes at least one additional treatment.

[0086] In some embodiments, administering an anti-CEACAM1 antibody for treating cancer or preventing cancer metastasis includes at least one additional anti-cancer therapy. According to some embodiments, the at least one additional anti-cancer therapy is selected from the group consisting of chemotherapy, radiation, surgery, and immunotherapy.

[0087] According to some embodiments, a method of treating cancer includes administering an anti-CEACAM1 antibody or fragment described herein and an additional anti-cancer agent. According to some embodiments, the additional anti-cancer agent is selected from the group consisting of immunomodulators, activated lymphocytes, immunocyte therapeutic agents, kinase inhibitors, and chemotherapeutic agents.

[0088] According to some embodiments, the additional immunomodulator is an inhibitor of an immune checkpoint molecule.

[0089] According to some embodiments, the immune checkpoint inhibitor inhibits the interaction between PD-1 and its ligand PD-L1. According to some embodiments, the inhibitor of the immune checkpoint molecule is an anti-PD-1 inhibitor, such as an antibody. In other embodiments, the immune checkpoint inhibitor is an anti-PD-L1 inhibitor, such as an antibody.

[0090] In another aspect, the present invention provides a method for inhibiting the formation of NETs in a subject, comprising administering to a patient an effective dose of an anti-CEACAM1 antibody, particularly CM24. In some embodiments, inhibiting the formation of NETs includes preventing the formation of NETs and / or reducing the likelihood that NETs will form in the subject. In some embodiments, inhibiting the formation of NETs includes inhibiting the growth or progression of pre-existing NETs and / or reducing the likelihood that pre-existing NETs will grow or progress in the subject. In some embodiments, the method of inhibiting the formation of NETs results in a reduction in the severity of symptoms associated with the development of NETs. In some embodiments, the symptoms associated with the development of NETs are thrombosis. In some embodiments, the subject being treated to inhibit the formation of NETs is a subject having or diagnosed with a cardiovascular condition. In some embodiments, the subject being treated to inhibit NET-mediated activity is a subject having or diagnosed with a condition that renders the subject prone to thrombosis (i.e., prothrombotic).

[0091] According to some embodiments, the pathological condition, i.e., the process or disorder involving NET-mediated activity, is a non-cancerous process or disorder. According to some embodiments, the NET-related disease or disorder is a non-malignant thrombotic disease or disorder.

[0092] According to some embodiments of the present invention, the anti-CEACAM1 mAb or antibody fragment inhibits the adhesion of non-cancerous cells to NET components.

[0093] Non-cancerous conditions, diseases, and disorders involving NET-mediated activity include, but are not limited to, thrombotic diseases, thrombosis, prothrombotic conditions, venous thromboembolism, arterial thromboembolism, thromboinflammatory conditions, hematological conditions, cardiovascular conditions, autoimmune diseases, autoinflammatory diseases or disorders, immune-mediated diseases, systemic inflammatory conditions. These and other NETs-related conditions are suitable for treatment with the compositions and methods of the present invention.

[0094] The compositions and methods of the present invention can also be referred to as thromboprophylactic. In some embodiments, the effect achieved by the compositions and methods of the present invention is the effect of primary thromboprophylaxis, i.e., aimed at directly minimizing the occurrence of thromboembolism, and in other embodiments, the effect is the effect of secondary thromboprophylaxis - preventing the recurrence of thromboembolism in subjects with a history of thrombosis-related events.

[0095] In some embodiments, the NET-related disease or disorder is a thrombotic cardiovascular disease.

[0096] In some embodiments, the thrombotic cardiovascular disease is myocardial infarction. In some embodiments, myocardial infarction is characterized by the abundance of NETs in the coronary artery thrombus of a subject. In additional embodiments, myocardial infarction is characterized by the presence of NETs in the coronary artery stent thrombus of a subject. In still more embodiments, myocardial infarction is characterized by polyp-mediated platelet-neutrophil interactions (which in turn trigger NET production).

[0097] In additional embodiments, the thrombotic cardiovascular disease is carotid atherosclerosis. In some embodiments, carotid atherosclerosis is characterized by elevated levels of myeloperoxidase (MPO), cell-free DNA, and MPO-DNA complexes (which are detected in the intraplaque hemorrhage segments of carotid atherosclerosis).

[0098] In additional embodiments, the thrombotic cardiovascular disease is cerebrovascular stroke. In some embodiments, cerebrovascular stroke is characterized by a positive correlation between NETotic markers and clot stability and resistance to endovascular therapy.

[0099] In additional embodiments, the thrombotic cardiovascular disease is deep vein thrombosis (DVT), portal vein thrombosis, or marantic endocarditis. In some embodiments, DVT is characterized by activated neutrophils and plasma nucleosomes / DNA.

[0100] In additional embodiments, the thrombotic cardiovascular disease is pulmonary embolism. In some embodiments, pulmonary embolism is characterized by NET-mediated thrombus organization and maturation.

[0101] In additional embodiments, the thrombotic cardiovascular disease is chronic thromboembolic pulmonary hypertension. In some embodiments, chronic thromboembolic pulmonary hypertension is characterized by the presence of NETs in the patient's plasma and pulmonary thrombi.

[0102] In yet other embodiments, the NET-related condition is a hematological disease or disorder.

[0103] In some embodiments, the hematological disease is thrombotic thrombocytopenic purpura (TTP). In some embodiments, TTP is characterized by impaired DNAse1-mediated degradation of NETs.

[0104] In additional embodiments, the hematological disease is heparin-induced thrombocytopenia or thrombosis. In some embodiments, heparin-induced thrombocytopenia or thrombosis is characterized by neutrophil activation (which leads to NETs-induced thrombosis).

[0105] In additional embodiments, the NET-related disease is an autoimmune disease.

[0106] In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE). In some embodiments, SLE is characterized by the demonstration of excessive cell death of neutrophils (which leads to NET formation).

[0107] In additional embodiments, the autoimmune disease is antiphospholipid syndrome (APS). In some embodiments, APS is characterized by antiphospholipid antibody-mediated induction of NET formation.

[0108] In additional embodiments, the autoimmune disease is rheumatoid arthritis (RA). In some embodiments, rheumatoid arthritis is characterized by increased NET formation in peripheral blood and synovium.

[0109] In additional embodiments, the autoimmune disease is psoriasis. In some embodiments, psoriasis is characterized by the correlation between the number of NETotic cells and the severity of the disease.

[0110] In additional embodiments, the autoimmune disease is ulcerative colitis. In some embodiments, ulcerative colitis is characterized by enhanced NET-mediated procoagulant activity.

[0111] In additional embodiments, the autoimmune disease is gout. In some embodiments, gout is characterized by monosodium urate (MSU) crystal-induced stimulation of neutrophils to produce NETs and IL-1β.

[0112] In additional embodiments, the autoimmune disease is systemic sclerosis.

[0113] In additional embodiments, the autoimmune disease is ANCA-associated vasculitis. In some embodiments, ANCA-associated vasculitis is characterized by NET formation, which triggers vasculitis and promotes an autoimmune response against neutrophil components.

[0114] In additional embodiments, the autoimmune disease is dermatomyositis. In some embodiments, dermatomyositis is characterized by an increase in NETs.

[0115] In additional embodiments, the autoimmune disease is polymyositis. In some embodiments, polymyositis is characterized by an increase in NETs.

[0116] In additional embodiments, the NET-related condition is systemic inflammatory response syndrome.

[0117] In some embodiments, the systemic inflammatory response syndrome is sepsis or septic shock. In other embodiments, the systemic inflammatory response syndrome is caused by a viral infection. According to some embodiments, the viral infection is SARS-CoV-2.

[0118] In additional embodiments, the systemic inflammatory response syndrome presents as disseminated intravascular coagulation (DIC).

[0119] In another aspect, a method of inhibiting NET formation in a subject is provided, the method comprising administering to the patient an effective dose of an anti-CEACAM1 antibody.

[0120] In another aspect, the present invention provides a kit for selecting a subject suitable for anti-CEACAM1 antibody treatment or for predicting a subject's response to an anti-CEACAM1 antibody, the kit comprising means for determining the level of at least one NET biomarker in a biological sample, means for comparing the expression level of the at least one NET biomarker with a reference value or a control sample value; and instructional material that guides the correlation between the ratio of the NET biomarker to the reference level. In some embodiments, the subject is diagnosed with cancer. In other embodiments, the subject is diagnosed with a non-malignant NET-related disease, disorder, or complication. In some embodiments, the subject is diagnosed with a thrombus-related condition. In still more embodiments, the subject is diagnosed with an autoimmune disease or a rheumatic disorder.

[0121] In some embodiments, the at least one NET biomarker is selected from the group consisting of myeloperoxidase (MPO), neutrophil elastase (NE), peptidylarginine deiminase 4 (PAD4), citrullinated histone H3 (Cit-H3), and cell-free DNA. According to some embodiments, the at least one NET biomarker is selected from MPO, NE, and DNA complexes. According to a particular embodiment, the NET biomarker is MPO. Each of these selections represents a separate embodiment of the present invention.

[0122] In some embodiments, the NET biomarker is MPO, and the present invention provides a kit for selecting a subject suitable for anti-CEACAM1 antibody treatment or for predicting a subject's response to an anti-CEACAM1 antibody, the kit comprising means for determining the level of MPO in a biological sample, means for comparing the expression level of MPO with a reference value or a control sample value; and instructional material that guides the correlation between the ratio of MPO to the reference level. In some embodiments, the subject is diagnosed with cancer. In other embodiments, the subject is diagnosed with a non-malignant NET-related disease, disorder, or complication. In other embodiments, the subject is diagnosed with a thrombus-related condition. In still more embodiments, the subject is diagnosed with an autoimmune disease or a rheumatic disorder.

[0123] In some embodiments, the subject is a cancer patient or the subject is suspected of having cancer, and thus, the present invention provides a kit for selecting a cancer subject suitable for anti-CEACAM1 antibody treatment or for predicting a cancer subject's response to an anti-CEACAM1 antibody, the kit comprising means for determining the level of MPO in a biological sample, means for comparing the expression level of MPO with a reference value or a control sample value; and instructional material that guides the correlation between the ratio of MPO to the reference level.

[0124] In some embodiments according to any aspect of the present invention, the anti-CEACAM1 mAb or a fragment thereof comprises a set of six CDR sequences, wherein HC-CDR1 comprises SEQ ID NO:1, HC-CDR2 comprises SEQ ID NO:2, HC-CDR3 comprises SEQ ID NO:3, LC-CDR1 comprises SEQ ID NO:4, LC-CDR2 comprises SEQ ID NO:5, and LC-CDR3 comprises SEQ ID NO:6.

[0125] In some embodiments according to any aspect of the present invention, the anti-CEACAM1 mAb or a fragment thereof comprises a set of six CDR sequences, wherein HC-CDR1 consists of SEQ ID NO:1, HC-CDR2 consists of SEQ ID NO:2, HC-CDR3 consists of SEQ ID NO:3, LC-CDR1 consists of SEQ ID NO:4, LC-CDR2 consists of SEQ ID NO:5, and LC-CDR3 consists of SEQ ID NO:6.

[0126] In some embodiments according to any aspect of the present invention, the anti-CEACAM1 mAb or a fragment thereof comprises a heavy chain variable region of SEQ ID NO:7 or a variant having at least 90% identity, and a light chain variable region of SEQ ID NO:8 or a variant having at least 90% identity.

