Anti-carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) antibodies for inhibition of neutrophil extracellular trap (NET)-mediated activity

Abstract

Pharmaceutical compositions containing anti-CEACAM 1 mAbs and their use in inhibiting NET-mediated activities and in the prevention and treatment of conditions associated with these activities are provided, exemplified by mAb CM24, which has also been shown to effectively inhibit cancer cell migration and NET-induced platelet aggregation. Additionally, prognostic methods based on NET biomarkers for use in patient selection and monitoring treatment efficacy are presented.

Description

[Technical Field]

[0001] The present invention is in the field of immunotherapy and cell biology and relates to CEACAM 1 targeting antibodies for the prevention and treatment of processes and disorders involving the activity of neutrophil extracellular traps (NETs). [Background technology]

[0002] Neutrophils are the most abundant circulating leukocytes in humans and are an essential component of the host response to pathogens. During infection, neutrophils migrate from peripheral blood to tissues in response to several chemotactic stimuli released within inflammatory sites. They can rapidly kill pathogens after phagocytosis but also through the release of their potent antimicrobial substances, including granule enzymes and proteins, oxidants (reactive oxygen species: ROS), and neutrophil extracellular traps (NETs). Neutrophils also infiltrate tumors and have been proposed as important mediators of neoplastic transformation, tumor progression, angiogenesis, and the regulation of immune responses.

[0003] Cancer-associated inflammation is a driving force for tumor initiation and progression, enabling cancer cells to escape immune surveillance. Infiltrating and resident immune cells in the tumor microenvironment (TME) play fundamental roles in tumor growth, metastasis formation, and response to immunotherapy. There is increasing evidence that circulating and infiltrating neutrophils also play multiple roles in tumor initiation and progression.

[0004] NETs are extracellular decondensed chromatin networks that may contain granule proteins, DNA, histones, and other materials. NETs are produced by neutrophils to phagocytose and kill pathogens and can form during infection, inflammation, and / or thrombosis. More specifically, when neutrophils detect pathogens, granule proteins, DNA, and / or histones may bind within the neutrophil. Neutrophils can then expel the bound granule proteins, DNA, and / or histones by disintegrating the nucleus and granule membrane, allowing the intracellular material to be "excreted" from the cell and form NETs. NETs can then capture, bind, phagocytose, and / or kill pathogens in a process called NETosis. Because NETs originate from neutrophils and play a role in fighting infection, they are commonly found extravascularly in inflamed or infected tissues.

[0005] Various markers have been tested to demonstrate NETosis. Methods to assess NETosis include the detection of colocalized neutrophil-derived proteins, extracellular DNA, and citrullinated histones, as well as the detection of NET remnants in fluid samples and flow cytometry detection of 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. Focusing on the specificity, objectivity, and quantification of NET markers, widely accepted NET markers used in clinical studies include 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) and the combination of cell-free DNA and CitH3 (Pranav et al. Front Immunol. 2019 Jan 24:10:28).

[0006] NETS also play an important role in the TME, contributing to cancer immunoediting, progression, and metastatic spread. NETs enhance tumor aggressiveness by enhancing cancer migration and invasion, and can trap circulating cancer cells, promoting the formation and spread of metastases.

[0007] 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 upregulated in T cells and NK cells upon activation, and its homophilic interaction leads to inhibition of lymphocyte cytotoxicity. Studies of several human tumor types suggest that exploitation of the CEACAM1 pathway may enable tumor immune evasion. CEACAM1 plays an important role in tumor immune evasion, metastasis, and angiogenesis, and its expression in primary cutaneous melanoma lesions strongly predicts the development of metastatic disease with poor prognosis. Furthermore, 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 blocking CEACAM1 interactions with monoclonal antibodies (mAbs) can enhance the immune response against tumors. CEACAM1 has also been suggested as a putative therapeutic target for preventing metastatic progression of colon cancer (Rayes et al., Immunol. 2020 April 15;204(8):2285-2294).

[0008] CEACAM1 is an intercellular adhesion regulator associated with angiogenesis and Fas-mediated apoptosis via its interaction with β-catenin, which enhances the cytotoxicity of natural killer cells against tumor cells. 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 through the inhibition of immune activity of tumor-infiltrating lymphocytes (TILs), reduced phosphorylation of immune receptors, and reduced SHP1 / 2 phosphorylation levels in T and NK cells.

[0009] International Patent Publication No. 2013 / 054331 discloses mAbs specific for human CEACAM1, which contain a specific set of complementarity-determining regions (CDRs). Chimeric antibodies are also disclosed, including the human / mouse antibody CM10.

[0010] International Patent Publication No. 2015166484 discloses humanized anti-CEACAM1 mAbs with a specific set of CDR sequences in a human framework and several back mutations. Among the claimed mAbs is a highly potent mAb called CM24.

[0011] CM24 is a humanized IgG4 mAb that binds specifically and with high affinity to the extracellular domain of CEACAM1. CM24 is a first-in-class clinical-stage mAb targeting CEACAM1 and has great potential for treating multiple cancers. Blockade of CEACAM1-CEACAM1 and CEACAM1-CEACAM5 interactions with CM24 is associated with antiangiogenesis, immune access, and checkpoint release mechanisms, enabling lymphocyte cytotoxic activity and tumor cell killing by T and NK cells.

[0012] CM24 is currently being studied in an open-label, multicenter, multidose-escalation and dose-expansion study (https: / / clinicaltrials.gov / ct2 / show / NCT04731467) in combination with nivolumab (anti-PD-1) in adults with selected advanced solid tumors (e.g., advanced, relapsed, refractory non-small cell lung cancer, metastatic pancreatic cancer).

[0013] There is an unmet need to provide effective agents that can inhibit NET-mediated activities for the prevention and treatment of a wide range of diseases and disorders associated with these activities. Summary of the Invention

[0014] The present invention provides pharmaceutical compositions comprising the humanized anti-CEACAM 1 mAb CM24, or mAbs comprising the same set of CDR sequences, and their use in inhibiting NET-mediated activities and in the prevention and treatment of conditions associated with these activities.

[0015] Because NET-mediated activity is involved in a wide range of pathologies, including cardiovascular, hematological, autoimmune, neoplastic, and inflammatory diseases, anti-CEACAM1 mAbs, and in particular CM24, can be effectively used to inhibit or delay NET-induced processes in these pathologies.

[0016] The present invention is based on the favorable results of utilizing CM24 in blocking several NET-mediated activities. In a recent dose-escalation portion of a Phase 1 / 2 trial of CM24 and nivolumab (NCT 04731467), and in an exploratory study conducted as part of this trial, it was unexpectedly found that CM24 therapy resulted in a significant reduction in serum NET levels in pancreatic cancer patients, and that this reduction remained significant for at least two weeks after CM24 / nivolumab treatment (a sustained effect). It was further advantageously found that pretreatment serum levels of myeloperoxidase (MPO), a NET marker, increased in patients who showed stable disease (SD) or partial response (PR) and survived longer, but not in patients who showed no 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 durable decrease in serum MPO was detected in patients who showed SD or PR, but not in patients who did not respond (PD). It is therefore disclosed for the first time that MPO can be used as a prognostic biomarker for selecting patients eligible for treatment with anti-CEACAM 1 antibodies and for monitoring the effectiveness of treatment with such antibodies.

[0017] In cancer, blocking, inhibiting, or slowing these NET-mediated activities results in blocking, inhibiting, or slowing intravasation into the vasculature, preventing cancer cell survival within the bloodstream, extravasation into organ parenchyma, and the formation of dormant cells or multicellular micro- and macrometastases.

[0018] Herein, for the first time, it is shown that CM24 effectively inhibits cancer cell migration and suppresses metastatic activity in vivo, thereby inhibiting disease progression. The present invention provides methods that utilize CM24, mAbs containing the same set of CDR sequences, or antibody fragments containing at least the binding site of CM24 to block the metastatic cascade driven by NETosis, thereby effectively disrupting the metastatic cascade and preventing the dissemination, spread, and subsequent exponential growth of distant metastatic colonies. The present invention is advantageous because it utilizes immunological methods for the treatment and, in some cases, even prevention of pathological conditions such as cancer and NET-associated thrombotic diseases and disorders, rather than relying on cytotoxic therapies, greatly reducing the adverse side effects associated with such treatments.

[0019] The present invention further provides a method for selecting subjects diagnosed with cancer for treatment with an anti-CEACAM 1 mAb or a fragment or conjugate thereof or for monitoring therapeutic effectiveness, wherein the level of a NET marker, e.g., myeloperoxidase (MPO), is used to optimize patient selection and monitor therapeutic effectiveness.

[0020] Thus, according to one aspect, the present invention provides a method for preventing or inhibiting NET-mediated activity, comprising utilizing a mAb or an active fragment or conjugate 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).

[0021] According to some embodiments, the 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 VINPGSDTNYNEKFKG (SEQ ID NO: 2), HC-CDR3 consists of GDYYGGFAVDY (SEQ ID NO: 3), LC-CDR1 consists of RTSQDIGNYLN (SEQ ID NO: 4), LC-CDR2 YTSRLHS consists of (SEQ ID NO: 5), and LC-CDR3 consists of QQGKSLPRT (SEQ ID NO: 6).

[0022] According to another aspect, the present invention also provides a method for preventing, inhibiting, or delaying a pathological process or condition associated with NET-mediated activity, comprising administering to a subject in need thereof a mAb or an 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 YTSRLHS consists of (SEQ ID NO: 5), and LC-CDR3 consists of QQGKSLPRT (SEQ ID NO: 6).

[0023] According to another aspect, the present invention also provides a pharmaceutical composition comprising a mAb or an active fragment thereof comprising a set of six CDR sequences, where HC-CDR1 consists of GYAFTNNLIE (SEQ ID NO: 1), HC-CDR2 consists of VINPGSDTNYNEKFKG (SEQ ID NO: 2), HC-CDR3 consists of GDYYGGFAVDY (SEQ ID NO: 3), LC-CDR1 consists of RTSQDIGNYLN (SEQ ID NO: 4), LC-CDR2 YTSRLHS consists of SEQ ID NO: 5, and LC-CDR3 consists of QQGKSLPRT (SEQ ID NO: 6), and a pharmaceutically acceptable salt, carrier, or excipient, for use in preventing or delaying NET-mediated activity and for preventing, inhibiting, or delaying pathological processes or disorders associated with NET-mediated activity.

[0024] In some embodiments, the anti-CAECAM1 antibody is a chimeric antibody. In other embodiments, the anti-CEACAM1 antibody is a humanized or partially humanized antibody.

[0025] In other embodiments, the anti-CEACAM1 comprises a heavy chain variable region comprising the sequence QVQLVQSGAEVKKPGASVKVSCKASGYAFTNNLIEWVRQAPGQGLEWIGVINPGSGDTNYNEKFKGRVTMTADKSISTAYMELSRLRSDDTAVYYCARGDYYGGFAVDYWGQGTTVTVSS (SEQ ID NO: 7), and a light chain variable region comprising the sequence DIQMTQSPSSLSASVGDRVTITCRTSQDIGNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQGKSLPRTFGGGTKVEIK (SEQ ID NO: 8), or an active fragment thereof comprising at least the binding site, or an antibody analog or derivative thereof having at least 90% identity to either of the chain sequences.

[0026] According to some embodiments, the heavy chain variable region of the antibody comprises an amino acid sequence at least about 95% identical to sequence SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence at least about 95% identical to sequence SEQ ID NO:8. According to some embodiments, the heavy chain variable region of the antibody comprises an amino acid sequence at least about 97% identical to sequence SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence at least about 97% identical to sequence SEQ ID NO:8. According to some embodiments, the heavy chain variable region of the antibody comprises an amino acid sequence at least about 99% identical to sequence SEQ ID NO:7, and the light chain variable region comprises an amino acid sequence at least about 99% identical to sequence SEQ ID NO:8. Each alternative represents a separate embodiment of the invention.

[0027] According to some embodiments, the antibody or fragment thereof is an IgG mAb. According to 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 kappa light chain constant region. Each option represents a separate embodiment of the present invention.

