Humanized and affinity matured anti-CEACAM1 antibodies

Abstract

The present invention relates to a humanized and affinity matured antibody against CEACAM1. Provided herein are recombinant antibodies, and antigen-binding fragments thereof, useful for binding and inhibiting carcinoembryonic antigen associated 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 generally relates 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] Multiple cellular activities have been attributed to the CEACAM1 protein, including roles in the differentiation and arrangement of tissue three-dimensional structure, angiogenesis, apoptosis, tumor suppression, metastasis, and the 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 multiple 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, distant metastasis, and shorter median overall survival and progression-free survival. CEACAM1 expression is also closely associated with distant metastasis of pancreatic cancer. CEACAM1 expression on tumors promotes the inhibition of CEACAM1-mediated T and NK cells. Thus, 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 glioblastoma is associated with poor prognosis. On T cells, CEACAM1 expression is mostly excluded from resting (naive) T cells, while the protein is expressed at high levels on activated T cells. CEACAM1-L is the major isoform expressed in most T cells and acts as an inhibitory receptor that downregulates T cell activation and suppresses T cell function. Thus, inhibition of CEACAM1 on T cells can restore T cell activity and increase anti-tumor responses.

[0008] CEACAM1 is also expressed on NK cells, which are lymphocytes involved in innate immunity and are involved in the early control of viral infections and the immune surveillance of tumors. When NK cells encounter cells expressing major histocompatibility complex (MHC) class I, inhibitory signals through receptor-ligand interactions prevent immune responses against these cells. However, when encountering cells in which MHC class I is downregulated, such as in virus-infected cells or cancer cells, NK cells are activated due to the lack of inhibitory signals, making the "diseased" cells vulnerable to NK cell-mediated killing. When CEACAM1 is present on the surface of NK and melanoma cells, CEACAM1:CEACAM1 interactions result in inhibition of NK-mediated killing, independent of MHC class I expression. Thus, disruption of this homophilic CEACAM1 interaction can be beneficial for restoring 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. Thus, inhibition of 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 opacity 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 has been identified as a Th1-specific cell surface protein that is expressed on subsets of activated T cells, dendritic cells, and macrophages, as well as 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, 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 functions as a cell 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 aegyptius, 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 through intact epithelial layers, thus allowing microbes to exploit CEACAM1 during mucosal colonization. Additionally, CEACAM1 is associated with infection by influenza virus H5N1 and filarial nematodes such as Wucheria bancrofti. Summary of the Invention

[0012] The present invention provides 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, 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. The 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 a subject in need thereof. The CEACAM1 antibodies and antigen-binding fragments thereof can also be used to treat patients resistant to checkpoint therapy. Also provided are methods of using the CEACAM1 antibodies and antigen-binding fragments thereof to reduce the colonization of bacteria expressing adhesins or Candida albicans on mammalian epithelium or to reduce the replication of influenza virus or the release of pro-inflammatory cytokines or chemokines associated with influenza virus infection.

[0013] In one aspect, the 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] wherein 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 (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2);

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

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

[0021] wherein X5 is F or P;

[0022] wherein X6 is D or F;

[0023] wherein X7 is A or Y; and

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

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

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

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

[0028] wherein X9 is W or N;

[0029] wherein X 10 is S or T;

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

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

[0032] wherein X 13 is P or F.

[0033] In one embodiment, the 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:

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

[0035] wherein X is any amino acid;

[0036] wherein 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 (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2);

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

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

[0042] wherein X5 is F or P;

[0043] wherein X7 is A or Y; and

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

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

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

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

[0048] wherein X9 is W or N;

[0049] wherein X 10 is S or T;

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

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

[0052] wherein X 13 is P or F.

[0053] In one embodiment, 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:

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

[0055] wherein 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] wherein X4 is D, G or P;

[0061] wherein X5 is F or P; and

[0062] wherein 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 comprises the sequence QQX9SSX 12 PX 13 T (SEQ ID NO:8);

[0066] wherein X9 is W or N;

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

[0068] wherein X 13 is P or F.

[0069] In one embodiment, 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

[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 contains the sequence RANSAVSYMY (SEQ ID NO:4);

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

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

[0076] In one embodiment, the 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

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

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

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

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

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

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

[0083] In one aspect, the 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 amino acid sequence of the heavy-chain variable region 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 the amino acid sequence of the light-chain variable region 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 an 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 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;

[0088] wherein the sequence of the heavy-chain variable region comprises a sequence that is at least 85% identical to the amino acid sequence of the heavy-chain variable region 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 amino acid sequence of the light-chain variable region 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] wherein X1 is A, D, N, or S;

[0093] wherein X2 is A or G; and

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

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

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

[0097] wherein X5 is F or P;

[0098] wherein X7 is A or Y; and

[0099] wherein 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 amino acid sequence of the heavy-chain variable region 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 amino acid sequence of the light-chain variable region 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] wherein:

[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 an 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 an isotype of IgG4.

[0114] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention contains an 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 deglycosylated.

[0116] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided by the present invention lacks a 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 label, and / or an imaging agent.

[0118] In another aspect, the present invention provides CEACAM1 antibodies and antigen-binding fragments thereof, characterized by the epitopes on CEACAM1 to which they bind. As described, such antibodies include, but are not limited to, the CEACAM1 antibodies and antigen-binding fragments thereof described herein by their structural features, including CDR motifs, CDR sequences, and heavy 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 provided herein selectively bind CEACAM1 compared to one or more CEACAM family members. In one embodiment, the CEACAM1 antibody or 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 located in the binding site of a heterologous interaction partner on CEACAM1, the heterologous interaction partner 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 virus (such as H5N1), and / or filarial nematodes such as Wuchereria bancrofti.

[0119] In one embodiment, the CEACAM1 antibody or antigen-binding fragment under consideration binds 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 CEACAM1 antibody or antigen-binding fragment thereof binds to the IgV-like N domain of CEACAM1 and binds to an epitope comprising one or more residues selected from the group consisting of residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and E99 of SEQ ID NO:17. In one embodiment, the epitope further comprises residue Q44 of SEQ ID NO:17. In one embodiment, the epitope further comprises one or more residues selected from the group consisting of residues S32, Q44, A49, I91, L95, and V96 of SEQ ID NO:17.

[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 present invention provides an antibody or antigen-binding fragment thereof that binds to CEACAM and partially or completely binds to the bacterial adhesin binding site on CEACAM1, the bacterial adhesins including but not limited to Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae Opa, Neisseria meningitidis Opa, Haemophilus influenzae OMP P1, Haemophilus aegyptius 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 a nucleic acid molecule encoding a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, and a vector comprising such a nucleic acid molecule. Also provided are cells comprising a vector encoding a CEACAM1 antibody or an antigen-binding fragment thereof as described herein, and cells expressing a CEACAM1 antibody or an antigen-binding fragment thereof as described herein. Also provided herein are chimeric antigen receptor T cells comprising a CDR of any of the antibodies or antigen-binding fragments disclosed herein.

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

[0129] In one embodiment, the present invention provides a method of inhibiting the binding of CEACAM1 to its interacting partner and / or reducing CEACAM1 activity 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. For example, embodiments of the present invention can be used to inhibit the interaction between CEACAM1 and CEACAM family members. 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 of inhibiting the binding of CEACAM1 to a bacterial adhesin using a CEACAM1 antibody or an antigen-binding fragment thereof described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or an antigen-binding fragment thereof described herein. In some embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae Opa, Neisseria meningitidis Opa, Haemophilus influenzae OMP P1, Haemophilus aegyptius OMP P1, or Moraxella adhesin OlpA. In one embodiment, the present invention provides a method of inhibiting the binding of CEACAM1 to Candida albicans using a CEACAM1 antibody or an antigen-binding fragment thereof described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or an antigen-binding fragment thereof described herein. In one embodiment, the present invention provides a method of inhibiting the binding of CEACAM1 to an influenza virus using a CEACAM1 antibody or an antigen-binding fragment thereof described herein, the method comprising contacting CEACAM1 with a CEACAM1 antibody or an antigen-binding fragment thereof described herein. In one embodiment, the influenza virus is H5N1.

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

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

[0134] In one embodiment, the present invention provides a method of reducing the replication of an influenza virus, the method comprising contacting CEACAM1 with a CEACAM1 antibody or an antigen-binding fragment thereof described herein. In one embodiment, the present invention provides a method of reducing the release of pro-inflammatory cytokines or chemokines associated with influenza virus infection, the method comprising contacting a cell population comprising epithelial cells with a CEACAM1 antibody or an antigen-binding fragment thereof described herein. In some embodiments, the influenza virus is H5N1.

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

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

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

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

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

[0140] In one embodiment, the present invention provides a method of treating a subject who is refractory (primary resistance) to checkpoint inhibitor therapy and a patient who was initially responsive to treatment but has subsequently become resistant to checkpoint inhibitor blockade (secondary or acquired resistance). Such methods of treatment include administering to the subject a CEACAM1 antibody or an antigen-binding fragment thereof described herein. In some embodiments, the subject has acquired resistance to therapy with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor. A resistant cancer may also be referred to as a refractory cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Patent 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. V H and the Vκ vector both contain genomic DNA fragments incorporating introns and polyA sequences. 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 positive for staining based on irrelevant hIgG4 is shown. There was no evidence of any staining of the HeLa-CEACAM3 or HeLa-CEACAM8 transfectants, and thus these data are not reported.

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

[0145] Figure 4 The chimeric CEACAM1 antibody V H 0 / Vκ0 is glycosylated in CDR1L. Introduction of the mutations N26Q and S29A eliminated this glycosylation. 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. V H and the Vκ domains are linked by a flexible glycine-serine (G4S) linker and fused in-frame to the M13 gene III phage coat protein. Expression of the single-chain variable fragment (scFv) is driven by the Lac promoter.

[0147] Figure 6 The binding of the phage to the CEACAM1 antigen is shown. Phages prepared from the parental V H 1 / Vκ8S29A scFv or an irrelevant scFv were serially diluted and incubated with plate-bound GST-CEACAM1. Binding of the phage to CEACAM1 was detected using an anti-M13 horseradish peroxidase (HRP) conjugate and a 3,3',5,5'-tetramethylbenzidine (TMB) substrate.

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

[0149] Fig. 8A 、 8B and 8C provide an overview of the library construction methods for generating a randomized phage library. Light chain CDR3 library ( Fig. 8A ), heavy chain CDR1 library ( Figure 8B ), and heavy chain CDR3 library ( Figure 8C ).

[0150] Fig.9A and 9B provide an overview of two different selection activities used during the affinity maturation of CEACAM1 antibodies. Fig.9A : Selection activity 1: Solid-phase panning of library phages with CEACAM5 / CEACAM6 depletion and multiple rounds of selection with decreasing concentrations of biotinylated soluble CEACAM1 before round 2. Fig. 9B : Selection activity 2: Panning selection with CEACAM1 in round 1, followed by 2 rounds of panning with CEACAM5 / CEACAM6 depletion and selection with decreasing concentrations of biotinylated soluble CEACAM1.

[0151] Fig.10 Shows an example of an scFv binding ELISA assay. 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 an anti-HIS6-HRP antibody and TMB. All variable light chains contain an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

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

[0153] Fig. 12A , 12B and 12C show that the CEACAM antibodies CP08H03 / Vκ8S29A (labeled "CP08_H03 / parental VL"), CP08H03 / CP08_F05, and V H 0 / Vк0 are selective for CEACAM1. CP08H03 / Vκ8S29A and CP08H03 / CP08F05 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). Fig. 12AShows the single-cycle kinetic sensing plots and fitting curves of purified lead humanized and affinity matured IgG4 variants. Increasing concentrations of different CEACAM family members were injected, and individual dissociation rates were determined by single-cycle kinetics (surface plasmon resonance, SPR). Fig. 12B Shows the chimeric antibody V H 0 / Vκ0 (labeled "chimeric"), three-point binding ELISA data of the purified lead humanized and affinity matured IgG4 variants binding to CEACAM1 and CEACAM3 family members. Three-point (high, medium, and low, concentrations based on the binding of chimeric antibody V H 0 / Vκ0 to CEACAM1) titrations were performed, and binding was detected using an anti-human κ-chain antibody and a TMB substrate. Fig. 12C Shows the chimeric antibody V H 0 / Vκ0 (labeled "chimeric") and three-point binding ELISA data of the purified lead humanized and affinity matured IgG4 variants binding to CEACAM1, 5, and 6 family members. Three-point (high, medium, and low, concentrations based on the binding of chimeric antibody V H 0 / Vκ0 to CEACAM-1) titrations were performed, and binding was detected using an anti-human κ-chain antibody and a TMB substrate.

[0154] Fig.13 Shows the sequence homology between the N domains of different CEACAM family members. CEACAM1 (C1, UniProtKB accession number P13688), CEACAM3 (C3, UniProtKB accession number P40198), CEACAM4 (C4, UniProtKB accession number O75871), CEACAM5 (C5, UniProtKB accession number P06731), CEACAM6 (C6, UniProtKB accession number P40199), CEACAM7 (C7, UniProtKB accession number Q14002), and CEACAM8 (C8, UniProtKB accession number P31997). The shown percentage identity matrix was created using Clustal 2.1. The specific residues analyzed for each CEACAM family member are shown.

[0155] Fig.14 Shows the results of CEACAM1 mutagenesis studies aimed at identifying the residues in CEACAM1 involved in binding to the shown CEACAM1 antibodies. CEACAM1-FLAG was expressed in human embryonic kidney (HEK) cells transfected with CEACAM1 containing the shown mutations (Y34C, V39A, G41A, N42A, R43A, Q44L, G47A, and Q89H), the proteins were resolved by SDS-PAGE, and then immunoblotted. Using the shown chimeric (VH The 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 Cα 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 including D40, N42, L95, V96, N97, and E99 that interact with the CP08H03 / Vκ8S29A Fab light chain are marked (see Fig.15 ) and CEACAM1 residues including F29, S32, Y34, Q44, T56, Q89, and I91 that interact with the CP08H03 / Vκ8S29A Fab heavy chain. 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 A close-up stereo image of the CP08H03 / Vκ8S29A Fab-CEACAM1 interaction is shown. CEACAM1 is drawn as a ribbon and the Fab chains (light and heavy chains) are drawn as Cα traces. Regions of 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. The side chains of interest are drawn as sticks and the hydrogen bonds are drawn as black dashed lines. Residue numbering is based on the primary amino acid sequences 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).

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

[0162] Fig.21 Shows the experimental setup for testing the ability of CEACAM1 antibodies to induce CD45 + cell proliferation in humanized non-obese diabetic (NOD) scidγ mice (NSG mice). At 38 days post-injection, engraftment of human peripheral blood mononuclear cells (PBMCs) adoptively transferred into NSG host mice by intraperitoneal injection was analyzed by fluorescence-activated cell sorting (FACS) for staining with human CD45 and a proliferation dye. At 24 days post-PBMC injection, mice were treated with a single injection of human IgG4 isotype control or the indicated concentrations of CP08H03 / Vκ8S29A (labeled "CP08_H03 / parental VL") or CP08H03 / CP08F05 antibody. At day 31, mice were treated with a second injection. At day 38, mice were sacrificed for data collection.

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

[0164] Fig.23 Shows that administration of the CEACAM1 antibody CP08H03 / Vκ8S29A (labeled "CP08_H03 / parental VL") or CP08H03 / CP08F05 respectively results in antibody-induced human CD45 +Increased expansion of immune cells. CP08H03 / Vκ8S29A induces the expansion of human CD45 PBMCs in vivo. On day 38, 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 splenocytes were isolated and collected for proliferation analysis. Ex vivo proliferation was performed under T cell stimulation conditions, where cells were cultured for 120 hours in soluble anti-CD3 (OKT3) (at designated concentrations of 10, 5, 2.5 μg / ml) and rIL-2 (40 units / ml). Dilution of the proliferation dye represents cell division / proliferation (when cells proliferate, double-stranded DNA is analyzed as a diluted signal). CP08H03 / CP08F05 contains the S29A mutation in CDR1L (Kabat numbering scheme, corresponding to the 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 A schematic diagram of the experimental protocol for generating Fig. 24B is provided. Fig. 24B shows the average tumor size after subcutaneous injection of 1x10 6 MALME-3M (human melanoma) cells together with 5×10 6 human PBMCs into NSG. Ten days later, palpable tumors were recorded, and the mice were randomly divided and treated intraperitoneally with the corresponding antibody concentrations on days 10, 13, 17, 20, and 24. Fig.24C shows the statistical comparison by linear regression between the hIgG4 control treatment group and three different CP08H03 / Vκ8S29A groups (2 mg / kg, 0.4 mg / kg, and 0.08 mg / kg).

[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 showed a decrease in the number of tumor cells and a decrease in the proliferation and an increase in the number of CD8- and CD4-positive T cells within the tumor. When examined ex vivo after stimulation with anti-CD3, these T cells showed increased proliferation. On the day of sacrifice, humanized NSG mice bearing melanoma tumors treated with isotype hIgG4 control (2 mg / kg), CP08H03 / Vκ8S29A (labeled "CP08_H03 / parental VL", 0.08 and 2 mg / kg) were sacrificed. Tumor cells as well as CD4 + and CD8 + tumor-infiltrating lymphocytes were collected for proliferation analysis. Tumor cells were identified as FSC Hi SSC Hi cells, which were negative for human CD45, and proliferation was quantified by evaluating the dilution of a commercially available dye (Becton-Dickinson) for proliferation. Human CD45 was identified by flow cytometry + CD4 + and CD45 + CD8 + T cells. Measurement of ex vivo T cell proliferation was performed under T cell stimulation conditions, where cells were cultured for 6 days in soluble anti-CD3 (2 μg / ml) and rIL-2 (40 units / ml).

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

[0168] Fig. 27CEACAM1 expression on primary CD4 + (upper) and CD8 + T (lower) T cells in TILs from naïve (left) and PD-1 and / or CTLA-4-resistant (right) melanoma patients is shown. Similar profiles of PD1 and TIM-3 expression are also shown.

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

[0170] Fig.29 It is shown that there is a relative decrease in central memory (T + ) compared to effector memory (T + ) cells in CD8 cm T cells isolated from patients resistant to anti-PD-1 and / or anti-CTLA-4 therapy compared to CD8 em T patients isolated from naïve patients. Tumor-associated cells from naïve patients and patients with acquired resistance were stained for central memory (CCR7 + CD62L + ) and effector memory (CCR7 - CD62L - ) markers in TACs derived from naïve patients and resistant patients.

[0171] Fig.30Shown is that the CEACAM1 antibody CP08H03 / Vκ8S29A (labeled "CP08") reverses T cell exhaustion in PD1 / CTLA-4 resistant tumors. Tumor-associated cells and PBMC were isolated from melanoma patients with secondary resistance to Pembrolizumab, Ipilimumab + Nivolumab, and Dabrafenib + Trametinib and stage IV disease. Tumor-associated cells and PBMC were stained for CEACAM1, PD1, or TIM-3, and the proportions of CD8 + and CD4 + T cells are shown expressing these markers (left). PBMC or tumor-associated cells ("tumor") cultured with soluble anti-CD3 (2 μg / ml) and rIL-2 (40 units / ml) in the presence of CP08H03 / Vκ8S29A or hIgG4 control antibody are shown in the right panel. Release of IFNγ and TNFα (measures for reversing T cell tolerance) was determined by ELISA.

[0172] Fig.31A and 31B Shown is flow cytometry analysis 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 : 5 x 10^4 of the indicated HeLa transfectants were washed with staining buffer and incubated with CP08H03 / Vκ8S29A (labeled "CP08", left) or CEACAM1 antibody CM-24 for 30 minutes at room temperature, 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. Live 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 the positive signal in the gate shown only in the HeLa CEACAM1 (C1) transfectant (left). In contrast, CM-24 (right panel) is not selective and cross-reacts with CEACAM1, CEACAM3, and CEACAM5. Fig.31B Shown is Fig.31A a different representation of the

[0173] Fig.32A 、32B And 32C shows 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 a primary Merkel cell carcinoma tumor were stained for CEACAM1, PD1, or TIM-3, and the CD8 + and CD4 + T cell ratios ( Fig.32A and 32B ) were shown. In the presence of CP08H03 / Vκ8S29A, CM-24, or hIgG4 control, respectively, the tumor-associated cells were incubated with soluble anti-CD3 (2

[0174] μg / ml) and rIL-2 (40 units / ml). The release of IFN-γ (a measure for reversing T cell tolerance) was measured ( Fig.32C ). *Comparing CP08 with hIgG4, P = 0.0138.

[0175] Fig.33A 、 33B 、33C and 33D show that compared with metastatic melanoma treated with CP08H03 / Vκ8S29A (labeled "CP08"), metastatic melanoma treated with CM-24 in NSG mice showed reduced TIL and increased tumor cells. Fig.33A Shows the experimental setup using a therapeutic tumor model in humanized NSG mice with human melanoma xenografts, which used four doses of 2 mg / kg of the corresponding antibody, including an hIgG4 control containing the same stable hinge mutation. Fig.33B Shows tumor-infiltrating CD4 Hi characterized by high FSC / SCC (FSC / SCC + ) and lack of the pan-leukocyte marker human CD45, T lymphocytes (gray), CD8 + T lymphocytes (black), and tumor cells (white) in a pie chart (left: control antibody. Middle: CEACAM1 antibody CP08H03 / Vκ8S29A. Right: CEACAM1 antibody CM-24). Fig.33C Shows the tumor cell proliferation of IgG4 control, CP08H03 / Vκ8S29A, and CM-24, indicating that CP08H03 / Vκ8S29A rather than CM-24 inhibits tumor proliferation. Fig.33D Shows increased proliferation of splenic CD4 + T cells in mice treated with CP08H03 / Vκ8S29A, and decreased proliferation of splenic CD4 + T cells in mice treated with CM-24.

[0176] Fig.34A and 34B and 34C show that the CEACAM1 antibody CM-24 is an agonist drug in a metastatic melanoma model. Shown are the absolute cell counts of tumor-infiltrating CD4 + T lymphocytes( Fig.34A ), CD8 + T lymphocytes( Fig.34B ) and tumor cells( Fig.34C ) characterized by forward / side scatter high (FSC / SCC Hi). Shown are the values obtained for each experimental mouse in each group (for IgG4, n = 9; for CP08, n = 8; for CM-24, n = 6). *P < 0.05; **P < 0.001. Statistical analysis refers to the data included in Fig.33B . Note that the number of TILs is increased( Fig.34A and Fig.34B ) and the number of tumor cells is decreased( Fig.34C ) in mice treated with CP08H03 / Vk8S29A (labeled "CP08") relative to mice treated with CM-24. This data indicates that CP08H03 / Vk8S29A is an antagonistic antibody and CM-24 is an agonist antibody.

