Humanized and affinity-matured Anti-ceacam1 antibodies

Abstract

To provide recombinant antibodies and antigen-binding fragments thereof useful for binding to and inhibiting carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1).SOLUTION: Provided are methods of using the disclosed CEACAM1 antibodies and antigen-binding fragments thereof for reducing T-cell tolerance and for the treatment of cancer.SELECTED DRAWING: Figure 36

Description

[Technical field]

[0001] The present invention relates generally to the fields of molecular biology and medicine. More specifically, the present invention provides monoclonal antibodies and antigen-binding fragments that bind to CEACAM1, and therapeutic compositions thereof, as well as methods of using such antibodies, including inhibiting homophilic and heterophilic interactions with CEACAM1, and methods of treating cancer and infectious diseases.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under NIH DK51362 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an immunoglobulin (Ig)-like transmembrane glycoprotein that is a member of the carcinoembryonic antigen (CEA) family. CEACAM family members are involved in cell-cell recognition and regulate cellular processes ranging from tissue architecture and angiogenesis to insulin homeostasis and regulation of T-cell proliferation.

[0004] A variety of cellular activities have been attributed to the CEACAM1 protein, including roles in differentiation and three-dimensional organization of tissues, angiogenesis, apoptosis, tumor suppression, metastasis, and regulation of innate and adaptive immune responses. Furthermore, several cell types express CEACAM1, including tumor cells, T cells, natural killer (NK) cells, and certain macrophages.

[0005] High expression of CEACAM1 occurs in various cancers, such as melanoma, colorectal cancer, gastric cancer, pancreatic cancer, bladder cancer, and thyroid cancer, and is associated with worse tumor progression, metastasis, and poor clinical prognosis. For example, non-small cell lung cancer (NSCLC) with high expression of CEACAM1 shows high microvascular density, distant metastasis, and reduced median overall survival and median progression-free survival. CEACAM1 expression was also strongly correlated with distant metastasis of pancreatic adenocarcinoma. CEACAM1 expression in tumors promotes CEACAM1-mediated inhibition of T cells and NK cells. As a result, tumor cell metastasis and cancer stem cell niche formation can be inhibited by inhibiting CEACAM1 activity.

[0006] CEACAM1 is also expressed in certain immune system cells and is involved in immune suppression and immune cell exhaustion. For example, high expression of CEACAM1 in tumor-infiltrating lymphocytes (TILs) and other tumor-infiltrating immune cells in gastric, lung, melanoma, colorectal cancer, and glioma is associated with poor prognosis. In T cells, CEACAM1 expression is largely excluded from resting (naive) T cells, but the protein is expressed at high levels in activated T cells. CEACAM1-L is the major isoform expressed in most T cells and functions as an inhibitory receptor that downregulates T cell activation and suppresses T cell function. Therefore, inhibiting CEACAM1 on T cells can restore T cell activity and increase antitumor responses.

[0007] CEACAM1 is further expressed on NK cells, lymphocytes involved in innate immunity, where they are involved in the early control of viral infections and in tumor immune surveillance. When NK cells encounter cells expressing major histocompatibility complex (MHC) class I, the immune response against these cells is prevented by inhibitory signals via receptor-ligand interactions. However, when they encounter cells in which MHC class I is downregulated, such as virus-infected or cancer cells, NK cells are activated by the lack of inhibitory signals, making the "disease" cells more susceptible to NK cell-mediated killing. When CEACAM1 is present on the surface of both NK cells and melanoma cells, the CEACAM1:CEACAM1 interaction inhibits NK-mediated killing, independent of MHC class I expression. Thus, disruption of this homophilic CEACAM1 interaction may be effective in restoring NK-mediated immune responses.

[0008] CEACAM1 expression on a subset of macrophages is further associated with fibrosis in the tumor microenvironment. CEACAM1 also regulates other stromal cells in the tumor microenvironment, such as vascular endothelium. Therefore, inhibiting the interaction of CEACAM1 with its binding partners can further inhibit fibrosis and angiogenesis.

[0009] CEACAM1 also mediates cell-cell adhesion through the IgV-like N-domain-containing CEACAM1 extracellular portion that is involved in homophilic (CEACAM1:CEACAM1) and heterophilic interactions (e.g., with CEA, CEACAM5, CEACAM8, T-cell immunoglobulin and mucin domain-containing 3 (TIM-3) protein, Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae / meningitidis opacity protein (OPA), Moraxella species, Opa-like protein OlpA, Haemophilus influenzae outer membrane protein (OMP) P1, Haemophilus aegyptius OMP P1, Candida albicans, influenza viruses such as H5N1). TIM-3 was identified as a Th1-specific cell surface protein expressed on activated T cells, a subset of dendritic cells and macrophages, as well as NK cells. TIM-3 is an activation-induced inhibitory molecule involved in tolerance and has been shown to induce T cell exhaustion in chronic viral infections and cancer. CEACAM1, which is also expressed on activated T cells, has been shown to interact with TIM-3, and this interaction is important for TIM-3-mediated T cell inhibition.

[0010] As shown above, CEACAM1 also functions as a cellular receptor on the apical membrane of mucosal cells for various Gram-negative bacterial pathogens associated with human mucosa, as well as fungal pathogens such as Candida albicans. For example, N. gonorrhoeae, N. meningitidis, Moraxella catarrhalis, H. influenza, H. aegyptius, and pathogenic Escherichia coli strains have well-characterized CEACAM1-binding adhesins. Binding of bacterial adhesins to CEACAM1 triggers endocytosis of bacteria into epithelial cells and transcytosis of microorganisms through intact epithelial layers, allowing microorganisms to utilize CEACAM1 during mucosal colonization. In addition, CEACAM1 is involved in the infection of influenza virus H5N1 and hybrid nematodes such as Wucheria bancrofti. Summary of the Invention

[0011] Provided herein are antibodies and antigen-binding fragments thereof that bind to CEACAM1 and block the interaction of CEACAM1 with one or more binding partners. Also provided are therapeutic compositions of such antibodies and antigen-binding fragments thereof, as well as methods of using these antibodies. By blocking the interaction of CEACAM1 with one or more binding partners, the antibodies and antigen-binding fragments thereof are useful for reducing, inhibiting, and / or reversing T cell tolerance and / or enhancing T cell proliferation. CEACAM1 antibodies and antigen-binding fragments thereof are further useful for treating cancer, reducing tumor growth, reducing tumor metastasis, and / or reducing cancer stemness in a subject in need thereof. CEACAM1 antibodies and antigen-binding fragments thereof are also useful for treating patients resistant to checkpoint therapy. Further provided are methods of using CEACAM1 antibodies and antigen-binding fragments thereof to reduce colonization of mammalian epithelia by bacteria expressing bacterial adhesins or Candida albicans, or to reduce influenza virus replication or release of proinflammatory cytokines or chemokines associated with influenza virus infection.

[0012] In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of the heavy chain variable region and the light chain variable region comprising CDR1, CDR2, and CDR3: The sequence of CDR1 (CDR1H) of the heavy chain variable region comprises the sequence X1HX2X3S (SEQ ID NO:1); X1 is A, D, N, or S; X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M; The sequence of CDR2 (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of the CDR3 (CDR3H) of the heavy chain variable region comprises the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO:3); X4 is D, G, or P; X5 is F or P; X6 is D or F; X7 is A or Y; and X8 is L, H, or F; The sequence of CDR1 (CDR1L) of the light chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2 (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); The sequence of the CDR3 of the light chain variable region (CDR3L) is QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6); X9 is W or N; X 10 is S or T; X 11 is A or an amino acid having a neutral hydrophilic side chain, including S, N, and T; X 12 is L, F, or N; and X 13 is P or F.

[0013] In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of which comprises CDR1, CDR2, and CDR3: The sequence of the variable heavy chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO:43); X is any amino acid; X1 is A, D, N, or S; X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M; and The sequence of CDR2 (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44); X4 is D, G, or P; X5 is F or P; X7 is A or Y; and X8 is L, H, or F; The sequence of CDR1 (CDR1L) of the light chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2 (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); The sequence of the CDR3 of the light chain variable region (CDR3L) is QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6); X9 is W or N; X 10 is S or T; X 11 is A or an amino acid having a neutral hydrophilic side chain, including S, N, and T; X 12 is L, F, or N; and X 13 is P or F.

[0014] In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of which comprises CDR1, CDR2, and CDR3: The sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO:1); X1 is A, D, N, or S; X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M; The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO:7); X4 is D, G, or P; X5 is F or P; and X8 is L, H, or F; The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of CDR3L is QQX9SSX. 12 PX 13 T (SEQ ID NO:8); X9 is W or N; X 12 is L, F, or N; and X 13 is P or F.

[0015] In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of the heavy chain variable region and the light chain variable region comprising a CDR1, a CDR2, and a CDR3; The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO:9); The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO: 10); The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of the CDR3L comprises the sequence QQWSSNPPT (SEQ ID NO:11).

[0016] In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of the heavy chain variable region and the light chain variable region comprising a CDR1, a CDR2, and a CDR3; The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO:9); The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO: 10); The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of the CDR3L comprises the sequence QQWTSNPPT (SEQ ID NO: 12).

[0017] In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the heavy chain variable region of SEQ ID NO: 13, and the sequence of the light chain variable region comprises a sequence that is at least 90% identical to a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0018] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region has SEQ ID NO: 13 and the sequence of the light chain variable region has an sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0019] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region has SEQ ID NO: 13 and the sequence of the light chain variable region has SEQ ID NO: 14.

[0020] In another embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the heavy chain variable region has the sequence of SEQ ID NO:13 and the light chain variable region has the sequence of SEQ ID NO:15.

[0021] In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 13; the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the light chain variable region amino acid sequence of SEQ ID NO: 14; The sequence of the variable heavy chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO:43); X is any amino acid; X1 is A, D, N, or S; X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M; and The sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44); X4 is D, G, or P; X5 is F or P; X7 is A or Y; and X8 is L, H, or F;

[0022] In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region comprises a sequence that is at least 85%, at least 90%, or at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO:13; the sequence of the light chain variable region comprises a sequence that is at least 85%, at least 90%, or at least 95% identical to the light chain variable region amino acid sequence of SEQ ID NO:14; Each heavy chain variable region and light chain variable region comprises CDR1, CDR2, and CDR3; and In that case: The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13, the sequence of CDR3H comprises residues D102, Y103, F104, P105, and Y106 of SEQ ID NO: 13; the sequence of CDR1L comprises residues A28, S30, and Y31 of SEQ ID NO: 14; The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO:14, and The sequence of CDR3L includes residues S91 and S92 of SEQ ID NO:14.

[0023] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention is a chimeric antibody, a CDR-grafted antibody, or a humanized antibody or antigen-binding fragment thereof.

[0024] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention is a multispecific or bispecific antibody or antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment is a bispecific antibody that contains complementary regions that bind to PD-1 or PD-L1.

[0025] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention is an scFv, Fv, Fab', Fab, F(ab')2, or diabody.

[0026] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention has the isotype IgG4.

[0027] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention comprises a S241P substitution in the constant region of the heavy chain.

[0028] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the invention is aglycosylated.

[0029] In one embodiment, a CEACAM1 antibody or antigen-binding fragment thereof provided by the invention lacks a C-terminal lysine in the heavy chain.

[0030] In one embodiment, a CEACAM1 antibody or antigen-binding fragment thereof provided by the invention is conjugated to one or more cytotoxins, fluorescent labels, and / or imaging agents.

[0031] In another aspect, the present invention provides CEACAM1 antibodies and antigen-binding fragments thereof characterized by the epitopes on CEACAM1 to which they bind. As described, such antibodies include, but are not limited to, CEACAM1 antibodies and antigen-binding fragments thereof described herein by their structural features, including CDR motifs, CDR sequences, and heavy and light chain variable sequences. In some embodiments, the present invention provides CEACAM1 antibodies and antigen-binding fragments thereof that bind to residues of the IgV-like N domain of CEACAM1. In another embodiment, the antibodies and antigen-binding fragments thereof provided herein also selectively bind to CEACAM1 relative to one or more CEACAM family members. In one embodiment, the CEACAM1 antibodies or antigen-binding fragments thereof do not show significant binding to other CEACAM family members, including CEACAM3, CEACAM5, CEACAM6, and / or CEACAM8. In some embodiments, the invention provides CEACAM1 antibodies and antigen-binding fragments that bind to epitopes on the N-domain of CEACAM1 that overlap or at least partially overlap the CEACAM1:CEACAM1 dimer interface, thereby blocking CEACAM1 homophilic interactions. In some embodiments, the invention provides CEACAM1 antibodies and antigen-binding fragments that bind to CEACAM1 residues that are located in binding sites on CEACAM1 for heterologous interaction partners, including, but not limited to, other CEACAM family members, TIM family members, bacterial adhesins (e.g., HopQ, OPA, OMP P1 and / or OlpA), Candida albicans, influenza viruses (e.g., H5N1) and / or nematodes, such as Wucheria bancrofti.

[0032] In one embodiment, the present invention relates to a contemplated CEACAM1 antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the sequence of SEQ ID NO: 13 and the light chain variable region having the sequence of SEQ ID NO: 14.

[0033] In one aspect, a contemplated CEACAM1 antibody or antigen-binding fragment thereof binds to the IgV-like N domain of CEACAM1 and binds to an epitope comprising one or more residues selected from the group consisting of residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and E99 of SEQ ID NO: 17. In one embodiment, the epitope further comprises residue Q44 of SEQ ID NO: 17. In one embodiment, the epitope further comprises one or more residues selected from the group consisting of residues S32, Q44, A49, I91, L95, and V96 of SEQ ID NO: 17.

[0034] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof binds to the IgV-like N domain of CEACAM1.

[0035] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof does not bind to one or more of CEACAM3, CEACAM5, CEACAM6, and CEACAM8.

[0036] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof binds at least partially to the TIM3 binding site on CEACAM1.

[0037] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof binds at least partially to a CEACAM1 binding site on CEACAM1 upon homodimerization.

[0038] In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM and partially or completely binds to a binding site on CEACAM1 for bacterial adhesins including, but not limited to, Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae Opa, Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1, and / or Moraxella species opa-like protein OlpA. In one aspect, the CEACAM1 antibody or antigen-binding fragment thereof binds to an epitope comprising one or more residues selected from the group consisting of residues F29, Y34, N42, Q89, and N97 of SEQ ID NO:17.

[0039] In one aspect, the CEACAM1 antibody or antigen-binding fragment thereof binds to an epitope that includes one or more residues selected from the group consisting of residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and N97 of SEQ ID NO: 17. In one embodiment, the epitope further includes residues F29, S32, D40, A49, T56, I91, L95, and E99 of SEQ ID NO: 17.

[0040] In one embodiment, the present invention provides a nucleic acid molecule encoding the CEACAM1 antibody or antigen-binding fragment thereof as described herein, as well as a vector comprising such a nucleic acid molecule.Also provided is a cell comprising a vector encoding the CEACAM1 antibody or antigen-binding fragment thereof as described herein, as well as a cell expressing the CEACAM1 antibody or antigen-binding fragment thereof as described herein.Further provided herein is a chimeric antigen receptor T cell comprising the CDR of any of the antibodies or antigen-binding fragments disclosed herein.

[0041] In one embodiment, the invention provides a composition comprising an antibody or antigen-binding fragment thereof described herein and a pharma- ceutically acceptable excipient.

[0042] In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of an interaction partner of CEACAM1 and / or to reduce CEACAM1 activity, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. For example, embodiments of the invention are useful for inhibiting the interaction between CEACAM1 and a member of the CEACAM family. In one embodiment, the CEACAM family member is CEACAM3, CEACAM5, CEACAM6, or CEACAM8. In some embodiments, the CEACAM family member is CEACAM1 itself.

[0043] In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of CEACAM1 to a member of the TIM family, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the TIM family member is TIM-3.

[0044] In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of CEACAM1 to a bacterial adhesin, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae Opa, Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1, or Moraxella species adhesin OlpA. In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of CEACAM1 to Candida albicans, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the present invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of CEACAM1 to an influenza virus, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the influenza virus is H5N1.

[0045] In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce colonization of mammalian epithelium harboring bacteria expressing a bacterial adhesin, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1, or Moraxella species adhesin OlpA.

[0046] In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce colonization of mammalian epithelium harboring Candida albicans, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein.

[0047] In one embodiment, the present invention provides a method for reducing influenza virus replication, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the present invention provides a method for reducing the release of proinflammatory cytokines or chemokines associated with influenza virus infection, the method comprising contacting a cell population comprising epithelial cells with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the influenza virus is H5N1.

[0048] In one embodiment, the invention provides methods of using the CEACAM1 antibodies or antigen-binding fragments thereof described herein to reduce T cell tolerance and / or enhance T cell proliferation or activation. These methods are useful for in vitro and in vivo applications.

[0049] In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce T cell tolerance and / or enhance T cell proliferation in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof.In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to treat cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof. In some embodiments, the cancer is glioma, glioblastoma, thymoma, mesothelioma, sarcoma, uterine carcinosarcoma, chromophobe renal cell carcinoma, adenoid cystic carcinoma, acute myeloid leukemia, melanoma, uveal melanoma, papillary renal cell carcinoma, clear cell renal carcinoma, bronchial carcinoma, lung adenocarcinoma, diffuse large B-cell lymphoma, pheochromocytoma and paraganglioma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cell carcinoma, breast cancer, prostate cancer, bladder cancer, gastric cancer, testicular germ cell carcinoma, ovarian cancer, head and neck cancer, uterine cancer, cervical cancer, or liver cancer. In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce tumor growth, reduce tumor metastasis, reduce tumor-associated fibrosis, and / or reduce cancer stemness in a subject in need thereof by administering an effective amount of the antibody or antigen-binding fragment to the subject. In some embodiments, the invention provides a method further comprising administering a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is a CTLA-4, PD-1, PD-L1, and PD-L2 inhibitor. In some embodiments, the invention provides methods further comprising administering one or more of an inhibitor of LAG3, TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226, or CD160. In some embodiments, the invention provides methods further comprising administering a TIM-3 inhibitor. In some embodiments, the inhibitor is administered simultaneously or sequentially with the antibody or antigen-binding fragment. In some embodiments, the inhibitor is administered separately or in a mixture with the antibody or antigen-binding fragment.

[0050] In one embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce colonization of an epithelium of a subject harboring bacteria expressing a bacterial adhesin in a subject in need thereof, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1, or Moraxella species OlpA.

[0051] In one embodiment, the present invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce epithelial colonization of a subject carrying Candida albicans in a subject in need of such reduction, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or antigen-binding fragment thereof described herein.

[0052] In one embodiment, the present invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce influenza virus replication in a subject in need thereof, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the present invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce proinflammatory cytokine or chemokine release associated with influenza virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the influenza virus is H5N1.

[0053] In one embodiment, the present invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to treat subjects who do not respond to treatment with checkpoint inhibitor therapy (primary resistance) and patients who initially respond to treatment but later become resistant to checkpoint inhibitor blockade (secondary or acquired resistance). Such a method of treatment comprises administering to said subject a CEACAM1 antibody or antigen-binding fragment thereof described herein. In some embodiments, the subject has acquired resistance to therapy with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor. Resistant cancers are also sometimes referred to as refractory cancers.

