CEACAM5 binding proteins and uses thereof

Abstract

The invention relates to the field of disease treatment, in particular to an anti-CEACAM5 antibody or an antigen binding fragment thereof, nucleic acid molecules encoding the anti-CEACAM5 antibody or the antigen binding fragment, and a method for preparing the anti-CEACAM5 antibody or the antigen binding fragment. The anti-CEACAM5 antibody or the antigen binding fragment of the anti-CEACAM5 antibody has high affinity binding and endocytosis activity on CEACAM5, and meanwhile, the anti-CEACAM5 antibody or the antigen binding fragment of the anti-CEACAM5 antibody has very good specificity. Thus, the invention further relates to the use of said antibodies or antigen-binding fragments thereof in the treatment and diagnosis of disease.

Description

Technical Field

[0001] This invention belongs to the field of therapeutic monoclonal antibodies, and more specifically, this invention relates to an antibody against CEACAM5; it also relates to the use of said antibody in the treatment and diagnosis of diseases. Technical Background

[0002] CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5), also known as CD66e, belongs to the large CEACAM subfamily of the immunoglobulin superfamily. It is a cell adhesion molecule, and its high expression may influence tumorigenesis and development. The CEACAM subfamily includes 12 glycoprotein members (CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and CEACAM21). CEACAM5 is primarily expressed on the cell membrane via GPI-anchored expression and can be hydrolyzed by GPI-PLD (glycosylphosphatidylinositol phospholipase D), detaching to form free CEACAM5 in the bloodstream. The CEACAM5 glycoprotein contains an Ig-like V-type (IgV) domain at its N-terminus, followed by six Ig-like C2-type (IgC2) domains and a GPI anchor. Its primary function in embryonic intestinal and colon tumors is to adhere to epithelial cells. Furthermore, it plays a crucial role in inhibiting colon cell differentiation and apoptosis; CEACAM5 can prevent tumor cells from undergoing anodic apoptosis.

[0003] CEACAM 5 expression is limited in normal adult tissues. In normal tissues, CEACAM 5 expression can be detected in the kidney, bladder, larynx, epiglottis, skin, submandibular gland, colon, esophagus, duodenum, parotid gland, and sublingual gland tissues. Conversely, it is not detected in the stomach, pancreas, and many other normal tissues. CEACAM 5 primarily functions as a cell adhesion molecule in normal tissues, mediating both homogeneous and heterogeneous adhesion. CEACAM 5 is expressed in gastric cancer, colonic adenocarcinoma, rectal adenocarcinoma, lung squamous cell carcinoma, bladder epithelial carcinoma, breast ductal carcinoma, ovarian endometriosis, prostate transitional cell carcinoma, pancreatic adenocarcinoma, and cervical squamous cell carcinoma, and has been found to be distributed throughout the cell surface and cytoplasm of cancer cells. CEA is a well-known biomarker for many types of malignant tumors, such as colorectal cancer and non-small cell lung cancer. High CEACAM5 expression has also been found in approximately 25% of patients with advanced non-squamous (NSq) non-small cell lung cancer (NSCLC), detectable by IHC (immunohistochemistry). Besides the increased CEACAM5 observed in tumor cells, studies have also shown changes in plasma free CEACAM5 levels; literature indicates that 64.7% (101 / 156) of CRC patients had sCEA levels higher than the normal range. Currently, clinical-stage ADCs targeting CEACAM5 include antibodies such as SAR408701. In previously published clinical lung cancer treatment studies, SAR408701 showed good efficacy against NSCLC, with an ORR between 20% and 30%.

[0004] Currently, there are no CEACAM5-targeting antibody drugs on the market. Therefore, it is urgent and necessary to develop antibodies targeting CEACAM5 with higher specificity, lower toxicity and side effects, better clinical efficacy, and more convenient administration methods, which will provide patients with more medication options. Summary of the Invention

[0005] In this application, the inventors have developed murine and humanized antibodies targeting CEACAM5 with excellent properties. These antibodies specifically recognize / bind to CEACAM5 and can be used to treat cancers expressing CEACAM5. Specifically, the antibodies of this invention can bind to membrane-bound CEACAM5 with high affinity while avoiding binding to soluble native CEACAM5 protein in plasma, thereby potentially preventing interference with antibody function from plasma proteins.

