Humanized and affinity matured anti-CEACAM1 antibody

Abstract

Provided herein are recombinant antibodies and antigen-binding fragments thereof that can be used to bind and inhibit carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Also provided are methods of reducing T cell tolerance and treating cancer using the disclosed CEACAM1 antibodies and antigen-binding fragments thereof.

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 for treating cancer and infectious diseases.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] 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 Art

[0004] Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the carcinoembryonic antigen (CEA) family of immunoglobulin (Ig)-like transmembrane glycoproteins. CEACAM family members are involved in cell-cell recognition and regulate cellular processes ranging from the shaping of tissue architecture and neovascularization to the regulation of insulin homeostasis and T-cell proliferation.

[0005] A variety of cellular activities have been attributed to the CEACAM1 protein, including roles in differentiation and arrangement of tissue three-dimensional architecture, angiogenesis, apoptosis, tumor suppression, metastasis, and regulation of innate and adaptive immune responses. In addition, several cell types express CEACAM1, including tumor cells, T cells, natural killer (NK) cells, and certain macrophages.

[0006] For example, high CEACAM1 expression occurs in a variety of cancers, such as melanoma, colorectal cancer, gastric cancer, pancreatic cancer, bladder cancer and thyroid cancer, and is associated with poor tumor progression, metastasis and poor clinical prognosis. For example, non-small cell lung cancer (NSCLC) with high CEACAM1 expression exhibits high microvessel density, distal metastasis and shorter median overall survival and progression-free survival. CEACAM1 expression is also closely related to distal metastasis of pancreatic cancer. CEACAM1 expression on tumors promotes CEACAM1-mediated inhibition of T and NK cells. Therefore, inhibiting CEACAM1 activity can inhibit tumor cell metastasis and the formation of cancer stem cell niches.

[0007] CEACAM1 is also expressed in certain immune system cells and plays a role in immunosuppression and immune cell exhaustion. For example, high CEACAM1 expression on tumor infiltrating lymphocytes (TILs) and other tumor infiltrating immune cells from gastric cancer, lung cancer, melanoma, colorectal cancer and glioma is associated with poor prognosis. On T cells, CEACAM1 expression is mostly excluded from resting (primary) T cells, while the protein is expressed at high levels on activated T cells. CEACAM1-L is the main isoform expressed in most T cells, and acts as an inhibitory receptor that downregulates T cell activation and inhibits T cell function. Therefore, CEACAM1 inhibition of T cells can restore T cell activity and increase anti-tumor response.

[0008] CEACAM1 is also expressed on NK cells, which are lymphocytes involved in innate immunity, which are involved in early control of viral infections and immune surveillance of tumors. When NK cells encounter cells expressing class I major histocompatibility complex (MHC), inhibitory signals through receptor-ligand interactions prevent immune responses against these cells. However, when encountering cells in which class I MHC is downregulated, such as in virally infected cells or cancer cells, NK cells are activated due to the lack of inhibitory signals, which makes "sick" cells susceptible to NK cell-mediated killing. When CEACAM1 is present on the surface of NK and melanoma cells, CEACAM1:CEACAM1 interactions lead to inhibition of NK-mediated killing, regardless of class I MHC expression. Therefore, the destruction of this homophilic CEACAM1 interaction can be beneficial to restore NK-mediated immune responses.

[0009] CEACAM1 expression on macrophage subsets is also 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 also inhibit fibrosis and angiogenesis.

[0010] CEACAM1 also mediates cell-cell adhesion through the extracellular portion of CEACAM1 containing an IgV-like N domain, which 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 opacifying protein (OPA), Moraxella sp. Opa-like protein OlpA, Haemophilus influenzae outer membrane protein (OMP) P1, Haemophilus aegyptius OMP P1, Candida albicans, and influenza viruses such as H5N1). TIM-3 was identified as a Th1-specific cell surface protein expressed on activated T cells, subsets of dendritic cells and macrophages, and NK cells. TIM-3 is an activation-induced inhibitory molecule that is involved in tolerance and has been shown to induce T cell exhaustion in chronic viral infections and cancer. CEACAM1 is also expressed on activated T cells and has been shown to interact with TIM-3, and this interaction is important for TIM-3-mediated T cell inhibition.

[0011] As described above, CEACAM1 also serves as a cellular receptor on the apical membrane of mucosal cells for various Gram-negative bacterial pathogens associated with human mucosa as well as with fungal pathogens such as Candida albicans. For example, Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus aegypti, and pathogenic Escherichia coli strains have well-characterized CEACAM1 binding adhesins. Engagement of CEACAM1 with bacterial adhesins triggers bacterial endocytosis into epithelial cells and microbial transcytosis transport through intact epithelial layers, thus allowing microorganisms to utilize CEACAM1 during mucosal colonization. In addition, CEACAM1 is associated with infection with influenza virus H5N1 and sub-nematodes such as Wucheria bancrofti. Summary of the invention

[0012] 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, and methods of using these antibodies. By blocking the interaction of CEACAM1 with one or more binding partners, the antibodies and antigen-binding fragments thereof can be used to reduce, inhibit and / or reverse T cell tolerance and / or to enhance T cell expansion. CEACAM1 antibodies and antigen-binding fragments thereof can also be used to treat cancer, reduce tumor growth, reduce tumor metastasis and / or reduce cancer stemness in subjects in need. CEACAM1 antibodies and antigen-binding fragments thereof can also be used to treat patients resistant to checkpoint therapy. Also provided are methods of using CEACAM1 antibodies and antigen-binding fragments thereof to reduce the colonization of mammalian epithelium by bacteria or Candida albicans expressing bacterial adhesins or to reduce the replication of influenza viruses or the release of proinflammatory cytokines or chemokines associated with influenza virus infection.

[0013] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein:

[0014] The CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X1HX2X3S (SEQ ID NO: 1);

[0015] Where X1 is A, D, N or S;

[0016] wherein X2 is A or G; and

[0017] wherein X3 is an amino acid having a hydrophobic side chain, including I or M;

[0018] The CDR2 sequence of the heavy chain variable region (CDR2H) comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0019] The CDR3 sequence of the heavy chain variable region (CDR3H) comprises the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO: 3);

[0020] Where X4 is D, G or P;

[0021] Where X5 is F or P;

[0022] Where X6 is D or F;

[0023] wherein X7 is A or Y; and

[0024] Where X8 is L, H or F;

[0025] The CDR1 sequence of the light chain variable region (CDR1L) comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0026] The CDR2 sequence of the light chain variable region (CDR2L) comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0027] The CDR3 sequence of the light chain variable region (CDR3L) comprises the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO: 6);

[0028] Where X9 is W or N;

[0029] Where X 10 is S or T;

[0030] Where X 11 is A or an amino acid with a neutral hydrophilic side chain including S, N and T;

[0031] Where X 12 is L, F or N; and

[0032] Where X 13 It is P or F.

[0033] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein:

[0034] The sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO: 43);

[0035] Wherein X is any amino acid;

[0036] Where X1 is A, D, N or S;

[0037] wherein X2 is A or G; and

[0038] wherein X3 is an amino acid having a hydrophobic side chain, including I or M; and

[0039] The CDR2 sequence of the heavy chain variable region (CDR2H) comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0040] The sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44);

[0041] Where X4 is D, G or P;

[0042] Where X5 is F or P;

[0043] wherein X7 is A or Y; and

[0044] Where X8 is L, H or F;

[0045] The CDR1 sequence of the light chain variable region (CDR1L) comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0046] The CDR2 sequence of the light chain variable region (CDR2L) comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0047] The CDR3 sequence of the light chain variable region (CDR3L) comprises the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO: 6);

[0048] Where X9 is W or N;

[0049] Where X 10 is S or T;

[0050] Where X 11 is A or an amino acid with a neutral hydrophilic side chain including S, N and T;

[0051] Where X 12 is L, F or N; and

[0052] Where X 13 It is P or F.

[0053] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein:

[0054] The sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO: 1);

[0055] Where X1 is A, D, N or S;

[0056] wherein X2 is A or G; and

[0057] wherein X3 is an amino acid having a hydrophobic side chain, including I or M;

[0058] The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0059] The sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO: 7);

[0060] Where X4 is D, G or P;

[0061] Where X5 is F or P; and

[0062] Where X8 is L, H or F;

[0063] The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0064] The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0065] The sequence of CDR3L contains the sequence QQX9SSX 12 PX 13 T (SEQ ID NO: 8);

[0066] Where X9 is W or N;

[0067] Where X 12 is L, F or N; and

[0068] Where X 13 It is P or F.

[0069] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein

[0070] The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO: 9);

[0071] The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0072] The sequence of CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO: 10);

[0073] The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0074] The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0075] The sequence of CDR3L comprises the sequence QQWSSNPPT (SEQ ID NO: 11).

[0076] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein

[0077] The sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO: 9);

[0078] The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0079] The sequence of CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO: 10);

[0080] The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0081] The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0082] The sequence of CDR3L comprises the sequence QQWTSNPPT (SEQ ID NO: 12).

[0083] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises 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 heavy chain variable region amino acid sequence of SEQ ID NO: 13, and wherein 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.

[0084] In one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 13, and wherein the sequence of the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0085] In another embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 13, and wherein the sequence of the light chain variable region comprises SEQ ID NO: 14.

[0086] In another embodiment, the present invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the sequence of the heavy chain variable region comprises SEQ ID NO:13, and wherein the sequence of the light chain variable region comprises SEQ ID NO:15.

[0087] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region;

[0088] wherein 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;

[0089] wherein 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;

[0090] wherein the sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO: 43);

[0091] Wherein X is any amino acid;

[0092] Where X1 is A, D, N or S;

[0093] wherein X2 is A or G; and

[0094] wherein X3 is an amino acid having a hydrophobic side chain, including I or M; and

[0095] wherein the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44);

[0096] Where X4 is D, G or P;

[0097] Where X5 is F or P;

[0098] wherein X7 is A or Y; and

[0099] Where X8 is L, H or F.

[0100] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region,

[0101] wherein 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,

[0102] wherein 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,

[0103] wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3; and

[0104] in:

[0105] The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13,

[0106] The sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO: 13,

[0107] The sequence of CDR1L comprises residues A28, S30 and Y31 of SEQ ID NO: 14,

[0108] The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO: 14, and

[0109] The sequence of CDR3L comprises residues S91 and S92 of SEQ ID NO:14.

[0110] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention is a chimeric antibody, a CDR-grafted antibody or a humanized antibody or an antigen-binding fragment thereof.

[0111] 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 comprising a complementary region that binds to PD-1 or PD-L1.

[0112] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention is scFv, Fv, Fab', Fab, F(ab')2 or diabody.

[0113] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention has the isotype IgG4.

[0114] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention comprises a S241P substitution in the heavy chain constant region.

[0115] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention is aglycosylated.

[0116] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention lacks the C-terminal lysine in the heavy chain.

[0117] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention is conjugated to one or more of a cytotoxin, a fluorescent marker and / or an imaging agent.

[0118] In another aspect, the present invention provides CEACAM1 antibodies and antigen-binding fragments thereof, characterized in that the epitopes on CEACAM1 to which they bind. As described, such antibodies include, but are not limited to, the CEACAM1 antibodies and antigen-binding fragments described herein by their structural features, including CDR motifs, CDR sequences, and heavy variable chains and light variable chain sequences. In some embodiments, the present invention provides CEACAM1 antibodies and antigen-binding fragments thereof that bind to residues in the IgV-like N domain of CEACAM1. In another embodiment, the antibodies and antigen-binding fragments thereof provided herein also selectively bind to CEACAM1 compared to one or more CEACAM family members. In one embodiment, the CEACAM1 antibody or its antigen-binding fragment does not exhibit significant binding to other CEACAM family members including CEACAM3, CEACAM5, CEACAM6 and / or CEACAM8. In some embodiments, the present invention provides CEACAM1 antibodies and antigen-binding fragments that bind to an epitope on the N domain of CEACAM1 that overlaps or at least partially overlaps with the CEACAM1:CEACAM1 dimer interface, thereby blocking CEACAM1 homophilic interactions. In some embodiments, the present invention provides CEACAM1 antibodies and antigen-binding fragments that bind to CEACAM1 residues that are located in the binding site of a heterologous interaction partner on CEACAM1, including but not limited to other CEACAM family members, TIM family members, bacterial adhesins (such as HopQ, OPA, OMP P1 and / or OlpA), Candida albicans, influenza viruses (such as H5N1), and / or sub-nematodes such as Wuchereria bancrofti.

[0119] In one embodiment, the contemplated CEACAM1 antibody or antigen-binding fragment thereof binds to the same epitope as an antibody or antigen-binding fragment having a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 13, and wherein the sequence of the light chain variable region comprises SEQ ID NO: 14.

[0120] In one aspect, the contemplated CEACAM1 antibodies or antigen-binding fragments thereof bind to the IgV-like N domain of CEACAM1 and bind 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.

[0121] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof binds to the IgV-like N domain of CEACAM1.

[0122] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof does not bind to one or more of CEACAM3, CEACAM5, CEACAM6, and CEACAM8.

[0123] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof at least partially binds to the binding site of TIM3 on CEACAM1.

[0124] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof at least partially binds to the binding site of CEACAM1 on CEACAM1 during homodimerization.

[0125] In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM and partially or completely binds to a bacterial adhesin binding site on CEACAM1, including but not limited to Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae Opa, Neisseria meningitidis Opa, Haemophilus influenzae OMP P1, Haemophilus aegypti OMPP1, and / or Moraxella 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.

[0126] 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 Y34, G41, N42, Q44, Q89, S93, D94, V96 and N97 of SEQ ID NO: 17. In one embodiment, the epitope further comprises residues F29, S32, D40, A49, T56, I91, L95 and E99 of SEQ ID NO: 17.

[0127] In one embodiment, the present invention provides nucleic acid molecules encoding CEACAM1 antibodies or antigen-binding fragments thereof described herein, and vectors comprising such nucleic acid molecules. Cells comprising vectors encoding CEACAM1 antibodies or antigen-binding fragments thereof described herein, and cells expressing CEACAM1 antibodies or antigen-binding fragments thereof described herein are also provided. Chimeric antigen receptor T cells comprising the CDRs of any of the antibodies or antigen-binding fragments disclosed herein are also provided herein.

[0128] In one embodiment, the invention provides a composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable excipient.

[0129] In one embodiment, the present invention provides a method for inhibiting the binding of CEACAM1 to its interaction partner and / or reducing the activity of CEACAM1 using the CEACAM1 antibody or antigen-binding fragment thereof described herein, the method comprising contacting CEACAM1 with the CEACAM1 antibody or antigen-binding fragment thereof described herein. For example, embodiments of the present invention can be used to inhibit the interaction between CEACAM1 and a CEACAM family member. In one embodiment, the CEACAM family member is CEACAM3, CEACAM5, CEACAM6 or CEACAM8. In some embodiments, the CEACAM family member is CEACAM1 itself.

[0130] In one embodiment, the present invention provides a method of inhibiting the binding of CEACAM1 to a TIM family member using a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the TIM family member is TIM-3.

[0131] In one embodiment, the present invention provides a method for inhibiting the binding of CEACAM1 to a bacterial adhesin using a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae Opa, Neisseria meningitidis Opa, Haemophilus influenzae OMP P1, Haemophilus aegypti OMPP1, or Moraxella adhesin OlpA. In one embodiment, the present invention provides a method for inhibiting the binding of CEACAM1 to Candida albicans using a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or an antigen-binding fragment thereof as described herein. In one embodiment, the present invention provides a method for inhibiting the binding of CEACAM1 to influenza virus using a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or an antigen-binding fragment thereof as described herein. In one embodiment, the influenza virus is H5N1.

[0132] In one embodiment, the present invention provides a method for reducing the colonization of mammalian epithelium by bacteria expressing bacterial adhesins using the CEACAM1 antibodies or antigen-binding fragments thereof described herein, the method comprising contacting CEACAM1 with the CEACAM1 antibodies or antigen-binding fragments thereof described herein. In some embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria meningitidis Opa, Haemophilus influenzae OMP P1, Haemophilus aegypti OMP P1, or Moraxella adhesin OlpA.

[0133] In one embodiment, the invention provides a method of reducing colonization of mammalian epithelium by Candida albicans using the CEACAM1 antibody or antigen-binding fragment thereof described herein, the method comprising contacting CEACAM1 with the CEACAM1 antibody or antigen-binding fragment thereof described herein.

[0134] In one embodiment, the invention provides a method for reducing the replication of influenza virus, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen binding fragment thereof as described herein. In one embodiment, the 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 as described herein. In some embodiments, the influenza virus is H5N1.

[0135] In one embodiment, the present invention provides methods for reducing T cell tolerance and / or enhancing T cell expansion or activation using the CEACAM1 antibodies or antigen-binding fragments thereof described herein. These methods can be used for in vitro and in vivo applications.

[0136] In one embodiment, the present invention provides a method of reducing T cell tolerance and / or enhancing T cell expansion in a subject in need thereof using a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof. In one embodiment, the present invention provides a method of treating cancer in a subject in need thereof using a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof. In some embodiments, the cancer is a glioma, a glioblastoma, a thymoma, a mesothelioma, a sarcoma, a uterine carcinosarcoma, a chromophobe renal cell carcinoma, adenoid cystic carcinoma, an acute myeloid leukemia, a melanoma, a uveal melanoma, a papillary renal cell carcinoma, a clear cell renal cell carcinoma, a bile duct carcinoma, a lung adenocarcinoma, a diffuse large B-cell lymphoma, a pheochromocytoma and a paraganglioma, a pancreatic cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a squamous cell carcinoma, a breast cancer, a prostate cancer, a bladder cancer, a gastric cancer, a testicular germ cell carcinoma, an ovarian cancer, a head and neck cancer, a uterine cancer, a cervical cancer or a liver cancer. In embodiments, the present invention provides a method of reducing tumor growth, reducing tumor metastasis, reducing tumor-associated fibrosis and / or reducing cancer stemness in a subject in need thereof by administering an effective amount of the antibody or antigen-binding fragment thereof to the subject. In some embodiments, the present 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 present invention provides a method that also includes administering one or more of an inhibitor of LAG3, TIGIT, LAP, Podoplanin, protein C receptor, ICOS, GITR, CD226 or CD160. In some embodiments, the present invention provides a method that also includes administering a TIM-3 inhibitor. In some embodiments, the inhibitor is administered simultaneously or continuously with an antibody or antigen-binding fragment. In some embodiments, the inhibitor is administered alone or as a mixture with an antibody or antigen-binding fragment.

[0137] In one embodiment, the present invention provides a method of reducing the colonization of the epithelium of a subject by bacteria expressing a bacterial adhesin in a subject in need thereof using a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or an antigen-binding fragment thereof as described herein. In some embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria meningitidis Opa, Haemophilus influenzae OMPP1, Haemophilus aegypti OMP P1, or Moraxella OlpA.

[0138] In one embodiment, the present invention provides a method of reducing epithelial colonization of a subject by Candida albicans in a subject in need thereof using a CEACAM1 antibody or antigen-binding fragment thereof described herein, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or antigen-binding fragment thereof described herein.

[0139] In one embodiment, the present invention provides a method of reducing the replication of influenza virus in a subject in need thereof using a CEACAM1 antibody or antigen binding fragment thereof as described herein, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or antigen binding fragment thereof as described herein. In one embodiment, the present invention provides a method of reducing the release of proinflammatory cytokines or chemokines associated with influenza virus infection in a subject in need thereof using a CEACAM1 antibody or antigen binding fragment thereof as described herein, the method comprising administering to the subject an effective amount of a CEACAM1 antibody or antigen binding fragment thereof as described herein. In some embodiments, the influenza virus is H5N1.

[0140] In one embodiment, the present invention provides a method for treating a subject who does not respond to a therapy of a checkpoint inhibitor therapy (primary resistance) using a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, and a patient who initially responds to treatment but later becomes resistant to checkpoint inhibitor blockade (secondary or acquired resistance). Such treatment methods include administering a CEACAM1 antibody or an antigen-binding fragment thereof as described herein to the subject. 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 may also be referred to as refractory cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0142] Figure 1The plasmid maps of the light chain expression vector pANTVκ and the heavy chain expression vector pANTVhG4(S241P) are shown. H Both Vκ and Vκ vectors contain a genomic DNA fragment with introns and poly A sequences incorporated. Expression of both chains is driven by the CMV promoter.

[0143] Figure 2 The selectivity of the CEACAM1 antibody variants is shown. Humanized variant intermediates were examined by flow cytometry on HeLa cells transfected with CEACAM1, 3, 5, 6 and 8. The proportion of cells that were positive based on irrelevant hIgG4 staining is shown. There was no evidence of any staining of HeLa-CEACAM3 or HeLa-CEACAM8 transfectants, so these data are not reported.

[0144] Figure 3A , 3B and 3C shows the heavy variable chain V H 1( Figure 3A )、Heavy variable chain CP08H03( Figure 3B ) and light variable chain Vκ8S29A ( Figure 3C ). The CDRs are shaded. The CDR residue numbers according to Kabat and according to the primary amino acid sequence are shown.

[0145] Figure 4 Chimeric CEACAM1 antibody V is shown H 0 / Vκ0 is glycosylated in CDR1L. The introduction of mutations N26Q and S29A abolished this glycosylation. The proteins were separated on SDS-PAGE under denaturing conditions. The molecular weights of the heavy chain, glycosylated light chain, and non-glycosylated light chain are shown. Residues N26 and S29 are numbered using the Kabat numbering scheme.

[0146] Figure 5 The plasmid map of the phagemid expression vector pANT43 is shown. H The Vκ domains are connected by a flexible glycine-serine (G4S) linker and fused in-frame to the M13 gene III bacteriophage coat protein. The expression of the single-chain variable fragment (scFv) is driven by the Lac promoter.

[0147] Figure 6 The binding of phage to CEACAM1 antigen is shown. H Phage prepared with 1 / Vκ8S29A scFv or irrelevant scFv were 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.

[0148] Figure 7 An overview of affinity maturation library design is provided. The CDRs (as defined by Kabat) are shown in bold, and the targeted positions are indicated with an X. A single position may contain all 20 amino acids or a subset thereof.

[0149] Fig. 8A , 8B An overview of library construction methods for generating randomized phage libraries is provided in and 8C. Fig. 8A ), heavy chain CDR1 library ( Figure 8B ) and heavy chain CDR3 library ( Figure 8C ).

[0150] Fig. 9A and 9B An overview of two different selection activities used during affinity maturation of CEACAM1 antibodies is provided. Fig. 9A Selection campaign 1: Solid phase panning of library phages CEACAM5 / CEACAM6 was deselected and prior to round 2, multiple rounds of selection were performed against decreasing concentrations of biotinylated soluble CEACAM1. Fig. 9B : Selection campaign 2: Panning selection with CEACAM1 was performed in round 1, followed by 2 rounds of panning CEACAM5 / CEACAM6 deselection and selection with decreasing concentrations of biotinylated soluble CEACAM1.

[0151] Fig.10 An example of a scFv binding ELISA assay is shown. Serial dilutions of purified parental scFv V H 1 / Vκ8S29A or affinity matured scFv variants were added to plates coated with GST-CEACAM1. Binding was detected using anti-HIS6-HRP antibody and TMB. All variable light chains contained an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0152] Fig.11The binding selectivity of different affinity-matured CEACAM1 antibodies is shown. Affinity-matured antibodies CP08H03 / Vк8S29A (labeled "CP08_H03 / parent VL"), CP08H03 / CP08F05, 8H3_9B3 / CP08F05, and CP08H03 / CP08E05 contain a phenylalanine (F) at CDR3H residue 104. Affinity-matured antibodies CP09B03 / CP08E05, CP09C02 / CP08E05, CP09C02 / CP08F05, and 9B3_9E5 / CP08E05 contain an aspartic acid residue (D) at CDR3H residue 104. HELA cells were transfected with vectors alone (HeLa-Neo) or vectors expressing CEACAM1, CEACAM3, CEACAM5, or CEACAM6, and stained with the indicated antibodies. The y-axis shows the % staining of the transfected cell groups for each antibody. hIgG4 = control antibody with the same stabilizing hinge mutation. MOPC = mouse IgG1 control antibody. Mouse antibodies as positive controls for transfected CEACAM isoforms: Col-1 = CEACAM3 and CEACAM5 antibodies. 9A6 = CEACAM6 antibody. T84.1 = CEACAM cross-reactive antibody and T84.66 = CEACAM5 antibody. 2nd FITC only = no primary antibody, only secondary FITC conjugated antibody. Col-1 and 9A6 are commercially available antibodies (Dako), and T84.1 and T84.66 have been described previously (Neumaier M, J Immunol 1985; 135: 3604-9). The data for affinity matured antibodies 9B3_8H3 / Vк8S29A, 8H3_9B3 / CP08_E05 and 8H3_9C2 / CPO08_F05, as well as the data for CEACAM8 antibody 80H3 were omitted from the figure due to abnormally high background signals.

[0153] Fig. 12A , 12B and 12C show the CEACAM antibodies CP08H03 / Vκ8S29A (labeled "CP08_H03 / parental VL"), CP08H03 / CP08_F05 and V H 0 / Vк0 is selective for CEACAM 1. CP08H03 / Vκ8S29A 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). Fig. 12ASingle cycle kinetic sensorgrams and fitted curves of purified lead humanized and affinity matured IgG4 variants are shown.Increasing concentrations of different CEACAM family members were injected and individual off-rates were determined by single cycle kinetics (surface plasmon resonance, SPR). Fig. 12B The chimeric antibody V H Three-point binding ELISA data for binding of the purified lead humanized and affinity-matured IgG4 variants to CEACAM1 and CEACAM3 family members. A three-point (high, medium and low, concentrations based on the chimeric antibody V H Binding of κB / Vκ0 to CEACAM1 was titrated and binding was detected using an anti-human κ chain antibody and TMB substrate. Fig. 12C The chimeric antibody V H Three-point binding ELISA data for binding of IgG4 variants of the chimeric antibody Vκ0 (labeled "chimeric") and purified lead humanized and affinity matured IgG4 variants to CEACAM1, 5, and 6 family members. A three-point (high, medium, and low, concentrations based on the chimeric antibody Vκ0) was performed. H Binding of κB / Vκ0 to CEACAM-1 was titrated and binding was detected using an anti-human κ chain antibody and TMB substrate.

[0154] Fig.13 The 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 created using Clustal 2.1. The specific residues analyzed for each CEACAM family member are shown.