[0028] In an exemplary embodiment, anti-CEACAM1 has the heavy chain sequence QVQLVQSGAEVKKPGASVKVSCKASGYAFTNNLIEWVRQAPGQGLEWIGVINPGSGDTNYNEKFKGRVTMTADKSISTAYMELSRLRSDDTAVYYCARGDYYGGFAVDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9) and light chain sequence DIQMTQSPSSLSASVGDRVTITCRTSQDIGNYLNWYQQKPGKAVKLLIYYTSRLHSGVPSRFSGSGSG TDYTLTISSLQPEDIATYFCQQGKSLPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10), or an active fragment thereof comprising at least the binding site, or an antibody analog or derivative thereof having at least 90% identity with any of the said chain sequences.

[0029] The antibodies of methods and compositions of the invention also include conjugates comprising the antibody or fragment thereof, which, according to some embodiments, may comprise the antibody or fragment thereof linked to a cytotoxic moiety, a radioactive moiety, or an affinity or labeling tag.

[0030] Any disease or disorder in which NETs are highly implicated in its pathogenesis or progression and / or in which inhibition of NET activity results in amelioration, delay, etc. of symptoms is eligible for treatment using the mAbs of the invention.

[0031] In some embodiments, the subject in need of treatment has been diagnosed with a neoplastic disease, i.e., cancer. In some embodiments, the neoplastic disease is a solid tumor. According to some embodiments, the cancer is a metastatic cancer or tumor. In some embodiments, the neoplastic disease is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, melanoma, cancer of unknown primary, skin, lung, thyroid, parathyroid, breast, heart, thymus, bone, soft tissue, brain, retinal, ophthalmic, head and neck, esophageal, gastric, colorectal, prostate, pancreatic, biliary, liver, bladder, adrenal, renal, genitourinary, testicular, cervical, fallopian tube, ovarian, uterine, vulvar, 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 option represents a separate embodiment of the present invention.

[0032] According to some embodiments, the cancer is selected from the group consisting of pancreatic cancer, lung cancer, and melanoma.

[0033] According to some embodiments, treatment with the anti-CEACAM1 antibodies or antibody fragments of the present invention 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, intravasation of cancer cells into the vasculature, survival of cancer cells within the bloodstream, extravasation of cancer cells into organ parenchyma, and formation of dormant cells or multicellular metastases.

[0034] In a further aspect, the present invention provides a method for preventing, delaying, or inhibiting the formation, migration, spreading, adhesion, or progression of metastases, comprising administering a mAb against CEACAM1 as defined above.

[0035] In some embodiments, the formation, migration or spread of metastases following tumor removal is prevented or inhibited.

[0036] 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.

[0037] The present invention further provides an anti-CEACAM 1 mAb for use in treating a NET-mediated disorder or complication in a subject in need of such treatment, the method comprising: (i) determining the level of at least one NET biomarker in a biological sample obtained from the subject; (ii) comparing the level of at least one NET biomarker to a reference value of a control sample value; (iii) administering an anti-CEACAM 1 antibody to the subject if the NET biomarker level in the sample is significantly higher than the reference value or control sample value.

[0038] According to some embodiments, the subject is a patient diagnosed with or suspected of having cancer.

[0039] According to some embodiments, the subject has been diagnosed with or is suspected of having a non-malignant NET-related disease, disorder, or complication, including a treatment-induced complication.

[0040] According to some embodiments, the levels of at least two NET biomarkers are measured.

[0041] Any marker known in the art for detecting NETosis can be used with the methods of the present invention. According to some embodiments, detection of NETosis includes detection of co-localized neutrophil-derived proteins and extracellular DNA and citrullinated histones, detection of NET remnants in fluid samples, and flow cytometry detection of cell-associated NET components. NET markers 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 marker is selected from MPO, NE, and DNA complexes.

[0042] According to some embodiments, the 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, the at least one NET marker is selected from MPO, NE, and DNA complexes. According to particular embodiments, the NET marker is MPO. Each option represents a separate embodiment of the present invention.

[0043] According to some particular embodiments, the NET biomarker is MPO.

[0044] Thus, the present invention, according to some embodiments, provides an anti-CEACAM 1 mAb for use in treating cancer in a subject in need thereof, the method comprising: (i) determining the level of myeloperoxidase (MPO) in a biological sample obtained from a subject diagnosed with cancer; (ii) comparing the MPO levels to a baseline of control sample values; (iii) administering an anti-CEACAM 1 antibody to the subject if the MPO level in the sample is significantly higher than the reference value or control sample value.

[0045] In some embodiments, a significantly elevated MPO level equates to an increase of at least about 100%, at least about 200%, or at least about 300% over the baseline or control sample value.

[0046] In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising: (i) determining the level of MPO in a biological sample obtained from a subject diagnosed with cancer; (ii) comparing the MPO level to a reference value or a control sample value; (iii) if the MPO level in the sample is significantly higher than the baseline or control sample level, administering anti-CEACAM1 to the subject.

[0047] In some embodiments, the biological sample is a blood sample. In yet 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 liquid biopsy, or particularly a tumor biopsy.

[0048] The present invention further provides a method for selecting a subject suitable for anti-CEACAM 1 antibody treatment, 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 control sample value, wherein a significant increase in the level of the NET biomarker relative to the reference value or control sample value indicates that the subject is likely to respond therapeutically to the anti-CEACAM 1 antibody.

[0049] According to some embodiments, the subject has been diagnosed with or is suspected of having cancer.

[0050] According to some embodiments, the subject has been diagnosed with or is suspected of having a non-malignant NET-related disease, disorder, or complication, including a treatment-induced complication.

[0051] According to some embodiments, the 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, the at least one NET marker is selected from MPO, NE, and DNA complexes. According to particular embodiments, the NET marker is MPO. Each option represents a separate embodiment of the present invention.

[0052] According to some embodiments, the levels of at least two NET markers are measured.

[0053] In some specific embodiments, the NET marker is MPO, and the present invention provides a method for selecting a cancer subject suitable for anti-CEACAM 1 antibody treatment, 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 control sample value, wherein a significant increase in the MPO level relative to the reference value or control sample value indicates that the subject is likely to respond therapeutically to the anti-CEACAM 1 antibody.

[0054] In some embodiments, the increase in MPO levels relative to the baseline or control sample value is equal to an increase of at least about 100%. In other embodiments, the increase is equal to at least about 200%. In yet other embodiments, the increase is equal to at least about 300%. In even more embodiments, an increased level of MPO identified in a patient characterizes the patient as predicted to develop a severe form of cancer.

[0055] In some embodiments, the anti-CEACAM 1 mAb or antibody fragment comprises the set of CDR sequences consisting of SEQ ID NOs: 1 to 6. In other embodiments, the anti-CEACAM 1 mAb is CM24.

[0056] In some embodiments, the subject selected for treatment has been diagnosed with a 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 has received anti-CEACAM 1 mAb therapy in combination with at least one other anti-cancer treatment, such as chemotherapy. In certain implementations, the patient has received anti-CEACAM 1 mAb therapy in combination with anti-PD-1 antibody therapy.

[0057] In another aspect, a method of inhibiting therapy-induced thrombosis is provided, comprising administering an anti-CEACAM1 mAb or a 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 thrombosis-inducing therapy is selected from immunotherapy, surgery, radiation, hormone therapy, and chemotherapy.

[0058] In a further aspect, a method of administering an anti-CEACAM 1 antibody to inhibit the invasiveness of cancer cells into the vasculature is provided.

[0059] In a further aspect, methods of administering anti-CEACAM 1 antibody therapy to inhibit the invasiveness of cancer cells into the surrounding extracellular matrix are provided.

[0060] In a further aspect, methods of administering anti-CEACAM 1 antibody therapy to inhibit cancer cell extravasation into organ parenchyma are provided.

[0061] In a further aspect, methods are provided for administering anti-CEACAM 1 antibody therapy to prevent dissemination and exponential growth of distant metastatic colonies.

[0062] Any administration route suitable for delivering a protein or antibody can be used with the compositions and methods of the present invention, and the administered composition will be formulated according to the mode of administration. In some embodiments, the mAb is administered parenterally. In 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.

[0063] Typically, intravenous (iv) administration by infusion or injection is used. In other embodiments, the anti-CEACAM 1 composition is administered via an intratumoral route. In other embodiments, the composition is administered during or after surgery.

[0064] Methods according to the present invention for treating cancer or a non-cancerous NET-associated disease or disorder, according to some embodiments, comprise administering to a subject in need thereof at least one dose of the above-described mAb against CEACAM1 in the range of 0.01 mg / kg to 50 mg / kg body weight.

[0065] According to some embodiments, 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.

[0066] According to some embodiments, the method comprises administering multiple doses of the mAb, wherein the multiple doses are the same or different. According to some embodiments, the method comprises administering multiple ascending doses. According to some embodiments, the method comprises administering at least one cycle of at least 12 weeks.

[0067] According to some embodiments, the treatment period is 2 to 60 weeks. According to other embodiments, the treatment period is 12 to 50 weeks. According to some specific embodiments, the treatment period is selected from the group consisting of 12 to 20 weeks, 20 to 30 weeks, and 30 to 50 weeks. According to still other embodiments, the treatment regimen comprises several administration cycles of at least 12 weeks each.

[0068] According to some embodiments, the treatment regimen comprises 1 to 8 cycles, each cycle comprising 2 to 6 infusions of the anti-CEACAM mAb over a period of at least 4 weeks. According to some embodiments, the treatment regimen comprises 2 to 6 cycles, each cycle comprising 4 infusions of the anti-CEACAM mAb over a period of at least 4 weeks.

[0069] According to some embodiments, administration is once every week, once every two weeks, once every three weeks, once every four weeks, or once every five weeks, with each possibility representing a separate embodiment of the present invention.

[0070] According to some embodiments, the treatment regimen comprises 1 to 10 cycles, each cycle comprising 2 to 5 infusions with the above-mentioned mAb every 1 to 4 weeks, followed by 2 to 8 weeks between each cycle.

[0071] According to some embodiments, a dose escalation regimen is provided that includes administration starting 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 yet other embodiments, the treatment regimen includes six cycles of four infusions, each administered every two weeks.

[0072] According to some embodiments, the subject is a human.

[0073] According to some embodiments, the human subject has been diagnosed with a NET-associated condition. According to some embodiments, the human subject has been diagnosed with cancer. According to some embodiments, the human subject is a cancer patient undergoing tumor removal surgery.

[0074] The methods and uses provided according to the present invention may involve sole treatment with an anti-CEACAM1 antibody or fragment, or may be included as part of a treatment regimen including at least one additional treatment.

[0075] In some embodiments, administration of an anti-CEACAM1 antibody for the treatment of cancer or prevention of 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.

[0076] According to some embodiments, a method of treating cancer comprises administering an anti-CEACAM1 antibody or fragment described herein and an additional anti-cancer agent, wherein the additional anti-cancer agent is selected from the group consisting of an immunomodulatory agent, an activated lymphocyte cell, an immunotherapy agent, a kinase inhibitor, and a chemotherapeutic agent.

[0077] According to some embodiments, the additional immunomodulatory agent is an inhibitor of an immune checkpoint molecule.

[0078] 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 an immune checkpoint molecule is an anti-PD-1 inhibitor, e.g., an antibody. In other embodiments, the immune checkpoint inhibitor is an anti-PD-L1 inhibitor, e.g., an antibody.

[0079] In another aspect, the invention provides methods for inhibiting NET formation in a subject, comprising administering to the patient an effective dose of an anti-CEACAM 1 antibody, particularly CM24. In some embodiments, inhibiting NET formation comprises preventing NET formation and / or reducing the likelihood that NETs will form in the subject. In some embodiments, inhibiting NET formation comprises inhibiting the growth or progression of existing NETs and / or reducing the likelihood that existing NETs will grow or progress in the subject. In some embodiments, methods of inhibiting NET formation result in a reduction in the severity of a symptom associated with the development of NETs. In some embodiments, the symptom associated with the development of NETs is thrombosis. In some embodiments, the subject receiving treatment to inhibit NET formation has or has been diagnosed with a cardiovascular condition. In some embodiments, the subject receiving treatment to inhibit NET-mediated activity has or has been diagnosed with a condition that predisposes the subject to thrombosis (i.e., prothrombotic).

[0080] According to some embodiments, the pathological condition, i.e., process or disorder involving NET-mediated activity, is a non-cancerous process or disorder. According to some embodiments, the NET-associated disease or disorder is a non-malignant thrombotic disease or disorder.

