Humanized and affinity matured anti-ceacam1 antibodies and methods of use thereof

Abstract

Provided herein are recombinant antibodies and antigen-binding fragments thereof useful for binding to and inhibiting carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Also provided are methods of using the disclosed CEACAM1 antibodies and antigen-binding fragments thereof to reduce T cell resistance and to treat cancer and infectious diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 316,140, ​​filed March 03, 2022, which is incorporated by reference in its entirety.

[0002] The present invention relates generally to the fields of molecular biology and medicine. More specifically, the present invention provides monoclonal antibodies and antigen-binding fragments that bind to CEACAM1, as well as therapeutic compositions thereof, and methods of using such antibodies, including inhibiting homophilic and heterophilic interactions with CEACAM1, and methods of treating cancer and infectious diseases.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under DK51362 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

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

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

[0006] High expression of CEACAM1 occurs in various cancers, such as melanoma, colorectal cancer, gastric cancer, pancreatic cancer, bladder cancer, and thyroid cancer, and is associated with worse tumor progression, metastasis, and poor clinical prognosis. For example, non-small cell lung cancer (NSCLC) with high expression of CEACAM1 shows high microvascular density, distant metastasis, and reduced median overall survival and median progression-free survival. CEACAM1 expression was also strongly correlated with distant metastasis of pancreatic adenocarcinoma. CEACAM1 expression in tumors promotes CEACAM1-mediated inhibition of T cells and NK cells. As a result, tumor cell metastasis and cancer stem cell niche formation can be inhibited by inhibiting CEACAM1 activity.

[0007] CEACAM1 is also expressed in certain immune system cells and is involved in immune suppression and immune cell exhaustion. For example, high expression of CEACAM1 in tumor-infiltrating lymphocytes (TILs) and other tumor-infiltrating immune cells in gastric, lung, melanoma, colorectal cancer, and glioma is associated with poor prognosis. In T cells, CEACAM1 expression is largely excluded from resting (naive) T cells, but the protein is expressed at high levels in activated T cells. CEACAM1-L is the major isoform expressed in most T cells and functions as an inhibitory receptor that downregulates T cell activation and suppresses T cell function. Therefore, inhibiting CEACAM1 on T cells can restore T cell activity and increase antitumor responses.

[0008] CEACAM1 is further expressed on NK cells, lymphocytes involved in innate immunity, where they are involved in the early control of viral infections and in tumor immune surveillance. When NK cells encounter cells expressing major histocompatibility complex (MHC) class I, the immune response against these cells is prevented by inhibitory signals via receptor-ligand interactions. However, when they encounter cells in which MHC class I is downregulated, such as virus-infected or cancer cells, NK cells are activated by the lack of inhibitory signals, making the "disease" cells more susceptible to NK cell-mediated killing. When CEACAM1 is present on the surface of both NK cells and melanoma cells, the CEACAM1:CEACAM1 interaction inhibits NK-mediated killing, independent of MHC class I expression. Thus, disruption of this homophilic CEACAM1 interaction may be effective in restoring NK-mediated immune responses.

[0009] CEACAM1 expression on a subset of macrophages is further associated with fibrosis in the tumor microenvironment in mouse models. CEACAM1 also regulates other stromal cells in the tumor microenvironment, such as vascular endothelium. Thus, inhibiting the interaction of CEACAM1 with its binding partners can further inhibit fibrosis and angiogenesis.

[0010] CEACAM1 also mediates homophilic (CEACAM1:CEACAM1) and heterophilic interactions (e.g., CEA, CEACAM5, CEACAM8, T-cell immunoglobulin and mucin domain-containing 3 (TIM-3) proteins, as well as programmed cell death protein 1 (PD-1), 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, Fusobacterium sp., Salmonella sp., Streptococcus agalactiae, and Candida It also mediates cell-cell adhesion through the IgV-like N-domain-containing CEACAM1 extracellular portion, which is involved in interactions with influenza viruses such as C. albicans. TIM-3 was identified as a Th1-specific cell surface protein expressed on activated T cells, a subset of dendritic cells and macrophages, as well as NK cells. TIM-3 is an activation-induced inhibitory molecule that is involved in tolerance and has been shown to induce T cell exhaustion in chronic viral infections and cancer. CEACAM1, which is also expressed on activated T cells, has been shown to interact with TIM-3, and this interaction is important for TIM-3-mediated T cell inhibition. PD-1 is the major checkpoint inhibitory pathway in humans.

[0011] As shown above, CEACAM1 also functions as a cellular receptor on the apical membrane of mucosal cells for various Gram-negative bacterial pathogens associated with human mucosa, as well as fungal pathogens such as Candida albicans. For example, N. gonorrhoeae, N. meningitidis, Moraxella catarrhalis, H. influenza, H. aegyptius, and pathogenic Escherichia coli strains have well-characterized CEACAM1-binding adhesins. Binding of bacterial adhesins to CEACAM1 triggers endocytosis of bacteria into epithelial cells and transcytosis of microorganisms through intact epithelial layers, allowing microorganisms to utilize CEACAM1 during mucosal colonization. In addition, CEACAM1 is involved in the infection of influenza virus H5N1 and filarial nematodes such as Wucheria bancrofti. Summary of the Invention

[0012] Provided herein are antibodies and antigen-binding fragments thereof that bind to CEACAM1 and block the interaction of CEACAM1 with one or more binding partners. Also provided are therapeutic compositions of such antibodies and antigen-binding fragments thereof, as well as methods of using these antibodies. By blocking the interaction of CEACAM1 with one or more binding partners, the antibodies and antigen-binding fragments thereof are useful for reducing, inhibiting, and / or reversing T cell tolerance and / or enhancing T cell proliferation. CEACAM1 antibodies and antigen-binding fragments thereof are further useful for treating cancer, reducing tumor growth, reducing tumor metastasis, and / or reducing cancer stemness in a subject in need thereof. CEACAM1 antibodies and antigen-binding fragments thereof are also useful for treating patients resistant to checkpoint therapy. Further provided are methods of using CEACAM1 antibodies and antigen-binding fragments thereof to reduce colonization of mammalian epithelia by bacteria expressing bacterial adhesins or Candida albicans.

[0013] In one aspect, an antibody or antigen-binding fragment thereof that binds to CEACAM1 is provided, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, Each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2, and CDR3, a) the sequence of CDR1H comprises the sequence DYYLY (SEQ ID NO: 1), b) the sequence of CDR2H comprises the sequence TISVGGGQTSYADSVKG (SEQ ID NO: 2), c) the sequence of the CDR3H comprises the sequence GLYYGPSWVAY (SEQ ID NO: 3), ARTYGPAWFAY (SEQ ID NO: 4), or ALTYGPAWLAY (SEQ ID NO: 5); d) the sequence of CDR1L comprises the sequence KSSQSLLNSANQKNYLA (SEQ ID NO: 6), e) the sequence of CDR2L comprises the sequence FASTRES (SEQ ID NO: 7), f) The sequence of CDR3L comprises the sequence QSHYPFYYT (SEQ ID NO: 8) or QSHFPYPLT (SEQ ID NO: 9).

[0014] In one embodiment, the sequence of the CDR3H comprises the sequence GLYYGPSWVAY (SEQ ID NO: 3) and the sequence of the CDR3L comprises the sequence QSHYPFYYT (SEQ ID NO: 8).

[0015] In one aspect, an antibody or antigen-binding fragment thereof that binds to CEACAM1 is provided, 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% identical to a sequence selected from the group consisting of SEQ ID NOs: 15 to 17, and the sequence of the light chain variable region comprises a sequence that is at least 90% identical to SEQ ID NO: 21 or SEQ ID NO: 22.

[0016] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a sequence that is at least 90% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 15, and a light chain variable region comprising a sequence that is at least 90% identical to any one of the light chain variable region amino acid sequences of SEQ ID NO:21.

[0017] In one embodiment, the heavy chain variable region comprises a sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-17, and the light chain variable region comprises a sequence at least 95% identical to SEQ ID NO: 21 or SEQ ID NO: 22. In one embodiment, the heavy chain variable region comprises a sequence at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 15, and the light chain variable region comprises a sequence at least 95% identical to any one of the light chain variable region amino acid sequences of SEQ ID NO: 21.

[0018] In an embodiment, the heavy chain variable region sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 15-17, and the light chain variable region sequence comprises SEQ ID NO: 21 or SEQ ID NO: 22. In one embodiment, the heavy chain variable region sequence comprises SEQ ID NO: 15, and the light chain variable region sequence comprises SEQ ID NO: 21.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody, a CDR-grafted antibody, or a humanized antibody or antigen-binding fragment thereof.

[0020] In some embodiments, the antibody or antigen-binding fragment is a multispecific or bispecific antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody that includes complementary regions that bind to PD-1, PD-L1, CTLA-4, TIM-3, epidermal growth factor receptor (EGFR), CD25, CD19, or an Fcγ receptor.

[0021] In some embodiments, the antibody or antigen-binding fragment is an scFv, Fv, Fab', Fab, F(ab')2, or a diabody.

[0022] In some embodiments, the antibody or antigen-binding fragment has an isotype of IgG4.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises a S241P substitution in the constant region of the heavy chain.

[0024] In some embodiments, the antibody or antigen-binding fragment is aglycosylated.

[0025] In some embodiments, the antibody or antigen-binding fragment lacks a C-terminal lysine in the heavy chain.

[0026] In some embodiments, the antibody or antigen-binding fragment is conjugated to one or more of a cytotoxin, a fluorescent label, and an imaging agent.

[0027] Provided herein is an antibody or antigen-binding fragment thereof that binds to the same epitope on CEACAM1 as the antibody or antigen-binding fragment thereof disclosed herein. Provided herein is an antibody or antigen-binding fragment thereof that binds to the IgV-like N domain of CEACAM1. In embodiments, the antibody or antigen-binding fragment thereof does not bind to one or more of CEACAM3, CEACAM5, CEACAM6, and CEACAM8. In some embodiments, the antibody or antigen-binding fragment thereof binds at least in part to a binding site on CEACAM1 for TIM-3 and / or binds at least in part to a binding site on CEACAM1 for PD-1. In some embodiments, the antibody or antigen-binding fragment thereof binds at least in part to a binding site on CEACAM1 for CEACAM1 during homodimerization.

[0028] Provided herein is a nucleic acid encoding the antibody or antigen-binding fragment thereof disclosed herein. In one embodiment, the nucleic acid is an isolated nucleic acid. Provided herein is a vector comprising the nucleic acid disclosed herein.

[0029] Provided herein is a cell comprising the vector disclosed herein.In one embodiment, the cell is an isolated cell.Provided herein is a cell expressing the antibody or antigen-binding fragment thereof disclosed herein.Provided herein is a T cell having a chimeric antigen receptor comprising the CDR of the antibody or antigen-binding fragment thereof disclosed herein.

[0030] Provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed herein and a pharma- ceutically acceptable excipient.

[0031] Provided herein is a method for inhibiting the binding of CEACAM1 to a member of the CEACAM family, comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.In some embodiments, the member of the CEACAM family is CEACAM5 or CEACAM8.In one embodiment, the member of the CEACAM family is CEACAM1.

[0032] A method for inhibiting the binding of CEACAM1 to a member of the TIM family is provided, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein. In one embodiment, the member of the TIM family is TIM-3.

[0033] A method for inhibiting the binding of CEACAM1 to PD-1 is provided, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0034] A method for inhibiting the binding of CEACAM1 to a bacterial adhesin, comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein. In an embodiment, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza outer membrane protein (OMP) P1, Haemophilus aegyptius OMP P1, Moraxella sp. Opa-like protein (OlpA), Fusobacterium sp. trimeric autotransporter adhesin CbpF, Salmonella sp. adhesin, or Streptococcus agalactiae IgI3-like β protein adhesin.

[0035] A method for inhibiting binding of CEACAM1 to Candida albicans is provided, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0036] A method for reducing colonization of mammalian epithelium by bacteria expressing a bacterial adhesin is provided, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein. In embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1 or Moraxella sp. OlpA, Fusobacterium sp. trimeric autotransporter adhesin CbpF, Salmonella sp. adhesin, or Streptococcus agalactiae IgI3-like β protein adhesin.

[0037] Provided herein is a method for reducing colonization of mammalian epithelium by Candida albicans, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0038] Provided herein is a method of reducing T cell tolerance, the method comprising contacting a cell population comprising T cells with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0039] Provided herein is a method of enhancing T cell proliferation, the method comprising contacting a cell population comprising T cells with an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0040] Provided herein is a method for reducing T cell tolerance in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0041] Provided herein is a method for enhancing T cell proliferation in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0042] Provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein. In embodiments, the cancer is melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cell carcinoma, prostate cancer, breast cancer, bladder cancer, or gastric cancer.

[0043] Provided herein is a method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0044] Provided herein is a method of reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0045] Provided herein is a method of reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0046] Provided herein is a method for reducing cancer stemness in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0047] Provided herein is 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 antibody or antigen-binding fragment thereof disclosed herein. In embodiments, the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1, Moraxella sp. OlpA, Fusobacterium sp. trimeric autotransporter adhesin CbpF, Salmonella sp. adhesin, or Streptococcus agalactiae IgI3-like β protein adhesin.

[0048] Provided herein is a method for reducing colonization of a subject's epithelium by Candida albicans in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein.

[0049] Provided herein is a method for reducing invasion of the lymphatic system of a subject by filarial parasites in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein. In one embodiment, the filarial parasite is Wucheria bancrofti.

[0050] Provided herein is a method of reducing invasion of the lymphatic system of a subject by cancer cells in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein. In embodiments, the method further comprises administering a checkpoint inhibitor. In embodiments, the checkpoint inhibitor is a CTLA-4, PD-1, PD-L1, and PD-L2 inhibitor.

[0051] In embodiments, the methods disclosed herein further comprise administering one or more of an inhibitor of LAG3, TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226, and / or CD160. In embodiments, the methods disclosed herein further comprise administering a TIM-3 inhibitor. In embodiments, the additional inhibitor is administered simultaneously or sequentially with the antibody or antigen-binding fragment or pharmaceutical composition. In embodiments, the additional inhibitor is administered separately or in admixture with the antibody or antigen-binding fragment thereof disclosed herein.

[0052] Methods of treating a subject in need of treatment are provided, the methods comprising administering to the subject an antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutical composition disclosed herein, wherein the subject has acquired resistance to treatment with checkpoint inhibitor therapy. In embodiments, the subject has acquired resistance to treatment with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. [Brief description of the drawings]

[0053] [Figure 1] Figure 2 shows the design of the affinity maturation library. The CDRs (Kabat definition) are underlined. X = position targeted for mutagenesis. Individual positions may contain all 20 amino acids or a subset thereof. VH CDR3 block CTRGLYYGPAWFAYW (SEQ ID NO: 25) for mutagenesis. VL CDR3 block CQQHYSTPWTF (SEQ ID NO: 26) for mutagenesis. [Diagram 2] Exemplary data from a perirep binding primary screen are shown. Wells with parental humanized scFv, irrelevant scFv, and media only are shown. Clones with at least 50% inhibition are indicated by arrows. [Figure 3A] Cross-reactivity ELISA results are shown for parental antibodies and 12 lead variants binding to CEACAM-1 / -3 / -5, and -6 (left to right) at three concentrations (1.67 μg / ml, 0.062 μg / ml, and 0.0069 μg / ml). [Figure 3B] Cross-reactivity ELISA results are shown for parental antibodies and 12 lead variants binding to CEACAM-1 / -3 / -5, and -6 (left to right) at three concentrations (1.67 μg / ml, 0.062 μg / ml, and 0.0069 μg / ml). [Figure 3C] Cross-reactivity ELISA results are shown for parental antibodies and 12 lead variants binding to CEACAM-1 / -3 / -5, and -6 (left to right) at three concentrations (1.67 μg / ml, 0.062 μg / ml, and 0.0069 μg / ml). [Figure 3D] Cross-reactivity ELISA results are shown for parental antibodies and 12 lead variants binding to CEACAM-1 / -3 / -5, and -6 (left to right) at three concentrations (1.67 μg / ml, 0.062 μg / ml, and 0.0069 μg / ml). [Figure 3E] Cross-reactivity ELISA results are shown for parental and pan-CEACAM antibody D14HD11 binding to CEACAM-1 / -3 / -5, and -6 (left to right) at three concentrations (1.67 μg / ml, 0.062 μg / ml, and 0.0069 μg / ml). [Figure 4] The specificity of antibody He14 / L1 as determined by single cycle surface plasmon resonance. Binding sensorgrams of increasing concentrations (70-280 nM) of antibody He14 / L1 (A) or control antibody CH14D11 (B) binding to hCEACAM1, hCEACAM3, hCEACAM5, and hCEACAM6 are shown. RU: resonance units. [Figure 5A] Shown is the structure of the CEACAM1:He14 / L1 Fab complex. Structure of the complex (L, H, and C chains) with the Fab heavy and light chains depicted as Cα traces and the antigen depicted as ribbons. [Figure 5B] The structure of the CEACAM1:He14 / L1 Fab complex is shown. The CEACAM1 molecule in the complex is superimposed on the CEACAM1 molecule in the dimer. The chains are labeled A through G in bold. [Figure 5C] Shows the structure of the CEACAM1:He14 / L1 Fab complex. Diagram of complexed CEACAM1 showing the epitope. The depicted molecular surface is drawn in semi-transparent light grey, the protein backbone is drawn as ribbons, and the relevant side chains are drawn as sticks and labelled. The region of the surface that interacts with the Fab is indicated. [Figure 5D] The structure of the CEACAM1:He14 / L1 Fab complex is shown. Schematic of He14 / L1 binding to the CEACAM1 GFCC' face. [Figure 5E]The structure of the CEACAM1:He14 / L1 Fab complex is shown. He14L1 specifically docks to both sides of CEACAM1 and may disrupt the conformation of CEACAM1. The GFCC' face is the main interacting face with the C'C"DE face potentially involved in the conformation. [Figure 6] Showing that the indicated CEACAM1 antibodies VH2 / Vκ4 (parental / parental), VH2 / L1 (parental / L1), He3 / L20, He17 / L1, and He14 / L1 block human CEACAM:human TIM-3 interactions. IgG4 = control antibody. [Figure 7] 1 shows the results of an in vitro assay of T cell function (proliferation assay). Ki67 expression indicates T cell proliferation in response to CEACAM1 antibodies He3 / L20, He14 / L1, H17 / L1, and VH2 / L1 (parental / L1) in subjects resistant to pembrolizumab treatment. [Figure 8A] Treatment with antibody He14 / L1 (Figure 8A) compared with control antibody (Figure 8B) results in increased B cell proliferation (164→347 cells) and T cell proliferation (394→913 cells) within 42 hours, indicating that He14 / L1 induces a broad induction of B and T cells, responses important for antitumor activity. [Figure 8B] Treatment with antibody He14 / L1 (Figure 8A) compared with control antibody (Figure 8B) results in increased B cell proliferation (164→347 cells) and T cell proliferation (394→913 cells) within 42 hours, indicating that He14 / L1 induces a broad induction of B and T cells, responses important for antitumor activity. [Figure 9]CEACAM1 expression of epitopes recognized by antibodies derived from parental antibodies, including He14 / L1, is associated with therapeutic resistance. A: CEACAM1 expression in metaclusters representing pre-plasmablastic B cells identified by Citrus analysis (metaclusters identified by Citrus C36175, C36194) shows that therapeutic resistance is associated with increased CEACAM1 expression in resistant (R) tumor samples compared to naive (N) tumor samples. Median levels of CEACAM1 expression determined by ANOVA. *p<0.05. B: CEACAM1 expression in metaclusters representing tumor-associated monocytic cells shows that therapeutic resistance is associated with increased CEACAM1 expression in resistant (R) tumor samples compared to naive (N) tumor samples in metaclusters identified by Citrus (C58750, C58749, C58832, C58843, C58885, C58888). Median levels of CEACAM1 expression determined by ANOVA. *Significance of p<0.05, **p<0.01. C: CEACAM1 expression in metaclusters representing dysfunctional CD8+ central memory T cells shows that therapy resistance is associated with increased CEACAM1 expression in therapy-resistant (R) tumor samples compared to naive (N) tumor samples in metaclusters identified by Citrus (C58736, C58786, C58789). Median levels of CEACAM1 expression determined by ANOVA. *Significance of p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] antibody

[0055] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and antigen-binding portions thereof (e.g., paratopes, CDRs), so long as they exhibit the desired biological activity and specificity.