[0155] Fig.14 Results of CEACAM1 mutagenesis studies designed to identify residues in CEACAM1 involved in binding to the indicated CEACAM1 antibodies are shown. CEACAM1-FLAG was expressed in human embryonic kidney (HEK) cells transfected with CEACAM1 containing the indicated mutations (Y34C, V39A, G41A, N42A, R43A, Q44L, G47A, and Q89H), and proteins were resolved by SDS-PAGE followed by immunoblotting.H 0 / Vκ0) and humanized CEACAM antibodies detect wild-type (WT) or mutant CEACAM1 proteins. A decrease in detection indicates that the mutated residue is involved in binding to the corresponding antibody used for detection.

[0156] Fig.15 The structure of the CEACAM1:CP08H03 / Vκ8S29A Fab complex is shown. In the structure of the complex, the Fab is shown as a Ca trace and the antigen is shown as a ribbon.

[0157] Fig.16 A view of the antigen (CEACAM1) towards the dimer interface is shown. CEACAM1 residues that interact with the CP08H03 / Vκ8S29A Fab light chain including D40, N42, L95, V96, N97 and E99 are labeled (see Fig.15 ) and CEACAM1 residues that interact with the CP08H03 / Vκ8S29A Fab heavy chain including F29, S32, Y34, Q44, T56, Q89 and I91. The relevant side chains are drawn as sticks.

[0158] Fig.17 The crystal structure showing the CEACAM1:CEACAM1 homodimer interface (PDB ID: 4QXW) is shown. One CEACAM1 monomer is shown on the left and the other on the right. Residues Y34, Q44, Q89 and N97 form the YQQN pocket.

[0159] Fig.18 Close-up stereo images of the CP08H03 / Vκ8S29A Fab-CEACAM1 interaction are shown. CEACAM1 is drawn as a ribbon and the Fab chains (light and heavy chains) are drawn as Cα traces. Regions of the Fab light chain residues (including S30, Y31, Y48, L49, S51, N52, W90, S91 and N93) and heavy chain residues S52, S53, T56, Y57, Y59, D102, Y103, F104, P105, Y106 that interact with CEACAM1 residues (including F29, S32, Y34, D40, N42, Q44, A49, T56, Q89, I91, L95, V96, N97 and E99) are marked. Side chains of interest are drawn as sticks and hydrogen bonds are drawn as black dashed lines. The residue numbering is based on the primary amino acid sequence of the antibody and CEACAM1.

[0160] Fig.19Comparison of CEACAM1 F29 and V49 or A49 residues in the CEACAM1 WT:CP08H03 / Vκ8S29A antibody crystal structure (left) or the CEACAM1 A49V / Q89H mutant crystal structure (right) is shown.

[0161] Fig. 20A and 20B Materials CEACAM1 antibody CP08H03 / Vκ8S29A (labeled as "CP08") blocks human CEACAM1: CEACAM1 ( Fig. 20A ) and CEACAM:human TIM-3 interaction ( Fig. 20B ). IgG4 = control antibody.

[0162] Fig.21 Figure 2 shows the results of a study to test the ability of CEACAM1 antibodies to induce CD45 in humanized non-obese diabetic (NOD) scidγ mice (NSG mice). + Experimental setup for the ability of cell proliferation. At 38 days after injection, the transplantation of human peripheral blood mononuclear cells (PBMCs) adoptively transferred to NSG host mice by intraperitoneal injection was analyzed by fluorescence activated cell sorter (FACS) for human CD45 and proliferation dye staining. At day 24 after PBMC injection, mice were treated with a single injection of human IgG4 isotype control or CP08H03 / Vκ8S29A (labeled "CP08_H03 / parent VL") or CP08H03 / CP08F05 antibodies at the indicated concentrations. At day 31, mice were treated with a second injection. At day 38, mice were sacrificed for data collection.

[0163] Fig. 22 It is shown that the CEACAM1 antibodies CP08H03 / Vκ8S29A (labeled "CP08_H03 / Parental") and CP08H03 / CP08F05 do not deplete transplanted human cells in humanized NSG mice. The mean percentage of human CD4 and CD8 T lymphocytes was assessed at day 38. CP08H03 / CP08F05 contains an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).

[0164] Fig.23 The results show that administration of CEACAM1 antibody CP08H03 / Vκ8S29A (labeled as "CP08_H03 / Parental VL") or CP08H03 / CP08F05, respectively, resulted in antibody-induced human CD45 in humanized NSG mice. +Increase of immune cell proliferation. CP08H03 / Vκ8S29A induces the expansion of human CD45 PBMC in vivo. On the 38th day, mice treated with isotype hIgG4 control (10 mg / kg), CP08H03 / Vκ8S29A (2 and 10 mg / kg) and CP08H03 / CP08F05 (2 and 10 mg / kg) were sacrificed, and spleen cells were separated and collected for proliferation analysis. In vitro proliferation was performed under T cell stimulation conditions, where cells were cultured for 120 hours under soluble anti-CD3 (OKT3) (specified concentrations of 10, 5, 2.5 μg / ml) and rIL-2 (40 units / ml). The dilution of the proliferation dye represents dividing cells / proliferation (when cells proliferate, double-stranded DNA is analyzed as a dilution signal). 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).

[0165] Fig.24A , 24B and 24C show that the CEACAM1 antibody CP08H03 / Vκ8S29A (labeled "CP08_H03 / Parental VL") reduces tumor growth in humanized mice. Fig.24A Provides the generation Fig. 24B Schematic diagram of the experimental scheme. Fig. 24B shows that the 1x10 6 MALME-3M (human melanoma) cells and 5×10 6 Mean tumor size after subcutaneous injection of individual PBMCs into NSG. After 10 days, palpable tumors were recorded and mice were randomized to be treated intraperitoneally with the corresponding antibody concentrations on days 10, 13, 17, 20 and 24. Fig.24C Shown is a statistical comparison of the hIgG4 control treatment group with three different CP08H03 / Vκ8S29A groups (2 mg / kg, 0.4 mg / kg and 0.08 mg / kg) by linear regression.

[0166] Fig.25It is shown that T cells from humanized mice transplanted with the human melanoma cell line MALME-3M and treated with the CEACAM1 antibody CP08H03 / Vκ8S29A (labeled "CP08_H03 / parental VL") as described in Figure 24 show a decrease in the number of tumor cells and a decrease in the proliferation and increase in the number of CD8 and CD4 positive T cells within the tumor. When examined ex vivo after anti-CD3 stimulation, these T cells showed increased proliferation. On the day of sacrifice, humanized NSG mice treated with isotype hIgG4 control (2 mg / kg), CP08H03 / Vκ8S29A (labeled "CP08_H03 / parental VL", 0.08 and 2 mg / kg) bearing melanoma tumors were sacrificed. Tumor cells and CD4 + and CD8 + Tumor infiltrating lymphocytes were used for proliferation analysis. Tumor cells were identified as FSC Hi SSC Hi Cells were negative for human CD45 and proliferation was quantified by assessing the dilution of a commercial dye (Becton-Dickinson) for proliferation. + CD4 + and CD45 + CD8 + T cells. Ex vivo T cell proliferation measurements were performed under T cell stimulation conditions, where cells were cultured for 6 days in the presence of soluble anti-CD3 (2 g / ml) and rIL-2 (40 units / ml).

[0167] Fig.26 Figure 24-25 shows the phenotypic changes of intratumoral memory CD8 T cells when CEACAM1 was blocked with the CEACAM1 antibody CP08H03 / Vκ8S29A (labeled as "CP08_H03 / parental VL") as described in Figures 24-25. Tumor-infiltrating CD3 T cells from humanized NSG mice bearing melanoma were analyzed using CD62L and CD44 cell markers. + CD8 + Flow cytometric analysis of T cell populations was performed to characterize central memory (CD62L + CD44 + ) and effector memory (CD62L - CD44 + CD3 + CD8 + ) T cell population. Treatment conditions were isotype hIgG4 control (2 mg / kg) and CP08H03 / Vκ8S29A (0.08, 0.4 and 2 mg / kg).

[0168] Fig. 27Figure 2 shows the expression of CEACAM1 in primary CD4 TILs from naive (left) and PD-1 and / or CTLA-4 resistant (right) melanoma patients. + (above) and CD8 + T (bottom) expression on T cells. Similar characterization of PD1 and TIM-3 expression is also shown.

[0169] Fig.28 It was shown that tumor-associated cells (TACs) from patients with acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy exhibited significantly higher CEACAM1 expression than TACs from patients not previously exposed to anti-PD-1 and / or anti-CTLA-4 therapy. TACs were obtained from patients with naive melanoma (not previously exposed to anti-PD-1 and / or anti-CTLA-4 therapy) or those melanoma patients who acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy (acquired resistance). TACs were obtained by culturing tumor tissue in DMEM medium, and floating cells were removed from the supernatant and analyzed. Cells were stained for CD3, CD4, and CD8, and CD3 was evaluated. + CD4 + Cells and CD3 + CD8 + CEACAM1 expression on cells. *, p=0.05; **, p<0.01.

[0170] Fig.29 It is shown that compared with CD8 + CD8 T cells isolated from patients resistant to anti-PD-1 and / or anti-CTLA-4 therapy were compared with those from patients with + Central memory in T cells (T cm ) relative to the effect memory (T em Tumor-associated cells from naive patients and patients with acquired resistance were subjected to central memory (CCR7 + CD62L + ) and effector memory (CCR7 - CD62L - ) marker staining.

[0171] Fig.30The CEACAM1 antibody CP08H03 / Vκ8S29A (labeled "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 CD8 + and CD4 + The proportion of T cells expressing these markers is shown (left). PBMCs or tumor-associated cells ("tumors") cultured with soluble anti-CD3 (2 g / ml) and rIL-2 (40 units / ml) in the presence of CP08H03 / Vκ8S29A or hIgG4 control antibodies are shown in the right figure. The release of IFNγ and TNFα (a measure for reversal of T cell tolerance) was determined by ELISA.

[0172] Fig.31A and 31B Shown are flow cytometric analyses of stable HeLa CEACAM1 (HeLa C1) transfectants, stable HeLa CEACAM3 transfectants (HeLa C3), stable HeLa CEACAM5 transfectants (HeLa C5), stable HeLa CEACAM6 transfectants (HeLa C6), and stable HeLa CEACAM8 transfectants (HeLa C8). Fig.31A : 5x10^4 of the indicated HeLa transfectants were washed with staining buffer and incubated with CP08H03 / Vκ8S29A (labeled "CP08", left) or CEACAM1 antibody CM-24 at room temperature for 30 minutes, washed twice with staining buffer, and stained with anti-human IgG4 fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 20 minutes at room temperature. Fluorescence intensity was determined by flow cytometry. Viable cells were determined by 4',6-diamidino-2-phenylindole (DAPI) staining, as shown on the y-axis. The corresponding CEACAM1 antibody staining is shown on the x-axis. For CP08H03 / Vκ8S29A, note that the positive signal in the gate is only shown in the HeLa CEACAM1 (C1) transfectant (left). In contrast, CM-24 (right) has no selectivity and cross-reacts with CEACAM1, CEACAM3, and CEACAM5. Fig.31B Shows Fig.31A Different representations of the data shown.

[0173] Fig.32A ,32B Figures 32 and 32C show that the CEACAM1 antibody CP08H03 / Vκ8S29A (labeled "CP08") is more effective than the CEACAM1 antibody CM-24 in reversing T cell tolerance in tumor-associated cells. Tumor-associated cells derived from primary Merkel cell carcinoma tumors were stained for CEACAM1, PD1, or TIM-3, and CD8 + and CD4 + The proportion of T cells ( Fig.32A and 32B ). Tumor-associated cells were incubated with soluble anti-CD3 (2 g / ml) and rIL-2 (40 units / ml) in the presence of CP08H03 / Vκ8S29A, CM-24, or hIgG4 control. IFN-γ release (a measure for reversal of T cell tolerance) was measured ( Fig.32C ). *Comparison of CP08 and hIgG4, P = 0.0138.

[0174] Fig.33A , 33B , 33C and 33D show that CM-24-treated metastatic melanomas in NSG mice exhibited decreased TILs and increased tumor cells compared to CP08H03 / Vκ8S29A (labeled "CP08")-treated metastatic melanomas. Fig.33A Shown is the experimental setup using a therapeutic tumor model in humanized NSG mice with human melanoma xenografts using four doses of 2 mg / kg of the respective antibody including a hIgG4 control containing the same stabilizing hinge mutations. Fig.33B The FSC / SCC high (FSC / SCC Hi ) and the pan-leukocyte marker human CD45 are characterized by the absence of tumor-infiltrating CD4 + T lymphocytes (grey), CD8 + Pie charts of the percentages of T lymphocytes (black) and tumor cells (white) (Left: control antibody. Middle: CEACAM1 antibody CP08H03 / Vκ8S29A. Right: CEACAM1 antibody CM-24). Fig.33C Tumor cell proliferation of IgG4 control, CP08H03 / Vκ8S29A, and CM-24 is shown, indicating that CP08H03 / Vκ8S29A, but not CM-24, inhibits tumor proliferation. Fig.33D Figure 2 shows the expression of splenic CD4 in CP08H03 / Vκ8S29A treated mice. + T cell proliferation was increased, and splenic CD4 + The proliferation of T cells is reduced.

[0175] Fig.34A, 34B 34C shows that the CEACAM1 antibody CM-24 is an agonist drug in a metastatic melanoma model. + T lymphocytes ( Fig.34A )、CD8 + T lymphocytes ( Fig.34B ) and tumor cells characterized by high forward / side scatter (FSC / SCC Hi) ( Fig.34C ) absolute cell counts. The values ​​obtained for each experimental mouse in each group are shown (for IgG4, n = 9; for CP08, n = 8; for CM-24, n = 6). *P < 0.05; **P < 0.001. Statistical analysis refers to the inclusion of Fig.33B Note that the number of TILs in mice treated with CP08H03 / Vk8S29A (labeled "CP08") was increased relative to that in mice treated with CM-24 ( Fig.34A and Fig.34B ) and the number of tumor cells decreased ( Fig.34C ). This data indicates that CP08H03 / Vk8 S29A is an antagonistic antibody and CM-24 is an agonistic antibody.

[0176] Fig.35A and 35B It is shown that the CEACAM1 antibody CP08H03 / Vκ8S29A covers the CEACAM1:HopQ binding interface and is expected to block CEACAM1:HopQ or CEACAM1:Opa protein interactions. Fig.35A The CEACAM1:HopQ binding interface based on analysis of three crystal structures (PDBID 6AW2, 6GBH and 6GBG) is shown. The CEACAM1 GFCC' face is formed by the interaction of the CEACAM1 CC' and FG loop' (see Huang et al., Nature. 2015 Jan 15; 517(7534): 386-90), participates in HopQ binding at CEACAM1 residues F29, Y34, N42, Q89 and N97, and generates various hydrogen bonds 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). Fig.35BShown is a superposition of the CP08H03 / Vκ8S29A:CEACAM1 crystal structure and the CEACAM1:HopQ crystal structure. The CP08H03 / Vκ8S29A antibody light and heavy chains are shown in surface illustration. The ribbon diagram shows the HopQ chain (three different crystal structures PDB ID 6AW2, 6GBH and 6GBG) and CEACAM1 from three different co-crystal structures with HopQ (PDB ID 6AW2, 6GBH, 6GBG), as well as CEACAM1 from a co-crystal structure with CP08H03 / Vκ8S29A to highlight the superposition of the CP08H03 / Vκ8S29A and HopQ binding epitopes.

[0177] Fig.36 It is shown that the CEACAM1 antibody CP08H03 / Vκ8S29A increases survival in tumor-bearing mice. NSG mice were injected with MALME-3M (human melanoma) cells and human PBMCs. Treatment was performed with the CEACAM1 antibody CP08H03 / Vκ8S29A or control human (h) IgG4 antibody on days 10, 13, 17, 20 and 24, respectively (see arrows). Shown is % survival. n=4 / group.

[0178] Fig.37A and 37B The CEACAM1 antibody CP08H03 / Vκ8S29A was shown to increase the expression of multiple factors involved in CD8 T cells derived from melanoma patients with secondary resistance to immunotherapy. + T cell immune response to cancer. Fig.37A A series of viSNE (Visually Distributed Stochastic Neighbor Embedding) plots presented in Cytobank using the Barnes-Hut implementation of the t-SNE algorithm are shown, depicting the expression of specific factors in CD8 as defined by mass cytometry on the left. + Intracellular expression in T cells. Quantification of heatmap levels of each indicated factor is shown to the right of the x-axis relative to the residues associated with each factor shown on the y-axis. Fig.37B Shows Fig.37A The indicated factors are depicted as fold changes in the intracellular responses in response to CP08H03 / Vκ8S29A relative to the hIgG4 control antibody set at 1.0.

[0179] Fig.38A and 38B The CEACAM1 antibody CP08H03 / Vκ8S29A was shown to re-enhance the ability of tumor-dissociated cells from two melanoma patients who had not been previously treated ( Fig.38B, Subject 189) or secondary resistance to immunotherapy ( Fig.38A , subject 185). In both cases, tumor samples were disrupted by mechanical dissociation (Miltenyi) and tumor dissociated cells were treated in vitro with only 2 μg / ml CP08H03 / Vκ8S29A or human IgG4 isotype control antibody. After 96 hours, significant levels of interferon-γ were detected in the supernatant of CP08H03 / Vκ8S29A but not in samples treated with human IgG4 isotype control antibody. *P<0.05 DETAILED DESCRIPTION

[0180] Antibody

[0181] 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.

[0182] As used herein, "antibody variable domain" refers to the portion of the light and heavy chains of an antibody molecule that includes 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 variable domain of the light chain. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat or according to Chothia. The term "framework region" (FR) refers to those variable domain residues other than the CDR residues.

[0183] As used herein, the term "complementarity determining region" (CDR) refers to the portion of an antibody variable domain that is (usually) involved in antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each CDR may comprise amino acid residues from a CDR as defined, for example, by Kabat (i.e., approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and approximately residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in 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 may also comprise amino acid residues from a "hypervariable loop" (i.e., approximately residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and approximately residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk 196 J. Mol. Biol. 901 (1987)). In some cases, CDR may include amino acids from CDR regions and hypervariable loops according to the Kabat definition. Kabat residue names do not always correspond directly to the linear numbering of amino acid residues (primary amino acid sequence). The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering, corresponding to shortening or insertion of structural components (whether framework or CDR) of the basic variable domain structure. For a given antibody or antigen-binding fragment thereof, the correct Kabat numbering of residues can be determined by aligning homologous residues in the sequence of the antibody or antigen-binding fragment thereof with the "standard" Kabat numbering sequence. An example of how Kabat numbering is related to the primary amino acid sequence of an antibody can be seen in Figure 3A , 3B and 3C. Alternatively, the CDRs may be defined according to the ImMunoGeneTics (IMGT) system (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)).

[0184] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises six CDRs, wherein:

[0185] (i) the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO: 9;

[0186] (ii) the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO: 2;

[0187] (iii) the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO: 10;

[0188] (iv) the CDR1 sequence of the light chain variable region comprises SEQ ID NO: 4;

[0189] (v) the CDR2 sequence of the light chain variable region comprises SEQ ID NO: 5; and

[0190] (vi) the CDR3 sequence of the light chain variable region comprises SEQ ID NO:11.

[0191] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises six CDRs, wherein:

[0192] (i) the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO: 9;

[0193] (ii) the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO: 2;

[0194] (iii) the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO: 10;

[0195] (iv) the CDR1 sequence of the light chain variable region comprises SEQ ID NO: 4;

[0196] (v) the CDR2 sequence of the light chain variable region comprises SEQ ID NO: 5; and

[0197] (vi) the CDR3 sequence of the light chain variable region comprises SEQ ID NO:12.

[0198] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises six CDRs, wherein:

[0199] (i) the CDR1 sequence of the heavy chain variable region comprises SEQ ID NO: 9

[0200] (ii) the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO: 2;

[0201] (iii) the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO: 10;

[0202] (iv) the CDR1 sequence of the light chain variable region comprises SEQ ID NO: 18;

[0203] (v) the CDR2 sequence of the light chain variable region comprises SEQ ID NO: 5; and

[0204] (vi) the CDR3 sequence of the light chain variable region comprises SEQ ID NO:11.

[0205] As shown in the Examples below, affinity maturation of CDR1H, CDR3H and CDR3L of a humanized, non-glycosylated CEACAM1 antibody yielded variants that conferred significantly improved binding affinity to CEACAM1. The obtained variants were examined and compared to the variability introduced into the affinity maturation library, indicating certain CDR positions at which the amino acid remained relatively unchanged and other CDR positions at which variation could be introduced, resulting in improved binding.

[0206] In one aspect, the present invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising CDR1H, wherein the CDR1H comprises residues 31-35 (Kabat definition, corresponding to, e.g., SEQ ID NO: 19 (see Figure 3A ) or SEQ ID NO: 13 (see Figure 3B ) and comprises the sequence X1HX2X3S (SEQ ID NO: 1),

[0207] wherein X1 of CDR1H is A, D, N or S;

[0208] wherein X2 of CDR1H is A or G; and

[0209] wherein X3 of CDR1H is an amino acid having a hydrophobic side chain including I or M.

[0210] Alternatively, the IMGT definition can be used to define CDR1H, wherein CDR1H comprises residues 26-33 of the CEACAM1 antibody (corresponding to, for example, SEQ ID NO: 19 (see Figure 3A ) or SEQ ID NO: 13 (see Figure 3B ) and comprises the sequence X14X15X16FX17X1HX2 (SEQ ID NO: 20),

[0211] Wherein X14 of CDR1H is G or E;

[0212] wherein X15 of CDR1H is an amino acid having an aromatic side chain including F or Y;

[0213] wherein X16 of CDR1H is T, S or I;

[0214] wherein X17 of CDR1H is an amino acid having a polar uncharged side chain including S, T or N;

[0215] wherein X1 of CDR1H is A, D, N or S; and

[0216] Wherein X2 of CDR1H is A or G.

[0217] In one embodiment, the CDR1H (Kabat definition) of the CEACAM1 antibody or antigen-binding fragment thereof comprises the sequence SHGMS (SEQ ID NO: 9).

[0218] In some embodiments, CDR1H (IMGT definition) comprises the sequence GFIFSHG (SEQ ID NO: 21).

[0219] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof comprising a CDR1H region, wherein the CDR1H comprises residues 26-35 (Kabat definition, corresponding to, e.g., SEQ ID NO: 19 (see Figure 3A ) or SEQ ID NO: 13 (see Figure 3B ) of the heavy variable chain primary amino acid sequence of residues 26 to 35), and comprising the sequence X 14 X 15 X 16 FX 17 X1HX2X3S (SEQ ID NO: 22),

[0220] Where X 14 is G or E;

[0221] Where X 15 is an amino acid having an aromatic side chain including F or Y;

[0222] Where X 16 is T, S or I;

[0223] Where X 17 is an amino acid having a polar uncharged side chain including S, T or N;

[0224] Where X1 is A, D, N or S;

[0225] wherein X2 is A or G; and

[0226] wherein X3 is an amino acid having a hydrophobic side chain, including I or M.

[0227] In one embodiment, the CDR1H region comprises the sequence GFIFSSHGMS (SEQ ID NO: 23).

[0228] In one aspect, the present invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising CDR3H, wherein the CDR3H comprises residues 95-102 (Kabat definition, corresponding to, e.g., SEQ ID NO: 19 (see Figure 3A ) or SEQ ID NO: 13 (see Figure 3B ) and comprises the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO: 3),

[0229] wherein X4 of CDR3H is D, G, or P;

[0230] wherein X5 of CDR3H is F or P;

[0231] wherein X6 of CDR3H is D or F;

[0232] wherein X7 of CDR3H is A or Y; and

[0233] wherein X8 of CDR3H is L, H or F.

[0234] In one embodiment, CDR3H comprises residues 95-102 (Kabat definition, corresponding to, e.g., SEQ ID NO: 19 (see Figure 3A ) or SEQ ID NO: 13 (see Figure 3B ) and comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO: 7),

[0235] wherein X4 of CDR3H is D, G, or P;

[0236] wherein X5 of CDR3H is F or P; and

[0237] wherein X8 of CDR3H is L, H or F.

[0238] In one embodiment, CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO: 10).

[0239] In one aspect, the present invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising a CDR3H region, wherein the CDR3H region comprises residues 94-102 (Kabat definition, corresponding to, e.g., SEQ ID NO: 19 (see Figure 3A ) or SEQ ID NO: 13 (see Figure 3B) and comprises the sequence X18HX4X5DYX6PX7WFAX8 (SEQ ID NO: 24),

[0240] Where X18 is R or K;

[0241] Where X4 is D, G or P;

[0242] Where X5 is F or P;

[0243] Where X6 is D or F;

[0244] wherein X7 is A or Y; and

[0245] Where X8 is L, H or F.

[0246] In one aspect, the CDR3H region comprises the sequence RHDFDYFPYWFAH (SEQ ID NO: 25).

[0247] In one aspect, the present invention provides a CEACAM1 antibody or antigen-binding fragment thereof comprising a CDR3L, wherein the CDR3L comprises residues 89-97 (Kabat definition, corresponding to, e.g., SEQ ID NO: 14 (see Figure 3C ) of the heavy variable chain primary amino acid sequence of residues 88 to 96) and comprising the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO: 6),

[0248] Where X9 is W or N;

[0249] Where X 10 is S or T;

[0250] Where X 11 is A or an amino acid with a neutral hydrophilic side chain including S, N and T;

[0251] Where X 12 is L, F or N; and

[0252] Where X 13 It is P or F.

[0253] In one embodiment, CDR3L comprises residues 89-97 (Kabat definition, corresponding to, e.g., SEQ ID NO: 14 (see Figure 3C ) of the heavy variable chain primary amino acid sequence of residues 88 to 96) and comprising the sequence QQX9SSX 12 PX 13 T (SEQ ID NO: 8),

[0254] Where X9 is W or N;

[0255] Where X 12 is L, F or N; and

[0256] Where X 13 It is P or F.

[0257] In one embodiment, the CDR3L comprises the sequence QQWSSNPPT (SEQ ID NO: 11) or the sequence QQWTSNPPT (SEQ ID NO: 12).