[0127] In an exemplary embodiment according to any aspect of the present invention, the anti-CEACAM1 antibody is CM24, which comprises the heavy chain sequence listed in SEQ ID NO:9 and the light chain sequence listed in SEQ ID NO:10, or an active fragment thereof that at least comprises the binding site, or an antibody analogue or derivative having at least 90% identity with any one of the chain sequences.

[0128] In some embodiments according to any aspect of the present invention, the antibody or a fragment thereof is an IgG mAb comprising a heavy chain constant region selected from IgG4, IgG1, and IgG2. In other embodiments, the antibody comprises a human IgG constant region selected from IgG1 and IgG4. In some embodiments, the anti-CEACAM1 antibody comprises a human κ light chain constant region. Each selection represents a separate embodiment of the present invention.

[0129] Additional embodiments and the full scope of the applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description. Brief Description of the Drawings

[0131] Some embodiments of the present invention are described herein by way of example with reference to the accompanying drawings. Now specifically referring in detail to the drawings, it should be emphasized that the details shown are by way of example and for the purpose of illustrative discussion of the embodiments of the present invention. In this regard, the description using the drawings makes it apparent to those skilled in the art how to practice the embodiments of the present invention.

[0132] Figures 1A - 1C . Illustrated is the inhibition of CM24-mediated in vitro NET-promoted cancer cell migration. The following human cancer cell lines were treated with CM24, isotype control, or untreated in serum-free medium with or without NETs: melanoma SK-MEL-28( Figure 1A )、non-small cell lung cancer (NSCLC) A549( Figure 1B ) and pancreatic cancer AsPC1( Figure 1C ). Serum was used as a chemotactic stimulus for cancer cell migration in a transwell Boyden chamber. Fluorescence of migrating cells was measured every 2 hours for 24 hours (SKMEL-28, A549) or 48 hours (AsPC1), and the mean AUC±SEM was presented.

[0133] The differences between untreated (no treatment) or isotype control and CM24-treated wells were calculated by two-way ANOVA statistical analysis. Significance was indicated by *p<0.05, ****p<0.0001, and ns indicated not significant.

[0134] Figures 2A - 2F Confocal microscopy images of NETs are depicted, showing the co-localization of CEACAM1 and NETs and the direct binding of CM24 to NETs, as shown by the overlapping regions of CM24 / MPO / extracellular DNA. DAPI staining was used to visualize the extracellular DNA component of NETs( Figures 2A - 2D 、 Figure 2F ), an anti-MPO antibody for NET markers( Figures 2A - 2C ), and CEACAM1 on the NET structure was stained with CM24 (Figures A, C, and E are emphasized by white arrows). Magnified pictures are shown in Figures 2C - 2E .

[0135] Figure 3 Serum MPO levels (ng / ml) measured in 30 healthy donors and 10 patients with pancreatic ductal adenocarcinoma (PDAC) before treatment are depicted (P<0.01).

[0136] Figures 4A - 4BThe figure shows the percentage reduction in serum MPO levels in patients after treatment with CM24 and nivolumab. MPO levels in serum samples from patients were analyzed in part A of the clinical study, including 10 patients with PDAC and 2 patients with colorectal cancer (CRC) ( Figure 4A ) or 10 patients with PDAC ( Figure 4B ). Measurements by ELISA were performed at five time points: before dosing at C1D1 (day 1 of cycle 1); end of CM24 infusion (EOI); 1.5 hours after EOI; before dosing at C1D15 (day 15 of cycle 1); and C1D15 EOI.

[0137] Figures 5A - 5B The figure depicts MPO levels in serum samples from PDAC patients before treatment with a combination of CM24, nivolumab, and the chemotherapy mixture Nal-irinotecan / 5FU / LV. Figure 5A The figure shows the MPO level, known survival time, and best response (PD = progressive disease, PR = partial response, SD = stable disease) for each patient. Figure 5B The mean serum MPO levels were compared between patients showing disease control (SD, PR) and patients with progression at the time of treatment (P < 0.05). The mean MPO level in healthy controls (N = 30) is depicted by the dashed line.

[0138] Figures 6A - 6B The figure depicts a significant reduction in serum MPO in patients showing disease control (PR or SD) compared to patients with progression (PD) two weeks after treatment with a combination of CM24, nivolumab, and Nal-irinotecan / 5FU / LV. Figure 6A The figure shows the percentage of MPO relative to before dosing for each patient, and Figure 6B shows the mean values for patients showing disease control and patients with progression (P = 0.0001).

[0139] Figure 7 The figure depicts the measurement of MPO levels per concentration (ng / uL) of NETs. MPO levels were measured by ELISA in different amounts of fresh or frozen NETs (5 ng / uL, 10 ng / uL, and 20 ng / uL). The MPO levels are represented as the mean of the quadrants.

[0140] Figure 8 The figure depicts dose-dependent NET-induced platelet aggregation. Platelets were combined with 5 ng / μl, 10 ng / μl, and 20 ng / μl NETs, and aggregation was quantified. Bars are the mean of N = 4 + / - SEM. Two-way ANOVA was used to determine significance (***p < 0.001, ****p < 0.0001).

[0141] Figure 9 Depicts the inhibition of adenosine diphosphate (ADP)-induced platelet aggregation by CM24. Platelet-rich plasma was incubated with CM24 or hIgG4 (as an isotype control) in the presence or absence of ADP as a platelet aggregation inducer for 30 minutes, and platelet aggregation was measured. Two-way ANOVA was used for selected comparisons (****p < 0.0001).

[0142] Figure 10 Depicts the inhibition of NET-induced platelet aggregation by CM24. Platelet-rich plasma was pretreated with CM24 or an isotype control antibody and added to fresh NETs at concentrations of 5 ng / μL and 10 ng / μL. The figure presents the mean AUC + / - SEM of aggregation measured over 30 min (N = 4). Two-way ANOVA and multiple comparisons were performed to determine statistical differences between treatments (****p < 0.0001).

[0143] Figure 11 Depicts the interference of CM24 with the adhesion of CEACAM1-expressing melanoma cells to NETs. After incubation with CM24 or an isotype control for 30 minutes, FACS analysis of CEACAM1-positive SK-MEL-28 cells relative to CEACAM1-negative Jurkat cells was performed. The figure presents NET-adherent cells (mean + / - SD, N = 3). Two-way ANOVA and multiple comparisons were performed to determine statistical differences between conditions (*p < 0.05). DETAILED DESCRIPTION OF THE INVENTION

[0145] The present invention relates to methods and compositions comprising anti-CAECAM1 antibodies for treating NET-related pathologies, including non-malignant thrombotic diseases and disorders, and for preventing, delaying, or inhibiting metastasis formation, migration, spread, and progression.

[0146] Without wishing to be bound by any theory or mechanism or action, it is proposed that inhibiting the capture of cells by the NETotic matrix prevents the pathological aggregation of cells, which, if not inhibited, can lead to the formation of thrombi or metastases. The present invention exemplifies the success of using the humanized anti-CEACAM1 antibody CM24 to inhibit NET-mediated cancer cell migration in vitro and metastasis in vivo.

[0147] The findings disclosed in the present invention also provide a better understanding of the crosstalk between cancer and NETs, which is crucial for developing novel therapeutic interventions that block cancer escape mechanisms and prevent the spread of metastases.

[0148] In addition, the present invention describes the utility of NET markers as effective biomarkers for selecting patients suitable for treatment with anti-CEACAM1 antibodies and for monitoring the effectiveness of treatment with such antibodies. In a non-limiting example, the NET marker is myeloperoxidase (MPO).

[0149] In the following description, to provide a thorough understanding of the various embodiments, certain specific details are set forth. However, those skilled in the art will understand that the provided embodiments may be practiced without these details. Unless the context otherwise requires, in the specification and the appended claims, the word "comprise" and its variations such as "comprises" and "comprising" shall be interpreted in an open, inclusive sense, i.e., "including, but not limited to". As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. It should also be noted that the term "or" is generally used in its sense that includes "and / or" unless the context clearly dictates otherwise. In addition, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0150] As used herein, the term "about" refers to an amount that approaches the recited amount with a difference of 10% or less.

[0151] The term "CEACAM1" is used to refer to the protein products of the human CEACAM1 gene such as NP_001020083.1, NP_001703.2. In humans, 11 different CEACAM1 splice variants have been detected to date. The individual CEACAM1 subtypes differ with respect to the number of extracellular immunoglobulin-like domains (e.g., CEACAM1 having four extracellular immunoglobulin-like domains is designated CEACAM1-4), membrane anchoring, and / or the length of its cytoplasmic tail region (e.g., CEACAM1-4 having a long cytoplasmic tail region is designated CEACAM1-4L and CEACAM1-4 having a short cytoplasmic tail region is designated CEACAM1-4S). The N-terminal domain of CEACAM1 begins immediately following the signal peptide and its structure is thought to be of the IgV type. For example, in CEACAM1 accession number P13688, the N-terminal IgV-type domain comprises 108 amino acids from amino acid 35 to 142. This domain has been identified as being responsible for homophilic binding activity (Watt et al. 2001, Blood. 98, 1469-79). All variants, including these splice variants, are included within the term "CEACAM1".

[0152] The terms "anti-CEACAM1 antibody", "antibody that recognizes CEACAM1", "antibody directed against CEACAM1", and "anti-CEACAM1 antibody" are interchangeable and are used herein to refer to an antibody that binds to the human CEACAM1 protein with sufficient affinity and specificity.

[0153] Specific anti-CEACAM1 antibodies are described in WO2010125571, which discloses, for example, a murine anti-human CEACAM1 antibody designated MRG1.

[0154] CM24 is a non-fully humanized mAb that is disclosed in detail in WO2015166484.

[0155] The unique properties of CM24 and similar antibodies included in the compositions and methods of the present invention confer several advantages for their use in humans, particularly in applications where long-term or repeated administration is required and other non-human antibodies cannot be administered due to concerns about eliciting an immunogenic response against the non-human antibody itself. Avoiding such an immune response becomes even more important when the person being treated is a patient suffering from a disease, where further deterioration of the patient's health should be avoided.

[0156] As used herein, the term "NET" or "NETs" refers to an extracellular complex of nucleosomes and proteins (e.g., proteins with antimicrobial activity). The extracellular complex can be derived from any myeloid or lymphoid cell, including neutrophils, macrophages, myeloid-derived suppressor cells, mast cells, eosinophils, basophils, dendritic cells, natural killer cells, monocytes, or B cells and T cells.

[0157] Neutrophils and macrophages are the major cell types known to cast extracellular traps that include DNA and histones (mostly in their citrullinated form) and are further decorated with diverse proteins. Like neutrophils, macrophages undergo a form of cell death called METosis, during which they cast extracellular traps (including proteins such as MPO). (Rahat et al. Front Immunol. 2023 Sep 26:14:1292819). Any aspect, embodiment, and claim of the present invention involving NET, NETs, NET markers, NET-related conditions, and NETosis also encompasses macrophage-derived extracellular traps, namely MET, METs, METosis, MET markers, MET-related conditions, and METosis.