[0006] The antibody of the present invention

[0007] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises complementarity-determining regions (CDRs) as follows:

[0008] (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:3 or 1; and / or, CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:4 or 2; or

[0009] (b) The heavy chain variable region (VH) containing CDR-H1, CDR-H2 and CDR-H3, and / or the light chain variable region (VL) containing CDR-L1, CDR-L2 and CDR-L3, wherein, compared with the heavy chain variable region (VH) and / or the light chain variable region (VL) described in (a), at least one CDR contains a mutation, said mutation being a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, said substitution is a conservative substitution.

[0010] In some embodiments, the antibody or its antigen-binding fragment includes complementarity-determining regions (CDRs) as follows:

[0011] (i) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:3; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:4;

[0012] (ii) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:1; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:2;

[0013] (iii) The heavy chain variable region (VH) containing CDR-H1, CDR-H2 and CDR-H3, and / or the light chain variable region (VL) containing CDR-L1, CDR-L2 and CDR-L3, wherein, compared with any of the heavy chain variable regions (i) and (ii) described below, at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains a mutation, said mutation being a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids).

[0014] In some implementations, the permutation is a conservative permutation.

[0015] In some implementations, the CDR is defined according to the IMGT, Kabat, Chothia, or AbM numbering system.

[0016] In some implementations, the antibody that specifically binds to CEACAM5 or its antigen-binding fragment includes the following complementarity-determining regions (CDRs):

[0017] (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:3; and CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:4; or

[0018] (b) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:1; and CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:2.

[0019] In some embodiments, the CEACAM5 includes human CEACAM5 and / or monkey CEACAM5. In some embodiments, the monkey is a cynomolgus monkey (Macaca fascicularis).

[0020] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the IMGT numbering system:

[0021] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 14 or a variant thereof; CDR-H2 with sequence SEQ ID NO: 24 or a variant thereof; CDR-H3 with sequence SEQ ID NO: 16 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 17 or a variant thereof; CDR-L2 with sequence SEQ ID NO: 18 or a variant thereof; CDR-L3 with sequence SEQ ID NO: 27 or a variant thereof; or

[0022] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 14 or a variant thereof; CDR-H2 with sequence SEQ ID NO: 15 or a variant thereof; CDR-H3 with sequence SEQ ID NO: 16 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 17 or a variant thereof; CDR-L2 with sequence SEQ ID NO: 18 or a variant thereof; CDR-L3 with sequence SEQ ID NO: 13 or a variant thereof;

[0023] The variant described in any of (1a) and (1b) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it originates; preferably, the substitution is a conservative substitution.

[0024] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the Chothia numbering system:

[0025] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 19 or a variant thereof; CDR-H2 with sequence SEQ ID NO: 25 or a variant thereof; CDR-H3 with sequence SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11 or a variant thereof; CDR-L2 with sequence SEQ ID NO: 12 or a variant thereof; CDR-L3 with sequence SEQ ID NO: 27 or a variant thereof; or

[0026] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 19 or a variant thereof; CDR-H2 with sequence SEQ ID NO: 20 or a variant thereof; CDR-H3 with sequence SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11 or a variant thereof; CDR-L2 with sequence SEQ ID NO: 12 or a variant thereof; CDR-L3 with sequence SEQ ID NO: 13 or a variant thereof;

[0027] The variant described in any of (2a) and (2b) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0028] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the Kabat numbering system:

[0029] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 21 or a variant thereof; CDR-H2 with sequence SEQ ID NO: 26 or a variant thereof; CDR-H3 with sequence SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11 or a variant thereof; CDR-L2 with sequence SEQ ID NO: 12 or a variant thereof; CDR-L3 with sequence SEQ ID NO: 27 or a variant thereof; or

[0030] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 21 or a variant thereof; CDR-H2 with sequence SEQ ID NO: 22 or a variant thereof; CDR-H3 with sequence SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11 or a variant thereof; CDR-L2 with sequence SEQ ID NO: 12 or a variant thereof; CDR-L3 with sequence SEQ ID NO: 13 or a variant thereof;

[0031] The variant described in any of (3a) and (3b) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it originates; preferably, the substitution is a conservative substitution.

[0032] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the AbM numbering system:

[0033] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 8 or a variant thereof; CDR-H2 with sequence SEQ ID NO: 23 or a variant thereof; CDR-H3 with sequence SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11 or a variant thereof; CDR-L2 with sequence SEQ ID NO: 12 or a variant thereof; CDR-L3 with sequence SEQ ID NO: 27 or a variant thereof; or

[0034] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 8 or a variant thereof; CDR-H2 with sequence SEQ ID NO: 9 or a variant thereof; CDR-H3 with sequence SEQ ID NO: 10 or a variant thereof; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11 or a variant thereof; CDR-L2 with sequence SEQ ID NO: 12 or a variant thereof; CDR-L3 with sequence SEQ ID NO: 13 or a variant thereof;

[0035] The variant described in any of (4a) and (4b) has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived; preferably, the substitution is a conservative substitution.