[0258] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein:

[0259] The CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X 14 X 15 X 16 FX 17 X1HX2X3S (SEQ ID NO: 22);

[0260] Where X 14 is G or E;

[0261] Where X 15 is an amino acid having an aromatic side chain including F or Y;

[0262] Where X 16 is T, S or I;

[0263] Where X 17 is an amino acid having a polar uncharged side chain including S, T or N;

[0264] Where X1 is A, D, N or S;

[0265] wherein X2 is A or G; and

[0266] wherein X3 is an amino acid having a hydrophobic side chain, including I or M;

[0267] The CDR2 sequence of the heavy chain variable region (CDR2H) comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0268] The CDR3 sequence of the heavy chain variable region (CDR3H) comprises the sequence HX4X5DYX6X19X7WFAX20 (SEQ ID NO: 45);

[0269] Where X4 is D, G or P;

[0270] Where X5 is F or P;

[0271] Where X6 is D or F;

[0272] Where X19 is P or A;

[0273] wherein X7 is A or Y; and

[0274] Where X20 is L, H, Y or F;

[0275] The CDR1 sequence of the light chain variable region (CDR1L) comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0276] The CDR2 sequence of the light chain variable region (CDR2L) comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0277] The CDR3 sequence of the light chain variable region (CDR3L) comprises the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO: 6);

[0278] Where X9 is W or N;

[0279] Where X 10 is S or T;

[0280] Where X 11 is A or an amino acid with a neutral hydrophilic side chain including S, N and T;

[0281] Where X 12 is L, F or N; and

[0282] Where X 13 is P or F; and

[0283] in

[0284] When X 19 A and / or X 20 When it is Y, X 10 is T, X4 is G or P, X1 is N, and / or X 16 It is T or S.

[0285] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein:

[0286] The CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X 14 FX 21 FX 22 X 23 HX2X3S (SEQ ID NO: 46);

[0287] Where X 14 is G or E;

[0288] Where X 21 is T or I;

[0289] Where X 22 is N or S;

[0290] Where X 23 A, D or S

[0291] wherein X2 is A or G; and

[0292] wherein X3 is an amino acid having a hydrophobic side chain, including I or M;

[0293] The CDR2 sequence of the heavy chain variable region (CDR2H) comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0294] The CDR3 sequence of the heavy chain variable region (CDR3H) comprises the sequence HX24FDYX6X19X7WFAX25 (SEQ ID NO: 47);

[0295] Where X24 is D or G;

[0296] Where X6 is D or F;

[0297] Where X19 is P or A;

[0298] wherein X7 is A or Y; and

[0299] Where X25 is H or Y;

[0300] The CDR1 sequence of the light chain variable region (CDR1L) comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0301] The CDR2 sequence of the light chain variable region (CDR2L) comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0302] The CDR3 sequence of the light chain variable region (CDR3L) comprises the sequence QQWX 10 X 10 NPPT (SEQ ID NO: 48);

[0303] Where X 10 is S or T;

[0304] in

[0305] When X21 is I, X6 is F, X19 is P and / or X7 is Y.

[0306] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein:

[0307] The CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X 14 FTFX 22 X 26 HAX3S (SEQ ID NO: 49);

[0308] Where X14 is G or E;

[0309] Where X 17 is S or N;

[0310] Where X 22 is N or S;

[0311] where X26 is A or D and

[0312] wherein X3 is an amino acid having a hydrophobic side chain, including I or M;

[0313] The CDR2 sequence of the heavy chain variable region (CDR2H) comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0314] The CDR3 sequence of the heavy chain variable region (CDR3H) comprises the sequence HX24FDYX6X19X7WFAX25 (SEQ ID NO: 47);

[0315] Where X24 is D or G;

[0316] Where X6 is D or F;

[0317] Where X19 is P or A;

[0318] wherein X7 is A or Y; and

[0319] Where X25 is H or Y;

[0320] The CDR1 sequence of the light chain variable region (CDR1L) comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0321] The CDR2 sequence of the light chain variable region (CDR2L) comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0322] The CDR3 sequence of the light chain variable region (CDR3L) comprises the sequence QQWX 10 X 10 NPPT (SEQ ID NO: 48);

[0323] Where X 10 It is S or T.

[0324] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1H, CDR2H and CDR3H (Kabat definition), wherein the light chain variable region comprises CDR1L, CDR2L and CDR3L (Kabat definition), and wherein:

[0325] The sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO: 1),

[0326] The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2),

[0327] The sequence of CDR3H comprises the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO: 3),

[0328] The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO: 4),

[0329] The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5), and

[0330] The sequence of CDR3L contains the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO: 6).

[0331] X1-X 18 Already defined previously.

[0332] In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3, and wherein:

[0333] The sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO: 43);

[0334] Wherein X is any amino acid;

[0335] Where X1 is A, D, N or S;

[0336] wherein X2 is A or G; and

[0337] wherein X3 is an amino acid having a hydrophobic side chain, including I or M; and

[0338] The CDR2 sequence of the heavy chain variable region (CDR2H) comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0339] The sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44);

[0340] Where X4 is D, G or P;

[0341] Where X5 is F or P;

[0342] wherein X7 is A or Y; and

[0343] Where X8 is L, H or F;

[0344] The CDR1 sequence of the light chain variable region (CDR1L) comprises the sequence RANSAVSYMY (SEQ ID NO: 4);

[0345] The CDR2 sequence of the light chain variable region (CDR2L) comprises the sequence LTSNRAT (SEQ ID NO: 5); and

[0346] The CDR3 sequence of the light chain variable region (CDR3L) comprises the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO: 6);

[0347] Where X9 is W or N;

[0348] Where X 10 is S or T;

[0349] Where X 11 is A or an amino acid with a neutral hydrophilic side chain including S, N and T;

[0350] Where X 12 is L, F or N; and

[0351] Where X 13 It is P or F.

[0352] 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 heavy chain variable region comprises CDR1H, CDR2H and CDR3H (Kabat definition), wherein the light chain variable region comprises CDR1L, CDR2L and CDR3L (Kabat definition), and wherein:

[0353] The sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO: 1),

[0354] The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2),

[0355] The sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO: 7),

[0356] The sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO: 4),

[0357] The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO: 5), and

[0358] The sequence of CDR3L contains the sequence QQX9SSX 12 PX 13 T (SEQ ID NO: 8).

[0359] X1-X 18 Already defined previously.

[0360] According to certain embodiments, the antibody and Fab thereof under consideration are also characterized by a humanized framework for reducing immunogenicity. In certain embodiments, the CDR of the antibody or Fab thereof under consideration is located in a framework obtained from a human antibody or Fab thereof. In other embodiments, the surface-exposed framework residues of the antibody or Fab thereof under consideration are replaced by framework residues of a human antibody or Fab thereof. CDR may also be located in a mouse or humanized framework connected to a human constant region (i.e., a chimeric antibody). In a preferred embodiment, the CDR of the antibody or Fab thereof under consideration is located in a framework as a complex of two or more human antibodies. In such embodiments, the contemplated antibody or antigen-binding fragment thereof comprises two or more sequence segments ("complexes") derived from the V regions of unrelated human antibodies, the V regions being selected to maintain the monoclonal antibody sequences important for antigen binding of the starting precursor anti-human CEACAM1 monoclonal antibody, and having all been filtered for the presence of potential T cell epitopes using "in silico tools" (Holgate and Baker, IDrugs. 2009 April; 12(4): 233-7). Tight fit of the human sequence segments to all portions of the starting antibody V regions and elimination of CD4 prior to synthesis of the antibody or antigen-binding fragment thereof is achieved. + T cell epitopes allow this technology to circumvent immunogenicity while maintaining optimal affinity and specificity through previously analyzed sequences essential for antigen specificity (Holgate and Baker, 2009).

[0361] Also provided herein are variable heavy chain and variable light chain sequences and pairs thereof that are similar but not identical to those disclosed in SEQ ID NOs: 13-16.

[0362] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence comprising SEQ ID NO:13.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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 in each sequence that is aligned for comparison purposes. For example, when a position in the nucleotide sequence being compared is occupied by the same base, then the molecules are identical at that position. The degree of identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at the shared position. For example, polypeptides having at least 85%, 90%, 95%, 98% or 99% identity to a particular polypeptide described herein and preferably exhibiting substantially the same function, and polynucleotides encoding such polypeptides are contemplated. Methods and computer programs for determining sequence identity and similarity are publicly available and include, but are not limited to, the GCG program package (Devereux et al., Nucleic Aci ds 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 can also be used to determine similarity. The BLAST program is publicly available from NC BI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 website http: / / www.ncbi.nlm.nih.gov / blast / ). These methods take into account various substitutions, deletions and other modifications when comparing sequences.

[0368] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0369] (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

[0370] (ii) 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.

[0371] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0372] (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;

[0373] (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

[0374] (iii) and in which:

[0375] The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13,

[0376] The sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO: 13,

[0377] The sequence of CDR1L comprises residues A28, S30 and Y31 of SEQ ID NO: 14,

[0378] The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO: 14, and

[0379] The sequence of CDR3L comprises residues S91 and S92 of SEQ ID NO:14.

[0380] The numbering of residues is based on the primary amino acid sequence of the antibody, see Figure 3A , 3B and 3C, such as heavy and light chain sequences.

[0381] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0382] (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;

[0383] (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

[0384] (iii) six CDRs, of which:

[0385] a. The CDR1 sequence of the heavy chain variable region comprises SEQ ID NO: 9;

[0386] b. the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO: 2;

[0387] c. the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO: 10;

[0388] d. the CDR1 sequence of the light chain variable region comprises SEQ ID NO: 4;

[0389] e. the CDR2 sequence of the light chain variable region comprises SEQ ID NO: 5; and

[0390] f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO:11.

[0391] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region;

[0392] wherein 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;

[0393] wherein 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;

[0394] wherein the sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO: 43);

[0395] Wherein X is any amino acid;

[0396] Where X1 is A, D, N or S;

[0397] wherein X2 is A or G; and

[0398] wherein X3 is an amino acid having a hydrophobic side chain, including I or M; and

[0399] wherein the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44);

[0400] Where X4 is D, G or P;

[0401] Where X5 is F or P;

[0402] wherein X7 is A or Y; and

[0403] Where X8 is L, H or F.

[0404] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0405] (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

[0406] (ii) 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.

[0407] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0408] (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;

[0409] (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: 15;

[0410] (iii) and in which:

[0411] The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13,

[0412] The sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO: 13,

[0413] The sequence of CDR1L comprises residues A28, S30 and Y31 of SEQ ID NO: 15,

[0414] The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO: 15, and

[0415] The sequence of CDR3L comprises residue S92 of SEQ ID NO:15.

[0416] The numbering of residues is based on the primary amino acid sequence of the antibody, see Figure 3A , 3B and 3C, such as heavy and light chain sequences.

[0417] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0418] (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;

[0419] (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

[0420] (vi) six CDRs, of which:

[0421] a. The CDR1 sequence of the heavy chain variable region comprises SEQ ID NO: 9;

[0422] b. the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO: 2;

[0423] c. the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO: 10;

[0424] d. the CDR1 sequence of the light chain variable region comprises SEQ ID NO: 4;

[0425] e. the CDR2 sequence of the light chain variable region comprises SEQ ID NO: 5; and

[0426] f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO:12.

[0427] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0428] (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

[0429] (ii) 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.

[0430] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0431] (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;

[0432] (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: 16;

[0433] (iii) and in which:

[0434] The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13,

[0435] The sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO: 13,

[0436] The sequence of CDR1L comprises residues S30 and Y31 of SEQ ID NO: 16,

[0437] The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO: 16, and

[0438] The sequence of CDR3L comprises residues S91 and S92 of SEQ ID NO:16.

[0439] The numbering of residues is based on the primary amino acid sequence of the antibody, see Figure 3A , 3B and 3C, such as heavy and light chain sequences.

[0440] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0441] (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;

[0442] (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

[0443] (ix) six CDRs, of which:

[0444] a. The CDR1 sequence of the heavy chain variable region comprises SEQ ID NO: 9;

[0445] b. the CDR2 sequence of the heavy chain variable region comprises SEQ ID NO: 2;

[0446] c. the CDR3 sequence of the heavy chain variable region comprises SEQ ID NO: 10;

[0447] d. the CDR1 sequence of the light chain variable region comprises SEQ ID NO: 18;

[0448] e. the CDR2 sequence of the light chain variable region comprises SEQ ID NO: 5; and

[0449] f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO:11.

[0450] Obviously, any of the frameworks described herein can be used in combination with any of the CDRs and CDR motifs described herein. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof utilizes

[0451] The framework described in Table 1.

[0452] In some embodiments of the aspects described herein, amino acid sequence modifications of antibodies or antigen-binding fragments thereof that bind to CEACAM1 described herein are contemplated. Amino acid sequence variants of antibodies or antigen-binding fragments thereof are prepared by introducing appropriate nucleotide changes into nucleic acids encoding the antibodies or antigen-binding fragments thereof or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibodies or antigen-binding fragments thereof. Any combination of deletions, insertions and substitutions is performed to obtain the final construct, provided that the final construct has the desired characteristics, such as binding specificity, inhibition of biological activity.

[0453] 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 by a different residue with similar side chain properties. Amino acids can be grouped according to the similarity of their side chain properties (see Lehninger, BIOCHEMISTRY (2nd Edition, Worth Publishers, New York, 1975):

[0454] (1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M);

[0455] (2) Non-charged polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q);

[0456] (3) Acidic: Asp (D), Glu (E);

[0457] (4) Basic: Lys (K), Arg (R), His (H).

[0458] Thus, a non-limiting example of a conservative amino acid substitution is replacement of a non-polar amino acid with another non-polar amino acid.

[0459] Alternatively, naturally occurring residues can be grouped based on common side-chain properties:

[0460] (1) Hydrophobicity: Ala (A), Val (V), Leu (L), Ile (I), Met (M);

[0461] (2) Neutral hydrophilicity: Ser (S), Thr (T), Cys (C), Asn (N), Gln (Q);

[0462] (3) Acidic: Asp (D), Glu (E);

[0463] (4) Basic: Lys (K), Arg (R), His (H);

[0464] (5) Residues that affect chain orientation: Gly (G), Pro (P);

[0465] (6) Aromatic: Phe (F), Trp (W), Tyr (Y).

[0466] Thus, a non-limiting example of a conservative amino acid substitution is replacement of a hydrophobic amino acid with another hydrophobic amino acid.

[0467] Amino acid sequence insertions are also contemplated, which may include amino and / or carboxyl terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies or antigen-binding fragments thereof having an N-terminal methionyl residue or antibodies or antigen-binding fragments thereof fused to a cytotoxic polypeptide. Other insertion variants of antibodies or antigen-binding fragments thereof include fusing the N or C terminus of the antibody or antigen-binding fragment thereof to an enzyme or polypeptide (e.g., biotin) that increases the serum half-life of the antibody or antigen-binding fragment thereof.

[0468] Any cysteine ​​residue that is not involved in maintaining the correct conformation of the antibody or antigen-binding fragment thereof that binds CEACAM1 may also be substituted, for example with serine or alanine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking.

[0469] 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).

[0470] In some embodiments, the antibody or its antigen-binding fragment has an amino acid change that changes the original glycosylation pattern of the antibody or its antigen-binding fragment. "Altering the original glycosylation pattern" means deleting one or more carbohydrate moieties present in the antibody or its antigen-binding fragment, and / or adding one or more glycosylation sites that are not present in the antibody or its antigen-binding fragment. The glycosylation of the antibody is usually N-connected or O-connected. N-connected refers to the connection of the carbohydrate moiety to the side chain of the 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 connection of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-connected glycosylation refers to the connection of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, the most common of which is serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Glycosylation sites are added to the antibody or antigen-binding fragment thereof that binds to CEACAM1 by altering the amino acid sequence so that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding or substituting one or more serine or threonine residues in the sequence of the original antibody or antigen-binding fragment thereof (for O-linked glycosylation sites).

[0471] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof provided herein is deglycosylated or non-glycosylated. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof contemplated lacks a C-terminal lysine in the heavy chain and / or contains an 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 the CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof lacks a NXS / T consensus sequence in the CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof has a mutation in CDR residues 26 and / or 29 (Kabat numbering) of the CDR1 of the variable light chain. When the antibody or antigen-binding fragment thereof comprises an Fc region, the carbohydrate attached thereto may be changed. For example, an antibody having a mature carbohydrate structure is described that lacks fucose attached to the Fc region of the antibody or antigen-binding fragment thereof. See, for example, U.S. Patent Publication Nos. 2003 / 0157108; 2004 / 0093621. Antibodies having bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody or antigen binding fragment thereof are cited in WO03 / 011878; U.S. Pat. No. 6,602,684. Antibodies having at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody or antigen binding fragment thereof are reported in WO 97 / 30087. See also WO 98 / 58964; WO 99 / 22764, which relate to antibodies having altered carbohydrates attached to their Fc region.

[0472] In some embodiments, it may be desirable to modify the antibodies or antigen-binding fragments thereof described herein that bind to CEACAM1 in terms of effector function, for example to enhance the 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 in the Fc region of the antibody or antigen-binding fragment thereof. Alternatively or additionally, one or more cysteine ​​residues may be introduced in the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibodies or antigen-binding fragments thereof thus produced may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., 176 J. Exp. Med. 1191 (1992); Shopes, 148 J. Immunol. 2918 (1992). As described by Wolff et al., 53 Cancer Res. 2560 (1993), heterobifunctional cross-linkers can also be used to prepare homodimeric antibodies with enhanced anti-tumor activity. Alternatively, antibodies or antigen-binding fragments thereof having dual Fc regions can be engineered and thus can have enhanced complement lysis and ADCC capabilities. See Stevenson et al., 3 Anti-Cancer Drug Design 219 (1989).

[0473] For example, WO 00 / 42072 describes an antibody with improved ADCC function in the presence of human effector cells, wherein the antibody comprises an amino acid substitution in its Fc region. Preferably, the antibody or its Fab with improved ADCC comprises a substitution (Eu numbering of residues) at positions 298, 333 and / or 334 in the Fc region. Typically, the Fc region of the change is a human IgG1 Fc region comprised of a substitution at one, two or three of these positions or consisting thereof. Such substitutions are optionally combined with substitutions that increase C1q binding and / or CDC. Substitutions include the Asn297Ala mutation in IgG1 Fc.

[0474] Antibodies with altered Clq binding and / or complement dependent cytotoxicity (CDC) are described in WO 99 / 51642, U.S. Pat. Nos. 6,194,551, 6,242,195, 6,528,624 and 6,538,124. The antibodies comprise amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333 and / or 334 of the Fc region thereof (Eu numbering of residues).

[0475] Antibodies with improved binding to the neonatal Fc receptor (FcRn) and increased half-life are described in WO 00 / 42072 and U.S. Patent Publication No. 2005 / 0014934. These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to CEACAM1. For example, the Fc region may 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). Antibody variants comprising preferred Fc regions with improved CEACAM1 binding comprise amino acid substitutions at one, two or three of positions 307, 380 and 434 of its Fc region (Eu numbering of residues). In one embodiment, the antibody or antigen-binding fragment thereof has 307 / 434 mutations. Engineered antibodies that bind to CEACAM1 with three or more (e.g., four) functional antigen-binding sites are also contemplated. See, e.g., U.S. Patent Publication No. US 2002 / 0004587.

[0476] Antibody fragments and types

[0477] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab fragment comprising the variable (V L ) and constant (C L ) domain and the variable domain of the heavy chain (V H ) and the first constant domain (C H 1) or consisting essentially of.

[0478] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab' fragment, which refers to a fragment of H Fab fragments having one or more cysteine ​​residues at the C-terminus of the 1 domain.

[0479] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a fragment comprising V H and C H 1 domain or an Fd fragment consisting essentially of the same.

[0480] In some embodiments of the aspects described herein, the CEACAM1 antibody portion comprises V H and C H 1 domain and in C H The Fd' fragment contains one or more cysteine ​​residues at the C-terminus of the 1 domain.

[0481] Single-chain Fv or scFv antibody fragments contain the V H and V L domains, such that these domains are present in a single polypeptide chain. H With V L The domains also contain a polypeptide linker that allows the scFv to form the desired structure for antigen binding. See, for example, Pluckthun, 113 Pharmacology Monoclonal Antibodies 269 (Rosenburg and Moore, eds., Springer-Verlag, New York, 1994). Thus, in some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a V comprising a single arm of the antibody. L and V H Fv fragments consisting essentially of or comprising a structural domain.

[0482] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is a diabody comprising two antigen binding sites, which comprises, in the same polypeptide chain, a light chain variable domain (V L )'s heavy chain variable domain (V H ).

[0483] In some embodiments of the aspects described herein, the CEACAM1 antibody portion comprises V H A dAb fragment consisting essentially of or comprising a domain.

[0484] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is a F(ab')2 fragment comprising a bivalent fragment comprising two Fab' fragments linked by a disulfide bond at the hinge region.

[0485] Linear antibodies are those described by Zapata et al., Protein Engin., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd fragments (V H -C H 1-V H -C H 1), which together with the complementary light chain polypeptide 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 is a fragment comprising a pair of tandem Fd fragments (V H -C H 1-V H -C H 1), the tandem Fd fragments and the complementary light chain polypeptides together form a pair of antigen binding regions.

[0486] A variety of techniques have been developed and can be used to produce antibody fragments. Traditionally, these fragments are 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 library discussed herein. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992). According to another method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for preparing antibody fragments are obvious to those skilled in the art. In other embodiments, the antibody fragment selected is a single-chain Fv fragment (scFv). See, for example, WO 93 / 16185.

[0487] In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds CEACAM1 and a complementary region that binds PD-1.

[0488] In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds CEACAM1 and a complementary region that binds PD-L1.

[0489] The contemplated antibodies or antigen-binding fragments can have constant regions of all types, 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 can be λ or κ. The antibody or antigen-binding fragment thereof can contain sequences from more than one class or isotype.

[0490] Also disclosed herein are chimeric antigen receptor T cells (CAR T cells) that bind CEACAM1. In one embodiment, one or more CDRs of the anti-CEACAM antibodies disclosed herein are transplanted onto a chimeric antigen receptor (CAR) on a T cell. Such genetically modified T cells utilize CAR (also referred to as chimeric T cell receptors) to target antigens expressed on tumor cells in a human leukocyte antigen-independent manner.

[0491] Antibody Binding

[0492] The human CEACAM1 gene produces 11 isoforms by alternative splicing. Each isoform has a variable (V)-like Ig domain at the amino (N) terminus of the protein. In addition to CEACAM1-1L and CEACAM1-1S isoforms, different isoforms also have 2 or 3 constant C2-like Ig domains. Eight CEACAM1 isoforms are anchored to the cell membrane through 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 (A) between the constant C2-like Ig domain and the transmembrane domain. Transmembrane CEACAM1 isoforms also have long (L) or short (S) cytoplasmic domains, which are determined by including or excluding CEACAM1 exon 7 in the messenger. The CEACAM1 L cytoplasmic domain has two ITIM motifs, which 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 features, which bind to the extracellular variable (V)-like Ig domain at the amino (N) terminus (N domain) of the protein of CEACAM1, i.e., a domain common to all isoforms of CEACAM1, including CEACAM1 isoforms 1L, 1S, 3L, 3S, 4L, 4S, 3A1, 3AS, 3, 4C1, and 4C2. In some embodiments, the antibodies and antigen-binding fragments thereof provided bind to human CEACAM1. In some embodiments, the antibodies and antigen-binding fragments thereof provided bind to mammalian CEACAM1. The sequence of the full-length form of CEACAM1 (NCBI reference sequence NP_001703.2; UNIPROT 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.

[0493] 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 specific residue on CEACAM1 includes selective interaction of the antibody or antigen-binding fragment thereof with CEACAM1. Thus, binding includes, for example, primary and secondary interactions, including hydrogen bonds, ionic interactions, salt bridges, and hydrophilic and hydrophobic interactions.

[0494] In certain embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are present in an amount of 10 -5 Up to 10 -12mol / l, 10 -6 Up to 10 -12 mol / l, 10 -7 Up to 10 -12 mol / l, 10 -8 Up to 10 -12 mol / l, 10 -9 Up to 10 -12 mol / l, 10 -10 Up to 10 -12 mol / l or 10 -11 Up to 10 -12 mol / l K D In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are present in an amount of 10 -5 Up to 10 -11 mol / l, 10 -6 Up to 10 -11 mol / l, 10 -7 Up to 10 -11 mol / l, 10 -8 Up to 10 -11 mol / l, 10 -9 Up to 10 -11 mol / l or 10 -10 Up to 10 -11 mol / l K D In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are present in an amount of 10 -5 Up to 10 -10 mol / l, 10 -6 Up to 10 -10 mol / l, 10 -7 Up to 10 -10 mol / l, 10 -8 Up to 10 -10 mol / l or 10 -9 Up to 10 -10 mol / l K D In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are present in an amount of 10 -5 Up to 10 -8 mol / l, 10 -6 Up to 10 -8 mol / l or 10 -7 Up to 10 -8 mol / l K D Binds to CEACAM1.

[0495] The term "specificity" 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, with little or no detectable reactivity with other parts of CEACAM1. Specificity can be relatively determined by competition assays or by epitope identification / characterization techniques described herein or their equivalents known in the art.

[0496] As used herein, an "epitope" can be formed by continuous amino acids or non-continuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed by continuous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. An epitope usually includes at least 3, more usually at least 5, about 9, or about 8-10 amino acids in a specific spatial conformation. An "epitope" includes a conventional sequence of an immunoglobulin V or V region. H / V L The epitope defines the minimum binding site of an antibody or antigen-binding fragment thereof and therefore represents the specific target of the antibody or antigen-binding fragment thereof. In the case of single-domain antibodies, the epitope represents the structural unit bound by the isolated variable domains.

[0497] In a specific embodiment, the contemplated antibody or antigen-binding fragment specifically binds to the same epitope as antibody CP08H03 / Vk8 S29A. In another embodiment, the contemplated antibody or antigen-binding fragment binds to the same epitope as CP08H03 / CP08F05.

[0498] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including antibodies described herein by their structural features, wherein the antibodies and antigen-binding fragments thereof specifically bind to at least a portion of the homophilic binding domain on CEACAM1 (i.e., a portion of the CEACAM1 protein involved in the formation of CEACAM1:CEACAM1 homodimers), thereby blocking CEACAM1 homophilic interactions. In certain embodiments, the antibodies or antigen-binding fragments thereof provided specifically bind to one or more of the CEACAM1 residues contained in the CC' and FG loops of CEACAM1 and including the YQQN pocket (i.e., Y34, Q44, Q89, N97 of SEQ ID NO: 17) at the CEACAM1:CEACAM1 dimer interface, see Huang et al., Nature. 2015 Jan 15; 517(7534): 386-90.

[0499] 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 and inhibits the activity of CEACAM1 and / or the binding of CEACAM1 to a heterologous binding partner (such as other CEACAM proteins or TIM-3). Inhibition of activity and inhibition of binding include partial inhibition. Methods for identifying CEACAM1 antibodies that block CEACAM1 homophilic and heterophilic interactions are described herein and are known to those skilled in the art. For example, competing antibodies, cross-blocking antibodies, and cross-blocked antibodies can be identified using any suitable method known in the art, including competitive ELISA or Assays in which binding of a competing or cross-blocking antibody to human CEACAM1 prevents binding of an antibody disclosed herein, and vice versa.