[0054] The patent or application file contains at least one drawing displayed in color. Copies of this patent or patent application publication with the color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0055] [Figure 1]Plasmid maps of the light chain expression vector pANTVK and the heavy chain expression vector pANTVhG4(S241P) are shown. Both the VH and Vκ vectors contain genomic DNA fragments incorporating introns and polyA sequences. Expression of both chains is driven by the CMV promoter. [Diagram 2] Selectivity of CEACAM1 antibody variants is shown. Humanized variant intermediates were examined by flow cytometry of HeLa cells transfected with CEACAM1, 3, 5, 6, and 8. The percentage of cells that were positive is shown based on irrelevant hIgG4 staining. There was no evidence of staining of HeLa-CEACAM3 or HeLa-CEACAM8 transfectants, so these data are not shown. [Figure 3A] Figure 1 shows the nucleotide and amino acid sequences of the heavy chain variable VH1. The CDRs are shaded. The numbering of the CDR residues according to Kabat and the primary amino acid sequence is shown. [Figure 3B] Figure 1 shows the nucleotide and amino acid sequences of the heavy chain variable CP08H03. The CDRs are shaded. The numbering of the CDR residues according to Kabat and the primary amino acid sequence is shown. [Figure 3C] Figure 1 shows the nucleotide and amino acid sequences of the light chain variable Vκ8 S29A. The CDRs are shaded. The numbering of the CDR residues according to Kabat and the primary amino acid sequence is shown. [Figure 4] FIG. 1 shows that the chimeric CEACAM1 antibody VH0 / Vκ0 is glycosylated in CDR1L. Introduction of the mutations N26Q and S29A abolishes this glycosylation. Proteins were separated by SDS-PAGE under denaturing conditions. The molecular weights of the heavy chain, glycosylated light chain, and aglycosylated light chain are shown. Residues N26 and S29 are numbered using the Kabat numbering scheme. [Diagram 5]Figure 1 shows the plasmid map of the phagemid expression vector pANT43. The VH and Vκ domains are linked via a flexible glycine-serine (G4S) linker and fused in-frame to the M13 gene III phage coat protein. Expression of the single chain variable fragment (scFv) is driven by the Lac promoter. [Figure 6] Phage binding to CEACAM1 antigen is shown. Phages were prepared from either the parental VH1 / Vκ8 S29A scFv or an irrelevant scFv, serially diluted, and incubated with plate-bound GST-CEACAM1. Phage binding to CEACAM1 was detected using anti-M13 horseradish peroxidase (HRP) conjugate and 3,3',5,5'-tetramethylbenzidine (TMB) substrate. [Figure 7] An outline of the design of the affinity maturation library is shown. The CDRS (as defined by Kabat) are shown in bold and the targeted positions are indicated with an X. Each position may include all 20 amino acids or a subset thereof. [Figure 8] 8A-8C show an overview of the library construction process for generating randomized phage libraries: light chain CDR3 library (FIG. 8A), heavy chain CDR1 library (FIG. 8B), and heavy chain CDR3 library (FIG. 8C). [Figure 9] Figure 9 shows an overview of the two different selection campaigns employed during affinity maturation of the CEACAM1 antibody. Figure 9A: Selection campaign 1: Deselection of solid-phase panning CEACAM5 / CEACAM6 was performed on the library phages and before round 2, a selection round was performed with decreasing concentrations of biotinylated soluble CEACAM1. Figure 9B: Selection campaign 2: Panning selection with CEACAM1 was performed in round 1, followed by deselection of panning CEACAM5 / CEACAM6 and two rounds of selection with decreasing concentrations of biotinylated soluble CEACAM1. [Figure 10]An example of an scFv binding ELISA assay is shown. A dilution series of purified parental scFvV H1 / Vκ8 S29A or affinity matured scFv variants was added to GST-CEACAM1 coated plates. Binding was detected using anti-HIS6-HRP antibody and TMB. All variable light chains contain an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain). [Figure 11] Binding selectivity of various affinity matured CEACAM1 antibodies is shown. Affinity matured antibodies CP08H03 / Vк8 S29A (denoted as "CP08_H03 / parental VL"), CP08H03 / CP08F05, 8H3_9B3 / CP08F05, and CP08H03 / CP08E05 contain a phenylalanine (F) at CDR3H residue 104. Affinity matured antibodies CP09B03 / CP08E05, CP09C02 / CP08F05, and 9B3_9E5 / CP08E05 contain an aspartic acid residue (D) at CDR3H residue 104. HELA cells were transfected with vector alone (HeLa-Neo) or vectors expressing CEACAM1, CEACAM3, CEACAM5, or CEACAM6, respectively, and stained with the indicated antibodies. The y-axis shows the percentage staining of each antibody with the transfected cell panel. hIgG4 = control antibody with the same stabilizing hinge mutation. MOPC = mouse IgG1 control antibody. Mouse antibodies as positive controls for the transfected CEACAM isoforms: Col-1 = CEACAM3 and CEACAM5 antibodies. 9A6 = CEACAM6 antibody. T84.1 = CEACAM cross-reactive antibody and T84.66 = CEACAM5 antibody. Secondary FITC only = no primary antibody, only secondary FITC-conjugated antibody. Col-1 and 9A6 are commercially available antibodies (Dako), T84.1 and T84.66 have been described previously (Neumaier M, J Immunol 1985;135:3604-9). Data for affinity matured antibodies 9B3_8H3 / Vк8 S29A, 8H3_9B3 / CP08_E05, and 8H3_9C2 / CPO08_F05, as well as data for CEACAM8 antibody 80H3, were omitted from the figures due to abnormally high background signals. [Figure 12A-1] CEACAM antibodies CP08H03 / Vκ8 S29A (denoted as "CP08_H03 / Parent VL"), CP08H03 / CP08_F05, and VH0 / Vκ0 are selective for CEACAM1. CP08H03 / Vκ8 S29A and CP08H03 / CP08F05 contain an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain). Figure 12A shows single cycle kinetic sensorgrams and fitting curves of purified lead humanized and affinity matured IgG4 variants. Increasing concentrations of different CEACAM family members were injected and single dissociation rates were measured by single cycle kinetics (surface plasmon resonance, SPR). [Figure 12A-2] Figure 12A shows single cycle kinetic sensorgrams and fitting curves of purified lead humanized and affinity matured IgG4 variants. Increasing concentrations of different CEACAM family members were injected and single cycle dissociation rates were measured by single cycle kinetics (surface plasmon resonance, SPR). [Figure 12B] Figure 12B shows three-point binding ELISA data for binding of chimeric antibody VH0 / Vκ0 (labeled "chimera"), a purified lead humanized and affinity matured IgG4 variant, to CEACAM1 and CEACAM3 family members. A three-point (high, medium and low, concentrations based on binding of chimeric antibody VH0 / Vκ0 to CEACAM1) titration was performed and binding was detected using an anti-human κ chain antibody and TMB substrate. [Figure 12C] Figure 12C shows three-point binding ELISA data for binding of chimeric antibody VH0 / Vκ0 (labeled "chimera") and purified lead humanized and affinity matured IgG4 variants to CEACAM1, 5 and 6 family members. Three-point (high, medium and low, concentrations based on binding of chimeric antibody VH0 / Vκ0 to CEACAM-1) titrations were performed and binding was detected using an anti-human κ chain antibody and TMB substrate. [Figure 13]Sequence homology between the N domains of different CEACAM family members is shown: CEACAM1 (C1, UniProtKB Accession No. P13688), CEACAM3 (C3, UniProtKB Accession No. P40198), CEACAM4 (C4, UniProtKB Accession No. O75871), CEACAM5 (C5, UniProtKB Accession No. P06731), CEACAM6 (C6, UniProtKB Accession No. P40199), CEACAM7 (C7, UniProtKB Accession No. Q14002), and CEACAM8 (C8, UniProtKB Accession No. P31997). The percent identity matrix shown was generated using Clustal2.1. The specific residues analyzed for each CEACAM family member are indicated. [Figure 14] Figure 1 shows the results of a CEACAM1 mutagenesis study aimed at identifying residues of CEACAM1 involved in binding to the indicated CEACAM1 antibodies. CEACAM1-FLAG was expressed in transfected human embryonic kidney (HEK) cells with CEACAM1 containing the indicated mutations (Y34C, V39A, G41A, N42A, R43A, Q44L, G47A, and Q89H), and proteins were separated by SDS-PAGE and then immunoblotted. Wild-type (WT) or mutant CEACAM1 proteins were detected using the indicated chimeric (VH0 / Vκ0) and humanized CEACAM antibodies. A decrease in detection indicates that the mutated residue is involved in binding to the respective antibody used for detection. [Figure 15] FIG. 1 shows the structure of the CEACAM1:CP08H03 / Vκ8 S29A Fab complex, with the Fab shown as a Cα trace and the antigen as a ribbon. [Figure 16]A diagram of the antigen (CEACAM1) facing dimer interface is shown. CEACAM1 residues that interact with the CP08H03 / Vκ8 S29A Fab light chain are labeled, including D40, N42, L95, V96, N97, and E99 (see FIG. 15), and CEACAM1 residues that interact with the CP08H03 / Vκ8 S29A Fab heavy chain are labeled, including F29, S32, Y34, Q44, T56, Q89, and I91. Relevant side chains are depicted as sticks. [Figure 17] CEACAM1: The crystal structure showing the CEACAM1 homodimer interface is shown (PDB ID: 4QXW). One CEACAM1 monomer is shown on the left and the other on the right. Residues Y34, Q44, Q89, and N97 form the YQQN pocket. [Figure 18] Stereo images of a close-up of the CP08H03 / Vκ8 S29A Fab-CEACAM1 interaction are shown. CEACAM1 is depicted as ribbons and the Fab chains (light and heavy) as Cα traces. Residues in the Fab light chain region (including S30, Y31, Y48, L49, S51, N52, W90, S91, and N93) and heavy chain region residues S52, S53, T56, Y57, Y59, D102, Y103, F104, P105, Y106 that interact with CEACAM1 residues include F29, S32, Y34, D40, N42, Q44, A49, T56, Q89, I91, L95, V96, N97, and E99 are labeled. Side chains of interest are depicted as sticks and hydrogen bonds are depicted as black dashed lines. Residue numbering is based on the primary amino acid sequences of the antibody and CEACAM1. [Figure 19] A comparison of CEACAM1 F29 with the V49 or A49 residues in the crystal structure of the CEACAM1 WT:CP08H03 / Vκ8 S29A antibody (left) or the crystal structure of the CEACAM1 A49V / Q89H mutant (right) is shown. [Figure 20] CEACAM1 antibody CP08H03 / Vκ8 S29A (designated "CP08") shows that it blocks human CEACAM1:CEACAM1 interactions (FIG. 20A) and CEACAM:human TIM-3 interactions (FIG. 20B). IgG4=control antibody. [Figure 21] 1 shows the experimental setup for testing the ability of CEACAM1 antibodies to induce CD45+ cell proliferation in humanized non-obese diabetic (NOD) scid gamma mice (NSG mice). Engraftment of human peripheral blood mononuclear cells (PBMCs) adoptively transferred into NSG host mice via intraperitoneal injection was analyzed by fluorescence activated cell sorter (FACS) for human CD45 and proliferation dye staining 38 days after injection. On day 24 after PBMC injection, mice were treated with a single injection of human IgG4 isotype control or the indicated concentrations of CP08H03 / Vκ8 S29A (denoted as "CP08_H03 / parental VL") or CP08H03 / CP08F05 antibodies. On day 31, mice were treated with a second injection. On day 38, mice were sacrificed for data acquisition. [Figure 22] Figure 1 shows that the CEACAM1 antibodies CP08H03 / Vκ8 S29A (denoted "CP08_H03 / parent") and CP08H03 / CP08F05 do not deplete human cells engrafted into humanized NSG mice. The mean percentage of human CD4 and CD8 T lymphocytes was assessed on day 38. CP08H03 / CP08F05 contains an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain). [Figure 23]Figure 2 shows that administration of CEACAM1 antibody CP08H03 / Vκ8 S29A (denoted as "CP08_H03 / Parent VL") or CP08H03 / CP08F05, respectively, results in increased antibody-induced human CD45+ immune cell proliferation in humanized NSG mice. CP08H03 / Vκ8 S29A induces proliferation of human CD45 PBMC in vivo. On day 38, isotype hIgG4 control (10 mg / kg), CP08H03 / Vκ8 S29A (2 and 10 mg / kg) and CP08H03 / CP08F05 (2 and 10 mg / kg) treated mice were sacrificed and ductal cells were isolated and collected for proliferation analysis. Ex vivo expansion was performed under T cell stimulatory conditions, cells were cultured for 120 hours in the presence of soluble anti-CD3 (OKT3) (at indicated concentrations 10, 5, 2.5 μg / ml) and rIL-2 (40 units / ml). Dilution of proliferation dye represents cell division / proliferation (double-stranded DNA was analyzed as a diluted signal as cells proliferated). CP08H03 / CP08F05 contains an S29A mutation in CDR1L (corresponding to the S28A mutation in the Kabat numbering scheme, primary amino acid sequence of the variable light chain). [Figure 24A] Figure 24A shows that CEACAM1 antibody CP08H03 / Vκ8 S29A (designated "CP08_H03 / parental VL") reduces tumor growth in humanized mice. Figure 24A shows a schematic diagram of the experimental protocol that led to Figure 24B. [Figure 24B] Figure 24B shows the average tumor size after subcutaneous injection of 1x106 MALME-3M (human melanoma) cells with 5x106 human PBMCs into NSG. Palpable tumors were recorded 10 days later and mice were randomized for treatment with respective antibody concentrations administered intraperitoneally on days 10, 13, 17, 20 and 24. [Figure 24C] FIG. 24C shows a statistical comparison by linear regression of the three different CP08H03 / Vκ8 S29A groups (2 mg / kg, 0.4 mg / kg and 0.08 mg / kg) versus the hIgG4 control treatment group. [Diagram 25]As described in FIG. 24, T cells from humanized mice engrafted with the human melanoma cell line MALME-3M and treated with the CEACAM1 antibody CP08H03 / Vκ8 S29A (designated "CP08_H03 / Parent VL") show reduced tumor cell load and reduced proliferation, as well as increased amounts of intratumoral CD8 and CD4 positive T cells. These T cells show increased proliferation when examined ex vivo after stimulation with anti-CD3. On the day of sacrifice, humanized NSG mice bearing melanoma tumors treated with isotype hIgG4 control (2 mg / kg), CP08H03 / Vκ8 S29A (designated "CP08_H03 / Parent VL", 0.08 and 2 mg / kg) were sacrificed. Tumor cells and CD4+ and CD8+ tumor infiltrating lymphocytes were isolated and collected for proliferation analysis. Tumor cells were identified as human CD45-negative FSCHiSSCHi cells, and proliferation was quantified by dilution of a commercially available dye (Becton-Dickinson) that assesses proliferation. Human CD45+CD4+ and CD45+CD8+ T cells were identified by flow cytometry. Ex vivo T cell proliferation measurements were performed under T cell stimulatory conditions, where cells were cultured for 6 days in the presence of soluble anti-CD3 (OKT3) (2 μg / ml) and rIL-2 (40 units / ml). [Figure 26] Phenotypic changes of intratumoral memory CD8 T cells upon blocking CEACAM1 with CEACAM1 antibody CP08H03 / Vκ8 S29A (denoted as "CP08_H03 / Parent VL") as described in Figures 24-25. Flow cytometric analysis of tumor-infiltrating CD3+CD8+ T cell populations from melanoma-bearing humanized NSG mice was performed using CD62L and CD44 cell markers for characterization of central memory (CD62L+CD44+) and effector memory (CD62L-CD44+CD3+CD8+) T cell populations. Treatment conditions were isotype hIgG4 control (2 mg / kg) and CP08H03 / Vκ8 S29A (0.08, 0.4 and 2 mg / kg). [Figure 27]CEACAM1 is shown to be expressed on primary CD4+ (top) and CD8+ T (bottom) T cells in TILs from naive (left) and PD-1 and / or CTLA-4 resistant (right) melanoma patients. Similar characterization of PD1 and TIM-3 expression is also shown. [Figure 28] We show that tumor-associated cells (TACs) from patients with acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy show significantly higher CEACAM1 expression compared to TACs from patients without previous exposure to anti-PD-1 and / or anti-CTLA-4 therapy. TACs were obtained from melanoma patients who were naive (no previous exposure to anti-PD-1 and / or anti-CTLA-4 therapy) or had acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy (acquired resistance). TACs were obtained and analyzed by culturing tumor tissue in DMEM medium and removing floating cells from the supernatant. Cells were stained for CD3, CD4, and CD8 to evaluate CEACAM1 expression in CD3+CD4+ and CD3+CD8+ cells. *, P=0.05; **, P<0.01. [Figure 29] Figure 1 shows the relative reduction in central memory (Tcm) versus effector memory (Tem) cells in CD8+ T cells isolated from patients resistant to anti-PD-1 and / or anti-CTLA-4 therapy compared to CD8+ T cells isolated from naive patients. Tumor associated cells from naive and resistant patients were stained for central memory (CCR7+CD62L+) and effector memory (CCR7-CD62L-) markers in TACs from naive and resistant patients. [Figure 30-1]CEACAM1 antibody CP08H03 / Vκ8 S29A (denoted "CP08") reverses T cell exhaustion in PD1 / CTLA-4 resistant tumors. Tumor associated cells and PBMCs were isolated from melanoma patients with secondary resistance to pembrolizumab, ipilimumab + nivolumab, and dabrafenib + trametinib and stage IV disease. Tumor associated cells and PBMCs were stained for CEACAM1, PD1, or TIM-3, and the percentage of CD8+ and CD4+ T cells showed expression of these markers (left). PBMCs or tumor associated cells ("tumor") cultured with soluble anti-CD3 (2 pg / ml) and rIL-2 (40 units / ml) in the presence of CP08H03 / Vκ8 S29A or hIgG4 control antibody are shown on the right. IFNγ and TNFα release, a measure of reversal of T cell resistance, was measured by ELISA. [Figure 30-2] CEACAM1 antibody CP08H03 / Vκ8 S29A (denoted "CP08") reverses T cell exhaustion in PD1 / CTLA-4 resistant tumors. Tumor associated cells and PBMCs were isolated from melanoma patients with secondary resistance to pembrolizumab, ipilimumab + nivolumab, and dabrafenib + trametinib and stage IV disease. Tumor associated cells and PBMCs were stained for CEACAM1, PD1, or TIM-3, and the percentage of CD8+ and CD4+ T cells showed expression of these markers (left). PBMCs or tumor associated cells ("tumor") cultured with soluble anti-CD3 (2 pg / ml) and rIL-2 (40 units / ml) in the presence of CP08H03 / Vκ8 S29A or hIgG4 control antibody are shown on the right. IFNγ and TNFα release, a measure of reversal of T cell resistance, was measured by ELISA. [Figure 31A]Flow cytometric analysis of stable HeLa CEACAM1 (HeLa C1), stable HeLa CEACAM3 (HeLa C3), stable HeLa CEACAM5 (HeLa C5), stable HeLa CEACAM6 (HeLa C6), and stable HeLa CEACAM8 (HeLa CEACAM8) transfectants (HeLa C8). 5x10^4 of the indicated HeLa transfectants were washed with staining buffer, incubated with CP08H03 / Vκ8 S29A (labeled "CP08"; left) or CEACAM1 antibody CM-24 for 30 min at room temperature, washed twice with staining buffer, and stained with anti-human IgG4 fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 20 min at room temperature. Fluorescence intensity was measured by flow cytometry. Live cells were determined by 4',6-diamidino-2-phenylindole (DAPI) staining shown on the y-axis. Staining with each CEACAM1 antibody is shown on the x-axis. Note that for CP08H03 / Vκ8 S29A, a positive signal in the gate is only shown in the HeLa CEACAM1(C1) transfectant (left). In contrast, CM-24 (right panel) is not selective and cross-reacts with CEACAM1, CEACAM3, and CEACAM5. [Figure 31B] 31B shows different representations of the data shown in FIG. 31A. [Diagram 32] We show that the CEACAM1 antibody CP08H03 / Vκ8 S29A (designated "CP08") is more effective at reversing T cell tolerance in tumor-associated cells than the CEACAM1 antibody CM-24. Tumor-associated cells from naive Merkel cell carcinoma tumors were stained with CEACAM1, PD1 or TIM-3 to show the percentage of CD8+ and CD4+ T cells (Figures 32A and 32B). Tumor-associated cells were cultured with soluble anti-CD3 (2 μg / ml) and rIL-2 (40 units / ml) in the presence of CP08H03 / Vκ8 S29A, CM-24 or hIgG4 control, respectively. IFN-γ release, a measure of reversal of T cell tolerance, was measured (Figure 32C). *, P=0.0138 comparing CP08 with hIgG4. [Diagram 33]Figure 33A shows that CM-24 treated metastatic melanoma in NSG mice shows a decrease in TILs and an increase in tumor cells compared to metastatic melanoma treated with CP08H03 / Vκ8 S29A (denoted as "CP08"). Figure 33A shows the experimental setup using a therapeutic tumor model in humanized NSG mice with human melanoma xenografts using four doses of each antibody at 2 mg / kg, including hIgG4 control, carrying the same stabilizing hinge mutation. Figure 33B shows a pie chart representation of the percentage of tumor infiltrating CD4+ T lymphocytes (grey), CD8+ T lymphocytes (black) and tumor cells (white) characterized by FSC / SCC High (FSC / SCCHi) and lack of pan-leukocyte marker human CD45 (left: control antibody; center: CEACAM1 antibody CP08H03 / Vκ8 S29A; right: CEACAM1 antibody CM-24). Figure 33C shows tumor cell proliferation for IgG4 control, CP08H03 / Vκ8 S29A, and CM-24, demonstrating inhibition of tumor growth by CP08H03 / Vκ8 S29A but not CM-24, and Figure 33D shows increased proliferation of splenic CD4+ T cells in CP08H03 / Vκ8 S29A-treated mice and decreased proliferation of splenic CD4+ T cells in CM-24-treated mice. [Diagram 34]CEACAM1 antibody CM-24 is an agonistic drug in a metastatic melanoma model. Absolute cell counts of tumor-infiltrating CD4+ T lymphocytes (Figure 34A), CD8+ T lymphocytes (Figure 34B), and tumor cells characterized by high forward / side scatter compared to metastatic melanoma (FSC / SCC Hi) (Figure 34C) are shown. Values ​​obtained for each experimental mouse are shown for each group (n=9 for IgG4; n=8 for CP08; n=6 for CM-24). *P<0.05; **P<0.001. Statistical analysis refers to the data included in Figure 33B. Note that the number of TILs was increased (Figures 34A and 34B) and the number of tumor cells was decreased (Figure 34C) in mice treated with CP08H03 / Vk8 S29A (denoted "CP08") and CM-24. This data indicates that CP08H03 / Vk8 S29A is a competitive and CM-24 is an antagonist antibody. [Diagram 35]Figure 35A shows that the CEACAM1 antibody CP08H03 / Vκ8 S29A covers the CEACAM1:HopQ binding interface and is predicted to block the CEACAM1:HopQ or CEACAM1:Opa protein interaction. Figure 35A shows the CEACAM1:HopQ binding interface based on the analysis of three crystal structures (PDB IDs 6AW2, 6GBH, and 6GBG). The CEACAM1 GFCC' face, formed by the interaction of the CEACAM1 CC' and FG loop' (see Huang et al., Nature. 2015 Jan 15; 517(7534): 386-90), is involved in HopQ binding at CEACAM1 residues F29, Y34, N42, Q89, and N97, generating various hydrogen bonding and hydrophobic interactions (Bonsor D, A. et. al. EMBO J. 2018 Jul 2; 37(13). pii: e98664; Moonens K et. al. EMBO J. 2018 Jul 2; 37(13). pii: e98665). Figure 35B shows the superposition of the CP08H03 / Vκ8 S29A:CEACAM1 crystal structure and the CEACAM1:HopQ crystal structure. The light and heavy chains of the CP08H03 / Vκ8 S29A antibody are shown in surface representation. The HopQ chain (three different crystal structures PDB IDs 6AW2, 6GBH, and 6GBG) and CEACAM1 from three different co-crystal structures with HopQ (PDB IDs 6AW2, 6GBH, 6GBG) as well as CEACAM1 from a co-crystal structure with CP08H03 / Vκ8 S29A are shown in ribbon diagrams highlighting the superposition of the CP08H03 / Vκ8 S29A with the HopQ binding epitope. [Diagram 36] Figure 1 shows that CEACAM1 antibody CP08H03 / Vκ8 S29A increases survival of tumor-bearing mice. NSG mice were injected with MALME-3M (human melanoma) cells and human PBMCs. Treatment with CEACAM1 antibody CP08H03 / Vκ8 S29A or a control human (h)IgG4 antibody was performed on days 10, 13, 17, 20, and 24, respectively (see arrows). Shown is survival rate. n=4 / group. [Figure 37A-1]We show that the CEACAM1 antibody CP08H03 / Vκ8 S29A increases the expression of various factors involved in the immune response to cancer by CD8+ T cells from melanoma patients with secondary resistance to immunotherapy. Figure 37A shows a series of viSNE (visualization by distributed stochastic neighbor embedding) maps rendered in Cytobank using the Barnes-Hut implementation of the t-SNE algorithm, describing the intracellular expression in CD8+ T cells of specific factors as defined by mass cytometry on the left. Quantification of the heatmap levels of each specified factor is shown on the right of the x-axis, relative to the residuals associated with each factor shown on the y-axis. [Figure 37A-2] Figure 37A shows a series of viSNE (visualization by distributed stochastic neighbor embedding) maps rendered in Cytobank using the Barnes-Hut implementation of the t-SNE algorithm, depicting the intracellular expression in CD8+ T cells of specific factors as defined by mass cytometry on the left. Quantification of the heatmap levels of each specified factor is shown on the right of the x-axis, relative to the residuals associated with each factor shown on the y-axis. [Figure 37B] FIG. 37B shows the fold dispersion of the intracellular responses of the indicated agents listed in FIG. 37A in response to CP08H03 / Vκ8 S29A relative to a hIgG4 control antibody, set as 1.0. [Figure 38] We show that the CEACAM1 antibody CP08H03 / Vκ8 S29A potentiates the interferon gamma (IFN-γ) secretion capacity of cells dissociated from tumors of two melanoma patients either without prior treatment (FIG. 38B, subject 189) or with secondary resistance to immunotherapy (FIG. 38A, subject 185). In both cases, tumor specimens were disrupted by mechanical dissociation (Miltenyi) and tumor dissociated cells were treated in vitro with 2 μg / ml of CP08H03 / Vκ8 S29A alone or with a human IgG4 isotype control antibody. After 96 hours, significant levels of interferon gamma were detected in the supernatants of CP08H03 / Vκ8 S29A but not in samples treated with the human IgG4 isotype control antibody. *P<0.05 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] antibody

[0057] 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), antibody fragments, and antigen-binding portions thereof (e.g., paratopes, CDRs), so long as they exhibit the desired biological activity and specificity.

[0058] As used herein, an "antibody variable domain" refers to the portion of the light and heavy chains of an antibody molecule that contains the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). H refers to the variable domain of the heavy chain. L refers to the light chain variable domain. The amino acid positions assigned to CDRs and FRs may be defined according to Kabat or Chothia. The term "framework region" (FR) refers to those variable domain residues other than the CDR residues.

[0059] As used herein, the term "complementarity determining region" (CDR) refers to the portion of an antibody variable domain that (typically) participates in antigen binding. Each variable domain usually has three CDR regions, identified as CDR1, CDR2 and CDR3. Each CDR may, for example, comprise amino acid residues from a CDR defined by Kabat (i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1987, 1991)). Each CDR is derived from a "hypervariable loop" (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) of the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) of the heavy chain variable domain (Chothia & Lesk 196 J. Mol. Biol. 901 (1987)) amino acid residues. In some cases, the CDRs can include amino acids from both the CDR regions and the hypervariable loops defined according to Kabat. The Kabat residue designations do not necessarily correspond directly to the linear numbering of the amino acid residues (primary amino acid sequence). The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering corresponding to the shortening or insertion of structural components, whether framework or CDR of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody or antigen-binding fragment thereof by aligning the homologous residues in the sequence of that antibody or antigen-binding fragment thereof with the "standard" Kabat numbered sequence. An example of how the Kabat numbering relates to the primary amino acid sequence of an antibody can be seen in Figures 3A, 3B, and 3C.Alternatively, the CDRs can be defined according to the ImMunoGeneTics (IMGT) system (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)).

[0060] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein has six CDRs: (i) the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO:9; (ii) the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; (iii) the sequence of CDR3 of the heavy chain variable region comprises SEQ ID NO: 10; (iv) the sequence of CDR1 of the light chain variable region comprises SEQ ID NO:4; (v) the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:5; and (vi) the sequence of CDR3 of the light chain variable region comprises SEQ ID NO:11.

[0061] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein has six CDRs: (i) the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO:9; (ii) the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; (iii) the sequence of CDR3 of the heavy chain variable region comprises SEQ ID NO: 10; (iv) the sequence of CDR1 of the light chain variable region comprises SEQ ID NO:4; (v) the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:5; and (vi) the sequence of CDR3 of the light chain variable region comprises SEQ ID NO:12.

[0062] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof has six CDRs: (i) the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO: 9; (ii) the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; (iii) the sequence of CDR3 of the heavy chain variable region comprises SEQ ID NO: 10; (iv) the sequence of CDR1 of the light chain variable region comprises SEQ ID NO: 18; (v) the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:5; and (vi) the sequence of CDR3 of the light chain variable region comprises SEQ ID NO:11.

[0063] As shown in the Examples below, affinity maturation of the CDR1H, CDR3H and CDR3L of a humanized, aglycosylated CEACAM1 antibody resulted in variants that conferred substantial improvements in CEACAM1 binding affinity. Inspection of the resulting variants and comparison of these variants with the variability introduced in the affinity maturation library indicated specific CDR positions where amino acids remained relatively unchanged and other CDR positions where changes could be introduced, resulting in improved binding.

[0064] In one aspect, the invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising a CDR1H, the CDR1H comprising residues 31-35 of a CEACAM1 antibody (corresponding to the Kabat definition, e.g., residues 31-35 in the primary amino acid sequence of the variable heavy chain of SEQ ID NO:19 (see FIG. 3A) or SEQ ID NO:13 (see FIG. 3B)), and comprising the sequence X1HX2X3S (SEQ ID NO:1); X1 of CDR1H is A, D, N, or S; X2 of CDR1H is A or G; and X3 of CDR1H is an amino acid with a hydrophobic side chain, including I or M.

[0065] Alternatively, CDR1H can be defined using the IMGT definition, where CDR1H comprises residues 26-33 of the CEACAM1 antibody (e.g., corresponding to residues 26-33 in the primary amino acid sequence of the variable heavy chain of SEQ ID NO: 19 (see FIG. 3A) or SEQ ID NO: 13 (see FIG. 3B)), and comprises the sequence X 14 X 15 X 16 FX 17 X1HX2 (SEQ ID NO: 20), CDR1H X 14 is G or E; CDR1H X 15 is an amino acid having an aromatic side chain, including F or Y; CDR1H X 16 is T, S, or I; CDR1H X 17 is an amino acid having a polar, uncharged side chain, including S, T, or N; X1 of CDR1H is A, D, N, or S; and X2 of CDR1H is A or G.

[0066] In one embodiment, the CDR1H (Kabat definition) of the CEACAM1 antibody or antigen-binding fragment thereof comprises the sequence SHGMS (SEQ ID NO:9).

[0067] In some embodiments, the CDR1H (IMGT definition) comprises the sequence GFIFSHG (SEQ ID NO:21).

[0068] In one aspect, the invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising a CDR1H region, the CDR1H comprising residues 26-35 of a CEACAM1 antibody (corresponding to the Kabat definition, e.g., residues 26-35 in the primary amino acid sequence of the variable heavy chain of SEQ ID NO: 19 (see FIG. 3A) or SEQ ID NO: 13 (see FIG. 3B)), and comprising the sequence X 14 X 15 X 16 FX 17 X1HX2X3S (SEQ ID NO: 22), X 14 is G or E; X 15 is an amino acid having an aromatic side chain, including F or Y; X 16 is T, S, or I; X 17 is an amino acid having a polar, uncharged side chain, including S, T, or N; X1 is A, D, N, or S; X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M.

[0069] In one embodiment, the CDR1H region comprises the sequence GFIFSSHSGMS (SEQ ID NO:23).

[0070] In one aspect, the invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising a CDR3H comprising residues 95-102 (corresponding to the Kabat definition, e.g., residues 99-110 in the primary amino acid sequence of the variable heavy chain of SEQ ID NO:19 (see FIG. 3A) or SEQ ID NO:13 (see FIG. 3B)), and comprising the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO:3); X4 of CDR3H is D, G, or P; X5 of CDR3H is F or P; X6 of CDR3H is D or F; X7 of CDR3H is A or Y; and X8 is L, H, or F.

[0071] In one embodiment, the CDR3H comprises residues 95 to 102 (corresponding to the Kabat definition, e.g., residues 99 to 110 in the primary amino acid sequence of the variable heavy chain of SEQ ID NO: 19 (see Figure 3A) or SEQ ID NO: 13 (see Figure 3B)) and comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO: 7); X4 of CDR3H is D, G, or P; X5 of CDR3H is F or P; and X8 of CDR3H is L, H, or F.

[0072] In one embodiment, the CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO: 10).

[0073] In one aspect, the invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising a CDR3H region, the CDR3H region comprising residues 94 to 102 (corresponding to the Kabat definition, e.g., residues 98 to 110 in the primary amino acid sequence of the variable heavy chain of SEQ ID NO:19 (see FIG. 3A) or SEQ ID NO:13 (see FIG. 3B)), and comprising the sequence X 18 HX4X5DYX6PX7WFAX8 (SEQ ID NO: 24), X 18 is R or K; X4 is D, G, or P; X5 is F or P; X6 is D or F; X7 is A or Y; and X8 is L, H, or F.

[0074] In one embodiment, the CDR3H region comprises the sequence RHDFDYFPYWFAH (SEQ ID NO:25).

[0075] In one aspect, the invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising a CDR3L comprising residues 89-97 (corresponding to the Kabat definition, e.g., residues 88-96 in the primary amino acid sequence of the variable heavy chain of SEQ ID NO: 14 (see FIG. 3C)) and having the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6), X9 is W or N; X 10 is S or T; X 11 is A or an amino acid having a neutral hydrophilic side chain, including S, N, and T; X 12 is L, F, or N; and X 13 is P or F.