[0158] The terms "NET - related conditions", "NET - related diseases", "NET - related disorders", "NET - driven disorders", and "NETopathies" are interchangeable and are used herein to refer to a pathology or condition involving an unwanted or uncontrolled NETosis process.

[0159] The term "NET biomarker" is used to denote a biological substance obtained from a subject that is quantitatively or qualitatively measured to detect NETosis. Detecting NETosis includes, but is not limited to, detecting NET formation, which includes detecting co - localized neutrophil - derived proteins and extracellular DNA and citrullinated histones, detecting NET remnants in fluid samples, and flow - cytometric detection of cell - associated NET components. Any biomarker known in the art for detecting NETosis can be used in the methods of the present invention. According to some embodiments, NET biomarkers include, but are not limited to: myeloperoxidase (MPO), neutrophil elastase (NE), peptidylarginine deiminase 4 (PAD4), citrullinated histone H3 (Cit - H3), and cell - free DNA. According to some embodiments, at least one biomarker is selected from MPO, NE, and DNA complexes. According to a particular embodiment, the NET biomarker is MPO.

[0160] According to some embodiments of the present invention, the term "biomarker" related to NETs or NETosis is used to describe, for example, a diagnostic or prognostic tool in patient selection.

[0161] According to some embodiments of the present invention, the terms "NET biomarker" and "NET - related biomarker" are used interchangeably.

[0162] The term control sample denotes a sample taken from one healthy subject or more than one healthy subject or a sample taken from an evaluable subject at different (e.g., earlier, pre - treatment) stages of a disease.

[0163] The term significantly or significant refers to a difference calculated by statistical methods known in the art to determine that a result or observation from a set of data is due to the inherent quality of the sample rather than random variance.

[0164] As used herein, the term "antigen" refers to a molecule or portion of a molecule capable of inducing antibody formation and being bound by an antibody. An antigen may have one or more epitopes. The specific reaction mentioned above is intended to indicate that an antigen will react with its corresponding antibody in a highly selective manner, rather than with multiple other antibodies that may be induced by other antigens. The antigen according to the present invention is the human CEACAM1 protein or a fragment thereof. According to some embodiments, the human programmed cell death 1 (PD-1) protein also serves as an antigen for the combination therapy according to the present invention.

[0165] As used herein, the term "antigenic determinant" or "epitope" refers to the region of an antigen molecule that reacts specifically with a particular antibody. Peptide sequences derived from an epitope can be used, either alone or in conjunction with a carrier moiety, to immunize animals and generate additional polyclonal or monoclonal antibodies using methods known in the art.

[0166] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that at least include the antibody-binding portion and exhibit the desired biological activity (i.e., against CEACAM1).

[0167] An antibody according to the present invention is a molecule that at least comprises the antigen-binding portion of an antibody. One or more antibodies according to the present invention include intact antibodies, such as polyclonal or monoclonal antibodies, and proteolytic fragments thereof, such as Fab or F(ab’)2 fragments. Other types of antibody fragments and constructs, as well as single-chain antibodies, also fall within the scope of the present invention.

[0168] As used herein, the terms "molecule having the antigen-binding portion of an antibody" and "antigen-binding fragment" are intended to include not only any isotype and intact immunoglobulin molecule produced by any animal cell line or microorganism, but also the antigen-binding reactive portion thereof, including but not limited to Fab fragments, Fab’ fragments, F(ab’)2 fragments, the variable portions of its heavy and / or light chains, Fab minibodies (see WO 93 / 15210, U.S. Patent Application 08 / 256,790, WO 96 / 13583, U.S. Patent Application 08 / 817,788, WO 96 / 37621, U.S. Patent Application 08 / 999,554, the entire contents of which are incorporated herein by reference), dimeric bispecific minibodies (see Muller et al. 1998), and single-chain antibodies incorporating such reactive moieties, as well as any other type of molecule that has been physically inserted into such antibody reactive moieties. Such molecules can be provided by any known technique, including but not limited to enzymatic cleavage, peptide synthesis, or recombinant techniques.

[0169] "Antibody fragment" contains only a part of a complete antibody and usually contains the antigen-binding site or part of the complete antibody and thus retains the ability to bind antigen. Examples of antibody fragments covered by this definition include: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab' fragments, which are Fab fragments having one or more cysteine residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CHI domains and one or more cysteine residues at the C-terminus of the CH1 domain; (v) Fv fragments having VL and VH domains of a single arm of an antibody; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 1989, 341, 544-546); (vii) isolated CDR regions; (viii) F(ab')2 fragments, bivalent fragments containing two Fab' fragments linked by a disulfide bridge in the hinge region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Huston et al., PNAS (USA) 1988, 85, 5879-5883); (x) "diabodies" having two antigen-binding sites, containing heavy-chain variable domains (VH) linked to light-chain variable domains (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" containing 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. Patent No. 5,641,870).

[0170] A single-chain antibody can be a single-chain composite polypeptide having antigen-binding ability and containing amino acid sequences homologous or similar to immunoglobulin light and heavy chain variable regions, i.e., linked VH-VL or single-chain Fv (scFv).

[0171] As used herein, the term "monoclonal antibody" 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. Further, in contrast to polyclonal antibody preparations which 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 that the antibody be made by any particular method. Monoclonal Abs can be obtained by methods known to those of skill in the art. For example, monoclonal antibodies useful in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 1991, 352, 624-628, or Marks et al., J. Mol. Biol., 1991, 222:581-597.

[0172] The mAbs of the invention can be of any immunoglobulin class, including IgG, IgM, IgE or IgA. In certain embodiments, the mAbs of the invention are IgG.

[0173] Humanized antibodies are antibodies from non-human species (e.g., murine antibodies) whose protein sequences have been modified to increase their similarity to antibody variants that occur naturally in humans. The process of "humanization" is generally applied to the development of monoclonal antibodies for administration to humans and is carried out when the process of developing a specific antibody involves production in a non-human immune system (such as in a mouse). Antibodies produced in this way have protein sequences that are different from the antibodies that occur naturally in humans and are thus immunogenic when administered to human patients. Humanized antibodies are considered to be different from chimeric antibodies, which have protein sequences similar to human antibodies but carry large segments of non-human protein.

[0174] It is possible to produce humanized antibodies without creating a chimeric intermediate. The direct creation of humanized antibodies can be accomplished by inserting appropriate CDR-encoding segments (responsible for the desired binding characteristics) into a human antibody scaffold, a process known as "CDR grafting". Typically, after an antibody has been developed to have the desired characteristics in a mouse (or another non-human animal), the DNA encoding the CDRs of that antibody can be sequenced. Once the exact sequences of the desired CDRs are known, these sequences are inserted into a construct containing the DNA for the human antibody framework.

[0175] Identification or determination of CDRs from a given heavy or light chain variable sequence is typically performed using one of several methods known in the art. For example, such determination is performed according to 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).

[0176] There are several known methods in the prior art for determining the CDR sequences of a given antibody molecule, but there is no standard and clear method. Determination of CDR sequences from the variable regions of the heavy and light chains of an antibody can be performed according to any method known in the art, including but not limited to the methods known as KABAT, Chothia, and IMGT. The selected CDR set can include sequences identified by more than one method, i.e., for example, some CDR sequences can be determined using KABAT, and some can be determined using IMGT. According to some embodiments, the CDR sequences of the mAb variable region are determined using the IMGT method.

[0177] When using the term "CDR having a certain sequence" or a similar term, it includes the option where the CDR contains the specified sequence and the option where the CDR consists of the specified sequence.

[0178] The antigen specificity of an antibody is based on the hypervariable regions (HVRs), i.e., the unique CDR sequences of both the light and heavy chains that together form the antigen-binding site.

[0179] Since the variable regions of some antibodies (e.g., CM24) included in the compositions and methods of the present invention differ from the variable regions of fully human antibodies by at least one amino acid, they are also labeled as "not fully humanized" antibodies. Thus, as used herein, the term "not fully humanized monoclonal antibody" refers to a monoclonal antibody having heavy and / or light chain variable domains in which the amino acid sequences flanking and / or adjacent to the CDRs are not fully human, i.e., are not identical to any known homologous or corresponding sequences taken from natural human antibodies.

[0180] Antibody sequences containing at least one amino acid substitution, deletion, and / or insertion, and up to about 10% of the chain sequence compared to the corresponding sequence are also included within the scope of the present invention. These substitutions are typically made within the "non-CDR sequences", i.e., the sequences of the constant domains or the sequences included in the variable region of the antibody, which are not the CDR sequences as described above. Alterations in the CDR sequences are less common but are also permitted as long as antibody binding is not affected.

[0181] When used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, the terms "homologous", "homology", "percent identity" or "percent homology" can be determined using the formulae described in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formulae are incorporated into the BLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990).

[0182] The present invention also provides conservative amino acid variants of the specifically disclosed antibodies and antibody fragments. Variants according to the invention that preserve the overall molecular structure of the encoded protein can also be prepared. Given the nature of the individual amino acids that make up the disclosed protein products, some reasonable substitutions will be recognized by those skilled in the art. Amino acid substitutions can be made based on, for example, similarities in the properties of the residues involved, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity, i.e., "conservative substitutions". As used herein, the term "antibody analogue" refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

[0183] Antibody variants and conjugates refer to any molecule comprising an antibody of the invention. For example, fusion proteins in which the antibody or its antigen-binding fragment is linked to another chemical entity are also within the scope of the present invention.

[0184] In some embodiments, the antibodies provided herein have a dissociation constant (K -8 M) of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -13 M to 10 -9 M, e.g., 10 -13 M) for human CEACAM1. K D can be measured by any suitable assay. In certain embodiments, K D can be measured using a surface plasmon resonance (SPR) assay (e.g., using D instrument). instrument).

[0185] The present invention is based in part on the results of clinical trials conducted to evaluate the safety and efficacy of the anti-CEACAM1 mAb CM24 (NCT04731467). Part A of the trial evaluated the safety of CM24 administration and involved administering a combination of CM24 and the anti-PD1 mAb nivolumab to patients with solid tumors in a dose-escalating manner. Part C also evaluated safety and consisted of two sub-parts - Part C1 and Part C2. During Part C1, 8 patients with pancreatic cancer were administered a combination of CM24 and nivolumab, as well as an additional therapeutic agent selected from gemcitabine and nab-paclitaxel; in Part C2, an additional 8 patients with pancreatic cancer were administered a combination of CM24 and nivolumab and the chemotherapy agent mixture Nal-IRI / 5FU / LV. Part D is the final stage of the clinical trial and evaluated the efficacy of the treatment by monitoring the progression of patients after the above-described treatment and comparing it to control patients who were administered standard therapy (gemcitabine / nab-paclitaxel or Nal-IRI / 5FU / LV) and did not receive antibody therapy. As used herein, "second-line therapy" refers to a subsequent treatment regimen after an unsuccessful or inadequate initial treatment regimen. Second-line therapy can include administering to the patient the same therapeutic agent as the first time, or a completely different therapeutic agent. In drugs and pharmaceutical formulations, the active agent is preferably used in combination with one or more pharmaceutically acceptable carriers and optionally any other therapeutic components. One or more carriers must be pharmaceutically acceptable in the sense that they are compatible with the other components of the formulation and not unduly harmful to its recipient. The active agent is provided in an amount effective to achieve the desired pharmacological effect as described above and in an amount suitable to achieve the desired daily dose.