[0056] As used herein, an "antibody variable domain" refers to the portion of the light and heavy chains of an antibody molecule that contains the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). H refers to the variable domain of the heavy chain. L refers to the light chain variable domain. The amino acid positions assigned to CDRs and FRs may be defined according to Kabat or Chothia. The term "framework region" (FR) refers to those variable domain residues other than the CDR residues.

[0057] As used herein, the term "complementarity determining region" (CDR) refers to the portion of an antibody variable domain that (typically) participates in antigen binding. Each variable domain usually has three CDR regions, identified as CDR1, CDR2 and CDR3. Each CDR may, for example, comprise amino acid residues from a CDR defined by Kabat (i.e., for residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1987, 1991)). Each CDR is derived from a "hypervariable loop" (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) of the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) of the heavy chain variable domain (Chothia & Lesk 196 J. Mol. Biol. 901 (1987)) amino acid residues. In some cases, CDRs can include amino acids from both the CDR regions and the hypervariable loops defined according to Kabat. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues (primary amino acid sequence). The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering corresponding to the shortening or insertion of structural components, whether framework or CDR of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody or antigen-binding fragment thereof by aligning the homologous residues in the sequence of that antibody or antigen-binding fragment thereof with the "standard" Kabat numbered sequence. Alternatively, CDRs can be defined according to the ImMunoGeneTics (IMGT) system (Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)).

[0058] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises a heavy variable chain comprising three CDRs: (i) the sequence of CDR1H comprises the sequence DYYLY (SEQ ID NO: 1); (ii) the sequence of CDR2H comprises the sequence TISVGGGQTSYADSVKG (SEQ ID NO: 2); (iii) the sequence of the CDR3H comprises the sequence GLYYGPSWVAY (SEQ ID NO: 3), ARTYGPAWFAY (SEQ ID NO: 4), or ALTYGPAWLAY (SEQ ID NO: 5).

[0059] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises a heavy variable chain comprising three CDRs: (i) the sequence of CDR1H comprises the sequence DYYLY (SEQ ID NO: 1); (ii) the sequence of CDR2H comprises the sequence TISVGGGQTSYADSVKG (SEQ ID NO: 2); (iii) the sequence of CDR3H comprises the sequence GLYYGPSWVAY (SEQ ID NO: 3).

[0060] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises a heavy variable chain comprising three CDRs: (i) the sequence of CDR1H comprises the sequence DYYLY (SEQ ID NO: 1); (ii) the sequence of CDR2H comprises the sequence TISVGGGQTSYADSVKG (SEQ ID NO: 2); (iii) the sequence of CDR3H comprises the sequence ARTYGPAWFAY (SEQ ID NO: 4).

[0061] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises a heavy variable chain comprising three CDRs: (i) the sequence of CDR1H comprises the sequence DYYLY (SEQ ID NO: 1); (ii) the sequence of CDR2H comprises the sequence TISVGGGQTSYADSVKG (SEQ ID NO: 2); (iii) the sequence of the CDR3H comprises the sequence ALTYGPAWLAY (SEQ ID NO:5).

[0062] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises a light variable chain comprising three CDRs: (i) the sequence of CDR1L comprises the sequence KSSQSLLNSANQKNYLA (SEQ ID NO: 6); (ii) the sequence of CDR2L comprises the sequence FASTRES (SEQ ID NO: 7), (iii) the sequence of CDR3L comprises the sequence QSHYPFYYT (sequence number 8) or QSHFPYPLT (sequence number 9).

[0063] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises a light variable chain comprising three CDRs: (i) the sequence of CDR1L comprises the sequence KSSQSLLNSANQKNYLA (SEQ ID NO: 6); (ii) the sequence of CDR2L comprises the sequence FASTRES (SEQ ID NO: 7), (iii) the sequence of CDR3L comprises the sequence QSHYPFYYT (sequence number 8).

[0064] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises a light variable chain comprising three CDRs: (i) the sequence of CDR1L comprises the sequence KSSQSLLNSANQKNYLA (SEQ ID NO: 6); (ii) the sequence of CDR2L comprises the sequence FASTRES (SEQ ID NO: 7), (iii) the sequence of CDR3L comprises the sequence QSHFPYPLT (sequence number 9).

[0065] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises six CDRs: (i) the sequence of CDR1H comprises the sequence DYYLY (SEQ ID NO: 1); (ii) the sequence of CDR2H comprises the sequence TISVGGGQTSYADSVKG (SEQ ID NO: 2); (iii) the sequence of the CDR3H comprises the sequence GLYYGPSWVAY (SEQ ID NO: 3), ARTYGPAWFAY (SEQ ID NO: 4), or ALTYGPAWLAY (SEQ ID NO: 5); (iv) the sequence of CDR1L comprises the sequence KSSQSLLNSANQKNYLA (SEQ ID NO: 6); (v) the sequence of CDR2L comprises the sequence FASTRES (SEQ ID NO:7); (vi) The sequence of CDR3L comprises the sequence QSHYPFYYT (sequence number 8) or QSHFPYPLT (sequence number 9).

[0066] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof provided herein comprises six CDRs: (i) the sequence of CDR1H comprises the sequence DYYLY (SEQ ID NO: 1); (ii) the sequence of CDR2H comprises the sequence TISVGGGQTSYADSVKG (SEQ ID NO: 2); (iii) the sequence of the CDR3H comprises the sequence GLYYGPSWVAY (SEQ ID NO: 3); (iv) the sequence of CDR1L comprises the sequence KSSQSLLNSANQKNYLA (SEQ ID NO: 6); (v) the sequence of CDR2L comprises the sequence FASTRES (SEQ ID NO:7); (vi) The sequence of CDR3L comprises the sequence QSHYPFYYT (sequence number 8).

[0067] According to certain embodiments, the contemplated antibodies and antigen-binding fragments thereof also feature a humanized framework to reduce immunogenicity. In certain embodiments, the CDRs of the contemplated antibodies or antigen-binding fragments thereof are located in a framework derived from a human antibody or antigen-binding fragment thereof. In other embodiments, surface-exposed framework residues of the contemplated antibodies or antigen-binding fragments thereof are replaced with framework residues of a human antibody or antigen-binding fragment thereof. The CDRs may also be placed in a murine or humanized framework linked to a human constant region (i.e., a chimeric antibody). In a preferred embodiment, the CDRs of the contemplated antibodies or antigen-binding fragments thereof are placed in a framework that is a composite of two or more human antibodies. In such an embodiment, the contemplated antibodies or antigen-binding fragments thereof comprise two or more sequence segments ("composite") derived from the V-regions of unrelated human antibodies selected to maintain the monoclonal antibody sequences important for antigen binding of the starting precursor anti-human CEACAM1 monoclonal antibody, which have all been filtered for the presence of potential T-cell epitopes using "in silico tools" (Holgate & Baker, IDrugs. 2009 Apr; 12(4): 233-7). Prior to synthesis of the antibody or antigen-binding fragment thereof, the human sequence segments are closely matched with all sections of the starting antibody V region to produce a CD4 + By removing T cell epitopes, this technology avoids immunogenicity while maintaining optimal affinity and specificity through prior analysis of sequences required for antigen specificity (Holgate & Baker, 2009).

[0068] Also provided herein are variable heavy and variable light chain sequences and pairs thereof, including sequences similar but not identical to the variable heavy and light chains and pairs thereof disclosed in SEQ ID NOs: 10-22.

[0069] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 10 to 17. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 15 to 17.

[0070] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 10-17. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof comprises a variable heavy chain amino acid sequence selected from the group consisting of SEQ ID NOs: 15-17.

[0071] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an array that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an array selected from the group consisting of SEQ ID NOs: 18-22.

[0072] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof comprises a light chain variable domain comprising an sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 or SEQ ID NO:22.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 22. In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain amino acid sequence comprising SEQ ID NO: 21 or SEQ ID NO: 22.

[0074] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 10 to 17; and (ii) comprises a light chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 18-22.

[0075] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-17; and (ii) comprises a light chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 or SEQ ID NO:22.

[0076] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising a sequence selected from the group consisting of SEQ ID NOs: 10 to 17; and / or (ii) comprises a light chain variable domain comprising a sequence selected from the group consisting of SEQ ID NOs: 18 to 22.

[0077] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising a sequence selected from the group consisting of SEQ ID NOs: 15 to 17; and / or (ii) comprises a light chain variable domain comprising SEQ ID NO:21 or SEQ ID NO:22.

[0078] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15; and (ii) comprises a light chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21.

[0079] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising SEQ ID NO: 15; and (ii) comprises a light chain variable domain comprising SEQ ID NO: 21.

[0080] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16; and (ii) comprises a light chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21.

[0081] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising SEQ ID NO: 16; and (ii) comprises a light chain variable domain comprising SEQ ID NO: 21.

[0082] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17; and (ii) comprises a light chain variable domain comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22.

[0083] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof (i) a heavy chain variable domain comprising SEQ ID NO: 17; and (ii) comprises a light chain variable domain comprising SEQ ID NO: 22.

[0084] As used herein, the term "identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions of each sequence that can be aligned for comparison purposes. For example, if the positions of the compared nucleotide sequences are occupied by the same base, the molecules are identical at that position. The degree of identity between nucleic acid sequences or amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at shared positions. For example, polypeptides having at least 85%, 90%, 95%, 98%, or 99% identity to the specific polypeptides described herein and preferably exhibiting substantially the same function, as well as polynucleotides encoding such polypeptides, are contemplated. Methods and computer programs for determining both sequence identity and similarity are publicly available, and include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12:387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990), and the ALIGN program (version 2.0). The well-known Smith-Waterman algorithm may also be used to determine similarity. BLAST programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at http: / / www.ncbi.nlm.nih.gov / blast / ). In comparing sequences, these methods take into account various substitutions, deletions, and other modifications.

[0085] In one embodiment, a CEACAM1 antibody or antigen-binding fragment thereof is provided, (i) a heavy chain variable domain comprising a sequence that is at least 80%, 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 of SEQ ID NO: 15; (ii) a light chain variable domain comprising a sequence that is at least 80%, 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 of SEQ ID NO: 21; and (iii) contains six CDRs; a. the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO:1; b. the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; c. the sequence of the CDR3 of the heavy chain variable region comprises SEQ ID NO:3; d. the sequence of CDR1 of the light chain variable region comprises SEQ ID NO:6; e. the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:7; f. The sequence of the CDR3 of the light chain variable region comprises SEQ ID NO:8.

[0086] In one embodiment, a CEACAM1 antibody or antigen-binding fragment thereof is provided, (i) a heavy chain variable domain comprising a sequence that is at least 80%, 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 of SEQ ID NO: 16; (ii) a light chain variable domain comprising a sequence that is at least 80%, 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 of SEQ ID NO: 21; and (iii) contains six CDRs; a. the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO:1; b. the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; c. the sequence of the CDR3 of the heavy chain variable region comprises SEQ ID NO:4; d. the sequence of CDR1 of the light chain variable region comprises SEQ ID NO:6; e. the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:7; f. The sequence of the CDR3 of the light chain variable region comprises SEQ ID NO:8.

[0087] In one embodiment, a CEACAM1 antibody or antigen-binding fragment thereof is provided, (i) a heavy chain variable domain comprising a sequence that is at least 80%, 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 of SEQ ID NO: 17; (ii) a light chain variable domain comprising a sequence that is at least 80%, 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 of SEQ ID NO: 22; and (iii) contains six CDRs; a. the sequence of CDR1 of the heavy chain variable region comprises SEQ ID NO:1; b. the sequence of CDR2 of the heavy chain variable region comprises SEQ ID NO:2; c. the sequence of the CDR3 of the heavy chain variable region comprises SEQ ID NO:5; d. the sequence of CDR1 of the light chain variable region comprises SEQ ID NO:6; e. the sequence of CDR2 of the light chain variable region comprises SEQ ID NO:7; f. The sequence of the CDR3 of the light chain variable region comprises SEQ ID NO:9.

[0088] It will be apparent that any of the frameworks described herein can be utilized in combination with any of the CDRs and CDR motifs described herein. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof utilizes a framework described in Table 1.

[0089] Some embodiments of the aspects described herein contemplate modification of the amino acid sequence of the antibody or antigen-binding fragment thereof that binds to CEACAM1 described herein. The amino acid sequence variants of the antibody or antigen-binding fragment thereof are prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antibody or antigen-binding fragment thereof or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody or antigen-binding fragment thereof. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final construct, provided that the final construct has the desired properties, e.g., binding specificity, inhibition of biological activity.

[0090] One type of variant is a conservative amino acid substitution variant. These variants have at least one amino acid residue in the antibody or antigen-binding fragment thereof replaced with a different residue having similar side chain properties. Amino acids can be grouped according to the similarity of their side chain properties (see Lehninger, BIOCHEMISTRY (2nd ed., Worth Publishers, New York, 1975): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).

[0091] Thus, a non-limiting example of a conservative amino acid substitution is one that replaces a non-polar amino acid with another non-polar amino acid.

[0092] Alternatively, naturally occurring residues can be grouped on the basis of common side chain properties. (1) Hydrophobicity: Ala(A), Val(V), Leu(L), Ile(I), Met(M); (2) Neutral hydrophilicity: Ser(S), Thr(T), Cys(C), Asn(N), Gln(Q); (3) Acidic: Asp(D), Glu(E); (4) Basic: Lys(K), Arg(R), His(H); (5) Residues that affect chain orientation: Gly (G), Pro (P); (6) Aromatic: Phe(F), Trp(W), Tyr(Y). Thus, a non-limiting example of a conservative amino acid substitution is one that replaces a hydrophobic amino acid with another hydrophobic amino acid.

[0093] Also contemplated are amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as amino acid sequence insertions which may include intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody or antigen-binding fragment thereof with an N-terminal methionyl residue, or an antibody or antigen-binding fragment thereof fused to a cytotoxic polypeptide. Other insertional variants of antibodies or antigen-binding fragments thereof include the fusion to the N- or C-terminus of the antibody or antigen-binding fragment thereof of an enzyme or a polypeptide which increases the serum half-life of the antibody or antigen-binding fragment thereof, such as biotin.

[0094] Cysteine ​​residues not involved in maintaining the proper conformation of an antibody or antigen-binding fragment thereof that binds CEACAM1 can also be substituted, e.g., to serine or alanine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0095] Conversely, cysteine ​​bond(s) may be added to the antibody or antigen-binding fragment thereof to improve its stability (particularly where the antibody or antigen-binding fragment thereof is an antibody fragment such as an Fv fragment).

[0096] In some embodiments, the described antibodies or antigen-binding fragments thereof have amino acid modifications that alter the original glycosylation pattern of the antibody or antigen-binding fragment thereof. By "altering the original glycosylation pattern" is meant deleting one or more carbohydrate moieties found in the antibody or antigen-binding fragment thereof and / or adding one or more glycosylation sites that are not present in the antibody or antigen-binding fragment thereof. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Addition of glycosylation sites to an antibody or antigen-binding fragment thereof that binds to CEACAM1 is accomplished by modifying the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). Modifications can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody or antigen-binding fragment thereof (for O-linked glycosylation sites).

[0097] In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof provided herein is deglycosylated or aglycosylated. In some embodiments, the contemplated CEACAM1 antibody or antigen-binding fragment thereof lacks a C-terminal lysine in the heavy chain and / or comprises a S241P substitution in the constant region of the heavy chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof lacks a glycosylation site in CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof lacks a NXS / T consensus sequence in CDR1 of the variable light chain. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof has a mutation at CDR residues 26 and / or 29 (Kabat numbering) of CDR1 of the variable light chain. If the antibody or antigen-binding fragment thereof comprises an Fc region, the carbohydrate(s) attached thereto can be modified. For example, antibodies have been described that have a mature carbohydrate structure that lacks fucose attached to the Fc region of the antibody or antigen-binding fragment thereof. See, for example, US Patent Publication Nos. 2003 / 0157108; 2004 / 0093621. Antibodies with bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody or antigen-binding fragment thereof are referenced in WO03 / 011878; US Patent No. 6,602,684. Antibodies with at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody or antigen-binding fragment thereof are reported in WO97 / 30087. See also WO98 / 58964; WO99 / 22764 for antibodies with modified carbohydrate attached to the Fc region.

[0098] In some embodiments, it may be desirable to modify an antibody or antigen-binding fragment thereof that binds CEACAM1 described herein with respect to effector function, for example to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody or antigen-binding fragment thereof. This can be accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody or antigen-binding fragment thereof. Alternatively or additionally, one or more cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody or antigen-binding fragment thereof thus generated may have improved internalization capability and / or increased complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., 176 J. Exp. Med. 1191 (1992); Shopes, 148 J. Immunol. 2918 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., 53 Cancer Res. 2560 (1993). Alternatively, an antibody or antigen-binding fragment thereof can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., 3 Anti-Cancer Drug Design 219 (1989).

[0099] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein are modified to exhibit reduced or eliminated effector function. This can be achieved, for example, by: (i) reducing or eliminating wild-type mammalian glycosylation of the antibody (e.g., by producing the antibody in an environment where such glycosylation does not occur, by mutating one or more carbohydrate attachment points so that the antibody cannot be glycosylated, or by chemically or enzymatically removing one or more carbohydrates from the antibody after it has been glycosylated); (ii) reducing or eliminating Fc receptor binding ability of the antibody (e.g., by mutation of the Fc region, by deletions within the Fc region, or by removal of the Fc region); or (iii) utilizing antibody isotypes known to have minimal or no effector function (i.e., including but not limited to IgG4). In some embodiments, the heavy chain constant region has one or more of the following mutations: S228P, N297Q, and T299A (numbering according to Kabat). In some embodiments, the heavy chain constant region has one or more of the following mutations: L234A, L235A, and P329G (Kabat EU index numbering).

[0100] For example, WO00 / 42072 describes an antibody with improved ADCC function in the presence of human effector cells, which comprises amino acid substitutions in its Fc region. Preferably, the antibody or antigen-binding fragment thereof with improved ADCC comprises substitutions at positions 298, 333, and / or 334 of the Fc region (Eu numbering of residues). Typically, the modified Fc region is a human IgG1 Fc region comprising or consisting of substitutions at one, two, or three of these positions. Such substitutions are optionally combined with substitution(s) that increase Clq binding and / or CDC. Substitutions include the Asn297Ala mutation of IgG1 Fc.