[0076] In one embodiment, the CDR3L comprises residues 89 to 97 (corresponding to the Kabat definition, e.g., residues 88 to 96 in the primary amino acid sequence of the variable heavy chain of SEQ ID NO: 14 (see FIG. 3C)) and has the sequence QQX9SSX 12 PX 13 T (SEQ ID NO:8); X9 is W or N; X 12 is L, F, or N; and X 13 is P or F.

[0077] In one embodiment, the CDR3L comprises the sequence QQWSSNPPT (SEQ ID NO: 11) or the sequence QQWTSNPPT (SEQ ID NO: 12).

[0078] In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of the heavy chain variable region and the light chain variable region comprising CDR1, CDR2, and CDR3: The sequence of CDR1 (CDR1H) of the heavy chain variable region is sequence X 14 X 15 X 16 FX 17 Contains X1HX2X3S (SEQ ID NO:22); X 14 is G or E; X 15 is an amino acid having an aromatic side chain, including F or Y; X 16 is T, S, or I; X 17 is an amino acid having a polar, uncharged side chain, including S, T, or N; X1 is A, D, N, or S; X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M; The sequence of CDR2 (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of the CDR3 (CDR3H) of the heavy chain variable region is HX4X5DYX6X 19 X7WFAX 20 (SEQ ID NO:45); X4 is D, G, or P; X5 is F or P; X6 is D or F; X 19 is P or A; X7 is A or Y; and X 20 is L, H, Y or F; The sequence of CDR1 (CDR1L) of the light chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2 (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of the CDR3 of the light chain variable region (CDR3L) is QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6); X9 is W or N; X 10 is S or T; X 11 is A or an amino acid having a neutral hydrophilic side chain, including S, N, and T; X 12 is L, F, or N; and X 13 is P or F; and X 19 is A and / or X 20 If Y, then X 10 is T, X4 is G or P, X1 is N, and / or X 16 is T or S.

[0079] In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of the heavy chain variable region and the light chain variable region comprising CDR1, CDR2, and CDR3: The sequence of CDR1 (CDR1H) of the heavy chain variable region is sequence X 14 FX 21 FX 22 X 23 HX2X3S (SEQ ID NO:46); X 14 is G or E; X 21 is T or I; X 22 is N or S; X 23 is A, D, or S, X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M; The sequence of CDR2 (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3 (CDR3H) of the heavy chain variable region is HX 24 FDYX6X 19 X7WFAX 25 (SEQ ID NO:47); X 24 is D or G; X6 is D or F; X 19 is P or A; X7 is A or Y; and X 25 is H or Y; The sequence of CDR1 (CDR1L) of the light chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2 (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); The sequence of the CDR3 of the light chain variable region (CDR3L) is QQWX 10 X 10 Contains NPPT (SEQ ID NO:48); X 10 is S or T; X 21 If is I, then X6 is F and X 19 is P and / or X7 is Y.

[0080] In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of the heavy chain variable region and the light chain variable region comprising CDR1, CDR2, and CDR3: The sequence of CDR1 (CDR1H) of the heavy chain variable region is sequence X 14 FTFX 22 X 26 containing HAX3S (SEQ ID NO:49); X 14 is G or E; X 17 is S or N; X 22 is N or S; X 26 is A or D, and X3 is an amino acid having a hydrophobic side chain, including I or M; The sequence of CDR2 (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3 (CDR3H) of the heavy chain variable region is HX 24 FDYX6X 19 X7WFAX 25 (SEQ ID NO:47); X 24 is D or G; X6 is D or F; X 19 is P or A; X7 is A or Y; and X 25 is H or Y; The sequence of CDR1 (CDR1L) of the light chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2 (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of the CDR3 of the light chain variable region (CDR3L) is QQWX 10 X 10 Contains NPPT (SEQ ID NO:48); X 10 is either S or T.

[0081] In one aspect, the invention provides a CEACAM1 antibody or antigen-binding fragment thereof, the CEACAM1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the chain variable region comprising CDR1H, CDR2H, and CDR3H (Kabat definition), and the light chain variable region comprising CDR1L, CDR2L, and CDR3L (Kabat definition): The sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO:1), The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2), The sequence of CDR3H comprises the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO:3), The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO: 4), The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5), and The sequence of CDR3L is QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6). X1~X 18 has been previously defined.

[0082] In one embodiment, the invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, each of which comprises CDR1, CDR2, and CDR3: The sequence of the heavy chain variable region comprises the sequence GXXXXX1HX2X3S (SEQ ID NO:43); X is any amino acid; X1 is A, D, N, or S; X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M; and The sequence of CDR2 (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44); X4 is D, G, or P; X5 is F or P; X7 is A or Y; and X8 is L, H, or F; The sequence of CDR1 (CDR1L) of the light chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2 (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); The sequence of the CDR3 of the light chain variable region (CDR3L) is QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6); X9 is W or N; X 10 is S or T; X 11 is A or an amino acid having a neutral hydrophilic side chain, including S, N, and T; X 12 is L, F, or N; and X 13 is P or F.

[0083] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the chain variable region comprises CDR1H, CDR2H, and CDR3H (Kabat definition), and the light chain variable region comprises CDR1L, CDR2L, and CDR3L (Kabat definition): The sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO:1); The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2), The sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO:7); The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO: 4), The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5), and The sequence of CDR3L is QQX9SSX. 12 PX 13 T (SEQ ID NO:8). X1~X 18 has been previously defined.

[0084] According to certain embodiments, the contemplated antibodies and antigen-binding fragments thereof also feature a humanized framework to reduce immunogenicity. In certain embodiments, the CDRs of the contemplated antibodies or antigen-binding fragments thereof are located in a framework derived from a human antibody or antigen-binding fragment thereof. In other embodiments, surface-exposed framework residues of the contemplated antibodies or antigen-binding fragments thereof are replaced with framework residues of a human antibody or antigen-binding fragment thereof. The CDRs may also be placed in a murine or humanized framework linked to a human constant region (i.e., a chimeric antibody). In a preferred embodiment, the CDRs of the contemplated antibodies or antigen-binding fragments thereof are placed in a framework that is a composite of two or more human antibodies. In such an embodiment, the contemplated antibodies or antigen-binding fragments thereof comprise two or more sequence segments ("composite") derived from the V-regions of unrelated human antibodies selected to maintain the monoclonal antibody sequences important for antigen binding of the starting precursor anti-human CEACAM1 monoclonal antibody, which have all been filtered for the presence of potential T-cell epitopes using "in silico tools" (Holgate & Baker, IDrugs. 2009 Apr; 12(4): 233-7). Prior to synthesis of the antibody or antigen-binding fragment thereof, the human sequence segments are closely matched with all sections of the starting antibody V region to produce a CD4 +By removing T cell epitopes, this technology avoids immunogenicity while maintaining optimal affinity and specificity through prior analysis of sequences required for antigen specificity (Holgate & Baker, 2009).

[0085] Also provided herein are similar, but non-identical, variable heavy and light chain sequences and pairs thereof disclosed in SEQ ID NOs: 13-16.

[0086] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID NO:13.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence comprising SEQ ID NO: 16. In other embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence comprising SEQ ID NO: 14. In other embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence comprising SEQ ID NO: 15.

[0088] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID NO:13 and a variable light chain amino acid sequence comprising SEQ ID NO:14.

[0089] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID NO:13 and a variable light chain amino acid sequence comprising SEQ ID NO:15.

[0090] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID NO:13 and a variable light chain amino acid sequence comprising SEQ ID NO:16.

[0091] As used herein, the term "identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions of each sequence that can be aligned for comparison purposes. For example, if the positions of the compared nucleotide sequences are occupied by the same base, the molecules are identical at that position. The degree of identity between nucleic acid sequences or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. For example, polypeptides having at least 85%, 90%, 95%, 98%, or 99% identity to the specific polypeptides described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are contemplated. Methods and computer programs for determining both sequence identity and similarity are publicly available, and include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990), and the ALIGN program (version 2.0). The well-known Smith-Waterman algorithm may also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at http: / / www.ncbi.nlm.nih.gov / blast / ). In comparing sequences, these methods take into account various substitutions, deletions, and other modifications.

[0092] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; and / or (ii) comprises a light chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO:14.

[0093] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; (ii) a light chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO: 14; (iii) In that case: The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13, the sequence of CDR3H comprises residues D102, Y103, F104, P105, and Y106 of SEQ ID NO: 13; the sequence of CDR1L comprises residues A28, S30, and Y31 of SEQ ID NO: 14; The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO:14, and The sequence of CDR3L includes residues S91 and S92 of SEQ ID NO:14. Residue numbering is based on the primary amino acid sequence of the antibody, see Figures 3A, 3B, and 3C for examples of heavy and light chain sequences.

[0094] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; (ii) a light chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO: 14; and (iii) a sequence comprising six CDRs: a. the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO:9; b. the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; c. the sequence of the CDR3 of the heavy chain variable region comprises SEQ ID NO:10; d. the sequence of CDR1 of the light chain variable region comprises SEQ ID NO:4; e. the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:5; and f. The sequence of the CDR3 of the light chain variable region comprises SEQ ID NO:11.

[0095] In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 13; the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the light chain variable region amino acid sequence of SEQ ID NO: 14; The sequence of the heavy chain variable region comprises the sequence GXXXXX1HX2X3S (SEQ ID NO:43); X is any amino acid; X1 is A, D, N, or S; X2 is A or G; and X3 is an amino acid having a hydrophobic side chain, including I or M; and The sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44); X4 is D, G, or P; X5 is F or P; X7 is A or Y; and X8 is L, H, or F;

[0096] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; and / or (ii) comprises a light chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO:15.

[0097] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; (ii) comprises a light chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO: 15; (iii) In that case: The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13, the sequence of CDR3H comprises residues D102, Y103, F104, P105, and Y106 of SEQ ID NO: 13; the sequence of CDR1L comprises residues A28, S30, and Y31 of SEQ ID NO: 15; The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO:15; and The sequence of CDR3L includes residue S92 of SEQ ID NO:15. Residue numbering is based on the primary amino acid sequence of the antibody, see Figures 3A, 3B, and 3C for examples of heavy and light chain sequences.

[0098] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (iv) a heavy chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; (v) a light chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO: 15; and (vi) a sequence comprising six CDRs: a. the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO:9; b. the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; c. the sequence of the CDR3 of the heavy chain variable region comprises SEQ ID NO:10; d. the sequence of CDR1 of the light chain variable region comprises SEQ ID NO:4; e. the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:5; and f. The sequence of the CDR3 of the light chain variable region comprises SEQ ID NO:12.

[0099] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; and / or (ii) comprises a light chain variable domain comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO: 16.

[0100] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (i) a heavy chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; (ii) comprises a light chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO: 16; (iii) In that case: The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13, the sequence of CDR3H comprises residues D102, Y103, F104, P105, and Y106 of SEQ ID NO: 13; The sequence of CDR1L comprises residues S30 and Y31 of SEQ ID NO: 16, The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO: 16; and The sequence of CDR3L includes residues S91 and S92 of SEQ ID NO:16. Residue numbering is based on the primary amino acid sequence of the antibody, see Figures 3A, 3B, and 3C for examples of heavy and light chain sequences.

[0101] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is (vii) a heavy chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable domain sequence of SEQ ID NO: 13; (viii) a light chain variable domain comprising a sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable domain sequence of SEQ ID NO: 16; and (ix) a sequence comprising six CDRs: a. the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO:9; b. the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; c. the sequence of the CDR3 of the heavy chain variable region comprises SEQ ID NO:10; d. the sequence of CDR1 of the light chain variable region comprises SEQ ID NO:18; e. the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:5; and f. The sequence of the CDR3 of the light chain variable region comprises SEQ ID NO:11.

[0102] It will be apparent that any of the frameworks described herein can be utilized in combination with any of the CDRs and CDR motifs described herein. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof utilizes a framework described in Table 1.

[0103] Some embodiments of the aspects described herein contemplate modification of the amino acid sequence of the antibody or antigen-binding fragment thereof that binds to CEACAM1 described herein. The amino acid sequence variants of the antibody or antigen-binding fragment thereof are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody or antigen-binding fragment thereof or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody or antigen-binding fragment thereof. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final construct, provided that the final construct has the desired properties, e.g., binding specificity, inhibition of biological activity.

[0104] One type of variant is a conservative amino acid substitution variant. These variants have at least one amino acid residue in the antibody or antigen-binding fragment thereof replaced with a different residue having similar side chain properties. Amino acids can be grouped according to the similarity of their side chain properties (see Lehninger, BIOCHEMISTRY (2nd ed., Worth Publishers, New York, 1975): (1) Nonpolar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M); (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp(D), Glu(E); (4) Basic: Lys(K), Arg(R), His(H). Thus, a non-limiting example of a conservative amino acid substitution is one that replaces a non-polar amino acid with another non-polar amino acid.

[0105] Alternatively, the naturally occurring residues can be grouped on the basis of common side chain properties. (1) Hydrophobicity: Ala(A), Val(V), Leu(L), Ile(I), Met(M); (2) Neutral hydrophilic: Ser(S), Thr(T), Cys(C), Asn(N), Gln(Q); (3) Acidic: Asp(D), Glu(E); (4) Basic: Lys(K), Arg(R), His(H); (5) Residues that affect chain orientation: Gly (G), Pro (P); (6) Aromatic: Phe(F), Trp(W), Tyr(Y). Thus, a non-limiting example of a conservative amino acid substitution is one that replaces a hydrophobic amino acid with another hydrophobic amino acid.

[0106] Also contemplated are amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as amino acid sequence insertions which may include intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody or antigen-binding fragment thereof with an N-terminal methionyl residue, or an antibody or antigen-binding fragment thereof fused to a cytotoxic polypeptide. Other insertional variants of antibodies or antigen-binding fragments thereof include the fusion to the N- or C-terminus of the antibody or antigen-binding fragment thereof of an enzyme or a polypeptide which increases the serum half-life of the antibody or antigen-binding fragment thereof, such as biotin.

[0107] Cysteine ​​residues not involved in maintaining the proper conformation of an antibody or antigen-binding fragment thereof that binds CEACAM1 can also be substituted, e.g., to serine or alanine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0108] Conversely, cysteine ​​bond(s) may be added to the antibody or antigen-binding fragment thereof to improve its stability (particularly where the antibody or antigen-binding fragment thereof is an antibody fragment such as an Fv fragment).

[0109] In some embodiments, the described antibodies or antigen-binding fragments thereof have amino acid modifications that alter the original glycosylation pattern of the antibody or antigen-binding fragment thereof. By "altering the original glycosylation pattern" is meant deleting one or more carbohydrate moieties found in the antibody or antigen-binding fragment thereof and / or adding one or more glycosylation sites that are not present in the antibody or antigen-binding fragment thereof. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Addition of glycosylation sites to an antibody or antigen-binding fragment thereof that binds to CEACAM1 is accomplished by modifying the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). Modifications can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody or antigen-binding fragment thereof (for O-linked glycosylation sites).

[0110] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof provided herein is deglycosylated or aglycosylated. In some embodiments, the contemplated CEACAM1 antibody or antigen-binding fragment thereof lacks a C-terminal lysine in the heavy chain and / or comprises a S241P substitution in the constant region of the heavy chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof lacks a glycosylation site in CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof lacks a NXS / T consensus sequence in CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof has a mutation at CDR residues 26 and / or 29 (Kabat numbering) of CDR1 of the variable light chain. If the antibody or antigen-binding fragment thereof comprises an Fc region, the carbohydrate(s) attached thereto can be modified. For example, antibodies have been described that have a mature carbohydrate structure that lacks fucose attached to the Fc region of the antibody or antigen-binding fragment thereof. See, for example, US Patent Publication Nos. 2003 / 0157108; 2004 / 0093621. Antibodies with bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody or antigen-binding fragment thereof are referenced in WO03 / 011878; US Patent No. 6,602,684. Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody or antigen-binding fragment thereof are reported in WO97 / 30087. See also WO98 / 58964; WO99 / 22764 for antibodies with modified carbohydrate attached to the Fc region.

[0111] In some embodiments, it may be desirable to modify an antibody or antigen-binding fragment thereof that binds CEACAM1 described herein with respect to effector function, for example to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody or antigen-binding fragment thereof. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody or antigen-binding fragment thereof. Alternatively or additionally, one or more cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody or antigen-binding fragment thereof thus generated may have improved internalization capability and / or increased complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., 176 J. Exp. Med. 1191 (1992); Shopes, 148 J. Immunol. 2918 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., 53 Cancer Res. 2560 (1993). Alternatively, an antibody or antigen-binding fragment thereof can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., 3 Anti-Cancer Drug Design 219 (1989).

[0112] For example, WO00 / 42072 describes an antibody with improved ADCC function in the presence of human effector cells, which comprises amino acid substitutions in its Fc region. Preferably, the antibody or antigen-binding fragment thereof with improved ADCC comprises substitutions at positions 298, 333, and / or 334 of the Fc region (Eu numbering of residues). Typically, the modified Fc region is a human IgG1 Fc region comprising or consisting of substitutions at one, two, or three of these positions. Such substitutions are optionally combined with substitution(s) that increase Clq binding and / or CDC. Substitutions include the Asn297Ala mutation of IgG1 Fc.

[0113] Antibodies with altered Clq binding and / or complement dependent cytotoxicity (CDC) are described in WO 99 / 51642, U.S. Patent Nos. 6,194,551, 6,242,195, 6,528,624, and 6,538,124. The antibodies contain amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333, and / or 334 of their Fc region (Eu numbering of residues).

[0114] Antibodies with improved binding to the neonatal Fc receptor (FcRn) and increased half-life are described in WO00 / 42072 and U.S. Patent Publication No. 2005 / 0014934. These antibodies comprise an Fc region having one or more substitutions that improve binding of the Fc region to CEACAM1. For example, the Fc region can have a substitution at one or more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, 428, or 434 (Eu numbering of residues). A preferred Fc region-containing antibody variant with improved CEACAM1 binding comprises amino acid substitutions (Eu numbering of residues) at one, two, or three of positions 307, 380, and 434 of its Fc region. In one embodiment, the antibody or antigen-binding fragment thereof has a 307 / 434 mutation. Modified antibodies that bind to CEACAM1 at three or more (e.g., four) functional antigen-binding sites are also contemplated. See, e.g., U.S. Patent Publication No. US2002 / 0004587.

[0115] Antibody fragments and types

[0116] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab fragment, which comprises the variable (V L ) domain and constant (C L ) domain and the variable domain of the heavy chain (V H ) and the first constant domain (C H 1) is included.

[0117] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab′ fragment, which is H This refers to a Fab fragment that has one or more cysteine ​​residues at the C-terminus of one domain.

[0118] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is H and C H An Fd fragment comprising, or consisting essentially of, one or more of the Fd domains.

[0119] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is H Domain and C H 1 domain and C H This is an Fd' fragment that has one or more cysteine ​​residues at the C-terminus of one domain.

[0120] Single-chain Fv or scFv antibody fragments are H Domain and V L Fv polypeptides comprise or consist essentially of V domains, and these domains are present in a single polypeptide chain. Generally, Fv polypeptides include V H Domain and V L The scFv may further comprise a polypeptide linker between the domains, which enables the scFv to form the desired structure for antigen binding. See, e.g., Pluckthun, 113 Pharmacology Monoclonal Antibodies 269 (Rosenburg & Moore, eds., Springer-Verlag, New York, 1994). Thus, in some embodiments of the aspects described herein, the CEACAM1 antibody fragment comprises a V domain of a single arm of the antibody. L Domain and V H An Fv fragment comprises or consists essentially of a domain.

[0121] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is a diabody that comprises two antigen-binding sites and includes a light chain variable domain (V L ) connected to a heavy chain variable domain (V H ) in the same polypeptide chain.

[0122] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is H A dAb fragment may be a dAb fragment comprising or consisting essentially of a domain.

[0123] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is a F(ab')2 fragment, which comprises a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region.

[0124] Linear antibodies refer to the antibodies described in Zapata et al., Protein Engin., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-V H -C H 1), which together with complementary light chain polypeptides form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. In some embodiments of the aspects described herein, the CEACAM1 antibody fragment comprises a pair of tandem Fd segments (V H -C H 1-V H -C H 1) is a linear antibody.

[0125] Various techniques have been developed and are available for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies. See, for example, Morimoto et al., 24 J. Biochem.Biophys.Meths.107 (1992); Brennan et al., 229 Science 81 (1985). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed herein. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody fragment of choice is a single chain Fv fragment (scFv). See, for example, WO93 / 16185.

[0126] In one embodiment, the antibody is a bispecific antibody that comprises a complementary region that binds to CEACAM1 and a complementary region that binds to PD-1.

[0127] In one embodiment, the antibody is a bispecific antibody that comprises a complementary region that binds to CEACAM1 and a complementary region that binds to PD-L1.

[0128] Contemplated antibodies or antigen-binding fragments may include all types of constant regions, including IgM, IgG, IgD, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, human isotype IgG1 is used. In another embodiment, human isotype IgG4 is used. The light chain constant region may be lambda or kappa. Antibodies or antigen-binding fragments thereof may include sequences from multiple classes or isotypes.

[0129] Also disclosed herein is a chimeric antigen receptor T cell (CAR T cell) that binds to CEACAM1. In one embodiment, one or more CDRs of the anti-CEACAM antibody disclosed herein are grafted onto a chimeric antigen receptor (CAR) on T cell. Such genetically modified T cell utilizes CAR, also known as chimeric T cell receptor, to target antigens expressed on tumor cells in a human leukocyte antigen-independent manner.

[0130] antibody binding

[0131] The human CEACAM1 gene generates 11 isoforms by alternative splicing. Each isoform has one variable (V)-like Ig domain at the amino (N) terminus of the protein. Except for the CEACAM1-1L and CEACAM1-1S isoforms, the various isoforms also have two or three constant C2-like Ig domains. Eight CEACAM1 isoforms are anchored to the cell membrane via a transmembrane domain, and three CEACAM1 isoforms (CEACAM1-4C1, -3, and -3C2) lack a transmembrane domain and are secreted. Two isoforms (CEACAM1-3AL and -3AS) have an Alu family repeat sequence (A) between the constant C2-like Ig domain and the transmembrane domain. The transmembrane CEACAM1 isoforms also have long (L) or short (S) cytoplasmic domains, determined by the inclusion or exclusion of CEACAM1 exon 7 in the message. The CEACAM1 L cytoplasmic domain has two ITIM motifs that are unique to CEACAM1 among the CEACAM family members. In one aspect, the present invention provides CEACAM1 antibodies or antigen-binding fragments thereof, including antibodies described herein by their structural feature of binding to an extracellular variable (V)-like Ig domain at the amino (N)-terminus (N-domain) of the CEACAM1 protein, i.e., a domain common to all isoforms of CEACAM1, including CEACAM1 isoforms 1L, 1S, 3L, 3S, 4L, 4S, 3Al, 3AS, 3, 4C1, and 4C2. In some embodiments, the provided antibodies and antigen-binding fragments thereof bind to human CEACAM1. In some embodiments, the provided antibodies and antigen-binding fragments thereof bind to mammalian CEACAM1. The sequence of the full-length form of CEACAM1 (NCBI Reference Sequence NP_001703.2; UNIPR.QT ID P13688) is provided as SEQ ID NO:26 (signal sequence: residues 1-34 of SEQ ID NO:26; Ig-V N domain: residues 35-142 of SEQ ID NO:26). The mature form of CEACAM1 (without signal sequence) is provided as SEQ ID NO:17.

[0132] As used herein, "binding" of an antibody or antigen-binding fragment thereof to CEACAM1, an epitope on CEACAM1, or, in certain embodiments described below, a particular residue on CEACAM1, includes selective interaction of the antibody or antigen-binding fragment thereof with CEACAM1. Thus, binding includes primary and secondary interactions including, for example, hydrophilic and hydrophobic interactions, as well as hydrogen bonds, ionic interactions, salt bridges.

[0133] In certain embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are -5 ~10 -12 mol / l, 10 -6 ~10 -12 mol / l, 10 -7 ~10 -12 mol / l, 10 -8 ~10 -12 mol / l, 10 -9 ~10 -12 mol / l, 10 -10 ~10 -12 mol / l, or 10 -11 ~10 -12 K in mol / l D In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein bind to CEACAM1 at 10 -5 ~10 -11 mol / l, 10 -6 ~10 -11 mol / l, 10 -7 ~10 -11 mol / l, 10 -8 ~10 -11 mol / l, 10 -9 ~10 -11 mol / l, or 10 -10 ~10 -11 K in mol / l D In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein bind to CEACAM1 at 10 -5 ~10 -10 mol / l, 10 -6 ~10 -10 mol / l, 10 -7 ~10 -10mol / l, 10 -8 ~10 -10 mol / l, or 10 -9 ~10 -10 K in mol / l D In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein bind to CEACAM1 at 10 -5 ~10 -8 mol / l, 10 -6 ~10 -8 mol / l, or 10 -7 ~10 -8 K in mol / l D and binds to CEACAM1.

[0134] The term "specificity" as used herein refers to the ability of an antibody or antigen-binding fragment thereof, such as an anti-CEACAM1 antibody or antigen-binding fragment thereof, to recognize an epitope within CEACAM1 but have little or no detectable reactivity with other parts of CEACAM1. Specificity can be relatively determined by competitive assays or by the epitope identification / characterization techniques described herein or their equivalents known in the art.

[0135] As used herein, an "epitope" can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least three, and more commonly at least five, about nine, or about eight to ten amino acids in a particular spatial conformation. An "epitope" is defined as a sequence that is unique to an immunoglobulin V protein. H / V L It comprises a structural unit that is conventionally bound by a variable domain pair. An epitope defines the minimal binding site of an antibody or antigen-binding fragment thereof and thus represents the target of specificity of the antibody or antigen-binding fragment thereof. In the case of a single domain antibody, the epitope represents the structural unit to which the variable domain binds alone.

[0136] In certain embodiments, contemplated antibodies or antigen-binding fragments specifically bind to the same epitope as antibody CP08H03 / Vk8 S29A, hi other embodiments, contemplated antibodies or antigen-binding fragments bind to the same epitope as CP08H03 / CP08F05.

[0137] In one aspect, the invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein by their structural features, which specifically bind to at least a portion of the homophilic binding domain on CEACAM1 (i.e., the portion of the CEACAM1 protein involved in the formation of the CEACAM1:CEACAM1 homodimer), thereby blocking the CEACAM1 homophilic interaction. In certain embodiments, the provided antibodies or antigen-binding fragments thereof specifically bind to one or more CEACAM1 residues (i.e., Y34, Q44, Q89, N97 of SEQ ID NO: 17) contained in the CC' and FG loops of CEACAM1 and comprising the YQQN pocket at the CEACAM1:CEACAM1 dimer interface (see Huang et al., Nature. 2015 Jan 15;517(7534):386-90).