[0186] The molecules of the present invention as active ingredients are dissolved, dispersed or mixed in well-known excipients that are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, etc. and combinations thereof. Other suitable carriers are well known to those skilled in the art. Additionally, if desired, the composition may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

[0187] The pharmaceutical composition can optionally be formulated to control the release of the active ingredient (a molecule comprising the antigen-binding portion of an antibody) or to prolong its presence in the patient's system. A number of suitable drug delivery systems are known and include, for example, implantable drug release systems, hydrogels, hydroxyethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release articles can be prepared by using polymers to complex or adsorb the molecules according to the present invention. For example, biocompatible polymers include matrices of ethylene-vinyl acetate copolymers and matrices of polyanhydride copolymers of dimeric stearic acid and sebacic acid. The release rate of the molecules according to the present invention, i.e., antibodies or antibody fragments, from such matrices depends on the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of the dispersed particles.

[0188] According to some embodiments, the pharmaceutical composition comprises 1-50 mg / ml of a humanized mAb against CEACAM1, such as CM24. According to some embodiments, the pharmaceutical composition comprises a basic amino acid. According to some embodiments, the pharmaceutical composition comprises a sugar. According to some embodiments, the pharmaceutical composition comprises a surfactant. According to some embodiments, the pharmaceutical composition comprises a basic amino acid, a sugar, and a surfactant. According to some embodiments, the pharmaceutical composition comprises (i) 1-10 mg / ml of a basic amino acid; (ii) 10 / 100 mg / ml of a sugar; (iii) 0.01-1 mg / ml of a surfactant; (iv) 1-50 mg / ml of a humanized mAb against CEACAM1 (such as CM24), 4-6 mg / ml of a basic amino acid, 70-100 mg / ml of a sugar, and 0.1-1 mg / ml of a nonionic surfactant; or (v) 10 mg / ml of CM24, 4.65 mg / ml of L-histidine, 82 mg / ml of sucrose, and 0.20 mg / ml of polysorbate 20.

[0189] The term "sugar" refers to monosaccharides, disaccharides, and polysaccharides, and examples of sugars include, but are not limited to, sucrose, trehalose, dextrose, and others.

[0190] According to some embodiments, the basic amino acid is selected from the group consisting of histidine, arginine, lysine, and ornithine. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the composition comprises 1-10, 2-9, 3-7, or 4-6 mg / ml of a basic amino acid. Each possibility represents a separate embodiment of the present invention.

[0191] According to some embodiments, the sugar is selected from the group consisting of sucrose, trehalose, glucose, dextrose, and maltose. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the composition comprises 10-200, 10-100, 50-150, or 70-100 mg / ml of a sugar. Each possibility represents a separate embodiment of the present invention.

[0192] According to yet other embodiments, the composition comprises a polyol, including but not limited to mannitol and sorbitol. Each possibility represents a separate embodiment of the invention.

[0193] According to some embodiments, the surfactant is nonionic. According to some embodiments, the surfactant is selected from the group consisting of: polysorbates, sorbitan esters, and poloxamers. Each possibility represents a separate embodiment of the invention. According to some embodiments, the surfactant is selected from the group consisting of: polysorbate 20, polysorbate 80. Each possibility represents a separate embodiment of the invention. According to some embodiments, the composition comprises 0.01 - 10, 0.01 - 1, 0.05 - 5, or 0.1 - 1 mg / ml of the surfactant. Each possibility represents a separate embodiment of the invention. According to some embodiments, the pharmaceutical composition comprises 4 - 6 mg / ml of a basic amino acid, 70 - 100 mg / ml of a sugar, and 0.1 - 1 mg / ml of a surfactant.

[0194] According to some embodiments, the pharmaceutical composition is in liquid form and comprises 1 - 50 mg / ml of CM24. According to other embodiments, the pharmaceutical composition is lyophilized. According to some embodiments, the pharmaceutical composition comprises: 10 mg / mL of CM24, 4.65 mg / mL of L - histidine, 82 mg / ml of sucrose, and 0.20 mg / ml of polysorbate 20.

[0195] According to some embodiments, the pharmaceutical composition comprises CM24 or a fragment as defined above, and an additional immunomodulator or kinase inhibitor. According to some embodiments, a pharmaceutical composition comprising at least one humanized mAb or fragment as defined above and a pharmaceutical composition comprising an additional immunomodulator or kinase inhibitor are used for the treatment of cancer by separate administration.

[0196] According to some specific embodiments, the additional immunomodulator is selected from the group consisting of: anti - human programmed cell death protein 1 (PD - 1), PD - L1, and PD - L2 antibodies, activated cytotoxic lymphocytes, lymphocyte activators, and RAF / MEK pathway inhibitors. Each possibility represents a separate embodiment of the invention. According to some specific embodiments, the additional immunomodulator is selected from the group consisting of: mAb against PD - 1, mAb against PD - L1, mAb against PD - L2, interleukin 2 (IL - 2), lymphokine - activated killer (LAK) cells.

[0197] The pharmaceutical composition of the present invention can be administered by any suitable means, including parenteral and enteral routes. The administration means include, but are not limited to, oral, topical, intranasal, subcutaneous, intramuscular, intravenous, intratumoral, intraarterial, intraarticular, intralesional, and transdermal. Generally, intravenous (i.v.) administration is used.

[0198] 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, on the administration schedule, the unit dose of the 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, and the judgment of the treating physician. As used herein, "therapeutically effective amount" means the amount of the molecule required to alleviate one or more symptoms associated with the disorder being treated over a period of time.

[0199] As used herein, the terms "individual", "patient", or "subject" refer to an individual diagnosed with, suspected of having, or at risk of developing at least one disease, for which the compositions and methods can be used for treatment. According to some embodiments, the individual is a mammal. According to some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. According to some embodiments, the individual is a human.

[0200] The term "therapeutically effective amount" refers to the amount of a drug effective for treating a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of a tumor; inhibit (e.g., slow to a certain extent and preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (e.g., slow to a certain extent and preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with the disorder to a certain extent. To the extent that a drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic or conjugated to a cytostatic and / or cytotoxic agent. For cancer treatment, in vivo efficacy can be measured, for example, by assessing duration of survival, time to disease progression, response rate, duration of response, and / or quality of life.

[0201] In some embodiments, administering an anti-CEACAM1 antibody for treating cancer or preventing cancer metastasis comprises at least one additional anti-cancer therapy. According to some embodiments, the at least one additional anti-cancer therapy is selected from the group consisting of chemotherapy, radiation, surgery, and immunotherapy.

[0202] As used herein, the terms "combination" or "combination therapy" can refer to the concurrent administration of the items to be combined or the sequential administration of the items to be combined. As described herein, when combination refers to the sequential administration of items, the items can be administered in any chronological order.

[0203] As used herein, "checkpoint inhibitor" refers to a drug that inhibits a biomolecule ("checkpoint molecule") produced by an organism that negatively regulates the anti-tumor / cancer activity of T cells in the organism. Checkpoint molecules include, but are not limited to, PD-1, PD-L-1, PD-L-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, PVR, TIGIT, IDO, KIR, A2AR, B7-H3, B7H4, and CD112R.

[0204] According to some embodiments, a method of treating cancer includes administering a pharmaceutical composition as part of a treatment regimen that includes administering at least one additional anti-cancer agent. According to certain embodiments, the anti-cancer composition comprises at least one chemotherapeutic agent. Chemotherapeutic agents that may be administered together with or separately from the antibody according to the invention may comprise any such agents known in the art to exhibit anti-cancer activity.

[0205] The terms "anti-cancer composition" and "anti-neoplastic composition" refer to compositions useful in the treatment of cancer that comprise at least one active therapeutic agent capable of inhibiting or preventing tumor growth or function, and / or causing tumor cells to be destroyed. Therapeutic agents suitable for use in anti-neoplastic compositions for treating cancer include, but are not limited to, chemotherapeutic agents, radioisotopes, toxins, cytokines such as interferons, and antagonists and immune checkpoint inhibitors that target cytokines, cytokine receptors, or antigens associated with tumor cells.

[0206] According to certain embodiments, the chemotherapeutic agent is selected from the group consisting of: alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthraquinone-substituted ureas, methylhydrazine derivatives, adrenocortical inhibitors, adrenocortical steroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of: 5-fluorouracil (5-FU), leucovorin (LV), irinotecan (including Nal-irinotecan), oxaliplatin, capecitabine, paclitaxel, and docetaxel. Two or more chemotherapeutic agents may be used in a mixture for co-administration in combination with the administration of the anti-CEACAM1 antibody. According to some embodiments, the chemotherapeutic agent mixture comprises Nal-irinotecan / 5FU / LV.

[0207] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those already suffering from a disorder, as well as those in whom the disorder is to be prevented. The term includes prophylactic treatment of a subject susceptible to a disease or disorder (e.g., a subject genetically or environmentally predisposed to cancer or another disease). In some embodiments, a biological sample is obtained from the subject prior to the course of treatment. A biological sample is any sample collected from an individual and includes, but is not limited to, samples of fluids (such as blood, urine, saliva, or cerebrospinal fluid) and tissue obtained by biopsy, surgical removal, or resection of a part of a limb or a benign or metastatic growth.

[0208] In certain embodiments, the methods described above include administering to a subject at least one dose of CM24 in the range of 0.01 mg / kg to 10 mg / kg body weight. In certain embodiments, the methods described above include administering (i) multiple identical or different doses of CM24; (ii) multiple escalating doses; or (iii) a pharmaceutical composition once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, or once every 5 weeks. In certain embodiments, the methods described above include 1 - 10 cycles of administration, each cycle comprising 2 - 5 infusions of CM24 every 1 - 4 weeks, followed by 2 - 8 weeks between each cycle. In certain embodiments, the above methods include administering CM24 as neoadjuvant, adjuvant, or maintenance therapy.

[0209] The terms "cancer," "cancerous," and "tumor" refer to a physiological condition in a mammal characterized by uncontrolled cell growth. Cancer is a class of diseases in which a group of cells exhibits uncontrolled or unwanted growth. Cancer cells can also spread to other locations, which may result in the formation of metastases. For example, the spread of cancer cells in the body can occur via the lymph or blood. Uncontrolled growth, invasion, and metastasis formation are also referred to as the malignant characteristics of cancer. These malignant properties distinguish cancer from benign tumors, which generally do not invade or metastasize.

[0210] Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include melanoma, lung cancer, thyroid cancer, breast cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, leukemia, lymphoma, myeloma, ovarian cancer, uterine cancer, sarcoma, gallbladder cancer, or endometrial cancer. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of: colon cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, glioblastoma, cervical cancer, prostate cancer, and lung cancer. In other embodiments, the cancer is selected from the group consisting of: melanoma, colorectal cancer, bladder cancer, lung cancer, non-small cell lung cancer (NSCLC), non-small cell lung adenocarcinoma (NSCLA), gastrointestinal cancer, pancreatic cancer, breast cancer, prostate cancer, thyroid cancer, gastric cancer, ovarian cancer, myeloma, and uterine cancer. Each possibility represents a separate embodiment of the invention.

[0211] According to some embodiments, the cancer comprises a solid tumor. According to some embodiments, the cancer is a metastatic cancer or tumor.