[0101] Antibodies with altered Clq binding and / or complement dependent cytotoxicity (CDC) are described in WO 99 / 51642, U.S. Patent Nos. 6,194,551, 6,242,195, 6,528,624, and 6,538,124. The antibodies contain amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333, and / or 334 of their Fc region (Eu numbering of residues).

[0102] Antibodies with improved binding to the neonatal Fc receptor (FcRn) and increased half-life are described in WO00 / 42072 and U.S. Patent Publication No. 2005 / 0014934. These antibodies comprise an Fc region having one or more substitutions that improve binding of the Fc region to CEACAM1. For example, the Fc region can have a substitution at one or more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, 428, or 434 (Eu numbering of residues). A preferred Fc region-containing antibody variant with improved CEACAM1 binding comprises amino acid substitutions (Eu numbering of residues) at one, two, or three of positions 307, 380, and 434 of its Fc region. In one embodiment, the antibody or antigen-binding fragment thereof has a 307 / 434 mutation. Modified antibodies that bind to CEACAM1 at three or more (e.g., four) functional antigen-binding sites are also contemplated. See, e.g., U.S. Patent Publication No. US2002 / 0004587.

[0103] Antibody Fragments and Types

[0104] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab fragment, which comprises the variable (V L ) domain and constant (C L ) domain and the variable domain of the heavy chain (V H ) and the first constant domain (C H 1) is included.

[0105] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is a Fab′ fragment, which is H This refers to a Fab fragment that has one or more cysteine ​​residues at the C-terminus of one domain.

[0106] In some embodiments of the aspects described herein, the CEACAM1 antibody fragment is H and C H An Fd fragment comprising, or consisting essentially of, one or more of the Fd domains.

[0107] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is H Domain and C H 1 domain and C H This is an Fd' fragment that has one or more cysteine ​​residues at the C-terminus of one domain.

[0108] Single-chain Fv or scFv antibody fragments are H Domain and V L Fv polypeptides comprise or consist essentially of V domains, and these domains are present in a single polypeptide chain. Generally, Fv polypeptides include V H Domain and V L The scFv may further comprise a polypeptide linker between the domains, which enables the scFv to form the desired structure for antigen binding. See, e.g., Pluckthun, 113 Pharmacology Monoclonal Antibodies 269 (Rosenburg & Moore, eds., Springer-Verlag, New York, 1994). Thus, in some embodiments of the aspects described herein, the CEACAM1 antibody fragment comprises the V domain of a single arm of the antibody. L Domain and V H An Fv fragment comprises or consists essentially of a domain.

[0109] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is a diabody that comprises two antigen-binding sites and includes a light chain variable domain (V L ) connected to a heavy chain variable domain (V H ) in the same polypeptide chain.

[0110] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is H A dAb fragment may be a dAb fragment comprising or consisting essentially of a domain.

[0111] In some embodiments of the aspects described herein, the CEACAM1 antibody portion is a F(ab')2 fragment, which comprises a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region.

[0112] Linear antibodies refer to the antibodies described in Zapata et al., Protein Engin., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-V H -C H 1), which together with complementary light chain polypeptides form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. In some embodiments of the aspects described herein, the CEACAM1 antibody fragment comprises a pair of tandem Fd segments (V H -C H 1-V H -C H 1) is a linear antibody.

[0113] Various techniques have been developed and are available for the production of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies. See, for example, Morimoto et al., 24 J.Biochem.Biophys.Meths.107 (1992); Brennan et al., 229 Science 81 (1985). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed herein. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody fragment of choice is a single chain Fv fragment (scFv). See, for example, WO93 / 16185.

[0114] In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds to CEACAM1 and a complementary region that binds to PD-1. In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds to CEACAM1 and a complementary region that binds to PD-L1. In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds to CEACAM1 and a complementary region that binds to TIM-3. In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds to CEACAM1 and a complementary region that binds to epidermal growth factor receptor (EGFR). In one embodiment, the antibody is a bispecific antibody comprising a complementary region that binds to CEACAM1 and a complementary region that binds to an Fc gamma receptor.

[0115] Contemplated antibodies or antigen-binding fragments may include all types of constant regions, including IgM, IgG, IgD, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, human isotype IgG1 is used. In another embodiment, human isotype IgG4 is used. The light chain constant region may be lambda or kappa. Antibodies or antigen-binding fragments thereof may include sequences from multiple classes or isotypes.

[0116] Also disclosed herein is a chimeric antigen receptor T cell (CAR T cell) that binds to CEACAM1. In one embodiment, one or more CDRs of the anti-CEACAM antibody disclosed herein are grafted onto a chimeric antigen receptor (CAR) on T cell. Such genetically modified T cell utilizes CAR, also known as chimeric T cell receptor, to target antigens expressed on tumor cells in a human leukocyte antigen-independent manner.

[0117] antibody binding

[0118] The human CEACAM1 gene generates 11 isoforms by alternative splicing. Each isoform has one variable (V)-like Ig domain at the amino (N) terminus of the protein. Except for the CEACAM1-1L and CEACAM1-1S isoforms, the various isoforms also have two or three constant C2-like Ig domains. Eight CEACAM1 isoforms are anchored to the cell membrane via a transmembrane domain, and three CEACAM1 isoforms (CEACAM1-4C1, -3, and -3C2) lack a transmembrane domain and are secreted. Two isoforms (CEACAM1-3AL and -3AS) have an Alu family repeat sequence (A) between the constant C2-like Ig domain and the transmembrane domain. The transmembrane CEACAM1 isoforms also have long (L) or short (S) cytoplasmic domains, determined by the inclusion or exclusion of CEACAM1 exon 7 in the message. The CEACAM1 L cytoplasmic domain has two ITIM motifs that are unique to CEACAM1 among the CEACAM family members. In one aspect, CEACAM1 antibodies or antigen-binding fragments thereof are provided, including antibodies described herein by their structural feature of binding to an extracellular variable (V)-like Ig domain at the amino (N)-terminus (N-domain) of the CEACAM1 protein, i.e., a domain common to all isoforms of CEACAM1, including CEACAM1 isoforms 1L, 1S, 3L, 3S, 4L, 4S, 3Al, 3AS, 3, 4C1, and 4C2. In some embodiments, the provided antibodies and antigen-binding fragments thereof bind to human CEACAM1. In some embodiments, the provided antibodies and antigen-binding fragments thereof bind to mammalian CEACAM1. The sequence of the full-length form of CEACAM1 (NCBI Reference Sequence NP_001703.2; UNIPR.QT ID P13688) is provided as SEQ ID NO:26 (signal sequence: residues 1-34 of SEQ ID NO:23; Ig-V N domain: residues 35-142 of SEQ ID NO:23). The mature form of CEACAM1 (without signal sequence) is provided as SEQ ID NO:24.

[0119] As used herein, "binding" of an antibody or antigen-binding fragment thereof to CEACAM1, an epitope on CEACAM1, or, in certain embodiments described below, a particular residue on CEACAM1, includes selective interaction of the antibody or antigen-binding fragment thereof with CEACAM1. Thus, binding includes primary and secondary interactions including, for example, hydrophilic and hydrophobic interactions, as well as hydrogen bonds, ionic interactions, salt bridges.

[0120] In certain embodiments, the CEACAM1 antibodies or antigen-binding fragments thereof described herein are -5 ~10 -12 mol / l, 10 -6 ~10 -12 mol / l, 10 -6 ~10 -9 mol / l, 10 -7 ~10 -12 mol / l, 10 -8 ~10 -12 mol / l, 10 -9 ~10 -12 mol / l, 10 -10 ~10 -12 mol / l, or 10 -11 ~10 -12 K in mol / l D In other embodiments, the CEACAM1 antibodies or binding fragments thereof described herein bind to CEACAM1 at 10 -5 ~10 -11 mol / l, 10 -6 ~10 -11 mol / l, 10 -7 ~10 -11 mol / l, 10 -8 ~10 -11 mol / l, 10 -9 ~10 -11 mol / l, or 10 -10 ~10 -11 K in mol / l D In other embodiments, the CEACAM1 antibodies or binding fragments thereof described herein bind to CEACAM1 at 10 -5 ~10 -10 mol / l, 10 -6 ~10 -10mol / l, 10 -7 ~10 -10 mol / l, 10 -8 ~10 -10 mol / l, or 10 -9 ~10 -10 K in mol / l D In other embodiments, the CEACAM1 antibodies or binding fragments thereof described herein bind to CEACAM1 at 10 -5 ~10 -8 mol / l, 10 -6 ~10 -8 mol / l, or 10 -7 ~10 -8 K in mol / l D and binds to CEACAM1.

[0121] The term "specificity" as used herein refers to the ability of an antibody or antigen-binding fragment thereof, such as an anti-CEACAM1 antibody or antigen-binding fragment thereof, to recognize an epitope within CEACAM1 but have little or no detectable reactivity with other parts of CEACAM1. Specificity can be relatively determined by competitive assays or by the epitope identification / characterization techniques described herein or their equivalents known in the art.

[0122] As used herein, an "epitope" can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least three, and more commonly at least five, about nine, or about eight to ten amino acids in a particular spatial conformation. An "epitope" is defined as a sequence that is unique to an immunoglobulin V protein. H / V LIt comprises a structural unit that is conventionally bound by a variable domain pair. An epitope defines the minimal binding site of an antibody or antigen-binding fragment thereof and thus represents the target of specificity of the antibody or antigen-binding fragment thereof. In the case of a single domain antibody, the epitope represents the structural unit to which the variable domain binds alone.

[0123] In certain embodiments, a contemplated antibody or antigen-binding fragment specifically binds to the same epitope as antibody He14 / L1.

[0124] In one aspect, the invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein by their structural features, which specifically bind to at least a portion of the homophilic binding domain on CEACAM1 (i.e., the portion of the CEACAM1 protein involved in the formation of the CEACAM1:CEACAM1 homodimer), thereby blocking the CEACAM1 homophilic interaction. The residues of CEACAM1 involved in homodimerization are shown in Table 1. [Table 1]

[0125] In some embodiments, the CEACAM antibody or antigen-binding fragment thereof binds to residues on the GFCC face of CEACAM1, such as F29, Y31, Q44, Y48, T56, Q89, D94, L95, and / or N97 of SEQ ID NO:24.

[0126] In some embodiments, the CEACAM antibody or antigen-binding fragment thereof binds to a residue on the ABED face of CEACAM1, such as M7, E16, Q27, Y48, Q53, Y68, or N70 of SEQ ID NO:24.

[0127] As used herein, a "blocking" antibody or antibody "antagonist" is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. For example, in some embodiments, a CEACAM1 antagonist antibody or antigen-binding fragment thereof binds to CEACAM1, inhibits the activity of CEACAM1, and / or inhibits the binding of CEACAM1 to other CEACAM proteins or heterologous binding partners such as TIM-3. Inhibition of activity and inhibition of binding include partial inhibition. Methods for identifying CEACAM1 antibodies that block homophilic and heterophilic interactions of CEACAM1 are described herein and known to those of skill in the art. For example, competing, cross-blocking, and cross-blocked antibodies can be identified using any suitable method known in the art, including competitive ELISA or BIACORE® assays, where the binding of a competing or cross-blocking antibody to human CEACAM1 prevents the binding of an antibody disclosed herein, or vice versa.

[0128] In certain embodiments, not all CDRs are directly involved in binding to antigen.In one embodiment, four of the six CDRs of CEACAM1 antibody or its antigen-binding fragment contact antigen.In one embodiment, five of the six CDRs of CEACAM1 antibody or its antigen-binding fragment contact antigen.In one embodiment, six of the six CDRs of CEACAM1 antibody or its antigen-binding fragment contact antigen.

[0129] CEACAM family members are widely expressed in various cell types, especially leukocytes, and affect a range of cellular functions. For example, CEACAM1 is expressed in epithelial cells, endothelial cells, lymphocytes, and myeloid cells, CEACAM3 is expressed in granulocytes and neutrophils, CEACAM5 is expressed in epithelial cells, and CEACAM6 is expressed in epithelial cells and granulocytes. However, the N-domain of CEACAM1 is approximately 90% similar to the N-domains of CEACAM family members 3, 5, and 6, making it difficult to selectively target CEACAM1.

[0130] Despite the high similarity of the N domains between CEACAM family members, in some embodiments, the antibodies or antigen-binding fragments provided herein, including those described herein by their structural features, are selective for CEACAM1. By selectively targeting CEACAM1, embodiments of the present invention may, for example, avoid undesirable interference with the broad activation function of CEACAM3.

[0131] As used herein, the terms "selective" and "selectivity" refer to the preferential binding of an antibody or antigen-binding fragment thereof (i.e., a CEACAM1 antibody or antigen-binding fragment thereof) to a particular region, target, or peptide; typically a region or epitope in CEACAM1, in contrast to one or more other biological molecules, including other CEACAM family members.

[0132] In some embodiments, contemplated CEACAM1 antibodies or antigen-binding fragments thereof do not exhibit significant binding to other CEACAM family members, including CEACAM3, CEACAM5, CEACAM6, and / or CEACAM8. In some embodiments, contemplated CEACAM1 antibodies or antigen-binding fragments thereof do not exhibit detectable binding to other CEACAM family members, including CEACAM3, CEACAM5, CEACAM6, and / or CEACAM8. In some embodiments, contemplated CEACAM1 antibodies or antigen-binding fragments thereof bind to CEACAM1 with an affinity that is at least 10-fold, e.g., at least 100-fold, and at least 1000-fold, and up to 10,000-fold or more stronger than the affinity with which a contemplated CEACAM1 antibody or antigen-binding fragment thereof binds to another target or polypeptide.

[0133] As used herein, the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (K D "Affinity," expressed by K), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding protein, such as an antibody or antibody fragment thereof.D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule. Alternatively, affinity can be expressed as the affinity constant (K A ), which can also be expressed as 1 / K D As will be clear to those skilled in the art, affinity can be measured in a manner known per se depending on the particular antigen of interest.

[0134] In one aspect, the invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein by their structural characteristics, which specifically bind to at least a portion of the binding site on CEACAM1 for one or more other CEACAM family members, thereby blocking the interaction of CEACAM1 with one or more other CEACAM family members, including, but not limited to, CEACAM5 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).

[0135] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein by their structural features, which specifically bind to at least a portion of the binding site on CEACAM1 for a TIM family member, thereby blocking the interaction of CEACAM1 with a TIM family member. In some embodiments, the TIM family member is TIM-1, TIM-3, or TIM-4. In some embodiments, the CEACAM1 antibody or antigen-binding fragment thereof specifically binds to one or more of CEACAM1 residues Y34, G41, N42, Q44, Q89, S93, D94, V96, and / or N97 of SEQ ID NO: 17, which have been shown to be involved in the binding of CEACAM1 to TIM-3 (Huang et al., Nature. 2015 Jan 15;517(7534):386-90).

[0136] In one aspect, the present invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein, which by their structural characteristics specifically bind to at least a portion of the binding site on CEACAM1 for a pathogen, thereby blocking the interaction between CEACAM1 and the pathogen.

[0137] In one aspect, the invention provides antibodies and antigen-binding fragments thereof, including the antibodies described herein by their structural features, which specifically bind to at least a portion of the binding site on CEACAM1 for a bacterial adhesive surface protein (adhesin), thereby blocking the interaction of CEACAM1 with the adhesin. In certain embodiments, the adhesin is expressed on the surface of CEACAM1-binding pathogenic bacteria, including, but not limited to, Escherichia coli, particularly diffusely adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, N. meningitidis, commensal Neisseria, Moraxella catarrhalis, Haemophilus influenza, Haemophilus aegyptius, Helicobacter pylori, Fusobacterium sp., Salmonella sp., and / or Streptococcus agalactiae. In one embodiment, the adhesin is the β protein from S. agalactiae.

[0138] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Streptococcus agalactiae (Group B Streptococcus). In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and the β protein from S. agalactiae. The interactions observed in the crystal structure of the BIgI domain of the β protein from S. agalactiae and human CEACAM1 are shown in Table 2. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts one or more of the interactions shown in Table 2. [Table 2]

[0139] 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 thereof specifically binds to one or more of CEACAM1 residues F29, Y34, N42, Q89, and N97, which are predicted to be involved in the binding of CEACAM1 to HopQ. The interactions observed in the crystal structure of HopQ and hCEACAM1 are shown in Table 3. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts one or more of the interactions shown in Table 3. [Table 3]

[0140] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof is 52 , Opa 65 , Opa 68 , Opa 70 , Opa 72 , Opa 73 , Opa 74 , and Opa 75In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to one or more of CEACAM1 residues Q44 and A49, which are predicted to be involved in binding of CEACAM1 to Neisserial Opa proteins.

[0141] 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.

[0142] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Haemophilus influenza OMP P1. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to one or more of CEACAM1 residues Q44 and A49, which are predicted to be involved in binding of CEACAM1 to Haemophilus influenza OMP P1.

[0143] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and Haemophilus aegyptius OMP P1. In one embodiment, the CEACAM1 antibody or antigen-binding fragment specifically binds to CEACAM1 residue F29, which is predicted to be involved in binding of CEACAM1 to Haemophilus aegyptius OMP P1.

[0144] In another embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and C. albicans.

[0145] In another embodiment, the invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of CEACAM1 to a filarial nematode, the method comprising contacting CEACAM1 with a CEACAM1 antibody or antigen-binding fragment thereof described herein. In one embodiment, the filarial nematode is Wucheria bancrofti.

[0146] antibody complex

[0147] In some embodiments of the aspects described herein, the antibody or antigen-binding fragment thereof that binds to CEACAM1 is conjugated to a functional moiety. Examples of useful functional moieties include, but are not limited to, blocking moieties, detectable moieties, diagnostic moieties, targeting moieties, and therapeutic moieties.

[0148] Exemplary blocking moieties include sufficient steric bulk and / or charged moieties such that reduced glycosylation occurs, for example, by blocking the ability of glycosidases to glycosylate the antibody or antigen-binding fragment thereof. Blocking moieties may additionally or alternatively reduce effector function, for example, by inhibiting the ability of the Fc region to bind to a receptor or complement protein. Preferred blocking moieties include cysteine ​​additions and PEG moieties.

[0149] In a preferred embodiment, the blocking moiety is a cysteine, preferably a cysteine ​​that is bound to a free cysteine, e.g., during or after translation of an Fc-containing polypeptide, e.g., in cell culture. Other blocked cysteine ​​adducts include cystine, mixed disulfide adducts, or disulfide bonds.

[0150] In another preferred embodiment, the blocking moiety is a polyalkylene glycol moiety, such as a PEG moiety, preferably a PEG-maleimide moiety. Preferred pegylated moieties (or related polymers) can be, for example, polyethylene glycol ("PEG"), polypropylene glycol ("PPG"), polyoxyethylated glycerol ("POG") and other polyoxyethylated polyols, polyvinyl alcohol ("PVA") and other polyalkylene oxides, polyoxyethylated sorbitol, or polyoxyethylated glucose. The polymer can be a homopolymer, random or block copolymer, a terpolymer based on the above monomers, linear or branched, substituted or unsubstituted, so long as it has at least one active sulfone moiety. The polymer moiety can be of any length or molecular weight, although these properties may affect the biological properties. The average molecular weight of polymers that are particularly useful for reducing clearance rates in pharmaceutical applications ranges from 2,000 to 35,000 daltons. Additionally, when two groups are linked to a polymer (one at each end), the length of the polymer can affect the effective distance and other spatial relationships between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or impart the desired biological activity. PEG is useful for biological applications for several reasons. PEG is typically clear, colorless, odorless, water-soluble, stable to heat, inert to many chemicals, non-hydrolyzable, and non-toxic. PEGylation can improve the pharmacokinetic performance of a molecule by increasing the apparent molecular weight of the molecule. An increase in apparent molecular weight reduces the rate of clearance from the body after subcutaneous or systemic administration. In many cases, PEGylation can reduce antigenicity and immunogenicity. Additionally, PEGylation can increase the solubility of biologically active molecules.