[0081] According to some embodiments of the present invention, the anti-CEACAM1 mAb or antibody fragment inhibits adhesion of non-cancerous cells to NET components.

[0082] Non-cancerous conditions, diseases, and disorders involving NET-mediated activity include, but are not limited to, thrombosis, thrombosis, pre-thrombotic conditions, venous thromboembolism, arterial thromboembolism, thrombo-inflammatory conditions, hematological conditions, cardiovascular conditions, autoimmune diseases, autoinflammatory diseases or disorders, immune-mediated diseases, and systemic inflammatory conditions. These and other NET-associated conditions are eligible for treatment using the compositions and methods of the present invention.

[0083] The compositions and methods of the present invention may also be referred to as thromboprophylaxis agents. In some embodiments, the effect achieved by the compositions and methods of the present invention is that of primary thromboprophylaxis, i.e., an effect aimed at directly minimizing the occurrence of thromboembolism, while in other embodiments, the effect is that of secondary thromboprophylaxis, i.e., prevention of recurrence of thromboembolism in subjects with a history of thrombotic events.

[0084] In some embodiments, the NET-associated disease or disorder is a thrombotic cardiovascular disease.

[0085] In some embodiments, the thrombotic cardiovascular disease is myocardial infarction. In some embodiments, the myocardial infarction is characterized by the presence of NETS in the subject's coronary thrombus. In additional embodiments, the myocardial infarction is characterized by the presence of NETS in the subject's coronary stent thrombus. In even more embodiments, the myocardial infarction is characterized by platelet-neutrophil interactions mediated through polyps, which in turn induce NET production.

[0086] In yet additional embodiments, the thrombotic cardiovascular disease is carotid artery sclerosis, hi some embodiments, the carotid artery sclerosis is characterized by elevated myeloperoxidase (MPO), cell-free DNA, and MPO-DNA complexes detected in intraplaque hemorrhagic segments of carotid artery sclerosis.

[0087] In further embodiments, the thrombotic cardiovascular disease is a cerebrovascular accident, hi some embodiments, a cerebrovascular accident is characterized by a positive correlation between NETotic markers and clot stability and resistance to endovascular treatment.

[0088] In further embodiments, the thrombotic cardiovascular disease is deep vein thrombosis (DVT), deep vein thrombosis, or cardiac pericarditis. In some embodiments, DVT is characterized by activated neutrophils and plasma nucleosomes / DNA.

[0089] In further embodiments, the thrombotic cardiovascular disease is pulmonary embolism, hi some embodiments, the pulmonary embolism is characterized by NET-mediated thrombus organization and maturation.

[0090] In further embodiments, the thrombotic cardiovascular disease is chronic pulmonary thromboembolic hypertension, hi some embodiments, chronic pulmonary thromboembolic hypertension is characterized by the presence of NETs and pulmonary thrombi in the patient's plasma.

[0091] In yet other embodiments, the NET-associated condition is a hematological disease or disorder.

[0092] In some embodiments, the hematological disorder is thrombotic thrombocytopenic purpura (TTP). In some embodiments, TTP is characterized by impaired DNase 1-mediated degradation of NETs.

[0093] In further embodiments, the hematological disorder is heparin-induced thrombocytopenia or thrombosis. In some embodiments, the heparin-induced thrombocytopenia or thrombosis is characterized by neutrophil activation leading to NET-induced thrombosis.

[0094] In a further embodiment, the NET-associated disease is an autoimmune disease.

[0095] In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE). In some embodiments, SLE is characterized by excessive cell death displayed by neutrophils, resulting in NET formation.

[0096] In further embodiments, the autoimmune disease is antiphospholipid syndrome (APS). In some embodiments, APS is characterized by antiphospholipid antibody-mediated induction of NET formation.

[0097] In further embodiments, the autoimmune disease is rheumatoid arthritis (RA). In some embodiments, rheumatoid arthritis is characterized by increased NET formation in the peripheral blood and synovial membrane.

[0098] In further embodiments, the autoimmune disease is psoriasis. In some embodiments, psoriasis is characterized by a correlation between the number of NETotic cells and the severity of the disease.

[0099] In further embodiments, the autoimmune disease is ulcerative colitis. In some embodiments, the ulcerative colitis is characterized by NET-mediated enhancement of procoagulant activity.

[0100] In further embodiments, the autoimmune disease is gout, hi some embodiments, gout is characterized by monosodium urate (MSU) crystal-induced stimulation of neutrophils to produce NETs and IL-1β.

[0101] In a further embodiment, the autoimmune disease is systemic sclerosis.

[0102] In additional embodiments, the autoimmune disease is ANCA-associated vasculitis, hi some embodiments, ANCA-associated vasculitis is characterized by NET formation, which induces vasculitis and promotes an autoimmune response against neutrophil components.

[0103] In further embodiments, the autoimmune disease is dermatomyositis. In some embodiments, the dermatomyositis is characterized by an increase in NETs.

[0104] In further embodiments, the autoimmune disease is polymyositis. In some embodiments, the polymyositis is characterized by an increase in NETs.

[0105] In a further embodiment, the NET-associated condition is systemic inflammatory response syndrome.

[0106] 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.

[0107] In a further embodiment, the systemic inflammatory response syndrome manifests as disseminated intravascular coagulation (DIC).

[0108] In another aspect, a method of inhibiting the formation of NETs in a subject is provided, comprising administering to the patient an effective dose of an anti-CEACAM 1 antibody.

[0109] In another aspect, the present invention provides a kit for selecting a subject suitable for anti-CEACAM 1 antibody therapy or predicting a subject's response to an anti-CEACAM 1 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 at least one NET biomarker to a reference value or control sample value; and instructional materials indicating a correlation between the ratio of the NET biomarker to the reference level. In some embodiments, the subject has been diagnosed with cancer. In other embodiments, the subject has been diagnosed with a non-malignant NET-associated disease, disorder, or complication. In some embodiments, the subject has been diagnosed with a thrombosis-related condition. In yet another embodiment, the subject has been diagnosed with an autoimmune disease or rheumatic disease.

[0110] In some embodiments, the 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, the at least one NET marker is selected from MPO, NE, and DNA complexes. According to particular embodiments, the NET marker is MPO. Each option represents a separate embodiment of the present invention.

[0111] In some embodiments, the NET biomarker is MPO, and the invention provides kits for selecting subjects suitable for anti-CEACAM 1 antibody treatment or predicting a subject's response to an anti-CEACAM 1 antibody, comprising: means for determining the level of MPO in a biological sample; means for comparing the expression level of MPO to a reference value or control sample value; and instructional materials indicating a correlation between the ratio of MPO to the reference level. In some embodiments, the subject has been diagnosed with cancer. In other embodiments, the subject has been diagnosed with a non-malignant NET-associated disease, disorder, or complication. In other embodiments, the subject has been diagnosed with a thrombosis-related condition. In yet another embodiment, the subject has been diagnosed with an autoimmune disease or rheumatic disease.

[0112] In some embodiments, the subject is a cancer patient or the subject is suspected of having cancer, and therefore the present invention provides a kit for selecting a cancer subject suitable for anti-CEACAM 1 antibody treatment or for predicting a cancer subject's response to an anti-CEACAM 1 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 control sample value; and instructional materials indicating the correlation between the ratio of MPO to the reference level.

[0113] According to some embodiments of any of the aspects of the present invention, the anti-CEACAM1 mAb or 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.

[0114] According to some embodiments of any of the aspects of the present invention, the anti-CEACAM1 mAb or 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.

[0115] According to some embodiments of any of the aspects of the present invention, the anti-CEACAM1 mAb or 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.

[0116] In an exemplary embodiment of any aspect of the invention, the anti-CEACAM1 antibody is CM24, which comprises the heavy chain sequence set forth in sequence SEQ ID NO:9 and the light chain sequence set forth in sequence SEQ ID NO:10, or an active fragment thereof comprising at least the binding site, or an antibody analog or derivative thereof having at least 90% identity to either of the chain sequences.

[0117] According to some embodiments of any of the aspects of the invention, the antibody or 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 kappa light chain constant region. Each option represents a separate embodiment of the invention.

[0118] Further embodiments and their full scope of applicability will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0119] Some embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings. With particular reference now to the drawings in detail, it is emphasized that the details shown are by way of example and are for the purpose of explaining the embodiments of the invention. In this regard, the description taken in conjunction with the drawings will make apparent to those skilled in the art how embodiments of the invention may be practiced.