[0212] According to some embodiments, the solid cancer is pancreatic cancer, lung cancer, colorectal cancer, or melanoma. According to some embodiments, the cancer is a metastatic cancer originating from a primary pancreatic, lung, colorectal, or melanoma tumor.

[0213] NETs provide a scaffold and stimulus for thrombus formation. Neutrophil release of NETs is associated with inflammation during sepsis and non-infectious diseases. Thus, as described herein, thrombotic conditions can be treated or prevented by disrupting NETs.

[0214] As used herein, the phrase "cardiovascular condition" or "cardiovascular disease or disorder" is intended to include all disorders characterized by insufficient, undesirable, or abnormal cardiac function, such as ischemic heart disease, hypertensive heart disease, and pulmonary hypertensive heart disease, valvular heart disease, congenital heart disease, and any condition leading to congestive heart failure in a subject, particularly a human subject. Also included are any vascular diseases and conditions leading to insufficient, undesirable, or abnormal cardiac function, such as stroke, thrombosis, ischemia, ischemic reperfusion, vascular occlusion, inflammation, etc. As used herein, cardiovascular conditions are not limited to those caused by atherosclerosis. Insufficient or abnormal cardiac function may be the result of disease, injury, and / or aging. In certain embodiments, the methods and compositions provided herein relate to treating or preventing a cardiovascular condition caused by or contributing to NETs activity by administering to a patient an effective dose of a composition comprising an anti-CEACAM1 antibody. In another embodiment, the cardiovascular condition is stroke, ischemic reperfusion, myocardial infarction, inflammation, or thrombosis. In certain embodiments, the cardiovascular condition to be treated is thrombosis. Clinically, inflammation and infection are associated with thrombosis. Thus, some embodiments provide methods and compositions for treating or preventing thrombosis in a patient, such as methods for treating or preventing a cardiovascular condition complicated by thrombosis. Thrombosis refers to the occurrence of a clot or inappropriate blood clot in a blood vessel at the site of vascular injury and depends on platelet adhesion, activation, and aggregation. Deep vein thrombosis (DVT) is commonly associated with inflammation and infection. A complication of thrombosis is that the clot will detach from the blood vessel wall where it formed and lodge elsewhere in the circulatory system, blocking blood flow and causing embolism. In certain embodiments, the cardiovascular condition to be treated is ischemia. In another embodiment, the cardiovascular condition to be treated is ischemic reperfusion. As used herein, the term "ischemia" refers to any local tissue ischemia resulting from reduced blood flow. Blood flow to a tissue can be reduced due to abnormalities in the blood vessels, such as thrombosis, embolism, or vasoconstriction. Reduced blood flow leads to local anemia, reduced oxygen levels, and ultimately tissue damage. Ischemia can also be caused by myocardial infarction, acute coronary syndrome, coronary artery bypass surgery, stroke, gastrointestinal ischemia, peripheral vascular disease, and surgery. In addition, the recruitment of leukocytes and / or platelets triggered by the initial tissue injury can limit blood flow in smaller capillaries, leading to a second wave of ischemia. The term "myocardial ischemia" refers to a subset of ischemia that encompasses circulatory disorders caused by coronary atherosclerosis and / or insufficient myocardial oxygen supply. For example, acute myocardial infarction represents irreversible ischemic damage to myocardial tissue. This damage results in an occlusive (e.g., thrombotic or embolic) event in the coronary circulation and creates an environment where myocardial metabolic demand exceeds the oxygen supply to the myocardial tissue. In certain embodiments, the cardiovascular condition to be treated is myocardial infarction.Myocardial infarction (i.e., heart attack) is the death of heart muscle due to a sudden blockage of a coronary artery by a blood clot. The coronary arteries are the blood vessels that supply blood and oxygen to the heart muscle. Blockage of a coronary artery deprives the heart muscle of blood and oxygen, causing damage to the heart muscle. Heart muscle damage causes chest pain and pressure. Inflammation (especially via the formation of atherosclerotic plaques) is known to contribute to the development of myocardial infarction. Disruption of a plaque can cause blood clot formation and lead to myocardial infarction. In some embodiments, the cardiovascular condition to be treated is stroke. Thromboembolic occlusion of an artery in the brain limits downstream blood flow and promotes the formation of a second arterial thrombus within the cerebral microvasculature. In some embodiments, the cardiovascular condition to be treated is thrombosis and the patient has systemic lupus erythematosus (SLE). Patients with SLE are also prone to developing venous thrombosis and have a reduced ability to degrade NETs. In some embodiments, the condition to be treated is sickle cell disease (SCD), a condition in which RBCs are deformed and rigid. The altered RBCs are more likely to restrict blood flow at certain points in the circulatory system, leading to a crisis. In patients with SCD, infection typically accelerates a fatal crisis.

[0215] In some embodiments, the formation of deep vein thrombosis (DVT) is prevented or inhibited. In some embodiments, the progression of one or more signs or symptoms of DVT is prevented or inhibited, e.g., the size of the thrombus does not increase. In some embodiments, the severity of one or more signs or symptoms of DVT is reduced, e.g., the size of the thrombus is reduced.

[0216] In another aspect, the methods described herein involve inhibiting the formation of NETs in a subject, including administering to the patient an effective dose of an anti-CEACAM1 antibody. In some embodiments, inhibiting the formation of NETs can include preventing the formation of NETs and / or reducing the likelihood that NETs will form in the subject. In some embodiments, inhibiting the formation of NETs can include inhibiting the growth or progression of pre-existing NETs and / or reducing the likelihood that pre-existing NETs will grow or progress in the subject. In some embodiments, the method of inhibiting the formation of NETs can reduce the severity of symptoms (e.g., thrombosis) associated with the development of NETs. In some embodiments, the subject being treated to inhibit the formation of NETs can be a subject having or diagnosed with a cardiovascular condition as described above. In some embodiments, the subject being treated to inhibit NET-mediated activity can be a subject having or diagnosed with a condition that renders the subject prone to thrombosis (i.e., prothrombotic). A condition that renders a subject prothrombotic can be any condition in which the subject is more likely to have or form NET-mediated thrombosis compared to a healthy subject.

[0217] The widely accepted crosstalk between inflammation and thrombosis has led to the introduction of the term thromboinflammation. Cells of the hematopoietic system, including neutrophils, platelets, and monocytes, have a major role in this process. Increasing evidence suggests that NET release is associated with the development of thrombosis in both veins and arteries. The methods and compositions of the present invention can be used for these conditions.

[0218] The role of NETs in the pathogenesis of autoimmune diseases has been previously suggested. Prolonged exposure to NET-related cascades is associated with autoimmunity and increases the chance of systemic organ damage. Several autoimmune and immune-mediated diseases are NET-related and are thus suitable for treatment using the compositions and methods of the present invention. This includes, for example, at least one of the following disease characteristics: increased NET formation, a correlation between the severity or progression of many NETotic diseases, induction of NETs by disease autoantibodies, enhanced procoagulant activity, stimulation of neutrophils to produce NETs.

[0219] Autoimmune and immune-mediated diseases associated with NETs include, but are not limited to: psoriasis, systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome, rheumatoid arthritis (RA), ulcerative colitis (UC), gout, ANCA-associated vasculitis, dermatomyositis, and polymyositis.

[0220] Autoinflammatory disorders, such as familial Mediterranean fever (FMF), are also included within the suitable diseases and conditions of the present invention. FMF is characterized by inflammatory attacks and neutrophil infiltration into the affected sites. During FMF attacks, neutrophils undergo excessive NET formation, which decreases after the inflammation subsides. These and other autoimmune and autoinflammatory disorders are suitable for treatment using the compositions and methods of the present invention.

[0221] Several systemic inflammatory responses and diseases are also suitable for treatment using the compositions of the present invention. These conditions include, but are not limited to: sepsis (septic shock) in which functional active tissue factor (TF) is found on peripheral NETs; and disseminated intravascular coagulation (DIC) in which NETs are associated with venous thromboembolism (VTE) and impaired fibrinolysis.

[0222] Several inflammatory lung diseases are characterized by the migration and detection of neutrophils and monocytes in the airway lumen and bronchoalveolar lavage fluid. NETs are associated with inflammatory diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), acute lung injury, acute respiratory distress syndrome, and asthma. These and other inflammatory diseases are also included within the scope of conditions suitable for treatment according to the present invention.

[0223] The following examples are presented to more fully illustrate some embodiments of the present invention. However, it should not be construed in any way as limiting the broad scope of the present invention. Those skilled in the art can readily envision many variations and modifications of the principles disclosed herein without departing from the scope of the present invention. Example

[0224] Example 1: Effects of NETs and CM24 on cancer cell migration

[0225] Table 1. Materials

[0226] Material Supplier Project Number Lot Number SKMEL - 28 ATCC HTB - 72 N / A A549 ATCC CRM - CCL - 185 N / A AsPC - 1 ATCC CRL - 1682 N / A PBS Corning 21 - 040 - CM 29320010 PMA (1 mM Stock Solution) Cayman 10008014 0584091-17 NETs Generated In - house with PMA hIgG4 BioXCell BE0349 82132101 Trypsin Corning 25-053-Cl 17621003 Accutase Innovative Cell Tech AT - 104 1A2005A CFDA - SE Cayman 14456 0587471-11 CytoTrace Red CMPTX Cayman 20698 0636212-1 Fluoroblok Migration Plate BD Falcon 351164 084502B

[0227] Cell culture

[0228] The human melanoma cell line SKMEL-28, the human lung adenocarcinoma cell line A549, and the human pancreatic adenocarcinoma cell line AsPC-1 were cultured according to ATCC recommendations and removed for passage and trypsin assay.

[0229] Migration assay

[0230] The cells were expanded to a maximum of 80% confluence for two generations. One day before treatment, the serum-containing medium was removed and serum-free medium was added. The next day, the cells were removed and stained with 5 μM carboxyfluorescein succinimidyl ester (CFDA-SE) dye at 37 °C for 10 minutes. The stained cells were washed twice with serum-containing medium to ensure that the excess dye was inactivated, and the cells were resuspended in serum-free medium and added to the top chamber of a Fluoroblok 96-well plate. Melanoma SKMEL-28 cells were added at 12,500 cells / well, NSCLC A549 cells and pancreatic cancer AsPC1 cells were added at 25,000 cells / well. NETs were prepared according to the method of Rayes et al. supra and added to the top chamber at a final concentration of 20 ng / μl. hIgG4 (as an isotype control) and CM24 were added to the appropriate wells of the plate at a final concentration of 500 μg / ml in serum-free medium. Serum-free medium or medium containing 10% serum was added to the feeder wells of the plate through the sample port. The plate was transferred to a cell imaging multimode reader (Cytation 5) set at CO2 and 37 °C, and imaged at 10X every 2 hours for a total of 24 hours using bright field and GFP cubes.