[0177] Fig.35A and 35B show 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 Shows the CEACAM1:HopQ binding interface based on analysis of three crystal structures (PDBID 6AW2, 6GBH, and 6GBG). The CEACAM1 GFCC' face is formed by the interaction of CEACAM1 CC' and the FG loop' (see Huang et al., Nature. January 15, 2015; 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. July 2, 2018; 37(13).pii:e98664; Moonens K et al. EMBO J. July 2, 2018; 37(13).pii:e98665). Fig.35BShows the superposition of the CP08H03 / Vκ8S29A:CEACAM1 crystal structure and the CEACAM1:HopQ crystal structure. The light and heavy chains of the CP08H03 / Vκ8S29A antibody are shown as surface diagrams. Ribbon diagrams show the HopQ chain (three different crystal structures PDB ID 6AW2, 6GBH, and 6GBG) and CEACAM1 from three co-crystal structures different from HopQ (PDB ID 6AW2, 6GBH, 6GBG), as well as CEACAM1 from the co-crystal structure with CP08H03 / Vκ8S29A, to highlight the superposition of the CP08H03 / Vκ8S29A and HopQ binding epitopes.

[0178] Fig.36 Shows that the CEACAM1 antibody CP08H03 / Vκ8S29A increases survival in tumor-bearing mice. NSG mice were injected with MALME-3M (human melanoma) cells and human PBMC. Treatment was performed with the CEACAM1 antibody CP08H03 / Vκ8S29A or a control human (h)IgG4 antibody on days 10, 13, 17, 20, and 24, respectively (see arrows). Shown as % survival. n = 4 / group.

[0179] Fig.37A and 38B Shows that the CEACAM1 antibody CP08H03 / Vκ8S29A increases the expression of multiple factors involved in the immune response of CD8 + T cells against cancer from melanoma patients with secondary resistance to immunotherapy. Fig.37A Shows a series of viSNE (visual distributed stochastic neighbor embedding) plots presented in Cytobank using the Barnes-Hut implementation of the t-SNE algorithm, which depict the intracellular expression of the indicated specific factors in CD8 + T cells as defined by mass cytometry in the left panel. The quantification of the heatmap levels of each indicated factor is shown on the right side of the x-axis relative to the residues associated with each factor shown on the y-axis. Fig.37B Shows Fig.37A the fold change of the intracellular response of the indicated factors described in

[0180] Fig.38A and 38B Shows that the CEACAM1 antibody CP08H03 / Vκ8S29A re-enhances the ability of tumor-dissociated cells from two melanoma patients who had not been previously treated to secrete interferon-γ (IFN-). Fig.38B, subject 189) or have 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 of CP08H03 / Vκ8S29A or human IgG4 isotype control antibody. After 96 hours, a significant level of interferon-γ was detected in the supernatant of CP08H03 / Vκ8S29A, but not in samples treated with human IgG4 isotype control antibody. *P<0.05 Detailed Description

[0181] Antibody

[0182] 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), provided that they exhibit the desired biological activity and specificity.

[0183] As used herein, "antibody variable domain" refers to the portions of the light and heavy chains of an antibody molecule that comprise the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the framework regions (FRs). V H Refers to the variable domain of the heavy chain. V L Refers to the variable domain of the light chain. The amino acid positions assigned to the 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.

[0184] As used herein, the term "complementary determining region" (CDR) refers to the portions of the variable domains of an antibody that (typically) participate in antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each CDR can contain amino acid residues from the CDRs 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 can also contain amino acid residues from "hypervariable loops" (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, a CDR can contain amino acids from both the CDR regions defined according to Kabat and the hypervariable loops. Kabat residue names do not always directly correspond to the linear numbering of amino acid residues (the primary amino acid sequence). The actual linear amino acid sequence can contain fewer or additional amino acids than in the strict Kabat numbering, corresponding to deletions or insertions in the structural components (whether framework or CDR) of the underlying variable domain structure. For a given antibody or antigen - binding fragment thereof, the correct Kabat numbering of residues can be determined by aligning the homologous residues in the sequence of the antibody or its antigen - binding fragment with a "standard" Kabat - numbered sequence. Examples of how Kabat numbering relates to the primary amino acid sequence of an antibody can be seen Figure 3A 、 3B and 3C. Alternatively, CDRs can be defined according to the ImMunoGeneTics (IMGT) system (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55 - 77 (2003)).

[0185] In one embodiment, the CEACAM1 antibody or antigen - binding fragment thereof provided herein contains six CDRs, wherein:

[0186] (i) the CDR1 sequence of the heavy - chain variable region contains SEQ ID NO:9;

[0187] (ii) The CDR2 sequence of the heavy chain variable region comprises SEQ ID NO:2;

[0188] (iii) The CDR3 sequence of the heavy chain variable region comprises SEQ ID NO:10;

[0189] (iv) The CDR1 sequence of the light chain variable region comprises SEQ ID NO:4;

[0190] (v) The CDR2 sequence of the light chain variable region comprises SEQ ID NO:5; and

[0191] (vi) The CDR3 sequence of the light chain variable region comprises SEQ ID NO:11.

[0192] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises six CDRs, wherein:

[0193] (i) The CDR1 sequence of the heavy chain variable region comprises SEQ ID NO:9;

[0194] (ii) The CDR2 sequence of the heavy chain variable region comprises SEQ ID NO:2;

[0195] (iii) The CDR3 sequence of the heavy chain variable region comprises SEQ ID NO:10;

[0196] (iv) The CDR1 sequence of the light chain variable region comprises SEQ ID NO:4;

[0197] (v) The CDR2 sequence of the light chain variable region comprises SEQ ID NO:5; and

[0198] (vi) The CDR3 sequence of the light chain variable region comprises SEQ ID NO:12.

[0199] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises six CDRs, wherein:

[0200] (i) The CDR1 sequence of the heavy chain variable region comprises SEQ ID NO:9

[0201] (ii) The CDR2 sequence of the heavy chain variable region comprises SEQ ID NO:2;

[0202] (iii) The CDR3 sequence of the heavy chain variable region comprises SEQ ID NO:10;

[0203] (iv) The CDR1 sequence of the light chain variable region comprises SEQ ID NO:18;

[0204] (v) The CDR2 sequence of the light chain variable region comprises SEQ ID NO:5; and

[0205] (vi) The CDR3 sequence of the light chain variable region comprises SEQ ID NO:11.

[0206] As shown in the Examples below, affinity maturation of CDR1H, CDR3H and CDR3L of the humanized non-glycosylated CEACAM1 antibody generates variants that confer a significant improvement in CEACAM1 binding affinity. Examination of the variants obtained and comparison of these variants with the variability introduced into the affinity maturation library indicates certain CDR positions (where the amino acids remain relatively invariant) and other CDR positions (where variability can be introduced), resulting in improved binding.

[0207] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof comprising CDR1H, wherein said CDR1H comprises residues 31-35 of the CEACAM1 antibody (Kabat definition, corresponding to residues 31 to 35 in the primary amino acid sequence of the heavy variable chain of, for example, SEQ ID NO:19 (see Figure 3A ) or SEQ ID NO:13 (see Figure 3B )) and comprises the sequence X1HX2X3S (SEQ ID NO:1),

[0208] wherein X1 of CDR1H is A, D, N or S;

[0209] wherein X2 of CDR1H is A or G; and

[0210] wherein X3 of CDR1H is an amino acid having a hydrophobic side chain including I or M.

[0211] Alternatively, the IMGT definition can be used to define CDR1H, wherein CDR1H comprises residues 26-33 of the CEACAM1 antibody (corresponding to residues 26 to 33 in the primary amino acid sequence of the heavy variable chain of, 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),

[0212] wherein X14 of CDR1H is G or E;

[0213] wherein X15 of CDR1H is an amino acid having an aromatic side chain including F or Y;

[0214] wherein X16 of CDR1H is T, S or I;

[0215] wherein X17 of CDR1H is an amino acid with a polar uncharged side chain including S, T or N;

[0216] wherein X1 of CDR1H is A, D, N or S; and

[0217] wherein X2 of CDR1H is A or G.

[0218] In one embodiment, CDR1H (Kabat definition) of a CEACAM1 antibody or an antigen-binding fragment thereof comprises the sequence SHGMS (SEQ ID NO:9).

[0219] In some embodiments, CDR1H (IMGT definition) comprises the sequence GFIFSHG (SEQ ID NO:21).

[0220] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof comprising a CDR1H region, wherein said CDR1H comprises residues 26-35 of the CEACAM1 antibody (Kabat definition, corresponding to residues 26 to 35 in the primary amino acid sequence of the heavy variable chain of, for example, SEQ ID NO:19 (see Figure 3A ) or SEQ ID NO:13 (see Figure 3B )) and comprises the sequence X 14 X 15 X 16 FX 17 X1HX2X3S (SEQ ID NO:22),

[0221] wherein X 14 is G or E;

[0222] wherein X 15 is an amino acid with an aromatic side chain including F or Y;

[0223] wherein X 16 is T, S or I;

[0224] wherein X 17 is an amino acid with a polar uncharged side chain including S, T or N;

[0225] wherein X1 is A, D, N or S;

[0226] wherein X2 is A or G; and

[0227] wherein X3 is an amino acid with a hydrophobic side chain including I or M.

[0228] In one embodiment, the CDR1H region comprises the sequence GFIFSSHGMS (SEQ ID NO:23).

[0229] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof comprising CDR3H, wherein said CDR3H comprises residues 95-102 (Kabat definition, corresponding to residues 99 to 110 in the primary amino acid sequence of the heavy variable chain of, for example, SEQ ID NO:19 (see Figure 3A ) or SEQ ID NO:13 (see Figure 3B )) and comprises the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO:3),

[0230] wherein X4 of CDR3H is D, G or P;

[0231] wherein X5 of CDR3H is F or P;

[0232] wherein X6 of CDR3H is D or F;

[0233] wherein X7 of CDR3H is A or Y; and

[0234] wherein X8 of CDR3H is L, H or F.

[0235] In one embodiment, CDR3H comprises residues 95-102 (Kabat definition, corresponding to residues 99 to 110 in the primary amino acid sequence of the heavy variable chain of, for example, SEQ ID NO:19 (see Figure 3A ) or SEQ ID NO:13 (see Figure 3B )) and comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO:7),

[0236] wherein X4 of CDR3H is D, G or P;

[0237] wherein X5 of CDR3H is F or P; and

[0238] wherein X8 of CDR3H is L, H or F.

[0239] In one embodiment, CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO:10).

[0240] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof comprising a CDR3H region, wherein said CDR3H region comprises residues 94-102 (Kabat definition, corresponding to residues 99 to 110 in the primary amino acid sequence of the heavy variable chain of, for example, SEQ ID NO:19 (see Figure 3A ) or SEQ ID NO:13 (see Figure 3B(residues 98 to 110 in the primary amino acid sequence of the heavy variable chain of

[0241] wherein X18 is R or K;

[0242] wherein X4 is D, G or P;

[0243] wherein X5 is F or P;

[0244] wherein X6 is D or F;

[0245] wherein X7 is A or Y; and

[0246] wherein X8 is L, H or F.

[0247] In one aspect, the CDR3H region comprises the sequence RHDFDYFPYWFAH (SEQ ID NO:25).

[0248] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof comprising CDR3L, wherein said CDR3L comprises residues 89 - 97 (Kabat definition, corresponding to, for example, residues 88 to 96 in the primary amino acid sequence of the heavy variable chain of SEQ ID NO:14 (see Figure 3C ) and comprises the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6),

[0249] wherein X9 is W or N;

[0250] wherein X 10 is S or T;

[0251] wherein X 11 is A or an amino acid having a neutral hydrophilic side chain including S, N and T;

[0252] wherein X 12 is L, F or N; and

[0253] wherein X 13 is P or F.

[0254] In one embodiment, CDR3L comprises residues 89 - 97 (Kabat definition, corresponding to, for example, residues 88 to 96 in the primary amino acid sequence of the heavy variable chain of SEQ ID NO:14 (see Figure 3C ) and comprises the sequence QQX9SSX 12 PX 13 T (SEQ ID NO:8),

[0255] wherein X9 is W or N;

[0256] wherein X 12 is L, F or N; and

[0257] wherein X 13 is P or F.

[0258] In one embodiment, CDR3L comprises the sequence QQWSSNPPT (SEQ ID NO:11) or the sequence QQWTSNPPT (SEQ ID NO:12).

[0259] In one aspect, the 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:

[0260] 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);

[0261] wherein X 14 is G or E;

[0262] wherein X 15 is an amino acid having an aromatic side chain including F or Y;

[0263] wherein X 16 is T, S or I;

[0264] wherein X 17 is an amino acid having a polar uncharged side chain including S, T or N;

[0265] wherein X1 is A, D, N or S;

[0266] wherein X2 is A or G; and

[0267] wherein X3 is an amino acid having a hydrophobic side chain including I or M;

[0268] The CDR2 sequence (CDR2H) of the heavy-chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2);

[0269] The CDR3 sequence (CDR3H) of the heavy-chain variable region comprises the sequence HX4X5DYX6X19X7WFAX20 (SEQ ID NO:45);

[0270] where X4 is D, G or P;

[0271] where X5 is F or P;

[0272] where X6 is D or F;

[0273] where X19 is P or A;

[0274] where X7 is A or Y; and

[0275] where X20 is L, H, Y or F;

[0276] The CDR1 sequence (CDR1L) of the light chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4);

[0277] The CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); and

[0278] The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6);

[0279] where X9 is W or N;

[0280] where X 10 is S or T;

[0281] where X 11 is A or an amino acid having a neutral hydrophilic side chain including S, N and T;

[0282] where X 12 is L, F or N; and

[0283] where X 13 is P or F; and

[0284] where

[0285] when X 19 is A and / or X 20 is Y, X 10 is T, X4 is G or P, X1 is N, and / or X 16 is T or S.

[0286] 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:

[0287] 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);

[0288] wherein X 14 is G or E;

[0289] wherein X 21 is T or I;

[0290] wherein X 22 is N or S;

[0291] wherein X 23 is A, D or S

[0292] wherein X2 is A or G; and

[0293] wherein X3 is an amino acid having a hydrophobic side chain including I or M;

[0294] The CDR2 sequence (CDR2H) of the heavy-chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2);

[0295] The CDR3 sequence (CDR3H) of the heavy-chain variable region comprises the sequence HX24FDYX6X19X7WFAX25 (SEQ ID NO:47);

[0296] wherein X24 is D or G;

[0297] wherein X6 is D or F;

[0298] wherein X19 is P or A;

[0299] wherein X7 is A or Y; and

[0300] wherein X25 is H or Y;

[0301] The CDR1 sequence (CDR1L) of the light-chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4);

[0302] The CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); and

[0303] The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQWX 10 X 10 NPPT (SEQ ID NO:48);

[0304] wherein X 10 is S or T;

[0305] wherein

[0306] when X21 is I, X6 is F, X19 is P and / or X7 is Y.

[0307] 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:

[0308] The CDR1 sequence (CDR1H) of the heavy chain variable region comprises the sequence X 14 FTFX 22 X 26 HAX3S (SEQ ID NO:49);

[0309] wherein X14 is G or E;

[0310] wherein X 17 is S or N;

[0311] wherein X 22 is N or S;

[0312] wherein X26 is A or D and

[0313] wherein X3 is an amino acid having a hydrophobic side chain including I or M;

[0314] The CDR2 sequence (CDR2H) of the heavy chain variable region comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2);

[0315] The CDR3 sequence (CDR3H) of the heavy chain variable region comprises the sequence HX24FDYX6X19X7WFAX25 (SEQ ID NO:47);

[0316] wherein X24 is D or G;

[0317] wherein X6 is D or F;

[0318] Wherein X19 is P or A;

[0319] Wherein X7 is A or Y; and

[0320] Wherein X25 is H or Y;

[0321] The CDR1 sequence (CDR1L) of the light chain variable region comprises the sequence RANSAVSYMY (SEQ ID NO:4);

[0322] The CDR2 sequence (CDR2L) of the light chain variable region comprises the sequence LTSNRAT (SEQ ID NO:5); and

[0323] The CDR3 sequence (CDR3L) of the light chain variable region comprises the sequence QQWX 10 X 10 NPPT (SEQ ID NO:48);

[0324] Wherein X 10 is S or T.

[0325] In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof, the 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:

[0326] The sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO:1),

[0327] The sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2),

[0328] The sequence of CDR3H comprises the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO:3), 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 comprises the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6).

[0331] X1-X 18Previously defined.

[0332] In one embodiment, the 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] wherein 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] wherein X4 is D, G, or P;

[0341] wherein X5 is F or P;

[0342] wherein X7 is A or Y; and

[0343] wherein 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] wherein X9 is W or N;

[0348] wherein X 10 is S or T;

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

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

[0351] wherein X 13 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), the sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4),

[0356] The sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5), and

[0357] The sequence of CDR3L comprises the sequence QQX9SSX 12 PX 13 T (SEQ ID NO:8).

[0358] X1-X 18 has been previously defined.

[0359] According to certain embodiments, the antibodies and antigen-binding fragments thereof contemplated are also characterized by a humanized framework for reducing immunogenicity. In certain embodiments, the CDRs of the antibody or antigen-binding fragment thereof contemplated are located in a framework obtained from a human antibody or antigen-binding fragment thereof. In other embodiments, the surface-exposed framework residues of the antibody or antigen-binding fragment thereof contemplated are replaced with framework residues of a human antibody or antigen-binding fragment thereof. The CDRs may also be located in a murine or humanized framework linked to a human constant region (i.e., a chimeric antibody). In a preferred embodiment, the CDRs of the antibody or antigen-binding fragment thereof contemplated are located in a framework that is a complex of two or more human antibodies. In such embodiments, the antibody or antigen-binding fragment thereof contemplated comprises two or more sequence segments ("complexes") derived from the V regions of unrelated human antibodies, which V regions have been selected to maintain the monoclonal antibody sequences important for antigen binding of the starting precursor anti-human CEACAM1 monoclonal antibody and have all been filtered for the presence of potential T cell epitopes using "computer tools" (Holgate and Baker, IDrugs. April 2009; 12(4):233-7). The close fit of the human sequence segments to all parts of the starting antibody V region and the elimination of CD4 + T cell epitopes allows this technique to avoid immunogenicity while maintaining optimal affinity and specificity by sequences necessary for the prior analysis of antigen specificity (Holgate and Baker, 2009).

[0360] Also provided herein are variable heavy chain and variable light chain sequences and pairings thereof that are similar but not identical to those disclosed in SEQ ID NOs: 13-16 and their pairings.

[0361] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence containing SEQ ID NO: 13.

[0362] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence containing SEQ ID NO: 16. In other embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence containing SEQ ID NO: 14. In other embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence containing SEQ ID NO: 15.

[0363] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence containing SEQ ID NO: 13 and a variable light chain amino acid sequence containing SEQ ID NO: 14.

[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:15.

[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:16.

[0366] As used herein, the term "identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each sequence that have been aligned for purposes of comparison. For example, a molecule is identical at a position when the position in the compared nucleotide sequences is occupied by the same base. 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 shared positions. For example, consider polypeptides having at least 85%, 90%, 95%, 98% or 99% identity to a specific polypeptide described herein and preferably exhibiting substantially the same function, and polynucleotides encoding such polypeptides. Methods and computer programs for determining sequence identity and similarity are publicly available, including but not limited to the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990) and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm can also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 website http: / / www.ncbi.nlm.nih.gov / blast / ). These methods take into account various substitutions, deletions and other modifications when comparing sequences.

[0367] In another aspect, the CEACAM1 antibody or antigen-binding fragment thereof comprises

[0368] (i) a variable heavy domain that 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 variable heavy domain sequence of SEQ ID NO:13; and / or

[0369] (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.

[0370] In another aspect, the CEACAM1 antibody or an antigen-binding fragment thereof comprises

[0371] (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;

[0372] (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

[0373] (iii) And wherein:

[0374] The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO:13,

[0375] The sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO:13,

[0376] The sequence of CDR1L comprises residues A28, S30 and Y31 of SEQ ID NO:14,

[0377] The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO:14, and

[0378] The sequence of CDR3L comprises residues S91 and S92 of SEQ ID NO:14.

[0379] The numbering of the residues is based on the primary amino acid sequence of the antibody, see Figure 3A 、 3B and 3C, such as the heavy and light chain sequences.

[0380] In another aspect, the CEACAM1 antibody or an antigen-binding fragment thereof comprises

[0381] (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;

[0382] (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

[0383] (iii) Six CDRs, wherein:

[0384] a. The CDR1 sequence of the heavy chain variable region comprises SEQ ID NO: 9;

[0385] b. The CDR2 sequence of the heavy chain variable region comprises SEQ ID NO: 2;

[0386] c. The CDR3 sequence of the heavy chain variable region comprises SEQ ID NO: 10;

[0387] d. The CDR1 sequence of the light chain variable region comprises SEQ ID NO: 4;

[0388] e. The CDR2 sequence of the light chain variable region comprises SEQ ID NO: 5; and

[0389] f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO: 11.

[0390] 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;

[0391] wherein the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the amino acid sequence of the heavy chain variable region of SEQ ID NO: 13;

[0392] wherein the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the amino acid sequence of the light chain variable region of SEQ ID NO: 14;

[0393] wherein the sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO: 43);

[0394] wherein X is any amino acid;

[0395] wherein X1 is A, D, N or S;

[0396] wherein X2 is A or G; and

[0397] wherein X3 is an amino acid having a hydrophobic side chain including I or M; and

[0398] wherein the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44);

[0399] wherein X4 is D, G, or P;

[0400] wherein X5 is F or P;

[0401] wherein X7 is A or Y; and

[0402] wherein X8 is L, H, or F.

[0403] In another aspect, the CEACAM1 antibody or an antigen-binding fragment thereof comprises

[0404] (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

[0405] (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.

[0406] In another aspect, the CEACAM1 antibody or an antigen-binding fragment thereof comprises

[0407] (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;

[0408] (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;

[0409] (iii) and wherein:

[0410] the sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO:13,

[0411] the sequence of CDR3H comprises residues D102, Y103, F104, P105, and Y106 of SEQ ID NO:13,

[0412] the sequence of CDR1L comprises residues A28, S30, and Y31 of SEQ ID NO:15,

[0413] the sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO:15, and

[0414] The sequence of CDR3L contains residue S92 of SEQ ID NO:15.

[0415] The numbering of residues is based on the primary amino acid sequence of the antibody, see Figure 3A 、 3B and 3C, such as the heavy and light chain sequences.

[0416] In another aspect, the CEACAM1 antibody or its antigen-binding fragment comprises

[0417] (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;

[0418] (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

[0419] (vi) six CDRs, wherein:

[0420] a. The CDR1 sequence of the heavy chain variable region comprises SEQ ID NO:9;

[0421] b. The CDR2 sequence of the heavy chain variable region comprises SEQ ID NO:2;

[0422] c. The CDR3 sequence of the heavy chain variable region comprises SEQ ID NO:10;

[0423] d. The CDR1 sequence of the light chain variable region comprises SEQ ID NO:4;

[0424] e. The CDR2 sequence of the light chain variable region comprises SEQ ID NO:5; and

[0425] f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO:12.