[0120] [Figure 1A] CM24-mediated inhibition of NET-promoted cancer cell migration in vitro is shown. The following human cancer cell lines were treated with CM24, an isotype control, or left untreated in serum-free medium with or without NETs: melanoma SK-MEL-28 (Figure 1A), non-small cell lung carcinoma (NSCLC) A549 (Figure 1B), and pancreatic cancer AsPC1 (Figure 1C). Serum was used as a chemotactic stimulus for cancer cell migration in Transwell-Boyden chambers. Fluorescence of migrated cells was measured every 2 h for 24 h (SKMEL-28, A549) or 48 h (AsPC1). The mean AUC ± SEM is shown. [Figure 1B] CM24-mediated inhibition of NET-promoted cancer cell migration in vitro is shown. The following human cancer cell lines were treated with CM24, an isotype control, or left untreated in serum-free medium with or without NETs: melanoma SK-MEL-28 (Figure 1A), non-small cell lung carcinoma (NSCLC) A549 (Figure 1B), and pancreatic cancer AsPC1 (Figure 1C). Serum was used as a chemotactic stimulus for cancer cell migration in Transwell-Boyden chambers. Fluorescence of migrated cells was measured every 2 h for 24 h (SKMEL-28, A549) or 48 h (AsPC1). The mean AUC ± SEM is shown. [Figure 1C]Figure 1 shows CM24-mediated inhibition of NET-promoted cancer cell migration in vitro. The following human cancer cell lines were treated with CM24, an isotype control, or left untreated in serum-free medium with or without NETs: melanoma SK-MEL-28 (Figure 1A), non-small cell lung carcinoma (NSCLC) A549 (Figure 1B), and pancreatic cancer AsPC1 (Figure 1C). Serum was used as a chemotactic stimulus for cancer cell migration in Transwell-Boyden chambers. Fluorescence of migrated cells was measured every 2 h for 24 h (SKMEL-28, A549) or 48 h (AsPC1). Mean AUC ± SEM is shown. Differences between untreated (untreated) or isotype control and CM24-treated wells were calculated by two-way ANOVA statistical analysis. Significance is indicated by *p<0.05, ****p<0.0001, and ns indicating no significance. [Figure 2A] Confocal microscopy images of NETs are shown demonstrating colocalization of CEACAM1 and NETs, ​​as well as direct CM24 binding to NETs, ​​as evidenced by areas of CM24 / MPO / extracellular DNA overlap. DAPI staining was used to visualize the extracellular DNA component of NETs (Figures 2A-2D, 2F), anti-MPO antibodies against the NET marker (Figures 2A-2C), and CM24 staining of CEACAM1 on NET structures (Figures 2A, 2C, and 2E are highlighted by white arrows). Magnified images are shown in Figures 2C-2E. [Figure 2B] Confocal microscopy images of NETs are shown demonstrating colocalization of CEACAM1 and NETs, ​​as well as direct CM24 binding to NETs, ​​as evidenced by areas of CM24 / MPO / extracellular DNA overlap. DAPI staining was used to visualize the extracellular DNA component of NETs (Figures 2A-2D, 2F), anti-MPO antibodies against the NET marker (Figures 2A-2C), and CM24 staining of CEACAM1 on NET structures (Figures 2A, 2C, and 2E are highlighted by white arrows). Magnified images are shown in Figures 2C-2E. [Figure 2C]Confocal microscopy images of NETs are shown demonstrating colocalization of CEACAM1 and NETs, ​​as well as direct CM24 binding to NETs, ​​as evidenced by areas of CM24 / MPO / extracellular DNA overlap. DAPI staining was used to visualize the extracellular DNA component of NETs (Figures 2A-2D, 2F), anti-MPO antibodies against the NET marker (Figures 2A-2C), and CM24 staining of CEACAM1 on NET structures (Figures 2A, 2C, and 2E are highlighted by white arrows). Magnified images are shown in Figures 2C-2E. [Figure 2D] Confocal microscopy images of NETs are shown demonstrating colocalization of CEACAM1 and NETs, ​​as well as direct CM24 binding to NETs, ​​as evidenced by areas of CM24 / MPO / extracellular DNA overlap. DAPI staining was used to visualize the extracellular DNA component of NETs (Figures 2A-2D, 2F), anti-MPO antibodies against the NET marker (Figures 2A-2C), and CM24 staining of CEACAM1 on NET structures (Figures 2A, 2C, and 2E are highlighted by white arrows). Magnified images are shown in Figures 2C-2E. [Figure 2E] Confocal microscopy images of NETs are shown demonstrating colocalization of CEACAM1 and NETs, ​​as well as direct CM24 binding to NETs, ​​as evidenced by areas of CM24 / MPO / extracellular DNA overlap. DAPI staining was used to visualize the extracellular DNA component of NETs (Figures 2A-2D, 2F), anti-MPO antibodies against the NET marker (Figures 2A-2C), and CM24 staining of CEACAM1 on NET structures (Figures 2A, 2C, and 2E are highlighted by white arrows). Magnified images are shown in Figures 2C-2E. [Figure 2F] Confocal microscopy images of NETs are shown demonstrating colocalization of CEACAM1 and NETs, ​​as well as direct CM24 binding to NETs, ​​as evidenced by areas of CM24 / MPO / extracellular DNA overlap. DAPI staining was used to visualize the extracellular DNA component of NETs (Figures 2A-2D, 2F), anti-MPO antibodies against the NET marker (Figures 2A-2C), and CM24 staining of CEACAM1 on NET structures (Figures 2A, 2C, and 2E are highlighted by white arrows). Magnified images are shown in Figures 2C-2E. [Figure 3]Serum MPO levels (ng / ml) measured before treatment in 30 healthy donors and 10 pancreatic ductal adenocarcinoma (PDAC) patients (P<0.01). [Figure 4A] Figure 4 shows the percent reduction in serum MPO levels in patients after treatment with CM24 and nivolumab. MPO levels were analyzed in serum samples from patients in Part A of the clinical trial, including 10 PDAC patients and 2 colorectal cancer (CRC) patients (Figure 4A) or 10 PDAC patients (Figure 4B). ELISA measurements were performed at five time points: pre-dose C1D1 (Cycle 1, Day 1); end of CM24 infusion (EOI); 1.5 hours after EOI; pre-dose C1D15 (Cycle 1, Day 15); and C1D15 EOI. [Figure 4B] Figures 4A-B show the percent reduction in serum MPO levels in patients after treatment with CM24 and nivolumab. MPO levels were analyzed in serum samples from patients in Part A of the clinical trial, including 10 PDAC patients and 2 colorectal cancer (CRC) patients (Figure 4A) or 10 PDAC patients (Figure 4B). ELISA measurements were performed at five time points: pre-administration of C1D1 (cycle 1, day 1); end of CM24 infusion (EOI); 1.5 hours after EOI; pre-administration of C1D15 (cycle 1, day 15); and C1D15 EOI. Figures 5A-B show MPO levels in serum samples from PDAC patients before treatment with a combination of CM24, nivolumab, and the chemotherapy cocktail Nal-irinotecan / 5FU / LV. [Figure 5A] MPO levels, known time alive and best response (PD = progressive disease, PR = partial response, SD = stable disease) for each patient are shown. [Figure 5B] Mean serum MPO levels are compared in patients who showed disease control (SD, PR) versus patients who progressed during treatment (P<0.05). The mean MPO levels in healthy controls (N=30) are shown by the dotted line. [Figure 6A]Figure 6 shows a significant decrease in serum MPO after 2 weeks of treatment with a combination of CM24, nivolumab, and Nal-irinotecan / 5FU / LV in patients who showed disease control (PR or SD) as opposed to patients who progressed (PD). Figure 6A shows the percentage of MPO relative to pre-dose for each patient, and Figure 6B shows the mean values ​​in patients who showed disease control versus patients who progressed (P=0.0001). [Figure 6B] Figure 6 shows a significant decrease in serum MPO after 2 weeks of treatment with a combination of CM24, nivolumab, and Nal-irinotecan / 5FU / LV in patients who showed disease control (PR or SD) as opposed to patients who progressed (PD). Figure 6A shows the percentage of MPO relative to pre-dose for each patient, and Figure 6B shows the mean values ​​in patients who showed disease control versus patients who progressed (P=0.0001). [Figure 7] Measurement of MPO levels (ng / uL) per concentration of NETs is shown. MPO levels were measured by ELISA in various amounts of fresh or frozen NETs (5, 10, and 20 ng / uL). MPO levels are shown as the mean of the quadrants. [Figure 8] Dose-dependent NET-induced platelet aggregation is shown. Platelets were combined with 5, 10, and 20 ng / μl of NETs, ​​and aggregation was quantified. Bars represent mean + / - SEM, N=4. Significance was determined using two-way ANOVA (***p<0.001, ****p<0.0001). [Figure 9] Figure 1 shows CM24-induced inhibition of adenosine diphosphate (ADP)-induced platelet aggregation. Platelet plasma was incubated with CM24 or hIgG4 (as an isotype control) for 30 minutes with or without ADP as an inducer of platelet aggregation, and platelet aggregation was measured. Two-way ANOVA was used for selected comparisons (****p<0.0001). [Figure 10]Figure 1 shows 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 and 10 ng / uL. The graph shows the mean AUC + / - SEM (N=4) of aggregation over the 30-minute period of the assay. A two-way ANOVA with multiple comparisons was performed to determine statistical differences between treatments (****p<0.0001). [Figure 11] Figure 1 shows interference of CEACAM1-expressing melanoma cell adhesion to NETs by CM24. FACS analysis of CEACAM1-positive SK-MEL-28 cells versus CEACAM1-negative Jurkat cells was performed after 30 minutes of incubation with CM24 or an isotype control. Graph shows NET-adherent cells (mean + / - SD, N=3). Two-way ANOVA with multiple comparisons was performed to determine statistical differences between conditions (*p<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0121] The present invention relates to methods and compositions comprising anti-CAECAM 1 antibodies for the treatment of NET-associated conditions, including non-malignant thrombotic diseases and disorders, and for preventing, slowing or inhibiting the formation, migration, spread and progression of metastases.

[0122] Without wishing to be bound by any theory, mechanism, or action, it is proposed that inhibiting cell entrapment by the NETotic matrix prevents the pathological aggregation of cells that, if uninhibited, can lead to the formation of thrombi or metastases. The present invention illustrates the successful use of CM24, a humanized anti-CEACAM 1 antibody, in inhibiting NET-mediated cancer cell migration in vitro and suppressing metastasis in vivo.

[0123] The findings disclosed in this invention also provide a better understanding of the crosstalk between cancer and NETs, ​​which is important for the development of novel therapeutic interventions that block cancer escape mechanisms and prevent metastatic spread.

[0124] Furthermore, the present invention describes the utility of NET markers as effective biomarkers for selecting patients eligible for treatment with anti-CEACAM 1 antibodies and for monitoring the effectiveness of treatment with such antibodies. In a non-limiting example, the NET marker is myeloperoxidase (MPO).

[0125] In the following description, specific details are set forth to provide a thorough understanding of various embodiments. However, one of ordinary skill in the art will understand that the provided embodiments may be practiced without these details. Throughout the following specification and claims, unless the context requires otherwise, the word "comprise" and variations thereof, such as "comprises" and "comprising," should be construed in an open and inclusive sense, i.e., "including, but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. It should also be noted that the term "or" is typically used in its sense to include "and / or" unless the content clearly dictates otherwise. Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0126] As used herein, the term "about" refers to an amount that is 10% or closer to the stated amount.

[0127] The term "CEACAM1" refers to the protein product of the human CEACAM1 gene, e.g., NP_001020083.1, NP_001703.2. Eleven different CEACAM1 splice variants have been detected in humans. Individual CEACAM1 isoforms differ with respect to the number of extracellular immunoglobulin-like domains (e.g., CEACAM1 with four extracellular immunoglobulin-like domains is known as CEACAM1-4), the membrane anchor, and / or the length of their cytoplasmic tails (e.g., CEACAM1-4 with a long cytoplasmic tail is known as CEACAM1-4L, and CEACAM1-4 with a short cytoplasmic tail is known as CEACAM1-4S). The N-terminal domain of CEACAM1 begins immediately after the signal peptide, and its structure is considered to be IgV-type. For example, in the CEACAM1 annotation P13688, the N-terminal IgV domain consists of 108 amino acids, from amino acids 35 to 142. This domain was identified as being involved in homophilic binding activity (Watt et al., 2001, Blood. 98, 1469-79). All variants, including these splice variants, are included within the term "CEACAM1."

[0128] The terms "anti-CEACAM1 antibody," "antibody recognizing CEACAM1," "antibody against CEACAM1," and "antibody against CEACAM1" are interchangeable and are used herein to refer to an antibody that binds to human CEACAM1 protein with sufficient affinity and specificity.

[0129] Specific anti-CEACAM1 antibodies are described, for example, in WO 2010125571, which discloses a mouse anti-human CEACAM1 antibody designated MRG1.

[0130] CM24 is a non-fully humanized mAb that is disclosed in detail in International Application Publication No. WO2015166484.

[0131] The unique properties of CM24 and similar antibodies included in the compositions and methods of the present invention confer several advantages to their use in humans, particularly in applications requiring long-term or repeated administration where other non-human antibodies cannot be administered due to the risk of eliciting an immunogenic response against the non-human antibody itself. Avoiding such immune responses becomes even more important when the person being treated is a patient suffering from a disease, and further deterioration of the patient's health should be avoided.

[0132] As used herein, the term "NET" refers to an extracellular complex of a nucleosome and a protein, e.g., a protein with antimicrobial activity. The extracellular complex may be derived from any myeloid or lymphoid cell, including neutrophils, macrophages, myeloid-derived suppressor cells, mast cells, eosinophils, basophils, dendritic cells, neutral killer cells, monocytes, or B cells.

[0133] Neutrophils and macrophages are the primary cell types known to form extracellular traps, which consist of DNA and histones (mostly in citrullinated form) and are further decorated with different proteins. Like neutrophils, macrophages undergo a process of cell death known as METosis, in which they form extracellular traps containing proteins such as MPO. (Rahat et al., Front Immunol. 2023 Sep 26:14:1292819). Any aspect, embodiment, or claim of the present invention related to NETs, ​​NETs, ​​NET markers, NET-associated conditions, and NETosis also encompasses macrophage-derived extracellular traps, i.e., MET, METs, METosis, MET markers, MET-associated conditions, and METosis.

[0134] The terms "NET-associated condition," "NET-associated disease," "NET-associated disorder," "NET-driven disorder," and "NETopathies" are used interchangeably and are used herein to refer to pathologies or conditions involving the unwanted or uncontrolled NETosis process.

[0135] The term "NET marker" refers to biological material obtained from a subject that is quantitatively or qualitatively measured to detect NETosis. Detection of NETosis includes, but is not limited to, detection of NET formation, including detection of co-localized neutrophil-derived proteins and extracellular DNA and citrullinated histones, detection of NET remnants in fluid samples, and flow cytometry detection of cell-associated NET components. Any marker known in the art for detecting NETosis can be used with the methods of the present invention. According to some embodiments, NET markers 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 marker is selected from MPO, NE, and DNA complexes. According to certain embodiments, the NET marker is MPO.

[0136] The term "biomarker" in relation to NETs or NETosis is used according to some embodiments of the present invention to describe a diagnostic or prognostic tool, for example in patient selection.

[0137] The terms "NET marker" and "NET biomarker" are used interchangeably according to some embodiments of the present invention.

[0138] The term control sample refers to a sample taken from a healthy subject or a sample taken from an assessed subject at a different (eg, earlier, pre-treatment) stage of the disease.

[0139] The terms significantly or significant refer 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 an inherent quality of the sample rather than random variance.

[0140] The term "antigen" as used herein refers to a molecule or a portion of a molecule that can induce antibody formation and be specifically bound by an antibody. An antigen may have one or more epitopes. The specific reaction referred to above means that the antigen reacts with the corresponding antibody in a highly selective manner, but does not react with many other antibodies that may be induced by other antigens. The antigen according to the present invention is human CEACAM1 protein or a fragment thereof. According to some embodiments, human programmed cell death 1 (PD-1) protein also serves as an antigen for the combination therapy according to the present invention.