[0036] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the IMGT numbering system:

[0037] (1a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 14; CDR-H2 of SEQ ID NO: 24; CDR-H3 of SEQ ID NO: 16; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 17; CDR-L2 of SEQ ID NO: 18; CDR-L3 of SEQ ID NO: 27; or

[0038] (1b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 14; CDR-H2 with sequence SEQ ID NO: 15; CDR-H3 with sequence SEQ ID NO: 16; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 17; CDR-L2 with sequence SEQ ID NO: 18; and CDR-L3 with sequence SEQ ID NO: 13.

[0039] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the Chothia numbering system:

[0040] (2a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 19; CDR-H2 of SEQ ID NO: 25; CDR-H3 of SEQ ID NO: 10; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 11; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO: 27; or

[0041] (2b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 19; CDR-H2 with sequence SEQ ID NO: 20; CDR-H3 with sequence SEQ ID NO: 10; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11; CDR-L2 with sequence SEQ ID NO: 12; and CDR-L3 with sequence SEQ ID NO: 13.

[0042] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the Kabat numbering system:

[0043] (3a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 21; CDR-H2 of SEQ ID NO: 26; CDR-H3 of SEQ ID NO: 10; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 11; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO: 27; or

[0044] (3b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 21; CDR-H2 with sequence SEQ ID NO: 22; CDR-H3 with sequence SEQ ID NO: 10; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11; CDR-L2 with sequence SEQ ID NO: 12; and CDR-L3 with sequence SEQ ID NO: 13.

[0045] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the AbM numbering system:

[0046] (4a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 8; CDR-H2 of SEQ ID NO: 23; CDR-H3 of SEQ ID NO: 10; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 11; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO: 27; or

[0047] (4b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 8; CDR-H2 with sequence SEQ ID NO: 9; CDR-H3 with sequence SEQ ID NO: 10; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 11; CDR-L2 with sequence SEQ ID NO: 12; and CDR-L3 with sequence SEQ ID NO: 13.

[0048] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0049] (a) A VH containing the sequence shown in SEQ ID NO:3 or a variant thereof and / or a VL containing the sequence shown in SEQ ID NO:4 or a variant thereof; or

[0050] (b) VH containing the sequence shown in SEQ ID NO:1 or a variant thereof and / or VL containing the sequence shown in SEQ ID NO:2 or a variant thereof;

[0051] The variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

[0052] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0053] (a) VH containing the sequence shown in SEQ ID NO:3 and VL containing the sequence shown in SEQ ID NO:4; or

[0054] (b) VH containing the sequence shown in SEQ ID NO:1 and VL containing the sequence shown in SEQ ID NO:2.

[0055] In some embodiments of the antibody or antigen-binding fragment disclosed herein, the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide. In some embodiments of the antibody or antigen-binding fragment thereof, the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.

[0056] As is known to those skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0057] In some embodiments, compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.

[0058] In some embodiments, the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification / storage.

[0059] In some embodiments, the N-terminal glutamine of the VH containing the sequence shown in SEQ ID NO:3 or 1 or a variant thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0060] In some embodiments, the antibody or its antigen-binding fragment described in any of the above embodiments further has a feature selected from the following:

[0061] (1) Specific binding membrane-bound CEACAM5 (e.g., human or monkey CEACAM5) and / or the extracellular domain ECD of CEACAM5 (e.g., human or monkey CEACAM5), for example by flow cytometry or biofilm interferometry (BLI) (e.g., ForteBio). ) Measurement;

[0062] (2) Non-binding or minimally binding to native CEACAM5, especially soluble native CEACAM5, for example via biofilm interference (BLI) techniques (such as ForteBio). ) Measurement;

[0063] (3) Does not bind or substantially does not bind to CEACAM1, CEACAM3, CEACAM7 and CEACAM8, for example, as determined by flow cytometry;

[0064] (4) It has no or reduced ADCC activity;

[0065] (5) Inducing CEACAM5 internalization, for example by flow cytometry;

[0066] (6) Inhibit cell (e.g., tumor cell) proliferation; and / or

[0067] (7) Inhibit tumor growth.