[0231] NET-induced migration was tested in a cell chemotaxis assay. In this assay, serum-starved, CFSE-labeled cancer cells were plated in the top chamber of a Fluoroblok migration plate, and whole serum medium was placed in the bottom chamber. Treatments (20 ng / μl NETs and 500 μg / ml antibody) were included in the top chamber with the cells. Images of the bottom of the membrane were taken every 2 hours for 24 hours (SKMEL-28, A549) or 48 hours (AsPC-1), and the total cell count of migrating cells over time was calculated ( Figures 1A - 1C ). NET-induced migration was calculated by subtracting the basal migration in the absence of NETs from the total migrating cancer cells towards the chemotactic stimulus in the presence of NETs. As described above, in the presence of NETs, the effect of CM24 on NET-induced migration was measured in the presence or absence of isotype control human anti-IgG4 or CM24, and the area under the curve (AUC) for each well was calculated (for SKMEL-28 and A549, N = 6 wells; for the AsPC-1 study, N = 3 wells). Statistical analysis using two-way ANOVA and multiple comparisons was used to test the significance of differences between treatment groups. Table 2 shows the percentage inhibition of NET-induced migration by CM24 compared to the isotype control.

[0232] Table 2. Percentage inhibition of cancer cell migration by CM24 (AUC of cell counts)

[0233]

[0234]

[0235] As Figures 1A - 1C and shown in Table 2, a significant increase in cancer cell migration was observed when NETs were added to the upper wells. The addition of the isotype control had no significant effect compared to the untreated wells. However, CM24 (500 μg / ml) significantly inhibited NET-induced migration in all three cancer cell lines compared to isotype control treatment (hIgG4 500 μg / ml) or untreated cells. In melanoma SKMEL-28 cells ( Figure 1A ), a 70% inhibition of NET-induced migration was observed with a p-value of 0.0197. In NSCLC A549 cells, CM24 inhibited NET-induced migration by 83% ( Figure 1B ), p-value < 0.0001. Finally, in pancreatic cancer AsPC-1 cells, a significant 49% inhibition of NET-induced migration was observed after treatment with CM24 compared to the isotype control (p-value < 0.0001) ( Figure 1C ). It was thus concluded that CM24 significantly attenuated cancer cell migration in response to NETs.

[0236] Example 2: Binding of CM24 to NETs

[0237] Table 3. Materials

[0238]

[0239] Sample Preparation & Immunofluorescence

[0240] Coat chamber slides with poly-D-lysine at room temperature and then wash thoroughly with water. Seed fresh neutrophils in poly-D-lysine-coated chamber slides (50K per chamber) and stimulate with 100 nM phorbol 12-myristate 13-acetate (PMA) for 4 hours to induce NETosis. Remove the medium, add 4% paraformaldehyde (PFA) and incubate at room temperature for 10 minutes. Remove the supernatant and then wash 2 times with wash buffer. Add blocking buffer and incubate at room temperature for 1 hour. Remove the blocking buffer, add the following primary antibodies in wash buffer and incubate at room temperature for 1 hour. a. Rabbit polyclonal anti-myeloperoxidase (MPO) 1:200; b. hIgG4 humanized mAb CM24 20 μg / ml; c. Human IgG4 isotype control at 20 μg / ml. Wash 3 times with wash buffer. Then, add secondary antibodies (a. anti-rabbit AF488 1:600; b-c. anti-human PE 1:200) in wash buffer and incubate at room temperature for 1 hour. Wash 3× with wash buffer. Add the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI) (1×) in PBS and image by confocal microscopy using the following LED / filter sets: a. Green fluorescent protein (GFP): Rabbit polyclonal anti-myeloperoxidase (MPO); b. Red fluorescent protein (RFP) phycoerythrin (PE): CEACAM1; c. DAPI. For chamber slides, aspirate the supernatant, then remove the chamber parts and fix with ProLong AntiFade fixative and glass coverslips. Image the slides using a Nikon A1 confocal with a Ti eclipse microscope and Nikon 40× Plan Fluor Oil DIC H N2 objective. Analyze the images using NIS elements version 5.12.03 and ImageJ Fiji.

[0241] Briefly, primary human neutrophils were stimulated with PMA to undergo NETosis in poly-D-lysine-coated chamber slides for 4 hours and then fixed with paraformaldehyde. For CM24 or isotype control, these slides were stained with anti-MPO (myeloperoxidase, a NET marker) and DAPI for extracellular DNA in the NET structure as well as nuclear DNA (in intact neutrophils), and imaged using confocal microscopy. MPO and DAPI showed typical NET morphology ( Figure 2A ). Specific binding of CM24 to the NET structure was observed compared to the isotype control that showed no detectable staining ( Figure 2B ).

[0242] When NETs were induced and directly stained in chamber slides, high-quality images of NETs were produced. MPO was used as a biomarker for NETs because it decorates the DNA structure of NETs. The binding of CM24 to NETs was tested, and specificity was ensured by parallel staining with the isotype control antibody hIgG4. Using confocal microscopy, clear and specific binding of CM24 to CEACAM1 on NETs was observed, suggesting involvement in NET-related diseases. The binding pattern suggested punctate surface staining of neutrophils undergoing NETosis, significantly distributed to the NETs themselves.

[0243] Example 3: Expression of CEACAM1 on cancer cell lines

[0244] To evaluate the binding potential of cancer cells to NETs through CEACAM1 interaction, the expression of CEACAM1 on different cancer cell lines used for migration assays (Example 1) and adhesion assays (Example 7) was measured. The binding of mouse anti-human CEACAM1 mAb MRG1 (described in WO2010125571) to the surface of several cell lines was tested by flow cytometry. Live cancer cells grown under normal culture conditions (e.g., maintained at less than 80% confluence) were incubated with mouse anti-human CEACAM1 antibody MRG1 or isotype control, followed by anti-mouse-PE staining. The samples were then stained for viability and run through a MACSQuant flow cytometer. Data were analyzed using FlowJo, and the percentage of CEACAM1-positive cells and the CEACAM1 expression intensity values for each cell line are shown in Table 4.

[0245] Table 4. Frequency (%) and mean fluorescence intensity (MFI) of MRG1-stained cancer cell lines.

[0246]

[0247]

[0248] Jurkat cells (a leukemia T cell line) that do not express CEACAM1 were used as a negative control. The human melanoma SK-MEL-28 cell line, the human pancreatic cancer AsPC-1 cell line, and the non-small cell lung cancer cell line A549, which were tested in the migration assay, showed expression of CEACAM1 as detected by MRG1, while Jurkat was negative. Both SK-MEL-28 and AsPC-1 cells showed a high degree and level of expression of CEACAM1, with 85% and nearly 100% of the cells being positive for CEACAM1, and the average MFI (a measure of the amount of antibody binding) being 48K and nearly 12K, respectively. Most (73.1%) of the A549 cells were positive when stained with MRG1, with a significantly lower intensity (MFI of 566). The expression of CEACAM1 in A549 depends on cell confluence, which is similar in all studies. Without being bound by any theory, anti-CEACAM1 antibodies can interfere with the interaction between NET-bound CEACAM1 and CEACAM1 on cancer cells or other cells, and thus affect NET-related diseases.

[0249] Example 4: Clinical trial results showing a reduction in MPO levels in patients treated with CM24 and nivolumab

[0250] Serum samples were collected from patients (clinical trial NCT04731467) treated with CM24 and nivolumab before and after treatment, and the levels of the NET marker MPO were tested. The pre-dose serum NET marker MPO levels in PDAC and CRC patients (n = 12) were >3.5-fold higher compared to healthy subjects (n = 30) (p < 0.001). PDAC patients (n = 10) showed a 3-fold higher serum MPO compared to healthy subjects ( Figure 3 ).

[0251] Figure 4A The average results of the 12 patients depicted in [figure] showed a significant reduction in serum MPO levels after treatment with CM24 on both day 1 and day 15 of the trial (p < 0.05). Figure 4B The same measurement of serum NET levels after CM24 treatment was illustrated only in PDAC patients (n = 10), showing a significant reduction in serum MPO two weeks after CM24 (p < 0.05).

[0252] In the dose-escalation arm of the trial, adult subjects with selected recurrent or metastatic solid tumors were treated with CM24 at doses of 10 mg / kg (3 patients), 15 mg / kg (3 patients), and 20 mg / kg (5 patients) every two weeks for 1-hour infusion, followed by 480 mg nivolumab per patient for 30-minute infusion. Blood samples were collected before treatment on Day 1 (pre-C1D1 dosing), at the end of CM24 administration (C1D1 EOI), and 1.5 hours after the end of nivolumab administration (C1D1 EOI 1.5HR). Two weeks later (Day 15), blood samples were collected before treatment (pre-C1D15 dosing) and at the end of CM24 dosing (C1D15 EOI). Serum samples were prepared immediately after each blood collection and stored in aliquots at -70 °C.

[0253] MPO of NET serum levels was measured by ELISA. The baseline level of MPO was represented by the average level measured in serum samples of 30 healthy volunteers. The percentage decrease in the increase of MPO detected in each sample relative to the baseline level before C1D1 dosing was calculated, and the mean and significance of 10 PDAC and 2 CRC patients were calculated ( Figure 4A ) and the mean and significance of only 10 PDAC patients were calculated ( Figure 4B ). After treatment with CM24 and nivolumab, a significant decrease in MPO levels was observed in patient sera, indicating a reduction in NETs levels.

[0254] Example 5: Clinical trial results showing significant inhibition of serum MPO in Part C2 patients with high pre-treatment levels and showing disease control after treatment with CM24, nivolumab, and chemotherapy

[0255] In Part C2 of clinical trial NCT04731467, patients were administered 20 mg / kg CM24, followed by 480 mg nivolumab per patient for 30-minute infusion. The dose of Nal-IRI was 70 mg / m2 intravenous injection for 90 minutes. At the end of Nal-IRI infusion, 200 - 400 mg / m2 leucovorin (LV) was administered via slow injection for at least 3 minutes, followed by initiation of infusion of 2400 mg / m2 5FU. Serum samples were collected before and after treatment, and MPO levels were tested by ELISA. In Part C2 of clinical trial NCT04731467, 6 out of 8 recruited patients received more than two treatments and were thus included in the analysis of serum MPO levels versus disease control. The results are presented in Figures 5 and 6. Figure 5AShows the MPO levels in the serum of patients before treatment. Compared with patients with progressive disease (PD) or healthy volunteers, patients showing disease control (including partial response (PR) or stable disease (SD)) after treatment had significantly higher pre-dose serum MPO levels. Figure 5B Depicts the mean MPO levels in the pre-treatment serum of patients showing PR or SD (241 ng / mL) and patients with progressive disease (63 ng / mL), showing a significant difference (p < 0.05). Figure 6 depicts the percentage reduction in MPO after treatment in each patient ( Figure 6A ) and as the mean change ( Figure 6B ), comparing patients showing disease control (SD, PR) and progression (PD). As can be observed in Figures 5A - 5B , the significant difference (p < 0.05) in pre-treatment serum MPO levels between patients showing disease control and higher survival of patients with progression suggests serum MPO as a potential biomarker for this treatment. Figures 6A - 6B Shows a significant reduction in serum MPO after treatment, only in patients showing disease control (P = 0.0001), but not in patients progressing at the time of treatment.

[0256] As shown in vitro, CM24 interferes with CEACAM1-mediated NET activity and results in a significant reduction in the levels of circulating NETs in patients. The ability to open the protective shield provided by NETs (allowing immune evasion, metastasis, thrombosis, and other pathological processes) provides the rationale for using CM24 against these life-threatening conditions.