[0141] As used herein, the term "antigenic determinant" or "epitope" refers to a region of an antigen molecule that specifically reacts with a particular antibody. Peptide sequences derived from epitopes can be used alone or conjugated to a carrier moiety to immunize animals to generate additional polyclonal or monoclonal antibodies, applying methods known in the art.

[0142] 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 contain at least an antibody-binding portion and exhibit the desired biological activity, i.e., activity against CEACAM1.

[0143] An antibody according to the invention is a molecule comprising at least an antigen-binding portion of an antibody. The antibody or antibodies according to the invention include intact antibodies, such as polyclonal or monoclonal antibodies, as well as proteolytic fragments thereof, such as Fab or F(ab')2 fragments. Other types of antibody fragments and constructs, as well as single-chain antibodies, are also within the scope of the invention.

[0144] As used herein, the terms "molecule comprising an antigen-binding portion of an antibody" and "antigen-binding fragment" are intended to include intact immunoglobulin molecules of any isotype and produced by any animal cell line or microorganism, as well as antigen-binding reactive fractions thereof, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, variable portions of the heavy and / or light chains thereof, Fab miniantibodies (see, e.g., WO 93 / 15210, U.S. Ser. No. 08 / 256,790, WO 96 / 13583, U.S. Ser. No. 08 / 817,788, WO 96 / 37621, U.S. Ser. No. 08 / 999,554, the entire contents of which are incorporated herein by reference), dimeric bispecific miniantibodies (see Muller et al., 1998) and single chain antibodies incorporating such reactive fractions, as well as any other type of molecule into which such an antibody reactive fraction is physically inserted. Such molecules may be provided by any known technique, including but not limited to enzymatic cleavage, peptide synthesis, or recombinant techniques.

[0145] An "antibody fragment" comprises only a portion of an intact antibody, generally including the antigen-binding site or portion of the intact antibody, and thus retains the ability to bind to the antigen. Examples of antibody fragments encompassed by this definition include: (i) a Fab fragment having the VL, CL, VH, and CH1 domains; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) an Fd fragment having the VH and CH1 domains; (iv) an Fd' fragment having the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) an Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 1989, 341, 544-546); (vii) isolated CDR regions; (viii) an F(ab')2 fragment, a bivalent fragment comprising two Fab' fragments linked by disulfide bonds at the hinge region; and (ix) a single-chain antibody molecule (e.g., a 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 comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448), and (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with complementary light chain polypeptides (Zapata et al., Protein Eng., 1995, 8, 1057-1062; and U.S. Patent No. 5,641,870).

[0146] A single-chain antibody can be a single-chain composite polypeptide, i.e., a combined VH-VL or single-chain Fv (scFv), which has antigen-binding ability and comprises amino acid sequences that are homologous or similar to the variable regions of immunoglobulin light and heavy chains.

[0147] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of nearly homogeneous antibodies; i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, 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" should not be construed as requiring production of the antibody by any particular method. Monoclonal Abs can be obtained by methods known to those skilled in the art. For example, monoclonal antibodies used in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or can be produced by recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597.

[0148] The mAbs of the invention may be of any immunoglobulin class, including IgG, IgM, IgE, or IgA, hi a specific embodiment, the mAb of the invention is an IgG.

[0149] A humanized antibody is an antibody derived from a non-human species (e.g., a mouse antibody) whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans. The "humanization" process is usually applied to monoclonal antibodies developed for administration to humans, and is carried out when the process of developing specific antibodies involves production in a non-human immune system (such as a mouse). The protein sequence of the antibody produced in this way is different from that of antibodies naturally occurring in humans, and therefore is immunogenic when administered to human patients. Humanized antibodies are considered to be different from chimeric antibodies, which have a protein sequence similar to that of human antibodies but contain large stretches of non-human proteins.

[0150] It is possible to produce humanized antibodies without creating chimeric intermediates. Direct generation of humanized antibodies can be achieved by inserting appropriate CDR-encoding segments (responsible for the desired binding properties) into a human antibody framework (a process known as "CDR grafting"). Generally, after an antibody is developed in a mouse (or another non-human animal) to have the desired properties, the DNA encoding the CDRs of the antibody can be sequenced. Once the precise sequences of the desired CDRs are known, these sequences are inserted into a construct containing the DNA of the human antibody framework.

[0151] Identifying or determining the CDRs from a given heavy or light chain variable sequence is typically done using one of a few methods known in the art, for example, according to Kabat (Wu TT and Kabat EA, J Exp Med, 1970;132:211-50) and IMGT (Lefranc MP, et al., Dev Comp Immunol, 2003,27:55-77).

[0152] Although several methods are known in the art for determining the CDR sequences of a given antibody molecule, there is no standard, defined method. Determination of CDR sequences from the heavy and light chain variable regions 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 set of selected CDRs can include sequences identified by more than one method; for example, some CDR sequences are determined using KABAT and some using IMGT. According to some embodiments, the CDR sequences of the mAb variable region are determined using the IMGT method.

[0153] When the term "CDR having a sequence" or similar term is used, it includes the option that the CDR comprises the specified sequence, and also the option that the CDR consists of the specified sequence.

[0154] The antigen specificity of an antibody is based on the hypervariable regions (HVRs), the unique CDR sequences of both the light and heavy chains that together form the antigen-binding site.

[0155] Some of the antibodies included in the compositions and methods of the invention, such as the variable region of CM24, differ by at least one amino acid from the variable region of a fully human antibody and are therefore also referred to as "non-fully humanized" antibodies. Thus, the term "non-fully humanized monoclonal antibody" as used herein refers to a monoclonal antibody having heavy and / or light chain variable domains in which the amino acid sequences adjacent to and / or immediately adjacent to the CDRs are not fully human, i.e., are not identical to any known homologous or corresponding sequences obtained from naturally occurring human antibodies.

[0156] Also included within the scope of the present invention are antibody sequences that contain at least one amino acid substitution, deletion, and / or insertion, and up to about 10% of the chain sequence, compared to the respective sequences. These substitutions are typically made in the "non-CDR sequences," i.e., sequences contained in the constant domain or variable region of the antibody that are not the CDR sequences disclosed above. Changes in the CDR sequences are less common but are tolerated as long as antibody binding is not affected.

[0157] The terms "homologous," "homology," "closest identity," or "percent homology," as used herein to describe an amino acid sequence or a nucleic acid sequence compared to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, as modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula has been incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990).

[0158] The present invention also provides conservative amino acid variants of the specifically disclosed antibodies and antibody fragments. Variants according to the present invention can also be generated that preserve the overall molecular structure of the encoded protein. Given the properties of the individual amino acids that make up the disclosed protein products, some rational substitutions will be recognized by those skilled in the art. Amino acid substitutions, i.e., "conservative substitutions," can be made, for example, based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. As used herein, the term "antibody analog" refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

[0159] Antibody variants and conjugates refer to any molecule comprising an antibody of the invention. For example, fusion proteins in which an antibody or antigen-binding fragment thereof is conjugated to another chemical entity are also within the scope of the invention.

[0160] In some embodiments, the antibodies provided herein have an activity against human CEACAM1 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 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D ) K D can be measured by any suitable assay. In certain embodiments, KD can be measured by a surface plasmon resonance (SPR) assay (e.g., using a BIACORE® instrument).

[0161] The present invention is based, in part, on the results of a clinical trial conducted to evaluate the safety and efficacy of the anti-CEACAM1 mAb CM24 (NCT04731467). Part A of the study evaluated the safety of CM24 administration and involved the administration of CM24 in combination with the anti-PD1 mAb nivolumab, at escalating doses, to patients with solid tumors. Part C also evaluated safety and consisted of two subparts: Part C1, during which eight patients with pancreatic cancer received CM24 in combination with nivolumab and an additional therapeutic agent selected from gemcitabine and nab-paclitaxel; and Part C2, during which eight additional patients with pancreatic cancer received CM24 in combination with nivolumab and the chemotherapy cocktail Nal-IRI / 5FU / LV. In the final phase of the clinical trial, Part D, the efficacy of the treatment will be evaluated by monitoring the progress of patients after the above treatment compared to control patients who received 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 may include the same therapeutic agent initially administered to the patient, or an entirely different therapeutic agent. In pharmaceutical and drug formulations, the active agent is preferably utilized with one or more pharmaceutically acceptable carriers and, optionally, any other therapeutic ingredients. The carrier must be pharmaceutically acceptable in terms of being compatible with the other ingredients of the formulation and not overly harmful to the recipient. The active agent, as described above, is provided in an amount effective to achieve the desired pharmacological effect and in an amount appropriate to achieve the desired daily dosage.

[0162] As is well known, the molecules of the present invention as active ingredients are dissolved, dispersed, or mixed in a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients include, for example, water, saline, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, and the like, and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the present compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like.

[0163] Alternatively, the pharmaceutical composition can 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. Many suitable drug delivery systems are known, including, for example, implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled-release preparations can be prepared through the use of polymers to complex or adsorb the molecules of the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of polyanhydride copolymers of stearic acid dimer and sebaric acid. The release rate of the molecules of 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 in the matrix, and the size of the dispersed particles.

[0164] 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 to 10 mg / mL of a basic amino acid, (ii) 10 / 100 mg / mL of a sugar, (iii) 0.01 to 1 mg / mL of a surfactant, (iv) 1 to 50 mg / mL of a humanized mAb against CEACAM1 (e.g., CM24), 4 to 6 mg / mL of a basic amino acid, 70 to 100 mg / mL of a sugar, and 0.1 to 1 mg / mL of a non-anionic 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.

[0165] The term "sugar" refers to monosaccharides, disaccharides, and polysaccharides, and examples of sugars include, but are not limited to, sucrose, trehalose, dextrose, and the like.

[0166] In 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. In some embodiments, the composition comprises 1-10, 2-9, 3-7, or 4-6 mg / ml of basic amino acid. Each possibility represents a separate embodiment of the present invention.

[0167] 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 sugar. Each possibility represents a separate embodiment of the present invention.

[0168] 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 present invention.

[0169] According to some embodiments, the surfactant is non-anionic. According to some embodiments, the surfactant is selected from the group consisting of polysorbate, sorbitan ester, and poloxamer. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the surfactant is selected from the group consisting of polysorbate 20 and polysorbate 80. Each possibility represents a separate embodiment of the present 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 present 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 the surfactant.

[0170] In some embodiments, the pharmaceutical composition is in liquid form and contains 1-50 mg / ml CM24. In other embodiments, the pharmaceutical composition is lyophilized. In some embodiments, the pharmaceutical composition contains 10 mg / ml CM24, 4.65 mg / ml L-histidine, 82 mg / ml sucrose, and 0.20 mg / ml polysorbate 20.

[0171] According to some embodiments, the pharmaceutical composition comprises CM24 or a fragment as defined above and an additional immunomodulatory agent or kinase inhibitor. According to some embodiments, the pharmaceutical composition comprising at least one humanized mAb or fragment as defined above and the pharmaceutical composition comprising an additional immunomodulatory agent or kinase inhibitor are administered separately for use in the treatment of cancer.

[0172] According to some specific embodiments, the additional immunomodulatory agent 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 present invention. According to some specific embodiments, the additional immunomodulatory agent is selected from the group consisting of mAb against PD-1, mAb against PD-L1, mAb against PD-L2, interleukin 2 (IL-2), and lymphokine-activated killer (LAK) cells.

[0173] The pharmaceutical composition of the present invention can be administered by any suitable means, including parenteral and enteral routes. Modes of administration include, but are not limited to, oral, topical, intranasal, subcutaneous, intramuscular, intravenous, intratumoral, intraarterial, intraarticular, intralesional, and transdermal. Intravenous (iv) administration is usually used.

[0174] It will be apparent to those skilled in the art that a therapeutically effective amount of a molecule according to the invention will depend, among other things, 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 administered molecule, and the judgment of the treating physician. As used herein, a "therapeutically effective amount" refers to the amount of a molecule required to alleviate one or more symptoms associated with the disorder being treated over a period of time.

[0175] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of suffering from, or is at risk of developing at least one disease for which the described compositions and methods are useful for treating. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In some embodiments, the individual is a human.

[0176] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent a drug can prevent the growth of cancer cells and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic or be conjugated to a cytostatic and / or cytotoxic agent. In the case of cancer treatment, in vivo efficacy can be measured, for example, by assessing survival time, time to disease progression, response rate, duration of response, and / or quality of life.