[0138] As used herein, a "blocking" antibody or antibody "antagonist" is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. For example, in some embodiments, a CEACAM1 antagonist antibody or antigen-binding fragment thereof binds to CEACAM1, inhibits the activity of CEACAM1, and / or inhibits the binding of CEACAM1 to other CEACAM proteins or heterologous binding partners such as TIM-3. Inhibition of activity and inhibition of binding include partial inhibition. Methods for identifying CEACAM1 antibodies that block homophilic and heterophilic interactions of CEACAM1 are described herein and known to those of skill in the art. For example, competing, cross-blocking, and cross-blocked antibodies can be identified using any suitable method known in the art, including competitive ELISA or BIACORE® assays, where the binding of a competing or cross-blocking antibody to human CEACAM1 prevents the binding of an antibody disclosed herein, or vice versa.

[0139] In one embodiment, the heavy chain of a contemplated antibody or antigen-binding fragment thereof specifically binds to CEACAM1 at residues F29, Y34, T56, Q89, S93, and / or D94 of SEQ ID NO: 17. In another embodiment, the heavy chain of a contemplated antibody or antigen-binding fragment thereof further specifically binds to CEACAM1 at residues S32, Q44, A49, and / or I91 of SEQ ID NO: 17.

[0140] In one embodiment, the light chain of a contemplated antibody or antigen-binding fragment thereof specifically binds to CEACAM1 at residues D40, G41, N42, N97, and / or E99 of SEQ ID NO: 17. In another embodiment, the light chain of a contemplated antibody or antigen-binding fragment thereof further specifically binds to CEACAM1 at residues L95 and / or V96 of SEQ ID NO: 17.

[0141] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof specifically binds to CEACAM1 at residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and / or E99 of SEQ ID NO: 17. In another preferred embodiment, the CEACAM1 antibody or antigen-binding fragment thereof further specifically binds to CEACAM1 at residues S32, Q44, A49, I91, L95, and / or V96 of SEQ ID NO: 17.

[0142] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof specifically binds to CEACAM1 at residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and E99 of SEQ ID NO:17.

[0143] In another embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to CEACAM1 at residues F29, S32, Y34, D40, G41, N42, Q44, A49, T56, Q89, I91, S93, D94, L95, V96, N97, and E99 of SEQ ID NO:17.

[0144] In certain embodiments, not all CDRs are directly involved in binding to the antigen. In one embodiment, four of the six CDRs of the CEACAM1 antibody or antigen-binding fragment thereof contact the antigen. In one embodiment, five of the six CDRs of the CEACAM1 antibody or antigen-binding fragment thereof contact the antigen. In one embodiment, six of the six CDRs of the CEACAM1 antibody or antigen-binding fragment thereof contact the antigen. In one embodiment, CDR2H, CDR3H, CDR1L, CDR2L, and CDR3L of the CEACAM1 antibody or antigen-binding fragment thereof are directly involved in binding to the antigen.

[0145] In one embodiment, the antibodies and antigen-binding fragments thereof provided herein specifically bind to an epitope of CEACAM1 located in the N-domain of CEACAM1. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to a CEACAM1 epitope that includes one or more CEACAM1 residues selected from F29, S32, D40, A49, and T56 of SEQ ID NO: 17. In a further embodiment, the CEACAM1 antibody specifically binds to a CEACAM1 epitope that includes residues F29, S32, D40, A49, T56, and I91 of SEQ ID NO: 17.

[0146] In one embodiment, the antibodies and antigen-binding fragments thereof provided herein specifically bind to an epitope of CEACAM1 located in the N-domain of CEACAM1. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to a CEACAM1 epitope that includes one or more CEACAM1 residues selected from S32, D40, A49, and I91 of SEQ ID NO: 17. In a further embodiment, the CEACAM1 antibody specifically binds to a CEACAM1 epitope that includes residues S32, D40, A49, and I91 of SEQ ID NO: 17.

[0147] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein binds to CEACAM1 and CDR2H residue Y57 binds to CEACAM1 at residue F29; CDR2H residue Y59 binds to CEACAM1 at residue S93; CDR3H residue D102 binds to CEACAM1 at residue T56; CDR3H residue Y103 binds to CEACAM1 at residues Y34 and / or Q89; CDR3H residue F104 binds to CEACAM1 at residue F29; CDR3H residue Y106 binds to CEACAM1 at residue D94; CDR1L residue S30 binds to CEACAM1 at residue E99; CDR1L residue Y31 binds to CEACAM1 at residue N97; CDR2L residue S51 binds to CEACAM1 at residue D40, and / or CDR2L residue N52 binds to CEACAM1 at residues G41 and / or N42. The numbering of the CDR residues is based on the primary amino acid sequence of the antibody, see Figures 3A, 3B, and 3C for examples of heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO:17.

[0148] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein binds to CEACAM1 and CDR2H residue Y57 binds to CEACAM1 at residue F29; CDR2H residue Y59 binds to CEACAM1 at residue S93; CDR3H residue D102 binds to CEACAM1 at residue T56; CDR3H residue Y103 binds to CEACAM1 at residues S32, Y34, Q44, and / or Q89; CDR3H residue F104 binds to CEACAM1 at residues F29 and / or A49; CDR3H residue P105 binds to CEACAM1 at residue I91; CDR3H residue Y106 binds to CEACAM1 at residue D94; CDR1L residue S30 binds to CEACAM1 at residue E99; CDR1L residue Y31 binds to CEACAM1 at residue N97; CDR2L residue S51 binds to CEACAM1 at residue D40; CDR2L residue N52 binds to CEACAM1 at residues G41 and / or N42; CDR3L residue S91 binds to CEACAM1 at residue L95, and / or CDR3L residue S92 binds to CEACAM1 at residue V96. Residue numbering is based on the primary amino acid sequence of the antibody, see Figures 3A, 3B, and 3C for examples of heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO:17.

[0149] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein binds to CEACAM1 and CDR2H residue Y57 binds to CEACAM1 at residue F29; CDR2H residue Y59 binds to CEACAM1 at residue S93; CDR3H residue D102 binds to CEACAM1 at residue T56; CDR3H residue Y103 binds to CEACAM1 at residues S32, Y34, Q44, and Q89; CDR3H residue F104 binds to CEACAM1 at residues F29 and A49; CDR3H residue P105 binds to CEACAM1 at residue I91; CDR3H residue Y106 binds to CEACAM1 at residue D94; CDR1L residue S30 binds to CEACAM1 at residue E99; CDR1L residue Y31 binds to CEACAM1 at residue N97; CDR2L residue S51 binds to CEACAM1 at residue D40; CDR2L residue N52 binds to CEACAM1 at residues G41 and N42; CDR3L residue S91 binds to CEACAM1 at residue L95, and CDR3L residue S92 binds to CEACAM1 at residue V96. Residue numbering is based on the primary amino acid sequence of the antibody, see Figures 3A, 3B, and 3C for examples of heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO:17.

[0150] CEACAM family members are widely expressed in various cell types, especially leukocytes, and affect a range of cellular functions. For example, CEACAM1 is expressed in epithelial cells, endothelial cells, lymphocytes, and myeloid cells, CEACAM3 is expressed in granulocytes and neutrophils, CEACAM5 is expressed in epithelial cells, and CEACAM6 is expressed in epithelial cells and granulocytes. However, the N-domain of CEACAM1 is approximately 90% similar to the N-domains of CEACAM family members 3, 5, and 6, making it difficult to selectively target CEACAM1.

[0151] Despite the high similarity of the N domains between CEACAM family members, in some embodiments, the antibodies or antigen-binding fragments provided herein, including those described herein by their structural features, are selective for CEACAM1. By selectively targeting CEACAM1, embodiments of the present invention may, for example, avoid undesirable interference with the broad activation function of CEACAM3.

[0152] As used herein, the terms "selective" and "selectivity" refer to the preferential binding of an antibody or antigen-binding fragment thereof (i.e., a CEACAM1 antibody or antigen-binding fragment thereof) to a particular region, target, or peptide; typically a region or epitope in CEACAM1, in contrast to one or more other biological molecules, including other CEACAM family members.

[0153] In one embodiment, a CEACAM1 antibody or antigen-binding fragment thereof does not exhibit significant binding to other CEACAM family members, including CEACAM3, CEACAM5, CEACAM6, and / or CEACAM8. In one embodiment, a CEACAM1 antibody or antigen-binding fragment thereof does not exhibit detectable binding to other CEACAM family members, including CEACAM3, CEACAM5, CEACAM6, and / or CEACAM8. In some embodiments, a contemplated CEACAM1 antibody or antigen-binding fragment thereof binds to CEACAM1 with an affinity that is at least 10-fold, e.g., at least 100-fold, and at least 1000-fold, and up to 10,000-fold or more stronger than the affinity with which a contemplated CEACAM1 antibody or antigen-binding fragment thereof binds to another target or polypeptide.

[0154] As used herein, the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (K D "Affinity," expressed by K), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding protein, such as an antibody or antibody fragment thereof. D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule. Alternatively, affinity can be expressed as the affinity constant (K A ), which can also be expressed as 1 / K D As will be clear to those skilled in the art, affinity can be measured in a manner known per se depending on the particular antigen of interest.

[0155] In one aspect, the invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein by their structural characteristics, which specifically bind to at least a portion of the binding site on CEACAM1 for one or more other CEACAM family members, thereby blocking the interaction of CEACAM1 with one or more other CEACAM family members, including, but not limited to, CEACAM3, CEACAM5, CEACAM6, and CEACAM8 (Ramani et al, Anal. Biochem. Jan. 15, 2012; 420(2); 127-38; Scheffrahn et al, J. Immunol. May. 15, 2002; 168(10); 5139-46).

[0156] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein by their structural features, which specifically bind to at least a portion of the binding site on CEACAM1 for a TIM family member, thereby blocking the interaction of CEACAM1 with a TIM family member. In some embodiments, the TIM family member is TIM-1, TIM-3, or TIM-4. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof specifically binds to one or more of CEACAM1 residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and / or N97 of SEQ ID NO: 17, which have been shown to be involved in the binding of CEACAM1 to TIM-3 (Huang et al., Nature. 2015 Jan 15;517(7534):386-90).

[0157] In one aspect, the invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein by their structural features, which specifically bind to at least a portion of the binding site on CEACAM1 for a bacterial adhesive surface protein (adhesin), thereby blocking the interaction of CEACAM1 with the adhesin. In certain embodiments, the adhesin is expressed on the surface of CEACAM1-binding pathogenic bacteria, including, but not limited to, Escherichia coli, particularly diffusely adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, N. meningitidis, commensal Neisseria, Moraxella catarrhalis, Haemophilus influenza, Haemophilus aegyptius, Helicobacter pylori, and / or Salmonella species.

[0158] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 expressed on the surface of Helicobacter pylori and HopQ. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to one or more of CEACAM1 residues F29, Y34, N42, Q89, and N97, which are predicted to be involved in the binding of CEACAM1 to HopQ.

[0159] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is 52 , Opa 65 , Opa 68 , Opav0, Opa 72 , Opa 73 , Opa 74 , and Opa 75In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to one or more of CEACAM1 residues Q44 and A49, which are predicted to be involved in binding of CEACAM1 to Neisserial Opa proteins.

[0160] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and the Opa-like protein OlpA expressed on the surface of Moraxella species.

[0161] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Haemophilus influenza OMP P1. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to one or more of CEACAM1 residues Q44 and A49, which are predicted to be involved in binding of CEACAM1 to Haemophilus influenza OMP P1.

[0162] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Haemophilus aegyptius OMP P1. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to CEACAM1 residue F29, which is predicted to be involved in binding of CEACAM1 to Haemophilus aegyptius OMP P1.

[0163] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and C. albicans.

[0164] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and influenza viruses, including but not limited to H5N1.

[0165] In another embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of CEACAM1 to a filarial nematode, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the filarial nematode is Wucheria bancrofti.

[0166] antibody complex

[0167] In some embodiments of the aspects described herein, the antibody or antigen-binding fragment thereof that binds to CEACAM1 is conjugated to a functional moiety. Examples of useful functional moieties include, but are not limited to, blocking moieties, detectable moieties, diagnostic moieties, targeting moieties, and therapeutic moieties.

[0168] Exemplary blocking moieties include sufficient steric bulk and / or charged moieties such that reduced glycosylation occurs, for example, by blocking the ability of glycosidases to glycosylate the antibody or antigen-binding fragment thereof. Blocking moieties may additionally or alternatively reduce effector function, for example, by inhibiting the ability of the Fc region to bind to a receptor or complement protein. Preferred blocking moieties include cysteine ​​additions and PEG moieties.

[0169] In a preferred embodiment, the blocking moiety is a cysteine, preferably a cysteine ​​that is bound to a free cysteine, e.g., during or after translation of an Fc-containing polypeptide, e.g., in cell culture. Other blocked cysteine ​​adducts include cystine, mixed disulfide adducts, or disulfide bonds.

[0170] In another preferred embodiment, the blocking moiety is a polyalkylene glycol moiety, such as a PEG moiety, preferably a PEG-maleimide moiety. Preferred pegylated moieties (or related polymers) can be, for example, polyethylene glycol ("PEG"), polypropylene glycol ("PPG"), polyoxyethylated glycerol ("POG") and other polyoxyethylated polyols, polyvinyl alcohol ("PVA") and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glucose. The polymer can be a homopolymer, random or block copolymer, a terpolymer based on the above monomers, linear or branched, substituted or unsubstituted, so long as it has at least one active sulfone moiety. The polymer moiety can be of any length or molecular weight, although these properties may affect the biological properties. The average molecular weight of polymers that are particularly useful for reducing clearance rates in pharmaceutical applications ranges from 2,000 to 35,000 daltons. Additionally, when two groups are linked to a polymer (one at each end), the length of the polymer can affect the effective distance and other spatial relationships between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or impart the desired biological activity. PEG is useful for biological applications for several reasons. PEG is typically clear, colorless, odorless, water-soluble, stable to heat, inert to many chemicals, non-hydrolyzable, and non-toxic. PEGylation can improve the pharmacokinetic performance of a molecule by increasing the apparent molecular weight of the molecule. An increase in apparent molecular weight reduces the rate of clearance from the body after subcutaneous or systemic administration. In many cases, PEGylation can reduce antigenicity and immunogenicity. Additionally, PEGylation can increase the solubility of biologically active molecules.

[0171] Examples of detectable moieties useful in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include detectable labels such as fluorescent moieties or fluorescent labels, imaging agents, radioisotope moieties, radio-opaque moieties, etc., such as biotin, fluorophores, chromophores, spin resonance probes, or radioactive labels. Exemplary fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, etc.) and other luminescent molecules (e.g., luminal). Fluorophores may be environmentally sensitive, such that their fluorescence changes when they are located near one or more residues in a modified protein that undergo a conformational change upon binding to a substrate (e.g., dansyl probe). Exemplary radioactive labels include atoms with one or more low sensitivity nuclei (e.g., 13 C. 15 N, 2 H, 125 I, 123 I, 99 Tc, 43 K, 52 Fe, 67 Ga, 68 Ga, 111 In). Other useful moieties are known in the art.

[0172] Examples of diagnostic moieties useful in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include detectable moieties suitable for revealing the presence of a disease or disorder. Typically, diagnostic moieties allow for the determination of the presence, absence, or level of a molecule, such as a target peptide, protein, or multiple proteins, associated with a disease or disorder. Such diagnostics are also suitable for predicting and / or diagnosing a disease or disorder and its progression.

[0173] Examples of therapeutic moieties useful in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include, for example, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, anti-infective agents, or generally therapeutic agents. A functional moiety can also have one or more of the above functions.

[0174] Exemplary therapeutic moieties include radionuclides with high energy ionizing radiation capable of causing multiple strand breaks in nuclear DNA and thus suitable for inducing cell death (e.g., cancer). Exemplary high energy radionuclides include: 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188 Re. These isotopes usually produce high-energy alpha or beta particles with short path lengths. Such radionuclides kill cells to which they are in close proximity, e.g., neoplastic cells to which the conjugate is attached or entering. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0175] Exemplary therapeutic moieties also include cytotoxic agents such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or crosslinking agents, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, and the like.

[0176] Exemplary therapeutic moieties also include alkylating agents such as the anthracycline family of drugs (e.g., adriamycin, carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenediones, and aziridines). In another embodiment, the chemotherapeutic moiety is a cytostatic drug, such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Exemplary DNA intercalators or crosslinkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0177] Exemplary therapeutic moieties also include transcriptional regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other exemplary cytostatic drugs compatible with the present invention include ansamycin benzoquinones, quinonoid derivatives (e.g., quinolones, genistein, bactacyclines), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazylquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylene phosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0178] Exemplary therapeutic moieties also include, for example, cytotoxic nucleosides, such as adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; taxoids (e.g., paclitaxel, docetaxel, taxanes), nocodazole, rhizoxin, dolastatins (e.g., dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD612, 6), tubulin-binding agents such as combretastatins (e.g., combretastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); antiangiogenic compounds such as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0179] Exemplary therapeutic moieties also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-alpha, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0180] Exemplary therapeutic moieties also include enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenz-imidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG34, and tyrphostin AG879.

[0181] Exemplary therapeutic moieties also include gene regulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, transretinal (vitamin A aldehyde), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0182] Exemplary therapeutic moieties also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and podophyllotoxins. Particularly useful members of these classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives, such as etoposide or etoposide phosphate, leurocidin, vindesine, leurosine, and the like.

[0183] Still other cytotoxic agents compatible with the teachings herein include auristatins (e.g., auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0184] Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies" in See, for example, "Monoclonal Antibodies For Cancer Detection And Therapy", Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

[0185] To increase the half-life of an antibody or polypeptide comprising an amino acid sequence described herein, a salvage receptor binding epitope can be attached to the antibody or antigen-binding fragment thereof (particularly an antibody fragment), for example as described in U.S. Pat. No. 5,739,277. The term "salvage receptor binding epitope" can refer to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule (e.g., Ghetie et al., 18 Ann. Rev. Immunol. 739 (2000). Antibodies having substitutions in their Fc region and increased serum half-lives are described in WO00 / 42072, WO02 / 060919; Shields et al., 276 J. Biol. Chem. 6591 (2001); Hinton, 279 J.Biol.Chem.6213-6216(2004). For example, a nucleic acid molecule encoding a salvage receptor binding epitope can be linked in frame to a nucleic acid encoding a polypeptide sequence described herein, such that the fusion protein expressed from this modified nucleic acid molecule comprises a salvage receptor binding epitope and a polypeptide sequence described herein. In another embodiment, serum half-life can also be increased, for example, by attaching other polypeptide sequences. For example, an antibody or antigen-binding fragment thereof useful in the method of the present invention is attached to serum albumin or a portion of serum albumin that binds to the CEACAM1 receptor or a serum albumin binding peptide, and serum albumin is attached to the antibody or antigen-binding fragment thereof (e.g., such polypeptide sequences are disclosed in WO01 / 45746). In one embodiment, the half-life of the Fab is increased by these methods. For additional serum albumin binding peptide sequences, see also Dennis et al.,277 J.Biol.Chem.35035(2002).

[0186] Other types of functional moieties are known in the art and can be readily used in the methods and compositions of the invention based on the teachings contained herein.

[0187] nucleic acid

[0188] Also provided herein are nucleic acids encoding CEACAM1 antibodies and their antigen-binding fragments, as well as vectors, host cells, and expression systems.As used herein, the term "nucleic acid" refers to any length of polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides.Thus, this term includes single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0189] Nucleic acids encoding CEACAM1 antibodies and antigen-binding fragments thereof can be, for example, DNA, cDNA, RNA, synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecules comprising any of these polynucleotides, alone or in combination. For example, expression vectors are provided that comprise a polynucleotide sequence encoding a CEACAM1 antibody or antigen-binding fragment thereof described herein, operably linked to expression control sequences suitable for expression in eukaryotic and / or prokaryotic host cells.

[0190] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. "Vector" includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acids. In some embodiments, the vectors used are vectors capable of autonomous replication (episomal vectors) and / or expression of nucleic acids to which they are linked (expression vectors). Many suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include negative-stranded RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses, i.e., AAV), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C, B, D viruses, HTLV-BLV complex, lentiviruses, and spumaviruses.

[0191] A variety of expression vectors have been developed for efficient synthesis of antibodies and their antigen-binding fragments in prokaryotic cells such as bacteria, and in eukaryotic systems, including but not limited to yeast and mammalian cell culture systems. Vectors can contain segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Also provided are cells that contain expression vectors for expressing the contemplated CEACAM1 antibody or its antigen-binding fragment.

[0192] Antibody preparation and expression system

[0193] The antibody or antigen-binding fragment thereof of the present invention is usually produced by recombinant expression. Nucleic acids encoding the light and heavy chain variable regions, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to regulatory sequences in the expression vector(s) to ensure expression of the immunoglobulin polypeptide. Expression regulatory sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression regulatory sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vectors are incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the recovery and purification of the cross-reactive antibodies.

[0194] These expression vectors are usually replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance or neomycin resistance) to allow detection of those cells transformed with the desired DNA sequence (see, e.g., Itakura et al., U.S. Patent No. 4,704,362).

[0195] Expression of antibodies and antigen-binding fragments contemplated by the present invention can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian and mammalian cells, either in vivo or in situ, or host cells of mammalian, insect, avian or yeast origin. Mammalian cells or tissues can be from humans, primates, hamsters, rabbits, rodents, cows, pigs, sheep, horses, goats, dogs or cats, although any other mammalian cells may be used.

[0196] E. coli is one prokaryotic host that is particularly useful for cloning the polynucleotides (e.g., DNA sequences) of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.

[0197] Other microorganisms, such as yeast, are also useful for expression. The genera Saccharomyces and Pichia are exemplary yeast hosts harboring suitable vectors, optionally with expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like. Common promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes involved in methanol, maltose, and galactose utilization, among others.

[0198] Furthermore, in vivo synthesis of ubiquitin transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion proteins thus generated can be processed in vivo or purified and processed in vitro to synthesize the CEACAM1 antibody or antigen-binding fragment thereof of the present invention having a specific amino-terminal sequence. Furthermore, problems associated with retention of the methionine residue from the initiation codon in direct yeast (or bacterial) expression can be avoided. Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).

[0199] The recombinant CEACAM1 antibody or peptide of the present invention can be produced using any of a series of yeast gene expression systems incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be used.

[0200] Production of CEACAM1 antibodies or antigen-binding fragments thereof in insects can be achieved, for example, by infecting an insect host with a baculovirus modified to express a transmembrane polypeptide by methods known to those skilled in the art (see Ausubel et al., 1987, 1993).

[0201] In addition to microorganisms, mammalian tissue culture may be used to express and produce the antibodies or antigen-binding fragments thereof (e.g., polynucleotides encoding immunoglobulins or fragments thereof) of the present invention. See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). In practice, eukaryotic cells are preferred, since many suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B cells, and hybridomas. Expression vectors for these cells may include expression control sequences such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary information processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0202] Alternatively, a nucleotide sequence encoding the antibody or antigen-binding fragment thereof can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al., U.S. Pat. No. 5,741,957; Rosen, U.S. Pat. No. 5,304,489; and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and / or heavy chains operably linked to a promoter and enhancer derived from a mammary gland-specific gene such as casein or beta-lactoglobulin.

[0203] Furthermore, plants have emerged as a convenient, safe and economical alternative mainstream expression system for recombinant antibody production based on large-scale culture of microorganisms or animal cells. Antibodies or antigen-binding fragments thereof can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be systemic, intracellular plastid-limited, or seed (endosperm)-limited. See, for example, U.S. Patent Publication No. 2003 / 0167531; U.S. Patent Nos. 6,080,560 and 6,512,162; and WO0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, for example, Biolex, NC).

[0204] Vectors carrying the polynucleotide sequences of interest (e.g., sequences encoding heavy and light chains and expression control sequences) can be transferred into host cells by well-known methods that vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection can be used for other cellular hosts. (See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Press, 2nd ed., 1989). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., supra). To generate transgenic animals, transgenes can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells and the nuclei of such cells transferred into enucleated oocytes.

[0205] The antibodies and antigen-binding fragments thereof of the invention can be expressed using a single vector or two vectors. If the heavy and light chains of the antibody are cloned into separate expression vectors, the vectors can be co-transfected to obtain expression and assembly of intact immunoglobulins. Once expressed, whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, (1982)). For pharmaceutical uses, substantially pure immunoglobulins of at least about 90-95% homogeneity are preferred, with 98-99% or greater homogeneity being most preferred.

[0206] Methods for modulating CEACAM1 activity

[0207] In one aspect, the invention provides methods of using the antibodies and antigen-binding fragments thereof described herein to reduce the interaction between CEACAM1 and another member of the CEACAM family, including, but not limited to, CEACAM1, CEACAM3, CEACAM5, CEACAM6, and CEACAM8. In some embodiments, the antibodies or antigen-binding fragments thereof disrupt homophilic interactions between CEACAM1 monomers.

[0208] In another aspect, the invention provides methods of using the antibodies and antigen-binding fragments thereof of the invention to reduce the interaction between CEACAM1 and members of the TIM family, including but not limited to TIM-1, TIM-3, and TIM-4. In some embodiments, the antibodies or antigen-binding fragments thereof disrupt the heterophilic interaction between CEACAM1 and TIM-3. Disruption of the interaction between CEACAM1 and TIM-3 by using the antibodies and antigen-binding fragments thereof contemplated by the invention may reverse the CEACAM1 inhibitory function while maintaining the TIM-3 activating function.

[0209] Embodiments of the invention are useful for immunosuppression, e.g., reducing T cell tolerance. By "reducing" is meant the ability to cause an overall decrease of about 20% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75%, 80%, 85%, 90%, 95% or more, compared to untreated controls. Immune suppression can be mediated by immune inhibitory receptors expressed on the surface of immune cells and their interaction with their ligands. For example, cytotoxic CD8 T cells can enter a state of "functional exhaustion" or "unresponsiveness," thereby expressing inhibitory receptors that prevent antigen-specific responses, such as proliferation and cytokine production. Thus, by inhibiting the activity and / or expression of such inhibitory receptors, the immune response against a cancer or tumor that is suppressed, inhibited, or unresponsive can be enhanced or not inhibited. Such enhancement or reversal of inhibition of immune response can result in greater T cell activity, responsiveness, and / or capacity or receptivity for activation.