[0426] In another aspect, the CEACAM1 antibody or its antigen-binding fragment comprises

[0427] (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

[0428] (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.

[0429] In another aspect, the CEACAM1 antibody or an antigen-binding fragment thereof comprises

[0430] (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;

[0431] (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;

[0432] (iii) And wherein:

[0433] The sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO: 13,

[0434] The sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO: 13,

[0435] The sequence of CDR1L comprises residues S30 and Y31 of SEQ ID NO: 16,

[0436] The sequence of CDR2L comprises residues S51 and N52 of SEQ ID NO: 16, and

[0437] The sequence of CDR3L comprises residues S91 and S92 of SEQ ID NO: 16.

[0438] The numbering of the residues is based on the primary amino acid sequence of the antibody, see Figure 3A 、 3B and 3C, such as the heavy and light chain sequences.

[0439] In another aspect, the CEACAM1 antibody or an antigen-binding fragment thereof comprises

[0440] (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;

[0441] (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

[0442] (ix) Six CDRs, wherein:

[0443] a. The CDR1 sequence of the heavy chain variable region comprises SEQ ID NO: 9;

[0444] b. The CDR2 sequence of the heavy chain variable region comprises SEQ ID NO: 2;

[0445] c. The CDR3 sequence of the heavy chain variable region comprises SEQ ID NO: 10;

[0446] d. The CDR1 sequence of the light chain variable region comprises SEQ ID NO: 18;

[0447] e. The CDR2 sequence of the light chain variable region comprises SEQ ID NO: 5; and

[0448] f. The CDR3 sequence of the light chain variable region comprises SEQ ID NO: 11.

[0449] 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 its antigen-binding fragment utilizes

[0450] the framework described in Table 1.

[0451] In some embodiments of the aspects described herein, amino acid sequence modifications of the antibody or its antigen-binding fragment that binds to CEACAM1 as described herein are contemplated. Amino acid sequence variants of the antibody or its antigen-binding fragment are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody or its antigen-binding fragment 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 antibody or its antigen-binding fragment. Any combination of deletions, insertions, and substitutions is made to obtain the final construct, provided that the final construct has the desired characteristics, such as binding specificity, inhibition of biological activity.

[0452] One type of variant is a conservative amino acid substitution variant. These variants have at least one amino acid residue in the antibody or its antigen-binding fragment replaced with a different residue having similar side chain characteristics. Amino acids can be grouped according to the similarity of their side chain characteristics (see Lehninger, BIOCHEMISTRY (2nd ed., Worth Publishers, New York, 1975):

[0453] (1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M);

[0454] (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q);

[0455] (3) Acidic: Asp (D), Glu (E);

[0456] (4) Basic: Lys (K), Arg (R), His (H).

[0457] Thus, a non-limiting example of a conservative amino acid substitution is the replacement of a non-polar amino acid with another non-polar amino acid.

[0458] Alternatively, naturally occurring residues can be grouped based on common side-chain properties:

[0459] (1) Hydrophobic: Ala (A), Val (V), Leu (L), Ile (I), Met (M);

[0460] (2) Neutral hydrophilic: Ser (S), Thr (T), Cys (C), Asn (N), Gln (Q);

[0461] (3) Acidic: Asp (D), Glu (E);

[0462] (4) Basic: Lys (K), Arg (R), His (H);

[0463] (5) Residues affecting chain orientation: Gly (G), Pro (P);

[0464] (6) Aromatic: Phe (F), Trp (W), Tyr (Y).

[0465] Thus, a non-limiting example of a conservative amino acid substitution is the replacement of a hydrophobic amino acid with another hydrophobic amino acid.

[0466] Amino acid sequence insertions are also contemplated, which can include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody or antigen-binding fragment thereof having an N-terminal methionyl residue, or an antibody or antigen-binding fragment thereof fused to a cytotoxic polypeptide. Other insertion variants of an antibody or antigen-binding fragment thereof include fusing the N or C terminus of the antibody or antigen-binding fragment thereof to an enzyme or polypeptide (such as biotin) that increases the serum half-life of the antibody or antigen-binding fragment thereof.

[0467] Any cysteine residues that do not participate in maintaining the correct conformation of an antibody or antigen-binding fragment thereof that binds CEACAM1 can also be replaced, for example, with serine or alanine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking.

[0468] Conversely, cysteine bonds can be added to an antibody or antigen-binding fragment thereof to increase its stability (particularly where the antibody or antigen-binding fragment thereof is an antibody fragment such as an Fv fragment).

[0469] In some embodiments, an antibody or antigen-binding fragment thereof has an amino acid alteration that changes the original glycosylation pattern of the antibody or antigen-binding fragment thereof. "Changing the original glycosylation pattern" means deleting one or more carbohydrate moieties present in the antibody or antigen-binding fragment thereof, and / or adding one or more glycosylation sites not present in the antibody or antigen-binding fragment thereof. Glycosylation of an antibody is generally N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of galactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Adding a glycosylation site to an antibody or antigen-binding fragment thereof that binds CEACAM1 is achieved by altering the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding or replacing one or more serine or threonine residues in the sequence of the original antibody or antigen-binding fragment thereof (for O-linked glycosylation sites).

[0470] In some embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof provided herein are deglycosylated or non-glycosylated. In some embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof contemplated lack a C-terminal lysine in the heavy chain and / or contain an S241P substitution in the constant region of the heavy chain. In some embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof lack a glycosylation site in CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof lack an N-X-S / T consensus sequence in CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof have a mutation in CDR residues 26 and / or 29 (Kabat numbering) of CDR1 of the variable light chain. When the antibody or antigen-binding fragment thereof comprises an Fc region, the carbohydrates linked thereto can be altered. For example, antibodies having a mature carbohydrate structure that lack fucose linked to the Fc region of the antibody or antigen-binding fragment thereof are described. See, e.g., U.S. Patent Publication Nos. 2003 / 0157108; 2004 / 0093621. Antibodies having a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrates linked to the Fc region of the antibody or antigen-binding fragment thereof are cited in WO 03 / 011878; U.S. Patent No. 6,602,684. Antibodies having at least one galactose residue in the oligosaccharides linked 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 linked to their Fc regions.

[0471] In some embodiments, it may be desirable to modify the CEACAM1-binding antibodies or antigen-binding fragments thereof described herein with respect to effector function, e.g., to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody or antigen-binding fragment. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody or antigen-binding fragment. Alternatively or additionally, one or more cysteine residues can be introduced in the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The resulting homodimeric antibodies or antigen-binding fragments thereof may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent 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), homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional crosslinkers. Alternatively, antibodies or antigen-binding fragments thereof having a dual Fc region can be engineered and can thus have enhanced complement lysis and ADCC capabilities. See Stevenson et al., 3 Anti-Cancer Drug Design 219 (1989).

[0472] For example, WO 00 / 42072 describes antibodies having improved ADCC function in the presence of human effector cells, wherein the antibodies contain amino acid substitutions in their Fc regions. Preferably, antibodies or antigen-binding fragments thereof having improved ADCC contain substitutions at positions 298, 333, and / or 334 in the Fc region (residue Eu numbering). Generally, the altered Fc region is a human IgG1 Fc region that contains substitutions at one, two, or three of these positions or consists thereof. Such substitutions are optionally combined with substitutions that increase Clq binding and / or CDC. Substitutions include the Asn297Ala mutation in IgG1 Fc.

[0473] Antibodies having altered Clq binding and / or complement-dependent cytotoxicity (CDC) are described in WO 99 / 51642, U.S. Patent Nos. 6,194,551, 6,242,195, 6,528,624, and 6,538,124. The antibodies contain amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333, and / or 334 in their Fc regions (residue Eu numbering).

[0474] Antibodies with improved binding to the neonatal Fc receptor (FcRn) and increased half-life are described in WO 00 / 42072 and US Patent Publication No. 2005 / 0014934. These antibodies contain an Fc region with one or more substitutions therein that improve the binding of the Fc region to CEACAM1. For example, the Fc region can have substitutions 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 containing a preferred Fc region with improved CEACAM1 binding contain amino acid substitutions at one, two, or three of positions 307, 380, and 434 in their Fc region (Eu numbering of residues). In one embodiment, the antibody or its antigen-binding fragment has 307 / 434 mutations. Engineered antibodies that bind CEACAM1 and have three or more (e.g., four) functional antigen-binding sites are also contemplated. See, e.g., US Patent Publication No. US2002 / 0004587.

[0475] Antibody fragments and types

[0476] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab fragment that comprises the variable (V L ) and constant (C L ) domains of the light chain and the variable domain (V H ) and the first constant domain (C H 1) of the heavy chain or consists essentially of the foregoing.

[0477] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab' fragment, which refers to a Fab fragment having one or more cysteine residues at the C-terminus of the C H 1 domain.

[0478] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is an Fd fragment that comprises V H and C H 1 domains or consists essentially of the foregoing.

[0479] In some embodiments of the aspects described herein, the CEACAM1 antibody moiety is an Fd' fragment that comprises V H and C H 1 domains and one or more cysteine residues at the C-terminus of the C H 1 domain.

[0480] Single-chain Fv or scFv antibody fragments contain the V H and V L domains or consist essentially thereof, such that these domains are present in a single polypeptide chain. Typically, the Fv polypeptide also contains a polypeptide linker between the V H and V L domains, which allows the scFv to form the required structure for antigen binding. See, e.g., 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 an Fv fragment that contains the V L and V H domains or consists essentially thereof of a single arm of the antibody.

[0481] In some embodiments of the aspects described herein, the CEACAM1 antibody moiety is a diabody that contains two antigen-binding sites and that contains a heavy-chain variable domain (V L ) linked to a light-chain variable domain (V H ) in the same polypeptide chain.

[0482] In some embodiments of the aspects described herein, the CEACAM1 antibody moiety is a dAb fragment that contains the V H domain or consists essentially thereof.

[0483] In some embodiments of the aspects described herein, the CEACAM1 antibody moiety is an F(ab')2 fragment that contains a bivalent fragment that contains two Fab' fragments linked by a disulfide bond in the hinge region.

[0484] Linear antibodies refer to the antibodies described by Zapata et al., Protein Engin., 8(10):1057-1062 (1995). Briefly, these antibodies contain a pair of tandem Fd fragments (V H -C H 1-V H -C H 1), which together with complementary light-chain polypeptides form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a linear antibody that contains a pair of tandem Fd fragments (V H -C H 1-V H -C H 1), and the tandem Fd fragments together with complementary light-chain polypeptides form a pair of antigen-binding regions.

[0485] A variety of techniques have been developed and can be used to generate antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies. See, e.g., 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 from recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed herein. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically conjugated 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 will be apparent to those skilled in the art. In other embodiments, the selected antibody fragment is a single-chain Fv fragment (scFv). See, e.g., WO 93 / 16185.

[0486] In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds CEACAM1 and a complementary region that binds PD-1.

[0487] In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds CEACAM1 and a complementary region that binds PD-L1.

[0488] The antibodies or antigen-binding fragments contemplated can have all types of constant regions, including IgM, IgG, IgD, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgG1 is used. In another embodiment, the human isotype IgG4 is used. The light chain constant region can be λ or κ. The antibody or its antigen-binding fragment can comprise sequences from more than one class or isotype.

[0489] 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 the CAR (also referred to as a chimeric T cell receptor) to target an antigen expressed on tumor cells in a human leukocyte antigen-independent manner.

[0490] Antibody binding

[0491] The human CEACAM1 gene generates 11 isoforms through alternative splicing. Each isoform has a variable (V)-like Ig domain at the amino (N)-terminal of the protein. Except for the 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 sequence (A) between the constant C2-like Ig domain and the transmembrane domain. The transmembrane CEACAM1 isoforms also have a long (L) or short (S) cytoplasmic domain, which is determined by including or excluding CEACAM1 exon 7 in the messenger. The CEACAM1L cytoplasmic domain has two ITIM motifs, which are unique to CEACAM1 among CEACAM family members. In one aspect, the present invention provides a CEACAM1 antibody or an antigen-binding fragment thereof, including the antibodies described herein by their structural features, which binds to the extracellular variable (V)-like Ig domain at the amino (N)-terminal (N domain) of the CEACAM1 protein, i.e., the domain common to all isoforms of CEACAM1, and the isoforms include CEACAM1 isoforms 1L, 1S, 3L, 3S, 4L, 4S, 3A1, 3AS, 3, 4C1, and 4C2. In some embodiments, the provided antibodies and their antigen-binding fragments bind to human CEACAM1. In some embodiments, the provided antibodies and their antigen-binding fragments 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-VN domain: residues 35-142 of SEQ ID NO:26). The mature form of CEACAM1 (without the signal sequence) is provided as SEQ ID NO:17.

[0492] As used herein, "binding" of an antibody or an antigen-binding fragment thereof to CEACAM1, an epitope on CEACAM1, or to certain specific residues on CEACAM1 in some embodiments below includes the selective interaction of the antibody or antigen-binding fragment with CEACAM1. Thus, binding includes, for example, primary and secondary interactions, including hydrogen bonds, ionic interactions, salt bridges, and hydrophilic and hydrophobic interactions.

[0493] In certain embodiments, the CEACAM1 antibody or an antigen-binding fragment thereof described herein is at 10 -5 to 10 -12mol / l, 10 -6 to 10 -12 mol / l, 10 -7 to 10 -12 mol / l, 10 -8 to 10 -12 mol / l, 10 -9 to 10 -12 mol / l, 10 -10 to 10 -12 mol / l or 10 -11 to 10 -12 mol / l of K D binds to CEACAM1. In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are at 10 -5 to 10 -11 mol / l, 10 -6 to 10 -11 mol / l, 10 -7 to 10 -11 mol / l, 10 -8 to 10 -11 mol / l, 10 -9 to 10 -11 mol / l or 10 -10 to 10 -11 mol / l of K D binds to CEACAM1. In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are at 10 -5 to 10 -10 mol / l, 10 -6 to 10 -10 mol / l, 10 -7 to 10 -10 mol / l, 10 -8 to 10 -10 mol / l or 10 -9 to 10 -10 mol / l of K D binds to CEACAM1. In other embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are at 10 -5 to 10 -8 mol / l, 10 -6 to 10 -8 mol / l or 10 -7 to 10 -8 mol / l of K D binds to CEACAM1.

[0494] As used herein, the term "specificity" refers to the ability of an antibody or an antigen-binding fragment thereof (such as an anti-CEACAM1 antibody or an antigen-binding fragment thereof) to recognize an epitope within CEACAM1 with little or no detectable reactivity with other parts of CEACAM1. Specificity can be determined relatively by competitive assays or by epitope identification / characterization techniques described herein or their equivalents known in the art.

[0495] As used herein, an "epitope" can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by contiguous amino acids generally remain upon exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope typically includes at least 3, more typically at least 5, about 9, or about 8-10 amino acids in a particular spatial conformation. An "epitope" includes the structural unit normally bound by an immunoglobulin V H / V L pair. An epitope defines the minimal binding site of an antibody or an antigen-binding fragment thereof and thus represents the specific target of the antibody or an antigen-binding fragment thereof. In the case of a single-domain antibody, the epitope represents the structural unit bound by the isolated variable domain.

[0496] In certain embodiments, the antibody or antigen-binding fragment under consideration specifically binds to the same epitope as the antibody CP08H03 / Vk8S29A. In another embodiment, the antibody or antigen-binding fragment under consideration binds to the same epitope as CP08H03 / CP08F05.

[0497] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including the 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 provided antibody or antigen-binding fragment thereof specifically binds to one or more of the CEACAM1 residues contained within 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.

[0498] 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 an antigen-binding fragment thereof binds 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 of skill in the art. For example, any suitable method known in the art can be used to identify competing antibodies, cross-blocking antibodies, and antibodies that are cross-blocked, including competitive ELISA or assays, in which the binding of a competing antibody or cross-blocking antibody to human CEACAM1 prevents the binding of the antibodies disclosed herein, and vice versa.

[0499] In one embodiment, the heavy chain of the antibody or antigen-binding fragment thereof being considered 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 antibody or antigen-binding fragment thereof being considered also specifically binds CEACAM1 at residues S32, Q44, A49, and / or I91 of SEQ ID NO:17.

[0500] In one embodiment, the light chain of the antibody or antigen-binding fragment thereof being considered 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 antibody or antigen-binding fragment thereof being considered also specifically binds CEACAM1 at residues L95 and / or V96 of SEQ ID NO:17.

[0501] In another embodiment, a CEACAM1 antibody or an 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, a CEACAM1 antibody or an antigen-binding fragment thereof also specifically binds CEACAM1 at residues S32, Q44, A49, I91, L95, and / or V96 of SEQ ID NO:17.

[0502] In another embodiment, the CEACAM1 antibody or an antigen-binding fragment thereof specifically binds to CEACAM1 at residues F29, Y34, D40, G41, N42, T56, Q89, S93, D94, N97, and E99 of SEQ ID NO:17.

[0503] In another embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to CEACAM1 at residues F29, S32, Y34, D40, G41, N42, Q44, A49, T56, Q89, I91, S93, D94, L95, V96, N97, and E99 of SEQ ID NO:17.

[0504] In certain embodiments, not all of the CDRs directly participate in binding to the antigen. In one embodiment, four of the six CDRs of the CEACAM1 antibody or an antigen-binding fragment thereof contact the antigen. In one embodiment, five of the six CDRs of the CEACAM1 antibody or an antigen-binding fragment thereof contact the antigen. In one embodiment, six of the six CDRs of the CEACAM1 antibody or an antigen-binding fragment thereof contact the antigen. In one embodiment, CDR2H, CDR3H, CDR1L, CDR2L, and CDR3L of the CEACAM1 antibody or an antigen-binding fragment thereof directly participate in binding to the antigen.

[0505] In one embodiment, the antibodies and antigen-binding fragments provided herein specifically bind to an epitope of CEACAM1 located on the N domain of CEACAM1. In one embodiment, the antibody or an 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.

[0506] In one embodiment, the antibodies and antigen-binding fragments provided herein specifically bind to an epitope of CEACAM1 located on the N domain of CEACAM1. In one embodiment, the antibody or an antigen-binding fragment thereof specifically binds 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.

[0507] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein binds CEACAM1, wherein

[0508] CDR2H residue Y57 binds CEACAM1 at residue F29,

[0509] CDR2H residue Y59 binds CEACAM1 at residue S93,

[0510] CDR3H residue D102 binds CEACAM1 at residue T56,

[0511] CDR3H residue Y103 binds CEACAM1 at residue Y34 and / or Q89,

[0512] CDR3H residue F104 binds CEACAM1 at residue F29,

[0513] CDR3H residue Y106 binds CEACAM1 at residue D94,

[0514] CDR1L residue S30 binds CEACAM1 at residue E99,

[0515] CDR1L residue Y31 binds CEACAM1 at residue N97,

[0516] CDR2L residue S51 binds CEACAM1 at residue D40, and / or

[0517] CDR2L residue N52 binds CEACAM1 at residue G41 and / or N42.

[0518] The numbering of CDR residues is based on the primary amino acid sequence of the antibody, see Figure 3A 、 3B and 3C, such as the heavy and light chain sequences. CEACAM1 residues are numbered according to SEQ ID NO:17.

[0519] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein binds CEACAM1, wherein

[0520] CDR2H residue Y57 binds CEACAM1 at residue F29,

[0521] CDR2H residue Y59 binds CEACAM1 at residue S93,

[0522] CDR3H residue D102 binds CEACAM1 at residue T56,

[0523] The CDR3H residue Y103 binds to CEACAM1 at residues S32, Y34, Q44, and / or Q89,

[0524] The CDR3H residue F104 binds to CEACAM1 at residues F29 and / or A49,

[0525] The CDR3H residue P105 binds to CEACAM1 at residue I91,

[0526] The CDR3H residue Y106 binds to CEACAM1 at residue D94,

[0527] The CDR1L residue S30 binds to CEACAM1 at residue E99,

[0528] The CDR1L residue Y31 binds to CEACAM1 at residue N97,

[0529] The CDR2L residue S51 binds to CEACAM1 at residue D40,

[0530] The CDR2L residue N52 binds to CEACAM1 at residues G41 and / or N42,

[0531] The CDR3L residue S91 binds to CEACAM1 at residue L95, and / or

[0532] The CDR3L residue S92 binds to CEACAM1 at residue V96.

[0533] The numbering of the residues is based on the primary amino acid sequence of the antibody, see Figure 3A 、 3B and 3C, such as the heavy and light chain sequences. The CEACAM1 residues are numbered according to SEQ ID NO:17.

[0534] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein binds to CEACAM1, wherein

[0535] The CDR2H residue Y57 binds to CEACAM1 at residue F29,

[0536] The CDR2H residue Y59 binds to CEACAM1 at residue S93,

[0537] The CDR3H residue D102 binds to CEACAM1 at residue T56,

[0538] The CDR3H residue Y103 binds to CEACAM1 at residues S32, Y34, Q44, and Q89,

[0539] The CDR3H residue F104 binds to CEACAM1 at residues F29 and A49,

[0540] The CDR3H residue P105 binds to CEACAM1 at residue I91,

[0541] The CDR3H residue Y106 binds to CEACAM1 at residue D94,

[0542] The CDR1L residue S30 binds to CEACAM1 at residue E99,

[0543] The CDR1L residue Y31 binds to CEACAM1 at residue N97,

[0544] The CDR2L residue S51 binds to CEACAM1 at residue D40,

[0545] The CDR2L residue N52 binds to CEACAM1 at residues G41 and N42,

[0546] The CDR3L residue S91 binds to CEACAM1 at residue L95, and

[0547] The CDR3L residue S92 binds to CEACAM1 at residue V96.

[0548] The numbering of the residues is based on the primary amino acid sequence of the antibody, see Figure 3A 、 3B and 3C, such as the heavy and light chain sequences. The CEACAM1 residues are numbered according to SEQ ID NO:17.

[0549] CEACAM family members are widely expressed on a variety of cell types, particularly leukocytes, thereby affecting the amplitude of cell function. 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.

[0550] Although the N domains are 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, the embodiments of the present invention can avoid unwanted interference, such as the broad activation functions of CEACAM3.

[0551] The terms "selective" and "selectivity" as used herein refer to the preferential binding of an antibody or an antigen-binding fragment thereof (i.e., a CEACAM1 antibody or an antigen-binding fragment thereof) to a specific region, target, or peptide; the region, target, or peptide is typically a region or epitope in CEACAM1 and not one or more other biomolecules, including other CEACAM family members.

[0552] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof being considered does not exhibit significant binding to CEACAM3, CEACAM5, CEACAM6, and / or CEACAM8. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof being considered does not exhibit detectable binding to CEACAM3, CEACAM5, CEACAM6, and / or CEACAM8. In some embodiments, the binding affinity of the CEACAM1 antibody or antigen-binding fragment thereof being considered for CEACAM1 is at least 10-fold, such as at least 100-fold, and at least 1000-fold, and up to 10,000-fold or greater than the binding affinity of the CEACAM1 antibody or antigen-binding fragment thereof being considered for another target or polypeptide.