[0500] In one embodiment, the heavy chain of the contemplated antibody or antigen-binding fragment thereof specifically binds CEACAM1 at residues F29, Y34, T56, Q89, S93 and / or D94 of SEQ ID NO: 17. In another embodiment, the heavy chain of the contemplated antibody or antigen-binding fragment thereof also specifically binds CEACAM1 at residues S32, Q44, A49 and / or I91 of SEQ ID NO: 17.

[0501] In one embodiment, the light chain of the contemplated antibody or antigen-binding fragment thereof specifically binds CEACAM1 at residues D40, G41, N42, N97 and / or E99 of SEQ ID NO: 17. In another embodiment, the light chain of the contemplated antibody or antigen-binding fragment thereof also specifically binds CEACAM1 at residues L95 and / or V96 of SEQ ID NO: 17.

[0502] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof specifically binds 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 also specifically binds CEACAM1 at residues S32, Q44, A49, I91, L95 and / or V96 of SEQ ID NO:17.

[0503] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof specifically binds CEACAM1 at residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97 and E99 of SEQ ID NO:17.

[0504] In another embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds 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.

[0505] 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 its antigen-binding fragment are in contact with the antigen. In one embodiment, five of the six CDRs of the CEACAM1 antibody or its antigen-binding fragment are in contact with the antigen. In one embodiment, six of the six CDRs of the CEACAM1 antibody or its antigen-binding fragment are in contact with the antigen. In one embodiment, CDR2H, CDR3H, CDR1L, CDR2L and CDR3L of the CEACAM1 antibody or its antigen-binding fragment are directly involved in binding to the antigen.

[0506] In one embodiment, the antibodies and antigen-binding fragments thereof provided herein specifically bind to an epitope of CEACAM1 located on the N domain of CEACAM1. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to a CEACAM1 epitope comprising one or more CEACAM1 residues selected from F29, S32, D40, A49 and T56 of SEQ ID NO: 17. In another embodiment, the CEACAM1 antibody specifically binds to a CEACAM1 epitope comprising residues F29, S32, D40, A49, T56 and I91 of SEQ ID NO: 17.

[0507] In one embodiment, the antibodies and antigen-binding fragments thereof provided herein specifically bind to an epitope of CEACAM1 located on the N domain of CEACAM1. In one embodiment, the antibodies or antigen-binding fragments thereof specifically bind to a CEACAM1 epitope comprising one or more CEACAM1 residues selected from S32, D40, A49 and I91 of SEQ ID NO: 17. In another embodiment, the CEACAM1 antibody specifically binds to a CEACAM1 epitope comprising residues S32, D40, A49 and I91 of SEQ ID NO: 17.

[0508] In one embodiment, the CEACAM1 antibodies or antigen-binding fragments thereof provided herein bind to CEACAM1, wherein

[0509] CDR2H residue Y57 binds CEACAM1 at residue F29,

[0510] CDR2H residue Y59 binds CEACAM1 at residue S93,

[0511] CDR3H residue D102 binds CEACAM1 at residue T56,

[0512] CDR3H residue Y103 binds CEACAM1 at residues Y34 and / or Q89,

[0513] CDR3H residue F104 binds CEACAM1 at residue F29,

[0514] CDR3H residue Y106 binds CEACAM1 at residue D94,

[0515] CDR1L residue S30 binds CEACAM1 at residue E99,

[0516] CDR1L residue Y31 binds CEACAM1 at residue N97,

[0517] CDR2L residue S51 binds CEACAM1 at residue D40, and / or

[0518] CDR2L residue N52 binds CEACAM1 at residues G41 and / or N42.

[0519] The numbering of CDR residues is based on the primary amino acid sequence of the antibody, see Figure 3A , 3B and 3C, eg, heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO:17.

[0520] In one embodiment, the CEACAM1 antibodies or antigen-binding fragments thereof provided herein bind to CEACAM1, wherein

[0521] CDR2H residue Y57 binds CEACAM1 at residue F29,

[0522] CDR2H residue Y59 binds CEACAM1 at residue S93,

[0523] CDR3H residue D102 binds CEACAM1 at residue T56,

[0524] CDR3H residue Y103 binds CEACAM1 at residues S32, Y34, Q44 and / or Q89,

[0525] CDR3H residue F104 binds CEACAM1 at residues F29 and / or A49,

[0526] CDR3H residue P105 binds CEACAM1 at residue I91,

[0527] CDR3H residue Y106 binds CEACAM1 at residue D94,

[0528] CDR1L residue S30 binds CEACAM1 at residue E99,

[0529] CDR1L residue Y31 binds CEACAM1 at residue N97,

[0530] CDR2L residue S51 binds CEACAM1 at residue D40,

[0531] CDR2L residue N52 binds CEACAM1 at residues G41 and / or N42,

[0532] CDR3L residue S91 binds CEACAM1 at residue L95, and / or

[0533] CDR3L residue S92 binds CEACAM1 at residue V96.

[0534] The numbering of residues is based on the primary amino acid sequence of the antibody, see Figure 3A , 3B and 3C, eg, heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO:17.

[0535] In one embodiment, the CEACAM1 antibodies or antigen-binding fragments thereof provided herein bind to CEACAM1, wherein

[0536] CDR2H residue Y57 binds CEACAM1 at residue F29,

[0537] CDR2H residue Y59 binds CEACAM1 at residue S93,

[0538] CDR3H residue D102 binds CEACAM1 at residue T56,

[0539] CDR3H residue Y103 binds CEACAM1 at residues S32, Y34, Q44, and Q89.

[0540] CDR3H residue F104 binds CEACAM1 at residues F29 and A49,

[0541] CDR3H residue P105 binds CEACAM1 at residue I91,

[0542] CDR3H residue Y106 binds CEACAM1 at residue D94,

[0543] CDR1L residue S30 binds CEACAM1 at residue E99,

[0544] CDR1L residue Y31 binds CEACAM1 at residue N97,

[0545] CDR2L residue S51 binds CEACAM1 at residue D40,

[0546] CDR2L residue N52 binds CEACAM1 at residues G41 and N42,

[0547] CDR3L residue S91 binds CEACAM1 at residue L95, and

[0548] CDR3L residue S92 binds CEACAM1 at residue V96.

[0549] The numbering of residues is based on the primary amino acid sequence of the antibody, see Figure 3A , 3B and 3C, eg, heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO:17.

[0550] CEACAM family members are widely expressed on a variety of cell types, especially leukocytes, thereby affecting the magnitude of cellular functions. For example, CEACAM1 is expressed on epithelial cells, endothelial cells, lymphocytes, and myeloid cells, CEACAM3 is expressed on granulocytes and neutrophils, CEACAM5 is expressed on epithelial cells, and CEACAM6 is expressed on epithelial cells and granulocytes. However, the N domain of CEACAM1 has approximately 90% similarity to the N domains of CEACAM family members 3, 5, and 6, making it difficult to selectively target CEACAM1.

[0551] Although the N domain is highly similar among CEACAM family members, in some embodiments, the antibodies or antigen-binding fragments thereof provided herein, including the antibodies described herein by their structural features, are selective for CEACAM1. By selectively targeting CEACAM1, embodiments of the present invention can avoid unwanted interference, such as the broad activation function of CEACAM3.

[0552] The terms "selective" and "selectivity" as used herein 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; the region, target or peptide is typically a region or epitope in CEACAM1 and not one or more other biological molecules, including other CEACAM family members.

[0553] In some embodiments, the contemplated CEACAM1 antibody or antigen-binding fragment thereof does not exhibit significant binding to CEACAM3, CEACAM5, CEACAM6 and / or CEACAM8. In some embodiments, the contemplated CEACAM1 antibody or antigen-binding fragment thereof does not exhibit detectable binding to CEACAM3, CEACAM5, CEACAM6 and / or CEACAM8. In some embodiments, the contemplated CEACAM1 antibody or antigen-binding fragment thereof binds to CEACAM1 with an affinity that is at least 10 times greater, such as at least 100 times greater, and at least 1000 times greater, and up to 10,000 times greater or stronger than the binding affinity of the contemplated CEACAM1 antibody or antigen-binding fragment thereof to another target or polypeptide.

[0554] As used herein, the equilibrium constant (K) for the dissociation of an antigen from an antigen binding protein is D "Affinity" as expressed in terms of affinity is a measure of the strength of binding 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, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule. Alternatively, affinity can also be expressed as an affinity constant (K A ), which is 1 / K D ). As is clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.

[0555] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including antibodies described herein by their structural features, wherein the antibodies and antigen-binding fragments thereof specifically bind to at least a portion of the binding site of one or more other members of the CEACAM family on CEACAM1, thereby blocking the interaction of CEACAM1 with one or more other members of the CEACAM family. These CEACAM family members include, but are 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).

[0556] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including antibodies described herein by their structural features, wherein the antibodies and antigen-binding fragments thereof specifically bind to at least a portion of the binding site of a TIM family member on CEACAM1, thereby blocking the interaction between CEACAM1 and a TIM family member. In some embodiments, this 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 residues 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).

[0557] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including antibodies described herein by their structural features, wherein the antibodies and antigen-binding fragments thereof specifically bind to at least a portion of the binding site of the bacterial adhesion surface protein (adhesin) on CEACAM1, thereby blocking the interaction between CEACAM1 and the adhesin. In certain embodiments, the adhesin is expressed on the surface of pathogenic bacteria that bind to CEACAM1, including but not limited to Escherichia coli, in particular diffusely adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria commensalis, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus aegypti, Helicobacter pylori and / or Salmonella sp.

[0558] 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 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 have been predicted to be involved in the binding of CEACAM1 to HopQ.

[0559] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and an opacity-associated (Opa) adhesin protein expressed on the surface of Neisseria species, including but not limited to Opas2, Opa65, Opa68, Opav0, Opa72, Opa73, Opa74, and Opa75. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to one or more of CEACAM1 residues Q44 and A49, which have been predicted to be involved in the binding of CEACAM1 to the Neisseria Opa protein.

[0560] 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.

[0561] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Haemophilus influenzae OMP P 1. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to one or more of CEACAM1 residues Q44 and A49, which have been predicted to be involved in the binding of CEACAM1 to Haemophilus influenzae OMP P 1.

[0562] In another embodiment, the CEACAM1 antibody or antigen binding fragment thereof disrupts the interaction between CEACAM1 and Haemophilus aegypti OMP P 1. In one embodiment, the CEACAM1 antibody or antigen binding fragment specifically binds to CEACAM1 residue F29, which has been predicted to be involved in the binding of CEACAM1 to Haemophilus aegypti OMP P 1.

[0563] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Candida albicans.

[0564] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and influenza virus (including but not limited to H5N1).

[0565] In another embodiment, the present invention provides a method for inhibiting the binding of CEACAM1 to a sub-nematode using the CEACAM1 antibody or antigen-binding fragment thereof described herein, the method comprising contacting CEACAM1 with the CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the sub-nematode is Wuchereria bancrofti.

[0566] Antibody conjugates

[0567] In some embodiments of the aspects described herein, the antibody or antigen-binding fragment thereof that binds 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.

[0568] Exemplary blocking moieties include moieties that have sufficient steric hindrance and / or charge to reduce the occurrence of glycosylation, for example by blocking the ability of glycosidases to glycosylate the antibody or antigen-binding fragment thereof. Additionally or alternatively, blocking moieties can reduce effector function, for example by inhibiting the ability of the Fc region to bind receptors or complement proteins. Preferred blocking moieties include cysteine ​​adducts and PEG moieties.

[0569] In a preferred embodiment, the blocking moiety is a cysteine, preferably a cysteine ​​that associates with a free cysteine, such as in cell culture, such as during or after translation of an Fc-containing polypeptide. Other blocking cysteine ​​adducts include cystine, mixed disulfide adducts, or disulfide bonds.

[0570] In another preferred embodiment, the blocking portion is a polyalkylene glycol portion, such as a PEG portion, and preferably a PEG-maleimide portion. Preferred pegylated portions (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 polyoxyalkylene oxides, polyoxyethylated sorbitol or polyoxyethylated glucose. The polymer can be a homopolymer, a random or block copolymer, a terpolymer based on the monomers listed above, a linear or branched, substituted or unsubstituted, as long as it has at least one active sulfone portion. The polymer portion can have any length or molecular weight, but these features can affect biological properties. The average molecular weight of polymers that are particularly useful for reducing clearance rates in pharmaceutical applications is in the range of 2,000 to 35,000 daltons. In addition, if two groups are attached to the polymer, one at each end, the length of the polymer can affect the effective distance and other spatial relationships between the two groups. Therefore, those skilled in the art can change the length of the polymer to optimize or confer the desired biological activity. PEG can be used in biological applications for several reasons. PEG is generally transparent, colorless, odorless, soluble in water, heat stable, inert to many chemical reagents, non-hydrolyzed and non-toxic. PEGylation can improve the pharmacokinetic properties of a molecule by increasing its apparent molecular weight. The increased apparent molecular weight reduces the clearance rate from the body after subcutaneous or systemic administration. In many cases, PEGylation can reduce antigenicity and immunogenicity. In addition, PEGylation can increase the solubility of biologically active molecules.

[0571] Examples of detectable moieties useful for the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include fluorescent moieties or labels, imaging agents, radioisotope moieties, radiopaque moieties, and the like, such as detectable labels, such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels. Exemplary fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, etc.) and other luminescent molecules (e.g., luminal). Fluorophores can be environmentally sensitive, such that if they are located near one or more residues in a modified protein that undergo structural changes upon binding to a substrate (e.g., a dansyl probe), their fluorescence changes. Exemplary radiolabels include proteins containing a low-sensitivity nucleus ( 13 C. 15 N. 2 H. 125 I. 123 I. 99 Tc, 43 K. 52 Fe, 67 Ga, 68 Ga, 111 In, etc.) Other useful moieties are known in the art.

[0572] Examples of diagnostic moieties useful for 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 condition. Typically, diagnostic moieties allow the presence, absence or level of a molecule (e.g., a target peptide, protein or proteins) associated with a disease or condition to be determined. Such diagnostics are also useful for predicting and / or diagnosing a disease or condition and its progression.

[0573] Examples of therapeutic moieties that can be used in the methods and antibodies and antigen-binding fragments thereof contemplated by the invention include, for example, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, anti-infective agents or therapeutic agents in general. Functional moieties may also have one or more of the above functions.

[0574] Exemplary therapeutic moieties include radionuclides with high energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA and are therefore useful for inducing cell death (e.g., of 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 188Re. These isotopes generally produce high energy alpha or beta particles with short path lengths. Such radionuclides kill cells in their immediate vicinity, such as neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on nonlocalized cells and are essentially nonimmunogenic.

[0575] Exemplary therapeutic moieties also include cytotoxic agents, such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or cross-linkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, and the like.

[0576] Exemplary therapeutic moieties also include alkylating agents, such as the anthracycline family (e.g., doxorubicin, carminomycin, cyclosporin-A, chloroquine, methotrexate, mithramycin, porphyrin, streptomycin, anthracenedione and aziridine). In another embodiment, the chemotherapeutic moiety is a cytostatic agent, 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-dichlorodiaminoplatinum (II) (cisplatin), melphalan, mitoxantrone and oxaliplatin.

[0577] Exemplary therapeutic moieties also include transcriptional regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine and idarubicin. Other exemplary cell growth inhibitors compatible with the present invention include ansamycin benzoquinone, quinone derivatives (e.g., quinolones, genistein, bactacyclin), busulfan, ifosfamide, nitrogen mustard, triimidazole quinone, diazocone, carboquinone, indolequinone EO9, divinyl iminobenzoquinone methyl DZQ, triethylene phosphoramide and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0578] Exemplary therapeutic moieties also include cytotoxic nucleosides, such as, for example, adenine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, tegafur, and 6-mercaptopurine; tubulin binding agents, such as taxanes (e.g., paclitaxel, docetaxel, taxanes), nocodazole, radix toxins, dolastatins (e.g., dolastatin 10, 11, or 15), colchicine and colchicine-like agents (e.g., ZD6126), combretastatins (e.g., combretastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (Navelbine)); anti-angiogenic compounds, such as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0579] Exemplary therapeutic moieties also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroxyprogesterone), 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-bisphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-alpha, rapamycin, sex hormone binding globulin, and thapsigargin.

[0580] Exemplary therapeutic moieties also include enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenzene-imidazole 1-β-D-ribofuranoside, etoposide, formestane, forsitracin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrosine phosphorylation inhibitor AG34, and tyrosine phosphorylation inhibitor AG 879.

[0581] Exemplary therapeutic moieties also include gene modulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehyde), retinoic acid, retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0582] Exemplary therapeutic moieties also include cytotoxic agents, such as, for example, pteridine family drugs, enediynes, and podophyllotoxins. Particularly useful members of these classes include, for example, methotrexate, podophyllotoxin or podophyllotoxin derivatives such as etoposide or etoposide phosphate, isovinabine, vindesine, vinblastine, etc.

[0583] Still other cytotoxins compatible with the teachings herein include auristatins (e.g., auristatin E and monomethyl auristatin E), calicheamicin, gramicidin D, maytansinoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, teniposide, colchicine, dihydroxyanthracenedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0584] The techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", 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", 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 Antibody In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); Therapy", 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).

[0585] In order to increase the half-life of an antibody or polypeptide containing an amino acid sequence described herein, a salvage receptor binding epitope may be linked to an antibody or antigen-binding fragment thereof (particularly an antibody fragment), as described, for example, in U.S. Pat. No. 5,739,277. The term "salvage receptor binding epitope" may refer to an epitope of 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-life are also described in WO 00 / 42072, WO 02 / 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 a fusion protein expressed by the engineered 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 linking to other polypeptide sequences. For example, an antibody or antigen-binding fragment thereof useful in the methods of the present invention can be linked to serum albumin or a portion of serum albumin that binds to a CEACAM1 receptor or a serum albumin binding peptide, such that serum albumin binds to the antibody or antigen-binding fragment thereof, such as WO 01 / 45746. In one embodiment, the half-life of the Fab is increased by these methods. See also Dennis et al., 277 J. Biol. Chem. 35035 (2002) for additional serum albumin binding peptide sequences.

[0586] Other types of functional moieties are known in the art and can be readily employed in the methods and compositions of the present invention based on the teachings contained herein.

[0587] Nucleic Acids

[0588] Also provided herein are nucleic acids encoding CEACAM1 antibodies and antigen-binding fragments thereof, as well as vectors, host cells and expression systems. As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, 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 derived nucleotide bases.

[0589] The nucleic acid encoding the CEACAM1 antibody and antigen-binding fragment thereof can be, for example, a DNA, cDNA, RNA, synthetically produced DNA or RNA, or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides alone or in combination. For example, an expression vector comprising a polynucleotide sequence encoding the CEACAM1 antibody or antigen-binding fragment thereof described herein is provided, the polynucleotide sequence being operably linked to an expression control sequence suitable for expression in a eukaryotic and / or prokaryotic host cell.

[0590] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto."Vector" includes but is not limited to viral vectors, plasmids, RNA vectors or linear or circular DNA or RNA molecules, which may be composed of chromosomes, non-chromosomes, semisynthetic or synthetic nucleic acids. In some embodiments, the vector used is a vector capable of autonomous replication (episomal vector) and / or expression of a nucleic acid connected thereto (expression vector). A large number of suitable vectors are known to those skilled in the art and are commercially available. Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses, AAV), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses include adenoviruses, herpes viruses (e.g., herpes simplex viruses type 1 and type 2, Epstein-Barr viruses, cytomegaloviruses) and poxviruses (e.g., vaccinia, fowlpox and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus and hepatitis virus.Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type, D-type viruses, HTLV-BLV group, lentivirus and foamy virus.

[0591] A variety of expression vectors have been developed for efficient synthesis of antibodies and antigen-binding fragments thereof in prokaryotic cells (such as bacteria) and in eukaryotic systems (including but not limited to yeast and mammalian cell culture systems). Vectors may contain segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Cells comprising expression vectors for expressing a desired CEACAM1 antibody or antigen-binding fragment thereof are also provided.

[0592] Antibody production and expression system

[0593] The antibody of the present invention or its antigen-binding fragment is usually produced by recombinant expression. The nucleic acid encoding the light chain and heavy chain variable region (optionally connected to the constant region) is inserted into the expression vector. The light chain and the heavy chain can be cloned in the same or different expression vectors. The DNA segment encoding the immunoglobulin chain is operably connected to the control sequence in the expression vector to ensure the expression of the immunoglobulin polypeptide. The expression control sequence includes but is not limited to a promoter (such as a naturally associated or heterologous promoter), a signal sequence, an enhancer element and a transcription termination sequence. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is integrated into a suitable host, the host is maintained under conditions suitable for high-level expression of nucleotide sequences and collection and purification of cross-reactive antibodies.

[0594] These expression vectors are usually replicable in the host organism as episomes or as an integral part of the host chromosomal DNA. Typically, the expression vector comprises 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).

[0595] The expression of antibodies and antigen-binding fragments contemplated by the present invention can occur in prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungi, bird and mammalian cells in vivo or in situ, or host cells of mammalian, insect, bird or yeast origin. Mammalian cells or tissues can be human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cells can be used.

[0596] Escherichia coli is a prokaryotic host particularly useful for cloning polynucleotides (e.g., DNA sequences) of the present invention. Other suitable microbial hosts include bacilli, such as Bacillus subtilus, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.

[0597] Other microorganisms such as yeast can also be used for expression. Saccharomyces and Pichia are exemplary yeast hosts, with suitable vectors having expression control sequences (e.g., promoters), replication origins, termination sequences, etc. as required. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, in particular, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.

[0598] In addition, by using, for example, the yeast ubiquitin hydrolase system, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved. The fusion proteins so produced can be processed in vivo or purified and processed in vitro, thereby allowing the synthesis of CEACAM1 antibodies or antigen-binding fragments thereof of the present invention having specific amino-terminal sequences. In addition, problems associated with retaining methionine residues derived from start codons in direct yeast (or bacteria) expression can be avoided. Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).

[0599] When yeast is grown in a medium rich in glucose, any of a series of yeast gene expression systems incorporating promoter and terminator elements of a large number of active expression genes encoding glycolytic enzymes can be used to obtain the recombinant CEACAM1 antibody or peptide of the present invention. Known glycolytic genes can also provide very effective transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be used.

[0600] Production of CEACAM1 antibodies or antigen-binding fragments thereof in insects can be achieved, for example, by infecting an insect host with a baculovirus engineered to express a transmembrane polypeptide by methods known to those skilled in the art. See Ausubel et al., 1987, 1993.

[0601] In addition to microorganisms, mammalian tissue culture can also be used to express and produce the antibodies or antigen-binding fragments thereof of the present invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Eukaryotic cells are actually preferred because many suitable host cell lines capable of secreting heterologous proteins (e.g., complete immunoglobulins) have been developed in the art, and the cell lines include CHO cell lines, various COS cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B cells and hybridomas. The expression vectors of these cells can include expression control sequences, such as replication origins, promoters and enhancers (Queen et al., Immunol. Rev. 89: 49 (1986)), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing 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).

[0602] Alternatively, the 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 subsequently expressed 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 promoters and enhancers from mammary gland-specific genes such as casein or beta lactoglobulin.

[0603] In addition, plants have become a convenient, safe and economical alternative mainstream expression system for recombinant antibody production, which is based on large-scale culture of microorganisms or animal cells. Antibodies or their antigen-binding fragments can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be systemic, limited to subcellular plastids or limited to seeds (endosperm). 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 a mature stage of development, including clinical trials (see, for example, Biolex, NC).

[0604] The vector containing the polynucleotide sequence of interest (for example, heavy chain and light chain coding sequence and expression control sequence) can be transferred to the host cell by well-known methods, depending on the type of cell host. For example, calcium chloride transfection is usually used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics or virus-based transfection can be used for other cell hosts. (generally referring to Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd edition, 1989). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection (generally referring to Sambrook et al., the same). In order to produce transgenic animals, transgenic microinjection can be carried out into fertilized oocytes, or the genome of embryonic stem cells can be incorporated, and the nucleus of such cells is transferred into enucleated oocytes.

[0605] Antibodies of the present invention and Fabs thereof can be expressed using a single vector or two vectors. When antibody heavy chains and light chains are cloned onto a separate expression vector, the vector is cotransfected to obtain expression and assembly of a complete immunoglobulin. Once expressed, the complete antibodies of the present invention, their dimers, separate light chains and heavy chains or other immunoglobulin forms can be purified according to the standard procedures of this area, and the standard procedures include ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, etc. (generally referring to Scopes, Protein Purification (Springer-Verlag, NY, (1982)). For pharmaceutical use, substantially pure immunoglobulins of at least about 90% to 95% homogeneity are preferred, and 98% to 99% or higher homogeneity are most preferred.

[0606] Methods of regulating CEACAM1 activity

[0607] In one aspect, the present 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.

[0608] On the other hand, the present invention provides a method for reducing the interaction between CEACAM1 and TIM family members (including but not limited to TIM-1, TIM-3 and TIM-4) using the antibodies and antigen-binding fragments thereof of the present invention. In some embodiments, the antibody or antigen-binding fragment thereof disrupts the heterophilic interaction between CEACAM1 and TIM-3. By using the antibodies and antigen-binding fragments thereof contemplated by the present invention to disrupt the interaction between CEACAM1 and TIM-3, the CEACAM1 inhibitory function can be reversed while maintaining the TIM-3 activation function.

[0609] Embodiments of the present invention can be used to reduce immunosuppression, such as T cell tolerance. "Reduction" refers to the ability to cause an overall reduction 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 an untreated control. Immunosuppression can be mediated by immunosuppressive receptors expressed on the surface of immune cells and their interactions with their ligands. For example, cytotoxic CD8 T cells can enter a "functional exhaustion" or "no response" state, whereby they express inhibitory receptors that prevent antigen-specific responses (such as proliferation and cytokine production). Therefore, by inhibiting the activity and / or expression of such inhibitory receptors, it is possible to enhance or not inhibit the suppressed, suppressed or unresponsive immune response to cancer or tumors. Such enhancement or reversal of immune response suppression can lead to higher T cell activity, responsiveness and / or the ability or receptivity of activation.