[0177] In some embodiments, administration of an anti-CEACAM1 antibody for the treatment of cancer or prevention of 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.

[0178] As used herein, the terms "combination" or "combination therapy" can refer to either simultaneous administration of the combined items or sequential administration of the combined items. As described herein, when combination refers to sequential administration of the items, the items can be administered in any temporal order.

[0179] As used herein, "checkpoint inhibitor" refers to a drug that inhibits biological molecules ("checkpoint molecules") produced by an organism that negatively regulate 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.

[0180] According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen that includes the administration of at least one additional anti-cancer agent. According to certain embodiments, the anti-cancer composition comprises at least one chemotherapeutic agent. The chemotherapeutic agent, which may be administered together with or separately from the antibody according to the invention, may include any such agent known in the art to exhibit anti-cancer activity.

[0181] The terms "anti-cancer" and "anti-tumor composition" refer to compositions useful for treating cancer that contain at least one active therapeutic agent capable of inhibiting or preventing tumor growth or function and / or causing destruction of tumor cells. Therapeutic agents suitable for anti-tumor compositions for treating cancer include, but are not limited to, chemotherapeutic agents, radioisotopes, toxins, cytokines such as interferons, and antagonists that target cytokines, cytokine receptors, or antigens associated with tumor cells, and immune checkpoint inhibitors.

[0182] According to certain embodiments, the chemotherapeutic agent is selected from 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, anthracenedione-substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, corticosteroids, 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 doxetaxel. Two or more chemotherapeutic agents can be used in a cocktail and administered in combination with the administration of an anti-CEACAM 1 antibody. According to some embodiments, the chemotherapy cocktail comprises Nal-irinotecan / 5FU / LV.

[0183] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder, as well as those in whom the disorder is to be prevented. The term includes prophylactic treatment of subjects who are predisposed to a disease or disorder, for example, those who are genetically or environmentally predisposed to cancer or another disease. In some embodiments, a course of treatment is preceded by obtaining a biological sample from the subject. A biological sample is any sample taken from an individual and includes, but is not limited to, fluid (e.g., blood, urine, saliva, or cerebrospinal fluid) and tissue samples obtained by biopsy, surgical removal or resection of a portion of a limb, or benign or metastatic growth.

[0184] In certain embodiments, the method comprises administering to the 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 method comprises administering (i) multiple doses of the same or different doses of CM24; (ii) multiple ascending doses; or (iii) a pharmaceutical composition once every week, once every two weeks, once every three weeks, once every four weeks, or once every five weeks. In certain embodiments, the method comprises 1 to 10 administration cycles, each cycle comprising 2 to 5 infusions with CM24 every 1 to 4 weeks, followed by 2 to 8 weeks between each cycle. In certain embodiments, the method comprises administering CM24 as a neoadjuvant, adjuvant, or maintenance treatment.

[0185] The terms "cancer," "cancerous," and "tumor" refer to a physiological condition in mammals characterized by unregulated 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 can result in the formation of metastases. The spread of cancer cells within the body can occur, for example, via the lymphatics or blood. Uncontrolled growth, invasion, and metastasis formation are also referred to as malignant characteristics of cancer. These malignant characteristics distinguish cancer from benign tumors, which typically do not invade or metastasize.

[0186] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include melanoma, lung, thyroid, breast, colon, prostate, liver, bladder, kidney, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterine, sarcoma, bile duct, 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 cancer (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 present invention.

[0187] According to some embodiments, the cancer comprises a solid tumor. According to some embodiments, the cancer is a metastatic cancer.

[0188] In some embodiments, the solid cancer is pancreatic cancer, lung cancer, colorectal cancer, or melanoma. In some embodiments, the cancer is a metastatic cancer that begins from a primary pancreatic, lung, colorectal, or melanoma tumor.

[0189] NETs provide a scaffold and stimulus for thrombus formation. The release of NETs from neutrophils is associated with inflammation during sepsis and non-infectious diseases. Therefore, as described herein, thrombotic conditions can be treated or prevented by disrupting NETs.

[0190] 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 disease, congenital heart disease, and any condition that results in congestive heart failure in a subject, particularly a human subject. Also included are any diseases and conditions of blood vessels that result in insufficient, undesirable, or abnormal cardiac function, such as stroke, thrombosis, ischemia, ischemia-reperfusion, vascular occlusion, inflammation, and the like. As used herein, cardiovascular conditions are not limited to conditions resulting from atherosclerosis. Insufficient or abnormal cardiac function can be the result of disease, injury, and / or aging. In certain embodiments, the methods and compositions provided herein are directed to the treatment or prevention of cardiovascular conditions caused by or contributed to by NET activity by administering to a patient an effective dose of a composition comprising an anti-CEACAM 1 antibody. In further embodiments, the cardiovascular condition is stroke, ischemic reperfusion, myocardial infarction, inflammation, or thrombosis. In certain embodiments, the cardiovascular condition being treated is thrombosis. Clinically, inflammation and infection are associated with thrombosis. Accordingly, some embodiments provide methods and compositions for treating or preventing thrombosis in a patient, e.g., methods for treating or preventing a cardiovascular condition complicated by thrombosis. Thrombosis is the development of an intravascular blood clot at the site of vascular injury or an inappropriate intravascular blood clot, and is dependent on platelet adhesion, activation, and aggregation. Deep vein thrombosis (DVT) is often associated with inflammation and infection. A complication of thrombosis is that the clot detaches from the vessel wall, forming a clot that lodges elsewhere in the circulatory system, blocking blood flow and causing an embolism. In certain embodiments, the cardiovascular condition being treated is ischemia. In another embodiment, the cardiovascular condition being treated is ischemic reperfusion. As used herein, the term "ischemia" refers to any localized tissue ischemia resulting from reduced blood inflow. Blood flow to a tissue can be reduced due to vascular abnormalities such as thrombosis, embolism, or vasoconstriction. Reduced blood flow results in localized anemia, reduced oxygen levels, and ultimately damage to the tissue.Ischemia can also be caused by myocardial infarction, acute coronary syndrome, coronary artery bypass surgery, stroke, gastrointestinal ischemia, peripheral vascular disease, and surgical treatment. Furthermore, recruitment of leukocytes and / or platelets caused by the initial tissue injury can restrict blood flow in smaller capillaries, resulting in a second wave of ischemia. The term "myocardial ischemia" refers to a subset of ischemia, encompassing circulatory disorders caused by coronary atherosclerosis and / or insufficient oxygen supply to the myocardium. For example, acute myocardial infarction represents irreversible ischemic injury to myocardial tissue. This injury results in an occlusive (e.g., thrombotic or embolic) event in the coronary circulation, creating an environment in which the metabolic demands of the myocardium exceed the supply of oxygen to the myocardial tissue. In certain embodiments, the cardiovascular condition being treated is myocardial infarction. Myocardial infarction (i.e., heart attack) is the death of the myocardium from sudden blockage of a coronary artery by a blood clot. Coronary arteries are blood vessels that supply blood and oxygen to the myocardium. Blockage of coronary arteries deprives the myocardium of blood and oxygen, causing damage to the myocardium. Damage to the myocardium causes chest pain and pressure. Inflammation is known to contribute to the development of myocardial infarction, particularly through the formation of atherosclerotic plaques. Plaque rupture can cause thrombosis, resulting in myocardial infarction. In certain embodiments, the cardiovascular condition being treated is stroke. Thromboembolic occlusion of an intracerebral artery restricts downstream blood flow and promotes the formation of a second myocardial infarction within the cerebral microvasculature. In certain embodiments, the cardiovascular condition being treated is thrombosis, and the patient has systemic lupus erythematosus (SLE). SLE patients are also prone to venous thrombosis and have a reduced ability to degrade NETs. In certain embodiments, the condition being treated is sickle cell disease (SCD), a condition in which RBCs are deformed and rigid. The altered RBCs restrict blood flow at certain points in the circulatory system, potentially leading to a crisis. In SCD patients, fatal crises are often precipitated by infection.

[0191] 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 thrombus does not increase in size. In some embodiments, the severity of one or more signs or symptoms of DVT is reduced, e.g., the thrombus size is reduced.

[0192] In another aspect, the methods described herein relate to inhibiting the formation of NETs in a subject, comprising administering to the patient an effective dose of an anti-CEACAM 1 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 existing NETs and / or reducing the likelihood that existing NETs will grow or progress in the subject. In some embodiments, methods of inhibiting the formation of NETs can reduce the severity of symptoms associated with the development of NETs, ​​such as thrombosis. In some embodiments, a subject receiving treatment to inhibit the formation of NETs can have or be diagnosed as having a cardiovascular condition, as described above. In some embodiments, a subject receiving treatment to inhibit NET-mediated activity can have or be diagnosed as having a condition that predisposes the subject to thrombosis (i.e., prothrombotic). A condition that predisposes a subject to prothrombotic behavior can be any condition in which a subject is more likely to have or form NET-mediated thrombosis compared to a healthy subject.

[0193] The widely accepted crosstalk between inflammation and thrombosis has led to the introduction of the term thrombotic inflammation. Cells of the hematopoietic system, including neutrophils, platelets, and monocytes, play a key role in this process. Increasing evidence links NET release to the development of both venous and arterial thrombosis. The methods and compositions of the present invention can be used for these conditions.

[0194] The role of NETs in the pathogenesis of autoimmune diseases has previously been suggested. Long-term 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 therefore eligible 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 number of NETotic disease events and the severity or progression of the disease, induction of NETs by disease autoantibodies, enhanced procoagulant activity, and stimulation of neutrophils to produce NETs.

[0195] NET-associated autoimmune and immune-mediated diseases include, but are not limited to, psoriasis, systemic lupus erythematosus (SLE), antiphospholipid syndrome, rheumatoid arthritis (RA), ulcerative colitis (UC), gout, ANCA-associated vasculitis, dermatomyositis, and polymyositis.

[0196] Autoinflammatory diseases, such as hereditary Familial Mediterranean Fever (FMF), are also included in the decipherable diseases and conditions of the present invention. FMF is characterized by inflammatory attacks and neutrophil infiltration into the affected area. During an FMF attack, neutrophils undergo excessive NET formation, which decreases after the inflammation resolves. These and other autoimmune and autoinflammatory diseases are suitable for treatment with the compositions and methods of the present invention.

[0197] Several systemic inflammatory responses and diseases are also suitable for treatment with the compositions of the present invention. These conditions include, but are not limited to, sepsis (septic shock), in which functionally 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.

[0198] Several inflammatory lung diseases are characterized by the migration and detection of neutrophils and monocytes in the airway lumen and bronchoalveolar lavage fluid. NETs have been 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 eligible for treatment by the present invention.

[0199] The following examples are presented to more fully illustrate some embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the invention. [Example]

[0200] Example 1: Effects of NETs and CM24 on cancer cell migration

[0201] [Table 1]

[0202] cell culture 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, passaged, and harvested for assay using trypsin.

[0203] Migration assay Cells were grown to 80% confluency for two passages. The day before treatment, serum-containing medium was removed and serum-free medium was added. The following day, cells were lifted and stained with 5 μM carboxyfluorescein succinimidyl ester (CFDA-SE) dye for 10 minutes at 37°C. Stained cells were washed twice with serum-containing medium to ensure excess staining was inactivated, resuspended in serum-free medium, and added to the upper chamber of a Fluoroblok 96-well plate. Melanoma SKMEL-28 cells were added at 12,500 cells / well, NSCLCA549 cells at 25,000 cells / well, and pancreatic cancer AsPC1 cells were added. NETs were prepared according to Rayes et al. (ibid.) and added to the upper 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 10% serum-containing medium was added to the feeder wells of the plate through the sample port. Plates were transferred to a cell imaging multimode reader (Cytation 5) with CO2 and a set point of 37°C and imaged every 2 hours at 10x using brightfield and a GFP cube for a total of 24 hours.