[0151] Examples of detectable moieties useful in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include detectable labels such as fluorescent moieties or fluorescent labels, imaging agents, radioisotope moieties, radio-opaque moieties, etc., such as biotin, fluorophores, chromophores, spin resonance probes, or radioactive labels. Exemplary fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, etc.) and other luminescent molecules (e.g., luminal). Fluorophores may be environmentally sensitive, such that their fluorescence changes when they are located near one or more residues in a modified protein that undergo a conformational change upon binding to a substrate (e.g., dansyl probe). Exemplary radioactive labels include atoms with one or more low sensitivity nuclei (e.g., 13 C. 15 N, 2 H, 125 I, 123 I, 99 Tc, 43 K, 52 Fe, 67 Ga, 68 Ga, 111 In). Other useful moieties are known in the art.

[0152] Examples of diagnostic moieties useful in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include detectable moieties suitable for revealing the presence of a disease or disorder. Typically, diagnostic moieties allow for the determination of the presence, absence, or level of a molecule, such as a target peptide, protein, or multiple proteins, associated with a disease or disorder. Such diagnostics are also suitable for predicting and / or diagnosing a disease or disorder and its progression.

[0153] Examples of therapeutic moieties useful in the methods and antibodies and antigen-binding fragments thereof contemplated by the present invention include, for example, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, or anti-infective agents. A functional moiety can also have one or more of the above functions.

[0154] Exemplary therapeutic moieties include radionuclides with high energy ionizing radiation capable of causing multiple strand breaks in nuclear DNA and thus suitable for inducing cell death (e.g., cancer). Exemplary high energy radionuclides include: 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, and 188 Examples of radioisotopes include Re. These isotopes usually produce high-energy alpha or beta particles with short path lengths. Such radionuclides kill cells to which they are in close proximity, e.g., neoplastic cells to which the conjugate is attached or entering. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0155] Exemplary therapeutic moieties also include cytotoxic agents such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or crosslinking agents, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, and the like.

[0156] Exemplary therapeutic moieties also include alkylating agents such as the anthracycline family of drugs (e.g., adriamycin, carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenediones, and aziridines). In another embodiment, the chemotherapeutic moiety is a cytostatic drug, such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Exemplary DNA intercalators or crosslinkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0157] Exemplary therapeutic moieties also include transcriptional regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other exemplary cytostatic drugs compatible with the present invention include ansamycin benzoquinones, quinonoid derivatives (e.g., quinolones, genistein, bactacyclines), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazylquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylene phosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0158] Exemplary therapeutic moieties also include, for example, cytotoxic nucleosides, such as adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; taxoids (e.g., paclitaxel, docetaxel, taxanes), nocodazole, rhizoxin, dolastatins (e.g., dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD612, 6), tubulin-binding agents such as combretastatins (e.g., combretastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); antiangiogenic compounds such as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0159] Exemplary therapeutic moieties also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-alpha, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0160] Exemplary therapeutic moieties also include enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenz-imidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG34, and tyrphostin AG879.

[0161] Exemplary therapeutic moieties also include gene regulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, transretinal (vitamin A aldehyde), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0162] Exemplary therapeutic moieties also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and podophyllotoxins. Particularly useful members of these classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives, such as etoposide or etoposide phosphate, leurocidin, vindesine, leurosine, and the like.

[0163] Still other cytotoxins compatible with the teachings of the present specification include auristatins (e.g., auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0164] Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled See, "Antibody In Cancer Therapy", in 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).

[0165] To extend the half-life of an antibody or polypeptide comprising the amino acid sequence described herein, a salvage receptor binding epitope can be attached to the antibody or its antigen-binding fragment (particularly an antibody fragment), for example as described in U.S. Pat. No. 5,739,277. The term "salvage receptor binding epitope" can refer to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule (e.g., Ghetie et al., 18 Ann.Rev.Immunol.739(2000)). Antibodies with substitutions in their Fc region and increased serum half-life are described in WO00 / 42072, WO02 / 060919; Shields et al., 276 J.Biol.Chem.6591(2001); Hinton, 279 J.Biol.Chem.6213-6216(2004). For example, a nucleic acid molecule encoding a salvage receptor binding epitope can be linked in frame to a nucleic acid encoding a polypeptide sequence described herein, such that the fusion protein expressed from this modified nucleic acid molecule comprises a salvage receptor binding epitope and a polypeptide sequence described herein. In another embodiment, serum half-life can also be increased, for example, by attaching other polypeptide sequences. For example, an antibody or antigen-binding fragment thereof useful in the method of the present invention is attached to serum albumin or a portion of serum albumin that binds to the CEACAM1 receptor or a serum albumin binding peptide, and serum albumin is attached to the antibody or antigen-binding fragment thereof (e.g., such polypeptide sequences are disclosed in WO01 / 45746). In one embodiment, the half-life of the Fab is increased by these methods. For additional serum albumin binding peptide sequences, see also Dennis et al., 277 J.Biol.Chem.35035 (2002).

[0166] 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.

[0167] nucleic acid

[0168] Also provided herein are nucleic acids encoding CEACAM1 antibodies and their antigen-binding fragments, as well as vectors, host cells, and expression systems.As used herein, the term "nucleic acid" refers to any length of polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides.Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0169] Nucleic acids encoding CEACAM1 antibodies and antigen-binding fragments thereof can be, for example, DNA, cDNA, RNA, synthetically produced DNA or RNA, or recombinantly produced chimeric nucleic acid molecules comprising any of these polynucleotides, alone or in combination. For example, expression vectors are provided that comprise a polynucleotide sequence encoding a CEACAM1 antibody or antigen-binding fragment thereof described herein, operably linked to expression control sequences suitable for expression in eukaryotic and / or prokaryotic host cells.

[0170] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. "Vector" includes, but is not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acids. In some embodiments, the vectors used are vectors capable of autonomous replication (episomal vectors) and / or expression of nucleic acids to which they are linked (expression vectors). Many suitable vectors are known to those of skill in the art and are commercially available. Viral vectors include negative-stranded RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses, i.e., AAV), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C, B, D viruses, HTLV-BLV complex, lentiviruses, and spumaviruses.

[0171] A variety of expression vectors have been developed for efficient synthesis of antibodies and their antigen-binding fragments in prokaryotic cells such as bacteria, and in eukaryotic systems, including but not limited to yeast and mammalian cell culture systems. Vectors can contain segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Also provided are cells that contain expression vectors for expressing the contemplated CEACAM1 antibody or its antigen-binding fragment.

[0172] Antibody preparation and expression system

[0173] The antibody or antigen-binding fragment thereof of the present invention is usually produced by recombinant expression. Nucleic acids encoding the light and heavy chain variable regions, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to regulatory sequences in the expression vector(s) to ensure expression of the immunoglobulin polypeptide. Expression regulatory sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression regulatory sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vectors are incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the recovery and purification of the cross-reactive antibodies.

[0174] These expression vectors are usually replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance or neomycin resistance) to allow detection of those cells transformed with the desired DNA sequence (see, e.g., Itakura et al., U.S. Patent No. 4,704,362).

[0175] Expression of antibodies and antigen-binding fragments contemplated by the present invention can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian and mammalian cells, either in vivo or in situ, or host cells of mammalian, insect, avian or yeast origin. Mammalian cells or tissues can be from humans, primates, hamsters, rabbits, rodents, cows, pigs, sheep, horses, goats, dogs or cats, although any other mammalian cells may be used.

[0176] E. coli is one prokaryotic host that is particularly useful for cloning the polynucleotides (e.g., DNA sequences) of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.

[0177] Other microorganisms, such as yeast, are also useful for expression. The genera Saccharomyces and Pichia are exemplary yeast hosts harboring suitable vectors, optionally with expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like. Common promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes involved in methanol, maltose, and galactose utilization, among others.

[0178] Furthermore, in vivo synthesis of ubiquitin transmembrane polypeptide fusion proteins can be achieved, for example, by using the yeast ubiquitin hydrolase system. The fusion proteins thus generated can be processed in vivo or purified and processed in vitro to synthesize the CEACAM1 antibody or antigen-binding fragment thereof of the present invention having a specific amino-terminal sequence. Furthermore, problems associated with retention of the methionine residue from the initiation codon in direct yeast (or bacterial) expression can be avoided. Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).

[0179] The recombinant CEACAM1 antibody or peptide of the present invention can be produced using any of a series of yeast gene expression systems incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be used.

[0180] Production of CEACAM1 antibodies or antigen-binding fragments thereof in insects can be achieved, for example, by infecting an insect host with a baculovirus modified to express a transmembrane polypeptide by methods known to those skilled in the art (see Ausubel et al., 1987, 1993).

[0181] In addition to microorganisms, mammalian tissue culture may be used to express and produce the antibodies or antigen-binding fragments thereof (e.g., polynucleotides encoding immunoglobulins or fragments thereof) of the present invention. See Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). In practice, eukaryotic cells are preferred, since many suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B cells, and hybridomas. Expression vectors for these cells may include expression control sequences such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary information processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0182] Alternatively, a nucleotide sequence encoding the antibody or antigen-binding fragment thereof can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al., U.S. Pat. No. 5,741,957; Rosen, U.S. Pat. No. 5,304,489; and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and / or heavy chains operably linked to a promoter and enhancer derived from a mammary gland-specific gene such as casein or beta-lactoglobulin.

[0183] Furthermore, plants have emerged as a convenient, safe and economical alternative mainstream expression system for recombinant antibody production based on large-scale culture of microorganisms or animal cells. Antibodies or antigen-binding fragments thereof can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be systemic, intracellular plastid-limited, or seed (endosperm)-limited. See, for example, U.S. Patent Publication No. 2003 / 0167531; U.S. Patent Nos. 6,080,560 and 6,512,162; and WO0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, for example, Biolex, NC).

[0184] Vectors carrying the polynucleotide sequences of interest (e.g., sequences encoding heavy and light chains and expression control sequences) can be transferred into host cells by well-known methods that vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection can be used for other cellular hosts (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Press, 2nd ed., 1989)). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally Sambrook et al., supra). To generate transgenic animals, transgenes can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.

[0185] The antibodies and antigen-binding fragments thereof of the invention can be expressed using a single vector or two vectors. If the heavy and light chains of the antibody are cloned into separate expression vectors, the vectors can be co-transfected to obtain expression and assembly of intact immunoglobulins. Once expressed, whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, (1982)). For pharmaceutical uses, substantially pure immunoglobulins of at least about 90-95% homogeneity are preferred, with 98-99% or greater homogeneity being most preferred.

[0186] Methods for modulating CEACAM1 activity

[0187] In one aspect, the invention provides methods of using the antibodies and antigen-binding fragments thereof described herein to reduce the interaction between CEACAM1 and another member of the CEACAM family, including but not limited to CEACAM1, CEACAM5, and CEACAM8. In some embodiments, the antibodies or antigen-binding fragments thereof disrupt homophilic interactions between CEACAM1 monomers.

[0188] In another aspect, the invention provides methods of using the antibodies and antigen-binding fragments thereof of the invention to reduce the interaction between CEACAM1 and members of the TIM family, including but not limited to TIM-1, TIM-3, and TIM-4. In some embodiments, the antibodies or antigen-binding fragments thereof disrupt the heterophilic interaction between CEACAM1 and TIM-3. Disruption of the interaction between CEACAM1 and TIM-3 by using the antibodies and antigen-binding fragments thereof contemplated by the invention may reverse the CEACAM1 inhibitory function while maintaining the TIM-3 activating function.

[0189] In another aspect, the invention provides methods of using the antibodies and antigen-binding fragments thereof of the invention to reduce the interaction between CEACAM1 and PD-1.

[0190] Embodiments of the invention are useful for immunosuppression, e.g., reducing T cell tolerance. By "reducing" is meant the ability to cause an overall decrease of about 20% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75%, 80%, 85%, 90%, 95% or more, compared to untreated controls. Immune suppression can be mediated by immune inhibitory receptors expressed on the surface of immune cells and their interaction with their ligands. For example, cytotoxic CD8 T cells can enter a state of "functional exhaustion" or "unresponsiveness," thereby expressing inhibitory receptors that prevent antigen-specific responses, such as proliferation and cytokine production. Thus, by inhibiting the activity and / or expression of such inhibitory receptors, the immune response against a cancer or tumor that is suppressed, inhibited, or unresponsive can be enhanced or not inhibited. Such enhancement or reversal of inhibition of immune response can result in greater T cell activity, responsiveness, and / or capacity or receptivity for activation.

[0191] Methods for measuring T cell activity are known in the art. As a non-limiting example, T cell tolerance can be induced by contacting T cells with recall antigen, anti-CD3 in the absence of costimulation, and / or ionomycin. For example, the levels of IL-27, LDH-A, RAB10, and / or ZAP70 (both intracellular and secreted) can be monitored to measure, for example, the degree of T cell tolerance (at the levels of IL-2, interferon-γ and TNF, which correlate with increased T cell tolerance). The response of cells pretreated with, for example, ionomycin to antigen can also be measured to determine the degree of T cell tolerance in a cell or cell population, for example, by monitoring the levels of secreted and / or intracellular IL-2 and / or TNF-α (see, for example, Macian et al. Cell 2002 109:719-731). Other characteristics of T cells that have undergone adaptive tolerance include increased levels of Fyn and ZAP-70 / Syk, Cbl-b, GRAIL, Ikaros, CREM (cAMP response element regulator), B-lymphocyte-derived maturation protein-1 (Blimp-1), PD-1, CD5, and SHP2; increased phosphorylation of ZAP-70 / Syk, LAT, PLCyl / 2, ERK, PKC-Θ / ΙΚΒΑ; increased activation of intracellular calcium levels; decreased histone acetylation, i.e., hypoacetylation, and / or increased CpG methylation at the IL-2 locus. Thus, in some embodiments, any one or more of these parameters can be assayed to determine whether an antibody or antigen-binding fragment thereof disclosed herein that inhibits CEACAM1 reduces immune tolerance. Reduction of T cell tolerance can also be assessed by examining tumor-infiltrating lymphocytes or T lymphocytes in lymph nodes draining established tumors. Such T cells are characterized as "exhausted" via, for example, reduced expression of cell surface molecules such as PD-1, TIM-3 or LAG-3, and secretion of cytokines such as interferon-γ.Thus, evidence of decreased T cell tolerance in the presence of a CEACAM1 antibody or antigen-binding fragment thereof includes, for example, an increase in the amount of T cells that have (a) antigen specificity for a tumor-associated antigen (e.g., as determined by a major histocompatibility complex class I or class II tetramer containing a tumor-associated peptide) and (b) the ability to secrete high levels of interferon-γ and cytolytic effector molecules, such as granzyme-B, compared to the ability observed in the absence of an inhibitor.

[0192] CEACAM1 antibodies and antigen-binding fragments thereof are further useful for enhancing T cell proliferation, activation, and proliferation.

[0193] In one aspect, the invention provides methods of using the antibodies and antigen-binding fragments thereof of the invention to reduce the interaction between CEACAM1 and a bacterial adhesin. In some embodiments, the antibodies and antigen-binding fragments thereof of the invention are effective in reducing and / or preventing colonization of mammalian epithelia. In some embodiments, the adhesin is expressed by Escherichia coli, particularly diffuse adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, N. meningitidis, commensal Neisseria, Moraxella catarrhalis, Haemophilus influenza, Haemophilus aegyptius, Helicobacter pylori, Fusobacterium sp., Salmonella sp., and / or Streptococcus agalactiae. 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 thereof disrupts the interaction between CEACAM1 and the opacity-associated (Opa) adhesin protein expressed on the surface of Neisseria species. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1, in embodiments, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between an OMP adhesin expressed on the surface of Haemophilus sp. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1, in embodiments, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and an adhesin expressed on the surface of Streptococcus agalactiae. In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and the Streptococcus agalactiae IgI3-like β protein adhesin.

[0194] In one embodiment, the CEACAM1 antibody or antigen-binding fragment thereof disrupts the interaction between CEACAM1 and C.albicans. In one embodiment, the present invention provides a method of using the CEACAM1 antibody or antigen-binding fragment thereof described herein to inhibit binding of CEACAM1 to a filarial nematode. In one embodiment, the filarial nematode is Wucheria bancrofti.

[0195] Treatment method

[0196] In one aspect, the invention provides CEACAM1 antibodies and antigen-binding fragments thereof, which are also useful for treating a subject in need of such treatment.

[0197] In the methods described herein, a therapeutically effective amount of an antibody or antigen-binding portion thereof described herein is administered to a mammal in need thereof. The antibodies or antigen-binding portions thereof described herein are particularly useful for administration to humans, but may also be administered to other mammals. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, farm animals, and livestock. A "therapeutically effective amount" refers to an amount of an antibody or antigen-binding portion thereof described herein that is effective to produce a desired therapeutic effect when administered to a mammal.

[0198] In some embodiments, the antibody or antigen-binding fragment thereof binds to CEACAM1 expressed by exhausted T cells or natural killer (NK) cells, thereby restoring T cell and NK cell activity and resulting in an increased anti-tumor response. In other embodiments, the antibody or antigen-binding fragment thereof binds to CEACAM1 expressed by tumor cells, thereby inhibiting tumor cell metastasis and the formation of cancer stem cell niches. In yet another embodiment, the antibody or antigen-binding fragment thereof binds to CEACAM1 expressed by macrophages associated with fibrosis in the tumor environment, thereby inhibiting fibrosis. In another embodiment, the antibody or antigen-binding fragment thereof binds to CEACAM1 expressed by other stromal cells in the tumor microenvironment, such as vascular endothelial cells, thereby inhibiting angiogenesis.

[0199] Accordingly, also provided herein are methods of treating a subject having cancer or a tumor and / or reducing tumor growth, comprising administering an effective amount of a CEACAM1 antibody or antigen-binding fragment thereof provided herein. "Reduction" includes inhibition and / or reversal, and may refer, for example, to symptoms of the disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or size of dormant tumors.

[0200] The term "cancer" refers to or describes a physiological condition in mammals that is usually characterized by unregulated cell growth. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Thus, the term "cancer" as used herein refers to unregulated cell growth that interferes with the normal function of bodily organs and systems, including cancer stem cells and tumor vascular niches. A subject with cancer is one in which objectively measurable cancer cells are present in the subject's body. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location and seed vital organs can ultimately lead to the death of the subject through the impaired function of the affected organ. Hematopoietic cancers, such as leukemia, can overpower the normal hematopoietic compartment of a subject, thereby causing hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) and ultimately leading to death.

[0201] By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. Individuals and patients are also subjects herein.