[0210] Methods for measuring T cell activity are known in the art. As a non-limiting example, T cell tolerance can be induced by contacting T cells with recall antigen, anti-CD3 in the absence of costimulation, and / or ionomycin. For example, the levels of IL-27, LDH-A, RAB10, and / or ZAP70 (both intracellular and secreted) can be monitored to measure, for example, the degree of T cell tolerance (at the levels of IL-2, interferon-γ and TNF, which correlate with increased T cell tolerance). The response of cells pretreated with, for example, ionomycin to antigen can also be measured to determine the degree of T cell tolerance in a cell or cell population, for example, by monitoring the levels of secreted and / or intracellular IL-2 and / or TNF-α (see, for example, Macian et al. Cell 2002 109:719-731). Other characteristics of T cells that have undergone adaptive tolerance include increased levels of Fyn and ZAP-70 / Syk, Cbl-b, GRAIL, Ikaros, CREM (cAMP response element regulator), B-lymphocyte-derived maturation protein-1 (Blimp-1), PD1, CD5, and SHP2; increased phosphorylation of ZAP-70 / Syk, LAT, PLCyl / 2, ERK, PKC-Θ / ΙΚΒΑ; increased activation of intracellular calcium levels; decreased histone acetylation, i.e., hypoacetylation and / or increased CpG methylation at the IL-2 locus. Thus, in some embodiments, any one or more of these parameters can be assayed to determine whether an antibody or antigen-binding fragment thereof disclosed herein that inhibits CEACAM1 reduces immune tolerance. Reduction of T cell tolerance can also be assessed by examining tumor-infiltrating lymphocytes or T lymphocytes in lymph nodes draining established tumors. Such T cells are characterized as "exhausted" via, for example, reduced expression of cell surface molecules such as PD1, TIM-3 or LAG-3, and secretion of cytokines such as interferon-γ.Thus, evidence of decreased T cell tolerance in the presence of a CEACAM1 antibody or antigen-binding fragment thereof includes, for example, an increase in the amount of T cells that have (a) antigen specificity for a tumor-associated antigen (e.g., as determined by a major histocompatibility complex class I or class II tetramer containing a tumor-associated peptide) and (b) the ability to secrete high levels of interferon-γ and cytolytic effector molecules, such as granzyme-B, compared to the ability observed in the absence of an inhibitor.

[0211] CEACAM1 antibodies and antigen-binding fragments thereof are further useful for enhancing T cell proliferation, activation, and proliferation.

[0212] In another aspect, the invention provides a method of using the antibodies and antigen-binding fragments thereof of the invention to reduce the interaction between CEACAM1 and a bacterial adhesin. In some embodiments, the antibodies and antigen-binding fragments thereof of the invention are effective in reducing and / or preventing colonization of mammalian epithelia. In some embodiments, the adhesin is expressed by Escherichia coli, particularly diffuse adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, N. meningitidis, commensal Neisseria, Moraxella catarrhalis, Haemophilus influenza, Haemophilus aegyptius, Helicobacter pylori, and / or Salmonella species. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and HopQ expressed on the surface of Helicobacter pylori. In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and opacity-associated (Opa) adhesin protein expressed on the surface of Neisseria species. In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and an OMP adhesin protein expressed on the surface of Haemophilus species.

[0213] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and C.albicans. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and influenza viruses, including but not limited to H5N1. In one embodiment, the invention provides a method of using the CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of CEACAM1 to filarial nematodes. In one embodiment, the filarial nematode is Wucheria bancrofti.

[0214] Treatment method

[0215] In one aspect, the invention provides CEACAM1 antibodies and antigen-binding fragments thereof, which are also useful for treating a subject in need of such treatment.

[0216] In the methods described herein, a therapeutically effective amount of an antibody or antigen-binding portion thereof described herein is administered to a mammal in need thereof. The antibodies or antigen-binding portions thereof described herein are particularly useful for administration to humans, but may also be administered to other mammals. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, farm animals, and livestock. A "therapeutically effective amount" refers to an amount of an antibody or antigen-binding portion thereof described herein that is effective to produce a desired therapeutic effect when administered to a mammal.

[0217] In some embodiments, the antibody or antigen-binding fragment thereof binds to CEACAM1 expressed by exhausted T cells or natural killer (NK) cells, thereby restoring T cell and NK cell activity and resulting in an increased anti-tumor response. In other embodiments, the antibody or antigen-binding fragment thereof binds to CEACAM1 expressed by tumor cells, thereby inhibiting tumor cell metastasis and the formation of cancer stem cell niches. In yet another embodiment, the antibody or antigen-binding fragment thereof binds to CEACAM1 expressed by macrophages associated with fibrosis in the tumor environment, thereby inhibiting fibrosis. In another embodiment, the antibody or antigen-binding fragment thereof binds to CEACAM1 expressed by other stromal cells in the tumor microenvironment, such as vascular endothelial cells, thereby inhibiting angiogenesis.

[0218] Accordingly, also provided herein are methods of treating a subject having cancer or a tumor and / or reducing tumor growth, comprising administering an effective amount of a CEACAM1 antibody or antigen-binding fragment thereof provided herein. "Reduction" includes inhibition and / or reversal, and may refer, for example, to symptoms of the disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or size of dormant tumors.

[0219] The term "cancer" refers to or describes a physiological condition in mammals that is usually characterized by unregulated cell proliferation. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Thus, the term "cancer" as used herein refers to unregulated cell proliferation that interferes with the normal function of bodily organs and systems, including cancer stem cells and tumor vascular niches. A subject with cancer is one in which objectively measurable cancer cells are present in the subject's body. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location and disseminate to vital organs can ultimately lead to the death of the subject through the impaired function of the affected organ. Hematopoietic cancers, such as leukemia, can overpower the normal hematopoietic compartment of a subject, thereby causing hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) and ultimately leading to death.

[0220] By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. Individuals and patients are also subjects herein.

[0221] As used herein, the terms "treat", "treated", "treating" or "treatment" refer to therapeutic treatment, the purpose of which is to slow down (alleviate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desirable clinical outcome. For purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of a pathology, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease state; delay in onset or slowing of progression of a pathology, disorder or disease; improvement of the condition, disorder or disease state; and remission (partial or complete), or enhancement or amelioration of a pathology, disorder or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival in the absence of treatment. The terms "prevent", "preventing" and the like refer to acting prior to the onset of overt disease or disorder to prevent the onset of a disease or disorder, minimize the extent of a disease or disorder, or delay its developmental process.

[0222] Embodiments of the invention may be used to treat metastasis, which is associated with the spread of cancer from its primary site to other locations in the body. Cancer cells can break away from the primary tumor, infiltrate lymphatic and blood vessels, circulate in the bloodstream, and grow (metastasize) in distant foci in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a series of processes that require tumor cells to divide from the primary tumor, travel through the bloodstream, and arrest at a distant site. At the new site, the cells establish a blood supply and may grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cells regulate this behavior, and interactions between tumor cells and host cells at distant sites are also important. Metastasis is most frequently detected by magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function tests, chest x-rays, and bone scans, alone or in combination, in addition to monitoring for certain symptoms.

[0223] Methods of reducing cancer stemness are also contemplated, including administration of the CEACAM1 antibody or antigen-binding fragment thereof disclosed herein. Cancer stemness may refer to the ability of cells to self-renew and generate additional cell types with distinct phenotypes. Cancer stem cells (CSCs) are cancer cells that exhibit stem cell-like properties. CSCs often exhibit at least one cancer characteristic and can generate at least one additional cell type with distinct phenotypes. Furthermore, cancer stem cells are capable of both asymmetric and symmetric replication. It is understood that cancer stem cells can result from differentiated cancer cells that acquire stem cell traits and / or stem cells that acquire phenotypes associated with cancer cells. Alternatively, cancer stem cells can reconstitute non-stromal cell types within a tumor.

[0224] CEACAM1 is expressed in many tumor types and CEACAM1 may regulate tumor growth and metastatic behavior. In another embodiment, CEACAM1 inhibition reduces tumor growth and metastasis.

[0225] Expression of CEACAM1 in a subset of macrophages is associated with fibrosis during carcinogenesis. In a further embodiment, CEACAM1 inhibition reduces tumor-associated fibrosis.

[0226] Cancers that can be treated by the compositions and methods contemplated by the present invention include tumors that are not vascularized or are not yet substantially vascularized, and vascularized tumors. Cancers can include non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma) or can include solid tumors. Types of cancers to be treated include, but are not limited to, benign and malignant tumors, as well as malignant tumors, such as sarcomas, carcinomas, and melanomas. Also included are adult tumors / cancers and pediatric tumors / cancers. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, etc.), cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and throat); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary cancer of the liver; vulvar cancer; and other carcinomas and sarcomas; and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nematoses, edema (such as that associated with brain tumors), and Meigs' syndrome. A patient may have more than one type of cancer.

[0227] The efficacy of the cancer treatment method, including the therapeutic formulation of the composition comprising the antibody and antigen-binding fragment thereof described herein, can be measured by various endpoints commonly used in the evaluation of cancer treatment, including, but not limited to, tumor regression, reduction in tumor weight or size, time to progression, survival time, progression-free survival, overall response rate, duration of response, and quality of life. In the case of cancer, a therapeutically effective amount of the recombinant CEACAM1 antibody or antigen-binding fragment thereof can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., delay to some extent, and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., delay 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. In the case of a patient with multiple types of cancer, a therapeutically effective amount of the recombinant CEACAM1 antibody or antigen-binding fragment thereof is an amount effective to treat at least one cancer. To the extent that the recombinant CEACAM1 antibody or antigen-binding fragment thereof acts to prevent proliferation and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, efficacy in vivo can be measured, for example, by assessing survival, progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life.

[0228] Checkpoint proteins interact with specific ligands that send signals to T cells and turn off or inhibit T cell function. Cancer cells can control the function of T cells entering the tumor microenvironment and suppress anti-cancer immune responses by expressing high levels of checkpoint proteins on their surface. The immune checkpoint protein Programmed Death-1 (PD-1) is an important immune checkpoint receptor expressed by activated T cells and B cells and mediates immune suppression. PD-1 is a member of the CD28 family of receptors that includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1, Programmed Death Ligand-1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), have been identified and are expressed on antigen-presenting cells and many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al., 2000; Latchman et al., 2001). Inhibition of PD-1 / PD-L1 interaction can promote strong antitumor activity. Examples of PD-1 inhibitors include, but are not limited to, pembrolizumab (MK-3475), nivolumab (MDX-1106), cemiplimab-rwlc (REGN2810), pidilizumab (CT-011), spartalizumab (PDR001), tislelizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI754091, JS001, LZM009, MEDI0680, MGA012, Sym021, and TSR-042. Examples of PD-L1 inhibitors include atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BGB-A333, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, and SHR-1316.

[0229] However, there is a significant population of cancer patients who undergo checkpoint inhibitor therapy and either (1) do not respond to this type of therapy (innate or primary resistance) or (2) respond initially but eventually develop disease progression (secondary or acquired resistance). Resistant cancers are sometimes referred to as refractory cancers. As shown in the example below, tumor-associated cells isolated from patients who have acquired resistance to PD-1 / PD-L1 inhibitors upregulate CEACAM1 expression compared to tumor-associated cells isolated from naive patients not exposed to PD-1 inhibitors. If CEACAM1 is expressed in the setting of acquired resistance, it is likely that CEACAM1-bearing cells are effector memory cells rather than central memory cells, consistent with the reduced anticancer response in resistant patients.

[0230] Thus, methods of using CEACAM1 antibodies and antigen-binding fragments thereof, including but not limited to, the specific CEACAM1 antibodies and antigen-binding fragments thereof provided herein, to treat patients with resistance to checkpoint inhibitors, such as inhibitors of PD-1, PD-L1, and / or CTLA-4, are also provided. In some embodiments, the CEACAM1 antibody used to treat patients with resistance to inhibitors of PD-1, PD-L1, and / or CTLA-4 is CP08H03 / Vk8 S29A or CP08H03 / CP08F05. In some embodiments, the resistance is congenital or primary resistance. In some embodiments, the resistance is secondary or acquired resistance. In some embodiments, the administered CEACAM1 antibody, including but not limited to, the CEACAM1 antibodies and antigen-binding fragments thereof provided herein, reverses T cell exhaustion in patients resistant to checkpoint inhibitor therapy. Cancers that exhibit resistance to PD-1, PDL-1, and / or CTLA-4 are suitable for treatment by the methods of the invention. In some embodiments, the CEACAM1 antibody or antigen-binding fragment is administered to a patient who has not previously received checkpoint inhibitor therapy.

[0231] In another aspect, the present invention relates to PD-L2, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (members of the CD2 family of molecules, all NK, gamma delta, and memory CD8 + Use of the CEACAM1 antibodies and antigen-binding fragments provided herein in the treatment of patients resistant to treatment with other checkpoint inhibitors, including but not limited to inhibitors of (expressed in αβ)T cells, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7) is provided.

[0232] In another aspect, the invention provides a method of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to treat a subject in need of reducing and / or preventing colonization of mammalian epithelia by Candida albicans and / or bacteria expressing bacterial adhesins (including, but not limited to, Escherichia coli, particularly diffuse adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, N. meningitidis, commensal Neisseria, Moraxella catarrhalis, Haemophilus influenza, Haemophilus aegyptius, Helicobacter pylori, and / or Salmonella species). In another aspect, the invention provides a method of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to reduce influenza virus replication and / or reduce the release of proinflammatory cytokines or chemokines associated with influenza virus infection. In some embodiments, the influenza virus is H5N1. In another aspect, the invention provides a method of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to treat a subject in need of reducing and / or preventing infection with a filarial nematode, such as Wucheria bancrofti. In another aspect, the invention provides a method of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to treat a subject in need of reducing and / or preventing the development of lymphedema and / or watervessels associated with infection with a filarial nematode, such as Wucheria bancrofti. In one embodiment, the invention provides a method of using the CEACAM1 antibodies or antigen-binding fragments thereof described herein to reduce invasion of the lymphatic system of a subject by filarial parasites in a subject in need of reducing invasion of the lymphatic system of the subject by filarial parasites. In one embodiment, the filarial nematode is Wucheria bancrofti.The subject may be infected with more than one of a bacteria expressing a bacterial adhesin, Candida albicans, influenza virus and / or a filarial nematode.

[0233] In another embodiment, the present invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce infiltration of a subject's lymphatic system by cancer cells in a subject in need of such reduction.

[0234] Screening Methods

[0235] Also provided herein are methods for identifying parent populations likely to respond to treatment with the CEACAM1 antibodies and antibody fragments provided herein, including, but not limited to, CP08H03 / Vk8 S29A and CP08H03 / CP08F05.

[0236] In some embodiments, cancer patients are screened for CEACAM1 expression in specific cell types, including T cells, NK cells, tumor cells, or other cells in the tumor microenvironment, such as macrophages. In some embodiments, cancer patients that show increased expression of CEACAM1 in specific cell types compared to controls are selected for treatment with the CEACAM1 antibodies and antibody fragments provided herein. A "control" level of CEACAM1 expression may refer to the level of CEACAM1 expression in one or more individuals without cancer. The levels may be measured on an individual basis or on an aggregate basis, such as an average. In some embodiments, the regulated levels of CEACAM1 expression from the same individual whose condition is being monitored are obtained at different times. In certain embodiments, a "control" level may refer to a level obtained from the same patient at an earlier time, for example, weeks, months, or years ago. In some embodiments, the control level is obtained from the patient before the patient undergoes cancer treatment. In some embodiments, the control level is obtained from the patient before the patient undergoes treatment with a checkpoint inhibitor.

[0237] In some embodiments, CEACAM1 expression is measured in patients resistant to checkpoint inhibitor therapy, including but not limited to, treatment with a PD-1 / PD-L1 / CTLA-4 inhibitor. In some embodiments, patients resistant to checkpoint inhibitor therapy and who show increased expression of CEACAM1 in certain cell types compared to controls are selected for treatment with the CEACAM1 antibodies and antibody fragments provided herein, including but not limited to, CP08H03 / Vk8 S29A and CP08H03 / CP08F05.

[0238] In some embodiments, the patient is assayed for allelic variants of human CEACAM1. Based on the allelic variants of human CEACAM1 expressed by the patient, the patient may be administered more or less anti-CEACAM1 antibody compared to patients expressing a wild-type variant of CEACAM1. In some embodiments, the patient is assayed for the presence of Y34C, Q44L, and / or Q89H allelic variants of CEACAM1. In some embodiments, patients found to express Y34C, Q44L, and / or Q89H allelic variants of CEACAM1 are administered higher and / or more frequent doses of anti-CEACAM1 antibody compared to patients expressing a wild-type variant of CEACAM1.

[0239] Pharmaceutical Compositions

[0240] In another aspect, the present invention provides a pharma- ceutically acceptable composition comprising a therapeutically effective amount of a CEACAM1 antibody or antigen-binding fragment thereof described herein, formulated with one or more pharma- ceutically acceptable excipients.

[0241] The dose of active agent(s) may vary depending on the reason for use, the individual subject, and the mode of administration. The dose may be adjusted based on the subject's weight, the subject's age and health, and the resistance to the compound(s) or composition. This may require, for example, 0.1, 1.0, 3.0, 6.0, or 10.0 mg / Kg of antibody or antigen-binding fragment thereof depending on the disease. For an IgG with a molecular weight of 150,000 g / mol (two binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μM, and 1.8 μM of binding sites for a 5 L blood volume.

[0242] The active agent and excipient(s) may be formulated into compositions and dosage forms according to methods known in the art. The pharmaceutical compositions of the present invention may be specially formulated in solid or liquid form, including forms adapted for parenteral administration, e.g., as a sterile solution or suspension, e.g., by subcutaneous, intratumoral, intramuscular or intravenous injection.

[0243] Therapeutic compositions comprising an antibody or antigen-binding fragment thereof that binds to CEACAM1 may be formulated with one or more pharma- ceutically acceptable excipients, which may be pharma- ceutically acceptable substances, compositions or vehicles, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, talc magnesium stearate, calcium stearate or zinc stearate, or stearic acid), solvents, or encapsulating substances, bulking agents, salts, surfactants and / or preservatives involved in carrying or transporting the therapeutic compound for administration to a subject. Some examples of substances that may act as pharma- ceutically acceptable excipients include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; gelatin; talc; waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as ethylene glycol and propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffers; and other non-toxic compatible substances used in the formulation.

[0244] Bulking agents are compounds that add mass to a formulation and contribute to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present invention include mannitol, glycine, polyethylene glycol and sorbitol.

[0245] The use of a surfactant can reduce aggregation of the reconstituted protein and / or reduce the formation of particles in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particles after reconstitution. Suitable surfactants according to the present invention include polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); Triton®; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, glyceryl ... -, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic®, PF68, etc.).

[0246] Preservatives may be used in the formulations of the present invention. Suitable preservatives for use in the formulations of the present invention include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl-dimethylammonium chlorides, where the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients are described in standard pharmaceutical texts, such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995).

[0247] The composition comprising the antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier may contain various concentrations of the CEACAM1 antibody or antigen-binding portion thereof described herein. For example, the composition may contain 10 mg / ml to 200 mg / ml, 25 mg / ml to 130 mg / ml, 50 mg / ml to 125 mg / ml, 75 mg / ml to 110 mg / ml, or 80 mg / ml to 100 mg / ml of the antibody or antigen-binding fragment thereof. The composition may also contain about 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, or 150 mg / ml of the antibody or antigen-binding fragment thereof.

[0248] In some embodiments, the composition comprising an antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier is lyophilized and provided in a composition for reconstitution prior to administration.

[0249] Method of administration

[0250] Therapeutic compositions comprising contemplated antibodies or antigen-binding fragments thereof may be administered in any convenient manner, including injection, transfusion, implantation or transplantation. The compositions described herein may be administered to patients by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intracranial, intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0251] In certain embodiments, the antibody or antigen-binding fragment thereof is administered to the mammal by intravenous infusion, i.e., introducing the antibody or antigen-binding fragment thereof into the vein of the mammal for a particular period of time, which in certain embodiments is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.

[0252] In certain embodiments, the compound or composition dose is administered to the subject daily, every other day, every 2 days, every 3 days, once a week, twice a week, three times a week, once every 2 weeks, or once a month. In other embodiments, the compound or composition dose is administered to the subject daily, every 2 days, every 3 days, once a week, once every 2 weeks, or once a month. In some embodiments, the compound or composition dose(s) is administered for 2, 3, 5, 7, 14, 21, or 28 days. In certain embodiments, the compound or composition dose is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or more.

[0253] Combination therapy

[0254] In one aspect, the present invention provides a CEACAM1 antibody or antigen-binding fragment thereof administered in combination with an additional therapeutic agent. Such additional agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, and anti-infective agents. The agents used in such combination therapy may fall into one or more of the above categories. The administration of the antibody or antigen-binding fragment thereof and the additional therapeutic agent may be simultaneous or sequential. The administration of the antibody or antigen-binding fragment thereof and the additional therapeutic agent may be separate or as a mixture. Furthermore, the method of treatment contemplated by the present invention may involve combination treatment with one or more cancer therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0255] Exemplary therapeutic moieties include radionuclides with high energy ionizing radiation capable of causing multiple strand breaks in nuclear DNA and thus suitable for inducing cell death (e.g., in cancer). Exemplary high energy radionuclides include: 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188 Re. These isotopes usually produce high-energy alpha or beta particles with short path lengths. Such radionuclides kill cells to which they are in close proximity, e.g., neoplastic cells to which the conjugate is attached or entering. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0256] Exemplary additional therapeutic agents also include cytotoxic agents such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or cross-linking agents, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, and the like.

[0257] Exemplary additional therapeutic agents also include alkylating agents such as the anthracycline family of drugs (e.g., adriamycin, carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenedione, and aziridine). In another embodiment, the chemotherapy moiety is a cytostatic drug, such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Exemplary DNA intercalators or crosslinkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0258] Exemplary additional therapeutic agents also include transcriptional regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other exemplary cytostatic agents compatible with the present invention include ansamycin benzoquinones, quinonoid derivatives (e.g., quinolones, genistein, bactacyclines), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazylquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylene phosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0259] Exemplary additional therapeutic agents also include, for example, cytotoxic nucleosides, such as adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; taxoids (e.g., paclitaxel, docetaxel, taxanes), nocodazole, rhizoxin, dolastatins (e.g., dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD61, 26), tubulin-binding agents such as combretastatins (e.g., combretastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); antiangiogenic compounds such as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0260] Exemplary additional therapeutic agents also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-alpha, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0261] Exemplary additional therapeutic agents also include enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenz-imidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG34, and tyrphostin AG879.

[0262] Exemplary additional therapeutic agents also include gene regulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, transretinal (vitamin A aldehyde), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0263] Exemplary additional therapeutic agents also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and podophyllotoxins. Particularly useful members of these classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives, such as etoposide or etoposide phosphate, leurocidin, vindesine, leurosine, and the like.

[0264] Still other additional therapeutic agents compatible with the teachings herein include auristatins (e.g., auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0265] In one embodiment, the CEACAM antibody or antigen-binding fragment thereof is administered in combination with an agent that is a checkpoint inhibitor. Such inhibitors may include small molecule inhibitors, or may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that may be targeted for blockade or inhibition include CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (a member of the CD2 family of molecules that inhibits all NK, gamma delta, and memory CD8 +(expressed on αβ)T cells), CD160 (also called BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM-3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, and Yervoy / ipilimumab (an anti-CTLA-4 checkpoint inhibitor), as well as the PD-1 and PD-L1 inhibitors listed above. Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0266] In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered in combination with a TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226, or CD160 inhibitor.

[0267] In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered in combination with a CTLA-4, PD-1, PD-L1, or PD-L2 inhibitor. In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered in combination with a TIM-3 inhibitor.

[0268] It is to be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. It is further to be understood that the disclosure of the present invention herein includes all possible combinations of such specific features. For example, if a specific feature is disclosed with respect to a specific aspect or embodiment of the present invention, or with respect to a specific claim, that feature can also be used, to the extent possible, in combination with and / or with other specific aspects and embodiments of the present invention, as well as in the present invention generally.

[0269] When reference is made herein to a method comprising two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes that possibility), and the method may include one or more other steps performed before any defined step, between two defined steps, or after all defined steps (unless the context excludes that possibility).

[0270] All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, to the extent that a definition or use of a term in a reference incorporated herein by reference contradicts or is contrary to the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0271] In order to facilitate a better understanding of the present invention, the following examples of specific embodiments are given, which should not be read as limiting or defining the entire scope of the invention. EXAMPLES

[0272] Example 1: Generation of a fully humanized CEACAM1 antibody

[0273] 1. Generation of Humanized Antibody Variants

[0274] Design of composite human antibody variable region sequences and expression of antibodies

[0275] First, a structural model of the V-region of the parental murine CEACAM1 antibody was generated using the Swiss PDB and analyzed to identify potential "constraint" amino acids in the V-region that may contribute to the binding properties of the antibody. In the regions outside the CDRs and adjacent regions, a broad selection of human sequence segments were identified as possible components of the novel humanized V-regions.

[0276] Based on the structural analysis, a large preliminary set of sequence segments that could be used to create humanized CEACAM antibody variants was selected and analyzed using iTope™ technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al, 2008. Drugs RD 9(6):385-396) and TCED™ for known antibody sequence-associated T cell epitopes (Bryson et al 2010, Biodrugs 21(1):1-8). Sequence segments that were identified as significant non-human germline binders to human MHC class II or scored significant hits against TCED™ were discarded. This analysis resulted in a reduced set of segments, and these combinations were analyzed again as described above to ensure that the junctions between the segments did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V region sequences lacking significant T cell epitopes. The heavy and light chains selected for gene synthesis, expression in mammalian cells, and activity testing are listed in Table 1.

[0277] Some of the heavy and light chains in Table 1 contained variations at positions that are considered part of a CDR according to the Kabat CDR definition, but not according to the IMGT CDR definition.

[0278] [Table 1]

[0279] Next, the parent mouse CEACAM1 antibody and the humanized CEACAM antibody variants V H The V and V sequences were synthesized with flanking restriction enzyme sites for cloning into the pANT expression vector system for IgG4(S241P) heavy chain and κ light chain (Figure 1). H The regions were cloned between the MluI and HindIII restriction sites, and the Vκ region was cloned between the BssHII and BamHI restriction sites. All constructs were verified by sequencing.

[0280] Thirty-nine heavy and light chain pairings were transiently transfected into HEK EBNA adherent cells using the PEI transfection method and incubated for 5–7 days after transfection. These 39 pairings included three controls: (1) chimeric antibody V H 0 / Vκ0 (mouse Vκ fused to the constant heavy chain region of human IgG4 H Area (V H (1) a chimeric VH heavy chain (V H 0) pairing with light chain variant Vκ1; and (3) V H 1 heavy chain paired with a chimeric Vκ light chain variant (Vκ0). The other 36 are V H and Vκ variants:V H 1 is paired with Vκ1 to Vκ12, and V H 2 is paired with Vκ1 to Vκ12, and V H 3 is paired with Vκ1 to Vκ6, and V H 4 was paired with Vκ1 to Vκ6.

[0281] Antibodies were purified from cell culture supernatants on a Protein A Sepharose column, buffer exchanged into PBS pH 7.4, and quantified at OD 280 nm using extinction coefficients based on the predicted amino acid sequence. One microgram of each antibody was analyzed by SDS-PAGE, and bands corresponding to typical antibody characteristics were observed. The size of the light chain and the presence of a faint band at 25 kDa indicate substantial utilization of the glycosylation motifs identified in the light chain.