[0610] The method for measuring T cell activity is known in the art. As a non-limiting example, T cell tolerance can be induced by contacting T cells with recall antigens, anti-CD3 (in the absence of costimulation) and / or ionomycin. The level of IL-27, LDH-A, RAB10 and / or ZAP70 (intracellular or secreted) can be monitored, for example, to determine the degree of T cell tolerance (wherein the level of IL-2, interferon-γ and TNF is related to the increased T cell tolerance). The response of cells to antigens pretreated with, for example, ionomycin can also be measured to determine the degree of T cell tolerance in cells or cell colonies, for example, by monitoring secreted and / or intracellular IL-2 and / or TNF-α levels (see, for example, Macian et al. Cell 2002 109: 719-731). Other characteristics of T cells with adaptive tolerance include increased levels of Fyn and ZAP-70 / Syk, Cbl-b, GRAIL, Ikaros, CREM (cAMP response element regulator), B lymphocyte-induced maturation protein-1 (Blimp-1), PD1, CD5 and SHP2; increased phosphorylation of ZAP-70 / Syk, LAT, PLCyl / 2, ERK, PKC-θ / ΙΒΑ; increased activation of intracellular calcium levels; decreased histone acetylation or hypoacetylation and / or increased CpG methylation at the IL-2 locus. Therefore, in some embodiments, one or more of any of these parameters can be determined to determine whether the antibodies or antigen-binding fragments thereof disclosed herein that inhibit CEACAM1 reduce immune tolerance. The reduction of T cell tolerance can also be assessed by examining tumor-infiltrating lymphocytes or T lymphocytes within lymph nodes draining from established tumors. Such T cells exhibit "exhaustion" characteristics through expression of cell surface molecules (such as PD1, TIM-3 or LAG-3) and reduced secretion of cytokines (such as interferon-γ). Thus, evidence that T cell tolerance has been reduced in the presence of a CEACAM1 antibody or antigen-binding fragment thereof includes, for example, an increase in the number of T cells observed in the absence of the inhibitor, the T cells having (a) antigenic specificity for tumor-associated antigens (e.g., as determined by major histocompatibility complex class I or class II tetramers containing tumor-associated peptides) and (b) the ability to secrete high levels of interferon-γ and cytolytic effector molecules (such as granzyme-B).

[0611] CEACAM1 antibodies and antigen-binding fragments thereof can also be used to enhance T cell expansion, activation and proliferation.

[0612] On the other hand, the present invention provides a method for reducing the interaction between CEACAM1 and bacterial adhesins using the antibodies and antigen-binding fragments thereof of the present invention. In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention effectively reduce and / or prevent colonization of mammalian epithelium. In some embodiments, the adhesin is expressed by Escherichia coli, particularly diffusely adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, Neisseria meningitidis, commensal Neisseria, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus aegypti, Helicobacter pylori and / or Salmonella. In one embodiment, the CEACAM1 antibody or its antigen-binding fragment disrupts the interaction between CEACAM1 and HopQ expressed on the surface of Helicobacter pylori. In another embodiment, the CEACAM1 antibody or its antigen-binding fragment disrupts the interaction between CEACAM1 and the turbidity-associated (Opa) adhesin protein expressed on the surface of Neisseria. In another embodiment, the CEACAM1 antibody or its antigen-binding fragment disrupts the interaction between CEACAM1 and the OMP adhesin protein expressed on the surface of Haemophilus.

[0613] In one embodiment, the CEACAM1 antibody or its antigen binding fragment disrupts the interaction between CEACAM1 and Candida albicans. In one embodiment, the CEACAM1 antibody or its antigen binding fragment disrupts the interaction between CEACAM1 and influenza virus (including but not limited to H5N1). In one embodiment, the present invention provides a method for inhibiting the binding of CEACAM1 to a sub-nematode using the CEACAM1 antibody or its antigen binding fragment described herein. In one embodiment, the sub-nematode is Wuchereria bancrofti.

[0614] Treatment

[0615] In one aspect, the present invention provides CEACAM1 antibodies and antigen-binding fragments thereof, which can also be used to treat a subject in need thereof.

[0616] 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. Although the antibodies or antigen-binding portions thereof described herein are particularly useful for administration to humans, they 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, domestic pets, and farm animals. "Therapeutically effective amount" means an amount effective to produce the desired therapeutic effect when an antibody or antigen-binding portion thereof described herein is administered to a mammal.

[0617] In some aspects, the antibody or its antigen-binding fragment binds to CEACAM1 expressed by exhausted T cells or natural killer (NK) cells, thereby restoring T cell and NK cell activity and leading to increased anti-tumor response. In other aspects, the antibody or its antigen-binding fragment binds to CEACAM1 expressed by tumor cells, thereby inhibiting tumor cell metastasis and the formation of cancer stem cell niches. In another aspect, the antibody or its antigen-binding fragment binds to CEACAM1 expressed by macrophages associated with fibrosis in the tumor environment, thereby inhibiting fibrosis. On the other hand, the antibody or its antigen-binding fragment binds to CEACAM1 expressed by other stromal cells (such as vascular endothelial cells) in the tumor microenvironment, thereby inhibiting angiogenesis.

[0618] Therefore, the present invention also provides a method for treating a subject with cancer or a tumor and / or reducing tumor growth, which comprises administering an effective amount of a CEACAM1 antibody or an antigen-binding fragment thereof provided herein. "Reducing" includes inhibiting and / or reversing, and can refer to, for example, the symptoms of the condition being treated, the presence or size of metastases or micrometastases, the size of primary tumors, the presence or size of dormant tumors.

[0619] The term "cancer" refers to or describes a physiological condition characterized by unregulated cell growth in mammals. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Therefore, as used herein, the term "cancer" refers to the uncontrolled growth of cells, which interferes with the normal function of body organs and systems (including cancer stem cells and tumor vascular niches). A subject suffering from cancer is a subject in which objectively measurable cancer cells are present in the subject. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that transfer from their original location and inoculate important organs can eventually lead to the death of the subject by functional degradation of the affected organs. Hematopoietic cancers, such as leukemia, can overcome the normal hematopoietic compartment of the subject, resulting in hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia), ultimately leading to death.

[0620] "Subject" means a mammal, including but not limited to a human or a non-human mammal, such as a cow, horse, dog, sheep or cat, etc. Individuals and patients are also subjects herein.

[0621] As used herein, the term "treat, treated, treating or treatment" refers to therapeutic treatment, wherein the purpose is to slow down (mitigate) an unwanted physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. For the purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, relieving symptoms; reducing the extent of a condition, disorder or disease; stabilizing (i.e., not worsening) the state of a condition, disorder or disease; delaying the onset of a condition, disorder or disease or slowing its progression; improving a condition, disorder or disease state; and alleviating (partially or completely), detectable or undetectable or enhancing or improving a condition, disorder or disease. Treatment includes causing a clinically significant response without an excessive level of side effects. Treatment also includes extending survival compared to the expected survival without treatment. The terms "prevent", "prevention" and the like refer to acting before an apparent disease or condition occurs to prevent the development of a disease or condition or to minimize the extent of a disease or condition, or to slow down its development process.

[0622] Embodiments of the present invention can be used to treat metastasis, which involves cancer spreading from its primary site to other parts of the body. Cancer cells can break away from the primary tumor, infiltrate lymph and blood vessels, circulate through the bloodstream, and grow in distant lesions (metastasis) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a continuous process, depending on tumor cells falling off from the primary tumor, passing through the bloodstream and stopping at a distant site. In the new site, cells establish a blood supply and can grow to form a life-threatening mass. Both the stimulation and inhibition of molecular pathways in tumor cells regulate this behavior, and the interaction between tumor cells and host cells at distant sites is also important. In addition to monitoring specific symptoms, metastasis is most often detected by using magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays, and bone scans alone or in combination.

[0623] Also contemplated is a method of reducing cancer stemness, comprising administering a CEACAM1 antibody or antigen-binding fragment thereof disclosed herein. Cancer stemness may refer to the ability of a cell to self-renew and produce additional phenotypically different cell types. Cancer stem cells (CSCs) are cancer cells that exhibit stem cell-like properties. CSCs typically exhibit at least one cancer marker and are capable of producing at least one additional phenotypically different cell type. In addition, cancer stem cells are capable of asymmetric and symmetric replication. It should be understood that cancer stem cells may be produced by differentiated cancer cells that acquire stemness traits and / or stem cells that acquire a phenotype associated with cancer cells. Alternatively, cancer stem cells may reconstruct non-stromal cell types within a tumor.

[0624] CEACAM1 is expressed by many tumor types, and CEACAM1 can regulate the growth and metastatic behavior of tumors. In another embodiment, CEACAM1 inhibition will reduce tumor growth and metastasis.

[0625] CEACAM1 expression on a subset of macrophages is associated with fibrosis during carcinogenesis. In another embodiment, CEACAM1 inhibition will reduce tumor-associated fibrosis.

[0626] Cancers that can be treated by the compositions and methods contemplated by the present invention include tumors that are not vascularized or not substantially vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as blood tumors, e.g., leukemias and lymphomas) or may include solid tumors. Types of cancer to be treated include, but are not limited to, benign and malignant tumors, and 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, but are not limited to, 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; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasm; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small 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 pharynx); 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; urological cancer; vulvar cancer; and other carcinomas and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin 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 non-lytic cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with pharyngopathy, edema (such as that associated with brain tumors), and Meigs syndrome. Patients may have more than one type of cancer.

[0627] The efficacy of cancer treatment methods including therapeutic formulations comprising compositions of antibodies and antigen-binding fragments thereof described herein can be measured by various endpoints commonly used to evaluate cancer treatments, including but not limited to tumor regression, tumor weight or size reduction, time to progression, survival duration, progression-free survival, overall response rate, response duration, and quality of life. In the case of cancer, a therapeutically effective amount of a recombinant CEACAM1 antibody or antigen-binding fragment thereof can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to some extent and preferably prevent) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down to some extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with the condition to some extent. In the case of a patient with more than one type of cancer, a therapeutically effective amount of a recombinant CEACAM1 antibody or antigen-binding fragment thereof is an amount that effectively treats at least one of the cancers. Insofar as a recombinant CEACAM1 antibody or antigen-binding fragment thereof is used to prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing survival duration, progression-free survival duration (PFS), response rate (RR), response duration, and / or quality of life.

[0628] Checkpoint proteins interact with specific ligands, sending signals to T cells and shutting off or inhibiting T cell function. By expressing high levels of checkpoint proteins on their surface, cancer cells can control the function of T cells entering the tumor microenvironment, thereby inhibiting anticancer immune responses. The immune checkpoint protein programmed death-1 (PD-1) is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands of PD-1, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), have been identified, which are expressed on antigen presenting cells and many human cancers and have been shown to downregulate T cell activation and cytokine secretion when binding to PD-1 (Freeman et al., 2000; Latchman et al., 2001). Inhibition of PD-1 / PD-L1 interaction can promote effective anti-tumor activity. Examples of PD-1 inhibitors include, but are not limited to, pembrolizumab (MK-3475), nivolumab (MDX-1106), cemiplizumab-rwlc (REGN2810), pidilizumab (CT-011), spartalizumab (PDR001), tisellizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI 754091, JS001, LZM009, MEDI0680, MGA012, Sym021, TSR-042. Examples of PD-L1 inhibitors include, but are not limited to, atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BGB-A333, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316.

[0629] However, there is a large population of cancer patients receiving checkpoint inhibitor therapy who (1) do not respond to this type of therapy (innate or primary resistance) or (2) initially respond but eventually develop disease progression (secondary or acquired resistance). Resistant cancers may also be referred to as refractory cancers. As shown in the examples below, tumor-associated cells isolated from patients with acquired resistance to PD-1 / PD-L1 inhibitors upregulate CEACAM1 expression relative to tumor-associated cells isolated from primary patients who were not exposed to PD-1 inhibitors. When CEACAM1 is expressed in the context of acquired resistance, cells carrying CEACAM1 are more likely to be effector memory cells rather than central memory cells, which is consistent with the reduction in anticancer responses in resistant patients.

[0630] Therefore, the present invention also provides a method for treating patients with resistance to checkpoint inhibitors (such as inhibitors of PD-1, PD-L1 and / or CTLA-4) using CEACAM1 antibodies and antigen-binding fragments thereof (including but not limited to specific CEACAM1 antibodies and antigen-binding fragments thereof provided herein). 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, resistance is innate or primary resistance. In some embodiments, resistance is secondary or acquired resistance. In some embodiments, the CEACAM1 antibody administered (including but not limited to CEACAM1 antibodies and antigen-binding fragments thereof provided herein) reverses T cell exhaustion in patients with resistance to checkpoint inhibitor therapy. Any cancer that exhibits resistance to PD-1, PDL-1 and / or CTLA-4 is suitable for treatment with the methods of the present invention. In some embodiments, the CEACAM1 antibody or antigen-binding fragment is administered to a patient who has not previously received checkpoint inhibitor therapy.

[0631] In another aspect, the present invention provides for the use of the CEACAM1 antibodies and antigen-binding fragments provided herein in treating patients resistant to therapy with other checkpoint inhibitors, including but not limited to PD-L2, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is present in all NK, γδ and memory CD8 + (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).

[0632] In another aspect, the present invention provides the use of the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to treat a subject in need of reducing and / or preventing the colonization of mammalian epithelium by Candida albicans and / or bacteria expressing bacterial adhesins (including but not limited to Escherichia coli, in particular diffusely adherent Escherichia coli (DAEC), Neisseria, Neisseria meningitidis, commensal Neisseria, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus aegypti, Helicobacter pylori and / or Salmonella). In another aspect, the present invention provides a method of reducing the replication of influenza virus and / or reducing the release of proinflammatory cytokines or chemokines associated with influenza virus infection using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein. In some embodiments, the influenza virus is H5N1. In another aspect, the present invention provides a method of treating a subject in need of reducing and / or preventing infection by a subnematode (such as Wuchereria bancrofti) using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein. In another aspect, the present invention provides a method for treating a subject in need of reducing and / or preventing the development of lymphedema and / or hydrocele associated with infection with a sub-nematode (such as Wuchereria bancrofti) using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein. In one embodiment, the present invention provides a method for reducing filarial invasion of the subject's lymphatic system in a subject in need thereof using the CEACAM1 antibodies or antigen-binding fragments thereof described herein. In one embodiment, the sub-nematode is Wuchereria bancrofti. The subject may be infected with more than one bacterial adhesin-expressing bacteria, Candida albicans, influenza virus, and / or sub-nematodes.

[0633] In another embodiment, the present invention provides a method of using the CEACAM1 antibodies or antigen-binding fragments thereof described herein to reduce invasion of the subject's lymphatic system by cancer cells in a subject in need thereof.

[0634] Screening methods

[0635] Also provided herein are methods of identifying a patient population that may respond to treatment with the CEACAM1 antibodies and antibody fragments provided herein, including but not limited to CP08H03 / Vk8 S29A and CP08H03 / CP08F05.

[0636] In some embodiments, the CEACAM1 expression on certain cell types of cancer patients is screened, and the cell types include T cells, NK cells, tumor cells, or other cells in the tumor microenvironment, such as macrophages. In some embodiments, cancer patients showing increased CEACAM1 expression on certain cell types compared to the control are selected for treatment with CEACAM1 antibodies and antibody fragments provided herein. The "control" level of CEACAM1 expression may refer to the level of CEACAM1 expression in one or more individuals who do not suffer from cancer. The level can be measured on an individual-by-individual basis, or on an aggregate basis such as an average value. In some embodiments, the control level of CEACAM1 expression obtained at different times from the same individual whose condition is monitored. In certain embodiments, the "control" level may refer to the level obtained from the same patient at an earlier time (e.g., weeks, months, or years ago). In some embodiments, the control level is obtained from the patient before the patient receives any cancer therapy. In some embodiments, the control level is obtained from the patient before the patient receives checkpoint inhibitor treatment.

[0637] In some embodiments, CEACAM1 expression is measured for patients who are resistant to checkpoint inhibitor therapy (including but not limited to therapy with PD-1 / PD-L1 / CTLA-4 inhibitors). In some embodiments, patients who are resistant to checkpoint inhibitor therapy and show increased CEACAM1 expression on certain cell types compared to controls are selected for treatment with CEACAM1 antibodies and antibody fragments provided herein (including but not limited to CP08H03 / Vk8 S29A and CP08H03 / CP08F05).

[0638] In some embodiments, the patient is determined for allelic variants of human CEACAM1. Based on the allelic variants of human CEACAM1 expressed by the patient, more or less anti-CEACAM1 antibodies can be administered to the patient compared to patients expressing wild-type variants of CEACAM1. In some embodiments, the presence of Y34C, Q44L and / or Q89H allelic variants of CEACAM1 in the patient is determined. In some embodiments, higher and / or more frequent doses of anti-CEACAM1 antibodies are administered to patients expressing Y34C, Q44L and / or Q89H allelic variants of CEACAM1 compared to patients expressing wild-type variants of CEACAM1.

[0639] Pharmaceutical composition

[0640] In another aspect, the present invention provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of a CEACAM1 antibody or antigen-binding fragment thereof described herein formulated together with one or more pharmaceutically acceptable excipients.

[0641] The dosage of the active agent may vary according to the reason for use, individual subjects and mode of administration. The dosage may be adjusted based on the subject's weight, the subject's age and health, and the tolerance of the compound or composition. For example, depending on the disease, for an antibody or its antigen-binding fragment, this may require 0.1, 1.0, 3.0, 6.0 or 10.0 mg / Kg. For an IgG (two binding sites) with a molecular weight of 150,000 g / mole, these dosages correspond to binding sites of about 18 nM, 180 nM, 540 nM, 1.08 μM and 1.8 μM for a volume of 5 L of blood.

[0642] Active agents and excipients can be formulated into compositions and dosage forms according to methods known in the art. The pharmaceutical compositions of the present invention can be particularly formulated into solid or liquid forms, including those suitable for parenteral administration, such as by subcutaneous, intratumoral, intramuscular or intravenous injection, such as as sterile solutions or suspensions.

[0643] Therapeutic compositions comprising antibodies or antigen-binding fragments thereof that bind to CEACAM1 can be formulated with one or more pharmaceutically acceptable excipients, which can be pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, talc, magnesium, calcium or zinc stearate, or stearic acid), solvents or encapsulating materials involved in carrying or transporting the therapeutic compound for administration to a subject, fillers, salts, surfactants and / or preservatives. Some examples of materials that can be used as pharmaceutically acceptable excipients include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffered solutions; and other nontoxic compatible substances used in pharmaceutical formulations.

[0644] Bulking agents are compounds that add mass to the pharmaceutical 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.

[0645] The use of surfactants can reduce the aggregation of reconstructed proteins and / or reduce the formation of particles in the reconstructed formulation. The amount of surfactant added should be able to reduce the aggregation of reconstructed proteins and minimize the formation of particles after reconstruction. 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 glucoside; Lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; Lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; Linoleyl-, myristyl- or cetyl-betaine; Lauramide propyl-, cocoamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitamidopropyl- or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmitamidopropyl- or isostearamidopropyl-dimethylamine; sodium methyl cocoyl or disodium methyl oleyl taurate; and polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.).

[0646] Preservatives can be used in the preparation of the present invention. Suitable preservatives for preparation of the present invention include octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride (a mixture of alkyl benzyl-dimethyl ammonium chloride, wherein 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 paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and meta-cresol. Other suitable excipients can be found in standard pharmacy textbooks, such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, the 19th edition Mack Publishing Company, Easton, Pa., (1995).

[0647] Compositions comprising antibodies or antigen-binding fragments thereof and pharmaceutically acceptable carriers may include various concentrations of CEACAM1 antibodies or antigen-binding portions thereof as described herein. For example, the composition may include 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 antibodies or antigen-binding fragments thereof. The composition may also include 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 antibodies or antigen-binding fragments thereof.

[0648] In some embodiments, the composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier is lyophilized and provided in the composition for reconstitution prior to administration.

[0649] Application method

[0650] The therapeutic composition comprising the contemplated antibody or antigen-binding fragment thereof can be administered in any convenient manner, including by injection, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intracranially, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

[0651] In certain embodiments, the antibody or its antigen-binding fragment is administered to a mammal by intravenous infusion, i.e., the antibody or its antigen-binding fragment is introduced into the vein of the mammal over a period of time. In certain embodiments, the period of time is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.

[0652] In certain embodiments, the dosage of the compound or composition is applied to the subject every day, every other day, every two days, every three days, once a week, twice a week, three times a week, once every two weeks, or once a month. In other embodiments, two, three or four doses of compounds or compositions are applied to the subject every day, every two days, every three days, once a week, once every two weeks, or once a month. In some embodiments, the dosage of the compound or composition is applied for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, or 28 days. In certain embodiments, the dosage of the compound or composition is applied for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or longer.

[0653] Combination therapy

[0654] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof administered with an additional therapeutic agent. Such additional agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitors, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, and anti-infective agents. The agents used in such combination therapies may belong to one or more of the aforementioned categories. The administration of the antibody or its antigen-binding fragment and the additional therapeutic agent may be simultaneous or continuous. The administration of the antibody or its antigen-binding fragment and the additional therapeutic agent may be separate or as a mixture. In addition, the therapeutic methods contemplated by the present invention may involve treatment in combination with one or more cancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiotherapy.

[0655] Exemplary additional therapeutic agents also include radionuclides with high energy ionizing radiation that can cause multiple strand breaks in nuclear DNA and are therefore suitable for inducing cell death (e.g., of 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 generally produce high energy alpha or beta particles with short path lengths. Such radionuclides kill cells in their immediate vicinity, such as neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on nonlocalized cells and are essentially nonimmunogenic.

[0656] Exemplary additional therapeutic agents also include cytotoxic agents, such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or cross-linkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, and the like.

[0657] Exemplary additional therapeutic agents also include alkylating agents, such as the anthracycline family (e.g., doxorubicin, carmine, cyclosporin-A, chloroquine, methotrexate, mithramycin, porphyrin, streptomycin, anthracenedione and aziridine). In another embodiment, the chemotherapeutic moiety is a cytostatic agent, 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-dichlorodiaminoplatinum (II) (cisplatin), melphalan, mitoxantrone and oxaliplatin.

[0658] Exemplary additional therapeutic agents also include transcriptional regulators, such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine and idarubicin. Other exemplary cell growth inhibitors compatible with the present invention include ansamycin benzoquinone, quinone derivatives (e.g., quinolones, genistein, bactacyclin), busulfan, ifosfamide, nitrogen mustard, triimidazole quinone, diazocone, carboquinone, indolequinone EO9, divinyl iminobenzoquinone methyl DZQ, triethylene phosphoramide and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0659] Exemplary additional therapeutic agents also include cytotoxic nucleosides, such as, for example, adenine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, tegafur, and 6-mercaptopurine; tubulin binding agents, such as taxanes (e.g., paclitaxel, docetaxel, taxanes), nocodazole, radixin, dolastatin (e.g., dolastatin 10, 11, or 15), colchicine, and colchicine analogs. Narcissines (e.g. ZD6126), combretastatins (e.g. combretastatin A-4, AVE-6032) and vinca alkaloids (e.g. vinblastine, vincristine, vindesine and vinorelbine (Navelbine)); anti-angiogenic compounds such as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine and (±)-thalidomide.

[0660] Exemplary additional therapeutic agents also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroxyprogesterone), 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, luteinizing hormone-releasing hormone, pifithrin-α, rapamycin, sex hormone binding globulin, and thapsigargin.

[0661] Exemplary additional therapeutic agents also include enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside, etoposide, formestane, forsitracin, milk echinopsin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mavinocine, trichostatin A, tyrosine phosphorylation inhibitor AG 34, and tyrosine phosphorylation inhibitor AG 879.

[0662] Exemplary additional therapeutic agents also include gene modulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehyde), retinoic acid, retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0663] Exemplary additional therapeutic agents also include cytotoxic agents, such as, for example, pteridine family drugs, enediynes, and podophyllotoxins. Particularly useful members of these classes include, for example, methotrexate, podophyllotoxin or podophyllotoxin derivatives such as etoposide or etoposide phosphate, isovinabine, vindesine, vinblastine, etc.

[0664] Still other additional therapeutic agents compatible with the teachings herein include auristatins (e.g., auristatin E and monomethyl auristatin E), calicheamicin, gramicidin D, maytansinoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, teniposide, colchicine, dihydroxyanthracenedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0665] In one embodiment, CEACAM antibodies or their antigen binding fragments are administered in combination with a medicament as a checkpoint inhibitor. Such inhibitors may include small molecule inhibitors or may include antibodies or their antigen binding fragments that bind and block or inhibit immune checkpoint receptors or antibodies that bind and block or inhibit immune checkpoint receptor ligands. Illustrative checkpoint molecules that can be targeted for blocking or inhibiting include but are not limited to CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (belonging to CD2 family molecules and in all NK, γδ and memory CD8 + (αβ) T cells), CD160 (also known as 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. Illustrative immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40 and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitors) as well as the above-mentioned PD-1 and PD-L1 inhibitors. Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0666] In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered with a TIGIT, LAP, Podoplanin, Protein C receptor, ICOS, GITR, CD226, or CD160 inhibitor.

[0667] In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered together 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 together with a TIM-3 inhibitor.

[0668] It should be understood that the present invention is not limited to the specific molecules, compositions, methods or schemes described, because these can change. Any methods and materials similar or equivalent to those described herein can be used to implement or test embodiments of the present invention. It should also be understood that the disclosure of the present invention in this specification includes all possible combinations of such specific features. For example, in the case where specific features are disclosed in the context of a specific aspect or embodiment of the present invention or a specific claim, the features may also be used in combination with other specific aspects and embodiments of the present invention to the extent possible and / or in the context of other specific aspects and embodiments of the present invention, and are generally used in the present invention.

[0669] Where 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 additional steps performed before any defined step, between two defined steps, or after all defined steps (unless the context excludes those possibilities).

[0670] All other cited patents and applications are incorporated herein by reference in their entirety. In addition, to the extent that a definition or use of a term in a reference incorporated herein by reference is inconsistent or contradictory to the definition of that term provided herein, the definition of that term provided herein shall prevail and the definition of that term in the reference shall no longer apply.

[0671] In order to facilitate a better understanding of the present invention, the following examples of specific embodiments are given. The following examples should not be construed as limiting or defining the entire scope of the present invention.

[0672] Example

[0673] Example 1: Generation of fully humanized CEACAM1 antibody

[0674] 1. Generation of humanized antibody variants

[0675] Design of composite human antibody variable region sequences and antibody expression

[0676] 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 "limiting" amino acids in the V region that might contribute to the antibody binding properties. For regions outside of and flanking the CDRs, a wide selection of human sequence segments were identified as possible components of new humanized V regions.