[0202] As used herein, the terms "treat", "treated", "treating" or "treatment" refer to therapeutic treatment, the purpose of which is to slow down (alleviate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of a pathology, disorder or disease; stabilization (i.e., not worsening) of the condition, disorder or disease state; delay in onset or slowing of progression of a pathology, disorder or disease; improvement of the condition, disorder or disease state; and remission (partial or complete), or enhancement or amelioration of a pathology, disorder or disease, whether detectable or undetectable. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes extending survival compared to the expected survival if not treated. The terms "prevent", "preventing" and the like refer to acting before the onset of overt disease or disorder to prevent the onset of a disease or disorder, minimize the extent of a disease or disorder, or delay its developmental process.

[0203] Embodiments of the invention may be used to treat metastasis, which is associated with the spread of cancer from its primary site to other locations in the body. Cancer cells can break away from the primary tumor, infiltrate lymphatic and blood vessels, circulate in the bloodstream, and grow (metastasize) in distant foci in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a series of processes that require tumor cells to divide from the primary tumor, travel through the bloodstream, and arrest at a distant site. At the new site, the cells establish a blood supply and may grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cells regulate this behavior, and interactions between tumor cells and host cells at distant sites are also important. Metastasis is most frequently detected by magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function tests, chest x-rays, and bone scans, alone or in combination, in addition to monitoring for certain symptoms.

[0204] Methods of reducing cancer stemness are also contemplated, including administration of the CEACAM1 antibody or antigen-binding fragment thereof disclosed herein. Cancer stemness may refer to the ability of cells to self-renew and generate additional cell types with distinct phenotypes. Cancer stem cells (CSCs) are cancer cells that exhibit stem cell-like properties. CSCs often exhibit at least one cancer characteristic and can generate at least one additional cell type with distinct phenotypes. Furthermore, cancer stem cells are capable of both asymmetric and symmetric replication. It is understood that cancer stem cells can result from differentiated cancer cells that acquire stem cell traits and / or stem cells that acquire phenotypes associated with cancer cells. Alternatively, cancer stem cells can reconstitute non-stromal cell types within a tumor.

[0205] CEACAM1 is expressed in many tumor types and CEACAM1 may regulate tumor growth and metastatic behavior. In another embodiment, CEACAM1 inhibition reduces tumor growth and metastasis.

[0206] Expression of CEACAM1 in a subset of macrophages is associated with fibrosis during carcinogenesis. In a further embodiment, CEACAM1 inhibition reduces tumor-associated fibrosis.

[0207] Cancers that can be treated by the compositions and methods contemplated by the present invention include tumors that are not vascularized or are not yet substantially vascularized, and vascularized tumors. Cancers can include non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma) or can include solid tumors. Types of cancers to be treated include, but are not limited to, benign and malignant tumors, as well as malignant tumors, such as sarcomas, carcinomas, and melanomas. Also included are adult tumors / cancers and pediatric tumors / cancers. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, etc.), alveolar lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., of the lips, tongue, mouth, and throat); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary system Cancer; vulvar cancer; vulvar sarcoma; and other carcinomas and sarcomas; and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nematoses, edema (such as that associated with brain tumors), and Meigs' syndrome. A patient may have more than one type of cancer.

[0208] The efficacy of the cancer treatment method, including the therapeutic formulation of the composition comprising the antibody and antigen-binding fragment thereof described herein, can be measured by various endpoints commonly used in the evaluation of cancer treatment, including, but not limited to, tumor regression, reduction in tumor weight or size, time to progression, survival time, progression-free survival, overall response rate, duration of response, and quality of life. In the case of cancer, a therapeutically effective amount of the recombinant CEACAM1 antibody or antigen-binding fragment thereof can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., delay to some extent, and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., delay to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. In the case of a patient with multiple types of cancer, a therapeutically effective amount of the recombinant CEACAM1 antibody or antigen-binding fragment thereof is an amount effective to treat at least one cancer. To the extent that the recombinant CEACAM1 antibody or antigen-binding fragment thereof acts to prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer treatment, efficacy in vivo can be measured, for example, by assessing survival, progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life.

[0209] Checkpoint proteins interact with specific ligands that send signals to T cells and turn off or inhibit T cell function. Cancer cells can control the function of T cells entering the tumor microenvironment and suppress anti-cancer immune responses by expressing high levels of checkpoint proteins on their surface. The immune checkpoint protein Programmed Death-1 (PD-1) is an important immune checkpoint receptor expressed by activated T cells and B cells and mediates immune suppression. PD-1 is a member of the CD28 family of receptors that includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1, Programmed Death Ligand-1 (PD-L1) and Programmed Death Ligand-2 (PD-L2), have been identified and are expressed on antigen-presenting cells and many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al., 2000; Latchman et al., 2001). Inhibition of PD-1 / PD-L1 interaction can promote strong antitumor activity. Examples of PD-1 inhibitors include, but are not limited to, pembrolizumab (MK-3475), nivolumab (MDX-1106), cemiplimab-rwlc (REGN2810), pidilizumab (CT-011), spartalizumab (PDR001), tislelizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI754091, JS001, LZM009, MEDI0680, MGA012, Sym021, and TSR-042. Examples of PD-L1 inhibitors include atezolizumab (MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BGB-A333, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, and SHR-1316.

[0210] However, there is a significant population of cancer patients who undergo checkpoint inhibitor therapy and either (1) do not respond to this type of therapy (innate or primary resistance) or (2) respond initially but eventually develop disease progression (secondary or acquired resistance). Resistant cancers are sometimes referred to as refractory cancers. As shown in the example below, tumor-associated cells isolated from patients who have acquired resistance to PD-1 / PD-L1 inhibitors upregulate CEACAM1 expression compared to tumor-associated cells isolated from naive patients not exposed to PD-1 inhibitors. If CEACAM1 is expressed in the setting of acquired resistance, it is likely that CEACAM1-bearing cells are effector memory cells rather than central memory cells, consistent with the reduced anticancer response in resistant patients.

[0211] Thus, also provided herein are methods of using CEACAM1 antibodies and antigen-binding fragments thereof, including but not limited to, the specific CEACAM1 antibodies and antigen-binding fragments thereof provided herein, to treat patients with resistance to checkpoint inhibitors, such as inhibitors of PD-1, PD-L1, and / or CTLA-4. In some embodiments, the CEACAM1 antibody used to treat patients with resistance to inhibitors of PD-1, PD-L1, and / or CTLA-4 is He14 / L1. In some embodiments, the resistance is congenital or primary resistance. In some embodiments, the resistance is secondary or acquired resistance. In some embodiments, the administered CEACAM1 antibody, including but not limited to, the CEACAM1 antibodies and antigen-binding fragments thereof provided herein, reverses T cell exhaustion in patients resistant to checkpoint inhibitor therapy. Any cancer that exhibits resistance to PD-1, PDL-1, and / or CTLA-4 is suitable for treatment by the methods of the invention. In some embodiments, the CEACAM1 antibody or antigen-binding fragment is administered to a patient who has not previously received checkpoint inhibitor therapy.

[0212] In another aspect, the present invention relates to PD-L2, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (members of the CD2 family of molecules, all NK, gamma delta, and memory CD8 + Use of the CEACAM1 antibodies and antigen-binding fragments provided herein in the treatment of patients resistant to treatment with other checkpoint inhibitors, including but not limited to inhibitors of (expressed in αβ)T cells, CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7) is provided.

[0213] In another aspect, the 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 colonization of mammalian epithelia by Candida albicans and / or bacteria expressing bacterial adhesins (including, but not limited to, Escherichia coli, particularly diffuse adherent Escherichia coli (DAEC), Neisseria gonorrhoeae, N. meningitidis, commensal Neisseria, Moraxella catarrhalis, Haemophilus influenza, Haemophilus aegyptius, Helicobacter pylori, Fusobacterium sp., Salmonella sp., and / or Streptococcus agalactiae).

[0214] In another aspect, the invention provides a method of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to treat a subject in need of reducing and / or preventing infection with a filarial nematode, such as Wucheria bancrofti. In another aspect, the invention provides a method of using the CEACAM1 antibodies and antigen-binding fragments thereof disclosed herein to treat a subject in need of reducing and / or preventing the development of lymphedema and / or watervessels associated with infection with a filarial nematode, such as Wucheria bancrofti. In one embodiment, the invention provides a method of using the CEACAM1 antibodies or antigen-binding fragments thereof described herein to reduce invasion of the lymphatic system of a subject by filarial parasites in a subject in need of such reduction. In one embodiment, the filarial nematode is Wucheria bancrofti. The subject may be infected with more than one of bacteria expressing a bacterial adhesin, Candida albicans, influenza virus and / or filarial nematodes.

[0215] In another embodiment, the present invention provides a method of using a CEACAM1 antibody or antigen-binding fragment thereof described herein to reduce invasion of cancer cells into the lymphatic system of a subject in a subject in need of such reduction.

[0216] Screening Methods

[0217] Also provided herein are methods for identifying parent populations likely to respond to treatment with the CEACAM1 antibodies and antibody fragments provided herein, including but not limited to He14 / L1.

[0218] In some embodiments, cancer patients are screened for CEACAM1 expression in specific cell types, including T cells, NK cells, B cells, tumor cells, fibroblasts, or other cells in the tumor microenvironment, such as macrophages. In some embodiments, cancer patients that show increased expression of CEACAM1 in specific cell types compared to controls are selected for treatment with the CEACAM1 antibodies and antibody fragments provided herein. A "control" level of CEACAM1 expression may refer to the level of CEACAM1 expression in one or more individuals without cancer. The levels may be measured on an individual basis or on an aggregate basis, such as an average. In some embodiments, the regulated levels of CEACAM1 expression from the same individual whose condition is being monitored are obtained at different times. In certain embodiments, a "control" level may refer to a level obtained from the same patient at an earlier time, for example, weeks, months, or years ago. In some embodiments, the control level is obtained from the patient before the patient undergoes cancer treatment. In some embodiments, the control level is obtained from the patient before the patient undergoes treatment with a checkpoint inhibitor.

[0219] In some embodiments, CEACAM1 expression is measured in patients resistant to checkpoint inhibitor therapy, including but not limited to, treatment with a PD-1 / PD-L1 / CTLA-4 inhibitor. In some embodiments, patients resistant to checkpoint inhibitor therapy and who show increased expression of CEACAM1 in specific cell types compared to controls are selected for treatment with the CEACAM1 antibodies and antibody fragments provided herein, including but not limited to, He14 / L1.

[0220] In some embodiments, the patient is assayed for allelic variants of human CEACAM1. Based on the allelic variants of human CEACAM1 expressed by the patient, the patient may be administered more or less anti-CEACAM1 antibody compared to patients expressing wild-type variants of CEACAM1. In some embodiments, the patient is assayed for the presence of Y34C, Q44L, and / or Q89H allelic variants of CEACAM1. In some embodiments, patients found to express Y34C, Q44L, A49V, and / or Q89H allelic variants of CEACAM1 are administered higher and / or more frequent doses of anti-CEACAM1 antibody compared to patients expressing wild-type variants of CEACAM1.

[0221] Pharmaceutical Compositions

[0222] In another aspect, the present invention provides a pharma- ceutically acceptable composition comprising a therapeutically effective amount of a CEACAM1 antibody or antigen-binding fragment thereof described herein, formulated with one or more pharma- ceutically acceptable excipients.

[0223] The dose of active agent(s) may vary depending on the reason for use, the individual subject, and the mode of administration. The dose may be adjusted based on the subject's weight, the subject's age and health, and the resistance to the compound(s) or composition. This may require, for example, 0.1, 1.0, 3.0, 6.0, or 10.0 mg / Kg of antibody or antigen-binding fragment thereof depending on the disease. For an IgG with a molecular weight of 150,000 g / mol (two binding sites), these doses correspond to approximately 18 nM, 180 nM, 540 nM, 1.08 μM, and 1.8 μM of binding sites for a 5 L blood volume.

[0224] The active agent and excipient(s) may be formulated into compositions and dosage forms according to methods known in the art. The pharmaceutical compositions of the present invention may be specially formulated in solid or liquid form, including forms adapted for parenteral administration, e.g., as a sterile solution or suspension, e.g., by subcutaneous, intratumoral, intramuscular or intravenous injection.

[0225] Therapeutic compositions comprising an antibody or antigen-binding fragment thereof that binds to CEACAM1 may be formulated with one or more pharma- ceutically acceptable excipients, which may be pharma- ceutically acceptable substances, compositions or vehicles, such as liquid or solid fillers, diluents, carriers, manufacturing aids (e.g., lubricants, talc magnesium stearate, calcium stearate or zinc stearate, or stearic acid), solvents, or encapsulating substances, bulking agents, salts, surfactants and / or preservatives involved in carrying or transporting the therapeutic compound for administration to a subject. Some examples of substances that may act as pharma- ceutically acceptable excipients include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, for example, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; gelatin; talc; waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as ethylene glycol and propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; water; isotonic saline; pH buffers; and other non-toxic compatible substances used in the formulation.

[0226] Bulking agents are compounds that add mass to a formulation and contribute to the physical structure of the formulation in lyophilized form. Suitable bulking agents according to the present invention include mannitol, glycine, polyethylene glycol and sorbitol.

[0227] The use of a surfactant can reduce aggregation of the reconstituted protein and / or reduce the formation of particles in the reconstituted formulation. The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particles after reconstitution. Suitable surfactants according to the present invention include polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, glyceryl ... -, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronic, PF68, etc.).

[0228] Preservatives may be used in the formulations of the present invention. Preservatives suitable for use in the formulations of the present invention include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl-dimethylammonium chlorides, where the alkyl group is a long-chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients are described in standard pharmaceutical texts, such as "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, 19th Ed. Mack Publishing Company, Easton, Pa., (1995).

[0229] The composition comprising the antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier may contain various concentrations of the CEACAM1 antibody or antigen-binding portion thereof described herein. For example, the composition may contain 10 mg / ml to 200 mg / ml, 25 mg / ml to 130 mg / ml, 50 mg / ml to 125 mg / ml, 75 mg / ml to 110 mg / ml, or 80 mg / ml to 100 mg / ml of the antibody or antigen-binding fragment thereof. The composition may also contain about 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, or 150 mg / ml of the antibody or antigen-binding fragment thereof.

[0230] In some embodiments, the composition comprising an antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier is lyophilized and provided in a composition for reconstitution prior to administration.

[0231] Method of administration

[0232] Therapeutic compositions comprising contemplated antibodies or antigen-binding fragments thereof may be administered in any convenient manner, including injection, transfusion, implantation or transplantation. The compositions described herein may be administered to patients by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intracranial, intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0233] In certain embodiments, the antibody or antigen-binding fragment thereof is administered to the mammal by intravenous infusion, i.e., introducing the antibody or antigen-binding fragment thereof into the vein of the mammal for a particular period of time, which in certain embodiments is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.

[0234] In certain embodiments, the compound or composition dose is administered to the subject daily, every other day, every 2 days, every 3 days, once a week, twice a week, three times a week, once every 2 weeks, or once a month. In other embodiments, the compound or composition dose is administered to the subject daily, every 2 days, every 3 days, once a week, once every 2 weeks, or once a month. In some embodiments, the compound or composition dose(s) is administered for 2, 3, 5, 7, 14, 21, or 28 days. In certain embodiments, the compound or composition dose is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months, or more.

[0235] Combination therapy

[0236] In one aspect, the present invention provides a CEACAM1 antibody or antigen-binding fragment thereof for administration with an additional therapeutic agent. Such additional agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, and anti-infective agents. The agents used in such combination therapy may fall into one or more of the above categories. The administration of the antibody or antigen-binding fragment thereof and the additional therapeutic agent may be simultaneous or sequential. The administration of the antibody or antigen-binding fragment thereof and the additional therapeutic agent may be separate or as a mixture. Furthermore, the method of treatment contemplated by the present invention may involve combination treatment with one or more cancer therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, and radiation therapy.

[0237] Exemplary additional therapeutic agents also include radionuclides with high-energy ionizing radiation suitable for inducing multiple strand breaks in nuclear DNA and thus inducing cell death (e.g., in cancer). Exemplary high-energy radionuclides include: 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, and 188 Examples of radioisotopes include Re. These isotopes usually produce high-energy alpha or beta particles with short path lengths. Such radionuclides kill cells to which they are in close proximity, e.g., neoplastic cells to which the conjugate is attached or entering. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0238] Exemplary additional therapeutic agents also include cytotoxic agents such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or cross-linking agents, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, and the like.

[0239] Exemplary additional therapeutic agents also include alkylating agents such as the anthracycline family of drugs (e.g., adriamycin, carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenedione, and aziridine). In another embodiment, the chemotherapy moiety is a cytostatic drug, such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. Exemplary DNA intercalators or crosslinkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0240] Exemplary additional therapeutic agents also include transcriptional regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other exemplary cytostatic agents compatible with the present invention include ansamycin benzoquinones, quinonoid derivatives (e.g., quinolones, genistein, bactacyclines), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazylquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylene phosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0241] Exemplary additional therapeutic agents also include, for example, cytotoxic nucleosides, such as adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; taxoids (e.g., paclitaxel, docetaxel, taxanes), nocodazole, rhizoxin, dolastatins (e.g., dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD61, 26), tubulin-binding agents such as combretastatins (e.g., combretastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); antiangiogenic compounds such as angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0242] Exemplary additional therapeutic agents also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-alpha, rapamycin, sex hormone-binding globulin, and thapsigargin.

[0243] Exemplary additional therapeutic agents also include enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenz-imidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG34, and tyrphostin AG879.

[0244] Exemplary additional therapeutic agents also include gene regulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, transretinal (vitamin A aldehyde), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0245] Exemplary additional therapeutic agents also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and podophyllotoxins. Particularly useful members of these classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives, such as etoposide or etoposide phosphate, leurocidin, vindesine, leurosine, and the like.

[0246] Additional other therapeutic agents compatible with the teachings herein include auristatins (e.g., auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0247] In one embodiment, the CEACAM antibody or antigen-binding fragment thereof is administered in combination with an agent that is a checkpoint inhibitor. Such inhibitors may include small molecule inhibitors, or may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that may be targeted for blockade or inhibition include CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM-3, VISTA, KIR, 2B4 (a member of the CD2 family of molecules that inhibits all NK, gamma delta, and memory CD8 +(expressed on αβ)T cells), CD160 (also called BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM-3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, and Yervoy / ipilimumab (an anti-CTLA-4 checkpoint inhibitor), as well as the PD-1 and PD-L1 inhibitors listed above. Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0248] In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered in combination with a TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226, or CD160 inhibitor.

[0249] In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered in combination with a CTLA-4, PD-1, PD-L1, or PD-L2 inhibitor. In some embodiments, the CEACAM1 antibodies and antigen-binding fragments thereof described herein are administered in combination with a TIM-3 inhibitor.

[0250] It should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention. Furthermore, it should be understood that the disclosure of the present invention herein includes all possible combinations of such specific features. For example, if a specific feature is disclosed in connection with a particular aspect or embodiment of the present invention, or in a particular claim, that feature can also be used in combination with and / or in connection with other specific aspects and embodiments of the present invention, and in the present invention generally, to the extent possible.

[0251] When reference is made herein to a method that includes two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes this possibility), and the method may include one or more other steps that occur before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes these possibilities).