[0204] NET-induced migration was tested in a cell chemotaxis assay. In this assay, serum-starved CFSE-labeled cancer cells were seeded in the upper chamber of a Fluoroblok migration plate, and full serum medium was placed in the lower chamber. Treatment (20 ng / μl NETs and 500 μg / ml antibody) was included in the upper chamber along with the cells. Images of the bottom of the membrane were captured every 2 h for 24 h (SKMEL-28, A549) or 48 h (AsPC-1), and the total number of migrated cells was calculated over time (Figures 1A-C). NET-induced migration was calculated by subtracting basal migration in the absence of NETs from the total cancer cells migrating in the presence of NETs toward the chemotactic stimulus. The effect of CM24 on NET-induced migration was measured in the presence of NETs with or without isotype control human anti-IgG4 or CM24, as described above, and the area under the curve (AUC) was calculated for each well (N = 6 wells for SKMEL-28 and A549; N = 3 wells for AsPC-1 studies). Statistical analysis using two-way ANOVA with multiple comparisons served to test the significance of differences between treatment groups. Table 2 shows the percent inhibition of NET-induced migration induced by CM24 relative to the isotype control.

[0205] [Table 2]

[0206] As shown in Figures 1A-1C and Table 2, a significant increase in cancer cell migration was observed when NETs were added to the upper well. Addition of the isotype control had no significant effect compared to untreated wells. However, NET-induced migration in all three cancer cell lines was significantly inhibited by CM24 (500 μg / ml) compared to isotype control-treated (hIgG4 500 μg / ml) or untreated cells. In melanoma SKMEL-28 cells (Figure 1A), 70% inhibition of NET-induced migration was observed with a p-value of 0.0197. In NSCLCA549 cells, CM24 inhibited NET-induced migration by 83% (Figure 1B), with a p-value of <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). Therefore, we conclude that CM24 significantly blunts cancer cell migration in response to NETs.

[0207] Example 2: Binding of CM24 to NETs

[0208] [Table 3]

[0209] Sample preparation and immunofluorescence Chamber slides were coated with poly-D-lysine at room temperature and then thoroughly washed with water. Fresh neutrophils were plated onto poly-D-lysine-coated chamber slides (50K per chamber) and stimulated with 100 nM phorbol-12-myristate-13-acetate (PMA) for 4 hours to induce neutrophil nephrosis. The medium was removed, and 4% paraformaldehyde (PFA) was added and incubated at room temperature for 10 minutes. The supernatant was removed and then washed twice with wash buffer. Blocking buffer was added and incubated at room temperature for 1 hour. The blocking buffer was removed, and the following primary antibodies were added in wash buffer and incubated 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. Washed three times with wash buffer. Secondary antibodies (a. anti-rabbit AF488 1:600, b-c. anti-human PE 1:200) were then added in wash buffer and incubated for 1 hour at room temperature. The sections were washed three times with wash buffer. The fluorescent stain 4',6-diamidino-2-phenylindole (DAPI) (1x) was added in PBS, and imaging was performed by confocal microscopy using the following LED / filter set: a. green fluorescent protein (GFP): rabbit polyclonal anti-myeloperxidase (MPO); b. red fluorescent protein (RFP): phycoerythrin (PE): CEACAM1; c. DAPI. The supernatant was aspirated using a chamber slide, and the chamber sections were then removed and mounted with ProLong AntiFade mounting medium and a glass coverslip. Slides were imaged using a Nikon A1 confocal microscope equipped with a Ti eclipse microscope and a Nikon 40X Plan Fluor Oil DIC H N2 objective. Images were analyzed using NIS elements version 5.12.03 and ImageJ Fiji.

[0210] Briefly, primary human neutrophils were stimulated with PMA to induce NETosis for 4 hours in poly-D-lysine-coated chamber slides and then fixed with paraformaldehyde. These slides were stained for extracellular DNA in NET structures and nuclear DNA (in intact neutrophils) with anti-MPO (myeloperoxidase, a NET marker) and DAPI, along with either CM24 or an isotype control, and imaged using confocal microscopy. MPO and DAPI demonstrated typical NET morphology (Figure 2A). Specific binding of CM24 to NET structures was observed compared to the isotype control, which showed no detectable staining (Figure 2B).

[0211] High-quality images of NETs were generated when NETs were induced in chamber slides and directly stained. MPO, which modifies the DNA structure of NETs, ​​was used as a biomarker for NETs. CM24 was tested for binding to NETs, ​​and specificity was confirmed 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 its involvement in NET-related diseases. The pattern of binding suggested punctate surface staining of neutrophils receiving NETs, ​​with significant distribution within the NETs themselves.

[0212] Example 3: Expression of CEACAM1 on cancer cell lines To assess the ability of cancer cells to bind to NETs via CEACAM1 interactions, we measured the expression of CEACAM1 on different cancer cell lines used in migration assays (Example 1) and adhesion assays (Example 7). The binding of the mouse anti-human CEACAM1 mAb MRG1 (described in International Patent Application Publication No. 2010125571) to the surface of several cell lines was examined by flow cytometry. Live cancer cells grown under normal culture conditions (e.g., maintaining less than 80% confluency) were stained with either the mouse anti-human CEACAM1 antibody MRG1 or an isotype control, followed by anti-mouse PE. Samples were then stained for viability and run on a MACSQuant flow cytometer. Data were analyzed using FlowJo, and the percentage of CEACAM1-positive cells and intensity of CEACAM1 expression values ​​for each cell line are shown in Table 4.

[0213] [Table 4]

[0214] Jurkat cells (a leukemic T cell line) that do not express CEACAM1 were used as a negative control. The human melanoma SK-MEL-28 cell line, human pancreatic cancer AsPC-1 cell line, and non-small cell lung cancer A549 cells tested in migration assays showed expression of CEACAM1 as detected by MRG1, whereas Jurkat cells were negative. Both SK-MEL-28 and AsPC-1 cells demonstrated high CEACAM1 expression levels, with 85% and nearly 100% of cells positive for CEACAM1, with mean MFIs (a measure of the amount of antibody bound) of 48K and nearly 12K, respectively. The majority of A549 cells (73.1%) stained positive with MRG1, albeit at a substantially lower intensity (MFI of 566). CEACAM1 expression in A549 cells depended on cell confluency, which was similar across all studies. Without being bound by any theory, anti-CEACAM1 antibodies may interfere with the interaction between NET-bound CEACAM1 and CEACAM1 on cancer cells or other cells, thus affecting NET-associated diseases.

[0215] Example 4: Clinical trial results showing decreased MPO levels in CM24 and nivolumab treated patients Serum samples from patients treated with CM24 and nivolumab (clinical trial NCT04731467) were collected pre- and post-treatment and tested for levels of the NET marker MPO. Pre-treatment serum levels of the NET marker MPO were >3.5-fold higher in PDAC and CRC patients (n=12) compared to healthy subjects (n=30) (p<0.001). PDAC patients (n=10) showed 3-fold higher serum MPO compared to healthy subjects (Figure 3).

[0216] The average results for 12 patients shown in Figure 4A demonstrate a significant decrease in serum MPO levels after treatment with CM24 on both days 1 and 15 of the study (p<0.05). Figure 4B shows the same measurements of serum NET levels after CM24 treatment in PDAC patients only (n=10), demonstrating a significant decrease in serum MPO after 2 weeks of CM24 (p<0.05).

[0217] In the dose-escalation arm of the study, selected adult subjects with 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) via a 1-hour infusion followed by a 30-minute infusion of nivolumab 480 mg / patient every 2 weeks. Blood samples were collected on Day 1 before treatment (C1D1 pre-dose), at the end of CM24 administration (C1D1 EOI), and 1.5 hours later at the end of nivolumab administration (C1D1 EOI 1.5 HR). Two weeks later (Day 15), blood samples were collected before treatment (C1D15 pre-dose) and at the end of CM24 administration (C1D15 EOI). Serum samples were prepared immediately after each blood draw and stored in aliquots at -70°C.

[0218] NET serum levels were measured by ELISA for MPO. Basal MPO levels are represented by the average levels measured in serum samples from 30 healthy volunteers. The recent decrease in MPO from the increase detected before C1D1 administration versus basal levels was calculated for each sample, and the average and significance were calculated for 10 PDAC patients and 2 CRC patients (Figure 4A), as well as for 10 PDAC patients only (Figure 4B). After treatment with CM24 and nivolumab, a significant decrease in MPO levels was observed in patient serum, indicating a decrease in NET levels.

[0219] Example 5: Clinical trial results showing high pre-treatment levels and significant suppression of serum MPO in Part C2 patients demonstrating disease control after treatment with CM24, nivolumab, and chemotherapy In Part C2 of Clinical Trial NCT04731467, patients received 20 mg / kg of CM24, followed by a 30-minute infusion of nivolumab at 480 mg / patient. The dose of Nal-IRI was 70 mg / m² intravenously over 90 minutes. Leucovorin (LV) at 200-400 mg / m² was administered by slow injection for at least 3 minutes at the end of the Nal-IRI infusion, after which a 2400 mg / m² 5FU infusion was initiated. Serum samples were collected before and after treatment and tested for MPO levels by ELISA. Of the eight patients recruited in Part C2 of Clinical Trial NCT04731467, six were treated more than twice and therefore were included in the analysis of serum MPO levels versus disease control. The results are shown in Figures 5 and 6. Figure 5A shows MPO levels in the patients' pretreatment serum. Patients who demonstrated disease control after treatment, including partial response (PR) or stable disease (SD), had significantly higher pretreatment serum MPO levels compared with patients demonstrating progressive disease (PD) or healthy volunteers. Figure 5B shows the mean pretreatment serum MPO levels (241 ng / mL) of patients who demonstrated PR or SD versus those who demonstrated progressive disease (63 ng / mL), demonstrating a significant difference (p<0.05). Figure 6 compares patients who demonstrated disease control (SD, PR) with those who progressed (PD), showing the percentage of MPO reduction after treatment in each patient (Figure 6A) and as a mean change (Figure 6B). As seen in Figures 5A-5B, a significant difference (p<0.05) in pretreatment serum MPO levels was observed between patients demonstrating disease control and a higher survival rate relative to patients who progressed, suggesting that serum MPO is a potential biomarker for this treatment. Figures 6A-6B show a significant decrease in serum MPO after treatment only in patients who demonstrated disease control (P=0.0001), but not in patients who progressed during treatment.

[0220] CM24, as demonstrated in vitro, interfered with CEACAM-1-mediated NET activity, resulting 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 a rationale for the use of CM24 to combat these life-threatening conditions.

[0221] Example 6: Effect of CM24 on NET-induced platelet aggregation Based on (Melissa V. Chan, Platelets; 2018; 29, 7:650-655), a platelet aggregation assay using freshly drawn blood was developed. Platelet-rich plasma (PRP) was incubated with the prothrombotic agent adenosine diphosphate (ADP) in a 96-well flat-bottom plate. The assay was read as absorbance at 595 nm over 30 minutes with intermittent shaking, and platelet aggregation was monitored as a decrease in absorbance. Wells of unprocessed PRP and platelet-poor plasma (PPP, platelet-pelleted plasma) were included on every plate (and read at every time point) so that aggregation could be expressed as a percentage, using PRP and PPP as 0% and 100%, respectively. Regarding NET stimulation of platelet aggregation, PRP was incubated with fresh NETs and frozen NETs (harvested from PMA-stimulated neutrophils).

[0222] [Table 5]

[0223] NET isolation Freshly collected neutrophils and polymorphonuclear cells (PMNs) were stimulated with 100 nM phorbol 12-myristate-13-acetate (PMA) for 4 h, and cell-free NETs were isolated, washed, and stored in PBS at 4°C for up to 24 h. Frozen NETs (frozen as acellular NET pellets) were thawed and resuspended in PBS immediately before use in the assay.

[0224] The basic assay involved the isolation of platelet-rich plasma (PRP) and plasma from healthy donors. NET-induced aggregation was assessed using fresh and frozen NETs. Using the optimal NET-inducing conditions identified above, CM24 (500 μg / ml) and an isotype control were added to the assay to assess the effect of the antibody on platelet aggregation.

[0225] Platelet-rich / platelet-poor plasma (PRP / PPP) preparation: 1. 40 ml of blood was drawn. a. Note: Donors must not be taking aspirin, antihistamines, antibiotics, or antiplatelet medications within 14 days prior to blood collection. 2. The blood was pooled and centrifuged at 175 xg for 20 minutes at 25°C in a swinging bucket rotor with a minimum brake. 3. The upper yellow platelet-rich plasma (PRP) was collected and platelets were counted using 2 μg / ml calcein AM, incubated at 37°C for 20 minutes, and green fluorescent cells were counted using a Nexcelom cell counter. 4. Approximately 1 ml of PRP was centrifuged at 15,000 x g for 2 minutes, and the supernatant, platelet-poor plasma (PPP), was collected.