[0677] Based on structural analysis, a large preliminary set of sequence segments that can be used to generate humanized CEACAM antibody variants were selected and analyzed in silico using iTope for peptides binding to human MHC class II alleles.TM Technology (Perry et al., 2008. Drugs RD 9(6):385-396) and the use of TCED TM The T cell epitopes associated with known antibody sequences (Bryson et al. 2010, Biodrugs 21(1):1-8) were analyzed. TM Sequence segments that obtained significant hit scores were discarded. This analysis resulted in a reduced set of segments, and the combination of these segments was analyzed again as described above to ensure that the connections between segments did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V region sequences that lacked important T cell epitopes. The heavy and light chains selected for gene synthesis, expression in mammalian cells, and activity testing are listed in

[0678] Table 1.

[0679] Some of the heavy and light chains in Table 1 contain variations at positions that are considered part of a CDR according to the Kabat CDR definition but not according to the IMGT CDR definition.

[0680] Table 1. Selection of heavy and light chains for gene synthesis.

[0681]

[0682] Next, V sequences of the parent murine CEACAM1 antibody and humanized CEACAM antibody variants were synthesized with flanking restriction enzyme sites. H and Vκ sequences for cloning into the pANT expression vector system for IgG4(S241P) heavy chain and κ light chain ( Figure 1 ). H The V region was cloned between MluI and HindIII restriction sites, and the Vκ region was cloned between BssHII and BamHI restriction sites. All constructs were confirmed by sequencing.

[0683] Thirty-nine heavy chain and light chain pairs were transiently transfected into HEK EBNA adherent cells using the PEI transfection method and incubated for 5-7 days after transfection. These 39 pairs included three controls: (1) chimeric antibody V H 0 / Vκ0 (mouse V H Area (V H 0) and a mouse Vκ region (Vκ0) fused to the constant light chain region of human IgG4); (2) 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 were composite IgG4 VH and Vκ variants: V H 1 pairs with Vκ1 to Vκ12, V H 2 pairs with Vκ1 to Vκ12, V H 3 pairs with Vκ1 to Vκ6, and V H 4 pairs with Vκ1 to Vκ6.

[0684] Antibodies were purified from cell culture supernatants on a protein A agarose column, the buffer was exchanged to PBS pH 7.4, and quantified by OD 280 nm using an extinction coefficient based on the predicted amino acid sequence. 1 μg of each antibody was analyzed by SDS-PAGE, and bands corresponding to a typical antibody profile were observed. The size of the light chain and the presence of a faint band at 25 kDa indicated that the glycosylation motif identified in the light chain was substantially utilized.

[0685] Competition ELISA analysis of humanized variants binding to CEACAM1

[0686] Binding of purified antibodies to human CEACAM1 was evaluated in a competition ELISA assay. Nunc ImmunoMaxiSorp 96-well flat-bottom microtiter plates were pre-coated with 1 μg / ml GST-CEACAM1 in 1x PBS overnight at 4°C. The next day, the plates were blocked with 2% BSA / PBS for 1 hour at room temperature ("RT") and then washed 3 times with PBST pH 7.4. Chimeric antibody V was added at 100 μg / ml to 0.07 or 0.002 μg / ml. H A 3-fold dilution series of 0 / Vκ0, irrelevant IgG4 antibody and humanized CEACAM1 antibody was premixed with a constant concentration of the parental mouse antibody (0.45 μg / ml final concentration), added to the plate and incubated for 1 hour at room temperature. After 3x PBST washing, the binding of the parental mouse CEACAM1 antibody was detected with anti-mouse-HRP and TMB substrate. The reaction was terminated with 3M HCl, and the absorbance was read at 450nm on a Dynex Technologies MRX TC II microplate reader and the binding curve was plotted. The binding of the humanized CEACAM antibody variants to CEACAM1 was compared with the chimeric antibody (V H Twelve of the 36 humanized CEACAM antibody variants showed no binding to CEACAM1 (including those of Vκ3, Vκ4, and Vκ5). H Variants that bind to CEACAM1 showed relative IC50 values ​​ranging from 0.9 to 5.2 compared to 0 / Vκ0. The data are summarized in Table 2.

[0687] Table 2. Summary of titer and binding data for humanized CEACAM antibody variants and control antibodies. Antibody expression titers (μg / ml) were obtained from static HEK EBNA transient transfections. IC50 values ​​obtained in competition assays were normalized to chimeric antibody V on the same plate. H 0 / Vκ0. Unbound antibodies are not included in the table. Antibodies in bold were used for multi-cycle kinetic analysis.

[0688] Variants Expression titer (μg / ml) Average relative IC50 value Number of experiments <![CDATA[V H 0 / Vκ0(control)]]> 22.9 1 4 <![CDATA[V H 0 / Vκ1(control)]]> 21.3 1.6 4 <![CDATA[V H 1 / Vκ0(control)]]> 30.5 0.9 4 <![CDATA[V H 1 / Vκ1]]> 20.6 2.5 4 <![CDATA[V H 1 / Vκ2]]> 23.1 1.8 4 <![CDATA[V H 1 / Vκ4]]> 27.2 2.7 4 <![CDATA[V H 1 / Vκ7]]> 34.6 1.6 2 <![CDATA[V H 1 / Vκ8]]> 33.3 1.7 2 <![CDATA[V H 1 / Vκ9]]> 39.2 1.5 2 <![CDATA[V H 1 / Vκ10]]> 35.4 1.6 2 <![CDATA[V H 1 / Vκ11]]> 38.0 2.2 2 <![CDATA[V H 1 / Vκ12]]> 44.5 1.4 2 <![CDATA[V H 2 / Vκ1]]> 10.6 2.8 4 <![CDATA[V H 2 / Vκ2]]> 43.9 2.0 4 <![CDATA[V H 2 / Vκ4]]> 30.2 3.0 4 <![CDATA[V H 2 / Vκ7]]> 41.7 1.3 2 <![CDATA[V H 2 / Vκ8]]> 44.9 1.6 2 <![CDATA[V H 2 / Vκ9]]> 39.6 1.7 2 <![CDATA[V H 2 / Vκ10]]> 35.6 1.4 2 <![CDATA[V H 2 / Vκ11]]> 32.0 1.4 2 <![CDATA[V H 2 / Vκ12]]> 40.9 1.5 2 <![CDATA[V H 3 / Vκ1]]> 17.1 3.5 4 <![CDATA[V H 3 / Vκ2]]> 17.0 2.5 4 <![CDATA[V H 3 / Vκ4]]> 21.0 5.2 4 <![CDATA[V H 4 / Vκ1]]> 13.7 4.0 4 <![CDATA[V H 4 / Vκ2]]> 29.7 3.3 4 <![CDATA[V H 4 / Vκ4]]> 30.1 4.0 4

[0689] Kinetic analysis of humanized variants binding to CEACAM1

[0690] As an alternative method to evaluate the binding of 36 antibody combinations and three control antibodies to CEACAM1, kinetic analysis was performed on a Biacore T200 (serial number 1909913) running Biacore T200 evaluation software V2.0.1 (Uppsala, Sweden). All experiments were performed at 25°C with HBS-P + running buffer (pH 7.4) (GE Healthcare, catalog number BR100671). All kinetic experiments were performed using His-tagged CEACAM1 as the analyte. For all experiments, the antibodies were immobilized on the surface of a S-series protein A sensor chip. For kinetic experiments, the amount of immobilized / captured ligand was limited to avoid mass transfer effects on the chip surface, where the surface ideally has an analyte binding level (R of 50-150 RU). max ). For all samples the antibody capture settings used a MW of 45 kDa for the CEACAM1 analyte, an antibody ligand of 150 kDa (estimated value for IgG), an Rmax of 50 RU, and a stoichiometry (Sm) of 2 since each antibody can bind 2 target molecules, a target response level of approximately 75 RU.

[0691] A single cycle analysis of 36 antibody combinations and three control antibodies was performed on purified antibodies (Vκ1 to Vκ6 variants) or supernatants of transiently transfected HEK EBNA cells (Vκ7 to Vκ12 variants). In some cases, when supernatants were not available, purified chimeric antibodies V H 0 / Vκ0 was added to HEK EBNA medium to serve as a positive control. The antibody was diluted to a concentration of 1 μg / ml in HBS-P+ (determined by IgG quantitative ELISA). At the beginning of each cycle, the antibody was loaded onto Fc2, Fc3, and Fc4 of the protein A chip and IgG was captured at a flow rate of 8 μl / min to obtain a RU of approximately 75. The surface was then stabilized. Single cycle kinetic data were obtained at a flow rate of 50 μl / min to minimize any potential mass transfer effects. H0 / Vκ0 were repeated multiple times to check the stability of the surface and analyte during the kinetic cycle. The signal from the reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3 and Fc4 to correct for differences in nonspecific binding to the reference surface. A 5-point 2-fold dilution range of 3.125 to 50 nM CEACAM1 was used without regeneration between concentrations. The association phase of 5 injections of increasing concentrations of CEACAM1 was monitored for 100 seconds and after the last injection of CEACAM1, a single dissociation phase was measured for 150 seconds. Regeneration of the protein A surface was performed using 2 injections of 10 mM glycine-HCl pH 1.5, followed by a 500-second stabilization period. The signal from each antibody blank run (no CEACAM1) was subtracted to correct for differences in surface stability. Single cycle kinetics (

[0692] Table 3) shows that 24 humanized variants bind to CEACAM1, while 12 variants do not. When combined with any humanized heavy chain, the light chains of Vκ3, Vκ5 and Vκ6 abolished CEACAM1 binding. These data are consistent with the competition ELISA data (Table 2).

[0693] Table 3. Single cycle kinetic parameters for binding of humanized CEACAM antibody variants and control antibodies to CEACAM1-HIS determined using Biacore T200. D Divide by the chimeric antibody V determined in the same experiment H K of 0 / Vκ0 D To calculate the chimeric antibody V H 0 / Vκ0 compared to the relative K D Antibodies in bold were used for multi-cycle kinetic analysis. S / N, supernatant. Non-binding variants are not included in the table.

[0694]

[0695] Among the 24 antibodies that bind to CEACAM1, the chimeric antibody V H Antibody variants that bound within 2-fold of Vκ0 / Vκ0 and had relative IC50s ranging from 0.9 to 1.8 were used for 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, VH 2 / Vκ10, V H 2 / Vκ11 and V H 2 / Vκ12 (see Table 2 and

[0696] Table 3, highlighted in bold).

[0697] For multi-cycle kinetic analysis, purified antibodies were fixed in HBS-P+ at a protein concentration of 1 μg / ml. At the beginning of each cycle, antibodies were captured on protein A to obtain about 75 RUs and stabilize the surface. Kinetic data were obtained at a flow rate of 80 μl / min to minimize any potential mass transfer effects. Multiple repetitions of blank (no CEACAM1) and repetitions of analytes of a single concentration were programmed into the kinetic run to check the stability of the surface and analyte in the kinetic cycle. For kinetic analysis, a 2-fold dilution range of 200 to 3.125 nM or 100 to 1.5625 nM CEACAM1 was selected. The association phase of CEACAM1 was monitored for 50 or 150 seconds, and the dissociation phase was measured for 100 seconds. At the end of each cycle, two injections of 10 mM glycine-HCL pH 1.5 were used to regenerate the protein A surface.

[0698] The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface, and a global Rmax parameter was used in a 1-to-1 binding model. D Divide by the chimeric antibody V on the same chip H K of 0 / Vκ0 D To calculate with V H 1 / Vκ0 compared to the relative K D The kinetic parameters measured for the interaction of CEACAM1 with humanized CEACAM antibody variants are shown in Table 4. Table 5 summarizes the average relative K values ​​obtained for the 12 antibody combinations using multi-cycle kinetic analysis. D .

[0699] Selectivity analysis of humanized variants binding to CEACAM1

[0700] The 24 humanized antibody variants binding to CEACAM1 (see Table 3) as well as the chimeric control antibody V H 0 / Vκ0 has a selective binding to CEACAM1. Figure 2 As shown, most variants were highly selective for CEACAM1 and showed little or no binding to CEACAM3, 5, 6, or 8. HAll 24 variants showed reduced binding to CEACAM5 compared to Vκ0 / Vκ0. There was no evidence of any staining of HeLa-CEACAM3 or HeLa-CEACAM8 transfectants. Therefore, this data is not reported. Vκ0 was selected due to its favorable affinity and selectivity for CEACAM1 and its favorable expression level. H 1 / Vκ8 serves as a framework for affinity maturation.

[0701] Table 4. Multi-cycle kinetic data of composite human antibody variants binding to CEACAM1-HIS using Biacore T200 (n=1). D Divide by the chimeric antibody V measured on the same chip H K of 0 / Vκ0 D To calculate the chimeric antibody V H 0 / Vκ0 compared to the relative K D .

[0702]

[0703] Table 5. Comparison of all humanized CEACAM antibody variants and chimeric antibody V obtained from multi-cycle kinetics determined using Biacore T200 H 0 / Vκ0 compared to the relative K D By comparing the K D Divide by the V of the chimeric antibody tested on each chip H K of 0 / Vκ0 D To calculate the chimeric antibody V H K compared to 0 / Vκ0 D The number of independent experiments for each variant is shown.

[0704]

[0705]

[0706] 2. Removal of N-linked / HEK-derived glycosylation

[0707] Sequence analysis revealed a potential N-linked glycosylation motif in the original mouse hybridoma light chain CDR1. The CDR1L of the parental murine antibody contained 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 light variable chain, see Figure 3C), which makes the N26 residue a target for N-linked glycosylation. To reduce potential glycosylation-related 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, such as Figure 4 shown.

[0708] Competition 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 with the antibody mutant N26Q, the antibody mutant S29A (Kabat numbering scheme) showed a higher expression level and a K value more similar to the non-mutated antibody. D (See Table 9) while maintaining high selectivity for CEACAM1. Therefore, during further development, the S29A mutation (Kabat numbering scheme) was incorporated into the CEACAM1 lead antibody.

[0709] Table 6. Results of competition ELISA experiments using aglycosylated chimeric antibodies. CDR1L residues are numbered according to the Kabat numbering scheme.

[0710]

[0711] Table 7. Multi-cycle kinetic analysis using aglycosylated chimeric antibodies. CDR1L residues are numbered according to the Kabat numbering scheme.

[0712]

[0713] Table 8 Selectivity analysis of non-glycosylated humanized variants. CDR1L residues are numbered according to the Kabat numbering scheme. The binding selectivity of the antibody variants was assessed by flow cytometry using HeLa cells transfected with vectors expressing CEACAM1, CEACAM5 and CEACAM6, respectively. Shown are the relative amounts of cells expressing the corresponding antigens bound by the indicated antibodies.

[0714] Variants CEACAM1 CEACAM5 CEACAM6 <![CDATA[V H 0 / Vκ0]]> 78.00 8.81 0.30 <![CDATA[V H 0 / Vκ0N26Q]]> 79.90 15.50 0.18 <![CDATA[V H 0 / Vκ0S29A]]> 77.90 9.82 0.00 Control IgG4 0.37 1.02 1.39

[0715] Table 9. Experimental summary of aglycosylated chimeric antibodies. CDR1L residues are numbered according to the Kabat numbering scheme.

[0716]

[0717] 3. Non-glycosylated CEACAM1 antibody VH1 / VK8 Affinity maturation of S29A Phage vector construction and parental V H 1 / VK8 S29A scFv binding assay

[0718] For the lead antibody VH 1 / VK8 affinity maturation, construct encoding V H The genes for VK1 and VK8 were amplified and converted into an scFv format using overlapping PCR, in which the heavy chain was linked 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 in the primary amino acid sequence of the light variable chain ( Figure 3C The scFv sequence was then cloned into the phagemid vector pANT43 using restriction enzymes Sfi I and Not I, allowing the scFv to be displayed 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 confirmed by sequencing. H Phages expressing 1 / VK8 S29A scFv or irrelevant scFv were tested for binding to GST-CEACAM1 ( Figure 6 ). Derived from parent V H The phage with the 1 / VK8 S29A sequence specifically bound to the antigen, as no binding was observed with the irrelevant phage.

[0719] Mutagenesis and library construction

[0720] To construct an affinity maturation library, semi-random codons were used to target aglycosylated humanized antibody V H Specific amino acids within CDR1H, CDR3H, and CDR3L of 1 / Vκ8S29A were used for "hotspot" mutagenesis. Sequence positions of possible contact residues were analyzed and ranked in order within each block. This information was used along with the amino acid preference for any given position in CDR3 and the crystal structure of the parental murine antibody. Where possible, the highest ranked contact residues within each block were prioritized.

[0721] Four different libraries were generated: one library for mutations in CDR1H (HC), two libraries for mutations in CDR3H, and one library for mutations in CDR3L (see Figure 7 ).

[0722] CDR1H was identified as being five amino acids in length (S31 to S35) (Kabat definition, corresponding to residues 31-35 of the primary amino acid sequence of the heavy variable chain, see Figure 3A ), where the IMGT CDR1H definition (G26 to G33) covers a more extended region. Taking all of this into account and combining it with the crystal data of the parental murine antibody, G26 to S35 were included in a single library, where each position included a subset of amino acids.

[0723] CDR3H was identified as being 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 heavy variable 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 heavy variable chain according to the Kabat definition, see Figure 3A and 3B ): Block 1 (R94 to D100 according to the Kabat definition, corresponding to residues 98 to 104 in the primary amino acid sequence of the heavy variable chain, see Figure 3A and 3B ) and block 2 (D100 to Y102 according to the Kabat definition, corresponding to residues 104 to 110 in the primary amino acid sequence of the heavy variable chain, see Figure 3A and 3B ), where each block contains a subset of amino acids in all positions. Including position R94 (corresponding to residue 98 of the primary amino acid sequence of the heavy variable chain according to the Kabat definition, see Figure 3A and 3B ) (Block 1) to allow for more diversity in the germline residues anchoring the CDRs.

[0724] CDR3L was identified as being 9 amino acids in length (Q89-T97) (corresponding to residues 88 to 96 of the primary amino acid sequence of the light variable chain according to the Kabat definition, see Figure 3C Region Q90 to P96 (corresponding to residues 89 to 97 of the primary amino acid sequence of the light variable chain according to the Kabat definition, see Figure 3C ) are contained in a single library, where each position includes a subset of amino acids. Kabat numbering is used for all protein sequence coordinates.

[0725] Fig. 8A , 8B 8C shows an overview of library construction. H 1 / Vκ8S29A non-expression plasmid with a truncated fragment of the parental scFv and two consecutive stop codons in the region to be randomized. The purpose of this step is to reduce the possibility that the parental scFv is produced and dominates the selection (as occasionally observed during affinity maturation), so that only recombinant antibody fragments generated by PCR can form functional scFv in the phagemid vector.

[0726] For the CDR3L library, randomization of CDR3L was performed by performing two PCRs. In the first PCR, a randomized 3′ primer and a V sequence containing a Sfi I restriction site were used. HThe FW1 specific 5' primer was used to amplify the majority of the scFv gene and introduce mutations into Vκ CDR3. A second PCR added the remainder of the scFv and appended a restriction site (Not I) for subcloning of the fragment.

[0727] For V H The V library was prepared by performing two PCRs using two templates containing the full-length parental scFv portion. H Initially, the Vκ light chain FW4 was amplified using a randomized 5′ library primer and a 3′ primer specific for the Vκ light chain FW4. H In a separate PCR, a 5′ primer based on the heavy chain FW1 region was added with a primer that was identical to the V H The 3′ primer complementary to a portion of the CDR randomized primer amplifies the V H The full-length V was then constructed by annealing the two amplified fragments and re-amplifying the scFv by PCR using primers that append two restriction sites (Sfi I or Not I) for subcloning of the fragments. H CDR randomized scFv library.

[0728] In order to evaluate the diversity of the generated library, the purified amplified DNA of all four libraries was then digested with Sfi I and Not I and connected to a similarly cut phagemid vector (pANT43). The connected 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. The library was electroporated multiple times to fully cover the theoretical library diversity. In all cases, 4.0 times or greater coverage was obtained. Single colonies from each of the four libraries were sequenced to confirm that the appropriate CDR blocks had mutated.

[0729] Bacteria from each library were inoculated into 150 ml 2TYCG (2%) cultures using an inoculum of at least 10x the observed library diversity. Cultures were grown to mid-log phase (OD 600nm ≈0.5-0.6), and estimated the total number of cells (based on OD 600nm 1≈5x10 8cells / ml). Helper phage was added and incubated for 1 hour, then centrifuged, resuspended in 2TYCK medium and grown overnight at 30°C. The next day, the culture supernatant was recovered by centrifugation and the phage was harvested using a 4 / 10x volume of cooled 20% PEG / 2.5M NaCl precipitation. After incubation on ice for 1 hour, the precipitated phage was recovered by centrifugation and the precipitate was resuspended in 1x PBS pH 7.4. The supernatant was centrifuged again to remove any cell debris and then the supernatant was reprecipitated as described above. The precipitated phage was resuspended in 1x PBS pH 7.4 and filter sterilized. In order to increase the chance of obtaining scFv with increased affinity, due to the relatively low affinity of the starting antibody, the multivalent superphage M13 K07ΔpIII helper phage was used for library recovery at a multiplicity of infection of 20. After the first round of selection, due to the enrichment of the expected antigen binders, the monovalent M13K07 helper phage with a multiplicity of infection of 10 was used.

[0730] Phage selection with improved affinity

[0731] Two separate selection strategies were implemented to increase the possibility of obtaining phages with improved affinity. CEACAM1 was biotinylated (for soluble selection) or not biotinylated (for solid phase panning) throughout the selection. Soluble selection (activity 1) or solid phase panning (activity 2) was used in different selection cascades in the first round to enrich functional binding phages and diversity. The deselection using closely related family members CEACAM5 and CEACAM6 was performed by separate panning of each protein at 1 μg / ml to try and reduce cross-reactivity. This was performed twice during each activity by either deselecting before any round of selection and before the second round (activity 1) or before the second and third rounds of selection (activity 2). For both activities, the four libraries were kept separate at all stages.

[0732] For soluble selection, each library was pre-blocked with PBSB, and then the phage was incubated with a decreasing concentration of biotinylated CEACAM1 antigen for up to three hours. After incubation, streptavidin paramagnetic beads (pre-blocked as above) were added to each selection and rotated upside down for 15 minutes. The streptavidin-antigen-phage complex was washed with an increasing number of PBST washes in each round of continuous selection, followed by PBS washings, and captured with a magnet between each step. Phage was eluted from the beads by adding 50mM HCl, and then neutralized by adding 1M Tris-HCl pH 9.0 solution.

[0733] Solid phase panning and all deselections were performed on Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates coated with antigen at 4°C overnight and then blocked with PBSB. For deselection, pre-blocked phage were incubated with CEACAM5 and then with CEACAM6, and then unbound phage were removed and used for subsequent selections. For CEACAM1 panning, pre-blocked phage were incubated with 8 μg / ml antigen and the plates were then washed with 3x PBST and 2x PBS. As with soluble selections, bound phage were eluted with 50 mM HCl. For soluble selections and panning, eluted phage were added to mid-log E. coli TG1 and cells were infected at 37°C for 1 hour, then plated on 2TYCG (2%) plates and grown overnight at 37°C. The next day, colonies were picked for screening, or plates were scraped and phages were revived as described above. Fig. 9A and 9B An overview of the different selection strategies used is shown.

[0734] Expression and initial testing of scFv

[0735] Soluble scFv was initially expressed and tested as a crude periplasmic extract. A single colony was picked into 1 ml of 2TYCG (0.1%) medium and grown by shaking at 37°C for 5 hours. The culture was induced by adding IPTG to a final concentration of 1 mM and then grown overnight at 30°C with shaking. The next day, the culture was 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 5mM MgSO4. The plate was then centrifuged and the supernatant containing the scFv was transferred to a fresh plate for determination.

[0736] Periplasmic extracts from colonies from different rounds of selection were screened for their ability to bind GST-CEACAM1 in a single-site binding assay. The parental scFv (V H 1 / Vκ8S29A) and irrelevant scFv were used for comparison.

[0737] Periplasmic extracts were blocked by diluting 1:1 with PBSB and then incubated for 1 hour at room temperature on Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates pre-coated with 1.0 μg / ml GST-CEACAM1. The plates were then washed and the binding of scFvs was detected with anti-HIS6-HRP antibody and TMB substrate. The reaction was stopped with 1 M HCl, the absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader, and the binding data were plotted.

[0738] Based on the relative expression of the parental scFv V in binding ELISA H 1 / Vκ8S29A (it contains mouse CDR) and the activity of unrelated scFv measured on the same plate to identify improved clones. Analyzed more than 4400 periplasmic extracts, 34 kinds of leader sequences that were at least 1.5 higher than the parent in two independent experiments were sequenced, and unique clones were identified. Inspection of the obtained sequence shows that the parent amino acid is found in several positions, but is encoded by a codon different from the parent. This shows that the selection occurs as expected, but the parent amino acid is the preferred amino acid at this position. Based on this sequence analysis, 19 kinds of unique CDR1H, three kinds of CDR3H block 1, three kinds of CDR3H block 2 and 9 kinds of unique CDR3L clones are used for large-scale scFv expression. Table 10 shows the summary of 34 kinds of leader sequences selected as purified scFv for further analysis and the CDR mutations of these mutants.

[0739] Tables 11, 12 and 13 highlight the conservation / variability of affinity matured CDRs in scFv variant lead sequences identified using GST-CEACAM1 binding ELISA.

[0740] Table 10. Summary of 34 scFv variant leader sequences identified using GST-CEACAM1 combined with ELISA. Summarizes the libraries of scFv sources and the rounds of deselection performed by scFv. Parent (Vκ8S29A) CDRs are shown at the top of the table. Mutations that differ from the parental sequence in CDR1H, CDR3H B1, CDR3H B2, and CDR3L are highlighted in bold. All variable light chains contain S29A mutations in CDR1L (Kabat numbering scheme, corresponding to the S28A mutations in the primary amino acid sequence of the variable light chain).