[0553] As used herein, the "affinity" represented by the equilibrium dissociation constant (K D ) of an antigen from an antigen-binding protein is a measure of the strength of binding between an epitope and an antigen-binding site on the antigen-binding protein (such as an antibody or an antibody fragment thereof). The smaller the K D value, the stronger the binding strength between the epitope and the antigen-binding molecule. Alternatively, the affinity can also be expressed as an association constant (K A ), which is 1 / K D ). As will be apparent to those skilled in the art, the affinity can be determined in a manner known per se, depending on the particular antigen of interest.

[0554] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including the 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 sites 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).

[0555] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including the 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 member of the TIM family on CEACAM1, thereby blocking the interaction between CEACAM1 and the member of the TIM family. In some embodiments, this member of the TIM family 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 the 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).

[0556] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including the 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 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 a pathogenic bacterium that binds CEACAM1, which pathogenic bacterium includes but is not limited to Escherichia coli, particularly diffusely adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria commensalis, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus aegyptius, Helicobacter pylori and / or Salmonella sp.

[0557] 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 the CEACAM1 residues F29, Y34, N42, Q89 and N97, which residues have been predicted to be involved in the binding of CEACAM1 to HopQ.

[0558] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and opacity-associated (Opa) adhesin proteins expressed on the surface of Neisseria species, said adhesin proteins 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 Neisseria Opa proteins.

[0559] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and the Opa-like protein OlpA expressed on the surface of Moraxella species.

[0560] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Haemophilus influenzae OMP P1. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to one or more of CEACAM1 residues Q44 and A49, which have been predicted to be involved in the binding of CEACAM1 to Haemophilus influenzae OMP P1.

[0561] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Haemophilus aegyptius OMP P1. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to CEACAM1 residue F29, which has been predicted to be involved in the binding of CEACAM1 to Haemophilus aegyptius OMP P1.

[0562] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Candida albicans.

[0563] 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).

[0564] In another embodiment, the present invention provides a method of inhibiting the binding of CEACAM1 to filarial nematodes using the CEACAM1 antibody or antigen-binding fragment thereof described herein, said method comprising contacting CEACAM1 with the CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the filarial nematode is Wuchereria bancrofti.

[0565] Antibody conjugate

[0566] In some embodiments of the aspects described herein, an antibody or an antigen-binding fragment thereof that binds to CEACAM1 is conjugated to a functional moiety. Examples of functional moieties that can be used include, but are not limited to, a blocking moiety, a detectable moiety, a diagnostic moiety, a targeting moiety, and a therapeutic moiety.

[0567] Exemplary blocking moieties include moieties that have sufficient steric hindrance and / or charge such that glycosylation of the antibody or antigen-binding fragment thereof is reduced, for example, by the ability to block glycosidase glycosylation. Additionally or alternatively, a blocking moiety can reduce effector function, for example, by the ability to inhibit Fc region-binding receptors or complement proteins. Preferred blocking moieties include cysteine adducts and PEG moieties.

[0568] In a preferred embodiment, the blocking moiety is cysteine, preferably cysteine that associates with free cysteine, for example, during or after translation of an Fc-containing polypeptide in cell culture. Other blocking cysteine adducts include cystine, mixed disulfide adducts, or disulfide bonds.

[0569] In another preferred embodiment, the blocking moiety is a polyalkylene glycol moiety, such as a PEG moiety, and preferably a PEG-maleimide moiety. Preferred polyethylene glycolylated moieties (or related polymers) can be, for example, polyethylene glycol (“PEG”), polypropylene glycol (“PPG”), polyoxyethylated glycerol (“POG”), and other polyoxyethylated polyols, polyvinyl alcohol (“PVA”), and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glucose. The polymer can be a homopolymer, a random or block copolymer, a terpolymer based on the monomers listed above, linear or branched, substituted or unsubstituted, provided that it has at least one reactive sulfone moiety. The polymer moiety can have any length or molecular weight, but these characteristics can affect biological properties. The average molecular weight of polymers that are particularly useful for reducing clearance in pharmaceutical applications ranges from 2,000 to 35,000 daltons. Additionally, if two groups are attached to the polymer, one at each end, the length of the polymer can affect the effective distance between the two groups and other spatial relationships. Thus, one of ordinary skill in the art can vary the length of the polymer to optimize or impart desired biological activity. PEG can be used in biological applications for several reasons. PEG is generally clear, colorless, odorless, water-soluble, heat-stable, inert to many chemical reagents, non-hydrolyzable, and non-toxic. Polyethylene glycolylation can improve the pharmacokinetic properties of a molecule by increasing the apparent molecular weight of the molecule. The increased apparent molecular weight reduces clearance from the body after subcutaneous or systemic administration. In many cases, polyethylene glycolylation can reduce antigenicity and immunogenicity. Additionally, polyethylene glycolylation can increase the solubility of a bioactive molecule.

[0570] Examples of detectable moieties that can be used in 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, etc., 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., luminol). The fluorophore can be environmentally sensitive such that its fluorescence is altered if it is located near one or more residues in the modified protein that undergo a conformational change upon binding to a substrate (e.g., dansyl probe). Exemplary radiolabels include small molecules containing atoms having one or more low-sensitivity nuclei ( 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.

[0571] Examples of diagnostic moieties that can be used in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include detectable moieties suitable for revealing the presence of a disease or disorder. Generally, the diagnostic moiety allows determination of the presence, absence or level of a molecule (e.g., a target peptide, protein or plurality of proteins) associated with a disease or disorder. Such diagnostics are also applicable to predicting and / or diagnosing a disease or disorder and its progression.

[0572] Examples of therapeutic moieties that can be used in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include, for example, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, anti-infective agents or therapeutic agents in general. The functional moiety can also have one or more of the above functions.

[0573] Exemplary therapeutic moieties include radionuclides having high-energy ionizing radiation, which are capable of causing multiple strand breaks in nuclear DNA and are thus suitable for inducing cell death (e.g., in cancer). Exemplary high-energy radionuclides include: 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188Re. These isotopes typically produce high-energy α or β particles with short path lengths. Such radionuclides kill the cells in their immediate vicinity, such as neoplastic cells to which the conjugate has been attached or has entered. They have little or no effect on non-targeted cells and are essentially non-immunogenic.

[0574] Exemplary therapeutic moieties also include cytotoxic agents such as cell growth inhibitors (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalating agents or cross-linking agents, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin-binding agents, hormones and hormone antagonists, anti-angiogenic agents, and the like.

[0575] Exemplary therapeutic moieties also include alkylating agents such as the anthracycline family of drugs (e.g., doxorubicin, carminomycin, cyclosporin-A, chloroquine, methotrexate, mithramycin, puromycin, streptozocin, anthramycin, and aziridine). In another embodiment, the chemotherapy moiety is a cell growth inhibitor 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 intercalating agents or cross-linking agents include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-dichlorodiammineplatinum(II) (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0576] Exemplary therapeutic moieties also include transcription 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., quercetin, genistein, bactacyclin), busulfan, ifosfamide, nitrogen mustard, triaziquone, diaziquone, carboquone, indoloquinone EO9, divinylenetriaminebenzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0577] Exemplary therapeutic moieties also include cytotoxic nucleosides such as vidarabine, cytarabine, cytarabine arabinoside, 5-fluorouracil, fludarabine, floxuridine, tegafur, and 6-mercaptopurine; microtubule-binding agents such as taxanes (e.g., paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatin (e.g., dolastatin 10, 11, or 15), colchicine and colchicine-like agents (e.g., ZD6126), combretastatin (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.

[0578] Exemplary therapeutic moieties also include hormones and hormone antagonists such as corticosteroids (e.g., prednisone), progesterones (e.g., hydroxyprogesterone or medroxyprogesterone), estrogens (e.g., diethylstilbestrol), anti-estrogens (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-diphosphonate, leuprolide, luteinizing hormone-releasing hormone, pifithrin-α, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0579] Exemplary therapeutic moieties also include enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-rotenone, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrosine phosphorylation inhibitor AG34, and tyrosine phosphorylation inhibitor AG 879.

[0580] 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, tretinoin, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0581] The exemplary therapeutic moiety also includes cytotoxic agents such as 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, vinorelbine, vindesine, vinblastine, etc.

[0582] Other cytotoxins compatible with the teachings herein include auristatins (e.g., auristatin E and monomethyl auristatin E), calicheamicin, gramicidin D, maytansine alkaloids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, teniposide, colchicine, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and their analogs or homologs.

[0583] Techniques for conjugating such therapeutic moieties to antibodies are well known, see, for example, 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", 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).

[0584] To increase the half-life of an antibody or polypeptide comprising the amino acid sequences described herein, a salvage receptor binding epitope can be linked to the antibody or an antigen-binding fragment thereof (especially an antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. The term "salvage receptor binding epitope" can refer to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule (e.g., Ghetie et al., 18 Ann. Rev. Immunol. 739 (2000)). Antibodies having substitutions in their Fc regions 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 ligated in-frame with a nucleic acid encoding a polypeptide sequence described herein such that the fusion protein expressed by the engineered nucleic acid molecule comprises the salvage receptor binding epitope and the polypeptide sequence described herein. In another embodiment, the serum half-life can also be increased, for example, by linking other polypeptide sequences. For example, an antibody or an antigen-binding fragment thereof useful in the methods of the invention can be linked to serum albumin or a portion of serum albumin that binds to the CEACAM1 receptor or a serum albumin binding peptide such that the serum albumin binds the antibody or an antigen-binding fragment thereof, such as the polypeptide sequences disclosed in WO 01 / 45746. In one embodiment, the half-life of a Fab is increased by these methods. For additional serum albumin binding peptide sequences, also see Dennis et al., 277 J. Biol. Chem. 35035 (2002).

[0585] Other types of functional moieties are known in the art and can be readily used in the methods and compositions of the invention based on the teachings contained herein.

[0586] Nucleic acid

[0587] 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, 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 comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0588] Nucleic acids encoding CEACAM1 antibodies and antigen-binding fragments thereof can be, for example, DNA, cDNA, RNA, synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecules comprising any one of those polynucleotides alone or in combination. For example, expression vectors are provided that contain polynucleotide sequences encoding CEACAM1 antibodies or antigen-binding fragments thereof as described herein, the polynucleotide sequences being operably linked to expression control sequences suitable for expression in eukaryotic and / or prokaryotic host cells.

[0589] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. "Vectors" include, but are not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules, which may consist of chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. In some embodiments, the vectors used are vectors capable of autonomous replication (episomal vectors) and / or expression of the nucleic acids linked thereto (expression vectors). 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 virus, AAV), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentiviruses, and foamy viruses.

[0590] A variety of expression vectors have been developed for the 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 are also provided that contain expression vectors for expressing the desired CEACAM1 antibody or antigen-binding fragment thereof.

[0591] Antibody Preparation and Expression Systems

[0592] The antibodies or antigen-binding fragments thereof of the present invention are generally produced by recombinant expression. Nucleic acids encoding the variable regions of the light and heavy chains (optionally linked to constant regions) are inserted into an expression vector. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector to ensure expression of the immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (e.g., native or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells. Once the vector is integrated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for collecting and purifying the cross-reactive antibody.

[0593] These expression vectors can generally replicate in the host organism either as episomes or as an integral part of the host chromosomal DNA. Typically, the expression vector contains a selectable 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, for example, Itakura et al., U.S. Patent No. 4,704,362).

[0594] Expression of the antibodies and antigen-binding fragments contemplated by the present invention can occur in prokaryotic or eukaryotic cells. Suitable hosts include bacteria or eukaryotic hosts, including yeast, insect, fungal, avian, and mammalian cells in vivo or in situ, or host cells derived from mammals, insects, birds, or yeast. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, but any other mammalian cells can be used.

[0595] Escherichia coli is a particularly useful prokaryotic host for cloning the polynucleotides (e.g., DNA sequences) of the present invention. Other suitable microbial hosts include bacilli, such as Bacillus subtilus, and other Enterobacteriaceae, such as the genus Salmonella, the genus Serratia, and various species of the genus Pseudomonas.

[0596] Other microorganisms such as yeast can also be used for expression. The genera Saccharomyces and Pichia are exemplary yeast hosts, where suitable vectors have expression control sequences (e.g., promoters), origins of replication, 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, iso-cytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.

[0597] In addition, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved by using, for example, the yeast ubiquitin hydrolase system. The fusion proteins so produced can be processed in vivo or purified and processed in vitro, thereby allowing synthesis of the CEACAM1 antibodies or antigen-binding fragments thereof of the present invention having a specific amino-terminal sequence. In addition, problems associated with retaining the methionine residue derived from the start codon in direct yeast (or bacterial) expression can be avoided. Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).

[0598] When yeast is grown in a glucose-rich medium, any of a series of yeast gene expression systems incorporating the promoter and terminator elements of the actively expressed genes encoding glycolytic enzymes produced in large amounts can be used to obtain the recombinant CEACAM1 antibodies or peptides of the present invention. The 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 utilized.

[0599] Production of CEACAM1 antibodies or antigen-binding fragments thereof in insects can be achieved. For example, by methods known to those skilled in the art, an insect host is infected with a baculovirus engineered to express a transmembrane polypeptide. See Ausubel et al., 1987, 1993.

[0600] In addition to microorganisms, mammalian tissue cultures can also be used to express and produce the antibodies or antigen-binding fragments thereof (e.g., polynucleotides encoding immunoglobulins or fragments thereof) of the present invention. See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Eukaryotic cells are actually preferred because many suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, and such 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 origins of replication, 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 papillomavirus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0601] Alternatively, a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al., U.S. Patent No. 5,741,957; Rosen, U.S. Patent No. 5,304,489; and Meade et al., U.S. Patent No. 5,849,992). Suitable transgenes include the coding sequences of the light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes such as casein or β-lactoglobulin.

[0602] In addition, plants have become a convenient, safe, and economical alternative to mainstream expression systems based on large-scale culturing of microorganisms or animal cells for the production of recombinant antibodies. Antibodies or antigen-binding fragments thereof can be expressed in plant cell cultures or in conventionally grown plants. Expression in plants can be systemic, restricted to subplastids, or restricted to seeds (endosperm). See, e.g., 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 an advanced stage of development, including clinical trials (see, e.g., Biolex, NC).

[0603] Vectors containing polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or virus-based transfection can be used for other cell hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally Sambrook et al., supra). To produce transgenic animals, the transgene can be microinjected into fertilized oocytes or incorporated into the genome of embryonic stem cells, and the nuclei of such cells can be transferred into enucleated oocytes.

[0604] The antibodies of the present invention and their antigen-binding fragments can be expressed using a single vector or two vectors. When the heavy and light chains of the antibody are cloned onto separate expression vectors, the vectors are co-transfected to obtain expression and assembly of the complete immunoglobulin. Once expressed, the complete antibodies of the present invention, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures in the art, which include ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982))). For pharmaceutical use, substantially pure immunoglobulins with at least about 90% to 95% homogeneity are preferred, and 98% to 99% or higher homogeneity is most preferred.

[0605] Methods for modulating CEACAM1 activity

[0606] In one aspect, the present invention provides methods for using the antibodies and their antigen-binding fragments 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 antibody or its antigen-binding fragment disrupts the homophilic interaction between CEACAM1 monomers.

[0607] In another aspect, the present invention provides methods for using the antibodies and their antigen-binding fragments of the present invention to reduce the interaction between CEACAM1 and members of the TIM family (including but not limited to TIM-1, TIM-3, and TIM-4). In some embodiments, the antibody or its antigen-binding fragment disrupts the heterophilic interaction between CEACAM1 and TIM-3. Disrupting the interaction between CEACAM1 and TIM-3 by using the antibodies and their antigen-binding fragments contemplated by the present invention can reverse the CEACAM1 inhibitory function while maintaining the TIM-3 activation function.

[0608] Embodiments of the present invention can be used to reduce immunosuppression, such as T cell tolerance. "Reduce" means the ability to cause an overall reduction of about 20% or greater, 30% or greater, 40% or greater, 45% or greater, 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, or 75%, 80%, 85%, 90%, 95% or greater compared to an untreated control. Immunosuppression can be mediated by immunosuppressive receptors expressed on the surface of immune cells and their interaction with their ligands. For example, cytotoxic CD8 T cells can enter a "functionally exhausted" or "non-responsive" state, whereby they express inhibitory receptors that prevent antigen-specific responses such as proliferation and cytokine production. Thus, by inhibiting the activity and / or expression of such inhibitory receptors, the immune response to cancer or tumors that has been blocked, inhibited or non-responsive can be enhanced or not inhibited. Enhancement or reversal of such immune response inhibition can lead to higher T cell activity, responsiveness and / or ability or receptivity regarding activation.

[0609] Methods of measuring T cell activity are known in the art. As non-limiting examples, T cell anergy can be induced by contacting T cells with recall antigens, anti-CD3 (in the absence of co-stimulation), and / or ionomycin. The levels of, for example, IL-27, LDH-A, RAB10, and / or ZAP70 (intracellular or secreted) can be monitored, for example, to determine the degree of T cell anergy induction (wherein the levels of IL-2, interferon-γ, and TNF are associated with increased T cell anergy). The response of cells pre-treated with, for example, ionomycin to an antigen can also be measured to determine the degree of T cell anergy in a cell or cell population, for example, by monitoring the levels of secreted and / or intracellular IL-2 and / or TNF-α (see, for example, Macian et al. Cell 2002 109:719-731). Other characteristics of anergic T cells include increased levels of Fyn and ZAP-70 / Syk, Cbl-b, GRAIL, Ikaros, CREM (cAMP response element modulator), B lymphocyte-induced maturation protein-1 (Blimp-1), PD1, CD5, and SHP2; increased phosphorylation of ZAP-70 / Syk, LAT, PLCγ1 / 2, ERK, PKC-θ / IκBα; increased activation of intracellular calcium levels; decreased histone acetylation or hypoacetylation and / or increased CpG methylation at the IL-2 locus. Thus, in some embodiments, one or more of any of these parameters can be determined to determine whether an antibody or an antigen-binding fragment thereof that inhibits CEACAM1 as disclosed herein reduces immune anergy. Reduction of T cell anergy can also be evaluated by examining tumor-infiltrating lymphocytes or T lymphocytes within lymph nodes draining an established tumor. Such T cells exhibit an "exhausted" phenotype by 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 anergy has been reduced in the presence of a CEACAM1 antibody or an antigen-binding fragment thereof includes, for example, an increased number of T cells relative to that observed in the absence of the inhibitor, said T cells having (a) antigen specificity for a tumor-associated antigen (e.g., as determined by major histocompatibility complex class I or II tetramers containing a tumor-associated peptide) and (b) the ability to secrete high levels of interferon-γ and cytolytic effector molecules such as granzyme-B.

[0610] CEACAM1 antibodies and antigen-binding fragments thereof can also be used to enhance T cell expansion, activation, and proliferation.

[0611] On the other hand, the present invention provides a method of using the antibodies and antigen-binding fragments thereof of the present invention to reduce the interaction between CEACAM1 and bacterial adhesins. In some embodiments, the antibodies and antigen-binding fragments thereof of the present invention effectively reduce and / or prevent colonization of mammalian epithelia. In some embodiments, the adhesin is expressed by Escherichia coli, particularly diffusely adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria commensalis, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus aegyptius, Helicobacter pylori, and / or Salmonella spp. 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 opacity-associated (Opa) adhesin protein expressed on the surface of Neisseria spp. 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 spp.

[0612] 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 of using the CEACAM1 antibody or its antigen-binding fragment described herein to inhibit the binding of CEACAM1 to filarial nematodes. In one embodiment, the filarial nematode is Wuchereria bancrofti.

[0613] Therapeutic methods

[0614] 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.

[0615] In the methods described herein, a therapeutically effective amount of the antibody or antigen-binding portion thereof described herein is administered to a mammalian subject in need thereof. Although the antibodies or antigen-binding portions thereof described herein are particularly useful for administration to humans, they can also be administered to other mammals. As used herein, the term "mammal" is intended to include, but not be limited to, humans, laboratory animals, domestic pets, and farm animals. "Therapeutically effective amount" means an amount that, when administered to a mammal, effectively produces the desired therapeutic effect.

[0616] In some aspects, an antibody or an antigen-binding fragment thereof binds to CEACAM1 expressed by exhausted T cells or natural killer (NK) cells, thereby restoring T cell and NK cell activity and resulting in an increased anti-tumor response. In other aspects, an antibody or an antigen-binding fragment thereof binds to CEACAM1 expressed by tumor cells, thereby inhibiting tumor cell metastasis and the formation of cancer stem cell niches. In yet another aspect, an antibody or an antigen-binding fragment thereof binds to CEACAM1 expressed by macrophages associated with fibrosis in the tumor microenvironment, thereby inhibiting fibrosis. In yet another aspect, an antibody or an antigen-binding fragment thereof binds to CEACAM1 expressed by other stromal cells (such as vascular endothelial cells) in the tumor microenvironment, thereby inhibiting angiogenesis.

[0617] Accordingly, the present disclosure also provides methods of treating a subject having cancer or a tumor and / or reducing tumor growth, which include 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 disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, or the presence or size of dormant tumors.

[0618] The term "cancer" refers to or describes a physiological condition in a mammal that is generally characterized by unregulated cell growth. This definition includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Thus, as used herein, the term "cancer" refers to the uncontrolled growth of cells that interferes with the normal function of body organs and systems, including cancer stem cells and tumor vascular niches. A subject having cancer is a subject in whom objectively measurable cancer cells are present. This definition includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that metastasize from their original location and seed vital organs can ultimately lead to the death of the subject through the degradation of the function of the affected organs. Hematopoietic cancers, such as leukemia, are capable of overwhelming the subject's normal hematopoietic compartment, resulting in hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia), ultimately leading to death.

[0619] "Subject" means a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat, etc. An individual and a patient are also subjects herein.

[0620] As used herein, the term "treat", "treated", "treating" or "treatment" refers to a therapeutic treatment in which the object is to slow down (alleviate) an unwanted physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms; reducing the extent of the condition, disorder or disease; stabilizing (i.e., not worsening) the state of the condition, disorder or disease; delaying the onset or slowing the progression of the condition, disorder or disease; improving the state of the condition, disorder or disease; and remission (partial or complete), detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without an excessive level of side effects. Treatment also includes prolonging survival as compared to the expected survival of an untreated subject. The terms "prevent", "prevention", etc. refer to acting before the onset of an overt disease or disorder to prevent the development of the disease or disorder or to minimize the extent of the disease or disorder, or to slow its course of development.