[0252] All other referenced patents and applications are incorporated herein by reference in their entirety. Furthermore, if a definition or use of a term in a reference incorporated herein by reference is inconsistent with or contradicts the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0253] To facilitate a better understanding of the present invention, the following examples of specific embodiments are given, which should not be construed as limiting or defining the entire scope of the invention. EXAMPLES

[0254] Example 1: Generation of a fully humanized CEACAM1 antibody

[0255] 1. Construction of Chimeric Antibodies

[0256] A murine anti-CEACAM1 antibody served as the starting point for humanization. Sequencing of the hybridoma expressing the parent antibody identified one heavy chain sequence and two kappa light chain sequences. To determine which light chain sequence encoded the CEACAM1 binding motif, VH and Vkappa sequences were synthesized with restriction enzyme sites for cloning into the pANT expression vector system for IgG4(S241P) heavy and kappa light chains. The VH region was cloned using the MluI and HindIII restriction sites, and the Vκ region was cloned using the BssHII and BamHI restriction sites.

[0257] 2. Expression and Purification of Chimeric Antibodies

[0258] Combinations of IgG4(S241P)VH and two possible Vκ chains encoding chimeric parent antibodies were transiently transfected into HEK EBNA adherent cells using PEI and incubated for 7 days post-transfection. Combinations of VH and one of the Vκ chains were not expressed, whereas combinations of VH and the second Vκ sequence produced significant levels of antibody. Expressed antibodies were purified from cell culture supernatants on a Protein A Sepharose column, buffer exchanged into PBS pH 7.4, and analyzed by the extinction coefficient (Ec) based on the predicted amino acid sequence. 0.1% = 1.53) and quantified at OD 280 nm.

[0259] 3. Competitive ELISA Analysis of Chimeric Antibodies Binding to Human CEACAM1

[0260] Binding of purified chimeric antibodies to human CEACAM1 was assessed by competitive ELISA assay. Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates were pre-coated with 1 μg / ml human GST-CEACAM1 in 1×PBS overnight at 4° C. The next day, plates were blocked with 2% BSA / PBS for 1 h at room temperature and then washed twice with PBS / 0.05% Tween 20 pH 7.4.

[0261] A 4-fold dilution series of chimeric antibodies or irrelevant control IgG4 antibodies from 50 μg / ml to 0.068 μg / ml was premixed with a fixed concentration of parental mouse antibody (final concentration 0.15 μg / ml), added to the plate and incubated for 1 h at room temperature. After washing three times with PBST, binding of mouse antibodies was detected with anti-mouse-HRP and TMB substrate. The reaction was stopped with 3 M HCl and absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader.

[0262] The expressed chimeric antibody was able to compete with the parental murine antibody for binding to GST-CEACAM1, confirming that the correct light chain had been identified. As expected, irrelevant human IgG4 did not compete. The heavy (VH0) and light (Vκ0) variable chains of the chimeric antibody are provided as SEQ ID NOs: 10 and 18, respectively.

[0263] 4. Design of Composite Human Antibody Variable Region Sequences

[0264] A structural model of the parental murine antibody V-region was created using the Swiss PDB and analyzed to identify key "constrained" amino acids in the V-region that may be essential for the binding properties of the antibody. Most residues contained within the CDRs (using both Kabat and Chothia definitions) as well as several framework residues were considered important. The VH and Vκ sequences of the parental murine antibodies contain common framework residues, and the CDR 1, 2, and 3 motifs are comparable to many murine antibodies. Sequence analysis also revealed potential N-linked glycosylation motifs at both position 27D (Kabat numbering) of the original murine hybridoma light chain CDR1 and position 56 of the heavy chain CDR2.

[0265] A composite human sequence of the parent antibody was created from multiple human antibodies that were combined to create CDRs similar or identical to those of the murine sequences. In the regions outside and adjacent to the CDRs, a broad selection of human sequence segments were identified as possible components of the new humanized V regions.

[0266] 5.CD4 + T cell epitope avoidance

[0267] Based on the structural analysis, a large preliminary set of sequence segments that could be used to create humanized variants was selected and analyzed using iTope™ technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al. 2008) and TCED™ of known antibody sequence-related T cell epitopes (Bryson et al. 2010). Sequence segments that were identified as significant non-human germline binders to human MHC class II or scored significant hits against TCED™ were discarded.

[0268] This analysis yielded a reduced set of segments, and these combinations were analyzed again as above to ensure that the junctions between the segments did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V region sequences that harbored significant T cell epitopes. Five heavy chain (VH1-VH5) and four preferred light chain (Vκ1-Vκ4) sequences were then selected for gene synthesis, expression in mammalian cells, and activity testing.

[0269] 6. Construction of Humanized Variants

[0270] Five humanized VH sequences (VH1-VH5) and four humanized Vκ sequences (Vκ1-Vκ4) were synthesized as described for the chimeric antibodies above and cloned into the pANT expression vector system for IgG4(S241P) heavy chain and kappa light chain. See Table 4. [Table 4]

[0271] 7. Expression and Purification of Humanized Variants

[0272] All combinations of composite IgG4(S241P) VH and Vκ chains (i.e., 20 pairings in total) as well as two control antibodies (chimeric VH (VH0) with variant Vκ1, and variant VH1 with chimeric Vκ (Vκ0)) were transiently transfected into HEK EBNA adherent cells using the PEI transfection method and incubated for 5-7 days post-transfection. Antibodies were purified from cell culture supernatants on a Protein A Sepharose column as described above. Antibodies were analyzed by OD 200 using extinction coefficients based on the predicted amino acid sequence. 280nm 1.5 μg of each antibody was analyzed by SDS-PAGE, and bands corresponding to typical antibody profiles were observed.

[0273] 8. Competitive ELISA Analysis of Humanized Variants Binding to CEACAM1

[0274] Binding of purified antibodies to human CEACAM1 was assessed in a competitive ELISA assay as described above. The ability of each variant to compete with the mouse parental antibody for binding to GST-CEACAM1 was compared to that of the chimeric antibody (VH0 / Vκ0) included on each plate.

[0275] The competitive ELISA data is summarized in Table 5. The binding of variants VH1 / Vκ1, VH1 / Vκ2, VH1 / Vκ3, VH2 / Vκ1, VH2 / Vκ2, and VH2 / Vκ3 to CEACAM1 was within 2-fold of the chimeric antibody, therefore these variants were subjected to Biacore multi-cycle kinetic analysis. [Table 5]

[0276] 9. Kinetic analysis of humanized variants binding to CEACAM1

[0277] As an alternative approach to assess binding of antibody combinations and control antibodies to CEACAM1, kinetic analysis was performed on a Biacore T200 running Biacore T200 Evaluation Software V2.0.1. All experiments were performed at 25°C using HBS-P+ running buffer (pH 7.4).

[0278] All kinetic experiments were performed using CEACAM1-HIS (Sino Biologics Inc.) as the analyte. For all experiments, antibodies were immobilized on the surface of a series S Protein A sensor chip. For kinetic experiments, the amount of ligand immobilized / captured must be limited to avoid mass transfer effects at the chip surface, and the surface should ideally support analyte binding levels (R max ) for the CEACAM1 analyte, 150 kDa for the antibody ligand (estimated for IgG), 50 RU for Rmax, and stoichiometry (S m ) was used as 2 to set a target response level of 100 RU to capture all sample antibodies.

[0279] Single cycle analysis

[0280] To evaluate the binding of all Composite Human Antibody™ variants, single cycle kinetic analysis was performed on the supernatants of all transiently transfected HEK EBNA cells. Purified chimeric antibody and purified chimeric antibody spiked into HEK EBNA medium were used as positive controls. Supernatants were diluted in HBS-P+ to a concentration of 1 μg / mL (measured by IgG quantification ELISA). At the start of each cycle, antibodies were loaded onto Fc2, Fc3, and Fc4 of a Protein A chip and captured at a flow rate of 8 μL / min to an RU of approximately 100. The surface was then allowed to stabilize. Single cycle kinetic data were acquired at a flow rate of 38 μl / min to minimize any potential mass transfer effects. Multiple repetitions of chimeric antibody were performed to check the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. 3. A five-point two-fold dilution range of 125-50 nM CEACAM1 was used with no regeneration between each concentration. For five injections of increasing concentrations of CEACAM1, the association phase was monitored for 150 s and a single dissociation phase was measured for 150 s after the last injection of CEACAM1. Regeneration of Protein A was performed using two injections of 10 mM glycine-HCL pH 1.5 followed by a stabilization period of 500 s.

[0281] In the raw sensorgrams of single cycle kinetics, the signal of the blank run (without CEACAM1) of each antibody was subtracted to correct for differences in surface stability. Single cycle kinetics (Table 6) showed significant differences in kinetic properties between the chimeric antibody and the humanized variants containing VH3, VH4, and VH5, both on and off kinetics. This change in kinetics correlates well with the inability of these variants to effectively compete with the chimeric antibody in competitive ELISA. The antibody variants containing Vκ4 were also unable to bind to CEACAM1, further supporting the results of the competitive ELISA. [Table 6]

[0282] Multi-cycle analysis

[0283] Six lead variants obtained from the competitive ELISA (VH1 / Vκ1, VH1 / Vκ2, VH1 / Vκ3, VH2 / Vκ1, VH2 / Vκ2, and VH2 / Vκ3) were tested by multi-cycle kinetic analysis.

[0284] For multi-cycle kinetic analysis, purified antibodies were immobilized in HBS-P+ at a protein concentration of 1 μg / mL. At the start of each cycle, antibodies were captured on Protein A to an RU of approximately 100 to stabilize the surface. Kinetic data were acquired at a flow rate of 35 μl / min to minimize any potential mass transfer effects. To check the stability of the surface and analyte over the kinetic cycles, multiple repeats of blanks (no CEACAM1) and repeats of a single concentration of analyte were incorporated into the kinetic run. For the kinetic analysis, a two-fold dilution series was chosen from 100 to 1.5625 nM CEACAM1. The binding phase of CEACAM1 was initially monitored for 180 s. In subsequent experiments, a longer binding time (275 s) was used to allow a more complete steady state to be reached. The dissociation phase was measured for 250 s. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle.

[0285] The signal of the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in non-specific binding to the reference surface, and the global Rmax parameter was used for the one-to-one binding model. Relative K compared to the chimeric parent antibody D is the K of the Composite Human Antibody™ variant D was calculated by dividing by that of the chimeric parent antibody on the same chip. The data are summarized in Table 7.

[0286] Composite Human Antibody variants VH1 / Vκ1, VH1 / Vκ2, VH1 / Vκ3, VH2 / Vκ1, VH2 / Vκ2, and VH2 / Vκ3 showed affinities within 2-fold of the chimeric parent antibody. See Table 8. [Table 7] [Table 8]

[0287] Example 2: Removal of N-linked glycosylation sites

[0288] Sequence analysis revealed potential N-linked glycosylation motifs at position N56 of the original murine hybridoma heavy chain and position S27 of the light chain (numbered according to the Kabat numbering system). These sites were mutated to eliminate the N-linked glycosylation sites (i.e., N56Q and S27(f)A). The deglycosylated residues are highlighted in bold in Table 19.

[0289] Example 3: Specificity of isolated Composite Human Antibody variants to CEACAM1

[0290] HeLa cells transfected with vectors expressing CEACAM1, CEACAM5, and CEACAM6, respectively, were used to evaluate the binding selectivity of selected antibody variants by flow cytometry. The resulting cells were used for flow cytometry experiments and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U / ml), and dihydrostreptomycin (100 μg / ml) at 37 °C and 5.0% CO2. Cell lines were stained with the indicated antibodies and then with oral tissue-conjugated monoclonal antibodies specific for the indicated antibody isotypes, such as human IgG4 or mouse IgG1, together with a cell viability dye (DAPI). Data were acquired on a Cytoflex flow cytometer (Invitrogen) and analyzed with FlowJo software (TreeStar, V7.6.5 for Windows).

[0291] The binding specificities of selected CEACAM1 antibodies are shown in Table 9. [Table 9]

[0292] Example 4: Proliferation of human hematopoietic cells induced by antibody treatment

[0293] The selected antibody is CD45 + To assess efficacy in stimulating proliferation of human cells, the HuNSG model was used (Fisher et al. Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity. Cancer Immunol Immunother. 2012 Oct;61(10):1721-33).

[0294] For this, human whole blood was collected from healthy volunteer donors and peripheral blood mononuclear cells (PBMCs) were isolated. Ten million freshly isolated human PBMCs were cultured in a 5%-native guinea pig with NSG (NOD.Cg-Prkdcscid Il2rg tm1Wjl PBMCs were adoptively transferred via intraperitoneal injection into (SzJ) host mice. Fifteen days after PBMC injection, animals were administered a single dose of the indicated humanized antibody (2 mg / kg via intraperitoneal injection). Five days later, PBMCs were stained with an antibody against human CD45 to identify hematopoietic cells and assessed via fluorescence-activated cell sorting (FACS). The results are summarized in Table 10. Lower values ​​indicate greater dilution of the dye inside the cells and less expression of human CD45. + This shows that cell proliferation is enhanced. [Table 10]

[0295] Example 5: Affinity maturation of VH2 N56Q / Vκ4 S27(f)A scFv

[0296] 1. Construction of phage vector and binding test of parental VH2 N56Q / Vκ4 S27(f)A scFv

[0297] The template for affinity maturation was a VH2 N56Q / Vκ4 S27(f)A aglycosylated humanized antibody. The genes encoding the starting antibody VH and Vκ were converted to scFv format using overlap PCR, linking the heavy chain to the light chain via a 15 amino acid (G4S)3 linker (SEQ ID NO:47). The scFv sequence was then cloned into the phagemid vector pANT65 using restriction enzymes NcoI and NotI, allowing display of the scFv on the phage surface as a gene III fusion protein. This plasmid is also capable of expressing a C-terminal Flag tag on the expressed scFv. The cloned scFv were transformed into E. coli (TG1) and all constructs were verified by sequencing.

[0298] M13 helper phages (monovalent) and hyperphages (multivalent) displaying the parental VH2 N56Q / Vκ4 S27(f)A scFv or an irrelevant scFv were prepared and tested for binding to CEACAM1 (Sino Biological). Phages derived from the parental VH2 N56Q / Vκ4 S27(f)A sequence bound specifically to the antigen, whereas no binding was observed with the irrelevant phage. As expected, the parental ScFv hyperphages bound at lower titers than the parental M13 helper phage, resulting in a large number of scFvs displayed on the phage surface.

[0299] 2. Library Design

[0300] The main determinants of antigen binding in antibodies are the CDRs (Wu and Kabat, 1970). Targeting mutations to the VH and Vκ CDR3s has been shown to be one of the most effective means of improving affinity by phage display (e.g., Shier et al., 1996).

[0301] To construct the affinity maturation library, specific amino acids within the VH and Vκ CDR3s of the humanized antibodies were targeted for "hot spot" mutagenesis using semi-randomized codons. Sequence positions were analyzed for potential contact residues (http: / / www.bioinf.org.uk / abs / MeanContacts.html) and ranked in order within each block. This information was used along with the amino acid preferences at any given position within the CDR3s of the heavy and light chains (http: / / www.abysis.org / ). Where possible, priority was given to the highest ranked contact residues within each block.

[0302] The VH CDR3 was identified as 11 amino acids long (Kabat definition) and divided into two libraries overlapping at G99: Block 1 (G95-G99) and Block 2 (G99-A101), each containing a subset of amino acids at every position. Position T93 was included in Block 1 to increase the diversity of germline residues anchoring the CDR. The Vκ CDR3 (Q90 to W96) was covered with a single library containing a subset of amino acids at each position. Kabat numbering is used for all protein sequence coordinates.

[0303] 3. Library Construction

[0304] All oligos were designed and synthesized as previously described (IDT (Integrated DNA Technologies)). Degenerate primers were mixed manually to reduce nucleotide bias during synthesis. An overview of library construction is shown in Figure 1.

[0305] A non-expressing plasmid containing the parental sequence of VH2 N56Q / Vκ4 S27(f)A configured as an scFv was designed to contain a stop codon to prevent the parental scFv from dominating the selection (as is often observed in affinity maturation programs). Using Quikchange mutagenesis (Agilent), each region of the sequence in the VH2 N56Q / VK4 S27(f)A scFv in pANT65 was randomized with the placement of a stop codon, ensuring that only mutated antibody variants in which the stop codon was replaced by a library-encoded amino acid were expressed. By using this approach, the unmutated parental sequence was truncated and therefore not displayed.

[0306] For the two VH CDR3 libraries (Block 1 and Block 2), randomization was performed by performing two PCRs using the stop codon as a template. First, a portion of the VH and Vκ were amplified with a randomized 5' library primer and a 3' primer specific for the Vκ light chain FW4. In a separate PCR, the remainder of the VH was amplified with a 5' primer based on the heavy chain FW1 region and a 3' primer complementary to a portion of the VH CDR randomization primer. A full-length VH CDR randomized scFv library was then constructed by annealing the two amplified fragments and re-amplifying the scFv by PCR with primers that added two restriction sites (either Nco I or Not I) for subcloning of the fragments.

[0307] For the Vκ CDR3 library, randomization of Vκ CDR3 was achieved by performing two PCRs: the first PCR used a randomized 3' primer and a VH FW1 specific 5' primer containing a Nco I restriction site to amplify most of the scFv gene and introduce mutations into Vκ CDR3, the second PCR added the rest of the scFv and added a restriction site (Not I) for subcloning of the fragments.

[0308] Purified and amplified DNA for all three libraries was digested with Nco I and Not I and ligated into a similarly cut phagemid vector (pANT65). The ligated DNA was precipitated, resuspended in nuclease-free water, and transformed into freshly prepared electrocompetent TG1 cells by electroporation. The following day, colonies were counted, plates were scraped, and glycerol stocks were prepared. Libraries were electroporated multiple times to fully cover the theoretical library diversity. The observed library sizes for each of the three libraries are shown in Table 11. In all cases, coverage of 4.95-fold or greater was obtained. Individual colonies from each of the three libraries were sequenced to confirm that the appropriate CDR blocks had been mutated. [Table 11]

[0309] 4. Rescue of Library Phages

[0310] Bacteria from each library were inoculated into 150 ml of 2TYCG (2%) cultures. The inoculum was used to be at least 100 times the observed diversity of the library. Cultures were grown to mid-logarithmic phase (OD 600nm ≈0.4) and the estimated total cell number (OD 600nm 1≒5×10 8 The cells were grown to a multiplicity of 100 (based on cells / ml). Hyperphages were added for 15 min (static) followed by incubation at 250 rpm for 45 min, then centrifuged, resuspended in 2TYCK medium and grown overnight at 30°C. The following day, phages were harvested by harvesting the culture supernatant by centrifugation followed by precipitation using 3 / 10 volume of chilled 20% PEG / 2.5M NaCl. After 1 h incubation on ice, precipitated phages were harvested by centrifugation and the pellet resuspended in 1x PBS pH 7.4. The supernatant was recentrifuged to remove any cell debris, after which the supernatant was reprecipitated as above. Precipitated phages were resuspended in 1x PBS pH 7.4. To increase the chances of obtaining scFvs with increased affinity, polyvalent hyperphage M13 K07ΔpIII helper phage was used at a multiplicity of infection of 20 for library rescue (due to the relatively low affinity of the starting antibody). Following the first round of selection, monovalent M13K07 helper phage (New England BioLabs) was used at a multiplicity of infection of 10, resulting in expected enrichment of antigen binders.

[0311] 5. Selection of phages with improved affinity

[0312] CEACAM1 was used for positive selection throughout. Soluble deselection using CEACAM3, CEACAM5, and CEACAM6 (Sino Biological) was performed at a final concentration of 50 nM each protein to reduce the chance of selecting affinity matured scFvs with CEACAM3, CEACAM5, and CEACAM6 cross-reactivity. This was performed twice during the campaign by deselecting before the second and third rounds. The three libraries were kept separate at all stages throughout the campaign.