[0741] Variants CDR1H CDR3H (Block 1) CDR3H (Block 2) CDR3L Parent GFIFSSHGMS RHDFD DAAWFAY QWSSNPP CP09E05 RHGFDY CP09F05 QNTALPF CP09F03 GFTFNNHGMS CP09A04 GFSFNAHAMS CP09E03 GFTFSAHAIS CP09D03 GFTFSSHAIS CP09B02 GFTFTSHAIS CP09C02 EFTFSDHAMS RHGFDY CP09B03 GFTFNAHAIS CP09G03 GFTFNAHAMS CP08G09 QWTAFPP CP08D02 QWTSFPP CP08G02 QWTNNPP CP08C08 QNTSLP CP08F05 QWTSNPP CP08E05 QWTTNPP CP08G01 QNTNLP CP08E01 QWTTFPP CP08B04 FPAWFAL CP08H03 FPYW CP08G10 FPAWFAF CP08H01 KHPPDY CP08B01 GFTFSAHAMS CP08A08 GFIFTNHGMS CP08A03 GFIFNNHAIS CP08B03 GFTFTAHAIS CP08D11 GYSFSAHGMS CP08B11 GFTFTNHGMS CP08C04 GFTFSSHGMS CP08B06 GFSFNSHAIS CP08F07 GFTFTDHAIS CP08C01 GYSFSNHGMS CP08A06 GYSFSSHGMS CP08D01 GFTFNAHGMS

[0742] Table 11. CDR motifs of heavy chain CDR1 in scFv variant leader sequences identified using GST-CEACAM1 binding ELISA. $The residue numbering is based on the Kabat numbering scheme. *The residue numbering is based on the primary amino acid sequence of the heavy variable chain. CDR1H comprises residues 31-35 according to the Kabat CDR definition and residues 26-33 according to the IMGT definition.

[0743]

[0744] Table 12. CDR motifs for heavy chain CDR3 in scFv variant leader sequences identified using GST-CEACAM1 binding ELISA. $ The residue numbering is based on the Kabat numbering scheme. *The residue numbering is based on the primary amino acid sequence of the heavy variable chain. # Not part of the CDR according to the Kabat definition. The residues were included in the mutagenesis to allow for more diversity in the germline residues anchoring the CDRs.

[0745]

[0746] Table 13 CDR motifs of light chain CDR3 in scFv variant leader sequences identified using GST-CEACAM1 binding ELISA. $ Residue numbering is based on the Kabat numbering scheme. *Residue numbering is based on the primary amino acid sequence of the light variable chain. #Residues were not mutated during affinity maturation.

[0747]

[0748] Large-scale ScFv expression and purification

[0749] The selected clones were expressed, purified and quantified to accurately test the scFv by binding ELISA. Briefly, a single colony was picked into 15 ml 2TYCG (2%) medium and grown overnight by shaking at 30°C. The starter culture was used to inoculate 500 ml 2TYCG (0.1%) and grown at 30°C until OD 600nm = ~ 0.8. The culture was induced by adding IPTG to a final concentration of 1mM and then grown overnight at 30°C with shaking. The next day, the culture was centrifuged and the supernatant discarded. The bacterial pellet was resuspended in 15ml TES and incubated on ice for 15 minutes. Then 22.5ml TES (diluted 1:5 in cold water) was added and incubated on ice for another 30 minutes. The cells were then centrifuged and the supernatant containing the scFv was transferred to a new test tube, and MgCl2, NaCl and imidazole were then added to final concentrations of 1mM, 300mM and 20mM, respectively, to reduce nonspecific binding. Ni-agarose beads were added and the scFv was bound by rotating and incubating for 2 hours at 4°C. The beads were precipitated by centrifugation and washed twice with wash buffer (25mMTris pH 7.4, 300mM NaCl, 20mM imidazole), and then eluted from the beads using elution buffer (25mM Tris pH 7.4, 300mM NaCl, 400mM imidazole). The samples were quantified by measuring OD 280nm and using the extinction coefficient based on the predicted amino acid sequence. About 1 μg of each scFv was analyzed by SDS-PAGE. Bands corresponding to typical scFv spectra were observed.

[0750] Evaluation of scFv binding to GST-CEACAM1 by ELISA assay

[0751] Binding of affinity matured purified scFv to human CEACAM1 was analyzed using GST-CEACAM1. NuncImmuno 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 H Two-fold dilution series of 1 / Vκ8S29A parental scFv or test scFv (50 μg / ml to 0.8 μg / ml) in PBSB were incubated on pre-coated ELISA plates at RT for 2 hours. Binding of scFv was detected with anti-HIS6-HRP antibody and TMB substrate. The reaction was stopped with 3M HCl, and the absorbance was read at 450nm on a Dynex Technologies MRX TC II microplate reader and binding curves were plotted. Exemplary binding assay data are shown in Fig.10 In each ELISA plate, the parent scFv (V H 1 / Vκ8S29A scFv) as a reference. An irrelevant scFv was included as a negative control on at least one plate. H Compared with the V1 / Vκ8S29A scFv, many scFvs have improved binding to CEACAM1. HImproved binding was observed for scFvs of Vκ and Vκ libraries. All 34 scFv variants were reformatted as complete IgG to provide higher accuracy for purity and quantification. Reformatting further allows analysis of the affinity component of antibody binding, which is affected by the bivalent nature of IgG. As used herein, "avidity" is a measure of the binding strength between an antigen binding molecule (such as an antibody or its antibody fragment described herein) and a related antigen.

[0752] Construction and testing of affinity-matured intact antibodies

[0753] scFv reformatted to full IgG

[0754] 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. The 25 affinity matured V H The variants were subcloned into the IgG4 S241P pANTVhG4 vector. Similarly, the nine affinity-matured Vκ sequences were subcloned into the κ light chain pANTVK vector using the BssH II and BamH I restriction sites. All constructs were confirmed by sequencing.

[0755] For expression, 25 lead humanized affinity-matured IgG4 V H The variants were combined with the parental humanized aglycosylated light chain (Vκ8S29A). The nine lead humanized affinity-matured κ light chains were combined with the parental humanized heavy chain (V H 1) Combinations. 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, the supernatant was harvested, quantified by ELISA and filtered for Biacore single-cycle kinetic analysis.

[0756] Single-cycle kinetic analysis of binding of humanized and affinity-matured lead IgG to CEACAM1

[0757] To evaluate the binding of the humanized affinity-matured reformatted lead IgG, single-cycle kinetic analysis of the crude supernatant was performed using a Biacore T200 running Biacore T200 control software V2.0.1 and Biacore T200 evaluation software V3.0. The antibody was diluted in HBS-P+ to a final concentration of 0.5 μg / ml. At the beginning of each cycle, the antibody was loaded onto the Fc2, Fc3, and Fc4 of the protein A chip. The IgG was captured at a flow rate of 10 μl / min to obtain an immobilization level (RL) of approximately 100 RU (the calculated R of approximately 50-150 RU after binding to the analyte) 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 any potential mass transfer effects. H 1 / Vκ8S29A) antibody was repeated multiple times to check the stability of the surface and the analyte in the kinetic cycle. The signal from the reference channel Fc1 (no antibody) was subtracted from the signal of Fc2, Fc3 and Fc4 to correct the difference in non-specific binding to the reference surface. A three-point two-fold dilution range of 70nM to 280nM CEACAM1 without regeneration between each concentration was used. The signal from each antibody blank run (no CEACAM1) was subtracted to correct the difference in surface stability. The dissociation phase of three increasing concentrations of CEACAM1 injections was monitored for 80 seconds each time and after the last injection of CEACAM1, a single dissociation phase was measured for 150 seconds. Two injections of 10mM glycine-HCL pH 1.5 were used to regenerate the protein A surface, followed by a 250-second stabilization period.

[0758] Single cycle kinetic constant (

[0759] Table 14) shows that all antibodies except one humanized affinity matured antibody bound to CEACAM1.

[0760] Table 14. Humanized and affinity matured variants and parent (V H The single-cycle kinetic constants for binding of the humanized and affinity-matured variants were calculated using the K D Divide by the K of the parent determined multiple times in the same experiment D To calculate the relative K compared to the parental IgG D The variants made are shown in bold. CDRs containing mutations are indicated by "+". All variable light chains contain the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain). The variants shown in bold have the same H1 / Vκ8S29A > twice the relative K D .

[0761]

[0762]

[0763] Eight humanized and affinity-matured heavy and light chain variants were identified that showed >twofold relative K compared to the parental D (exist

[0764] These include three V H variants (CP08H03, CP09B03 and CP09C02) and five kappa light chain variants (CP08E01, CP08E05, CP08F05, CP08D02 and CP08G09).

[0765] CP09C02 (which was derived from the CDR1H library) contained an additional point mutation in CDR3H B1 (D96G), which was most likely introduced by PCR during the library construction phase (see "Example 1, 1. Generation of humanized antibody variants" section). Therefore, an additional heavy chain clone CP09E05 was also obtained because it was identified as H CDR3 B1 has only this single point mutation and may therefore help identify which region is involved in the observed increase in affinity. H and five Vκ variants were used to determine whether recombinant affinity-matured heavy and light chains could have improved effects.

[0766] Expression of combined lead heavy and light chain antibodies

[0767] Four humanized affinity-matured IgG4 V proteins identified after expression with the parental light chain H Each of the variants (CP08H03, CP09B03, CP09C02, and CP09E05) was combined with five lead humanized affinity-matured κ light chains (CP08E01, CP08E05, CP08F05, CP08D02, and CP08G09) (i.e., a total of 20 pairings, see Table 15). As a control, the humanized affinity-matured IgG4 V H The variants were combined with the parental light chain (Vκ8S29A), and the five lead humanized affinity-matured κ light chains were combined with the parental heavy chain (V H1) Combination (i.e., a total of 10 control antibodies, see Table 15). As described above, the combination was transiently transfected into HEK EBNA adherent cells in 6-well plates using the PEI transfection method and incubated for 5-7 days after transfection. The supernatant was harvested, quantified by ELISA and filtered for single-cycle kinetic analysis on Biacore.

[0768] Table 15. Parent V H or lead humanized affinity matured IgG4 V H Combination of variants with parent Vκ or lead humanized affinity-matured Vκ light chains. Transfected antibodies with recombinant affinity-matured heavy and light chains (black), affinity-matured antibodies and parent antibodies expressed with parent heavy or light chains. All variable light chains contain S29A mutations in CDR1L (Kabat numbering scheme, corresponding to S28A mutations in the primary amino acid sequence of variable light chains).

[0769]

[0770]

[0771] Single-cycle kinetic analysis of combined lead heavy and light chain antibodies

[0772] Single-cycle kinetics using transient HEK supernatants were performed as described previously. The fitted data for single-cycle kinetics are shown.

[0773] Table 16. Fifteen heavy and light chain combinations with at least twofold relative K compared to the parent D Among them, six combinations (CP08H03 / CP08E05, CP08H03 / CP08F05, CP08H03 / Vк8S29A, CP09B03 / CP08E05, CP09C02 / CP08E05, and CP09C02 / CP08F05) obtained K D More than four times larger than the parent (in

[0774] Highlighted in bold in Table 16). These six variants were used for large-scale production and protein A purification for further analysis. Single cycle kinetics also revealed that three variants were found to be non-functional when combined.

[0775] Table 16. Leading V H The single-cycle kinetic constants for the binding of the Vκ and Vκ combination antibodies and the parental antibodies to CEACAM1. H K of Vκ and Vκ combination variants D Divide by the K of the parent determined in the same experiment D To calculate the relative K compared to the parent D The variant highlighted in bold is KD Lead combinations that are >4 times that of the parent. CDRs containing mutations are indicated by "+". All variable light chains contain the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the S28A mutation in the primary amino acid sequence of the variable light chain).

[0776]

[0777]

[0778] Recombination of four affinity matured heavy chain CDRs to generate six additional heavy chain variants

[0779] Six combinations with greater than four times improvement are included in four different V H Three unique heavy chains with mutations in the CDRs: CP08H03 (CDRH3 B2); CP09B03 (CDR1H) and CP09C02 (CDR3H B2 and a single mutation in CDR3H B1 that is also uniquely present in CP08E05), see Table 17. To determine if further improvement could be obtained, four mutations V H Recombination of CDRs (Table 18). Using scFv-specific primers, penetration PCR was used to recombine each V H The CDRs were then cloned into the IgG4 S241P heavy chain expression vector using Mlu I and Hind III restriction sites to generate six new V H Variants (8H3_9B3, 8H3_9C2, 8H3_9E5, 9B3_9E5, 8H3_9C2 (CDR1) and 9B3_9E5_8H3).

[0780] Table 17 VH CDRs used for recombination.

[0781]

[0782] Table 18 Recombinant heavy chain clones. H The cloned individual CDRs were recombined to generate six recombinant affinity matured heavy chains. Mutations in CDR1H, CDR3H B1 and CDR3H B2 that differ from the parental sequence are highlighted in bold. * Residue 104 was selected based on the sequence selected for CDR3H B2.

[0783]

[0784]

[0785] Recombinant V H Expression of CDR1 and CDR3 heavy chain and leader light chain:

[0786] The six recombinant V H CDR1 and CDR3 variants (see Table 18) were combined with (1) parental light chain (Vκ8S29A), (2) light chain CP08E05 or (3) light chain CP08F05. When combined with affinity-matured heavy chains, the latter two light chains previously gave improved effects (see Table 16). 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 after transfection. The supernatant was harvested, quantified by ELISA and filtered for single-cycle kinetic analysis on Biacore.

[0787] Table 19 Recombinant V H CDR1 and CDR3 lead humanized affinity matured IgG4V H Variant and parental Vκ or two leading humanized affinity matured κ light chains. Combinations with two affinity matured light chains are indicated by "AM", and combinations with parental light chains are indicated by "P". All variable light chains contain S29A mutations in CDR1L (Kabat numbering scheme, corresponding to S28A mutations in the primary amino acid sequence of variable light chains).

[0788]

[0789] Recombinant V H With leading V L Single cycle kinetic analysis of antibodies:

[0790] Single cycle kinetics using transient HEK supernatants were performed as described previously. The fitted data for single cycle kinetics are shown in Table 20.

[0791] Eight variants were found to have a relative K greater than fourfold compared to the parent. D Among them, five obtained K D More than six times that of the parent (bold in Table 20). Five antibodies (8H3_9B3 / CP08E05, 8H3_9B3 / CP08F05, 8H3_9B3 / Vк8S29A, 8H3_9C2 / CP08F05 and 9B3_9E5 / CP08E05) were used for large-scale production and protein A purification for further analysis.

[0792] Table 20 Recombinant lead humanized affinity matured IgG4 V H The single-cycle kinetic constants of the variants with the parental Vκ or one of the two lead humanized affinity-matured κ light chains. The K D Divide by the K of the parent determined in the same experiment DTo calculate the relative K compared to the parent D Variants with > six-fold variation compared to the parent are highlighted in bold. CDRs containing mutations are indicated with a "+". All variable light chains contained an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0793]

[0794] Expression, purification and testing of lead antibodies

[0795] The six most improved combination variants (CP08H03 / CP08E05, CP08H03 / CP08F05, CP08H03 / Vк8S29A, CP09B03 / CP08E05, CP09C02 / CP08E05, and CP09C02 / CP08F05) were quantified using the PEI method.

[0796] Highlighted in bold in Table 16) and the five most improved V H CDR1 and V H CDR3 recombinant variants (8H3_9B3 / CP08E05, 8H3_9B3 / CP08F05, 8H3_9B3 / Vк8S29A, 8H3_9C2 / CP08F05 and 9B3_9E5 / CP08E05, highlighted in bold in Table 20) were transiently transfected into HEK EBNA adherent cells in Erlenmeyer flasks and incubated for 5-7 days post-transfection. Antibodies were purified from cell culture supernatants on a protein A agarose column, the buffer was exchanged to PBS pH 7.2, and the extinction coefficients based on the predicted amino acid sequences were calculated by OD 280nm Quantification was performed. 2 μg of each antibody was analyzed by SDS-PAGE, and bands corresponding to the typical antibody spectrum were observed.

[0797] Single-cycle kinetic analysis of purified lead humanized and affinity matured antibodies (using purified protein)

[0798] Single cycle kinetics were performed as described above using purified antibodies instead of HEK supernatant. The fitted data for single cycle kinetics are shown in Table 21. The expression levels of the individual mutants are provided in Table 22.

[0799] All 11 lead variants had > four-fold binding compared to the parent antibody (see Table 21). The data obtained using purified IgG were consistent with those previously obtained using supernatants.

[0800] Table 21 Single cycle kinetic constants of purified lead humanized affinity matured antibodies. DDivide by the K of the parent determined in the same experiment D To calculate the relative K compared to the parent D Mutations in CDR1H, CDR3H B1, CDR3H B2 or CDR1L are indicated with a "+" where applicable. All variable light chains contained an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0801]

[0802] Table 22 Expression levels of purified lead humanized affinity matured antibodies. Mutations in CDR CDR1H, CDR3H B1, CDR3H B2 or CDR1L are indicated by "+" when applicable. All variable light chains contain S29A mutations in CDR1L (Kabat numbering scheme, corresponding to S28A mutations in the primary amino acid sequence of the variable light chain).

[0803]

[0804] Removal of potential CD4+ T cell epitopes

[0805] Use of iTope for in silico analysis of peptides binding to human MHC class II alleles TM technique (Perry et al. 2008) and using the TCED of T cell epitopes associated with known antibody sequences TM (Bryson et al. 2010) analyzed the sequences of 11 lead antibodies (see Table 21) to ensure that no significant T cell epitopes were introduced during affinity maturation. The CDR1 mutation (G26E, according to the CDR definition of IMGT) present in the heavy chain of CP09C02 was associated with the introduction of no promiscuous high epitopes observed in the parental sequence (see Table 21).

[0806] Selectivity analysis of lead antibodies

[0807] Initial selectivity analysis of several lead antibodies showed that antibodies with phenylalanine (F) at CDR3H position 104 exhibited, on average, increased selectivity for CEACAM1 compared to antibodies with aspartic acid (D) at CDR3H position 104 ( Fig.11 ).

[0808] Multi-cycle kinetic analysis

[0809] Variants CP08H03 / Vк8S29A and CP08H03 / CP08F05 were further analyzed using a Biacore T200 instrument running Biacore T200 evaluation software V3.0.1 using multi-cycle kinetic analysis. The purified antibodies were diluted to a concentration of 1 μg / ml in HBS-P+. At the beginning of each cycle, each antibody was captured on the protein A surface to obtain a RL of about 100RU. After capture, the surface was stabilized. Kinetic data were obtained using a flow rate of 80 μl / min to minimize any potential mass transfer effects. Multiple repetitions of blanks (no CEACAM1) and repetitions of a single concentration of analyte were programmed into the kinetic run to check the stability of the surface and analyte in the kinetic cycle. For kinetic analysis, a two-fold dilution range from 100 to 1.56 nM CEACAM1 was selected. The association phase of CEACAM1 was monitored for 150 seconds, and the dissociation phase was measured for 150 seconds. At the end of each cycle, regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5.

[0810] The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface, and a global Rmax parameter was used in a 1-to-1 binding model. D Divide by the K of the parent on the same chip D To calculate the parent (V H 1 / Vκ8S29A) compared to the relative K D The kinetic parameters measured for the interaction of CEACAM1 with the affinity matured CEACAM1 antibody variants CP08H03 / Vк8S29A and CP08H03 / CP08F05 are shown in Table 23. H Both affinity-matured CEACAM1 antibody variants exhibited >fourfold affinity improvement compared to the 1 / Vκ8S29A parent.

[0811] Table 23 Antibody V determined using Biacore T200 H Figure 1 Multi-cycle kinetic data of binding of 1 / Vκ8S29A (parent), chimeric antibody (VH0 / VK0) and two affinity-matured lead sequences to CEACAM1. The K D Divide by the K of the parent determined on the same chip D To calculate the parent (V H 1 / Vκ8S29A) compared to the relative K D All variable light chains contained an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0812]

[0813]

[0814] Example 2: Selectivity of CEACAM1 Antibodies

[0815] To further evaluate the CEACAM1 antibody V H 0 / Vκ0, CP08H03 / Vк8S29A, CP08H03 / CP08F05 CEACAM1 binding selectivity relative to other proteins, using a single cycle kinetic analysis performed as described above to compare the binding affinity to CEACAM1, CEACAM3, CEACAM5 and CEACAM6. Single cycle kinetics were performed using CEACAM concentrations ranging from 280nM to 70nM. The antibodies were loaded onto the chip at the following concentrations (taking into account the different analyte MWs): 100RU for CEACAM1, 375RU for CEACAM3, 71.4RU for CEACAM5, and 150RU for CEACAM6. No significant differences were observed for the three CEACAM1 antibodies CP08H03 / Vк8S29A, CP08H03 / CP08F05 and Vκ0 for CEACAM3, CEACAM5 and CEACAM6. H 0 / Vκ0 significant binding (see Fig. 12A ).

[0816] These results are consistent with the data obtained by measuring antibody specificity using ELISA. For 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 PBS. CP08H03 / Vκ8S29A, CP08H03 / CP08F05, or Vκ8S29A were prepared in 2% BSA / PBS. H 0 / Vк0 1:3 dilution series (50 μg / mL starting concentration). 100 μL of sample was added to the pre-coated plate and incubated for 1 hour at RT. CEACAM antibodies were detected using anti-human Igκ chain-peroxidase secondary antibody (AP502P). The plate was developed with TMB and stopped with 3M HCl. The results were analyzed by subtracting the background. Essentially no CEACAM1 antibodies CP08H03 / Vκ8S29A, CP08H03 / CP08F05 and V H Binding of 0 / Vκ0 to CEACAM3, CEACAM5 or CEACAM6 (see Fig. 12B and 12C ).

[0817] This high degree of selectivity was observed despite the high homology of the N domains of the different CEACAMs: 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 in the percent identity matrix generated using Clustal2.1 (see Fig.13 ).

[0818] Example 3: Epitope analysis of CEACAM1 antibodies

[0819] To determine which residues on CEACAM1 are involved in binding to certain 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 antibodies VH0 / VK0 (chimeric antibody), VH1 / VK8, VH2 / VK4, VH3 / VK1 and VH4 / VK1 were used as detection antibodies. 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 CEACAM1 antibodies, indicating that these CEACAM1 residues may be involved in binding (see Fig.14 ).

[0820] Example 4: Crystal structures of CEACAM antibodies and CEACAM1

[0821] To more precisely delineate the binding interface between CEACAM1 and CP08H03 / VK8 S29A, the crystal structure of human CEACAM1 in complex with the CP08H03 / VK8 S29A Fab fragment was determined.

[0822] CEACAM1 was expressed by E. coli transformed with a pET21D-based plasmid expressing an untagged form of CEACAM1. The protein was refolded in an arginine-containing buffer and purified. Fab fragments were prepared by antibody digestion, concentrated to about 18 mg / ml, and then purified by protein A affinity and gel filtration chromatography using immobilized papain resin. Purified CEACAM1 and Fab were mixed in a 1:1 molar ratio before crystallization screening. Initial crystallization hits for CEACAM1:Fab complexes were determined and subsequently optimized. Diffraction quality crystals were grown at room temperature under conditions containing 18-20% PEG 6000, 50 mM potassium dihydrogen phosphate, 20 mM Tris pH 7.0, and 1% β-octyl glucoside. SDS-PAGE analysis and silver staining of washed crystals were used to confirm the crystallization of the complex. X-ray data for many crystals were collected from beamline NE-CAT 24-ID-E at the Advanced Photon Source of Argonne National Laboratory. The best data from two non-twinned isomorphous crystals are combined to produce A highly redundant dataset of 1000 Å was used for structure determination and refinement. The structure of the complex was solved by molecular replacement and refined to a final R and R 自由 The values ​​are 24.9% and 32.8% respectively.

[0823] The structure of the CEACAM1:CP08H03 / Vκ8S29A Fab complex was determined to be Resolution. CP08H03 / Vκ8S29A Fab binds CEACAM1 with a 1:1 stoichiometry (see Fig.15 ). Fig.16 Molecular surface representation of CEACAM1 in Figure 24 shows part of the epitope of the Fab fragment on CEACAM1. The major and minor interactions between the Fab molecules and CEACAM1 are listed in Tables 24 and 25.

[0824] Table 24. Major interactions between CEACAM1 CP08H03 / Vκ8S29A Fab #Residue numbering is based on the Kabat numbering scheme. *Residue numbering is based on the primary amino acid sequence of the heavy variable chain.

[0825]

[0826]

[0827] Table 25. Minor interactions between CEACAM1 CP08H03 / Vκ8S29A Fab #Residue numbering is based on the Kabat numbering scheme. *Residue numbering is based on the primary amino acid sequence of the heavy variable chain.

[0828]

[0829] Referring to the existing structure of CEACAM1 dimer ( Fig.17 ), it is clear that Fab binds to the interface of CEACAM1 involved in self-association. It is speculated that this competitive interaction leads to the dissociation of dimers in solution. The residues targeting CEACAM1 include four residues (Y34, Q44, Q89, N97) that form the YQQN pocket at the CEACAM1:CEACAM1 dimer interface. It is worth noting that several residues on CEACAM1 that bind 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. 2015Jan 15; 517(7534): 386-90).

[0830] In the Fab light chain, residues in CDR1, CDR2, and CDR3 ( Fig.18 ) interact primarily with residues in two loops between the β strands of the major β sheet in CEACAM1 and also with residues of β strands in said fold. In the Fab heavy chain, residues of CDR2 and CDR3 interact primarily with residues distributed on four different β strands of the central β sheet.

[0831] The interacting surfaces had a shape complementarity of 0.5 and complex formation buried the total solvent accessible surface of 1607A1. No interaction was observed between the antigen and the Fab heavy chain CDR1.

[0832] Alignment of the 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. In addition, human CEACAM5 contains a histidine at position 89. Polymorphisms of these residues in hCEACAM-1 include Ala49Val (rs8110904) and Gln89His (rs8111468). To further examine the selective properties of the CEACAM1 antibody CP08H03 / Vκ8S29A, the human CEACAM1 A49V / Q89H mutant was expressed and purified as described above. It is noted that there is a natural human allelic variant of human CEACAM1 that converts Q89 to H89, as described by Huang et al., Nature. 2015 Jan 15; 517(7534):386-90. The structure of the CEACAM1 A49V / Q89H mutant was determined to Resolution and comparison with the CEACAM1 wild-type CP08H03 / Vκ8S29A Fab complex. As described above, CDR3H residue F104 of CP08H03 / Vκ8S29A contacts residue F29 in wild-type CEACAM1 (see Fig.19 , left). Note that 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 / Vκ8S29A blocks the F29-F29 interaction. CEACAM1 residue A49 is located near the F104 / F29 interaction site. Due to the increased hydrophobicity of valine in non-CEACAM1 family members compared to alanine in human CEACAM1, mutation of human CEACAM1 residue A49 to valine results in movement of the hydrophobic CEACAM1 residue F29 closer to the CEACAM1 V49 residue. This rotameric shift of F29 is also observed in the human CEACAM5 (PDB code 2QSQ) and human CEACAM3 (PDB code 6AW1) crystal structures and is predicted to clash with CDR3H residue F104 (see Fig.19 , right). This is illustrated by the orientation change exhibited by the CEACAM1 F29 loop, which moves closer to the space previously occupied by CDR3H residue F104 (see Fig.19 , right). These data suggest that this steric hindrance caused by the A49V mutation interferes with the binding of the CEACAM1 antibody CP08H03 / Vκ8S29A to other CEACAM1 family members containing valine at position 49, and therefore its major effect on antibody selectivity. It is predicted that this rotational isomeric shift of the F29 loop will also affect the interaction between CDR2H residue Y57 and F29. In addition, the CEACAM1 Ala49Val polymorphism (rs8110904) is associated with lymphedema caused by Wuchereria bancrofti (a filarial worm that invades the lymphatic system) (Debrah L, B. et al. Hum Genomics. 2017Nov 9; 11(1):26). The development of the disease is associated with the Ala49Val polymorphism, which marks the alanine 49 residue found to be involved in binding to the CP08H03 / Vκ8S29A antibody. Therefore, it is expected that CP08H03 / VK8S29A may also interfere with W. bancrofti and other relevant pathogens or cancer processes where phenotypic worms interact with lymphatic vessels, such as tumor invasion.