[0621] Embodiments of the present invention can be used to treat metastasis, which involves the spread of cancer from its primary site to other parts of the body. Cancer cells can detach from the primary tumor, infiltrate the lymph and blood vessels, circulate through the bloodstream, and grow in distant foci (metastases) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a continuous process that depends on the tumor cells detaching from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form life-threatening masses. Both stimulatory and inhibitory molecular pathways within the tumor cells regulate this behavior, and the interaction between the tumor cells and the host cells at the distant site is also important. In addition to monitoring for specific symptoms, metastasis is most commonly 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, either alone or in combination.

[0622] Methods of reducing cancer stemness are also contemplated, which include administering a CEACAM1 antibody or an antigen-binding fragment thereof disclosed herein. Cancer stemness can refer to the ability of cells to self-renew and give rise to additional phenotypically distinct cell types. Cancer stem cells (CSCs) are cancer cells that exhibit stem cell-like properties. CSCs typically exhibit at least one cancer hallmark and are capable of giving rise to at least one additional phenotypically distinct cell type. In addition, cancer stem cells are capable of asymmetric and symmetric replication. It should be understood that cancer stem cells can be generated from differentiated cancer cells that have acquired stemness traits and / or stem cells that have acquired a phenotype associated with cancer cells. Alternatively, cancer stem cells can reconstitute non-stromal cell types within the tumor.

[0623] CEACAM1 is expressed by many tumor types, and CEACAM1 can regulate tumor growth and metastatic behavior. In another embodiment, CEACAM1 inhibition will reduce tumor growth and metastasis.

[0624] CEACAM1 expression on macrophage subsets is associated with fibrosis during carcinogenesis. In another embodiment, CEACAM1 inhibition will reduce tumor-associated fibrosis.

[0625] Cancers that can be treated by the compositions and methods contemplated by the present invention include tumors that are non-vascularized or not yet substantially vascularized, as well as vascularized tumors. Cancers can include non-solid tumors (such as hematologic tumors, e.g., leukemia and lymphoma) or can include solid tumors. The types of cancers 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, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. 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 cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric 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 lymphoma and non-Hodgkin 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 cancers; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system cancers; vulvar cancer; and other carcinomas and sarcomas; and B cell lymphoma (including low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocyte (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved 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 myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with pharyngopathy, edema (such as edema associated with brain tumors), and Meigs syndrome. A patient can have more than one type of cancer.

[0626] The efficacy of a cancer treatment method using a therapeutic preparation comprising a composition containing the 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, reduction in tumor weight or size, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, and quality of life. In the case of cancer, a therapeutically effective amount of a recombinant CEACAM1 antibody or an antigen-binding fragment thereof can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and preferably prevent to some extent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and preferably prevent to some extent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with the disorder to some extent. In the case where a patient has more than one type of cancer, a therapeutically effective amount of a recombinant CEACAM1 antibody or an antigen-binding fragment thereof is an amount effective to treat at least one of the cancers. With respect to the use of a recombinant CEACAM1 antibody or an antigen-binding fragment thereof 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 duration of survival, progression-free survival duration (PFS), response rate (RR), duration of response, and / or quality of life.

[0627] Checkpoint proteins interact with specific ligands, sending signals to T cells and cutting 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, thus suppressing the anti-cancer immune response. The immune checkpoint protein programmed death-1 (PD-1) is a key immune checkpoint receptor expressed by activated T and B cells and mediates immune suppression. 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 have been identified, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), which are expressed on antigen-presenting cells as well as on many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al., 2000; Latchman et al., 2001). Inhibiting the 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), cemiplimab-rwlc (REGN2810), pidilizumab (CT-011), spartalizumab (PDR001), tislelizumab (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.

[0628] However, there is a large population of cancer patients receiving checkpoint inhibitor therapy who (1) do not respond to this type of therapy (congenital or primary resistance) or (2) initially respond but eventually develop disease progression (secondary or acquired resistance). Resistant cancers can 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 naïve patients 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 reduced anti-cancer response in resistant patients.

[0629] Accordingly, the present invention also provides methods of treating patients resistant 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 the specific CEACAM1 antibodies and antigen-binding fragments provided herein). In some embodiments, the CEACAM1 antibody for treating patients resistant to inhibitors of PD-1, PD-L1, and / or CTLA-4 is CP08H03 / Vk8S29A or CP08H03 / CP08F05. In some embodiments, the resistance is congenital or primary resistance. In some embodiments, the resistance is secondary or acquired resistance. In some embodiments, the administered CEACAM1 antibody (including but not limited to the CEACAM1 antibodies and antigen-binding fragments provided herein) reverses T cell exhaustion in patients resistant to checkpoint inhibitor therapy. Any cancer that exhibits resistance to PD-1, PDL-1, and / or CTLA-4 is suitable for treatment by 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.

[0630] In another aspect, the present invention provides the use of the CEACAM1 antibodies and antigen-binding fragments provided herein in treating patients resistant to therapies using other checkpoint inhibitors, said other checkpoint inhibitors including but not limited to PD-L2, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (which belongs to the CD2 molecule family and is expressed on 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).

[0631] In another aspect, the present invention provides methods of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to treat a mammal in need of reducing and / or preventing colonization of Candida albicans and / or bacteria expressing adhesins (including but not limited to Escherichia coli, particularly diffusely adherent Escherichia coli (DAEC), Neisseria spp., Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus aegyptius, Helicobacter pylori, and / or Salmonella spp.) on the epithelium. In another aspect, the present invention provides methods of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to reduce influenza virus replication and / or reduce the release of pro-inflammatory cytokines or chemokines associated with influenza virus infection. In some embodiments, the influenza virus is H5N1. In another aspect, the present invention provides methods of using the CEACAM1 antibody agents and antigen-binding fragments thereof disclosed herein to treat a subject in need of reducing and / or preventing infection with a filarial nematode such as Wuchereria bancrofti. In another aspect, the present invention provides methods of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to treat a subject in need of reducing and / or preventing the development of lymphedema and / or hydrocele associated with infection with a filarial nematode such as Wuchereria bancrofti. In one embodiment, the present invention provides a method of using the CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce invasion of a subject's lymphatic system by filarial nematodes in a subject in need thereof. In one embodiment, the filarial nematode is Wuchereria bancrofti. The subject may be infected with more than one bacterium expressing adhesins, Candida albicans, influenza virus, and / or filarial nematode.

[0632] In another embodiment, the present invention provides a method of using the CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce invasion of a subject's lymphatic system by cancer cells in a subject in need thereof.

[0633] Screening methods

[0634] Also provided herein are methods of identifying a population of patients likely to respond to treatment with the CEACAM1 antibodies and antibody fragments provided herein (including but not limited to CP08H03 / Vk8S29A and CP08H03 / CP08F05).

[0635] In some embodiments, CEACAM1 expression on certain cell types of cancer patients is screened, said cell types including 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 a control are selected for treatment with the CEACAM1 antibodies and antibody fragments provided herein. The "control" level of CEACAM1 expression can refer to the level of CEACAM1 expression in one or more individuals without cancer. The level can be measured on an individual-by-individual basis or on an aggregated basis such as an average. In some embodiments, the control level of CEACAM1 expression obtained from the same individual whose condition is being monitored but at different times. In certain embodiments, the "control" level can 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 therapy.

[0636] In some embodiments, CEACAM1 expression is determined for patients resistant to checkpoint inhibitor therapy (including but not limited to therapy with PD-1 / PD-L1 / CTLA-4 inhibitors). In some embodiments, patients resistant to checkpoint inhibitor therapy and showing increased CEACAM1 expression on certain cell types compared to a control are selected for treatment with the CEACAM1 antibodies and antibody fragments provided herein (including but not limited to CP08H03 / Vk8S29A and CP08H03 / CP08F05).

[0637] In some embodiments, the allelic variants of human CEACAM1 of a patient are determined. Based on the allelic variants of human CEACAM1 expressed by the patient, more or less anti-CEACAM1 antibody can be administered to the patient compared to a patient expressing the wild-type variant of CEACAM1. In some embodiments, the presence of the Y34C, Q44L, and / or Q89H allelic variants of CEACAM1 in the patient is determined. In some embodiments, a higher and / or more frequent dose of anti-CEACAM1 antibody is administered to a patient expressing the Y34C, Q44L, and / or Q89H allelic variants of CEACAM1 compared to a patient expressing the wild-type variant of CEACAM1.

[0638] Drug composition

[0639] 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 as described herein formulated with one or more pharmaceutically acceptable excipients.

[0640] The dosage of the active agent can vary depending on the reason for use, the individual subject, and the mode of administration. The dosage can be adjusted based on the body weight of the subject, the age and health of the subject, and the tolerance of the compound or composition. For example, depending on the disease, for an antibody or antigen-binding fragment thereof, 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 / mol, these dosages correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μM, and 1.8 μM of binding sites in a 5 L blood volume.

[0641] The active agent 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 specifically formulated in solid or liquid forms, including those suitable for parenteral administration, such as by subcutaneous, intratumoral, intramuscular, or intravenous injection, for example as a sterile solution or suspension.

[0642] A therapeutic composition comprising an antibody or antigen-binding fragment thereof that binds 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 (such as lubricants, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), solvents, or encapsulating materials, which are involved in carrying or transporting a 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 carboxymethyl cellulose, 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; diols, 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 buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations.

[0643] Fillers are compounds that increase the mass of a pharmaceutical formulation and contribute to the physical structure of the formulation in freeze-dried form. Suitable fillers according to the present invention include mannitol, glycine, polyethylene glycol, and sorbitol.

[0644] The use of surfactants can reduce the aggregation of refolded proteins and / or reduce the formation of particles in the refolding formulation. The amount of surfactant added should be sufficient to reduce the aggregation of refolded proteins and minimize the formation of particles after refolding. 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; lauramidopropyl-, cocamidopropyl, linoleamidopropyl-, myristamidopropyl-, palmitamidopropyl- or isostearamidopropyl-betaine (e.g., lauramidopropyl); myristamidopropyl-, palmitamidopropyl- or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or sodium methyl oleoyl taurate; and copolymers of polyethylene glycol, polypropylene glycol, and ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.).

[0645] Preservatives can be used in the formulations of the present invention. Suitable preservatives for the formulations of the present invention include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl-dimethyl ammonium chlorides, where the alkyl is a long-chain compound) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl esters of p-hydroxybenzoic acid such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. Other suitable excipients can be found in standard pharmaceutical textbooks, such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Edition Mack Publishing Company, Easton, Pa., (1995).

[0646] Compositions comprising an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier can comprise various concentrations of the CEACAM1 antibodies or antigen-binding portions thereof described herein. For example, the composition can comprise from 10 mg / ml to 200 mg / ml, 25 mg / ml to 130 mg / ml, 50 mg / ml to 125 mg / ml, 75 mg / ml to 110 mg / ml, or 80 mg / ml to 100 mg / ml of the antibody or antigen-binding fragment thereof. The composition can also comprise about 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, or 150 mg / ml of the antibody or antigen-binding fragment thereof.

[0647] In some embodiments, a composition comprising an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier is lyophilized and provided in the composition for reconstitution prior to administration.

[0648] Method of Administration

[0649] The therapeutic composition comprising the antibody or antigen-binding fragment thereof under consideration can be administered in any convenient manner, including by injection, infusion, implantation, or transplantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intracranially, by intravenous or intralymphatic injection, or intraperitoneally to a patient. In one embodiment, the cell composition of the invention is preferably administered by intravenous injection.

[0650] In certain embodiments, the antibody or antigen-binding fragment thereof is administered to a mammal by intravenous infusion, i.e., introducing the antibody or antigen-binding fragment thereof into a 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.

[0651] In certain embodiments, the dose of the compound or composition is administered to a subject once daily, 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 doses, three doses, or four doses of the compound or composition are administered to a subject once daily, every two days, every three days, once a week, once every two weeks, or once a month. In some embodiments, the dose of the compound or composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, or 28 days. In certain embodiments, the dose of the compound or composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or longer.

[0652] Combination therapy

[0653] 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.

[0654] 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.

[0655] 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.

[0656] Exemplary additional therapeutic agents also include alkylating agents such as the anthracycline family of drugs (e.g., doxorubicin, carminomycin, cyclosporin-A, chloroquine, methotrexate, mithramycin, puromycin, streptozotocin, anthramycin and aziridine). In another embodiment, the chemotherapeutic moiety is a cell growth inhibitor 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 intercalating agents or cross-linking agents include but are not limited to bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-dichlorodiammineplatinum(II) (cisplatin), melphalan, mitoxantrone and oxaliplatin.

[0657] 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 quinone, quinone derivatives (e.g., quinolone, genistein, bactacyclin), busulfan, ifosfamide, nitrogen mustard, triaziquone, diaziquone, carboquone, indoloquinone EO9, divinyliminobenzoquinone methyl DZQ, triethylenephosphoramide and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0658] Exemplary additional therapeutic agents also include cytotoxic nucleosides such as vidarabine, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, tegafur and 6-mercaptopurine; tubulin-binding agents such as taxanes (e.g., paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatin (e.g., dolastatin 10, 11 or 15), colchicine and colchicine-like agents (e.g., ZD6126), combretastatin (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.

[0659] 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.

[0660] 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.

[0661] 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.

[0662] 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.

[0663] 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.

[0664] In one embodiment, the CEACAM antibody or an antigen-binding fragment thereof is administered in combination with an agent that is a checkpoint inhibitor. Such inhibitors can include small molecule inhibitors or can include antibodies or antigen-binding fragments thereof that bind and block or inhibit an immune checkpoint receptor or antibodies that bind and block or inhibit an immune checkpoint receptor ligand. Illustrative checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (which belongs to the CD2 family of molecules and is expressed on 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. B-7 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, biotherapeutic agents, or small molecules that bind 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 (a CTLA-4 blocking antibody), anti-OX40, and Yervoy / ipilimumab (an anti-CTLA-4 checkpoint inhibitor), and the aforementioned 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.

[0665] In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered together with a TIGIT, LAP, Podoplanin, protein C receptor, ICOS, GITR, CD226, or CD160 inhibitor.

[0666] 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.

[0667] It should be understood that the present invention is not limited to the specific molecules, compositions, methods or protocols described, as these may vary. 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, where a specific feature is disclosed in the context of a particular aspect or embodiment of the present invention or a particular claim, that feature can also be used in combination with and / or in the context of other particular aspects and embodiments of the present invention to the extent possible, and is generally used in the present invention.

[0668] In the case of methods mentioned herein that include two or more defined steps, the defined steps can be carried out in any order or simultaneously (unless the context excludes that possibility), and the method can include one or more other steps carried out before any defined step, between two defined steps or after all defined steps (unless the context excludes those possibilities).

[0669] All other cited patents and applications are incorporated herein by reference in their entirety. Additionally, when the definitions and uses of terms in the references incorporated herein by reference are inconsistent or contradictory to the definitions of such terms provided herein, the definitions of such terms provided herein shall prevail and the definitions of such terms in the references shall no longer apply.

[0670] For 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.

[0671] Examples

[0672] Example 1: Generation of a fully humanized CEACAM1 antibody

[0673] 1. Generation of humanized antibody variants

[0674] Design of a composite human antibody variable region sequence and expression of the antibody

[0675] First, a structural model of the V region of the parental murine CEACAM1 antibody was generated using Swiss PDB and analyzed to identify potential "restricted" amino acids in the V region that may contribute to the antibody binding properties. For regions outside the CDRs and flanking the CDRs, a widely selected human sequence segment was identified as a possible component of the new humanized V region.

[0676] Based on the structural analysis, a large set of preliminary sequence segments were selected that can be used to generate humanized CEACAM antibody variants, and iTope, which is used for computer analysis of peptides binding to human MHC class II alleles, was usedTM techniques (Perry et al., 2008. Drugs RD 9(6):385 - 396) and the T - cell epitopes associated with known antibody sequences (Bryson et al 2010, Biodrugs 21(1):1 - 8) were analyzed. Sequences identified as important non - germline binders of human MHC class II or those that obtained significant hit scores for TCED TM were discarded. This analysis yielded a reduced set of segments, and the combinations of these segments were analyzed again as described above to ensure that the junctions between segments did not contain potential T - cell epitopes. The selected sequence segments were assembled into full 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 TM Table 1. Some of the heavy and light chains in Table 1 contain variations at positions that are considered part of the CDR according to the Kabat CDR definition but not according to the IMGT CDR definition.

[0677] Table 1. Heavy and light chains selected for gene synthesis.

[0678]

[0679]

[0680]

[0681] Figure 1 Next, the V and Vκ sequences of the parental murine CEACAM1 antibody and the humanized CEACAM antibody variants with flanking restriction enzyme sites were synthesized for cloning into the pANT expression vector system of IgG4(S241P) heavy and κ light chains ( H ). The V region was cloned between the MluI and HindIII restriction sites, and the Vκ region was cloned between the BssHII and BamHI restriction sites. All constructs were confirmed by sequencing. Figure 1 H The 39 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) the chimeric antibody V

[0682] 0 / Vκ0 (composed of the murine V region (V H 0) fused to the constant heavy - chain region of human IgG4 and the murine Vκ region (Vκ0) fused to the constant light - chain region of human IgG4); (2) the pairing of the chimeric VH heavy chain (V H 0) with the light - chain variant Vκ1; and (3) the pairing of the V H 1 heavy chain with the chimeric Vκ light - chain variant (Vκ0). The other 36 were composite IgG4V H 0) and the murine Vκ region (Vκ0) fused to the constant light - chain region of human IgG4); (2) the pairing of the chimeric VH heavy chain (V H 1 heavy chain with the chimeric Vκ light - chain variant (Vκ0). The other 36 were composite IgG4V​H Combinations with 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.

[0683] Antibodies were purified from cell culture supernatants on a Protein A Sepharose column, the buffer was changed to PBS pH 7.4, and quantification was performed by OD 280nm using the 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 motifs identified in the light chain were substantially utilized.

[0684] Competitive ELISA analysis of humanized variants that bind CEACAM1

[0685] The binding of the purified antibodies to human CEACAM1 was evaluated in a competitive 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"), then washed 3 times with PBST pH 7.4. A 3-fold dilution series of the chimeric antibody V H 0 / Vκ0, an irrelevant IgG4 antibody, and humanized CEACAM1 antibodies at 100 μg / ml to 0.07 or 0.002 μg / ml were pre-mixed with a constant concentration of the parental murine antibody (final concentration 0.45 μg / ml), added to the plates and incubated for 1 hour at room temperature. After washing with 3x PBST, the binding of the parental murine CEACAM1 antibody was detected with anti-mouse-HRP and TMB substrate. The reaction was terminated with 3M HCl, the absorbance was read at 450 nm on a Dynex Technologies MRX TC II microplate reader and the binding curves were plotted. The binding of the humanized CEACAM antibody variants to CEACAM1 was compared to the chimeric antibody (V H 0 / Vκ0) included on each plate. Twelve of the 36 humanized CEACAM antibody variants showed no binding to CEACAM1 (including those with Vκ3, Vκ4, and Vκ5). The variants that bind CEACAM1 showed a range of relative IC50 values from 0.9 to 5.2 compared to the chimeric antibody V H 0 / Vκ0. The data are summarized in Table 2.

[0686] Table 2. Summary of titers and binding data for humanized CEACAM antibody variants and control antibodies. Antibody expression titers (μg / ml) were from static HEK EBNA transient transfection. IC50 values obtained in competition assays were normalized to chimeric antibody V H 0 / Vκ0 on the same plate. Unbound antibodies were not included in the table. Antibodies shown in bold were used for multi-cycle kinetic analysis.

[0687] 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

[0688] Kinetic analysis of humanized variants that bind CEACAM1

[0689] 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 carried out at 25 °C with HBS-P+ running buffer (pH 7.4) (GE Healthcare, catalog number BR100671). His-tagged CEACAM1 was used as the analyte for all kinetic experiments. For all experiments, antibodies were immobilized on the surface of an 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 had an analyte binding level of 50 - 150 RU (R max ). For the capture settings of all sample antibodies, 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 because each antibody can bind 2 target molecules, and a target response level of approximately 75 RU were used.

[0690] 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 antibody V H 0 / Vκ0 was added to HEK EBNA medium to be used as a positive control. Antibodies were diluted to a concentration of 1 μg / ml in HBS-P+ (determined by IgG quantification ELISA). At the start of each cycle, antibodies were loaded onto Fc2, Fc3, and Fc4 of the Protein A chip and IgG was captured at a flow rate of 8 μl / min to obtain approximately 75 RU. Then the surface was stabilized. Single-cycle kinetic data were obtained at a flow rate of 50 μl / min to minimize any potential mass transfer effects. For chimeric antibody V H0 / Vκ0 was repeated multiple times to examine the stability of the surface and the analyte during the kinetic cycles. The signals from Fc2, Fc3, and Fc4 were subtracted by the signal from the reference channel Fc1 (without antibody) to correct for differences in non-specific binding to the reference surface. A 5-point 2-fold dilution range of 3.125 to 50 nM CEACAM1 without regeneration between concentrations was used. The association phase of 5 increasing concentrations of CEACAM1 injections was monitored for 100 s and after the last injection of CEACAM1, a single dissociation phase of 150 s was measured. The protein A surface was regenerated using 2 injections of 10 mM glycine-HCl pH 1.5 followed by a 500 s stabilization period. The signals from each antibody blank run (without CEACAM1) were subtracted to correct for differences in surface stability. Single-cycle kinetics (

[0691] Table 3) showed that 24 humanized variants bound to CEACAM1 while 12 variants did not. When combined with any of the humanized heavy chains, the light chains of Vκ3, Vκ5, and Vκ6 abolished CEACAM1 binding. These data were consistent with the competitive ELISA data (Table 2).

[0692] Table 3. Single-cycle kinetic parameters for the binding of humanized CEACAM antibody variants and control antibodies to CEACAM1-HIS determined using Biacore T200. The K D of the humanized CEACAM1 antibody variants was divided by the K H of chimeric antibody V D 0 / Vκ0 determined in the same experiment to calculate the relative K H compared to chimeric antibody V D . Antibodies shown in bold were used for multi-cycle kinetic analysis. S / N, supernatant. Non-binding variants were not included in the table.

[0693]

[0694] Among the 24 antibodies that bound to CEACAM1, antibody variants that exhibited binding within 2-fold of chimeric antibody V H 0 / Vκ0 and had a relative IC50 in the range of 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 Tables 2 and

[0695] Table 3, highlighted in bold).

[0696] For the multi-cycle kinetic analysis, the purified antibody was immobilized in HBS-P+ at a protein concentration of 1 μg / ml. At the start of each cycle, the antibody was captured on Protein A to obtain approximately 75 RU and the surface was stabilized. Kinetic data were obtained at a flow rate of 80 μl / min to minimize any potential mass transfer effects. Multiple replicates of the blank (no CEACAM1) and replicates of a single concentration of the analyte were programmed into the kinetic run to examine the stability of the surface and the analyte during the kinetic cycle. For the kinetic analysis, a 2-fold dilution range of CEACAM1 from 200 to 3.125 nM or 100 to 1.5625 nM was selected. The association phase of CEACAM1 was monitored for 50 or 150 s and the dissociation phase was measured for 100 s. At the end of each cycle, the Protein A surface was regenerated using two injections of 10 mM glycine-HCL pH 1.5.