[0257] Example 6: Effect of CM24 on NET-induced platelet aggregation

[0258] A platelet aggregation assay was developed using freshly drawn blood, based on (Melissa V. Chan, Platelets; 2018; 29:7, 650 - 655). In a 96-well flat-bottom plate, platelet-rich plasma (PRP) was incubated with the thrombogenic agent adenosine diphosphate (ADP). The assay was read as the absorbance at 595 nm by intermittent shaking and reading within 30 minutes, and platelet aggregation was observed as a decrease in absorbance. Un-treated PRP and platelet poor plasma (PPP, plasma from which platelets have been precipitated) wells were included on each plate (and read at each time point) such that aggregation could be expressed as a percentage, using PRP and PPP as 0% and 100% respectively. For NET stimulation of platelet aggregation, PRP was incubated with fresh and frozen NETs (collected from PMA-stimulated neutrophils).

[0259] Material

[0260]

[0261] NET isolation

[0262] Freshly drawn neutrophils and polymorphonuclear cells (PMNs) were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 4 hours, and cell-free NETs were isolated, washed, and stored in PBS at 4 °C for up to 24 hours. Before use in assays, frozen NETs (frozen as cell-free NET pellet) were thawed and resuspended in PBS.

[0263] The basic assay included isolation of platelet-rich plasma (PRP) and plasma from healthy donors. NET-induced aggregation was evaluated using fresh and frozen NETs. Using the optimal NET-inducing conditions identified above, CM24 (500 μg / ml) and isotype control were added to the assay to evaluate the effect of the antibody on platelet aggregation.

[0264] Platelet-rich / poor plasma (PRP / PPP) preparation:

[0265] 1. Collect 40 ml of blood.

[0266] a. Note: Donors should not take aspirin, antihistamines, antibiotics, or antiplatelet drugs 14 days before blood draw.

[0267] 2. Pool the blood and centrifuge at 175 × g for 20 minutes at 25 °C in a swinging-bucket rotor with minimal braking.

[0268] 3. Collect the top yellow platelet-rich plasma (PRP), and count platelets using 2 μg / ml calcein AM, incubate at 37 °C for 20 minutes, and count green fluorescent cells on a Nexcelom cell counter.

[0269] 4. Centrifuge approximately 1 ml of PRP at 15,000 × g for 2 minutes. Collect the supernatant, platelet-poor plasma (PPP).

[0270] Assay setup:

[0271] 1. Add the antibodies (hIgG4 and CM24) to PRP and incubate at 37 °C for 30 minutes.

[0272] 2. Add 5 μl of NETs to the appropriate wells of a clear-bottom 96-well plate. Dilute in Tyrodes-HEPES buffer.

[0273] 3. Add 95 μl of PPP / PRP / PRP + antibody and read the absorbance at 595 nm.

[0274] a. Oscillate between readings at 37 °C at 807 rpm in a plate reader or at room temperature at 1000 rpm in a microplate reader.

[0275] At each time point, calculate the percentage of aggregation for each well using the following formula, using only PRP as 0% and PPP as 100%:

[0276]

[0277] Figure 8 And the results shown in Table 5 indicate that both fresh and frozen NETs at 5 ng / μL, 10 ng / μL, and 20 ng / μL are able to effectively induce platelet aggregation in a dose-dependent manner.

[0278] Table 5. NET concentration-dependent platelet aggregation (AUC).

[0279]

[0280] To evaluate the in vitro antithrombotic effect of CM24, CM24 or its isotype control was added to the ADP-induced platelet aggregation assay described above. PRP was incubated with 500 μg / ml of CM24 or hIgG4 at 37 °C for 30 minutes. The pretreated platelets were combined with ADP in the assay plate, and aggregation was quantified as described in the method section. As Figure 9 And as shown in Table 6, CM24 significantly inhibited ADP-induced platelet aggregation compared to the isotype control.

[0281] Table 6. CM24 or isotype control in the ADP-induced platelet aggregation assay.

[0282]

[0283] To evaluate the in vitro antithrombotic effect of CM24, CM24 and its isotype control were added to the NET-induced platelet aggregation assay. PRP was incubated with 500 μg / ml of CM24 or hIgG4 at 37 °C for 30 minutes. The pretreated platelets were combined with 5 ng / μl and 10 ng / μl NETs in the assay plate, and aggregation was quantified as described in the method section. As Figure 10 And as shown in Table 7, in the absence of treatment, NETs induced platelet aggregation. CM24 significantly inhibited NET-induced platelet aggregation compared to untreated and isotype control.

[0284] Summary data showing the percentage of aggregation at the assay endpoint are shown in Table 8, while Figure 10 and Table 7 shows the AUC of the percentage of aggregation throughout the assay. At fresh NET concentrations of 5 ng / μl and 10 ng / μl, addition of CM24 almost completely blocked NET-induced platelet aggregation. The isotype control antibody reduced platelet aggregation to a lesser extent, but the difference between CM24 and the isotype control was highly significant when using fresh NETs. The inhibition of platelet aggregation by CM24 showed complete blockade at 5 ng / μl NET and significant inhibition at 10 ng / μl NET. Based on the MPO levels measured in patient serum samples, 5 ng / μL NETs is the most appropriate concentration for use in vitro, as Figure 7 illustrated in

[0285] Table 7. Platelet aggregation induced by fresh NETs. Inhibition by CM24 is shown compared to isotype and untreated controls. Data are mean AUC (% aggregation relative to time) values in a 30-minute experiment.

[0286]

[0287] Table 8. Platelet aggregation induced by fresh NETs and inhibitor-treated. Data are percentage of aggregation at the end of the study (30 minutes).

[0288]

[0289] Example 7: CM24 interferes with the adhesion of cancer cells expressing CEACAM1 to NETs.

[0290] Materials

[0291]

[0292] Fresh NETs were isolated and resuspended in PBS one day before the experiment and stored at 4°C. Frozen NETs were also used and resuspended in PBS. On the day of the experiment, both fresh and frozen NETs were stained.

[0293] Cancer cells were cultured according to the recommendations of ATCC. Cells were removed using trypsin and stained with CytoTell Red (CTR) 650 in PBS and washed thoroughly to remove excess stain. Some cells were left unstained to serve as compensation controls. After CTR staining, the cells were incubated in a U-bottom polypropylene 96-well plate with 500 μg / ml CM24 or isotype control at 37 °C for 30 minutes. Then freshly prepared and frozen NETs were added to the wells such that the final concentration in each well was 4 ng / μl. The NETs and cells were shaken at 500 rpm at 37 °C for 2 hours. The cells were maintained in buffer in the presence of BSA and absence of serum. After incubation, the cells were washed vigorously and then stained for nuclei blue in PBS + 1% BSA and then acquired on a MASCQuant flow cytometer. Data were analyzed using FlowJo (TreeStar, Inc).

[0294] As detailed in Example 3 and Table 4 above, adhesion of CEACAM1-positive human melanoma SK-MEL-28 cells to NETs was shown by FACS analysis compared to adhesion of Jurtkat cells that do not express CEACAM1. Inhibition of this adhesion was further tested by using CM24 or isotype control. Figure 11 The results depicted in showed that CM24 significantly reduced the adhesion of SK-MEL-28 cancer cells to NETs, while no significant effect of the isotype control was observed.

[0295] Conclusion of experimental results

[0296] In vitro assays were used to show NET-induced platelet aggregation in a dose-dependent manner. CM24 significantly inhibited NET-induced platelet aggregation. Compared to the isotype control, at 5 ng / μl and 10 ng / μl of freshly prepared NETs, addition of CM24 reduced platelet aggregation by 70% and 53%, respectively.

[0297] In vitro platelet aggregation studies showed the potential of CM24 to inhibit both NET-induced platelet aggregation and platelet aggregation induced by other stimuli such as ADP. These results suggest that blocking CEACAM-1 using CM24 can effectively inhibit blood coagulation, suggesting the use of CM24 for inhibiting thrombosis and other non-malignant NET-related conditions as well as cancer-related thrombosis and metastasis.

[0298] Treatment of cancer cells with CM24 in vitro results in reduced binding of cancer cells to NETs and decreased NET-induced cell migration. In the dose-escalation part of a clinical trial study (NCT 04731467), patients with advanced solid tumors (mainly PDAC and CRC patients) who had received 2 prior lines of therapy were administered 10 mg / kg, 15 mg / kg, and 20 mg / kg of CM24 every other week, and 480 mg of nivolumab every four weeks. In serum samples collected from the patients, the levels of MPO (a known NET marker) were significantly reduced after treatment.

[0299] The expanded clinical phase of CM24 in combination with nivolumab and Nal-IRI / 5FU / LV was followed in patients with pancreatic adenocarcinoma. In serum samples collected from these patients, high pre-treatment levels of serum MPO were detected in patients who subsequently showed disease control (PR, SD), while patients with no response (PD) showed low pre-treatment levels of MPO similar to those observed in healthy donors. In addition, a significant reduction in MPO levels was observed in patients who showed disease control (PR, SD) to CM24 but not in those who did not (PD), suggesting that CM24 is a potential treatment for NET-related diseases and complications and for inhibiting cancer invasion and metastatic spread.

[0300] The above observations suggest that serum NET markers, such as MPO, can be used as biomarkers for selecting patients for anti-CEACAM1 treatment and for monitoring and staging patients during treatment.

[0301] 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 such specific embodiments for various applications without undue experimentation and without departing from the general concept, and, therefore, such adaptations and modifications are intended to be and should be embraced within the meaning and range of the 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. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A pharmaceutical composition comprising an anti-CEACAM1 mAb or an active fragment thereof, and a pharmaceutically acceptable salt, carrier or diluent, for use in preventing, inhibiting or delaying a pathological process, condition or disorder involving NET-mediated activity in a subject, wherein the mAb or active fragment comprises a set of six CDR sequences comprising SEQ ID No. 1-6.

2. A method of preventing, inhibiting or delaying a pathological process, condition or disorder involving NET-mediated activity in a subject, the method comprising administering to the subject a therapeutically effective dose of an anti-CEACAM1 mAb or an active fragment thereof, wherein the mAb or antibody fragment comprises a set of CDR sequences comprising SEQ ID No. 1-6.

3. The method or pharmaceutical composition according to any one of claims 1 and 2, wherein the formation of NETs in the subject is inhibited.

4. The method or pharmaceutical composition for use according to any one of claims 1 and 2, wherein the pathological process or condition involving NET-mediated activity is cancer or a tumor.

5. The method or pharmaceutical composition for use according to claim 4, wherein the cancer comprises solid tumors.

6. The method or pharmaceutical composition for use according to any one of claims 4 and 5, wherein the cancer is metastatic cancer or a tumor.

7. The method or pharmaceutical composition for use according to any one of claims 4-6, wherein the cancer is selected from the group consisting of: epithelial cancer, lymphoma, blastoma, sarcoma, melanoma, cancer of unknown primary, skin cancer, lung cancer, thyroid cancer, parathyroid cancer, breast cancer, heart cancer, thymic cancer, bone cancer, soft tissue cancer, brain cancer, retinal cancer, ophthalmic cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, pancreatic cancer, biliary tract cancer, liver cancer, bladder cancer, adrenal cancer, kidney cancer, urogenital cancer, testicular cancer, cervical cancer, fallopian tube cancer, ovarian cancer, uterine cancer, vulvar cancer or endometrial cancer.

8. The method or pharmaceutical composition for use according to claim 4, wherein the cancer is selected from pancreatic cancer, colorectal cancer, lung cancer and melanoma.