[0068] In some implementations, the Native CEACAM5 may be the CEACAM5 protein shed from the surface of tumor cells in cancer patients, which can shed into tumor tissue or enter plasma or other tissues.

[0069] In some embodiments, the antibody or its antigen-binding fragment described in any of the above embodiments may contain a constant region derived from or derived from human immunoglobulins.

[0070] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from or originating from human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region having altered effector functions (e.g., reduced ADCC activity) compared to the wild-type Fc region. In some exemplary embodiments, the antibody or its antigen-binding fragment of the present invention comprises a variant of the human IgG1 heavy chain constant region having the following substitutions compared to its derived wild-type sequence: Leu234Ala, Leu235Ala, and Gly237Ala (according to the EU numbering system). In such embodiments, the antibody or its antigen-binding fragment of the present invention has reduced ADCC activity. In some embodiments, the antibody or its antigen-binding fragment comprises a variant of the human IgG1 heavy chain constant region as shown in SEQ ID NO: 7. In some embodiments, the heavy chain constant region (CH) as shown in SEQ ID NO: 7 or its variant lacks a C-terminal lysine. In some embodiments, the heavy chain of the antibody or its antigen-binding fragment comprises the sequence shown in SEQ ID NO:5 or a variant thereof, the variant having up to 20 conserved substitutions compared to the antibody (e.g., up to 15, 10, or 5 conserved substitutions; e.g., 1, 2, 3, 4, or 5 conserved substitutions). In some embodiments, the heavy chain constant region (CH) of the heavy chain shown in SEQ ID NO:5 or a variant thereof lacks a C-terminal lysine.

[0071] In some embodiments, the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region derived from or originating from human immunoglobulins (e.g., κ or λ). In some embodiments, the light chain of the antibody or its antigen-binding fragment comprises a sequence as shown in SEQ ID NO:6 or a variant thereof, the variant having up to 20 conserved substitutions compared to it (e.g., up to 15, 10, or 5 conserved substitutions; e.g., 1, 2, 3, 4, or 5 conserved substitutions).

[0072] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0073] (1) A heavy chain including the VH shown in SEQ ID NO:3 and the heavy chain constant region (CH) shown in SEQ ID NO:7, and a light chain including the VL shown in SEQ ID NO:4 and the light chain constant region (CL) shown in SEQ ID NO:6;

[0074] (2) A heavy chain including the VH shown in SEQ ID NO:3 and the heavy chain constant region (CH) shown in SEQ ID NO:5, and a light chain including the VL shown in SEQ ID NO:4 and the light chain constant region (CL) shown in SEQ ID NO:6;

[0075] (3) A heavy chain comprising the VH region shown in SEQ ID NO:1 and the heavy chain constant region (CH) shown in SEQ ID NO:5, and a light chain comprising the VL region shown in SEQ ID NO:2 and the light chain constant region (CL) shown in SEQ ID NO:6; or

[0076] (4) A heavy chain including the VH shown in SEQ ID NO:1 and the heavy chain constant region (CH) shown in SEQ ID NO:7, and a light chain including the VL shown in SEQ ID NO:2 and the light chain constant region (CL) shown in SEQ ID NO:6.

[0077] In some embodiments, the antibody of the present invention comprises a heavy chain having the sequence shown in SEQ ID NO:28 and a light chain having the sequence shown in SEQ ID NO:29.

[0078] In some embodiments, the N-terminal glutamine of the heavy chain having the sequence shown in SEQ ID NO:28 undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.

[0079] In some embodiments, the heavy chain having the sequence shown in SEQ ID NO:28 is lacking a C-terminal lysine residue.

[0080] In some embodiments, the N-terminal glutamine having the sequence shown in SEQ ID NO:28 undergoes cyclization to form pyroglutamic acid or pyroglutamate, and the C-terminal lysine of the heavy chain having the sequence shown in SEQ ID NO:28 is deficient.

[0081] In some embodiments, the antibody of the present invention comprises a heavy chain having the sequence shown in SEQ ID NO:30 and a light chain having the sequence shown in SEQ ID NO:29.

[0082] In some embodiments, the antibody of the present invention comprises a heavy chain having the sequence shown in SEQ ID NO:31 and a light chain having the sequence shown in SEQ ID NO:29.

[0083] In some embodiments, the antibody of the present invention comprises a heavy chain having the sequence shown in SEQ ID NO:32 and a light chain having the sequence shown in SEQ ID NO:29.