[0833] Example 5: CEACAM1 Antibodies Block CEACAM1:CEACAM1 Interactions

[0834] The ability of CEACAM1 antibody to block CEACAM1 homodimerization was tested. CEACAM1-CEACAM1 competition ELISA studies were performed in triplicate to determine the ability of CP08H03 / Vκ8S29A antibody (concentration range 0-1000nM) to inhibit the binding of human CEACAM1 IgV domain untagged protein (1pg / ml) and human CEACAM1-GST protein (37.5pg / ml). In addition, IgG4 antibody was used as a control (0-1000nM). Goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) was used and measured by adding TMB solution (Life technologies). OD values ​​were read at 450nm on a microplate reader. Data were plotted in Graphpad and the best fit IC-50 value was determined.

[0835] CEACAM1 antibody CP08H03 / Vκ8S29A was shown to block CEACAM1:CEACAM1 homophilic interactions (see Fig. 20A ).

[0836] Example 6: CEACAM1 Antibodies Block CEACAM1:TIM-3 Interaction

[0837] The ability of CEACAM1 antibodies to reduce the binding of CEACAM1 to TIM-3 was detected. CEACAM1 / TIM-3 competition ELISA studies were performed in triplicate to determine the ability of CP08H03 / Vκ8S29A antibody (concentration range 0-300nM) to inhibit the binding of human TIM-3IgV domain untagged protein (3pg / ml) and human CEACAM1-GST protein (37.5pg / ml). In addition, human IgG4 antibody was used as a control (0-1000nM). A goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) was used and assayed by adding TMB solution (Life technologies). OD values ​​were read at 450nm on a microplate reader. Data were plotted in Graphpad and the best fit IC-50 value was determined. Fig. 20B As shown, CEACAM antibody CP08H03 / Vκ8S29A blocks CEACAM1:TIM-3 heterophilic interaction.

[0838] Example 7: CEACAM1 antibody induces T cell proliferation

[0839] The ability of CEACAM antibodies CP08H03 / Vκ8S29A and CP08H03 / CP08F05 to induce T cell proliferation was studied in humanized NOD scidγ mice (NSG mice). Fig.21Freshly isolated human PBMC (5x10^6) were transferred into NOD.Cg-PrMc by intraperitoneal (ip) injection. scld I / 2rg tm1Wjl / SzJ (NSG) mice. 21 days after PBMC injection, human immune cell engraftment in NSG animals was examined by tail bleeding. 24 and 31 days after PBMC injection, the first and second doses of the specified concentrations of CEACAM1 antibody or isotype control antibody were administered to humanized NSG mice by intraperitoneal injection. After the study was terminated (34 days after PBMC injection), the mice were killed and the spleens were surgically dissected for further analysis. Single cell suspensions from transplanted mice were stained with cell proliferation dyes and cultured in vitro for another 2 days in complete RPMI medium in the presence of soluble anti-human CD3 stimulation (2 μg / ml, OKT3 clone) and rIL-2 (40U / ml). The cells were maintained at a concentration of 10^7 cells / ml. After in vitro stimulation, the cells were stained with antibodies against the human CD45 pan-leukocyte marker and evaluated by flow cytometry.

[0840] No antibody-dependent cell-mediated cytotoxicity (ADCC) was observed in any of the groups tested (see Fig. 22 Administration of CEACAM antibodies CP08H03 / VK8 S29A or CP08H03 / CP08F05, respectively, resulted in an increase in antibody-induced T cell expansion in vivo (see Fig.23 ).

[0841] Example 8: CEACAM1 Antibody Reduces Tumor Growth in a Melanoma Model

[0842] To evaluate the ability of CEACAM1 antibodies to reduce tumor growth, 1x 10 6 MALME-3M (human melanoma) and 5x10 6 Individual PBMCs were injected subcutaneously into 7-8 week old male NSG (NOD.Cg-PrMc scld I / 2rg tm1Wjl / SzJ) mice. 600E ) cell line was established in 1975 from the metastatic site (lung) of a 43-year-old Caucasian male with metastatic melanoma. On days 7-9, all mice were confirmed to exhibit reconstituted T cell populations (experimental setup see Fig.24A ). Animals were treated intraperitoneally on days 10, 13, 17, 20 and 24 with CEACAM antibody CP08H03 / VK8 S29A or hIgG4 control antibody.

[0843] 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) in a 43-year-old Caucasian male with metastatic melanoma harboring BRAFV600E. 2x10^7 MALME-3M cells were injected subcutaneously (sc) into NOD.Cg-PrMc scld I / 2rg tm1Wjl / SzJ (NSG) mice. After a 30-minute adaptation period, freshly isolated human PBMCs (1x10^8) were then transferred into tumor-bearing NSG mice by intraperitoneal (ip) injection. 7 to 9 days after PBMC injection, NSG animals were examined for human immune cell engraftment by tail bleeding. At 10, 13, 17, 20, and 24 days after human cell injection, tumor-bearing humanized NSG mice received a total of five doses of CEACAM1 antibodies or isotype control antibodies at specified concentrations by intraperitoneal injection. After the study was terminated (34 days after human cell injection), the mice were sacrificed and surgically dissected.

[0844] The CEACAM1 antibody CP08H03 / VK8 S29A effectively reduced tumor growth and proliferation at various concentrations (see Fig. 24B , 24C and 25) without depleting the T cell population (see Fig. 22 In addition, administration of CEACAM1 antibody restored human CD4 + and CD8 + Proliferative capacity of tumor-infiltrating lymphocytes, see Fig.25 It was also observed that CD8 T memory cells were mainly biased towards central memory T cells when treated with CP08H03 / Vκ8S29A in vivo ( Fig.26 ). Note that the CEACAM1 antibody CP08H03 / VK8 S29A increased T relative to that observed in control treated animals. cm With T em The relative proportions of α and β are consistent with the enhanced anticancer response.

[0845] Example 9: CEACAM1 antibodies can be used to treat cancers resistant to checkpoint inhibitors

[0846] CEACAM1 is expressed on the majority of TILs derived from patients with naive melanoma or melanoma resistant to anti-PD-1 and / or anti-CTLA-4 therapy; and CEACAM1 expression levels are greater than those of PD-1 or TIM-3 (see Fig. 27 ). About 80% of the samples had more than 20% of the CD4 isolated from TILs. +CEACAM1 expression was shown on T cell populations. To compare CEACAM1 expression in patients with acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy with those not previously exposed to anti-PD-1 and / or anti-CTLA-4 therapy, tumor-associated cells (TACs) were obtained from patients with primary melanoma (not previously exposed to anti-PD-1 and / or anti-CTLA-4 therapy) or those with acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy (acquired resistance). TACs 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 was evaluated. + CD4 + and CD3 + CD8 + These studies showed that tumor-associated cells deprived of the tumor microenvironment in acquired resistance upregulated CEACAM1 expression relative to that observed in naive patients (see Fig.28 ), suggesting that patients resistant to anti-PD-1 and / or anti-CTLA-4 therapy may benefit from anti-CEACAM1 antibodies or antigen-binding fragments thereof, such as those contemplated in the present disclosure.

[0847] As expected, CD8 + Central memory in T cells (T cm ) cells relative to effector memory (T em ) is relatively reduced, which is consistent with the reduced anticancer response in resistant patients (see Fig.29 Tumor-associated cells from patients with naive melanoma (untreated patients) or melanoma patients resistant to immune checkpoint inhibitors (treatment failure patients) were stained for CD44, CCR7, and CD62L, and central memory T cells were stained for CD44, CCR7, and CD62L. cm (CD44 高 、CD62L 高 、CCR7 高 ) and effect memory T em (CD44 高 、CD62L 低 、CCR7 低 ) were expressed as a percentage of total CD8 T cells present in the bulk tumor.

[0848] 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 and stage IV disease to pembrolizumab (PD-1 inhibitor), ipilimumab (CTLA-4 inhibitor) + nivolumab (PD-1 inhibitor), and dabrafenib (B-Raf inhibitor) + trametinib (MEK inhibitor). Tumor-associated cells and PBMCs were stained for CEACAM1, PD1, or TIM-3, and CD8 + and CD4 + The proportion of T cells (see Fig.30 , left). Tumor biopsies were subjected to enzymatic digestion or a commercial mechanical / enzymatic dissociation system (GentleMACS dissociator, Miltenyi Biotec). Enzymatic digestion was based on previously established methods for generating melanoma TILs (Dudley et al., 2003, 2008). Briefly, tumor biopsies were cut into small fragments of approximately 2-3 mm in length and placed in a 100 U ml -1 DNA enzyme, 10 mg ml -1Collagenase VIII (Sigma-Aldrich) was used for the enzyme digestion mixture, and the mixture was incubated at 37 ° C for 45 minutes with continuous rotation. GentleMACS was dissociated according to the manufacturer's protocol. In brief, the tumor was cut into small fragments of about 2-3 mm in length and placed in a C tube (Miltenyi Biotech) with RPMI1640 (Lonza, Slough, UK) and solution 1, 2 and 3 (all from Miltenyi Biotec) according to the manufacturer's recommendation; then the digestion mixture containing the tumor was subjected to three 36-second mechanical disaggregation steps (program h_tumor_01.01, 02.01 and 03.01) in the GentleMACS dissociator, and two 30-minute incubations at 37 ° C were interspersed after the first and second disaggregation steps, respectively. After disaggregation, the TILs dissociated from the enzyme digestion and GentleMACS were passed through a 100 μm filter for further analysis. Dissociated tumor cells and autologous PBMCs were stained with the following antibodies according to standard procedures: fluorescent dye-conjugated monoclonal antibodies specific for human CD3, CD4, CD8, TIM-3, PD1, CEACAM1, CD45, and active dyes. Data were acquired with a Cytoflex flow cytometer (Invitrogen) and analyzed with FlowJo software (TreeStar, V7.6.5 for Windows). In 96-well plates, PBMCs or tumor-associated cells were incubated with soluble anti-CD3 (2 pg / ml) and rIL-2 (40 units / ml) in the presence of CP08H03 / VK8 S29A or hIgG4 controls in a 96-well plate supplemented with 10% fetal calf serum (FCS), 1% glutamine, 100 IU ml -1 Penicillin, 100 μg ml -1 The cells were cultured in complete medium (RPMI 1640 (Lonza)) containing streptomycin (Life Technologies) and 25 mM HEPES (Sigma-Aldrich). After 96 hours, the cell culture supernatant was collected for further TNF- and IFN-ELISA (BD) analysis according to the manufacturer's method. CP08H03 / VK8 S29A reversed T cell exhaustion in PD-1 / CTLA-4 resistant tumors, as demonstrated by increased TNF-α and IFN-γ production in both tumor-associated cells and PBMCs (see Fig.30 , right).

[0849] Example 10: CEACAM1 antibodies contemplated by the present invention exhibit improved efficacy compared to previously known CEACAM1 antibodies

[0850] The properties of antibody CP08H03 / VK8 S29A were compared with those of anti-CEACAM1 antibody CM-24 (WO2015 / 166484). Unlike the CEACAM1 antibody CP08H03 / VK8 S29A disclosed herein, CM-24 (i) binds CEACAM1 away from the dimer interface based on modeling, (ii) exhibits cross-reactivity with CEACAM3 and CEACAM5, (iii) shows limited ability to reverse T cell tolerance in TILs, and (iv) acts as an agonistic antibody rather than an antagonistic antibody in a mouse model of metastatic melanoma.

[0851] CP08H03 / Vκ8S29A was selective for CEACAM1 and did not show 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 ( Fig.31A and 31B ). The cervical adenocarcinoma cell line HeLa (ATCC No CCL-2) and transfected cell lines HeLaCEACAM1, HeLaCEACAM3, HeLaCEACAM5, HeLaCEACAM6 and HeLaCEAC AM8 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 pg / ml) at 37°C, 5.0% CO2. The cell lines were stained with the indicated antibodies and then with a fluorescent dye-conjugated monoclonal antibody specific for the indicated antibody isotype (such as human IgG4 or mouse IgG1) and a live dye (DAPI). Data were acquired with a Cytoflex flow cytometer (Invitrogen) and analyzed with FlowJo software (TreeStar, V7.6.5 for Windows).

[0852] In addition, CM-24 showed limited ability to reverse T cell tolerance in tumor-associated cells. Compared with CM-24 in primary Merkel cell carcinoma tumor cells, incubation of tumor-associated cells with antibody CP08H03 / Vκ8S29A resulted in a more extensive reversal of T cell tolerance over a range of antibody concentrations ( Fig.32A , 32Band 32C). According to the manufacturer's protocol, Merkel cell carcinoma biopsies were subjected to a commercial mechanical / enzymatic dissociation system (GentleMACS dissociator, Miltenyi Biotec). In brief, the tumor was cut into small fragments of about 2-3 mm in length and placed in a C tube (Miltenyi Biotech) with RPMI 1640 (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-second mechanical disaggregation steps (programs h_tumor_01.01, 02.01 and 03.01) in the GentleMACS dissociator, interspersed with two 30-minute incubations at 37°C after the first and second disaggregation steps, respectively. After disaggregation, the TILs from enzymatic digestion and GentleMACS dissociation were passed through a 100 pm filter for further analysis. In vitro assay of T cell function in the tumor environment: Dissociated tumor cells and autologous PBMCs were plated in 96-well plates in a 5% culture medium supplemented with 10% fetal calf serum (FCS), 1% glutamine, 100 IU ml -1 Penicillin, 100 μg ml -1 Streptomycin (Life Technologies), 25 mM HEPES (Sigma-Aldrich) in complete medium (RPMI 1640 (Lonza)) and 40 IU ml -1 Recombinant IL-2 (NIH) and soluble CD3 (2 g / ml) were co-cultured. After 96 h, cell culture supernatants were collected and used for further TNF- and IFN-ELISA (BD) analysis according to the manufacturer's procedures.

[0853] In a metastatic melanoma model (experimental setup see Fig.33A ), mice treated with CP08H03 / Vκ8S29A in vivo showed a significant reduction in tumor cells and TIL CD4 + and CD8 + Lymphocytes increased significantly ( Fig.33B , 34A , 34B and 34C). On the other hand, mice treated with CM-24 in vivo showed a large increase in tumor cells and an almost complete absence of TIL CD4 compared to control mice treated with hIgG4 control. + and CD8 + Lymphocytes ( Fig.33B , 34A, 34B and 34C). In addition, tumor cells showed reduced proliferation in animals treated with CP08H03 / Vκ8S29A relative to animals treated with hIgG4 control or CM24 ( Fig.33C In addition, compared with hIgG4-treated animals or CM24-treated mice, CP08H03 / Vκ8S29A-treated animals had significantly higher CD4 + T cells showed increased proliferation ( Fig.33D In contrast to CP08H03 / Vκ8S29A treated animals, CM24 treated animals showed decreased splenic CD4 + T cell proliferation ( Fig.33D The human melanoma cell line MALME-3M was provided by Dr. Nicole Beauchemin (McGill University). MALME-3M was isolated in 1975 from a patient with BRAF V600E The metastatic site (lung) of a 43-year-old white male with metastatic melanoma was established. 2x10^7 MALME-3M was injected subcutaneously (sc) into NSG mice. After 30 minutes, the mice were made to absorb tumor cells, and then freshly separated human PBMC (1x10^8) was transferred to the NSG mice carrying tumors by intraperitoneal (ip) injection. 7-9 days after PBMC injection, the human immune cell implantation of NSG animals was checked by tail bleeding. On the 14th day, palpable tumor nodules were detected. Starting from the 17th day after human cell injection, humanized NSG mice carrying tumors received a total of four doses of CP08H03 / Vκ8S29A antibody (2mg / kg), CM-24 (2mg / kg) or isotype control antibody (2mg / kg) twice a week by intraperitoneal injection. After the study was terminated (30 days after human cell injection), mice were killed and surgically dissected. Metastatic tumors were stored together with spleen, lungs and liver for further analysis. Total cell counts and proliferation and frequencies of CD4, CD8, and tumor cells were characterized by high FSC and SSC and negative expression of the human pan-leukocyte marker CD45.

[0854] Fig.34A , 34B and 34C through Fig.33A The scheme shown provides Fig.33B Statistical comparison of the results shown. Animals treated with CM-24 showed larger tumors and no evidence of tumor-associated T cells. On the other hand, an increase in the amount of infiltrating T cells and a decrease in tumor cells were observed in mice treated with CP08H03 / Vκ8S29A ( Fig.33B As shown, evaluation of tumor cell proliferation showed that CP08H03 / Vκ8S29A, but CM-24, did not inhibit tumor proliferation ( Fig.33CIn addition, splenic CD4 + Increased proliferation of T cells and increased expression of splenic CD4 + Decreased proliferation of T cells ( Fig.33D ).

[0855] Example 11: CEACAM1 antibody blocks the interaction between CEACAM1 and HopQ.

[0856] 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 shown to promote gastric colonization and H. pylori-induced pathology, for example by enabling translocation of bacterial virulence factors into host cells and enhancing the release of pro-inflammatory mediators.

[0857] Published crystal structure data (PDB ID 6AW2, 6GBH, 6GBG, see Bonsor, D, et al. EMBO J. 2018 Jul 2; 37(13) and Moonens K et al. EMBO J. 2018 Jul 2; 37(13)) indicate that the GFCC loop of CEACAM1 is involved in binding to HopQ and that CEACAM1 residues F29, Y34, N42, Q89 and N97 make various hydrogen bonds and hydrophobic interactions with HopQ residues (see Fig.35A Modeling based on CECAM1:HopQ cocrystal and CEACAM1:CP08H03 / Vκ8S29A Fab cocrystal indicated that the CEACAM1 antibody CP08H03 / Vκ8S29A covers the CEACAM1 binding site of HopQ (see Fig.35B ) and are therefore able to disrupt the CEACAM1:HopQ interaction.

[0858] Example 12: CEACAM1 Antibody Promotes Long-term Survival

[0859] The ability of the CEACAM1 antibody CP08H03 / Vκ8S29A to promote long-term survival of tumor-bearing mammals was investigated using a mouse melanoma model.

[0860] 10 6 MALME-3M (human melanoma) cells and 5x 10 6 Personal PBMC (from HLA-A2 matched donors) were injected subcutaneously into NSG mice. On day 10, tumors reached 2-2.5 mm 3The mice were randomly divided into groups (n=4 / group). Anti-CEACAM1 antibody CP08H03 / Vκ8S29A 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 time surviving animals showing strong clinical activity were sacrificed (arrows).

[0861] like Fig.36 As shown, treatment with anti-CEACAM1 antibodies significantly increased the survival of tumor-bearing mice. In addition, at necropsy, antibody-treated animals exhibited localized tumors without visible metastases, consistent with disease control. This data suggests that the anti-CEACAM1 antibodies and fragments thereof disclosed herein can be used to treat cancer and increase survival.

[0862] Example 13: CP08H03 / Vκ8S29A increases immune responses in tumor cells of naive patients or patients with secondary resistance to immunotherapy

[0863] Isolated tumor samples were used to examine the ability of the CEACAM1 antibody CP08H03 / Vκ8S29A to increase immune responses in tumors from de novo melanoma patients or melanoma patients who exhibited secondary resistance to immunotherapy.

[0864] In one example, isolated tumor specimens from patients with secondary resistance were disrupted by mechanical dissociation, and the dissociated cells were treated with CP08H03 / Vκ8S29A or hIgG4 control antibody (2 μg / ml) in culture in the presence of 2 μg / ml anti-CD3 and 40 units / ml recombinant IL-2 for 4 days. The cells were then examined by mass cytometry using the following antibodies to detect CD8 using standard techniques + Multiple intracellular factors in T cells related to the immune response 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); intercalator (103Rh).

[0865] like Fig.37A and 37B As shown, treatment with CP08H03 / Vκ8S29A antibody resulted in a significant induction of CD8 +These results directly indicate that CP08H03 / Vκ8S29A antibody can inhibit the expression of cytokines in CD8 T cells. + Induces the production of multiple factors in T cells that are potentially involved in productive anti-tumor immune responses.

[0866] In another example, tumor samples from two melanoma patients who had either not been previously treated (subject 189) or had secondary resistance to immunotherapy (subject 185) were disrupted by mechanical dissociation (Miltenyi). 5 Dissociated tumor cells were placed in culture dishes at 2 μg / ml. Freshly isolated tumor dissociated cells were exposed to 2 μg / ml CP08H03 / Vκ8S29A or human IgG4 isotype control antibody alone. After 96 hours, the supernatant was removed and ELISA analysis was performed in triplicate to detect the presence of interferon-γ.

[0867] like Fig.38A and 38B As shown in , treatment with the CP08H03 / Vκ8S29A antibody induced significant levels of cytokine interferon-γ secretion into cells isolated from patients with secondary resistance to immunotherapy treatment ( Fig.38A , subject 185) or immunotherapy-naive patients ( Fig.38B , subject 189) in the supernatant of tumor dissociated cells.

[0868] Taken together, these data suggest that the anti-CEACAM1 antibodies and fragments thereof disclosed herein may be used to treat de novo cancer patients and those with secondary resistance to immunotherapy.

[0869] Sequence Overview

[0870]

[0871]

[0872]

[0873]

[0874]

[0875] While the above written description of the invention enables one of ordinary skill in the art to make and use what is presently believed to be the best mode thereof, those of ordinary skill in the art will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiments, methods, and examples herein.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CEACAM1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3; and in: The sequence of CDR1H is shown as sequence SHGMS (SEQ ID NO: 9); The sequence of CDR2H is shown in the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2); The sequence of CDR3H is shown as the sequence HDFDYFPYWFAH (SEQ ID NO: 10); The sequence of CDR1L is shown as the sequence RANSAVSYMY (SEQ ID NO: 4); The sequence of CDR2L is shown in the sequence LTSNRAT (SEQ ID NO: 5); and The sequence of CDR3L is shown as the sequence QQWSSNPPT (SEQ ID NO: 11) or QQWTSNPPT (SEQ ID NO: 12).

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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 the light chain variable region amino acid sequence of SEQ ID NO:14 or SEQ ID NO:

15.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein 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 the light chain variable region amino acid sequence of SEQ ID NO:14 or SEQ ID NO:

15.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 13; and wherein the sequence of the light chain variable region comprises SEQ ID NO:

14.

5. The antibody or antigen-binding fragment thereof according to claim 3, wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 13; and Wherein the sequence of the light chain variable region comprises SEQ ID NO:

15.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody or a CDR-grafted antibody.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is a multispecific or bispecific antibody or antigen-binding fragment thereof.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is a scFv, Fv, Fab', Fab, F(ab')2 or a diabody.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof has an isotype of IgG1, IgG2, IgG3 or IgG4. The antibody or antigen-binding fragment thereof according to claim 10 , wherein the antibody or antigen-binding fragment thereof has the isotype IgG4.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof comprises an Fc region and wherein the Fc region comprises a modification that increases the half-life of the antibody.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof contains an S241P substitution in the constant region of the heavy chain.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is aglycosylated.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof lacks a C-terminal lysine in the heavy chain.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more of polyethylene glycol (PEG), an Fc region or albumin.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more of a cytotoxin, a fluorescent marker and an imaging agent.

18. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-16. A vector comprising the nucleic acid according to claim 18 .

20. A cell comprising the vector according to claim 19.

21. A cell expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16.

22. A T cell having a chimeric antigen receptor comprising the CDRs of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16.

23. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, and a pharmaceutically acceptable excipient.

24. An in vitro method of inhibiting the binding of CEACAM1 to CEACAM1, the method comprising contacting CEACAM1 with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

25. An in vitro method for inhibiting the binding of CEACAM1 to TIM3, the method comprising contacting CEACAM1 with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

26. An in vitro method for inhibiting the binding of CEACAM1 to Helicobacter pylori adhesin HopQ, the method comprising contacting CEACAM1 with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

27. An in vitro method of reducing colonization of mammalian epithelium by Helicobacter pylori bacteria expressing the Helicobacter pylori adhesin HopQ, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

28. An in vitro method for reducing T cell tolerance, the method comprising contacting a cell population comprising T cells with an antibody or antigen binding fragment thereof according to any one of claims 1 to 17.

29. An in vitro method for enhancing T cell expansion, the method comprising contacting a cell population comprising T cells with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17.

30. Use of an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for treating Merkel cell carcinoma or melanoma in a subject in need thereof.

31. Use of an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for reducing tumor growth in a subject in need thereof, wherein the tumor is Merkel cell carcinoma or melanoma.

32. Use of an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for reducing tumor metastasis in a subject in need thereof, wherein the tumor is Merkel cell carcinoma or melanoma.

33. The use according to any one of claims 30 to 32, further comprising administering a checkpoint inhibitor.

34. The use according to claim 33, wherein the checkpoint inhibitor is a CTLA-4, PD-1, PD-L1 or PD-L2 inhibitor.

35. The use according to any one of claims 30 to 32, further comprising administering one or more inhibitors of LAG3, TIGIT, LAP, Podoplanin, Protein C receptor, ICOS, GITR, CD226 and / or CD160.

36. The use according to any one of claims 30 to 32, further comprising administering a TIM-3 inhibitor.

37. The use of claim 33, wherein the additional inhibitor is administered simultaneously or sequentially with the antibody or antigen-binding fragment thereof.

38. The use according to claim 33, wherein the additional inhibitor is administered alone or as a mixture with the antibody or antigen-binding fragment thereof.

39. Use of an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 in the preparation of a medicament for treating Merkel cell carcinoma or melanoma in a subject in need thereof, wherein the subject has acquired resistance to treatment with a checkpoint inhibitor therapy.

40. The use of claim 39, wherein the subject has acquired resistance to therapy with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

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