[0313] For selection, each library was pre-blocked with 3% BSA / PBS, after which the phages were incubated with decreasing concentrations of biotinylated soluble CEACAM1 antigen for up to 2 hours. After incubation, streptavidin paramagnetic beads (pre-blocked as above) were added to each selection and rotated for 10 minutes. Streptavidin-antigen-phage complexes were washed with increasing numbers of washes with PBST in each successive round of selection, followed by PBS washes, and captured with a magnet between each step. For the VH B2 library, the output titers in the first round were low, so a higher CEACAM1 concentration was used in the second round to increase the number of selected phage binders. Only for the light chain library, the output titers in the third round were relatively large, so a fourth round of selection was performed to further enrich for positive binding phages. Phages were eluted from the beads by addition of 50 mM HCl, after which the solution was neutralized by addition of 1 M Tris-HCl pH 9.0.

[0314] 6. Expression of soluble scFv (periprep)

[0315] Soluble scFvs were first expressed and tested as crude periplasmic extracts. Individual colonies from the selection output were picked into 1 ml of 2TYCG (0.1%) medium and grown by shaking at 37°C for 5 hours. Cultures were induced by adding IPTG to a final concentration of 1 mM and then grown overnight at 30°C with shaking. The next day, cultures were centrifuged and the supernatants discarded. Bacterial pellets were resuspended in TES buffer pH 7.4 and incubated on ice for 30 minutes. Plates were then centrifuged and the supernatants containing scFvs were transferred to new plates for assay.

[0316] 7. High-throughput Competitive Screening of Periprep

[0317] Peripreps of colonies derived from different rounds of selection were screened for their ability to compete with parental RSB02 IgG for binding to CEACAM1 in a single-point competition assay. For comparison, the parental RSB02 scFv and an irrelevant scFv were included on each assay plate.

[0318] Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates were coated overnight with 0.0625 μg / ml CEACAM1. Plates were then washed and blocked with 3% BSA / PBS for 1 h at room temperature. Periplasmic extracts were diluted 1:1 with 0.246 μg / mL VH2 N56Q / Vk4 S27(f)A IgG (parental RSB02 IgG) in 6% BSA / PBS. The diluted periplasmic extracts were then added (100 μL per well) to the blocked plates and samples were incubated on the plates for 1 h at room temperature. Plates were then washed and binding of VH2 N56Q / Vk4 S27(f)A IgG was detected with anti-human-HRP antibody (Sigma, Gillingham, UK) and TMB substrate (Invitrogen). The reaction was stopped with 1M HCl, the absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader, and binding curves were plotted.

[0319] Improved clones were identified based on activity in a competitive ELISA against the parental RSB02 scFv assayed on the same plate. A total of over 7300 peripreps from all three libraries were analyzed. Positive hits were picked onto "cherry" plates, retested in competitive ELISA to confirm results, and sequenced. Dominant single sequence clones were identified in both the VH B1 (approximately 41% of clones sequenced after round 3 were single dominant clones) and VL libraries (approximately 20% of clones sequenced in rounds 3 and 4 were single dominant clones). The VL library appeared to contain scFvs with higher inhibitory potential. As a result, a higher cutoff point for positive hits was used (at least 50% inhibition). See Figure 2.

[0320] To select lead VH and VL to be combined for IgG production, the positive hits (from primary and secondary screening) were ranked to determine those with the highest average percentage inhibition compared to the parent. Five VH B1 clones, three VH B2 clones, and eleven VL clones were selected. Upon sequence analysis, L4 was identified as a mixed VL clone consisting of two different sequences. These were cloned separately to generate clones L4#1 and L4#2, resulting in a total of 12 VL clones being advanced. After reformatting, it was subsequently shown that the light chain L4#2 had the same sequence as L17. He3, originally derived from the VH B1 library, in addition to containing changes in B1, also contained a single mutation in B2. A summary of the 20 lead clones and their CDR sequences is shown in Table 12. [Table 12]

[0321] Example 6: Construction and testing of affinity matured whole antibodies

[0322] 1. Reformatting the Lead scFv into an Antibody

[0323] The 20 variants identified by scFv screening were PCR amplified using primers that introduced flanking restriction enzyme sites for cloning into the IgG4 S241P pANTVhG4 vector and the kappa light chain pANTVK vector. Eight affinity matured VH variants were subcloned into the IgG4 S241P pANTVhG4 vector using the MluI and HindIII restriction sites. Similarly, 12 affinity matured Vkappa sequences were subcloned into the kappa light chain pANTVK vector using the BssHII and BamHI restriction sites.

[0324] 2. Expression of 20 lead reformatted scFvs as whole IgG

[0325] For expression, 8 lead humanized affinity matured IgG4 VH variants and the parental humanized heavy chain (VH2 N56Q) were combined with the parental humanized light chain (Vκ4 S27(f)A) and 12 lead humanized affinity matured kappa light chains to obtain a total of 117 combinations. These combinations were transiently transfected into HEK EBNA adherent cells (LGC Standards) in 12-well plates using the PEI transfection method. Seven days after transfection, supernatants were harvested, quantified by ELISA, and filtered for Biacore single cycle kinetic analysis.

[0326] Example 7: Single-cycle kinetic analysis of humanized and affinity matured lead IgG binding to CEACAM1

[0327] To evaluate the binding of humanized affinity matured IgG, single cycle kinetic analysis was performed on crude supernatant using a Biacore T200 running Biacore T200 Control Software V2.0.1 and Biacore T200 Evaluation Software V3.0 (Uppsala, Sweden). Antibodies were diluted in HBS-P+ (GE Healthcare) to a final concentration of 1.0 μg / ml. At the start of each cycle, antibodies were loaded onto Fc2, Fc3, and Fc4 of a Protein A chip (GE Healthcare). IgG was captured at a flow rate of 10 μl / min to obtain an immobilization level (RL) of approximately 100 RU (a level calculated to give an Rmax of approximately 50-150 RU upon analyte binding). The surface was then stabilized. Single cycle kinetic data were acquired using CEACAM1 as the analyte at a flow rate of 35 μl / min to minimize any potential mass transfer effects. Multiple replicates with the parental (VH2 N56Q / Vκ4 S27(f)A) antibody were performed to check the stability of the surface and analyte over the kinetic cycles. The signal of the reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. A five-point two-fold dilution range of 25 nM to 100 nM CEACAM1 was used without regeneration between each concentration. The signal of a blank run (no CEACAM1) for each antibody was subtracted to correct for differences in surface stability. For three injections of increasing concentrations of CEACAM1, the association phase was monitored for 200 s each time, and a single dissociation phase was measured for 300 s after the last injection of CEACAM1. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5, followed by a stabilization period of 240 s.

[0328] 117 combinations were tested in two experiments. Table 13. Single cycle kinetic constants showed that all affinity matured antibodies bound to CEACAM1. [Table 13-1] [Table 13-2]

[0329] After analysis of single cycle data, 12 variants were selected based on improved affinity and differences in observed kinetic profiles, including faster apparent association and dissociation rates observed for He3 / L20, He17 / L1, and He17 / L16, which included multiple heavy and light chains with different fold improvements to reduce the risk of cross-reactivity with other CEACAM family members (bold in Table 13).

[0330] Example 8: Testing of lead antibodies

[0331] 1. Expression, Purification, and Testing of Lead Antibody

[0332] The 12 lead compound variants, together with the parent IgG (variants are shown in bold in Table 13), were transiently transfected into HEK EBNA adherent cells in triple flasks using the PEI method and incubated for 5-7 days post-transfection. Antibodies were purified from cell culture supernatants on a Protein A Sepharose column (GE Healthcare), buffer exchanged into PBS pH 7.2, and the OD was measured using extinction coefficients based on the predicted amino acid sequence. 280nm 2 μg of each reduced antibody was analyzed by SDS-PAGE, and bands corresponding to the general antibody profile were observed.

[0333] 2. Cross-reactivity ELISA of 12 lead variants binding to CEACAM1 / -3 / -5 / -6

[0334] Binding experiments using parental IgG previously demonstrated specific binding to CEACAM1 with no cross-reactivity to CEACAM3 / 5 and 6. Cross-reactivity of the 12 lead variants and parental IgG with CEACAM3 / 5 and 6 was assessed using a binding ELISA assay. Binding to CEACAM1 in the same assay was used as a positive control. Mouse pan-CEACAM antibody D14HD11 (Abcam) was used as a positive control to confirm the integrity of CEACAM1- / -3 / -5 and -6.

[0335] Nunc Immuno MaxiSorp 96-well flat-bottom microtiter plates (ThermoFisher) were precoated overnight at 4°C with CEACAM1 at 0.5 μg / ml in 1× PBS. The next day, plates were blocked with 2% BSA / PBS for 1 h at room temperature and then washed twice with PBS / 0.05% Tween 20 pH 7.4. Binding curves of parental IgG and D14HD1 were generated to identify appropriate antibody concentrations for evaluation of cross-reactivity. A 3-fold dilution series was performed from 5.0 μg / ml to 0.0069 μg / ml, which were added to the plate and incubated for 1 h at room temperature. After three PBST washes, wells containing parental IgG were incubated with goat anti-human IgG kappa chain-HRP antibody (Merck Millipore). Wells containing D14HD11 were incubated with anti-mouse peroxidase IgG (Sigma Aldrich). Binding was detected using TMB substrate (Invitrogen) and the reaction was stopped with 3M HCl. Absorbance was read at 450 nm on a Dynex Technologies MRX TC II plate reader and used to plot binding curves. Perfect sigmoidal curves were obtained for both the RSB02 parent antibody and D14HD11.

[0336] To test for cross-reactivity, three concentrations were selected for testing, corresponding to the top (1.67 μg / ml), midpoint (0.062 μg / ml), and bottom (0.0069 μg / ml) of the binding curve generated for parental IgG CEACAM1 binding. All 12 variants were tested at 1.67 μg / ml, 0.062 μg / ml, and 0.0069 μg / ml for binding to CEACAM1 / -3 / -5 and -6 (all coated at 0.5 μg / ml in 1×PBS overnight at 4° C.). The absorbance of parental IgG binding to CEACAM1 at 1.67 μg / ml was set to 100% binding. The percentage of CEACAM1 / -3 / -5 and -6 binding of all variants was then calculated relative to the absorbance of CEACAM1 binding of parental IgG at 1.67 μg / ml on the same plate. A plot of the binding data achieved for the 12 variants is shown in FIG.

[0337] As expected, RSB02 parental IgG and all 12 variants tested bound to CEACAM1 at all concentrations tested (Figure 3). As a positive control, D14HD11 was tested and shown to bind to CEACAM1 / -3 / -5 and -6 (Figure 3E).

[0338] This high degree of selectivity can be observed despite the fact that the N-domains of different CEACAMs share a high degree of homology (the N-domains of CEACAM1 and CEACAM3 are 88% identical, the N-domains of CEACAM1 and CEACAM5 are 89% identical, and the N-domains of CEACAM1 and CEACAM6 are 90% identical, as shown by a percent identity matrix generated using Clustal2.1; see Figure 13 of PCT Publication No. WO2020 / 118295).

[0339] 3. Cross-reactivity analysis of 12 leads by single-cycle kinetics

[0340] To further evaluate the cross-reactivity of the humanized affinity matured lead IgGs, single cycle Biacore kinetic analysis was performed using CEACAM3, 5, and 6 with CEACAM1 as a positive control.

[0341] Antibodies were diluted in HBS-P+ to a final concentration of 1.0 μg / ml. Antibodies were loaded onto Fc2, Fc3, and Fc4 of the Protein A chip at the start of each cycle. IgG was captured at a flow rate of 10 μl / min to give an Rmax of 50-150 after CEACAM binding. Multiple replicates with parental (VH2 N56Q / Vκ4 S27(f)A) antibodies were performed to check surface and analyte stability over kinetic cycles. The signal of reference channel Fc1 (no antibody) was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. A four-point two-fold dilution range of CEACAM1 / -3 / -5 / -6 from 25 nM to 200 nM was used with no regeneration between each concentration. The signal of a blank run (no CEACAM) for each antibody was subtracted to correct for differences in surface stability. The association step was monitored for 275 s each for four injections of increasing concentrations of each CEACAM, and a single dissociation step was measured for 250 s after the last injection of each CEACAM. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5, followed by a stabilization period.

[0342] Mouse pan-CEACAM antibody D14HD11 was used as a positive control to measure functionality of CEACAM1 / -3 / -5 / -6. Because capture of D14HD11 was insufficient using Protein A sensor chips, an additional run was performed using an anti-mouse capture kit (GE Healthcare). D14HD11 was diluted to 5 μg / ml in HBS P+ and loaded onto Fc2 of the anti-mouse sensor chip at a flow rate of 10 μl / min to obtain the required immobilization level for each CEACAM. The signal of the reference channel Fc1 (no antibody) was subtracted from the signal of Fc2 to correct for differences in nonspecific binding to the reference surface. A three-point two-fold dilution range of CEACAM1 / -3 / -5 / -6 from 70 nM to 280 nM was used with no regeneration between each concentration. The signal of a blank run (no CEACAM) for each antibody was subtracted to correct for differences in surface stability. The association phase was monitored for 275 s each for three injections of increasing concentrations of CEACAM, and a single dissociation phase was measured for 1800 s (30 min) after the last injection of CEACAM. Regeneration of the anti-mouse sensor surface was performed using one injection of 10 mM glycine-HCL pH 1.7, followed by a stabilization period.

[0343] All variants tested bound to CEACAM1, but no binding to CEACAM3 / 5 / 6 was observed by Biacore, consistent with the binding observed with the parent IgG. D14HD11 showed binding to all CEACAMs tested.

[0344] 4. Multicycle kinetics of 12 lead variants in CEACAM1

[0345] A multi-cycle kinetic analysis was performed on 12 lead antibodies to establish their exact affinity for CEACAM1 using a Biacore T200 instrument running Biacore T200 Evaluation Software V3.0.1 (Uppsala, Sweden). Purified antibodies were diluted in HBS-P+ to a concentration of 1 μg / ml. At the start of each cycle, each antibody was captured on the Protein A surface to a RL of approximately 100 RU. After capture, the surface was allowed to stabilize. Kinetic data were acquired using a flow rate of 35 μl / min to minimize any potential mass transfer effects. CEACAM1 was used in the kinetic analysis. To check for surface and analyte stability over kinetic cycles, multiple repeats of blank (CEACAM1) and repeats of a single concentration of analyte were incorporated into the kinetic run. A two-fold dilution range was selected from 200 to 3.125 nM CEACAM1 for the kinetic analysis. The binding phase of CEACAM1 was monitored for 275 s and the dissociation phase was measured for 250 s. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle.

[0346] The signal of the reference channel Fc1 was subtracted from the signals of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surfaces, and the global Rmax parameter was used for the one-to-one binding model. Relative K D RSB02 parent K D was calculated by dividing by that of the humanized and affinity matured variants on the same chip. The kinetic parameters measured for the interaction of CEACAM1 with the RSB02 affinity matured variants are shown in Table 14. All humanized affinity matured variants showed improved affinity compared to oya (VH2 N56Q / Vκ4 S27(f)A).

[0347] The He3 / L20, He17 / L1, and He17 / L16 variants displayed significantly faster on / off rates compared to the parental IgG. [Table 14-1] [Table 14-2]

[0348] Example 9: Specificity of affinity matured CEACAM1 antibodies measured by flow cytometry

[0349] HeLa cells transfected with vectors expressing CEACAM1, CEACAM3, CEACAM5, CEACAM6, or CEACAM8, respectively, were used to evaluate the binding selectivity of selected antibody variants by flow cytometry. The resulting cells were used for flow cytometry experiments and were cultured at 37°C and 5.0% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U / ml), and dihydrostreptomycin (100 μg / ml). Cell lines were stained with the indicated antibodies and then with oral tissue-conjugated monoclonal antibodies specific for the indicated antibody isotypes, such as human IgG4 or mouse IgG1, together with a cell viability dye (DAPI). Data were acquired on a Cytoflex flow cytometer (Invitrogen) and analyzed with FlowJo software (TreeStar, V7.6.5 for Windows).

[0350] The binding specificities of selected CEACAM1 antibodies are shown in Table 15. [Table 15]

[0351] Example 10: Specificity of affinity matured CEACAM1 antibody He14 / L1 by surface plasmon resonance (SPR)

[0352] The binding specificity of antibody He14 / L1 to hCEACAM1, hCEACAM3, hCEACAM5, and hCEACAM6 was demonstrated by single cycle surface plasmon resonance (SPR).

[0353] Single cycle kinetic analysis was performed to evaluate the binding of He14 / L1 to hCEACAM1, hCEACAM3, hCEACAM5, and hCEACAM6. Kinetic experiments were performed at 25°C on a Biacore T200 running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (Cytiva, Uppsala, Sweden). HBS-P+ (Cytiva, Uppsala, Sweden) was used for the running buffer as well as for diluting the ligand and analyte. At the start of each cycle, antibodies were loaded onto a Protein A sensor chip (Cytiva, Uppsala, Sweden). IgG was captured at a flow rate of 10 μl / min to a predefined immobilization level taking into account the different molecular weights of the analytes (100 RU for CEACAM1, 375 RU for hCEACAM3, 71 RU for hCEACAM5, and 150 RU for hCEACAM6). The surface was then allowed to stabilize. A three-point two-fold dilution range from 70 to 280 nM was used for antigen in running buffer with no regeneration between each concentration. For each of three injections of increasing concentrations of antigen, the association step was monitored for 80 s and a single dissociation step was measured for 150 s after the last injection of antigen. A similar method was used for the anti-pan-hCEACAM control antibody, mouse D14HD11, except in this case a CM5 chip immobilized with anti-mouse capture reagent (Cytiva, Uppsala, Sweden) was used, and association was monitored for 275 s and dissociation was monitored for 1800 s. Data were double-referenced and subtracted.

[0354] As shown in FIG. 4, the antibody He14 / L1 binds to hCEACAM1 with high specificity.

[0355] Example 11: Crystal structure of CEACAM antibody and CEACAM1

[0356] To precisely map the binding interface between CEACAM1 and the humanized affinity matured aglycosylated antibody He14 / L1, the crystal structure of human CEACAM1 in complex with the He14 / L1 Fab fragment was determined at atomic resolution (Table 16). The co-crystal structure has been deposited in the RCSB Protein Data Bank using the PDB code 7N3W. [Table 16]

[0357] Tagless CEACAM1 was expressed in E. coli, refolded in arginine-containing buffer, and purified. He14 / L1 Fab was prepared by digesting the provided antibody with immobilized papain resin and then purified on a 1 ml HiTrap MabSelect SuRe column (GE Healthcare) followed by 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:He14 / L1 Fab complex were identified and subsequently optimized. Diffraction-quality crystals were grown at room temperature under conditions containing 20% ​​PEG8000 and 0.1M HEPES pH 7.5. SDS-PAGE analysis and silver staining of washed crystals revealed all the predicted components of the complex.

[0358] The structure of the CEACAM1:VH14 / VL1 Fab complex was determined at 1.85 Å resolution. The complex was crystallized in the prototypical monoclinic space group P21, and two copies of the complex were found in the asymmetric unit. In the structural coordinate file, chains A and L correspond to the Fab light chain, chains B and H correspond to the Fab heavy chain, and chains C and D correspond to a molecule of CEACAM1. The structure of the complex (chains C, L, and H) is shown in Figure 5A.