[0697] The signal from reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to the reference surface, and the global Rmax parameter was used in a 1:1 binding model. By dividing the K D of the humanized CEACAM antibody variants by the K H of the chimeric antibody V D 0 / Vκ0 on the same chip, the relative K H compared to V D 1 / Vκ0 was calculated. The kinetic parameters measured for the interaction of CEACAM1 with the humanized CEACAM antibody variants are shown in Table 4. Table 5 summarizes the average relative K D obtained using 12 antibody combinations in the multi-cycle kinetic analysis.

[0698] Selectivity analysis of humanized variants that bind CEACAM1

[0699] The binding selectivity of 24 humanized antibody variants that bind CEACAM1 (see Table 3) and the chimeric control antibody V H 0 / Vκ0 to CEACAM1 was tested by flow cytometry on HeLa cells transfected with CEACAM1, 3, 5, 6, and 8. As Figure 2 shown, most of the variants were highly selective for CEACAM1 and showed little or no binding to CEACAM3, 5, 6, or 8. Compared to the chimeric control antibody V HCompared to 0 / Vκ0, all 24 variants showed reduced binding to CEACAM5. There was no evidence of any staining of the HeLa-CEACAM3 or HeLa-CEACAM8 transfectants. Therefore, this data was not reported. Due to its favorable affinity and selectivity for CEACAM1 and its favorable expression level, V H 1 / Vκ8 was selected as the framework for affinity maturation.

[0700] Table 4. Multicycle kinetic data (n = 1) of human antibody variants binding to CEACAM1-HIS measured using Biacore T200. By dividing the K D of the humanized CEACAM antibody variant by the K H of the chimeric antibody V D 0 / Vκ0 measured on the same chip, the relative K H compared to the chimeric antibody V D 0 / Vκ0 was calculated.

[0701]

[0702] Table 5. Summary of the relative K H compared to the chimeric antibody V D 0 / Vκ0 for all humanized CEACAM antibody variants obtained from multicycle kinetics measured using Biacore T200. The fold difference in K D compared to the chimeric antibody V H 0 / Vκ0 was calculated by dividing the K D of the test antibody variant by the K H of the chimeric antibody V D 0 / Vκ0 tested on each chip. The number of independent experiments for each variant is shown.

[0703]

[0704]

[0705] 2. Removal of N-linked / HEK-derived glycosylation

[0706] Sequence analysis showed a potential N-linked glycosylation motif in the original murine hybridoma light chain CDR1. The CDR1L of the parental murine antibody contains the N-X-S / 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 the potential glycosylation-related immunogenicity, two CDR mutations were designed to remove the N-X-S / T consensus sequence (glycosylation site): N26Q and S29A (Kabat numbering scheme). Mutation of either residue eliminates glycosylation, as Figure 4 shown.

[0707] Competitive ELISA experiments (see Table 6), multi-cycle kinetic analyses (see Table 7), and selectivity analyses (see Table 8) were performed to confirm the binding of the mutant chimeras to CEACAM1. Compared to the antibody mutant N26Q, the antibody mutant S29A (Kabat numbering scheme) exhibited a higher expression level and a K D more similar to that of the unmutated antibody (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.

[0708] Table 6. Results of competitive ELISA experiments using non-glycosylated chimeric antibodies. CDR1L residues are numbered according to the Kabat numbering scheme.

[0709]

[0710] Table 7. Multi-cycle kinetic analyses using non-glycosylated chimeric antibodies. CDR1L residues are numbered according to the Kabat numbering scheme.

[0711]

[0712] Table 8. Selectivity analyses of non-glycosylated humanized variants. CDR1L residues are numbered according to the Kabat numbering scheme. Binding selectivity of antibody variants was evaluated by flow cytometry using HeLa cells transfected with vectors expressing CEACAM1, CEACAM5, and CEACAM6, respectively. Shown is the relative amount of cells expressing the corresponding antigen bound by the indicated antibody.

[0713] 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

[0714] Table 9. Experimental summary of non-glycosylated chimeric antibodies. CDR1L residues are numbered according to the Kabat numbering scheme.

[0715]

[0716] 3. Affinity maturation of non-glycosylated CEACAM1 antibody VH1 / VK8S29A Phage vector construction and binding test of parental V H 1 / VK8S29A scFv

[0717] For the lead antibody V HAffinity maturation of one of 1 / VK8, constructing genes encoding V H 1 and VK8 and converting them into scFv form using overlap PCR, where the heavy chain is linked to the light chain via a 15 - amino - acid (G4S)3 linker. The 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 ). Then the scFv sequence is cloned into the phagemid vector pANT43 using the restriction enzymes Sfi I and Not I such that the scFv is displayed on the phage surface as a gene III fusion protein ( Figure 5 ). The cloned scFv is transformed into Escherichia coli (TG1) and all constructs are confirmed by sequencing. Phages containing the parental V H 1 / VK8S29A scFv or an irrelevant scFv are prepared and tested for binding to GST - CEACAM1 ( Figure 6 ). Phages derived from the parental V H 1 / VK8S29A sequence bind antigen - specifically, as no binding was observed for the irrelevant phages.

[0718] Mutagenesis and library construction

[0719] To construct an affinity - matured library, semi - random codons are used to target specific amino acids within CDR1H, CDR3H, and CDR3L of the non - glycosylated humanized antibody V H 1 / Vκ8S29A for "hot - spot" mutagenesis. The sequence positions of possible contact residues are analyzed and sorted in order within each block. This information is used together with the amino - acid preferences at any given position in CDR3 and the crystal structure of the parental murine antibody. Where possible, the highest - ranked contact residues within each block are preferred.

[0720] Four different libraries are generated: one library for mutagenesis of CDR1H (HC), two libraries for mutagenesis of CDR3H, and one library for mutagenesis of CDR3L (see Figure 7 ).

[0721] CDR1H is identified as being five amino acids in length (S31 to S35) (Kabat definition, corresponding to residues 31 - 35 in 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 everything into account and combining with the crystal data of the parental murine antibody, G26 to S35 are included in a single library, where each position includes a subset of amino acids.

[0722] The CDR3H was identified as 12 amino acids in length (H95 - Y102 according to Kabat definition, corresponding to residues 99 - 110 of the primary amino acid sequence of the heavy variable chain, see Figure 3A ). For mutagenesis, the CDR3H was divided into two libraries that overlapped at D100 (corresponding to residue 104 of the primary amino acid sequence of the heavy variable chain according to Kabat definition, see Figure 3A and 3B ): Block 1 (R94 to D100 according to 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 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 contained a subset of amino acids at all positions. Position R94 (corresponding to residue 98 of the primary amino acid sequence of the heavy variable chain according to Kabat definition, see Figure 3A and 3B )(Block 1) was included to allow for more diversity in the germline residues that anchor the CDR.

[0723] The CDR3L was identified as 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 Kabat definition, see Figure 3C ). The region Q90 to P96 (corresponding to residues 89 to 97 of the primary amino acid sequence of the light variable chain according to Kabat definition, see Figure 3C ) was included in a single library, where each position included a subset of amino acids. Kabat numbering was used for all protein sequence coordinates.

[0724] Fig. 8A 、 8B and 8C show an overview of library construction. A non - expressing plasmid containing a truncated fragment of the V H 1 / Vκ8S29A parental scFv and two consecutive stop codons in the region to be randomized was prepared. The purpose of this step was to reduce the likelihood that the parental scFv would be produced and dominate the selection (as occasionally observed during affinity maturation), such that only recombinant antibody fragments generated by PCR could form functional scFvs in the phagemid vector.

[0725] For the CDR3L library, randomization of CDR3L was performed by carrying out two PCRs. In the first PCR, a randomized 3′ primer and V HFW1-specific 5′ primers were used to amplify most of the scFv gene and introduce mutations into VκCDR3. A second PCR added the remaining part of the scFv and appended a restriction site (Not I) for subcloning of the fragment.

[0726] For the V H library, PCR of the V H library was carried out by two rounds of PCR using two templates containing the full-length parental scFv part. Initially, the V H was amplified with a randomized 5′ library primer and a 3′ primer specific for Vκ light chain FW4. In a separate PCR, the remainder of the V H was amplified with a 5′ primer based on the heavy chain FW1 region plus a 3′ primer complementary to a part of the V H CDR randomization primer. Then the full-length V H CDR randomized scFv library was constructed by annealing the two amplified fragments and re-amplifying the scFv by PCR using a primer that appended two restriction sites (Sfi I or Not I) for subcloning of the fragment.

[0727] To assess the diversity of the generated libraries, the purified amplified DNA of all four libraries was then digested with Sfi I and Not I and ligated into a similarly cut phagemid vector (pANT43). The ligated DNA was precipitated, resuspended in nuclease-free water, and transformed into freshly prepared electrocompetent TG1 cells by electroporation. The next day, the colonies were counted, the plates were scraped and glycerol stocks were prepared. The libraries were electroporated multiple times to adequately cover the theoretical library diversity. In all cases, a coverage of 4.0-fold or greater was obtained. Individual colonies from each of the four libraries were sequenced to confirm that the appropriate CDR blocks had been mutated.

[0728] Bacteria from each library were inoculated into 150 ml of 2TYCG (2%) culture using an inoculum of at least 10x the observed library diversity. The cultures were grown to mid-log phase (OD 600nm ≈0.5 - 0.6), and the total cell number was estimated (based on OD 600nm of 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 then the phage was harvested using 4 / 10x volume of cold 20% PEG / 2.5 M NaCl precipitation. After incubation on ice for 1 hour, the precipitated phage was recovered by centrifugation and the pellet 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 filtered through a sterile filter. To increase the chance of obtaining scFv with increased affinity, due to the relatively low affinity of the starting antibody, the multivalent hyperphage M13K07ΔpIII helper phage was used at a multiplicity of infection of 20 for library rescue. After the first round of selection, due to the expected enrichment of antigen binders, the monovalent M13K07 helper phage with a multiplicity of infection of 10 was used.

[0729] Phage selection with improved affinity

[0730] Two separate selection strategies were implemented to increase the likelihood of obtaining phage with improved affinity. CEACAM1 was biotinylated (for soluble selection) or not biotinylated (for solid-phase panning) throughout the selection. In round 1, soluble selection (activity 1) or solid-phase panning (activity 2) was used in different selection cascades to enrich for functional binding phage and diversity. The negative selection using closely related family members CEACAM5 and CEACAM6 was carried out by individual panning at 1 μg / ml of each protein to try and reduce cross-reactivity. This was done twice during each activity either before any round of selection and before round 2 (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.

[0731] For soluble selection, each library was pre-blocked with PBSB, and then the phage was incubated with decreasing concentrations of biotinylated CEACAM1 antigen for up to three hours. After incubation, streptavidin paramagnetic beads (pre-blocked as above) were added to each selection and inverted and rotated for 15 minutes. The streptavidin-antigen-phage complex was washed with increasing numbers of PBST washes followed by PBS washes in each round of successive selection, and captured with a magnet between each step. The phage was eluted from the beads by adding 50 mM HCl, and then the solution was neutralized by adding 1 M Tris-HCl pH 9.0.

[0732] Solid-phase panning and all de-selections were carried out overnight at 4 °C on Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates coated with the antigen, and then blocked with PBSB. For de-selection, the pre-blocked phages were incubated with CEACAM5 and then with CEACAM6, and then the unbound phages were removed and used for subsequent selection. For CEACAM1 panning, the pre-blocked phages were incubated with 8 μg / ml antigen, and then the plates were washed with 3x PBST and 2x PBS. As with soluble selection, the bound phages were eluted with 50 mM HCl. For soluble selection and panning, the eluted phages were added to mid-log Escherichia coli TG1 and the cells were infected for 1 h at 37 °C, then plated on 2TYCG (2%) plates and grown overnight at 37 °C. The next day, colonies were picked for screening, or the plates were scraped and the phages were rescued as described above. Fig.9A and 9B shows an overview of the different selection strategies used.

[0733] Expression and initial testing of scFv

[0734] Soluble scFv was initially expressed and tested as a crude periplasmic extract. Single colonies were picked into 1 ml of 2TYCG (0.1%) medium and grown by shaking at 37 °C for 5 h. The cultures were induced by adding IPTG to a final concentration of 1 mM and then grown overnight by shaking at 30 °C. The next day, the cultures were centrifuged and the supernatant was discarded. The bacterial pellet was resuspended in tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer pH 7.4 and incubated on ice for 30 min. The cells were then centrifuged and the supernatant was discarded. The pellet was resuspended in ice-cold 5 mM MgSO4. The plates were then centrifuged and the supernatant containing the scFv was transferred to fresh plates for assay.

[0735] The periplasmic extracts of colonies from different rounds of selection were screened in a single-point binding assay for their ability to bind GST-CEACAM1. The parental scFv (V H 1 / Vκ8S29A) and an irrelevant scFv were included on each assay plate for comparison.

[0736] The periplasmic extract was diluted 1:1 with PBSB and then incubated for 1 hour at room temperature on a Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plate pre-coated with 1.0 μg / ml GST-CEACAM1. The plate was subsequently washed and the binding of the scFv was detected with anti-HIS6-HRP antibody and TMB substrate. The reaction was terminated with 1M HCl, the absorbance was read at 450 nm on a Dynex Technologies MRX TC II microplate reader, and the binding data were plotted.

[0737] Based on the activity in the binding ELISA relative to the parental scFv V H 1 / Vκ8S29A (which contains murine CDRs) and an irrelevant scFv assayed on the same plate, improved clones were identified. More than 4400 periplasmic extracts were analyzed, 34 leading sequences with a binding at least 1.5-fold higher than the parental in two independent experiments were sequenced, and unique clones were identified. Examination of the obtained sequences showed that parental amino acids were found at several positions but were encoded by codons different from the parental. This indicates that selection occurred as expected, but the parental amino acids are the preferred amino acids at this position. Based on this sequence analysis, 19 unique CDR1H, three CDR3H block 1, three CDR3H block 2, and nine unique CDR3L clones were used for large-scale scFv expression. Table 10 shows a summary of the 34 leading sequences selected as purified scFvs for further analysis and the CDR mutations of these mutants.

[0738] Tables 11, 12, and 13 highlight the conservation / variability of affinity-matured CDRs in the scFv variant leading sequences identified using GST-CEACAM1 binding ELISA.

[0739] Table 10. Summary of 34 scFv variant leading sequences identified using GST-CEACAM1 binding ELISA. The library from which the scFv was derived and the rounds of de-selection performed on the scFv are summarized. The parental (Vκ8S29A) CDRs are shown at the top of the table. Mutations different from the parental sequence in CDR1H, CDR3H B1, CDR3H B2, and CDR3L are highlighted in bold. All variable light chains contain an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0740] 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

[0741] Table 11. Identification of CDR motifs in heavy chain CDR1 of scFv variant leading sequences 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 heavy variable chain. CDR1H contains residues 31 - 35 according to the Kabat CDR definition and residues 26 - 33 according to the IMGT definition.

[0742]

[0743] Table 12. CDR motifs of CDR3 in the heavy chain of the scFv variant leader sequence 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 heavy variable chain. # Not part of the CDR defined by Kabat. Residues were included in mutagenesis to allow for more diversity in the germline residues that anchor the CDR.

[0744]

[0745] Table 13 CDR motifs of CDR3 in the light chain of the scFv variant leader sequence 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.

[0746]

[0747] Large-scale ScFv expression and purification

[0748] The selected clones were expressed, purified, and quantified to accurately test the scFv by binding ELISA. Briefly, single colonies were picked into 15 ml of 2TYCG (2%) medium and grown overnight by shaking at 30 °C. The starter culture was used to inoculate 500 ml of 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 1 mM and then grown overnight with shaking at 30 °C. The next day, the culture was centrifuged and the supernatant was discarded. The bacterial pellet was resuspended in 15 ml of TES and incubated on ice for 15 minutes. Then 22.5 ml of TES (diluted 1:5 in cold water) was added and incubated on ice for an additional 30 minutes. The cells were then centrifuged and the supernatant containing the scFv was transferred to a new tube, and then MgCl2, NaCl, and imidazole were added to final concentrations of 1 mM, 300 mM, and 20 mM, respectively, to reduce non-specific binding. Ni-agarose beads were added and the scFv was allowed to bind by rotating incubation at 4 °C for 2 hours. The beads were pelleted by centrifugation and washed twice with wash buffer (25 mM Tris pH 7.4, 300 mM NaCl, 20 mM imidazole), and then the scFv was eluted from the beads using elution buffer (25 mM Tris pH 7.4, 300 mM NaCl, 400 mM imidazole). The samples were quantified by measuring OD 280 nm and using the extinction coefficient based on the predicted amino acid sequence. Approximately 1 μg of each scFv was analyzed by SDS-PAGE. Bands corresponding to the typical scFv profile were observed.

[0749] Assessment of the binding of scFv to GST-CEACAM1 by ELISA

[0750] Analysis of the binding of affinity matured purified scFv to human CEACAM1 using GST-CEACAM1. Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates were pre-coated overnight at 4 °C with 1.0 μg / ml GST-CEACAM1. The next day, a two-fold dilution series of V H 1 / Vκ8S29A parental scFv or test scFv (50 μg / ml to 0.8 μg / ml) in PBSB was incubated on the pre-coated ELISA plates at RT for 2 hours. Binding of the scFv was detected using anti-HIS6-HRP antibody and TMB substrate. The reaction was terminated with 3M HCl, the absorbance was read at 450 nm on a Dynex Technologies MRX TC II microplate reader and the binding curves were plotted. Exemplary binding assay data are shown in Figure 10 In each ELISA plate, the parental scFv (V H 1 / Vκ8S29A scFv) was included as a reference. An irrelevant scFv was included as a negative control on at least one plate. Many scFvs were observed to have improved binding to CEACAM1 compared to the parental V H 1 / Vκ8S29A scFv. Derived from V HImproved binding was observed with the scFv of the Vκ library. All 34 scFv variants were reformatted into full IgG to provide higher accuracy regarding purity and quantification. The reformatting further allowed analysis of the avidity component of antibody binding, which is affected by the bivalent nature of IgG. As used herein, "avidity" is a measure of the strength of binding between an antigen-binding molecule, such as an antibody or an antibody fragment described herein, and a relevant antigen.

[0751] Construction and testing of affinity-matured full antibodies

[0752] scFv reformatted into full IgG

[0753] Thirty-four variants identified by scFv screening were PCR amplified using primers that introduced flanking restriction enzyme sites for cloning into the IgG4S241P pANTVhG4 vector and the κ light chain pANTVK vector. Twenty-five affinity-matured V H variants were subcloned into the IgG4S241P pANTVhG4 vector using the Mlu I and HindIII restriction sites. Similarly, 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.

[0754] For expression, twenty-five leader-humanized affinity-matured IgG4V H variants were combined with the parental humanized non-glycosylated light chain (Vκ8S29A). Nine leader-humanized affinity-matured κ light chains were combined with the parental humanized heavy chain (V H 1). These combinations were transiently transfected into HEK EBNA adherent cells (in 6-well plates) using the PEI transfection method. Five to seven days after transfection, the supernatants were harvested, quantified by ELISA and filtered for Biacore single-cycle kinetics analysis.

[0755] Single-cycle kinetics analysis of the binding of humanized and affinity-matured lead IgG to CEACAM1

[0756] To evaluate the binding of the reformatted lead IgG with humanized affinity maturation, single-cycle kinetic analysis of the crude supernatant was performed using a Biacore T200 running BiacoreT200 control software V2.0.1 and Biacore T200 evaluation software V3.0. The antibody was diluted to a final concentration of 0.5 μg / ml in HBS-P+. At the start of each cycle, the antibody was loaded onto Fc2, Fc3, and Fc4 of a Protein A chip. IgG was captured at a flow rate of 10 μl / min to obtain an immobilization level (RL) of approximately 100 RU (an R max level of approximately 50 - 150 RU was obtained after calculating the bound analyte). Then the surface was stabilized. CEACAM1 was used as the analyte to obtain single-cycle kinetic data at a flow rate of 80 μl / min to minimize any potential mass transfer effects. Multiple repeats were performed with the parental (V H 1 / Vκ8S29A) antibody to check the stability of the surface and analyte during the kinetic cycles. The signal from the reference channel Fc1 (without antibody) was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to the reference surface. A three-point two-fold dilution range of 70 nM to 280 nM CEACAM1 without regeneration between concentrations was used. The signal from each antibody blank run (without CEACAM1) was subtracted to correct for differences 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. The Protein A surface was regenerated using two injections of 10 mM glycine-HCL pH 1.5, followed by a 250-second stabilization period.

[0757] The single-cycle kinetic constants (

[0758] Table 14) indicate that all antibodies except one humanized antibody with affinity maturation bind CEACAM1.

[0759] Table 14. Single-cycle kinetic constants of humanized and affinity-matured variants and parental (V H 1 / Vκ8S29A) antibodies binding to CEACAM1. The relative K D compared to the parental IgG was calculated by dividing the K D of the humanized and affinity-matured variants by the K D of the parental determined multiple times in the same experiment. The variants performed are shown in bold. CDRs containing mutations are indicated by “+”. All variable light chains contain an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain). The variants shown in bold have the same as the parental V H1 / Vκ8S29A has a relative K more than twice that of the parent D 。

[0760]

[0761]

[0762] Eight humanized and affinity matured heavy and light chain variants were identified that showed a relative K more than twice that of the parent D (highlighted in bold in

[0763] Table 14). These included three V H variants (CP08H03, CP09B03 and CP09C02) and five κ light chain variants (CP08E01, CP08E05, CP08F05, CP08D02 and CP08G09).

[0764] CP09C02 (which was derived from the CDR1H library) contains an additional point mutation in CDR3H B1 (D96G), which was most likely introduced by PCR during library construction (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 having only this single point mutation in V H CDR3B1 and may thus help to identify which region is involved in the observed increase in affinity. Subsequently, four V H and five Vκ variants were obtained to determine whether recombinant affinity matured heavy and light chains could have an improved effect.

[0765] Expression of the combined leading heavy and light chain antibodies

[0766] Each of the four humanized affinity matured IgG4V H variants (CP08H03, CP09B03, CP09C02 and CP09E05) identified after expression with the parental light chain was combined with five leading 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 IgG4V H variant was combined with the parental light chain (Vκ8S29A), and the five leading humanized affinity matured κ light chains were combined with the parental heavy chain (V H1) Combinations (i.e., a total of 10 control antibodies, see Table 15). As described above, the combinations were transiently transfected into HEK EBA adherent cells in 6-well plates using the PEI transfection method and incubated for 5 - 7 days after transfection. The supernatants were harvested, quantified by ELISA, and filtered for single-cycle kinetics analysis on Biacore.