[0226] Assay setup: 1. Antibodies (hIgG4 and CM24) were added to PRP and incubated at 37°C for 30 minutes. 2. 5 μl of NET was added to the appropriate wells of a clear, flat-bottom 96-well plate. Dilutions were made in Tyrode's-HEP ES buffer. 3. 95 μl of PPP / PRP / PRP+antibody was added and the absorbance was read at 595 nm. a. Shaking between readings was either 807 rpm at 37° C. in a plate reader or 1000 rpm at room temperature in a microplate reader.

[0227] The percentage of aggregation was calculated for all wells at all time points using the following formula, with PRP alone as 0% and PPP as 100%:

[0228]

number

[0229] The results shown in Figure 8 and Table 5 indicate that both fresh and frozen NETs at 5, 10, and 20 ng / μl were able to efficiently induce platelet aggregation in a dose-dependent manner.

[0230] [Table 6]

[0231] To evaluate the antithrombotic effect of CM24 in vitro, 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. Pretreated platelets were combined with ADP in the assay plate, and aggregation was quantified as described in the Methods section. As shown in Figure 9 and Table 6, CM24 significantly inhibited ADP-induced platelet aggregation compared to the isotype control.

[0232] [Table 7]

[0233] To evaluate the antithrombotic effect of CM24 in vitro, CM24 and its isotype control were added to a NET-induced platelet aggregation assay. PRP was incubated with 500 μg / ml of CM24 or hIgG4 for 30 minutes at 37°C. Pretreated platelets were combined with 5 and 10 ng / μl of NETs in an assay plate, and aggregation was quantified as described in the Methods section. As shown in Figure 10 and Table 7, untreated NETs induced platelet aggregation. CM24 significantly inhibited NET-induced platelet aggregation compared to untreated and isotype controls.

[0234] Summary data showing the percentage of aggregation at the assay endpoint are shown in Table 8, while Figure 10 and Table 7 demonstrate the AUC of the percentage of aggregation over the course of the entire assay. At fresh NET concentrations of 5 and 10 ng / μl, the addition of CM24 almost completely blocked NET-induced platelet aggregation. While the isotype control antibody reduced platelet aggregation to a lesser extent, the difference between CM24 and the isotype control was highly significant when fresh NET was used. Inhibition of platelet aggregation by CM24 showed complete blockage at 5 ng / μl NET and significant inhibition at 10 ng / μl NET. 5 ng / μL NET is the most relevant concentration for in vitro use, based on the levels of MPO measured in patient serum samples, as illustrated in Figure 7.

[0235] [Table 8]

[0236] [Table 9]

[0237] Example 7: CM24 prevents adhesion of CEACAM1-expressing cancer cells to NETs.

[0238] [Table 10]

[0239] Fresh NETs were isolated the day before the experiment, resuspended in PBS, and stored at 4°C. Frozen NETs were also used and resuspended in PBS. Both fresh and frozen NETs were stained on the day of the experiment.

[0240] Cancer cells were cultured according to ATCC recommendations. Cells were lifted using trypsin, stained with CytoTell Red (CTR) 650 in PBS, and thoroughly washed to remove excess stain. Some cells were left unstained for use as compensation controls. After CTR staining, cells were incubated with 500 μg / ml CM24 or isotype control in a U-bottom polypropylene 96-well plate at 37°C for 30 minutes. Stained fresh and frozen NETs were then added to the wells to a final concentration of 4 ng / μl in each well. The NETs and cells were shaken at 500 rpm for 2 hours at 37°C. Cells were maintained in buffer in the presence of BSA and in the absence of serum. After incubation, cells were vigorously washed and then stained for Nuclear Blue in PBS + 1% BSA. Data were then acquired using a MASCQuant flow cytometer. Data were analyzed using FlowJo (TreeStar, Inc.).

[0241] As detailed in Example 3 and Table 4 above, adhesion of CEACAM1-positive human melanoma SK-MEL-28 cells to NETs was demonstrated by FACS analysis compared to adhesion to Jurtkat cells, which do not express CEACAM1. Inhibition of this adhesion was further tested by utilizing CM24 or an isotype control. The results, shown in Figure 11, demonstrate that CM24 significantly reduced adhesion of SK-MEL-28 cancer cells to NETs, ​​while no significant effect of the isotype control was observed.

[0242] Conclusion of the experimental results NET-induced platelet aggregation was demonstrated in a dose-dependent manner using an in vitro assay. CM24 significantly inhibited NET-induced platelet aggregation. At 5 ng / μl and 10 ng / μl of fresh NETs, ​​the addition of CM24 reduced platelet aggregation by 70% and 53%, respectively, compared to the isotype control.

[0243] In vitro platelet aggregation studies demonstrated 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 blockade of CEACAM-1 using CM24 can effectively inhibit blood coagulation, suggesting the use of CM24 to inhibit thrombosis and other non-malignant NET-associated conditions, as well as cancer-associated thrombosis and metastasis.

[0244] Treatment of cancer cells with CM24 in vitro resulted in decreased cancer cell binding to NETs and decreased NET-induced cell migration. In the CM24 dose-escalation portion of a clinical trial study (NCT 04731467), patients with advanced solid tumors (primarily PDAC and CRC) who had received two prior lines of therapy received 10, 15, and 20 mg / kg of CM24 every other week and 480 mg of nivolumab every four weeks. In serum samples collected from patients, levels of MPO, a known NET marker, were significantly reduced after treatment.

[0245] We followed an expanded clinical phase of CM24 in combination with nivolumab and Nal-IRI / 5FU / LV in patients with pancreatic adenocarcinoma. In serum samples collected from these patients, high pretreatment serum MPO levels were detected in patients who subsequently demonstrated disease control (PR, SD), while non-responders (PD) showed low pretreatment MPO levels similar to those observed in healthy donors. Furthermore, a significant decrease in MPO levels was observed in patients who demonstrated disease control in response to CM24 (PR, SD) as opposed to patients who did not (PD), suggesting that CM24 is a potential treatment for NET-related disease and complications and for inhibiting cancer invasion and metastatic spread.

[0246] The above observations suggest that serum NET markers, such as MPO, can be used as biomarkers for selecting patients for anti-CEACAM1 therapy and for monitoring and staging treated patients.

[0247] The foregoing description of specific embodiments fully reveals the general nature of the present invention, so that others, by applying their current knowledge, can easily modify and / or adapt such specific embodiments to various uses without undue experimentation and without departing from the general concept; therefore, such adaptations and modifications should be understood within the meaning and range of equivalents of the embodiments of the present disclosure, and are so intended. It should be understood that the phraseology or terminology used herein is for purposes of description and not limitation. The means, materials, and steps for carrying out the various functions of the present disclosure 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 for use in preventing, inhibiting or delaying a pathological process, condition or disorder associated with NET-mediated activity in a subject, wherein said mAb or active fragment comprises a set of six CDR sequences comprising SEQ ID NOs: 1-6, and a pharmaceutically acceptable salt, carrier or diluent.

2. 1. A method of preventing, inhibiting, or delaying a pathological process, condition, or disorder associated with NET-mediated activity in a subject, said method comprising administering to said subject a therapeutically effective dose of an anti-CEACAM 1 mAb or an active fragment thereof, said mAb or antibody fragment comprising a set of CDR sequences comprising SEQ ID NOs: 1-6.

3. 3. The method or pharmaceutical composition of claim 1 or 2, wherein the formation of NET in the subject is inhibited.

4. 3. The method or pharmaceutical composition for use according to claim 1 or 2, wherein the pathological process or condition associated with NET-mediated activity is cancer or tumor.

5. 5. The pharmaceutical composition for the method or use of claim 4, wherein the cancer comprises a solid tumor.

6. 6. The pharmaceutical composition for the method or use of claim 4 or 5, wherein the cancer is a metastatic cancer or tumor.

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

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

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

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

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

12. 12. The method or pharmaceutical composition for use of 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 to 12, wherein the subject is a cancer patient undergoing tumor removal surgery.

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

15. 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. 13. 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 an immunomodulatory agent, an immunocellular therapeutic agent, a kinase inhibitor, and a chemotherapeutic agent.

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

18. 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. 1. A pharmaceutical composition comprising an anti-CEACAM 1 mAb or an active fragment thereof for use in treating a NET-related disease, disorder, or complication in a subject in need thereof, said use comprising: (i) determining the level of at least one NET marker in a biological sample obtained from a subject diagnosed with cancer; (ii) comparing the level of said at least one NET marker with a reference value or a control sample value; (iii) administering to said subject an anti-CEACAM 1 antibody if the level of said at least one NET marker in said sample is significantly higher than said reference value or said control sample value.

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

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

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

23. 1. A method of treating a NET-related disease, disorder, or complication in a subject in need of such treatment, said method comprising: (i) determining the level of at least one MPO biomarker in a biological sample obtained from said subject; (ii) comparing the level of said at least one NET biomarker to a reference value or a control sample value; (iii) administering to said subject an anti-CEACAM 1 mAb or an active fragment thereof if the level of said at least one NET biomarker in said sample is significantly higher than said reference value or said control sample value.

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

25. 24. The method of claim 23, wherein the NET-associated disease, disorder or complication is a non-malignant condition.

26. The method of any one of claims 23 to 25, wherein the NET biomarker is MPO.

27. Provided is a method for selecting a cancer patient suitable for treatment with an anti-CEACAM1 mAb or an active fragment thereof, 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 an increase in the level of MPO relative to the reference value or the control sample value indicates that the subject is likely to respond therapeutically to the anti-CEACAM1 mAb or an active fragment thereof.

28. 1. A pharmaceutical composition comprising an anti-CEACAM1 mAb or active fragment for use in treating a subject diagnosed with or suspected of having cancer, said use comprising selecting a cancer patient suitable for an anti-CEACAM1 antibody, said selection comprising the steps of: (i) providing a biological sample from said subject; (ii) determining the level of MPO in said sample of step (i); and (iii) comparing said MPO level with a reference value or a control sample value, wherein an increase in the level of MPO relative to said reference value or said control sample value indicates that said subject is likely to respond therapeutically to said anti-CEACAM1 mAb or active fragment.

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

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

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

32. 32. The method or pharmaceutical composition for use of any one of claims 19 to 31, wherein an active fragment of the anti-CEACAM 1 mAb comprises 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).

33. 33. The method or pharmaceutical composition for use of any one of claims 19 to 32, wherein the anti-CEACAM 1 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 for use or pharmaceutical composition for use according to any one of claims 19 to 33, wherein the anti-CEACAM 1 mAb is CM24.

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

36. 36. The method for use or pharmaceutical composition for use of claim 35, wherein the non-malignant condition is a disease or disorder selected from thrombosis, thrombus, disease-related thrombosis, prothrombotic condition, thrombo-inflammatory condition, venous thromboembolism, arterial thromboembolism, cardiovascular condition, autoimmune disease, autoinflammatory disease or disorder, immune-mediated disease, systemic inflammatory condition.

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

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

39. 39. The method or pharmaceutical composition for use of claim 38, wherein the thrombotic cardiovascular disease is selected from myocardial infarction, carotid artery sclerosis, cerebrovascular accident, deep vein thrombosis (DVT), portal vein thrombosis, cardiac pericarditis, pulmonary embolism, and chronic pulmonary thromboembolic hypertension.

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

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

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

43. 43. The method or pharmaceutical composition for use of 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. 38. The method or pharmaceutical composition for use according to claim 37, wherein the non-malignant thrombotic disorder is systemic inflammatory response syndrome.

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

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

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

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

50. 1. A kit for predicting a subject's response to anti-CEACAM 1 antibody therapy, 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 or control sample value; and instructions for use indicating a correlation between the ratio of the NET biomarker and the reference or control level.

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

52. 51. The kit of claim 50, wherein the subject has been diagnosed with a non-malignant NET-associated disease, disorder, or complication.

53. 53. The method of any one of claims 50 to 52, wherein said at least one NET biomarker is MPO.

54. 54. The method, pharmaceutical composition for use, or kit of any one of claims 1 to 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 YTSRLHS (SEQ ID NO: 5), and LC-CDR3 consists of QQGKSLPRT (SEQ ID NO: 6).

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

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

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

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

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