[0282] Competitive ELISA analysis of humanized variants binding to CEACAM1

[0283] Binding of purified antibodies to human CEACAM1 was assessed in a competitive ELISA assay. Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates were pre-coated with 1 μg / ml GST-CEACAM1 in 1×PBS overnight at 4° C. The next day, plates were blocked with 2% BSA / PBS for 1 hour at room temperature ("RT") and then washed three times with PBST pH 7.4. Chimeric antibody V H Three-fold dilution series of humanized CEACAM1 antibodies from 100 μg / ml to 0.07 or 0.002 μg / ml were premixed with a fixed concentration of parental mouse antibody (final concentration 0.45 μg / ml), added to the plate, and incubated for 1 h at room temperature. After washing three times with PBST, binding of the parental mouse CEACAM1 antibody was detected with anti-mouse HRP and TMB substrate. The reaction was stopped with 3 M HCl, absorbance was read at 450 nm on a Dynex Technologies MRX TCII plate reader, and binding curves were plotted. Binding of the humanized CEACAM antibody variants to CEACAM1 was measured using the chimeric antibody (Vκ0) included on each plate (Vκ0 / Vκ0). H Twelve of the 36 humanized CEACAM antibody variants showed no binding to CEACAM1 (variants including Vκ3, Vκ4, and Vκ5). The variants that bound to CEACAM1 were the chimeric antibody V H The relative IC50 values ​​ranged from 0.9 to 5.2 compared to Vκ0 / Vκ0. The data are summarized in Table 2.

[0284] [Table 2]

[0285] Kinetic analysis of humanized variants binding to CEACAM1

[0286] As an alternative approach to evaluate the binding of 36 antibody combinations and three control antibodies to CEACAM1, kinetic analyses were performed on a Biacore T200 (serial no. 1909913) running Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden). All experiments were performed at 25 °C using HBS-P+ running buffer (pH 7.4) (GE Healthcare, catalog no. BR100671). All kinetic experiments were performed using His-tagged CEACAM1 as the analyte. For all experiments, antibodies were immobilized on the surface of a Series S Protein A sensor chip. For kinetic experiments, the amount of ligand immobilized / captured was limited to avoid mass transfer effects at the surface of the chip and the surface was ideally designed to support analyte binding levels (R max A target response level of approximately 75 RU was set to capture all sample antibodies using a MW of 45 kDa for the CEACAM1 analyte, 150 kDa for the antibody ligand (estimated for IgG), an Rmax of 50 RU, and a stoichiometry (Sm) of 2 since each antibody has the capacity to bind two target molecules.

[0287] Single cycle analysis of 36 antibody combinations and three control antibodies was performed with purified antibodies (Vκ1–Vκ6 variants) or supernatants of transiently transfected HEK EBNA cells (Vκ7–Vκ12 variants). In some cases, when supernatants were not available, purified chimeric antibodies V H0 / Vκ0 was added to HEK EBNA culture medium to serve as a positive control. Antibodies were diluted in HBS-P+ to a concentration of 1 μg / ml (measured by IgG quantification ELISA). At the start of each cycle, antibodies were loaded onto Fc2, Fc3 and Fc4 of the Protein A chip and IgG was captured at a flow rate of 8 μl / min to an RU of approximately 75. The surface was then allowed to stabilize. Single cycle kinetic data were acquired at a flow rate of 50 μl / min to minimize potential mass transfer effects. Chimeric antibody V H Multiple repeats of 0 / Vκ0 were performed to check the stability of the surface and analyte over the kinetic cycles. The signal of the reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surface. A 5.2-fold dilution range of 3.125-50 nM CEACAM1 was used without regeneration between each concentration. For five injections of increasing concentrations of CEACAM1, the association phase was monitored for 100 s and a single dissociation phase was measured for 150 s after the last injection of CEACAM1. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCl pH 1.5 followed by a stabilization period of 500 s. The signal of a blank run (no CEACAM1) for each antibody was subtracted to correct for differences in surface stability. Single cycle kinetics (

[0288] (Table 3) showed that 24 humanized variants bound to CEACAM1, while 12 variants did not. Vκ3, Vκ5, and Vκ6 light chains abolished CEACAM1 binding when combined with humanized heavy chains. These data are consistent with the competition ELISA data (Table 2).

[0289] [Table 3]

[0290] Among the 24 antibodies that bound to CEACAM1, chimeric antibody V HAntibody variants that showed binding within 2-fold of Vκ0 / Vκ0 and had relative IC50s in the range of 0.9-1.8 were advanced to multi-cycle kinetic analysis using Biacore:V H 1 / Vκ2, V H 1 / Vκ7, V H 1 / Vκ8, V H 1 / Vκ9, V H 1 / Vκ10, V H 1 / Vκ12, V H 2 / Vκ7, V H 2 / Vκ8, V H 2 / Vκ9, V H 2 / Vκ10, V H 2 / Vκ11 and V H 2 / Vκ12 (see Tables 2 and 3 (highlighted in bold)).

[0291] For multi-cycle kinetic analysis, purified antibodies were immobilized in HBS-P+ at a protein concentration of 1 μg / ml. At the start of each cycle, antibodies were captured on Protein A to achieve an RU of approximately 75 and to stabilize the surface. Kinetic data were acquired at a flow rate of 80 μl / min to minimize potential mass transfer effects. To check the stability of both the surface and analyte over the kinetic cycles, multiple repeats of blanks (no CEACAM1) and single concentration analyte were incorporated into the kinetic studies. For the kinetic analysis, a two-fold dilution range was selected from either 200–3.125 nM or 100–1.5625 nM CEACAM1. The binding phase of CEACAM1 was monitored for 50 s or 150 s, and the dissociation phase was measured for 100 s. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle.

[0292] The signal of the reference channel Fc1 was subtracted from the signals of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surfaces, and the global Rmax parameter was used for the one-to-one binding model. H Relative K compared to 1 / Vκ0 D is the K of the humanized CEACAM antibody variant D, and chimeric antibody V on the same chip. H The kinetic parameters measured for the interaction of CEACAM1 with the humanized CEACAM antibody variants are shown in Table 4. The average relative K obtained for the 12 antibody combinations analyzed using multi-cycle kinetics. D An overview is shown in Table 5.

[0293] Analysis of selectivity of humanized variants binding to CEACAM1

[0294] The binding selectivity of CEACAM1 was evaluated using 24 humanized antibody variants (see Table 3) that bound to CEACAM1, as well as the chimeric control antibody V H We tested Vκ0 / Vκ0 by flow cytometry on HeLa cells transfected with CEACAM1, 3, 5, 6, and 8. As shown in Figure 2, the majority of the variants are highly selective for CEACAM1, showing little or no binding to CEACAM3, 5, 6, or 8. All 24 variants were highly selective for the chimeric control antibody Vκ0 / Vκ0. H Vκ0 / Vκ1 showed lower binding to CEACAM5 compared to Vκ0 / Vκ0. There was no evidence of staining of HeLa-CEACAM3 or HeLa-CEACAM8 transfectants; therefore, this data was not reported. Due to its favorable affinity and selectivity for CEACAM1 and its favorable expression levels, Vκ0 / Vκ1 showed lower binding to CEACAM5 compared to Vκ0 / Vκ0. There was no evidence of staining of HeLa-CEACAM3 or HeLa-CEACAM8 transfectants; therefore, this data was not reported. H 1 / Vκ8 was selected as the framework for affinity maturation.

[0295] [Table 4]

[0296] [Table 5]

[0297] 2. Removal of N-linked / HEK-derived glycosylation

[0298] Sequence analysis revealed potential N-linked glycosylation motifs in the original murine hybridoma light chain CDR1. CDR1L of the parental murine antibody contains a NXS / T consensus sequence (N26 and S29 according to Kabat numbering, corresponding to residues 26 and 28 in the primary amino acid sequence of the variable light chain, see FIG. 3C), making the N26 residue a target for N-linked glycosylation. To reduce potential glycosylation-associated immunogenicity, two CDR mutations were designed to remove the NXS / T consensus sequence (glycosylation site): N26Q and S29A (Kabat numbering scheme). Mutation of either residue abolishes glycosylation, as shown in FIG. 4.

[0299] Competitive ELISA experiments (see Table 6), multi-cycle kinetic analysis (see Table 7), and selectivity analysis (see Table 8) were performed to confirm the binding of the mutant chimeras to CEACAM1. Compared to antibody mutant N26Q, antibody mutant S29A (Kabat numbering scheme) showed higher expression levels and similar K D Showed (

[0300] (See Table 9) while maintaining high selectivity for CEACAM1. Therefore, the S29A mutation (Kabat numbering scheme) was incorporated into the CEACAM1 lead antibody during further development.

[0301] [Table 6]

[0302] [Table 7]

[0303] [Table 8]

[0304] [Table 9]

[0305] 3. Aglycosylated CEACAM1 Antibody V H Affinity maturation of 1 / Vκ8 S29A

[0306] Construction of phage vectors and parent V H Testing the binding of 1 / Vκ8 S29A scFv

[0307] One of the lead antibodies, V H For affinity maturation of Vκ1 / Vκ8, H Genes encoding Vκ1 and Vκ8 were constructed and converted to scFv format using overlap PCR linking the heavy chain to the light chain via a 15 amino acid (G4S)3 linker. CDR1L residue S29 is numbered according to the Kabat numbering scheme and corresponds to residue 28 of the primary amino acid sequence of the variable light chain (Figure 3C). The scFv sequence was then cloned into the phagemid vector pANT43 using the restriction enzymes SfiI and NotI, allowing display of the scFv on the phage surface as a gene III fusion protein (Figure 5). The cloned scFvs were transformed into E. coli (TG1) and all constructs were verified by sequencing. The parent Vκ1 and Vκ8 sequences were identified using overlap PCR, linking the heavy chain to the light chain via a 15 amino acid (G4S)3 linker. CDR1L residue S29 is numbered according to the Kabat numbering scheme and corresponds to residue 28 of the primary amino acid sequence of the variable light chain (Figure 3C). The scFv sequence was then cloned into the phagemid vector pANT43 using the restriction enzymes SfiI and NotI, allowing display of the scFv on the phage surface as a gene III fusion protein (Figure 5). The cloned scFvs were transformed into E. coli (TG1) and all constructs were verified by sequencing. H Phage containing either the 1 / Vκ8 S29A scFv or an irrelevant scFv were prepared and tested for binding to GST-CEACAM1 (Figure 6). No binding was observed with the irrelevant phage, indicating that the parent Vκ8 S29A scFv was the only phage that was capable of binding to GST-CEACAM1. H Phage derived from the 1 / Vκ8 S29A sequence bound specifically to the antigen.

[0308] Mutagenesis and library construction

[0309] To construct the affinity maturation library, aglycosylated humanized antibody V HSpecific amino acids within the CDR1H, CDR3H and CDR3L of .1 / Vκ8 S29A were subject to "hot spot" mutagenesis using semi-randomized codons. The sequence positions were analyzed for potential contact residues and ranked in order within each block. This information was used along with amino acid preferences at given positions within CDR3 and the crystal structure of the parental murine antibody. Where possible, priority was given to the highest ranked contact residues within each block.

[0310] Four different libraries were generated: one CDR1H (HC) mutation library, two CDR3H mutation libraries, and one CDR3L mutation library (see FIG. 7).

[0311] The CDR1H was identified as being 5 amino acids in length (S31-S35) (Kabat definition, corresponding to residues 31-35 of the primary amino acid sequence of the variable heavy chain, see Figure 3A) and (G26-G33) in the IMGT CDR1H definition, which covers a more extended region. Combined with the crystallographic data of the parent mouse antibody, G26-S35 were covered in a single library containing a subset of the amino acids contained at each position.

[0312] CDR3H was identified to be 12 amino acids in length (H95-Y102 according to the Kabat definition, corresponding to residues 99-110 of the primary amino acid sequence of the variable heavy chain, see Figure 3A). For mutagenesis, CDR3H was divided into two libraries overlapping at D100 (corresponding to residue 104 of the primary amino acid sequence of the variable heavy chain according to the Kabat definition, see Figures 3A and 3B): Block 1 (R94-D100 according to the Kabat definition, corresponding to residues 98-104 of the primary amino acid sequence of the variable heavy chain, see Figures 3A and 3B) and Block 2 (D100-Y102 according to the Kabat definition, corresponding to residues 104-110 of the primary amino acid sequence of the variable heavy chain, see Figures 3A and 3B), each of which contained a subset of amino acids at all positions. Position R94 (corresponding to residue 98 of the primary amino acid sequence of the variable heavy chain according to the Kabat definition, see Figures 3A and 3B) (Block 1) was included to increase the diversity of germline residues anchoring the CDRs.

[0313] The CDR3L was identified to be 9 amino acids in length (Q89-T97) (Kabat definition, corresponding to residues 88-96 of the primary amino acid sequence of the variable light chain, see Figure 3C). The region Q90-P96 (Kabat definition, corresponding to residues 89-97 of the primary amino acid sequence of the variable light chain, see Figure 3C) was covered with a single library containing a subset of amino acids at each position. Kabat numbering has been used for all protein sequence coordinates.

[0314] An overview of library construction is shown in Figures 8A, 8B, and 8C. H A truncated fragment of the 1 / Vκ8 S29A parental scFv was prepared containing two consecutive stop codons in the region to be randomized. The purpose of this step was to reduce the chance that a parental scFv would be generated and dominate the selection (as is sometimes observed during affinity maturation), so that only recombinant antibody fragments generated by PCR would be able to form functional scFvs in the phagemid vector.

[0315] For the CDR3L library, randomization of the CDR3L was performed by performing two PCRs. The first PCR used a randomized 3' primer and a V H A FW1 specific 5' primer was used to amplify most of the scFv gene and introduce a mutation in the Vκ CDR3. A second PCR added the remainder of the scFv and added a restriction enzyme site (NotI) for subcloning of the fragment.

[0316] V H For libraries, V H PCR on the repertoire was carried out by carrying out two PCRs using two templates containing portions of the full-length parental scFv. H was amplified using a randomized 5' library primer and a 3' primer specific for Vκ light chain FW4. H The remainder of the sequence was synthesized using a 5' primer based on the heavy chain FW1 region and a V H The full-length V was then amplified using a 3′ primer that was complementary to a portion of the CDR randomization primer. H A CDR-randomized scFv library was constructed by annealing the two amplified fragments and reamplified the scFv by PCR using primers that added two restriction enzyme sites (either SfiI or NotI) for subcloning of the fragments.

[0317] To assess the diversity of the generated libraries, purified and amplified DNA for all four libraries was then digested using SfiI and NotI and ligated into a similarly cut phagemid vector (pANT43). The ligated DNA was precipitated, resuspended in nuclease-free water, and transformed into freshly prepared electrocompetent TG1 cells by electroporation. The next day, colonies were counted, plates were scraped, and glycerol stocks were prepared. Libraries were electroporated multiple times to fully cover the theoretical library diversity. In all cases, a coverage of 4.0x or greater was obtained. Individual colonies from each of the four libraries were sequenced to confirm that the appropriate CDR blocks had been mutated.

[0318] Bacteria from each library were inoculated into 150 ml of 2TYCG (2%) cultures using an inoculum of at least 10 times the observed diversity of the library. Cultures were grown to mid-logarithmic phase (OD 600nm ≒0.5-0.6) and the estimated total cell number (OD 600nm 1≒5×10 8The cells were grown to a multiplicity of 10 (based on cells / ml). Helper phage was added and incubated for 1 h, then centrifuged, resuspended in 2TYCK medium, and grown overnight at 30 °C. The following day, phages were harvested by collecting the culture supernatant by centrifugation, followed by precipitation using 4 / 10x volume of chilled 20% PEG / 2.5M NaCl. After 1 h incubation on ice, precipitated phages were collected by centrifugation and the pellet was resuspended in 1x PBS pH 7.4. The supernatant was recentrifuged to remove cell debris, after which the supernatant was reprecipitated as above. Precipitated phages were resuspended in 1x PBS pH 7.4 and filter sterilized. To increase the chances of obtaining scFvs with increased affinity, due to the relatively low affinity of the starting antibody, polyvalent hyperphage M13 K07ΔpIII helper phage was used at a multiplicity of infection of 20 for library rescue. Following the first round of selection, monovalent M13K07 helper phage at a multiplicity of infection of 10 was used as a result of expected enrichment of antigen binders.

[0319] Selection of phages with improved affinity

[0320] Two separate selection strategies were performed to increase the probability of obtaining phages with improved affinity. CEACAM1 was used through either biotinylated (for soluble selection) or non-biotinylated (for solid-phase panning) selection. Soluble selection (campaign 1) or solid-phase panning selection (campaign 2) were used in round 1 of the different selection cascades to enrich for functional binding phages and diversity. Deselection with closely related family members CEACAM5 and CEACAM6 was performed by panning each protein separately at 1 μg / ml to try and reduce cross-reactivity. This was performed twice during each campaign, either by deselecting before any round of selection and before round 2 (campaign 1) or before both the second and third rounds of selection (campaign 2). In both campaigns, the four libraries were kept separate at all stages.

[0321] For soluble selections, each library was pre-blocked with PBSB, after which phages were incubated with decreasing concentrations of biotinylated CEACAM1 antigen for up to 3 h. After incubation, streptavidin paramagnetic beads (pre-blocked as above) were added to each selection and rotated for 15 min. Streptavidin-antigen-phage complexes were washed with increasing numbers of washes with PBST in each successive round of selection, followed by PBS washes and captured on a magnet between each step. Phages were eluted from the beads by addition of 50 mM HCl, after which the solution was neutralized by addition of 1 M Tris-HCl pH 9.0.

[0322] Solid-phase panning selections and all deselections were performed on Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates that were coated with antigen overnight at 4°C and then blocked with PBSB. For deselection, preblocked phage were incubated with CEACAM5 followed by CEACAM6, after which unbound phage were removed and used for subsequent selections. For CEACAM1 panning selections, preblocked phage were incubated with 8 μg / ml antigen, after which plates were washed 3× PBST and 2× PBS. Bound phage were eluted with 50 mM HCl as for soluble selections. For both soluble and panning selections, eluted phage were added to mid-logarithmic phase E. coli TG1 and allowed to infect cells for 1 h at 37°C before plating on 2TYCG (2%) plates and growing overnight at 37°C. The next day, colonies were picked for screening or plates were scraped and phage were rescued as above. The different selection strategies used are outlined in Figures 9A and 9B.

[0323] Expression and initial testing of scFv

[0324] Soluble scFvs were first expressed and tested as crude periplasmic extracts. Individual colonies were picked into 1 ml of 2TYCG (0.1%) medium and grown at 37°C with shaking for 5 hours. Cultures were induced by adding IPTG to a final concentration of 1 mM and then grown overnight at 30°C with shaking. The next day, cultures were centrifuged and the supernatant discarded. The bacterial pellet was resuspended in Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer pH 7.4 and incubated on ice for 30 minutes. The cells were then centrifuged and the supernatant discarded. The pellet was resuspended in ice-cold 5 mM MgSCE. The plates were then centrifuged and the supernatant containing the scFvs was transferred to a new plate for assay.

[0325] Periplasmic extracts of colonies derived from different rounds of selection were screened for their ability to bind GST-CEACAM1 in a single-point binding assay. For comparison, the parental scFv (V H 1 / Vκ8 S29A) and an irrelevant scFv were included in each assay plate. Periplasmic extracts were blocked by diluting 1:1 in PBSB and then incubated for 1 h at room temperature on NuncImmuno MaxiSorp 96-well flat-bottom microtiter plates pre-coated with 1.0 μg / ml GST-CEACAM1. Plates were then washed and binding of scFv was detected with anti-HIS6-HRP antibody and TMB substrate. Reactions were stopped with 1 M HCl, absorbance was read at 450 nm on a Dynex Technologies MRX TCII plate reader, and binding data were plotted.

[0326] Parental scFvV assayed on the same plate HImproved clones were identified based on activity in binding ELISA compared to 1 / Vκ8 S29A (containing mouse CDRs) and an unrelated scFv. Over 4400 periplasmic extracts were analyzed and 34 leads with binding at least 1.5-fold greater than the parent in two separate experiments were sequenced and unique clones were identified. Examination of the resulting sequences showed that at some positions the parent amino acid was found but encoded by a different codon than the parent. This indicates that the selection was as expected, but the parent amino acid was the preferred amino acid at this position. Based on this sequence analysis, 19 unique CDR1H, 3 CDR3H block 1, 3 CDR3H block 2 and 9 unique CDR3L clones were progressed to large-scale scFv expression. An overview of the 34 leads selected for further analysis as purified scFvs is shown in Table 10 along with the CDR mutations of these variants.

[0327] Tables 11, 12, and 13 highlight the conservation / diversity of affinity matured CDRs in lead scFv variants identified using the GST-CEACAM1 binding ELISA.

[0328] [Table 10-1] [Table 10-2]

[0329] [Table 12]

[0330] [Table 13]

[0331] Large-scale expression and purification of ScFv

[0332] Selected clones were expressed, purified, and quantified to accurately test the scFv by binding ELISA. Briefly, individual colonies were picked into 15 ml of 2TYCG (2%) medium and grown overnight at 30° C. with shaking. The seed was used to inoculate 500 ml of 2TYCG (0.1%) and OD 600 The cells were grown at 30°C until the chromatin concentration reached 0.8 nm. The cultures were induced by adding IPTG to a final concentration of 1 mM and then grown overnight at 30°C with shaking. The next day, the cultures were centrifuged and the supernatant discarded. The bacterial pellet was resuspended in 15 ml TES and incubated on ice for 15 minutes. Then, 22.5 ml TES (diluted 1 in 5 with cold water) was added and incubated on ice for an additional 30 minutes. The cells were then centrifuged and the supernatant containing the scFv was transferred to a new tube, after which MgCl2, NaCl, and imidazole were added to final concentrations of 1 mM, 300 mM, and 20 mM, respectively, to reduce non-specific binding. Ni-agarose beads were added and the scFv was allowed to bind with rotation for 2 hours at 4°C. The beads were pelleted by centrifugation and washed twice with wash buffer (25 mM Tris pH 7.4, 300 mM NaCl, 20 mM imidazole) before the scFvs were eluted from the beads using elution buffer (25 mM Tris pH 7.4, 300 mM NaCl, 400 mM imidazole). OD280nm was measured and samples were quantified using extinction coefficients based on the predicted amino acid sequence. Approximately 1 μg of each scFv was analyzed by SDS-PAGE. Bands corresponding to typical scFv characteristics were observed.

[0333] Assessment of scFv binding to GST-CEACAM1 measured by ELISA

[0334] Binding of affinity matured purified scFv to human CEACAM1 was analyzed using GST-CEACAM1. Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates were pre-coated with 1.0 μg / ml GST-CEACAM1 overnight at 4° C. The next day, V HTwo-fold dilution series (50 μg / ml to 0.8 μg / ml) of 1 / Vκ8 S29A parental scFv or test scFvs were incubated on the pre-coated ELISA plates for 2 hours at room temperature. Binding of scFvs was detected with anti-HIS6-HRP antibody and TMB substrate. Reactions were stopped with 3M HCl, absorbance was read at 450 nm on a Dynex Technologies MRX TCII plate reader, and binding curves were plotted. An example of binding assay data is shown in Figure 10. Parental scFv (V H 1 / Vκ8 S29A scFv) was included on each ELISA plate as a reference. An irrelevant scFv was included on at least one plate as a negative control. H Improved binding to CEACAM1 was observed for some scFvs compared to Vκ1 / Vκ8 S29A scFv. H Improved binding was observed for scFvs derived from both the VK and VK libraries. All 34 scFv variants were reformatted to whole IgG, providing greater precision in terms of purity and quantification. Reformatting further permitted analysis of the avidity component of antibody binding, which is influenced by the bivalent nature of IgG. As used herein, "avidity" is a measure of the strength of binding between an antigen-binding molecule (e.g., an antibody or antibody fragment thereof described herein) and an associated antigen.

[0335] Construction and testing of affinity matured whole antibodies

[0336] Reformatting scFv to whole IgG

[0337] The 34 variants identified by scFv screening were PCR amplified using primers that introduced flanking restriction enzyme sites for cloning into the IgG4 S241P pANTVhG4 vector and the kappa light chain pANTVK vector. HThe variants were subcloned into the IgG4 S241P pANTVhG4 vector using the MluI and HindIII restriction enzyme sites. Similarly, the nine affinity matured Vκ sequences were subcloned into the kappa light chain pANTVK vector using the BssHII and BamHI restriction sites. All constructs were verified by sequencing.

[0338] 25 leads humanized affinity matured IgG4 V for expression H The variants were combined with the parental humanized aglycosylated light chain (Vκ8 S29A). The nine lead humanized affinity matured kappa light chains were combined with the parental humanized heavy chain (V H 1). These combinations were transiently transfected into HEK EBNA adherent cells (in 6-well plates using the PEI transfection method. Five to seven days after transfection, supernatants were harvested, quantified by ELISA, and filtered for Biacore single cycle kinetics analysis.

[0339] Single-cycle kinetic analysis of humanized and affinity matured lead IgG binding to CEACAM1

[0340] To evaluate the binding of the humanized affinity matured reformatted lead IgG, single cycle kinetic analysis was performed on the crude supernatant using Biacore T200 running Biacore T200 Control Software V2.0.1 and Biacore T200 Evaluation Software V3.0. Antibodies were diluted in HBS-P+ to a final concentration of 0.5 μg / ml. At the start of each cycle, antibodies were loaded onto Fc2, Fc3 and Fc4 of the Protein A chip. IgG was captured at a flow rate of 10 μl / min to a RL of approximately 100 RU (RL of approximately 50-150 RU upon analyte binding). max The surface was then stabilized. Single cycle kinetic data were acquired using CEACAM1 as the analyte at a flow rate of 80 μl / min to minimize potential mass transfer effects. HMultiple replicates using the (Vκ8 S29A) antibody were performed to check the stability of the surface and analyte over the kinetic cycles. The signal of the reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surface. A 3.2-fold dilution range of 70 nM to 280 nM CEACAM1 was used without regeneration between each concentration. The signal of a blank run (no CEACAM1) for each antibody was subtracted to correct for differences in surface stability. For three injections of increasing concentrations of CEACAM1, the association phase was monitored for 80 seconds each time, and a single dissociation phase was measured for 150 seconds after the last injection of CEACAM1. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5 followed by a stabilization period of 250 seconds.

[0341] Single cycle kinetic constants (Table 14) showed that all but one of the humanized affinity matured antibodies bound to CEACAM1.

[0342] [Table 14-1] [Table 14-2]

[0343] Eight humanized and affinity matured heavy and light chain variants were identified and their relative K D These included three V H variants (CP08H03, CP09B03, and CP09C02) and five kappa light chain variants (CP08E01, CP08E05, CP08F05, CP08D02, and CP08G09).

[0344] CP09C02 (derived from the CDR1H library) contained an additional point mutation in CDR3H B1 (D96G), which was likely introduced by PCR during the library construction step (see section "Example 1, 1. Generation of humanized antibody variants"). H An additional heavy chain clone, CP09E05, was also progressed as it was identified to have only this single point mutation in CDR3 B1, which could potentially help identify the region responsible for the observed increase in affinity. H and five Vκ variants were developed to determine whether the efficacy could be improved by recombining affinity matured heavy and light chains.