[0767] Table 15. Parental V H or leader humanized affinity matured IgG4 V H variants in combination with parental Vκ or leader humanized affinity matured Vκ light chains. Transfected antibodies with recombinant affinity matured heavy and light chains (black), affinity matured antibodies expressed with parental heavy or light chains, and parental antibodies. All variable light chains contain an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0768]

[0769]

[0770] Single-cycle kinetics analysis of leader heavy and light chain antibody combinations

[0771] Single-cycle kinetics using transient HEK supernatants was performed as described previously. Fitted data for single-cycle kinetics are shown

[0772] Table 16. Fifteen heavy and light chain combinations have at least twice the relative K D . Among them, six combinations (CP08H03 / CP08E05, CP08H03 / CP08F05, CP08H03 / Vκ8S29A, CP09B03 / CP08E05, CP09C02 / CP08E05, and CP09C02 / CP08F05) obtained a K D more than four times greater than the parental (highlighted in bold in

[0773] Table 16). These six variants were used for large-scale production and protein A purification for further analysis. Single-cycle kinetics also revealed that when combined, three variants were found to be non-functional.

[0774] Table 16. Leader V H and Vκ combination antibodies and parental antibodies' single-cycle kinetics constants for binding to CEACAM1. The relative K H compared to the parental was calculated by dividing the K D of the leader V D and Vκ combination variants by the K D of the parental measured in the same experiment. Variants highlighted in bold are KD is a quadruple leader combination of the parental. Mutated CDRs are indicated with a "+". All variable light chains contain an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0775]

[0776]

[0777] Recombination of four affinity matured heavy chain CDRs to generate six additional heavy chain variants

[0778] Six combinations with greater than quadruple improvement are contained in four different V H Three unique heavy chains with mutations in the CDRs: CP08H03 (CDRH3B2); CP09B03 (CDR1H) and CP09C02 (CDR3H B2 and a single mutation in CDR3H B1, which is also uniquely present in CP08E05), see Table 17. To determine if further improvement could be obtained, four mutant V H CDRs were recombined (Table 18). Using scFv specific primers, individual V H CDRs were recombined using overlap extension PCR and subsequently cloned into the IgG4S241P 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).

[0779] Table 17 VH CDRs used for recombination.

[0780]

[0781] Table 18 Recombinant heavy chain clones. Individual CDRs from four leading V H clones were recombined to generate six recombinant affinity matured heavy chains. Mutations that differ from the parental sequence in CDR1H, CDR3H B1 and CDR3H B2 are highlighted in bold. *Residue 104 was selected based on the sequence selected for CDR3H B2.

[0782]

[0783]

[0784] Recombinant V H Expression of CDR1 and CDR3 heavy chains with the leading light chain:

[0785] Six recombinant V H CDR1 and CDR3 variants (see Table 18) were combined with (1) the parental light chain (Vκ8S29A), (2) the light chain CP08E05, or (3) the 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 supernatants were harvested, quantified by ELISA, and filtered for single-cycle kinetic analysis on Biacore.

[0786] Table 19 Recombinant V H CDR1 and CDR3 leading humanized affinity-matured IgG4V H variants combined with the parental Vκ or two leading humanized affinity-matured κ light chains. Combinations with the two affinity-matured light chains are denoted by "AM", and combinations with the parental light chain are denoted by "P". 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).

[0787]

[0788] Recombinant V H combined with leading V L Single-cycle kinetic analysis of antibodies:

[0789] Single-cycle kinetics using transient HEK supernatants were performed as described previously. The fitted data for single-cycle kinetics are shown in Table 20.

[0790] Eight variants were found to have a relative K greater than four-fold compared to the parental D . Among them, five obtained K D were more than six-fold that of the parental (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.

[0791] Table 20 Single-cycle kinetic constants of recombinant leading humanized affinity-matured IgG4V H variants combined with the parental Vκ or one of two leading humanized affinity-matured κ light chains. By dividing the K D of the humanized and affinity-matured variants by the K DTo calculate the relative K compared to the parental antibody D Variants that are > six-fold compared to the parental antibody are highlighted 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).

[0792]

[0793] Expression, purification, and testing of lead antibodies

[0794] Six of the most improved combinatorial variants (CP08H03 / CP08E05, CP08H03 / CP08F05, CP08H03 / Vκ8S29A, CP09B03 / CP08E05, CP09C02 / CP08E05, and CP09C02 / CP08F05,

[0795] highlighted in bold in Table 16) and five of the most improved V H CDR1 and V H CDR3 recombinant variants (8H3_9B3 / CP08E05, 8H3_9B3 / CP08F05, 8H3_9B3 / Vκ8S29A, 8H3_9C2 / CP08F05, and 9B3_9E5 / CP08E05, highlighted in bold in Table 20) were transiently co-transfected into HEK EBNA adherent cells in a shake flask and incubated for 5 - 7 days post-transfection. Antibodies were purified from the cell culture supernatant on a Protein A agarose column, the buffer was changed to PBS pH 7.2, and quantification was performed by OD 280nm using the extinction coefficient based on the predicted amino acid sequence. 2 μg of each antibody was analyzed by SDS-PAGE, and bands corresponding to a typical antibody profile were observed.

[0796] Single-cycle kinetics analysis of purified lead humanized and affinity matured antibodies (using purified proteins)

[0797] Single-cycle kinetics was 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 individual mutants are provided in Table 22.

[0798] The binding of all 11 lead variants was > four-fold compared to the parental antibody (see Table 21). The data obtained using purified IgG were consistent with the data previously obtained using supernatant.

[0799] Table 21 Single-cycle kinetic constants of purified lead humanized affinity matured antibodies. By comparing the K DDivided by the K of the parent determined in the same experiment D to calculate the relative K compared to the parent D . Mutations in CDR CDR1H, CDR3H B1, CDR3H B2 or CDR1L are indicated by "+" when applicable. All variable light chains contain an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0800]

[0801] 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 an S29A mutation in CDR1L (Kabat numbering scheme, corresponding to an S28A mutation in the primary amino acid sequence of the variable light chain).

[0802]

[0803] Removal of potential CD4+ T cell epitopes

[0804] Using iTope, a computer analysis for peptides binding to human MHC class II alleles TM technique (Perry et al. 2008) and TCED of T cell epitopes related to known antibody sequences TM (Bryson et al. 2010) to analyze 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 no introduction of confounding high epitopes observed in the parental sequence (see Table 21).

[0805] Selectivity analysis of lead antibodies

[0806] Initial selectivity analysis of several lead antibodies showed that antibodies with phenylalanine (F) at CDR3H position 104 on average showed an increased selectivity for CEACAM1 compared to antibodies with aspartic acid (D) at CDR3H position 104 Figure 11 ).

[0807] Multi-cycle kinetic analysis

[0808] Using a Biacore T200 instrument running Biacore T200 evaluation software V3.0.1, the variants CP08H03 / Vк8S29A and CP08H03 / CP08F05 were further analyzed using multi-cycle kinetic analysis. The purified antibody was diluted to a concentration of 1 μg / ml in HBS-P+. At the start of each cycle, each antibody was captured on the Protein A surface to obtain an RL of approximately 100 RU. 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 replicates of the blank (without CEACAM1) and replicates of a single concentration of analyte were programmed into the kinetic run to check the stability of the surface and analyte during 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, the Protein A surface was regenerated using two injections of 10 mM glycine-HCL pH 1.5.

[0809] The signal from the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to the reference surface and the global Rmax parameter was used in the 1:1 binding model. By dividing the K D of the affinity-matured composite human antibody variant by the K D of the parental on the same chip H the relative K D compared to the parental (V H 1 / Vκ8S29A) was calculated. 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. Compared to the V H 1 / Vκ8S29A parental, the two affinity-matured CEACAM1 antibody variants showed > four-fold improvement in affinity.

[0810] Table 23 Multi-cycle kinetic data for the binding of antibody V H 1 / Vκ8S29A (parental), chimeric antibody (VH0 / VK0), and two affinity-matured leads to CEACAM1 measured using Biacore T200. The relative K D compared to the parental was calculated by dividing the K D of the affinity-matured variant by the K H of the parental measured on the same chip D . 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).

[0811]

[0812]

[0813] Example 2: Selectivity of CEACAM1 Antibody

[0814] To further evaluate the binding selectivity of CEACAM1 antibodies V H 0 / Vκ0, CP08H03 / Vк8S29A, CP08H03 / CP08F05 for CEACAM1 relative to other proteins, the binding affinities for CEACAM1, CEACAM3, CEACAM5, and CEACAM6 were compared using the single-cycle kinetic analysis performed as described above. Single-cycle kinetics were performed using CEACAM concentrations from 280 nM to 70 nM. The antibodies were loaded onto the chip at the following concentrations (taking into account the different analyte MWs): 100 RU for CEACAM1, 375 RU for CEACAM3, 71.4 RU for CEACAM5, and 150 RU for CEACAM6. For CEACAM3, CEACAM5, and CEACAM6, no significant binding of the three CEACAM1 antibodies CP08H03 / Vк8S29A, CP08H03 / CP08F05, and V H 0 / Vκ0 was observed (see Figure 12A ).

[0815] These results are consistent with the data obtained by measuring antibody specificity using ELISA. For the ELISA experiment, 96-well plates were coated with 0.5 or 1.0 μg / ml of CEACAM1. Nonspecific binding was blocked with 2% BSA / Dulbecco PBS. 1:3 dilution series (starting concentration 50 μg / mL) of CP08H03 / Vκ8S29A, CP08H03 / CP08F05, or V H 0 / Vк0 were prepared in 2% BSA / PBS. 100 μL of the samples were added to the pre-coated plates and incubated for 1 hour at RT. CEACAM antibodies were detected using an anti-human Igκ chain-peroxidase secondary antibody (AP502P). The plates were developed with TMB and terminated with 3M HCl. The results were analyzed by subtracting the background. Essentially no binding of the three CEACAM1 antibodies CP08H03 / Vκ8S29A, CP08H03 / CP08F05, and V H 0 / Vκ0 to CEACAM3, CEACAM5, or CEACAM6 was observed (see Figure 12B and 12C ).

[0816] Although the N domains of different CEACAMs are highly homologous, this high selectivity can be observed: 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 identity percentage matrix generated using Clustal 2.1 (see Figure 13 ).

[0817] Example 3: Epitope analysis of CEACAM1 antibodies

[0818] 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. Forty-eight hours after transfection, a Western blot of the CEACAM1 protein was performed. The CEACAM1 antibodies VH0 / VK0 (chimeric antibody), VH1 / VK8, VH2 / VK4, VH3 / VK1, and VH4 / VK1 were used as detection antibodies. Mutations in CEACAM1 residues Y34, V39, G41, N42, R43, Q44, G47, and Q89 (which are part of the CEACAM1 GFCC face) resulted in a decrease in the binding of CEACAM1 to the CEACAM1 antibodies, indicating that these CEACAM1 residues may be involved in binding (see Figure 14 ).

[0819] Example 4: Crystal structure of CEACAM antibody and CEACAM1

[0820] To more precisely delineate the binding interface between CEACAM1 and CP08H03 / VK8S29A, the crystal structure of human CEACAM1 complexed with the CP08H03 / VK8S29A Fab fragment was determined.

[0821] CEACAM1 was expressed by Escherichia coli transformed with a pET21D-based plasmid expressing CEACAM1 in unlabeled form. The protein was refolded in arginine-containing buffer and purified. Fab fragments were prepared by antibody digestion, concentrated to approximately 18 mg / ml, and then purified using immobilized papain resin, followed by protein A affinity and gel filtration chromatography. Purified CEACAM1 and Fab were mixed in a 1:1 molar ratio prior to crystallization screening. Initial crystallization hits of the CEACAM1:Fab complex were identified and subsequently optimized. Diffraction-quality crystals were grown at room temperature in conditions containing 18 - 20% PEG 6000, 50 mM potassium dihydrogen phosphate, 20 mM Tris pH 7.0, and 1% β-octylglucoside. SDS-PAGE analysis and silver staining of the washed crystals were used to confirm crystallization of the complex. X-ray data of 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 were merged to generate a highly redundant data set for structure determination and refinement. The structure of the complex was solved by molecular replacement and refined to final R and R 自由 values of 24.9% and 32.8%, respectively.

[0822] The structure of the CEACAM1:CP08H03 / Vκ8S29A Fab complex was determined to a resolution. CP08H03 / Vκ8S29A Fab binds to CEACAM1 in a 1:1 stoichiometry (see Figure 15 ). Figure 16 The molecular surface representation of CEACAM1 in

[0823] shows a portion of the epitope of the Fab fragment on CEACAM1. The major and minor interactions between the Fab molecule and CEACAM1 are listed in Tables 24 and 25. Table 24. Major interactions between CEACAM1 and CP08H03 / Vκ8S29A Fab (interaction distance <

[0824]

[0825]

[0826] ). #Residue numbering is based on the Kabat numbering scheme. *Residue numbering is based on the primary amino acid sequence of the heavy variable chain. #Residue numbering is based on the Kabat numbering scheme. *Residue numbering is based on the primary amino acid sequence of the heavy variable chain.

[0827]

[0828] Referring to the existing structure of the CEACAM1 dimer ( Figure 17 ), it is clear that the Fab binds to the interface of CEACAM1 involved in self-association. It is speculated that this competitive interaction leads to the dissociation of the dimer in solution. The residues targeting CEACAM1 include four residues (Y34, Q44, Q89, N97) that form the YQQN pocket at the CEACAM1:CEACAM1 dimer interface. Notably, several residues on CEACAM1 that bind the antibody are also predicted to be involved in binding to TIM-3, including CEACAM1 residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and / or N97 (Huang et al., Nature. 2015 Jan 15; 517(7534):386-90).

[0829] In the Fab light chain, the residues in CDR1, CDR2, and CDR3 ( Figure 18 ) mainly interact with the residues in two loops between the β-strands of the major β-sheet in CEACAM1, and also interact with the residues of the β-strands in said sheet. In the Fab heavy chain, the residues of CDR2 and CDR3 mainly interact with the residues distributed on four different β-strands of the central β-sheet.

[0830] The interacting surface has a shape complementarity of 0.5, and complex formation buries the total solvent-accessible surface of 1607A1. No interaction was observed between the antigen and CDR1 of the Fab heavy chain.

[0831] Alignment of human CEACAM family members indicates 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. The polymorphisms of these residues in hCEACAM-1 include Ala49Val (rs8110904) and Gln89His (rs8111468). To further examine the selectivity properties of the CEACAM1 antibody CP08H03 / Vκ8S29A, the human CEACAM1 A49V / Q89H mutant was expressed and purified as described above. Note that there are natural human allelic variants of human CEACAM1 that convert 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 The resolution was compared with the CEACAM1 wild-type CP08H03 / Vκ8S29A Fab complex. As described above, the CDR3H residue F104 of CP08H03 / Vκ8S29A contacts the residue F29 in wild-type CEACAM1 (see Figure 19 , left panel). It was noted that F29 of one CEACAM1 monomer binds F29 of the second monomer at the CEACAM1:CEACAM1 homodimer interface. Binding of the CDR3H residue F104 of CP08H03 / Vκ8S29A blocks the F29-F29 interaction. The CEACAM1 residue A49 is located near the F104 / F29 interaction site. Since the hydrophobicity of valine is increased in non-CEACAM1 family members compared to alanine in human CEACAM1, mutation of the human CEACAM1 residue A49 to valine causes the hydrophobic CEACAM1 residue F29 to move closer to the CEACAM1 V49 residue. This rotamer shift of F29 was also observed in the crystal structures of human CEACAM5 (PDB accession 2QSQ) and human CEACAM3 (PDB accession 6AW1) and is predicted to clash with the CDR3H residue F104 (see Figure 19 , right panel). This is illustrated by the change in orientation shown by the CEACAM1 F29 loop, which moves closer to the space previously occupied by the CDR3H residue F104 (see Figure 19 , right panel). 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 thus has a major effect on antibody selectivity. This rotamer shift of the F29 loop is also predicted to affect the interaction between the 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. 2017 Nov 9;11(1):26). The development of the disease is associated with the Ala49Val polymorphism, and the 49 residue alanine was found to be involved in binding to the CP08H03 / Vκ8S29A antibody. Thus, CP08H03 / Vκ8S29A is expected to also interfere with Wuchereria bancrofti and other related pathogens or cancer processes such as tumor invasion that interact with lymphatic vessels in phenotypic worms.

[0832] Example 5: CEACAM1 antibody blocks CEACAM1:CEACAM1 interaction

[0833] The ability of CEACAM1 antibodies to block CEACAM1 homodimerization was tested. CEACAM1-CEACAM1 competitive ELISA studies were performed in triplicate to determine the ability of the CP08H03 / Vκ8S29A antibody (concentration range 0 - 1000 nM) to inhibit the binding of human CEACAM1 IgV domain unlabeled protein (1 pg / ml) and human CEACAM1-GST protein (37.5 pg / ml). In addition, IgG4 antibody was used as a control (0 - 1000 nM). Goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) was used, and the assay was developed by adding TMB solution (Life technologies). OD values were read at 450 nm on a microplate reader. Data were plotted in Graphpad and the best-fit IC-50 value was determined.

[0834] The CEACAM1 antibody CP08H03 / Vκ8S29A was shown to block CEACAM1:CEACAM1 homophilic interaction (see Figure 20A ).

[0835] Example 6: CEACAM1 antibody blocks CEACAM1:TIM-3 interaction

[0836] The ability of CEACAM1 antibodies to reduce the binding of CEACAM1 to TIM-3 was examined. CEACAM1 / TIM-3 competitive ELISA studies were performed in triplicate to determine the ability of the CP08H03 / Vκ8S29A antibody (concentration range 0 - 300 nM) to inhibit the binding of human TIM-3 IgV domain unlabeled protein (3 pg / ml) and human CEACAM1-GST protein (37.5 pg / ml). In addition, human IgG4 antibody was used as a control (0 - 1000 nM). Goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) was used, and the assay was developed by adding TMB solution (Life technologies). OD values were read at 450 nm on a microplate reader. Data were plotted in Graphpad and the best-fit IC-50 value was determined. As Figure 20B shown, the CEACAM antibody CP08H03 / Vκ8S29A blocks the CEACAM1:TIM-3 heterophilic interaction.

[0837] Example 7: CEACAM1 antibody induces T cell proliferation

[0838] The ability of the CEACAM antibodies CP08H03 / Vκ8S29A and CP08H03 / CP08F05 to induce T cell proliferation was investigated in humanized NOD scidγ mice (NSG mice). See Figure 21. Freshly isolated human PBMC (5x10^6) were transferred into NOD.Cg-PrMc scld I / 2rg tm1Wjl / SzJ (NSG) mice by intraperitoneal (i.p.) injection. Twenty-one days after PBMC injection, human immune cell engraftment in NSG animals was examined by tail bleeding. Twenty-four and 31 days after PBMC injection, the first and second doses of the designated concentration of CEACAM1 antibody or isotype control antibody were administered to humanized NSG mice by intraperitoneal injection. After study termination (34 days after PBMC injection), the mice were sacrificed and the spleens were surgically dissected for further analysis. Single cell suspensions from transplanted mice were stained with a cell proliferation dye and cultured in vitro for 2 days in complete RPMI medium in the presence of soluble anti-human CD3 stimulation (2 μg / ml, OKT3 clone) and rIL-2 (40 U / ml). The cells were maintained at a concentration of 10^7 cells / ml. After in vitro stimulation, the cells were stained with an antibody against the human CD45 pan-leukocyte marker and evaluated by flow cytometry.

[0839] Antibody-dependent cell-mediated cytotoxicity (ADCC) was not observed in any of the test groups (see Figure 22 ). Administration of the CEACAM antibodies CP08H03 / VK8S29A or CP08H03 / CP08F05 separately resulted in increased antibody-induced T cell expansion in vivo (see Figure 23 ).

[0840] Example 8: CEACAM1 Antibodies Reduce Tumor Growth in a Melanoma Model

[0841] To evaluate the ability of CEACAM1 antibodies to reduce tumor growth, 1 x 10 6 MALME-3M (human melanoma) cells were subcutaneously injected into 7-8 week old male NSG (NOD.Cg-PrMc 6 I / 2rg scld / SzJ) mice together with 5 x 10 tm1Wjl human PBMC. The MALME-3M (BRAFV 600E ) cell line was established in 1975 from a metastatic site (lung) of a 43-year-old white male with metastatic melanoma. On days 7 - 9, all mice were confirmed to exhibit a reconstituted T cell population (experimental setup see Figure 24A ). The animals were treated intraperitoneally with the CEACAM antibody CP08H03 / VK8S29A or hIgG4 control antibody on days 10, 13, 17, 20, and 24.

[0842] The human melanoma cell line MALME-3M was a gift from Dr. Nicole Beauchemin (McGill University, Montreal, Canada). MALME-3M was established in 1975 from a metastatic site (lung) of a 43-year-old white male with metastatic melanoma harboring BRAFV600E. 2 x 10^7 MALME-3M cells were injected subcutaneously (s.c.) into NOD.Cg-PrMc scld I / 2rg tm1Wjl / SzJ (NSG) mice. After a 30-minute acclimation period, freshly isolated human PBMCs (1 x 10^8) were then transferred into tumor-bearing NSG mice by intraperitoneal (i.p.) injection. Seven to nine days after PBMC injection, human immune cell engraftment in NSG animals was examined 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 antibody or isotype control antibody at the designated concentration by intraperitoneal injection. After the study was terminated (34 days after human cell injection), the mice were sacrificed and subjected to surgical dissection.

[0843] The CEACAM1 antibody CP08H03 / VK8S29A effectively reduced tumor growth and proliferation at various concentrations (see Figure 24B , 24C and 25), while not depleting the T cell population (see Figure 22 ). In addition, the proliferative capacity of human CD4 + and CD8 + tumor-infiltrating lymphocytes was restored by administration of the CEACAM1 antibody, see Figure 25 . A major bias of CD8 T memory cells towards central memory T cells was also observed upon in vivo treatment with CP08H03 / Vκ8S29A ( Figure 26 ). It was noted that the CEACAM1 antibody CP08H03 / VK8S29A increased the relative proportion of T cm to T em relative to that observed in control-treated animals, consistent with enhanced anti-cancer responses.

[0844] Example 9: CEACAM1 antibodies can be used to treat cancers resistant to checkpoint inhibitors

[0845] CEACAM1 is expressed on the majority of TILs derived from primary melanoma patients or melanoma patients resistant to anti-PD-1 and / or anti-CTLA-4 therapies; and the CEACAM1 expression level is greater than the expression levels of PD-1 or TIM-3 (see Figure 27 ). Approximately 80% of the samples had greater than 20% of CD4 +CEACAM1 expression is exhibited on T cell populations. To compare CEACAM1 expression in patients with acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy with patients who have not been previously exposed to anti-PD-1 and / or anti-CTLA-4 therapy, tumor-associated cells (TAC) were obtained from naïve melanoma patients (not previously exposed to anti-PD-1 and / or anti-CTLA-4 therapy) or those patients with acquired resistance to anti-PD-1 and / or anti-CTLA-4 therapy (acquired resistance). TAC were obtained by culturing tumor tissue in DMEM medium and removing floating cells from the supernatant. Cells were stained for CD3, CD4, and CD8, and CEACAM1 expression on CD3 + CD4 + and CD3 + CD8 + was evaluated. These studies showed that tumor-associated cells deprived of the tumor microenvironment in acquired resistance upregulated CEACAM1 expression relative to that observed in naïve patients (see Figure 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.