[0359] GFCC' plane bond

[0360] He14 / L1Fab binds to the CEACAM1 symmetric mate in the asymmetric unit of the crystal through the GFCC' interface with a 1:1 stoichiometry (Figure 5D). At this interface, the GFCC' residues of CEACAM1, especially F29, Q44, T56, Q89, and N97, contribute to tight binding with the CDR residues of both the heavy and light chains (Table 17), and the CC' and FG loops of CEACAM1 show strong involvement in the complex formation. These GFCC' face residues are also important for the homodimerization of human CEACAM1, as well as for the interaction with various ligands such as TIM-3 and HopQ. Superimposing CEACAM1 in the Fab complex with the protomer of CEACAM1 in the dimer yielded a root mean square deviation of 0.453A across the 81 aligned Cα positions. The GFCC' binding interface has a higher shape complementarity score of 0.74, which is the preferred binding surface.

[0361] ABED surface bonding

[0362] He14 / L1 Fab binds with a 1:1 stoichiometry to CEACAM1 present in the crystal asymmetric unit (Table 18). Upon complex formation, the Fab appears to perturb the structure of CEACAM1. In particular, there are significant structural differences in the β-strands C' and C'', which are involved in self-association, as well as the C'-C'' loop. In the complex, there is a reduction in intramolecular hydrogen bond interactions within this perturbed region, which results in a significant shortening of the C'' strand and a change in the orientation of C', as the residues that appeared to form the C'' strand in the dimer became part of an extended loop (Figure 5B). Superimposition of the CEACAM1 in the Fab complex and the protomers of CEACAM1 in the dimer gave a mean square deviation of 1.32 A over the 81 aligned Cα positions. As shown, the perturbed loops appear to hinder dimerization and the binding surface is modified by Fab binding.

[0363] The epitopes on CEACAM1 are shown in the diagram of the molecular surface of CEACAM1 (Figure 5C). In the He14 / L1 Fab light chain, CDR1 interacts primarily with CEACAM1 residues in the C' strand and the C'-C" loop, and CDR3 interacts primarily with residues in the D and E strands and the DE loop. In the Fab heavy chain, CDR1 and CDR2 interact with chains A and B and the connecting interconnecting loop. In addition, there may be a weak electrostatic interaction between the side chain of heavy chain Lys65 and CEACAM1 Glu5 (located 4.5A away and not depicted). The shape complementarity of the interaction surface is 0.58. Light chain CDR2 and heavy chain CDR3 do not interact with CEACAM1.

[0364] The crystal structure of the human CEACAM1 homodimer (PDB 4QXW) highlights the importance of the GFCC' face in homodimer formation. Furthermore, the crystal structure of the ABED face (PDB 2GK2) supports the role of the ABED face as a secondary minor interface in CEACAM1 oligomerization. Both the GFCC' and ABED faces bind within the same crystal lattice in the He14 / L1 CEACAM1 complex crystal structure, indicating that He14 / L1 exhibits a unique dual binding mode with human CEACAM1 through the GFCC' and ABED faces (Figure 5E).

[0365] [Table 17] [Table 18]

[0366] Example 12: CEACAM1 antibodies block CEACAM1:TIM-3 interaction

[0367] The ability of selected CEACAM1 antibodies to reduce the binding of CEACAM1 to TIM-3 was examined. CEACAM1 / TIM-3 competitive ELISA assays were performed in triplicate to measure the ability of selected antibodies (concentration range 0-250 nM) to inhibit the binding of human TIM-3 IgV domain tagless protein and human CEACAM1-GST protein. Plates were coated with 3 μg / ml hTIM-3 tagless protein by overnight incubation, after which wells were washed and resuspended in Tris-buffered saline buffer containing 10 mM CaCl2 (TBS-Ca 2+ Blocking was performed with 2% BSA in 100 mM NaCl buffer (pH 7.5). Blocking of hTIM-3 binding to hCEACAM1-GST protein was performed in the presence of antibodies using 4 μM hCEACAM1-GST tagged protein at concentrations ranging from 0 to 250 nM. Human IgG4 antibody was used as a control (0 to 250 nM). Goat polyclonal anti-GST-HSP antibody from Abcam (1:2000) was used to detect human CEACAM1-GST protein and the assay was developed by adding TMB solution (Life technologies). OD values ​​at 450 nm were read on a plate reader. Data were plotted in Graphpad Prism and best-fit IC-50 values ​​were determined. As shown in Figure 6, the indicated CEACAM antibodies blocked the CEACAM1:TIM-3 heterophilic interaction.

[0368] Example 13: CEACAM1 antibodies reverse T cell function in tumor dissociated cells derived from patients resistant to checkpoint inhibitor therapy

[0369] 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, peripheral blood mononuclear cells (PBMCs) were isolated from a melanoma patient (CY123.1) with secondary resistance to pembrolizumab (PD-1 inhibitor). The PBMCs were cultured in triplicate at 0.5 × 10 cells / mL in 200 ml of complete medium (RPMI1640, Lonza) supplemented with 10% fetal calf serum (FCS), 1% glutamine, 100 IU / ml penicillin, 100 μg / ml streptomycin (Life Technologies), and 25 mM HEPES (Sigma-Aldrich) in 96-well U-bottom plates under T cell stimulation conditions. 5 Cells were resuspended and cultured at a concentration of 100 IU / ml recombinant IL-2 (NIH) and soluble CD3 (UCHT1, Abcam) at 1 mg / ml in the presence of various concentrations (0.3-30 nM) of soluble chimeric (parental) antibodies, or humanized affinity matured heavy chain (He3, He17, He14) and / or light chain ((L20, L1)) variant antibodies, or hIgG4 isotype control antibodies. After 96 h of culture, CD3 + CD8 + Cell proliferation was measured by assessing intracellular Ki67 expression in the T cell fraction in triplicates.

[0370] As shown in Figure 7, CEACAM1 antibodies reverse T cell exhaustion in PD-1 resistant tumors.

[0371] Example 14: CEACAM1 antibodies induce B and T cell induction in CD45-positive cells derived from metastatic melanoma

[0372] We investigated the effect of the CEACAM1 antibody He14 / L1 on CD45-positive cells derived from metastatic melanoma.

[0373] Equal numbers of sorted CD45 positive cells from three metastatic melanomas (treatment naive, n=1; treatment resistant, n=2) were treated in vitro with He14 / L1 (5mg / ml) or human IgG4 isotype control (5mg / ml) in culture medium at 37°C for 42 hours and single-cell sequencing was performed. Briefly, cells were processed using Chromium Controller and Chromium Single Cell 5'Library & Gel Bead kits from 10X Genomics according to the manufacturer's protocol. Samples were sequenced on an Illumina NovaSeq S2-100 instrument. Demultiplexing, barcoding, single-cell 5' unique molecular identifier (UMI), transcript alignment, and counting were performed using the Cell Ranger pipeline. Data were subsequently imported into R by the Read10x() command function in Seurat for analysis. The number of cells and the number of UMIs / transcripts per cell (nUMIs) in the merged datasets were plotted, evaluated, and quality control metrics were applied. Datasets were merged, canonical correlation analysis was used to establish similar cells between groups, and cells were defined by applying principal component analysis scores that visualized metaclusters in a 2D UMAP. Cluster identification was based on signature genes. This identified B cells in the metacluster (M2) and T cells in four metaclusters (M0, M20, M23, M25) in the isotype (ISO) and He14 / L1 samples.

[0374] As shown in Figure 8, treatment with antibody He14 / L1 resulted in increased proliferation of B cells (164 to 347 cells) and T cells (394 to 913 cells) within 42 hours compared to control antibody, indicating that He14 / L1 induces a broad range of B and T cell responses that are important for antitumor activity.

[0375] Example 15: CEACAM1 expression in cells from patients resistant to therapy

[0376] We investigated the expression of CEACAM1 in tumor-associated B cells, tumor-associated monocytic cells, and T cells isolated from treatment-resistant and naive patients.

[0377] Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD, n=5), treatment-naive melanoma patients (PBMC-N, n=5), and treatment-resistant melanoma patients (PBMC-R, n=5), or mechanically dissociated metastatic tumors from treatment-naive melanomas (Tumor-N, n=9) or treatment-resistant melanomas (Tumor-R, n=16) were stained with anti-human CEACAM1 monoclonal antibody labeled with Tb159 (parent antibody for He14 / L1 generation) or a panel of heavy metal-labeled antibodies for immune profiling (Maxpar® Direct™ Immune Profiling Assay™). Stained cells were analyzed on a CyTOF2 mass cytometer. Sample acquisition for comprehensive multiplexed single-cell phenotyping, file processing using R, and mass cytometry data analysis using Cytobank were performed by standard methods. Cluster identification, characterization, and regression (Citrus) analysis was performed to establish associations between markers and clinical phenotypes. Cells matched to B-cell, monocytic, and CD8 T-cell fractions based on markers defined by the manufacturer (Fludigm) were analyzed for median levels of human CEACAM1 expression detected by Tb159-labeled parental monoclonal antibody in treatment-naive and treatment-resistant samples associated with metaclusters identified by Citrus analysis. Statistical analysis was performed by Kruskal-Wallis test followed by Dunn's multiple comparison test. *p 0.05, **p < 0.01.

[0378] As shown in Figure 9, tumor-associated B cells, monocytic cells, and CD8 + Expression of CEACAM1 in metaclusters representing T cells is increased in treatment-resistant tumor samples compared to cells isolated from treatment-naive tumor samples. [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4] [Table 19-5] [Table 19-6]

[0379] While the foregoing written description of the invention enables one of ordinary skill in the art to make and use what is currently believed to be the best mode thereof, those of ordinary skill in the art will understand and recognize the existence of variations, permutations, and equivalents of the specific embodiments, methods, and examples herein.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; each of the heavy chain variable region and the light chain variable region comprises CDR1, CDR2, and CDR3; a) the sequence of CDR1H comprises the sequence DYYLY (SEQ ID NO:1), b) the sequence of CDR2H is 【Chemistry 1】 Including, c) the sequence of CDR3H comprises the sequence GLYYGPSWVAY (SEQ ID NO: 3), ARTYGPAWFAY (SEQ ID NO: 4), or ALTYGPAWLAY (SEQ ID NO: 5); d) the sequence of CDR1L is 【Chemistry 2】 Including, e) the sequence of CDR2L comprises the sequence FASTRES (SEQ ID NO:7); f) The antibody or antigen-binding fragment thereof, wherein the sequence of CDR3L comprises the sequence QSHYPFYYT (SEQ ID NO: 8) or QSHFPYPLT (SEQ ID NO: 9).

2. a) the sequence of the CDR3H comprises the sequence GLYYGPSWVAY (SEQ ID NO: 3); b) The antibody or antigen-binding fragment thereof according to claim 1, wherein the sequence of CDR3L comprises the sequence QSHYPFYYT (sequence number 8).

3. An antibody or antigen-binding fragment thereof that binds to CEACAM1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; a) the sequence of the heavy chain variable region comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-17; b) The antibody or antigen-binding fragment thereof, wherein the sequence of the light chain variable region comprises a sequence that is at least 90% identical to SEQ ID NO:21 or SEQ ID NO:

22.

4. a) the sequence of the heavy chain variable region comprises a sequence that is at least 90% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 15; b) The antibody or antigen-binding fragment thereof of claim 3, wherein the sequence of the light chain variable region comprises a sequence that is at least 90% identical to the light chain variable region amino acid sequence of any one of SEQ ID NOs:

21.

5. a) the sequence of the heavy chain variable region comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-17; b) The antibody or antigen-binding fragment thereof of claim 3, wherein the sequence of the light chain variable region comprises a sequence that is at least 95% identical to SEQ ID NO:21 or SEQ ID NO:

22.

6. a) the sequence of the heavy chain variable region comprises a sequence that is at least 95% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 15; b) The antibody or antigen-binding fragment thereof of claim 5, wherein the sequence of the light chain variable region comprises a sequence that is at least 95% identical to the light chain variable region amino acid sequence of any one of SEQ ID NOs:

21.

7. a) the sequence of the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 15-17; b) The antibody or antigen-binding fragment thereof of claim 3, wherein the sequence of the light chain variable region comprises SEQ ID NO:21 or SEQ ID NO:

22.

8. a) the sequence of the heavy chain variable region comprises SEQ ID NO: 15; b) The antibody or antigen-binding fragment thereof of claim 7, wherein the sequence of the light chain variable region comprises SEQ ID NO:

21.

9. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment is a chimeric antibody, a CDR-grafted antibody, or a humanized antibody or antigen-binding fragment thereof.

10. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment is a multispecific or bispecific antibody or antigen-binding fragment thereof.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the antibody or antigen-binding fragment is a bispecific antibody that contains complementary regions that bind to PD-1, PD-L1, CTLA-4, TIM-3, epidermal growth factor receptor (EGFR), CD25, CD19, or an Fcγ receptor.

12. The antibody or antigen-binding fragment is selected from the group consisting of scFv, Fv, Fab', Fab, F(ab') ２ 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, which is a mAb, a mAb, or a diabody.

13. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment has the isotype IgG4.

14. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a S241P substitution in the constant region of the heavy chain.

15. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment is deglycosylated.

16. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment is missing the C-terminal lysine of the heavy chain.

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

18. 10. An antibody or antigen-binding fragment thereof that binds to the same epitope on CEACAM1 as the antibody or antigen-binding fragment thereof according to any one of the preceding claims.

19. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment binds to the IgV-like N domain of CEACAM1.

20. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment does not bind to one or more of CEACAM3, CEACAM5, CEACAM6, and CEACAM8.

21. the antibody or antigen-binding fragment thereof a) binds at least partially to the binding site on CEACAM1 for TIM-3, and / or b) An antibody or antigen-binding fragment thereof according to any one of the preceding claims, which at least partially binds to the binding site on CEACAM1 for PD-1.

22. 2. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment at least partially binds to a binding site on CEACAM1 for CEACAM1 during homodimerization.

23. 10. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of the preceding claims.

24. A vector comprising the nucleic acid of claim 23.

25. 25. An isolated cell comprising the vector of claim 24.

26. A cell expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 22.

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

28. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 22 and a pharma- ceutically acceptable excipient.

29. A method for inhibiting the binding of CEACAM1 to a member of the CEACAM family, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

30. 30. The method of claim 29, wherein the member of the CEACAM family is selected from the group consisting of CEACAM1, CEACAM5, and CEACAM8.

31. 30. The method of claim 29, wherein the member of the CEACAM family is CEACAM1.

32. A method for inhibiting the binding of CEACAM1 to a member of the TIM family, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

33. The method of claim 32, wherein the member of the TIM family is TIM-3.

34. A method for inhibiting the binding of CEACAM1 to PD-1, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

35. A method for inhibiting the binding of CEACAM1 to a bacterial adhesin, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

36. 36. The method of claim 35, wherein the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza outer membrane protein (OMP) P1, Haemophilus aegyptius OMP P1, Moraxella sp. Opa-like protein (OlpA), Fusobacterium sp. trimeric autotransporter adhesin CbpF, Salmonella sp. adhesin, or Streptococcus agalactiae adhesin.

37. A method for inhibiting the binding of CEACAM1 to Candida albicans, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

38. A method for reducing colonization of mammalian epithelium by bacteria expressing a bacterial adhesin, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

39. 39. The method of claim 38, wherein the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza (OMP) P1, Haemophilus aegyptius OMP P1, or Moraxella sp. OlpA, Fusobacterium sp. trimeric autotransporter adhesin CbpF, Salmonella sp. adhesin, or Streptococcus agalactiae adhesin.

40. A method for reducing colonization of mammalian epithelium by Candida albicans, the method comprising contacting CEACAM1 with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

41. A method for reducing T cell tolerance, the method comprising contacting a cell population comprising T cells with the antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or the pharmaceutical composition of claim 28.

42. A method for enhancing T cell proliferation, the method comprising contacting a cell population comprising T cells with the antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or the pharmaceutical composition of claim 28.

43. A method for reducing T cell tolerance in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, or a pharmaceutical composition according to claim 28.

44. A method for enhancing T cell proliferation in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, or a pharmaceutical composition according to claim 28.

45. A method for enhancing B cell proliferation, the method comprising contacting a cell population comprising T cells with the antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or the pharmaceutical composition of claim 28.

46. A method for enhancing B cell proliferation in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, or a pharmaceutical composition according to claim 28.

47. A method for enhancing monocyte proliferation, the method comprising contacting a cell population comprising T cells with the antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or the pharmaceutical composition of claim 28.

48. A method for enhancing monocyte proliferation in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

49. 29. A method of treating cancer in a subject in need of such treatment, the method comprising administering to the subject the antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or the pharmaceutical composition of claim 28.

50. 50. The method of claim 49, wherein the cancer is melanoma, pancreatic cancer, thyroid cancer, lung cancer, colorectal cancer, squamous cell carcinoma, prostate cancer, breast cancer, bladder cancer, or gastric cancer.

51. A method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, or a pharmaceutical composition according to claim 28.

52. A method for reducing tumor metastasis in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

53. A method for reducing tumor-associated fibrosis in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

54. A method for reducing cancer stemness in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

55. A method for reducing colonization of a subject's epithelium by bacteria expressing a bacterial adhesin in a subject in need of such reduction, the method comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

56. 56. The method of claim 55, wherein the bacterial adhesin is Helicobacter pylori adhesin HopQ, Neisseria gonorrhoeae opacity protein (Opa), Neisseria meningitidis Opa, Haemophilus influenza OMP P1, Haemophilus aegyptius OMP P1, Moraxella sp. OlpA, Fusobacterium sp. trimeric autotransporter adhesin CbpF, Salmonella sp. adhesin, or Streptococcus agalactiae adhesin.

57. A method for reducing colonization of a subject's epithelium by Candida albicans in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

58. A method for reducing colonization of a subject's epithelium by filarial parasites in a subject in need of such reduction, the method comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

59. 59. The method of claim 58, wherein the filarial parasite is Wucheria bancroftii.

60. A method for reducing invasion of the lymphatic system of a subject by cancer cells in a subject in need of such reduction, the method comprising administering to the subject an antibody or antigen-binding fragment thereof described in any one of claims 1 to 22, or a pharmaceutical composition described in claim 28.

61. 55. The method of any of claims 43-54, further comprising administering a checkpoint inhibitor.

62. 62. The method of claim 61, wherein the checkpoint inhibitor is a CTLA-4, PD-1, PD-L1, and PD-L2 inhibitor.

63. 55. The method of any of claims 43-54, further comprising administering one or more of an inhibitor of LAG3, TIGIT, LAP, podoplanin, protein C receptor, ICOS, GITR, CD226, and / or CD160.

64. 55. The method of any of claims 43-54, further comprising administering a TIM-3 inhibitor.

65. The method of any of claims 61-64, wherein the additional inhibitor is administered simultaneously or sequentially with the antibody or antigen-binding fragment.

66. The method of any of claims 61 to 64, wherein the additional inhibitor is administered separately or in a mixture with the antibody or antigen-binding fragment.

67. 29. A method of treating a subject in need of treatment, said method comprising administering to said subject an antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, or a pharmaceutical composition according to claim 28, wherein said subject has acquired resistance to treatment with checkpoint inhibitor therapy.

68. 68. The method of claim 67, wherein the subject has acquired resistance to therapy with one or more of a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

Images