[0345] Expression of lead heavy and light chain antibody combinations

[0346] Four humanized affinity matured IgG4 Vs identified after co-expression with the parental light chain H Each of the variants (CP08H03, CP09B03, CP09C02, and CP09E05) was paired with five lead humanized affinity matured kappa light chains (CP08E01, CP08E05, CP08F05, CP08D02, and CP08G09) (i.e., 20 pairings in total, see Table 15). As a control, a humanized affinity matured IgG4 V H The variants were combined with the parent light chain (Vκ8 S29A) and five lead humanized affinity matured kappa light chains were synthesized from the parent heavy chain (V H 1) (i.e., a total of 10 control antibodies, see Table 15). The combinations were transiently transfected into HEK EBNA adherent cells in 6-well plates using the PEI transfection method as described above and incubated for 5-7 days post-transfection. Supernatants were harvested, quantified by ELISA, and filtered for single cycle kinetic analysis on a Biacore.

[0347] [Table 15]

[0348] Single cycle kinetic analysis of lead heavy and light chain combination antibodies

[0349] Single cycle kinetic analysis using transient HEK supernatants was performed as previously described. Fitted data of single cycle kinetics are shown.

[0350] Table 16. Fifteen heavy and light chain combinations have relative K values ​​that are at least two times better than the parent. D Of these, six combinations (CP08H03 / CP08E05, CP08H03 / CP08F05, CP08H03 / Vк8S29A, CP09B03 / CP08E05, CP09C02 / CP08E05, and CP09C02 / CP08F05) had more than four times the K D (highlighted in bold in Table 16)

[0351] These six variants were progressed to larger scale production and Protein A purification for further analysis. Single cycle kinetic analysis also revealed that three variants did not function in combination.

[0352] [Table 16-1] [Table 16-2]

[0353] Recombination of the four affinity-matured heavy chain CDRs to generate six additional heavy chain variants

[0354] Six combinations with over 4x improvement were achieved with four different V HThree unique heavy chains with mutations in the CDRs were included: CP08H03 (CDRH3 B2); CP09B03 (CDR1H) and CP09C02 (CDR3H B2 and a single mutation in CDR3H B1, which is also uniquely present in CP08E05) (see Table 17). To determine whether further improvements could be obtained, four mutations V H CDR recombination was performed (Table 18). Pull-through PCR was used to isolate individual VFvs using scFv-specific primers. H The CDRs were recombined and subsequently cloned into the IgG4 S241P heavy chain expression vector using the MluI and HindIII restriction enzyme sites to generate six novel V H The variants (8H3_9B3, 8H3_9C2, 8H3_9E5, 9B3_9E5, 8H3_9C2(CDR1) and 9B3_9E5_8H3) were generated.

[0355] [Table 17]

[0356] [Table 18]

[0357] Recombinant V H Expression of CDR1 and CDR3 heavy chain and lead light chain:

[0358] Six recombinant V H The CDR1 and CDR3 variants (see Table 18) were combined with (1) the parent light chain (Vκ8 S29A), (2) the light chain CP08E05, or (3) the light chain CP08F05, the latter two light chains having previously shown improving effects when combined with affinity matured heavy chains (see Table 16).

[0359] The resulting 18 combinations are summarized in Table 19. These combinations were transiently transfected into HEK EBNA adherent cells in 6-well plates using the PEI transfection method and incubated for 5-7 days post-transfection. Supernatants were harvested, quantified by ELISA, and filtered for single cycle kinetic analysis on a Biacore.

[0360] [Table 19]

[0361] Recombinant V H and Reed V L Single cycle kinetic analysis of antibodies:

[0362] Single cycle kinetic analysis using transient HEK supernatants was performed as previously described. Single cycle kinetic fitting data is shown in Table 20.

[0363] Eight variants had a relative K >4-fold higher than the parent D Of these, five had more than six-fold superior K D was achieved (bold in Table 20). Five antibodies, 8H3_9B3 / CP08E05, 8H3_9B3 / CP08F05, 8H3_9B3 / Vк8 S29A, 8H3_9C2 / CP08F05 and 9B3_9E5 / CP08E05, were progressed to large-scale production and Protein A purification for further analysis.

[0364] [Table 20]

[0365] Expression, purification and testing of lead antibodies

[0366] The six most improved combination variants (CP08H03 / CP08E05, CP08H03 / CP08F05, CP08H03 / Vк8S29A, CP09B03 / CP08E05, CP09C02 / CP08E05, and CP09C02 / CP08F05,

[0367] The five most improved V H CDR1 and V H The CDR3 recombinant variants (8H3_9B3 / CP08E05, 8H3_9B3 / CP08F05, 8H3_9B3 / Vк8 S29A, 8H3_9C2 / CP08F05, and 9B3_9E5 / CP08E05, highlighted in bold in Table 20) were transiently transfected into HEK EBNA adherent cells in triple flasks using the PEI method and incubated for 5-7 days after transfection. Antibodies were purified from cell culture supernatants on a Protein A Sepharose column, buffer exchanged into PBS pH 7.2, and measured at OD using extinction coefficients based on the predicted amino acid sequence. 280nm Two micrograms of each antibody was analyzed by SDS-PAGE, and bands corresponding to typical antibody characteristics were observed.

[0368] Single cycle kinetic analysis of purified lead humanized and affinity matured antibodies (using purified proteins)

[0369] Single cycle kinetic analysis was performed as above using purified antibody instead of HEK supernatant. Single cycle kinetic fitting data is shown in Table 21. Expression levels of individual mutants are shown in Table 22.

[0370] All 11 lead variants bound >4-fold better than the parent antibody (see Table 21). Data obtained using purified IgG were consistent with data previously obtained using supernatants.

[0371] [Table 21]

[0372] [Table 22]

[0373] Elimination of potential CD4+ T cell epitopes

[0374] The sequences of 11 lead antibodies (see Table 21) were analyzed using iTope™ technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al 2008) and using TCED™ of known antibody sequence-associated T cell epitopes (Bryson et al 2010) to confirm that no significant T cell epitopes were introduced during the affinity maturation process. The CDR1 mutation (G26E, CDR definition by IMGT) found in the heavy chain of CP09C02 is associated with the introduction of a highly promiscuous epitope not observed in the parental sequence (see Table 21).

[0375] Selectivity analysis of lead antibodies

[0376] Initial selectivity analysis of several lead antibodies showed that antibodies containing phenylalanine (F) at position 104 of CDR3H showed, on average, higher selectivity for CEACAM1 compared to antibodies containing aspartic acid (D) at position 104 of CDR3H (Figure 11).

[0377] Multi-cycle reaction rate analysis

[0378] Variants CP08H03 / Vк8 S29A and CP08H03 / CP08F05 were further analyzed using multi-cycle kinetic analysis using a Biacore T200 instrument running Biacore T200 Evaluation Software V3.0.1. Purified antibodies were diluted in HBS-P+ to a concentration of 1 μg / ml. At the start of each cycle, each antibody was captured on the Protein A surface to a RL of approximately 100 RU. After capture, the surface was allowed to stabilize. Kinetic data were acquired using a flow rate of 80 μl / min to minimize potential mass transfer effects. To check the stability of both the surface and the analyte over the kinetic cycles, multiple repeats of blanks (no CEACAM1) and repeats of a single concentration of analyte were incorporated into the kinetic studies. For the kinetic analysis, a 2-fold dilution range was selected from 100 to 1.56 nM CEACAM1. The binding phase of CEACAM1 was monitored for 150 s and the dissociation phase was measured for 150 s. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle.

[0379] The signal of the reference channel Fc1 was subtracted from the signals of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surfaces, and the global Rmax parameter was used for the one-to-one binding model. H Relative K compared to 1 / Vκ8 S29A D is the K of the affinity matured composite human antibody variant D The kinetic parameters measured for the interaction of CEACAM1 with affinity matured CEACAM1 antibody variants CP08H03 / Vк8 S29A and CP08H03 / CP08F05 are shown in Table 23. Both affinity matured CEACAM1 antibody variants showed a V H 1 / Vκ8 S29A showed a >4-fold improvement in affinity compared to the parent.

[0380] [Table 23]

[0381] Example 2: Selectivity of CEACAM1 antibodies

[0382] CEACAM1 antibody V H To further evaluate the binding selectivity of CP08H03 / Vκ0, CP08H03 / Vκ8 S29A, and CP08H03 / CP08F05 to CEACAM1 versus other proteins, their binding affinities to CEACAM1, CEACAM3, CEACAM5, and CEACAM6 were compared using single cycle kinetic analysis performed as described above. Single cycle kinetic analysis was performed using CEACAM concentrations from 280 nM to 70 nM. Antibodies were loaded onto the chip at the following concentrations (taking into account the MW of the various analytes): 100 RU for CEACAM1, 375 RU for CEACAM3, 71.4 RU for CEACAM5, and 150 RU for CEACAM6. The three CEACAM1 antibodies, CP08H03 / Vκ8 S29A, CP08H03 / CP08F05, and Vκ0, were loaded onto the chip at the following concentrations (taking into account the MW of the various analytes): 100 RU for CEACAM1, 375 RU for CEACAM3, 71.4 RU for CEACAM5, and 150 RU for CEACAM6. H No significant binding of Vκ0 / Vκ0 was observed with CEACAM3, CEACAM5, and CEACAM6 (see FIG. 12A).

[0383] These results were consistent with the data obtained by measuring the specificity of the antibodies by ELISA. In the ELISA experiments, 96-well plates were coated with 0.5 or 1.0 μg / ml of CEACAM1. Nonspecific binding was blocked with 2% BSA / Dulbecco's PBS. CP08H03 / Vκ8 S29A, CP08H03 / CP08F05, or V H A 1:3 dilution series of 0 / Vк0 (starting concentration 50 μg / mL) was prepared in 2% BSA / PBS. 100 μL of sample was added to the pre-coated plate and incubated for 1 h at room temperature. Anti-human Ig κ chain-peroxidase secondary antibody (AP502P) used to detect CEACAM antibodies. Plates were developed with TMB and stopped with 3M HCl. Results were analyzed by background subtraction. Three CEACAM1 antibodies, CP08H03 / Vк8 S29A, CP08H03 / CP08F05, and Vк8 S29A were used. HEssentially no binding of 0 / Vκ0 to CEACAM3, CEACAM5, or CEACAM6 was observed (see Figures 12B and 12C).

[0384] This high degree of selectivity can be observed despite the fact that the N domains of different CEACAMs share a high degree of homology: the N domains of CEACAM1 and CEACAM3 are 88% identical, the N domains of CEACAM1 and CEACAM5 are 89% identical, and the N domains of CEACAM1 and CEACAM6 are 90% identical, as shown by a percent identity matrix generated using Clustal2.1 (see Figure 13).

[0385] Example 3: Epitope analysis of CEACAM1 antibody

[0386] To determine which residues on CEACAM1 are involved in binding to the specific CEACAM1 antibodies contemplated by the present invention, single point mutations were introduced into FLAG-tagged CEACAM1. Each FLAG-tagged CEACAM1 mutant was transfected into 293T cells. 48 hours after transfection, CEACAM1 protein was subjected to Western blotting. CEACAM1 antibody V H 0 / Vκ0 (chimeric antibody), V H 1 / Vκ8, V H 2 / Vκ4, V H 3 / Vκ1, and V H 4 / Vκ1 was used as the detection antibody. Mutation of CEACAM1 residues Y34, V39, G41, N42, R43, Q44, G47, and Q89, which are part of the CEACAM1 GFCC' face, resulted in reduced binding of CEACAM1 to the CEACAM1 antibody, indicating that these CEACAM1 residues may be involved in binding (see FIG. 14).

[0387] Example 4: Crystal structure of CEACAM antibody and CEACAM1

[0388] To more precisely map the binding interface between CEACAM1 and CP08H03 / Vκ8 S29A, we determined the crystal structure of human CEACAM1 complexed with the CP08H03 / Vκ8 S29A Fab fragment.

[0389] CEACAM1 was expressed from E. coli transformed with a pET21D-based plasmid expressing an untagged version of CEACAM1. The protein was refolded and purified in an arginine-containing buffer. After concentration to approximately 18 mg / ml using immobilized papain resin, Fab fragments were prepared by digesting the antibody and purified by protein A affinity chromatography and gel filtration chromatography. Purified CEACAM1 and Fab were mixed in a 1:1 molar ratio prior to crystallization screening. An initial crystallization hit of the CEACAM1:Fab complex was identified and subsequently optimized. Diffraction-quality crystals were grown at room temperature in conditions containing 18–20% PEG6000, 50 mM potassium dihydrogen phosphate, 20 mM Tris pH 7.0, and 1% β-octylglucoside. SDS-PAGE analysis of washed crystals and silver staining were used to confirm the crystallization of the complex. X-ray data from multiple crystals were collected at beamline NE-CAT24-ID-E at the Advanced Photon Source at Argonne National Laboratory. The best data from two untwinned isomorphic crystals were merged to create a highly redundant data set at 3.3 Å for structure determination and refinement. The structure of the complex was solved by molecular replacement with final R and R values ​​of 24.9% and 32.8%, respectively. free Refined to value.

[0390] The structure of the CEACAM1:CP08H03 / Vκ8 S29A Fab complex was determined at 3.3 Å resolution. CP08H03 / Vκ8 S29A Fab binds to CEACAM1 with a 1:1 stoichiometry (see FIG. 15). Some of the epitopes on CEACAM1 of the Fab fragment are shown in the molecular surface representation of CEACAM1 in FIG. 16. The primary and secondary interactions between the Fab molecule and CEACAM1 are shown in Tables 24 and 25.

[0391] [Table 24]

[0392] [Table 25]

[0393] With reference to the existing structure of the CEACAM1 dimer (Figure 17), it is clear that Fab binds to the interface of CEACAM1 involved in self-association. Presumably, this competitive interaction leads to dissociation of the dimer in solution. Residues that target CEACAM1 include four residues (Y34, Q44, Q89, N97) that form the YQQN pocket at the CEACAM1:CEACAM1 dimer interface. Notably, several residues on CEACAM1 that bind to antibodies are also predicted to be involved in binding to TIM-3, including CEACAM1 residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and / or N97 (Huang et al., Nature. 2015 Jan 15;517(7534):386-90).

[0394] In the Fab light chain, residues of CDR1, CDR2, and CDR3 (Figure 18) interact mainly with residues in the two loops between the β-strands of the main β-sheet of CEACAM1 and also with residues in the β-strands of the sheet. In the Fab heavy chain, residues of CDR2 and CDR3 interact mainly with residues distributed in the four different β-strands of the central β-sheet.

[0395] The interacting surfaces have a shape complementarity of 0.5 and the complex formation obscures 1607 A1 of the total solvent exposed surface. No interaction is observed between the antigen and the Fab heavy chain CDR1.

[0396] Alignment of human CEACAM family members showed that CEACAM3, 5, 6, 7, and 8 all contain a valine residue at position 49, while human CEACAM1 contains an alanine at this position. Additionally, human CEACAM5 contains a histidine at position 89. Polymorphisms of hCEACAM-1 at these residues include Ala49Val (rs8110904) and Gln89His (rs8111468). To further explore the selective nature of CEACAM1 antibody CP08H03 / Vκ8 S29A, human CEACAM1 A49V / Q89H mutant was expressed and purified as described above. It should be noted that there are naturally occurring human allelic variants of human CEACAM1 that convert Q89 to H89, as described in Huang et al., Nature. 2015 Jan 15; 517(7534): 386-90. The structure of the CEACAM1 A49V / Q89H mutant was determined at 1.7 Å resolution and compared to the CEACAM1 wild-type:CP08H03 / Vκ8 S29A Fab complex. As described above, CDR3H residue F104 of CP08H03 / Vκ8 S29A makes contact with residue F29 of wild-type CEACAM1 (see FIG. 19, left panel). In particular, F29 of one CEACAM1 monomer binds to F29 of the second monomer at the CEACAM1:CEACAM1 homodimer interface. Binding of CDR3H residue F104 of CP08H03 / Vκ8 S29A blocks the F29-F29 interaction. CEACAM1 residue A49 is located near the F104 / F29 interaction site. Due to the higher hydrophobicity of valine in non-CEACAM1 family members compared to alanine in human CEACAM1, mutation of human CEACAM1 residue A49 to valine brings the hydrophobic CEACAM1 residue F29 closer to the CEACAM1 V49 residue. This rotamer shift of F29 is also observed in the crystal structures of human CEACAM5 (PDB code 2QSQ) and human CEACAM3 (PDB code 6AW1), where it is predicted to clash with CDR3H residue F104 (see FIG. 19, right panel).This is illustrated by the change in orientation displayed by the CEACAM1 F29 ring, which approaches the space previously occupied by CDR3H residue F104 (see Figure 19, right panel). These data indicate that this steric hindrance caused by the A49V mutation prevents binding of the CEACAM1 antibody CP08H03 / Vκ8 S29A to other CEACAM1 family members that contain a valine at position 49, and as such contributes significantly to the selectivity of the antibody. It is predicted that this rotamer shift of the F29 ring may also affect the interaction between CDR2H residues Y57 and F29. Furthermore, the CEACAM1 Ala49Val polymorphism (rs8110904) has been linked to lymphedema caused by Wuchereria bancrofti (Debrah L, B.et.al Hum Genomics.2017 Nov 9;11(1):26) - a filarial parasite that invades the lymphatic system. Disease onset is associated with the Ala49Val polymorphism, which marks the alanine 49 residue found to be involved in binding to the CP08H03 / Vκ8 S29A antibody, and it is therefore anticipated that CP08H03 / Vκ8 S29A may also interfere with Wucheria bancrofti and other related pathogens that phenocopy parasite interactions with the lymphatic system or with cancer processes such as tumor invasion.

[0397] Example 5: CEACAM1 antibodies block CEACAM1:CEACAM1 interactions

[0398] The ability of CEACAM1 antibodies to block CEACAM1 homodimerization was tested. CEACAM1-CEACAM1 competitive ELISA tests were performed in triplicate to measure the ability of CP08H03 / Vκ8 S29A antibodies (concentration range 0-1000 nM) to inhibit the binding of human CEACAM1 IgV domain untagged protein (1 μg / ml) and human CEACAM1-GST protein (37.5 μg / ml). Additionally, an IgG4 antibody was used as a control (0-1000 nM). The assay was performed using a goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) and with the addition of TMB solution (Life Technologies). OD values ​​were read at 450 nm on a plate reader. Data were plotted in Graphpad and optimal IC-50 values ​​were determined.

[0399] The CEACAM1 antibody CP08H03 / Vκ8 S29A was shown to block the CEACAM1:CEACAM1 homophilic interaction (see FIG. 20A).

[0400] Example 6: CEACAM1 antibodies block CEACAM1:TIM-3 interaction

[0401] The ability of CEACAM1 antibodies to decrease the binding of CEACAM1 to TIM-3 was examined. CEACAM1 / TIM-3 competitive ELISA tests were performed in triplicate to measure the ability of CP08H03 / Vκ8 S29A antibody (concentration range 0-300 nM) to inhibit the binding of human TIM-3 IgV domain untagged protein (3 μg / ml) and human CEACAM1-GST protein (37.5 μg / ml). Additionally, a human IgG4 antibody was used as a control (0-1000 nM). The assay was performed using a goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) and with the addition of TMB solution (Life Technologies). OD values ​​were read at 450 nm on a plate reader. Data were plotted in Graphpad and the optimal IC-50 value was determined. As shown in Figure 20B, the CEACAM antibody CP08H03 / Vκ8 S29A blocks the CEACAM1:TIM-3 heterophilic interaction.

[0402] Example 7: CEACAM1 antibodies induce T cell proliferation

[0403] The ability of CEACAM antibodies CP08H03 / Vκ8 S29A and CP08H03 / CP08F05 to induce T cell proliferation was examined in humanized NOD scid gamma mice (NSG mice). See Figure 21 for the experimental set-up. Freshly isolated human PBMCs (5x10^6) were injected intraperitoneally (ip) into NOD.Cg-Prkdc mice. scid Il2rg tm1Wjl / SzJ (NSG) mice. 21 days after PBMC injection, NSG animals were examined for human immune cell engraftment by tail tip bleeding. 24 and 31 days after PBMC injection, humanized NSG mice were administered the first and second doses of the indicated concentrations of CEACAM1 antibody or isotype control antibody via intraperitoneal injection. At the end of the study (34 days after PBMC injection), mice were sacrificed and spleens surgically dissected for further analysis. Single cell suspensions from engrafted mice were stained with a cell proliferation dye and cultured in complete RPMI medium in the presence of soluble anti-human CD3 stimulator (2ug / ml, OKT3 clone) and rIL-2 (40U / ml) for an additional 2 days in vitro. Cells were maintained at a concentration of 10^7 cells / ml. After in vitro stimulation, cells were stained with an antibody against the human CD45 pan-leukocyte marker and evaluated by flow cytometry.

[0404] No antibody-dependent cellular cytotoxicity (ADCC) was observed in any of the groups tested (see FIG. 22). Administration of the CEACAM antibodies CP08H03 / Vκ8 S29A or CP08H03 / CP08F05, respectively, increases antibody-induced T cell proliferation in vivo (see FIG. 23).

[0405] Example 8: CEACAM1 antibodies reduce tumor growth in melanoma models

[0406] To assess the ability of CEACAM1 antibodies to reduce tumor growth, 1 × 106 MALME-3M (human melanoma) at 5 × 10 6 7-8 week-old male NSG (NOD.Cg-Prkdc) mice were cultured with human PBMCs. scid Il2rg tm1Wjl / SzJ) mice were subcutaneously injected with MALME-3M (BRAFV 600E The .) cell line was established in 1975 from a metastatic site (lung) derived from a 43 year old Caucasian male with metastatic melanoma. On days 7-9, all mice were confirmed to exhibit reconstituted T cell populations (see FIG. 24A for experimental setup). Animals were treated intraperitoneally with CEACAM antibody CP08H03 / Vκ8 S29A or hIgG4 control antibody on days 10, 13, 17, 20 and 24. The human melanoma cell line MALME-3M was kindly provided by Dr. Nicole Beauchemin (McGill University, Montreal, Canada). MALME-3M was established in 1975 from a metastatic site (lung) of a 43-year-old Caucasian male with metastatic melanoma involving BRAFV600E. 2x10^7 MALME-3M was cloned into NOD.Cg-Prkdc scid Il2rg tm1Wjl CEACAM1 antibodies were injected subcutaneously (sc) into cancer-bearing NSG mice. After 30 min of acclimation, freshly isolated human PBMCs (1x10^8) were then transferred into the cancer-bearing NSG mice by intraperitoneal (ip) injection. Seven to nine days after PBMC injection, NSG animals were examined for human immune cell engraftment by tail-tip bleeding. At 10, 13, 17, 20, and 24 days after human cell injection, cancer-bearing humanized NSG mice were administered the indicated concentrations of CEACAM1 antibody or isotype control antibody by ip injection for a total of five doses. At the end of the study (34 days after human cell injection), mice were sacrificed and surgically dissected.

[0407] The CEACAM1 antibody CP08H03 / Vκ8 S29A was effective in reducing tumor growth at various concentrations and proliferations (see Figures 24B, 24C and 25), while not depleting T cell populations (see Figure 22). Furthermore, human CD4 + and CD8 +The proliferation capacity of tumor-infiltrating lymphocytes was restored by administration of CEACAM1 antibody (see FIG. 25). Furthermore, in vivo treatment with CP08H03 / Vκ8 S29A resulted in a bias from CD8 T memory cells to primarily central memory T cells (FIG. 26). Consistent with the augmentation of the anti-cancer response, CEACAM1 antibody CP08H03 / Vκ8 S29A enhanced the proliferation of T em T for cm Note that the relative proportion of IL-1, IL-2, and IL-3 was increased compared to that observed in control treated animals.

[0408] Example 9: CEACAM1 antibodies are useful in treating cancers resistant to checkpoint inhibitors

[0409] CEACAM1 is expressed in a significant proportion of TILs derived from melanoma patients who are naive or resistant to anti-PD-1 and / or anti-CTLA-4 therapy; the expression levels of CEACAM1 are greater than those of PD-1 or TIM-3 (see Figure 27). Approximately 80% of the samples showed CD4 + More than 20% of the T cell population shows CEACAM1 expression. To compare the expression of CEACAM1 in patients who have acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy with patients who have not previously received anti-PD-1 and / or anti-CTLA-4 therapy, tumor associated cells (TAC) were obtained from melanoma patients who were naive (not previously received anti-PD-1 and / or anti-CTLA-4 therapy) or who have acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy (acquired resistance). TAC were obtained by culturing tumor tissue in DMEM medium and removing floating cells from the supernatant. Cells were stained for CD3, CD4, and CD8, and CD3 + CD4 + and CD3 + CD8 +These studies demonstrated that tumor-associated cells deprived of the tumor microenvironment in acquired resistance upregulate CEACAM1 expression relative to that observed in naive patients (see FIG. 28), indicating that patients resistant to anti-PD-1 and / or anti-CTLA-4 therapy may benefit from an anti-CEACAM1 antibody or antigen-binding fragment thereof, such as an antibody or antigen-binding fragment thereof contemplated in the present disclosure.

[0410] As expected, CD8 + T cells showed increased effector memory (T em ) cells to central memory (T cm Tumor-associated cells from either naive (treatment-naive patients) or immune checkpoint inhibitor-resistant (treatment-failed patients) melanoma patients were stained for CD44, CCR7, and CD62L to identify central memory T cells, consistent with a reduced anti-cancer response in resistant patients (see FIG. 29). cm (CD44 high , CD62L high , CCR7 high ) and Effector Memory T em (CD44 high , CD62L low , CCR7 low ) were expressed as a percentage of the total CD8 T cells present in the bulk tumor.