[0846] As expected, compared to patients not previously exposed to anti-PD-1 and / or anti-CTLA-4 therapy, central memory T + cells in CD8 cm T cells were relatively decreased in patients resistant to anti-PD-1 and / or anti-CTLA-4 therapy relative to effector memory T em cells, which was consistent with the decreased anti-cancer response in resistant patients (see Figure 29 ). Tumor-associated cells from naïve melanoma patients (untreated patients) or melanoma patients resistant to immune checkpoint inhibitors (treatment failure patients) were stained for CD44, CCR7, and CD62L, and the relative amounts of central memory T cm (CD44 高 , CD62L 高 , CCR7 高 ) and effector memory T em (CD44 高 , CD62L 低 , CCR7 低 ) were expressed as a percentage of the total CD8 T cells present in the bulk tumor.

[0847] To evaluate the ability of CEACAM1 antibodies to reverse T cell exhaustion in patients resistant to treatment with checkpoint inhibitors such as PD-1 / PD-L1 and CTLA-4 inhibitors, PBMCs and tumor-associated cells were isolated from melanoma patients with secondary resistance to pembrolizumab (PD-1 inhibitor), ipilimumab (CTLA-4 inhibitor) + nivolumab (PD-1 inhibitor), and dabrafenib (B-Raf inhibitor) + trametinib (MEK inhibitor) and stage IV disease. Tumor-associated cells and PBMCs were stained for CEACAM1, PD1, or TIM-3, and the percentages of CD8 + and CD4 + T cells were shown (see Figure 30 , left panel). Tumor biopsies were subjected to enzymatic digestion or a commercial mechanical / enzymatic dissociation system (GentleMACS dissociator, Miltenyi Biotec). Enzymatic digestion was based on a previously established method for generating melanoma TILs (Dudley et al., 2003, 2008). Briefly, tumor biopsies were cut into small fragments approximately 2-3 mm in length and placed in a solution containing 100 U ml -1 DNase, 10 mg ml -1In an enzyme digestion mixture consisting of collagenase VIII (Sigma-Aldrich), and incubated with continuous rotation at 37 °C for 45 minutes. GentleMACS dissociation was performed according to the manufacturer's protocol. Briefly, the tumor was cut into small fragments approximately 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; then the digestion mixture containing the tumor was subjected to three 36-second mechanical depolymerization steps (programs h_tumor_01.01, 02.01, and 03.01) in a GentleMACS dissociator, with two 30-minute incubations at 37 °C interspersed after the first and second depolymerization steps. After depolymerization, the TILs from enzyme digestion and GentleMACS dissociation 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 a viability dye. Data were acquired using a Cytoflex flow cytometer (Invitrogen) and analyzed using FlowJo software (TreeStar, V7.6.5 for Windows). In 96-well plates, PBMCs or tumor-associated cells were cultured with soluble anti-CD3 (2 pg / ml) and rIL-2 (40 units / ml) in complete medium (RPMI 1640 (Lonza)) 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). After 96 hours, cell culture supernatants were collected for further TNF-α and IFN-γ ELISA (BD) analysis according to the manufacturer's method. CP08H03 / VK8S29A 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 Figure 30 , right panel).

[0848] Example 10: Compared with previously known CEACAM1 antibodies, the CEACAM1 antibodies contemplated by the present invention exhibit improved efficacy

[0849] The properties of the antibody CP08H03 / VK8S29A were compared to the anti-CEACAM1 antibody CM-24 (WO2015 / 166484). Different from the CEACAM1 antibody CP08H03 / VK8S29A disclosed herein, CM-24 (i) binds CEACAM1 based on modeling away from the dimer interface, (ii) exhibits cross-reactivity with CEACAM3 and CEACAM5, (iii) shows limited ability to reverse T cell tolerance in TILs, and (iv) functions as an agonistic antibody rather than an antagonistic antibody in a mouse model of metastatic melanoma.

[0850] CP08H03 / Vκ8S29A is selective for CEACAM1 and does not show significant binding to CEACAM3, CEACAM5, CEACAM6 or CEACAM8. On the other hand, CM-24 shows significant cross-reactivity with CEACAM3 and CEACAM5 at higher antibody concentrations ( Figure 31A and 31B ). The cervical adenocarcinoma cell line HeLa (ATCC No CCL-2) and transfected cell lines HeLaCEACAM1, HeLaCEACAM3, HeLaCEACAM5, HeLaCEACAM6 and HeLaCEACAM8 used for flow cytometry experiments were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U / ml) and dihydrostreptomycin (100 μg / ml) at 37 °C, 5.0% CO2. The cell lines were stained with the indicated antibodies and then with monoclonal antibodies conjugated with fluorescent dyes specific for the indicated antibody isotypes (such as human IgG4 or mouse IgG1) and a live dye (DAPI). Data were obtained using a Cytoflex flow cytometer (Invitrogen) and analyzed using FlowJo software (TreeStar, V7.6.5 for Windows).

[0851] In addition, CM-24 shows limited ability to reverse T cell tolerance in tumor-associated cells. Incubation of tumor-associated cells with the antibody CP08H03 / Vκ8S29A results in a more extensive reversal of T cell tolerance within a certain antibody concentration range compared to CM-24 in primary Merkel cell carcinoma tumor cells ( Figure 32A 、 32Band 32C). According to the manufacturer's protocol, Merkel cell carcinoma biopsies were subjected to a commercial mechanical / enzyme dissociation system (GentleMACS dissociator, Miltenyi Biotec). Briefly, tumors were cut into small fragments approximately 2 - 3 mm in length and placed into C tubes (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 tumors was then subjected to three 36 - second mechanical depolymerization steps (programs h_tumor_01.01, 02.01, and 03.01) in the GentleMACS dissociator, with two 30 - minute incubations at 37°C interspersed after the first and second depolymerization steps. After depolymerization, TILs from enzymatic digestion and GentleMACS dissociation were passed through a 100 - pm filter for further analysis. In vitro determination of T - cell function in the tumor microenvironment: Dissociated tumor cells and autologous PBMCs were cultured in 96 - well plates in complete medium (RPMI 1640 (Lonza)) 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) together with 40 IU / ml -1 recombinant IL - 2 (NIH) and soluble CD3 (2 μg / ml). After 96 hours, cell culture supernatants were collected and used for further TNF - and IFN - ELISA (BD) analysis according to the manufacturing procedures.

[0852] In a metastatic melanoma model (experimental setup see Figure 33A ), mice treated in vivo with CP08H03 / Vκ8S29A showed a significant reduction in tumor cells and a significant increase in TIL CD4 + and CD8 + lymphocytes compared to control mice treated in vivo with human IgG4 (hIgG4) ( Figure 33B , 34A , 34B and 34C). On the other hand, mice treated in vivo with CM - 24 showed a substantial increase in tumor cells and an almost complete absence of TIL CD4 + and CD8 + lymphocytes compared to control mice treated with hIgG4 control ( Figure 33B , 34A, 34B, and 34C). Additionally, tumor cells showed decreased proliferation in animals treated with CP08H03 / Vκ8S29A relative to animals treated with hIgG4 control or CM24 ( Figure 33C ). Additionally, CD4 + T cells in the spleens of animals treated with CP08H03 / Vκ8S29A showed increased proliferation relative to animals treated with hIgG4 or mice treated with CM24 ( Figure 33D ). In contrast to animals treated with CP08H03 / Vκ8S29A, animals treated with CM24 showed decreased proliferation of splenic CD4 + T cells ( Figure 33D ). The human melanoma cell line MALME-3M was provided by Dr. Nicole Beauchemin (McGill University). MALME-3M was established in 1975 from a metastatic site (lung) of a 43-year-old white male with metastatic melanoma bearing BRAF V600E . 2 x 10^7 MALME-3M cells were injected subcutaneously (s.c.) into NSG mice. The mice were allowed to uptake the tumor cells for 30 minutes, and then freshly isolated human PBMCs (1 x 10^8) were transferred into the tumor-bearing NSG mice by intraperitoneal (i.p.) injection. Seven to nine days after PBMC injection, human immune cell engraftment in NSG animals was examined by tail bleeding. At day 14, palpable tumor nodules were detected. Starting on day 17 after human cell injection, tumor-bearing humanized NSG mice received a total of four doses of CP08H03 / Vκ8S29A antibody (2 mg / kg), CM-24 (2 mg / kg), or isotype control antibody (2 mg / kg) twice a week by intraperitoneal injection. After the study was terminated (30 days after human cell injection), the mice were sacrificed and surgically dissected. Metastatic tumors were preserved along with the spleen, lung, and liver for further analysis. Total cell counts, 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.

[0853] Figure 34A , 34B and 34C provided a statistical comparison of the results shown in Figure 33A by the protocol shown. Animals treated with CM-24 showed larger tumors and no evidence of tumor-associated T cells. On the other hand, an increased amount of infiltrating T cells and decreased tumor cells were observed in mice treated with CP08H03 / Vκ8S29A ( Figure 33B ). As shown, assessment of tumor cell proliferation showed that CP08H03 / Vκ8S29A inhibited tumor proliferation, but CM-24 did not inhibit tumor proliferation ( Figure 33B ). As shown, assessment of tumor cell proliferation showed that CP08H03 / Vκ8S29A inhibited tumor proliferation, but CM-24 did not inhibit tumor proliferation ( Figure 33C)。In addition, increased proliferation of splenic CD4 + T cells was observed in mice treated with CP08H03 / Vκ8S29A, while decreased proliferation of splenic CD4 + T cells was observed in mice treated with CM-24( Figure 33D ).

[0854] Example 11: The CEACAM1 antibody blocks the interaction between CEACAM1 and HopQ.

[0855] HopQ is expressed on the surface of Helicobacter pylori, a bacterium that specifically colonizes human gastric epithelium and is a major pathogen in the development of ulcer disease and gastric cancer. The HopQ-CEACAM1 interaction has been shown to promote gastric colonization and Hp-induced pathology, for example, by enabling the translocation of bacterial virulence factors into host cells and enhancing the release of pro-inflammatory mediators.

[0856] Published crystal structure data (PDB IDs 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)) show that the GFCC loop of CEACAM1 is involved in binding to HopQ, and CEACAM1 residues F29, Y34, N42, Q89, and N97 make various hydrogen bond and hydrophobic interactions with HopQ residues (see Figure 35A ). Modeling based on the CEAM1:HopQ co-crystal and the CEACAM1:CP08H03 / Vκ8S29A Fab co-crystal shows that the CEACAM1 antibody CP08H03 / Vκ8S29A covers the CEACAM1 binding site of HopQ (see Figure 35B ) and can thus disrupt the CEACAM1:HopQ interaction.

[0857] Example 12: CEACAM1 antibody promotes long-term survival

[0858] The ability of the CEACAM1 antibody CP08H03 / Vκ8S29A to promote long-term survival of tumor-bearing mammals was investigated using a mouse melanoma model.

[0859] Ten 6 MALME-3M (human melanoma) cells and 5x10 6 human PBMCs (from an HLA-A2 matched donor) were subcutaneously injected into NSG mice. On day 10, the tumors reached 2 - 2.5 mm 3, and the mice were randomly grouped (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 the surviving animals showing strong clinical activity (arrows) were sacrificed.

[0860] As Figure 36 shown, treatment with anti-CEACAM1 antibody significantly increased the survival rate of tumor-bearing mice. In addition, at autopsy, the antibody-treated animals showed local tumors without visible metastases, which was consistent with the control of the disease. This data indicates that the anti-CEACAM1 antibodies and fragments thereof disclosed herein can be used to treat cancer and increase survival.

[0861] Example 13: CP08H03 / Vκ8S29A increases the immune response in tumor cells of primary patients or patients with secondary resistance to immunotherapy

[0862] The ability of the CEACAM1 antibody CP08H03 / Vκ8S29A to increase the immune response in tumors from primary melanoma patients or melanoma patients showing secondary resistance to immunotherapy was examined using isolated tumor samples.

[0863] In one instance, isolated tumor samples 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 medium for 4 days in the presence of 2 μg / ml anti-CD3 and 40 units / ml recombinant IL-2. The cells were then examined by mass cytometry using the following antibodies to detect multiple intracellular cytokines associated with the immune response to tumors in CD8 + T cells: 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).

[0864] As Figure 37A and 37B shown, treatment with the CP08H03 / Vκ8S29A antibody led to a significant induction of CD8 +Factors for intracellular indication in T cells. These results directly indicate that the CP08H03 / Vκ8S29A antibody induces the production of multiple factors in CD8 + T cells, and these factors are potentially associated with productive anti-tumor immune responses.

[0865] In another example, tumor samples associated with two melanoma patients who had not been previously treated (subject 189) or had secondary resistance to immunotherapy (subject 185) were disrupted by mechanical dissociation (Miltenyi). 8 x 10 5 dissociated tumor cells per ml were placed in a culture dish. The freshly isolated tumor-dissociated cells were exposed only to 2 μg / ml of CP08H03 / Vκ8S29A or a human IgG4 isotype control antibody. After 96 hours, the supernatant was removed, and ELISA analysis was performed in triplicate to detect the presence of interferon-γ.

[0866] As Figure 38A and 38B shown, treatment with the CP08H03 / Vκ8S29A antibody induced a significant level of cytokine interferon-γ secretion into the supernatant of tumor-dissociated cells isolated from patients with secondary resistance to immunotherapy treatment ( Figure 38A , subject 185) or patients who had not been treated with immunotherapy ( Figure 38B , subject 189), as compared to that observed with the control human IgG4 antibody.

[0867] In summary, these data indicate that the anti-CEACAM1 antibodies and fragments thereof disclosed herein can be used to treat patients with primary cancer and those with secondary resistance to immunotherapy.

[0868] Sequence summary

[0869]

[0870]

[0871]

[0872]

[0873]

[0874] Although the above written description of the present invention enables one of ordinary skill in the art to make and use those currently considered to be the best mode thereof, one 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.

[0875] 1. An antibody or antigen-binding fragment thereof that binds to CEACAM1, said antibody or antigen-binding fragment comprising a heavy-chain variable region and a light-chain variable region;

[0876] wherein each of said heavy-chain variable region and said light-chain variable region comprises CDR1, CDR2, and CDR3; and

[0877] wherein:

[0878] the sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO:1);

[0879] wherein X1 is A, D, N, or S;

[0880] wherein X2 is A or G; and

[0881] wherein X3 is an amino acid with a hydrophobic side chain including I or M;

[0882] the sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2);

[0883] the sequence of CDR3H comprises the sequence HX4X5DYX6PX7WFAX8 (SEQ ID NO:3);

[0884] wherein X4 is D, G, or P;

[0885] wherein X5 is F or P;

[0886] wherein X6 is D or F;

[0887] wherein X7 is A or Y; and

[0888] wherein X8 is L, H, or F;

[0889] the sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4);

[0890] the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and

[0891] the sequence of CDR3L comprises the sequence QQX9X 10 X 11 X 12 PX 13 T (SEQ ID NO:6); wherein X9 is W or N;

[0892] wherein X 10 is S or T;

[0893] wherein X 11is A or an amino acid having a neutral hydrophilic side chain including S, N, and T;

[0894] wherein X 12 is L, F, or N; and

[0895] wherein X 13 is P or F.

[0896] 2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein

[0897] the sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO: 43);

[0898] wherein X is any amino acid;

[0899] wherein X1 is A, D, N, or S;

[0900] wherein X2 is A or G; and

[0901] wherein X3 is an amino acid having a hydrophobic side chain including I or M; and

[0902] the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO: 44);

[0903] wherein X4 is D, G, or P;

[0904] wherein X5 is F or P;

[0905] wherein X7 is A or Y; and

[0906] wherein X8 is L, H, or F.

[0907] 3. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein

[0908] the sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO: 1);

[0909] wherein X1 is A, D, N, or S;

[0910] wherein X2 is A or G; and

[0911] wherein X3 is an amino acid having a hydrophobic side chain including I or M;

[0912] the sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO: 2);

[0913] the sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO: 7);

[0914] where X4 of CDR3H is D, G or P;

[0915] where X5 of CDR3H is F or P; and

[0916] where X8 of CDR3H is L, H or F;

[0917] the sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4);

[0918] the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and

[0919] the sequence of CDR3L comprises the sequence QQX9SSX 12 PX 13 T (SEQ ID NO:8);

[0920] where X9 is W or N;

[0921] where X 12 is L, F or N; and

[0922] where X 13 is P or F.

[0923] 4. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein

[0924] the sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO:9);

[0925] the sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2);

[0926] the sequence of CDR3H comprises the sequence HDFDYFPYWFAH (SEQ ID NO:10);

[0927] the sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4);

[0928] the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and

[0929] the sequence of CDR3L comprises the sequence QQWSSNPPT (SEQ ID NO:11).

[0930] 5. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein

[0931] the sequence of CDR1H comprises the sequence SHGMS (SEQ ID NO:9);

[0932] The sequence of CDR2H contains the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2);

[0933] The sequence of CDR3H contains the sequence HDFDYFPYWFAH (SEQ ID NO:10);

[0934] The sequence of CDR1L contains the sequence RANSAVSYMY (SEQ ID NO:4);

[0935] The sequence of CDR2L contains the sequence LTSNRAT (SEQ ID NO:5); and

[0936] The sequence of CDR3L contains the sequence QQWTSNPPT (SEQ ID NO:12).

[0937] 6. An antibody or antigen-binding fragment thereof that binds to CEACAM1, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region;

[0938] wherein the sequence of the heavy chain variable region comprises a sequence that is at least 90% identical to the amino acid sequence of the heavy chain variable region of SEQ ID NO:13; and

[0939] wherein the sequence of the light chain variable region comprises a sequence that is at least 90% identical to the amino acid sequence of a light chain variable region selected from the group consisting of:

[0940] SEQ ID NO:14,

[0941] SEQ ID NO:15 and

[0942] SEQ ID NO:16.

[0943] 7. The antibody or antigen-binding fragment thereof according to embodiment 6,

[0944] wherein the sequence of the heavy chain variable region comprises a sequence that is at least 95% identical to the amino acid sequence of the heavy chain variable region of SEQ ID NO:13; and

[0945] wherein the sequence of the light chain variable region comprises a sequence that is at least 95% identical to the amino acid sequence of a light chain variable region selected from the group consisting of:

[0946] SEQ ID NO:14,

[0947] SEQ ID NO:15 and

[0948] SEQ ID NO:16.

[0949] 8. The antibody or antigen-binding fragment thereof according to embodiment 7,

[0950] wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 13; and

[0951] wherein the sequence of the light chain variable region comprises a sequence selected from the group consisting of:

[0952] SEQ ID NO: 14,

[0953] SEQ ID NO: 15, and

[0954] SEQ ID NO: 16.

[0955] 9. The antibody or antigen-binding fragment thereof according to embodiment 8,

[0956] wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 13; and

[0957] wherein the sequence of the light chain variable region comprises SEQ ID NO: 14.

[0958] 10. The antibody or antigen-binding fragment thereof according to embodiment 8,

[0959] wherein the sequence of the heavy chain variable region comprises SEQ ID NO: 13; and

[0960] wherein the sequence of the light chain variable region comprises SEQ ID NO: 15.

[0961] 11. An antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region;

[0962] wherein the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the amino acid sequence of the heavy chain variable region of SEQ ID NO: 13;

[0963] wherein the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the amino acid sequence of the light chain variable region of SEQ ID NO: 14;

[0964] wherein the sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO: 43);

[0965] wherein X is any amino acid;

[0966] wherein X1 is A, D, N, or S;

[0967] wherein X2 is A or G; and

[0968] wherein X3 is an amino acid having a hydrophobic side chain including I or M; and

[0969] wherein the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO:44);

[0970] wherein X4 is D, G or P;

[0971] wherein X5 is F or P;

[0972] wherein X7 is A or Y; and

[0973] wherein X8 is L, H or F.

[0974] 12. An antibody or antigen-binding fragment thereof that binds to CEACAM1, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region;

[0975] wherein the sequence of the heavy chain variable region comprises a sequence that is at least 85% identical to the amino acid sequence of the heavy chain variable region of SEQ ID NO:13;

[0976] wherein the sequence of the light chain variable region comprises a sequence that is at least 85% identical to the amino acid sequence of the light chain variable region of SEQ ID NO:14;

[0977] wherein each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2 and CDR3; and

[0978] wherein:

[0979] the sequence of CDR2H comprises residues Y57 and Y59 of SEQ ID NO:13,

[0980] the sequence of CDR3H comprises residues D102, Y103, F104, P105 and Y106 of SEQ ID NO:13,

[0981] the sequen...

Claims

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

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the sequence of the heavy variable chain comprises the sequence GXXXXX1HX2X3S (SEQ ID NO:43); wherein X is any amino acid; wherein X1 is A, D, N, or S; wherein X2 is A or G; and wherein X3 is an amino acid with a hydrophobic side chain including I or M; and the sequence of CDR3H comprises the sequence HX4X5DYFPX7WFAX8 (SEQ ID NO:44); wherein X4 is D, G, or P; wherein X5 is F or P; wherein X7 is A or Y; and wherein X8 is L, H, or F.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the sequence of CDR1H comprises the sequence X1HX2X3S (SEQ ID NO:1); wherein X1 is A, D, N, or S; wherein X2 is A or G; and wherein X3 is an amino acid with a hydrophobic side chain including I or M; the sequence of CDR2H comprises the sequence TISSGGTYTYYPDSVKG (SEQ ID NO:2); the sequence of CDR3H comprises the sequence HX4X5DYFPYWFAX8 (SEQ ID NO:7); wherein X4 of CDR3H is D, G, or P; wherein X5 of CDR3H is F or P; and wherein X8 of CDR3H is L, H, or F; the sequence of CDR1L comprises the sequence RANSAVSYMY (SEQ ID NO:4); the sequence of CDR2L comprises the sequence LTSNRAT (SEQ ID NO:5); and The sequence of CDR3L contains the sequence QQX9SSX 12 PX 13 T (SEQ ID NO:8); wherein X9 is W or N; where X 12 is L, F or N; and where X 13 is P or F.

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

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

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

16.

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

16.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the sequence of the heavy chain variable region contains SEQ ID NO:13; and wherein the sequence of the light chain variable region contains a sequence selected from the group consisting of: SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:

16.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the sequence of the heavy chain variable region contains SEQ ID NO:13; and wherein the sequence of the light chain variable region contains SEQ ID NO:

14.

10. The antibody or antigen-binding fragment thereof according to claim 8, 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.

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