9. The method or pharmaceutical composition for use according to any one of claims 4-8, wherein administration of the anti-CEACAM1 mAb or antibody fragment results in the prevention, inhibition or delay of at least one of the following: formation of metastases, migration or spread of metastases, adhesion of metastases, progression or growth of metastases, intravasation of cancerous cells into the vasculature, invasion of cancer cells into the surrounding extracellular matrix, survival of cancer cells in the bloodstream, extravasation of cancer cells into the organ parenchyma, formation of dormant cells or multicellular metastases, and seeding and exponential growth of distant metastasis colonies.

10. The method or pharmaceutical composition for use according to claim 9, wherein the formation, migration or spread of metastases after tumor resection surgery is prevented or inhibited.

11. The method or pharmaceutical composition for use according to any one of claims 4-10, comprising administering at least one additional anti-cancer therapy.

12. The method or pharmaceutical composition for use according to claim 11, wherein the at least one additional anti-cancer therapy is selected from the group consisting of chemotherapy, radiation, surgery, and immunotherapy.

13. The method or pharmaceutical composition for use according to any one of claims 1-12, wherein the subject is a cancer patient who has undergone tumor resection surgery.

14. The method or pharmaceutical composition for use according to claim 13, wherein the subject who has undergone surgery has been treated with at least one additional anti-cancer therapy.

15. The method or pharmaceutical composition for use according to claim 14, wherein the therapy is chemotherapy, and administration of the mAb results in inhibition of chemotherapy-induced thrombosis.

16. The method or pharmaceutical composition for use according to claim 12, wherein the additional anti-cancer therapy comprises administration of an anti-cancer agent selected from the group consisting of immunomodulators, immunocytotherapy agents, kinase inhibitors, and chemotherapeutic agents.

17. The method or pharmaceutical composition for use according to claim 16, wherein the immunomodulator is an inhibitor of an immune checkpoint molecule.

18. The method or pharmaceutical composition for use according to claim 17, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

19. A pharmaceutical composition comprising an anti-CEACAM1 mAb or an active fragment thereof for use in treating NET-related diseases, disorders, or complications in a subject in need of such treatment, the use comprising: (i) determining the level of at least one NET biomarker in a biological sample from a subject diagnosed with cancer; (ii) comparing the level of the at least one NET biomarker with a reference value or a control sample value; and (iii) if the level of at least one NET biomarker in the sample is significantly higher than the reference value or the control sample value, administering an anti-CEACAM1 antibody to the subject.

20. The pharmaceutical composition for use according to claim 19, wherein the NET-related disease is cancer.

21. The pharmaceutical composition for use according to claim 19, wherein the NET-related disease, disorder, or complication is a non-malignant condition.

22. The pharmaceutical composition for use according to any one of claims 19-21, wherein the NET biomarker is myeloperoxidase (MPO).

23. A method of treating NET-related diseases, disorders, or complications in a subject in need of such treatment, the method comprising: (i) determining the level of at least one MPO biomarker in a biological sample obtained from the subject; (ii) comparing the level of the at least one NET biomarker with a reference value or a control sample value; and (iii) if the level of at least one NET-biomarker in the sample is significantly higher than the reference value or the control sample value, administering an anti-CEACAM1 mAb or an active fragment thereof to the subject.

24. The method according to claim 23, wherein the subject is diagnosed with cancer or suspected of having cancer.

25. The method according to claim 23, wherein the NET-related disease, disorder or complication is a non-malignant condition.

26. The method according to claims 23-25, wherein the NET biomarker is MPO.

27. A method for selecting cancer patients suitable for treatment with an anti-CEACAM1 mAb or an active fragment thereof, comprising the steps of: (i) Provide a biological sample from the subject; (ii) Determine the level of MPO in the sample of step (i), and (iii) Compare the MPO level with a reference value or a control sample value, wherein an increase in the MPO level relative to the reference value or the control sample value indicates that the subject may have a therapeutic response to the anti-CEACAM1 mAb or an active fragment thereof.

28. A pharmaceutical composition comprising an anti-CEACAM1 mAb or an active fragment, for use in treating a subject diagnosed with cancer or suspected of having cancer, wherein the use comprises selecting cancer patients suitable for the anti-CEACAM1 antibody, and the selection comprises the following steps: (i) Provide a biological sample from the subject; (ii) Determine the level of MPO in the sample of step (i), and (iii) Compare the MPO level with a reference value or a control sample value, wherein an increase in the MPO level relative to the reference value or the control sample value indicates that the subject may have a therapeutic response to the anti-CEACAM1 mAb or an active fragment thereof.

29. The method or pharmaceutical composition for use according to any one of claims 19-28, wherein a significantly higher MPO level corresponds to an increase of at least about 100%, at least about 200% or at least about 300% relative to the reference value or the control sample value.

30. The method or pharmaceutical composition for use according to any one of claims 19-29, wherein the biological sample is a blood sample.

31. The method or pharmaceutical composition for use according to any one of claims 19-29, wherein the biological sample is a biopsy.

32. The method or pharmaceutical composition for use according to any one of claims 19 - 31, wherein the anti-CEACAM1 mAb or active fragment comprises a set of six CDR sequences, wherein the heavy chain CDR1 (HC-CDR1) comprises the sequence GYAFTNNLIE (SEQ ID NO:1), the heavy chain CDR2 (HC-CDR2) comprises the sequence VINPGSGDTNYNEKFKG (SEQ ID NO:2), the heavy chain CDR3 (HC-CDR3) comprises the sequence GDYYGGFAVDY (SEQ ID NO:3), the light chain CDR1 (LC-CDR1) comprises the sequence RTSQDIGNYLN (SEQ ID NO:4), the light chain CDR2 (LC-CDR2) comprises the sequence YTSRLHS (SEQ ID NO:5), and the light chain CDR3 (LC-CDR3) comprises the sequence QQGKSLPRT (SEQ ID NO:6).

33. The method or pharmaceutical composition for use according to any one of claims 19 - 32, wherein the anti-CEACAM1 mAb or active fragment comprises the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:

8.

34. The method or pharmaceutical composition for use according to any one of claims 19 - 33, wherein the anti-CEACAM1 mAb is CM24.

35. The method or pharmaceutical composition for use according to any one of claims 1 - 3, wherein the pathological process or condition involving NET-mediated activity is a non-malignant condition.

36. The method or pharmaceutical composition for use according to claim 35, wherein the non-malignant condition is a disease or disorder selected from the following: thrombotic diseases, thrombosis, disease-related thrombosis, prothrombotic conditions, thromboinflammatory conditions, venous thromboembolism, arterial thromboembolism, cardiovascular conditions, autoimmune diseases, autoinflammatory diseases or disorders, immune-mediated diseases, systemic inflammatory conditions.

37. The method or pharmaceutical composition for use according to claim 35, wherein the non-malignant condition is a non-malignant thrombotic disease or disorder.

38. The method or pharmaceutical composition for use according to claim 37, wherein the non-malignant thrombotic disease is a thrombotic cardiovascular disease.

39. The method or pharmaceutical composition for use according to claim 38, wherein the thrombotic cardiovascular disease is selected from: myocardial infarction, carotid atherosclerosis, cerebrovascular stroke, deep vein thrombosis (DVT), portal vein thrombosis, marantic endocarditis, pulmonary embolism, and chronic thromboembolic pulmonary hypertension.

40. The method or pharmaceutical composition for use according to claim 37, wherein the non-malignant thrombotic disease is a hematological disease.

41. The method or pharmaceutical composition for use according to claim 40, wherein the hematological disease is thrombotic thrombocytopenic purpura (TTP), or heparin-induced thrombocytopenia or thrombosis.

42. The method or pharmaceutical composition for use according to claim 37, wherein the non-malignant thrombotic disease is an autoimmune disease.

43. The method or pharmaceutical composition for use according to claim 42, wherein the autoimmune disease is selected from: systemic lupus erythematosus (SLE), antiphospholipid syndrome (APS), rheumatoid arthritis (RA), psoriasis, ulcerative colitis, gout, systemic sclerosis, ANCA-associated vasculitis, dermatomyositis, and polymyositis.

44. The method or pharmaceutical composition for use according to claim 37, wherein the non-malignant thrombotic disease is a systemic inflammatory response syndrome.

45. The method or pharmaceutical composition for use according to claim 44, wherein the systemic inflammatory response syndrome is COVID-19 infection, sepsis, or septic shock.

46. A method of preventing, delaying, or inhibiting thrombosis in a subject, comprising administering an anti-CEACAM1 mAb or an active fragment thereof to a subject in need thereof.

47. The method according to claim 46, wherein the thrombosis is treatment-induced thrombosis.

48. The method according to claim 47, wherein the treatment is selected from immunotherapy, surgery, hormone therapy, and chemotherapy.

49. The method according to claim 47, wherein treatment-induced thrombosis is inhibited, delayed, or prevented.

50. A kit for predicting the response of a subject to anti-CEACAM1 antibody treatment, the kit comprising means for determining the level of at least one NET-biomarker in a biological sample, means for comparing the expression level of the NET-biomarker with a reference value or control sample value; and instructional material for guiding the correlation between the ratio of the NET biomarker to the reference or control level.

51. The kit according to claim 50, wherein the subject is diagnosed with cancer or suspected of having cancer.

52. The kit according to claim 50, wherein the subject is diagnosed with a non-malignant NET-related disease, disorder, or complication.

53. The kit according to any one of claims 50-52, wherein the at least one NET-biomarker is MPO.

54. The method, pharmaceutical composition for use, or kit according to any one of claims 1-53, wherein the anti-CEACAM1 mAb or fragment thereof comprises a set of six CDR sequences, wherein HC-CDR1 consists of GYAFTNNLIE (SEQ ID NO:1), HC-CDR2 consists of VINPGSGDTNYNEKFKG (SEQ ID NO:2), HC-CDR3 consists of GDYYGGFAVDY (SEQ ID NO:3), LC-CDR1 consists of RTSQDIGNYLN (SEQ ID NO:4), LC-CDR2 consists of YTSRLHS (SEQ ID NO:5), and LC-CDR3 consists of QQGKSLPRT (SEQ ID NO:6).

55. The method, pharmaceutical composition for use, or kit according to any one of claims 1-54, wherein the anti-CAECAM1 mAb is selected from chimeric antibodies, humanized antibodies, and partially humanized antibodies.

56. The method, pharmaceutical composition for use, or kit according to any one of claims 1-55, wherein the anti-CEACAM1 mAb comprises a heavy chain variable region containing the sequence listed in SEQ ID NO:7 and a light chain variable region containing the sequence listed in SEQ ID NO:8, or an active fragment thereof, or an antibody analogue or derivative having at least 90% identity with any of the chain sequences.

57. The method, pharmaceutical composition for use, or kit according to any one of claims 1-56, wherein the mAb has a heavy chain constant region selected from human IgG4 and human IgG1 and a human κ light chain constant region.

58. The method, pharmaceutical composition for use, or kit according to any one of claims 1-57, wherein the mAb is CM24, comprising the heavy chain sequence listed in SEQ ID NO:9 and the light chain sequence listed in SEQ ID NO:10, or an active fragment thereof, or an antibody analogue or derivative having at least 90% identity with any of the chain sequences.

Images