[0411] To evaluate the ability of CEACAM1 antibodies to reverse T cell exhaustion in patients resistant to treatment with checkpoint inhibitors such as PD-1 / PD-L1 and CTLA-4 inhibitors, PBMCs and tumor-associated cells were isolated from melanoma patients with secondary resistance to pembrolizumab (PD-1 inhibitor), ipilimumab (CTLA-4 inhibitor) + nivolumab (PD-1 inhibitor) and dabrafenib (B-Raf inhibitor) + trametinib (MEK inhibitor) and stage IV disease. Tumor-associated cells and PBMCs were stained for CEACAM1, PD1, or TIM-3 and CD8+ T cells were stained for CD8+ T cells. + and CD4 + The percentage of T cells was shown (see Figure 30, left panel). Tumour biopsies were subjected to either enzymatic digestion or a commercially available mechanical / enzymatic dissociation system (GentleMACS dissociator, Miltenyi Biotec). Enzymatic digestion was based on a previously established methodology for the generation of melanoma TILs (Dudley et al, 2003, 2008). Briefly, tumour biopsies were cut into small pieces of approximately 2–3 mm in length and incubated with 100 U ml -1 DNAse, 10mg ml -1The tumors were placed in an enzymatic digestion mixture consisting of collagenase VIII (Sigma-Aldrich) and incubated for 45 min at a temperature of 37 °C under continuous rotation. GentleMACS dissociation was performed according to the manufacturer's protocol. Briefly, tumors were cut into small pieces of approximately 2-3 mm in length and placed in C-tubes (Miltenyi Biotech) containing RPMI1640 (Lonza, Slough, UK) and solutions 1, 2 and 3 (all from Miltenyi Biotec) according to the manufacturer's recommendations; the digestion mixture containing the tumor was then subjected to three 36 s mechanical dissociation steps (programs h_tumor_01.01, 02.01 and 03.01) in a GentleMACS dissociator, followed by two 30 min incubations at 37 °C, sandwiched after the first and second dissociation steps, respectively. After dissociation, the TILs obtained from enzymatic digestion and GentleMACS dissociation were passed through a 100 μm strainer for further analysis. Dissociated tumor cells and autologous PBMCs were stained with the following antibodies according to standard procedures: fluorochrome-conjugated monoclonal antibodies specific for human CD3, CD4, CD8, TIM-3, PD1, CEACAM1, CD45, and cell viability dyes. Data were acquired using a Cytoflex flow cytometer (Invitrogen) and analyzed using FlowJo software (TreeStar, V7.6.5 for Windows). Tumor cells were cultured in 96-well plates in complete medium (10% fetal calf serum (FCS), 1% glutamine, 100 IU ml -1 Penicillin, 100 μg ml -1PBMCs or tumor-associated cells were cultured with soluble anti-CD3 (2 pg / ml) and rIL-2 (40 units / ml) in the presence of CP08H03 / Vκ8 S29A or hIgG4 control in RPMI1640 (Lonza) supplemented with streptomycin (Life Technologies), 25 mM HEPES (Sigma-Aldrich). After 96 hours, cell culture supernatants were harvested for further TNF-a and IFN-γ ELISA (BD) analysis according to the manufacturer's protocol. CP08H03 / Vκ8 S29A reverses T cell exhaustion in PD-1 / CTLA-4 resistant tumors as evidenced by increased production of TNF-α and IFN-γ in both tumor-associated cells and PBMCs (see Figure 30, right panel).

[0412] Example 10: CEACAM1 antibodies contemplated by the present invention exhibit improved potency over previously known CEACAM1 antibodies.

[0413] The properties of antibody CP08H03 / Vκ8 S29A were compared to the anti-CEACAM1 antibody CM-24 (WO2015 / 166484). Unlike the CEACAM1 antibody CP08H03 / Vκ8 S29A disclosed herein, CM-24 (i) binds CEACAM1 away from the dimer interface based on modeling, (ii) shows cross-reactivity with CEACAM3 and CEACAM5, (iii) has limited ability to reverse T cell resistance in TILs, and (iv) functions as an agonist antibody rather than an antagonist antibody in mouse models of metastatic melanoma.

[0414] CP08H03 / Vκ8 S29A was selective for CEACAM1 and showed no significant binding to CEACAM3, CEACAM5, CEACAM6, or CEACAM8. On the other hand, CM-24 showed significant cross-reactivity with CEACAM3 and CEACAM5 at higher antibody concentrations (FIGS. 31A and 31B). The cervical adenocarcinoma cell line HeLa (ATCC number CCL-2) and the transfected cell lines HeLaCEACAM1, HeLaCEACAM3, HeLaCEACAM5, HeLaCEACAM6, and HeLaCEACAM8 used for flow cytometry experiments were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U / ml), and dihydrostreptomycin (100 μg / ml) at 37° C. and 5.0% CO2. The cell lines were incubated with the indicated antibodies followed by monoclonal antibodies conjugated with fluorescent dyes specific for the indicated antibody isotypes, such as human IgG4 or mouse IgG1. Cells were stained with local antibodies along with a cell viability dye (DAPI). Data were acquired using a Cytoflex flow cytometer (Invitrogen) and analyzed using FlowJo software (TreeStar, V7.6.5 for Windows).

[0415] Furthermore, CM-24 had limited ability to reverse T cell resistance in tumor-associated cells. Incubation of tumor-associated cells with antibody CP08H03 / Vκ8 S29A resulted in a more extensive reversal of T cell resistance across a range of antibody concentrations compared to CM-24 in naive Merkel cell carcinoma tumor cells (Figures 32A, 32B, and 32C). Merkel cell carcinoma biopsies were subjected to a commercially available mechanical / enzymatic dissociation system (GentleMACS dissociator, Miltenyi Biotec) according to the manufacturer's protocol. Briefly, tumors were cut into pieces of approximately 2-3 mm in length and placed in C-tubes (Miltenyi Biotech) containing RPMI1640 (Lonza, Slough, UK) and solutions 1, 2 and 3 (all from Miltenyi Biotec) according to the manufacturer's recommendations; the digestion mixture containing tumors was then subjected to three 36 s mechanical dissociation steps (programs h_tumor_01.01, 02.01 and 03.01) in a GentleMACS dissociator, with two 30 min incubations at 37°C, sandwiched after the first and second dissociation steps, respectively. After dissociation, TILs obtained from enzymatic digestion and GentleMACS dissociation were passed through a 100 μm strainer for further analysis. In vitro assay of T cell function in the tumor environment: Dissociated tumor cells and autologous PBMCs were cultured in 96-well plates in complete medium (10% fetal calf serum (FCS), 1% glutamine, 100 IU ml -1 Penicillin, 100 μg ml -1 40 IU ml in RPMI 1640 (Lonza) supplemented with streptomycin (Life Technologies), 25 mM HEPES (Sigma-Aldrich), and in the presence of various concentrations of antibody or relevant isotype control. -1 They were cultured with recombinant IL-2 (NIH) and soluble CD3 (2 pg / ml). After 96 hours, cell culture supernatants were harvested for further TNF-a and IFN-gamma ELISA (BD) analysis according to the manufacturer's protocol.

[0416] In a metastatic melanoma model (see FIG. 33A for experiment), mice treated with CP08H03 / Vκ8 S29A in vivo showed a significant reduction in tumor cells and TIL CD4 expression compared to control mice treated with human IgG4 (hIgG4) in vivo. + and CD8 + On the other hand, mice treated with CM-24 in vivo showed a significant increase in tumor cells and TIL CD4 cells compared to control mice treated with hIgG4 control. + and CD8 + The tumor cells showed a near complete lack of lymphocytes (Figures 33B, 34A, 34B, and 34C). Furthermore, tumor cells showed reduced proliferation in CP08H03 / Vκ8 S29A-treated animals compared to hIgG4 control or CM24-treated animals (Figure 33C). Furthermore, the CD4 + T cells showed increased proliferation compared to hIgG4- or CM24-treated animals (FIG. 33D). In contrast to CP08H03 / Vκ8 S29A-treated animals, CM24-treated animals showed increased proliferation of splenic CD4 + The human melanoma cell line MALME-3M was kindly provided by Dr. Nicole Beauchemin (McGill University). MALME-3M was derived from a BRAF mutant line in 1975. V600EThe tumor was established from a metastatic site (lung) in a 43-year-old Caucasian male with metastatic melanoma with pulmonary fibrosis. 2x10^7 MALME-3M was injected subcutaneously (sc) into NSG mice. After 30 min to allow the mice to absorb the tumor cells, freshly isolated human PBMCs (1x10^8) were transferred into the tumor-bearing NSG mice by intraperitoneal (ip) injection. 7–9 days after PBMC injection, NSG animals were examined for engraftment of human immune cells by tail-tip bleeding. On day 14, palpable tumor nodules were detected. Beginning on day 17 after human cell injection, cancer-bearing humanized NSG mice were administered CP08H03 / Vκ8 S29A antibody (2mg / kg), CM-24 (2mg / kg) or isotype control antibody (2mg / kg) by intraperitoneal injection twice weekly for a total of four doses. At the end of the study (30 days after human cell injection), mice were sacrificed and surgically dissected. The metastatic tumors, along with the spleen, lungs, and liver, were preserved for further analysis. Total cell number and proliferation, as well as CD4, CD8, and tumor cell frequency, were characterized by high FSC and SSC and negative expression of the human pan-leukocyte marker CD45.

[0417] Figures 34A, 34B, and 34C provide a statistical comparison of the results shown in Figure 33B with the protocol shown in Figure 33A. CM-24 treated animals showed larger tumors with no evidence of tumor associated T cells. On the other hand, an increased amount of infiltrating T cells and a decrease in tumor cells was observed in CP08H03 / Vκ8 S29A treated mice (Figure 33B). As previously mentioned, evaluation of tumor cell proliferation showed inhibition of tumor growth by CP08H03 / Vκ8 S29A but not by CM-24 (Figure 33C). Furthermore, splenic CD4 + Increased proliferation of T cells was observed, while splenic CD4 + A decrease in T cell proliferation was observed (Figure 33D).

[0418] Example 11: CEACAM1 antibodies block the interaction between CEACAM1 and HopQ.

[0419] HopQ is expressed on the surface of Helicobacter pylori, a bacterium that specifically colonizes the human gastric epithelium and is a major causative agent in the development of ulcer disease and gastric cancer. HopQ-CEACAM1 interaction has been suggested to promote gastric colonization and Hp-induced pathology, for example, by enabling the translocation of bacterial virulence factors into host cells and promoting the release of proinflammatory mediators.

[0420] Published crystal structure data (see PDB IDs 6AW2, 6GBH, 6GBG, Bonsor,D,et.al. EMBO J.2018 Jul 2;37(13) and Moonens K et al. EMBO J.2018 Jul 2;37(13)) show that the GFCC' loop of CEACAM1 is involved in binding to HopQ, with CEACAM1 residues F29, Y34, N42, Q89, and N97 forming various hydrogen bond and hydrophobic interactions with HopQ residues (see Figure 35A). Modeling based on CECAM1:HopQ cocrystals and CEACAM1:CP08H03 / Vκ8 S29A Fab cocrystals indicates that the CEACAM1 antibody CP08H03 / Vκ8 S29A can cover the CEACAM1 binding site of HopQ (see Figure 35B) and disrupt the CEACAM1:HopQ interaction.

[0421] Example 12: CEACAM1 antibodies promote long-term survival

[0422] The ability of the CEACAM1 antibody CP08H03 / Vκ8 S29A to promote long-term survival in cancer-bearing mammals was investigated using a mouse melanoma model.

[0423] 10 6 MALME-3M (human melanoma) cells and 5 × 10 6 Human PBMCs (from HLA-A2-matched donors) were injected subcutaneously into NSG mice. On day 10, tumors grew to 2–2.5 mm 3After reaching 1 h of survival, mice were randomized (n=4 / group). Anti-CEACAM1 antibody CP08H03 / Vκ8 S29A or control human IgG4 antibody was administered intraperitoneally on days 10, 13, 17, 20, and 24, respectively. Survival was monitored for 104 days, at which point surviving animals showing active clinical activity were sacrificed (arrows).

[0424] As shown in Figure 36, treatment with anti-CEACAM1 antibody significantly increased the survival rate of cancer-bearing mice. Furthermore, at autopsy, the antibody-treated animals showed local tumors without visible metastases, which is consistent with disease control. This data indicates that the anti-CEACAM1 antibody and fragments thereof disclosed herein are useful for treating cancer and increasing survival.

[0425] Example 13: CP08H03 / Vκ8 S29A increases immune responses in tumor cells derived from naive patients or from patients with secondary resistance to immunotherapy

[0426] The ability of the CEACAM1 antibody CP08H03 / Vκ8 S29A to increase immune responses in tumors derived from melanoma patients who were naïve or had secondary resistance to immunotherapy was examined using isolated tumor specimens.

[0427] In one example, tumor specimens isolated from patients with secondary resistance were disrupted by mechanical dissociation and dissociated cells were treated with either CP08H03 / Vκ8 S29A or hIgG4 control antibody (2 μg / ml) in culture medium for 4 days in the presence of 2 μg / ml anti-CD3 and 40 units / ml recombinant IL-2. The cells were then examined by mass cytometry using the following antibodies: CD8 +We detected various intracellular factors associated with the immune response of T cells to tumors: IFNγ (clone B27; 168Er), IL-17A (clone N49-653; 164Dy), IL-17F (clone SHLR17; 166Er); granzyme B (clone GB11; 171Yb); perforin (clone B-D48; 175Lu); MIP1β (clone D21-1351; 150Nd); TNFα (clone Mab11; 152Sm), CD3 (clone UCHT1; 170Er); CD8 (clone RPA78; 146Nd); and intercalator (103Rh).

[0428] As shown in Figures 37A and 37B, treatment with CP08H03 / Vκ8 S29A antibody significantly reduced CD8 + These results suggest that the CP08H03 / Vκ8 S29A antibody induces a significant increase in the expression of intracellular factors in CD8 T cells. + We have directly shown that it induces the production of a variety of factors in T cells that may be associated with a productive anti-tumor immune response.

[0429] In another example, tumor specimens associated with two melanoma patients who were either previously untreated (subject 189) or had secondary resistance to immunotherapy (subject 185) were disrupted by mechanical dissociation (Miltenyi). 5 Tumor cells were plated in culture dishes at 2 μg / ml. Freshly isolated tumor dissociated cells were exposed to 2 μg / ml of CP08H03 / Vκ8 S29A alone or human IgG4 isotype control antibody. After 96 hours, supernatants were removed and ELISA assays were performed in triplicate to detect the presence of interferon-γ.

[0430] As shown in Figures 38A and 38B, treatment with CP08H03 / Vκ8 S29A antibody induced secretion of the cytokine interferon-γ into the supernatants of tumor dissociated cells isolated from patients who were either secondary resistant to immunotherapy treatment (Figure 38A, subject 185) or naive to immunotherapy treatment (Figure 38B, subject 189) at significant levels compared to those observed with a control human IgG4 antibody.

[0431] Taken together, these data indicate that the anti-CEACAM1 antibodies and fragments thereof disclosed herein are useful for treating naive cancer patients and patients with secondary resistance to immunotherapy.

[0432] [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4]

[0433] While the foregoing written description of the invention enables one of ordinary skill in the art to make and use what is currently believed to be the best mode thereof, those of ordinary skill in the art will understand and recognize the existence of variations, permutations, and equivalents of the specific embodiments, methods, and examples herein.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2, and CDR3; and The sequence of CDR1H is sequence X 1 HX 2 X 3 S (SEQ ID NO:1); X 1 is A, D, N, or S; X 2 is A or G; and X 3 is an amino acid having a hydrophobic side chain, including I or M; The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H is the sequence HX 4 X 5 D.Y.X. 6 P.X. 7 WFAX 8 (SEQ ID NO: 3), X 4 is D, G, or P; X 5 is F or P; X 6 is D or F; X 7 is A or Y; and X 8 is L, H, or F; The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of CDR3L is the sequence QQX 9 X 10 X 11 X 12 P.X. 13 T (SEQ ID NO:6); X 9 is W or N; X 10 is S or T; X 11 is A or an amino acid having a neutral hydrophilic side chain, including S, N, and T; X 12 is L, F, or N; and X 13 is P or F; An antibody or an antigen-binding fragment thereof.

2. The sequence of the heavy chain variable region is 1 HX 2 X 3 S (SEQ ID NO:43); X is any amino acid; X 1 is A, D, N, or S; X 2 is A or G; and X 3 is an amino acid having a hydrophobic side chain, including I or M; and The sequence of CDR3H is the sequence HX 4 X 5 DYFPX 7 WFAX 8 (SEQ ID NO:44); X 4 is D, G, or P; X 5 is F or P; X 7 is A or Y; and X 8 is L, H, or F; The antibody or antigen-binding fragment thereof described in claim 1.

3. The sequence of CDR1H is sequence X 1 HX 2 X 3 S (SEQ ID NO:1); X 1 is A, D, N, or S; X 2 is A or G; and X 3 is an amino acid having a hydrophobic side chain, including I or M; The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H is the sequence HX 4 X 5 DYFPYWFAX 8 (SEQ ID NO:7); CDR3H X 4 is D, G, or P; CDR3H X 5 is F or P; and CDR3H X 8 is L, H, or F; The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of CDR3L is the sequence QQX 9 SSX 12 P.X. 13 T (SEQ ID NO:8); X 9 is W or N; X 12 is L, F, or N; and X 13 is P or F; The antibody or antigen-binding fragment thereof described in claim 1.

4. The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO:9); The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO: 10); The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of CDR3L comprises the sequence QQWSSNPPTT (SEQ ID NO: 11), The antibody or antigen-binding fragment thereof described in claim 1.

5. The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO:9); The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); The sequence of CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO: 10); The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4); The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of CDR3L comprises the sequence QQWTSNPPT (SEQ ID NO: 12), The antibody or antigen-binding fragment thereof described in claim 1.

6. An antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region comprises a sequence that is at least 90% identical to the heavy chain variable region amino acid sequence of SEQ ID NO:13; and the sequence of the light chain variable region comprises a sequence that is at least 90% identical to a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; An antibody or an antigen-binding fragment thereof.

7. the sequence of the heavy chain variable region comprises a sequence that is at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO:13; and the sequence of the light chain variable region comprises a sequence that is at least 95% identical to a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; The antibody or antigen-binding fragment thereof described in claim 6.

8. the sequence of the heavy chain variable region comprises SEQ ID NO:13; and the sequence of the light chain variable region has a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; The antibody or antigen-binding fragment thereof described in claim 7.

9. the sequence of the heavy chain variable region comprises SEQ ID NO:13; and the sequence of the light chain variable region comprises SEQ ID NO: 14; The antibody or antigen-binding fragment thereof described in claim 8.

10. the sequence of the heavy chain variable region comprises SEQ ID NO:13; and the sequence of the light chain variable region comprises SEQ ID NO: 15; The antibody or antigen-binding fragment thereof described in claim 8.

11. An antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of SEQ ID NO:13; the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the light chain variable region amino acid sequence of SEQ ID NO: 14; The sequence of the variable heavy chain is 1 HX 2 X 3 S (SEQ ID NO:43); X is any amino acid; X 1 is A, D, N, or S; X 2 is A or G; and X 3 is an amino acid having a hydrophobic side chain, including I or M; and The sequence of CDR3H is the sequence HX 4 X 5 DYFPX 7 WFAX 8 (SEQ ID NO:44); X 4 is D, G, or P; X 5 is F or P; X 7 is A or Y; and X 8 is L, H, or F; An antibody or an antigen-binding fragment thereof.

12. An antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region; the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the heavy chain variable region amino acid sequence of SEQ ID NO:13; the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the light chain variable region amino acid sequence of SEQ ID NO: 14; each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2, and CDR3; and the sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13; the sequence of CDR3H comprises residues D102, Y103, F104, P105, and Y106 of SEQ ID NO: 13; the sequence of CDR1L comprises residues A28, S30, and Y31 of SEQ ID NO:14; the sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO:14; and the sequence of CDR3L comprises residues S91 and S92 of SEQ ID NO: 14; An antibody or an antigen-binding fragment thereof.

13. the sequence of the heavy chain variable region comprises a sequence that is at least 90% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 13; the sequence of the light chain variable region comprises a sequence that is at least 90% identical to the light chain variable region amino acid sequence of SEQ ID NO: 14; The antibody or antigen-binding fragment thereof described in claim 12.

14. the sequence of the heavy chain variable region comprises a sequence that is at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 13; the sequence of the light chain variable region comprises a sequence that is at least 95% identical to the light chain variable region amino acid sequence of SEQ ID NO: 14; The antibody or antigen-binding fragment thereof described in claim 13.

15. The antibody or antigen-binding fragment of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is a chimeric antibody, a CDR-grafted antibody, or a humanized antibody or an antigen-binding fragment thereof.

16. The antibody or antigen-binding fragment of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment is a multispecific or bispecific antibody or an antigen-binding fragment thereof.

17. 17. The antibody or antigen-binding fragment of claim 16, wherein the antibody or antigen-binding fragment is a bispecific antibody that contains a complementary region that binds to PD-1 or PD-L1.

18. The antibody or antigen-binding fragment is selected from the group consisting of scFv, Fv, Fab', Fab, F(ab') 2 18. The antibody or antigen-binding fragment of any one of claims 1 to 17, which is a mAb, a mAb or a diabody.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment has the isotype IgG4.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, wherein the antibody or antigen-binding fragment thereof comprises a S241P substitution in the constant region of the heavy chain.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, wherein the antibody or antigen-binding fragment is aglycosylated.

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment lacks the C-terminal lysine of the heavy chain.

23. The antibody or antigen-binding fragment of any one of claims 1 to 22, wherein the antibody or antigen-binding fragment is conjugated to one or more of a cytotoxin, a fluorescent label and an imaging agent.

24. An antibody or antigen-binding fragment that binds to the same epitope on CEACAM1 as the antibody or antigen-binding fragment of claim 9.

25. An antibody or antigen-binding fragment thereof that binds to the IgV-like N domain of CEACAM1, wherein the antibody or antigen-binding fragment thereof binds to an epitope that includes one or more residues selected from the group consisting of residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and E99 of SEQ ID NO:

17.

26. 26. The antibody or antigen-binding fragment of claim 25, wherein the epitope further comprises residue Q44 of SEQ ID NO:

17.

27. 26. The antibody or antigen-binding fragment of claim 25, wherein the epitope further comprises one or more residues selected from the group consisting of residues S32, Q44, A49, I91, L95, and V96 of SEQ ID NO:

17.

28. The antibody or antigen-binding fragment of any one of claims 25 to 27, wherein the antibody or antigen-binding fragment binds to the IgV-like N domain of CEACAM1.

29. 28. The antibody or antigen-binding fragment of any one of claims 25 to 27, wherein the antibody or antigen-binding fragment does not bind to one or more of CEACAM3, CEACAM5, CEACAM6, and CEACAM8.

30. The antibody or antigen-binding fragment of any one of claims 25 to 27, wherein the antibody or antigen-binding fragment at least partially binds to a binding site on CEACAM1 for TIM3.

31. The antibody or antigen-binding fragment of any one of claims 25 to 27, wherein the antibody or antigen-binding fragment at least partially binds to a binding site on CEACAM1 for CEACAM1 during homodimerization.

32. An antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment binds to an epitope that includes one or more residues selected from the group consisting of residues F29, Y34, N42, Q89, and N97 of SEQ ID NO:

17.

33. An antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment thereof binds to an epitope that includes one or more residues selected from the group consisting of residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and N97 of SEQ ID NO:

17.

34. 34. The antibody or antigen-binding fragment of claim 33, wherein the epitope further comprises residues F29, S32, D40, A49, T56, I91, L95, and E99 of SEQ ID NO:

17.

35. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 34.

36. A vector comprising the nucleic acid of claim 35.

37. A cell comprising the vector of claim 36.

38. A cell expressing the antibody or antigen-binding fragment of any one of claims 1 to 34.

39. A T cell having a chimeric antigen receptor comprising the CDR of the antibody or antigen-binding fragment of any one of claims 1 to 34.

40. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 34 and a pharma- ceutically acceptable excipient.

41. A method for inhibiting binding of CEACAM1 to a member of the CEACAM family, said method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.

42. 42. The method of claim 41 , wherein the member of the CEACAM family is selected from the group consisting of CEACAM3, CEACAM5, CEACAM6, and CEACAM8.

43. 42. The method of claim 41, wherein the member of the CEACAM family is CEACAM1.

44. A method for inhibiting binding of CEACAM1 to a member of the TIM family, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.

45. 45. The method of claim 44, wherein the member of the TIM family is TIM3.

46. A method for inhibiting binding of CEACAM1 to a bacterial adhesin, said method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.

47. 47. The method of claim 46, wherein the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza outer membrane protein (OMP) P1, Haemophilus aegyptius OMP P1, or Moraxella species Opa-like protein (OlpA).

48. A method for inhibiting the binding of CEACAM1 to Candida albicans, said method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.

49. A method for inhibiting binding of CEACAM1 to influenza virus, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.

50. 50. The method of claim 49, wherein the influenza virus is H5N1.

51. A method for reducing colonization of mammalian epithelium by bacteria expressing a bacterial adhesin, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.

52. 52. The method of claim 51, wherein the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1, or Moraxella species OlpA.

53. A method of reducing colonization of mammary epithelium by Candida albicans, said method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.

54. A method of reducing influenza virus replication, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment of any one of claims 1 to 34.

55. 35. A method of reducing the release of proinflammatory cytokines or chemokines associated with influenza virus infection, the method comprising contacting a cell population comprising epithelial cells with the antibody or antigen-binding fragment of any one of claims 1 to 34.

56. 56. The method of claim 54 or 55, wherein the influenza virus is H5N1.

57. 35. A method of reducing T cell resistance, the method comprising contacting a cell population comprising T cells with an antibody or antigen-binding fragment of any one of claims 1 to 34.

58. 35. A method of enhancing T cell proliferation, the method comprising contacting a cell population comprising T cells with the antibody or antigen-binding fragment of any one of claims 1 to 34.

59. 35. A method of reducing T cell resistance in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

60. 35. A method of enhancing T cell proliferation in a subject in need thereof, said method comprising administering to said subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

61. 35. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.

62. 62. The method of claim 61, wherein the cancer is melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cell carcinoma, prostate cancer, breast cancer, bladder cancer, or gastric cancer.

63. 35. A method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

64. 35. A method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34.

65. 35. A method of reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

66. 35. A method for reducing cancer stemness in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

67. 35. A method for reducing colonization of an epithelium of a subject by bacteria expressing a bacterial adhesin in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

68. 68. The method of claim 67, wherein the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1, or Moraxella species OlpA.

69. 35. A method of reducing epithelial colonization of a subject by Candida albicans in a subject in need thereof, said method comprising administering to said subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

70. 35. A method of reducing influenza virus replication in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

71. 35. A method for reducing the release of proinflammatory cytokines or chemokines associated with influenza virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

72. 72. The method of claim 70 or 71, wherein the influenza virus is H5N1.

73. A method for reducing invasion of the lymphatic system of a subject by filarial parasites in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

74. 74. The method of claim 73, wherein the filarial parasite is Wucheria bancroftii.

75. 35. A method of reducing infiltration of the lymphatic system of a subject by cancer cells in a subject in need thereof, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment of any one of claims 1 to 34.

76. 67. The method of any one of claims 59-66, further comprising administering a checkpoint inhibitor.

77. 77. The method of claim 76, wherein the checkpoint inhibitor is a CTLA-4, PD-1, PD-L1, and PD-L2 inhibitor.

78. 67. The method of any one of claims 59 to 66, further comprising administering one or more of an inhibitor of LAG3, TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226 and / or CD160.

79. 67. The method of any one of claims 59 to 66, further comprising administering a TIM-3 inhibitor.

80. 80. The method of any one of claims 76 to 79, wherein the additional inhibitor is administered simultaneously or sequentially with the antibody or antigen-binding fragment.

81. 80. The method of any one of claims 76 to 79, wherein the additional inhibitor is administered separately or in a mixture with the antibody or antigen-binding fragment.

82. 35. A method of treating a subject in need of treatment, said method comprising administering to said subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 34, wherein said subject has acquired resistance to treatment with checkpoint inhibitor therapy.

83. 83. The method of claim 82, wherein the subject has acquired resistance to therapy with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor.

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