[0084] In some embodiments, the antibody or its antigen-binding fragment described in any of the above embodiments is a murine antibody, a chimeric antibody, or a humanized antibody.

[0085] In some embodiments, the variable region of the antibody or its antigen-binding fragment described in any of the above embodiments is of human origin.

[0086] In some embodiments, the antibody or its antigen-binding fragment described in any of the above embodiments is selected from ScFv, Fab, Fab', F(ab')2, Fab'-SH, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody, and multispecific antibody.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof described in any of the above embodiments is labeled. In some embodiments, the antibody or antigen-binding fragment thereof is labeled with a detectable marker, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0088] The present invention also provides for the use of antibodies or antigen-binding fragments or pharmaceutical compositions thereof provided herein for the treatment of tumors.

[0089] Derived antibodies

[0090] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to CEACAM5 (particularly human CEACAM5). Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., an avidin or a multihistidine tag) capable of mediating the binding of the antibody or antigen-binding fragment to another molecule.

[0091] As a derivative of antibodies, the present invention provides a conjugate comprising the antibody or its antigen-binding fragment of the present invention and a conjugation portion.

[0092] In some embodiments, the coupling portion is selected from detectable markers. The detectable markers described in this invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads (e.g., The label may include thermometric markers such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.) and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioisotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0093] In some embodiments, the coupling portion is selected from therapeutic agents. In some embodiments, the therapeutic agent is preferably an antitumor agent, such as a cytotoxic agent, cytokine, toxin, or radionuclide.

[0094] In some embodiments, the conjugation moiety is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups.

[0095] As a derivative of antibodies, the present invention provides a multispecific antibody comprising the antibody of the present invention or its antigen-binding fragment.

[0096] In some embodiments, the multispecific antibody comprises the antibody of the present invention or its antigen-binding fragment as a first antigen-binding domain, and further comprises at least one second antigen-binding domain targeting other targets.

[0097] In some embodiments, each antigen-binding domain of the multispecific antibody retains its original binding specificity.

[0098] In some embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0099] As a derivative of the antibody, the present invention provides a chimeric antigen receptor comprising the antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the chimeric antigen receptor comprises the antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv) as an extracellular antigen-binding domain specifically binding to CEACAM5, as well as a transmembrane domain and one or more intracellular T cell signaling domains. The present invention also provides host cells (e.g., immune cells such as T lymphocytes, NK cells, DC cells, macrophages) containing or expressing the chimeric antigen receptor.

[0100] Antibody preparation

[0101] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0102] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10:163-167 (1992)). Furthermore, the Fv, Fab, or F(ab')2 fragments can also be directly isolated from the recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0103] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or a variable region of the heavy chain and / or the variable region of the light chain of the present invention. According to codon degeneracy in the art, in some embodiments, the nucleotide sequence may be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0104] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present invention. In some embodiments, the vector of the present invention is, for example, a plasmid, granule, bacteriophage, lentivirus, etc. In some embodiments, the vector is capable of expressing the antibody or antigen-binding fragment of the present invention in a subject (e.g., a mammal, such as a human).

[0105] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of an antibody or antigen-binding fragment of the present invention and a second nucleotide sequence encoding a light chain or a light chain variable region thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector containing the first nucleotide sequence and a second vector containing the second nucleotide sequence.

[0106] In some embodiments, the antibody or antigen-binding fragment of the present invention can be used to construct a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain (e.g., ScFv) that specifically binds to CEACAM5, a transmembrane domain, and one or more intracellular T-cell signaling domains. In such embodiments, the isolated nucleic acid molecule of the present invention may comprise a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence encoding the chimeric antigen receptor further comprising a nucleotide sequence encoding an antibody or antigen-binding fragment (e.g., ScFv) of the present invention. In some embodiments, the isolated nucleic acid molecule of the present invention encodes a chimeric antigen receptor comprising an antigen-binding fragment (e.g., ScFv) of an antibody of the present invention.

[0107] In some embodiments, the antibodies or antigen-binding fragments of the present invention can be used to construct chimeric antigen receptor-modified immune cells, the chimeric antigen receptor-modified immune cells comprising chimeric antigen receptors (CARs) and immune cells (e.g., T lymphocytes, NK cells, dendritic cells, macrophages).

[0108] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. The host cell may be a eukaryotic cell (e.g., mammalian cell, insect cell, yeast cell) or a prokaryotic cell (e.g., *E. coli*). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of the present invention is a mammalian cell, such as a CHO (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44, CHO-EBNA).

[0109] In some embodiments, the host cell of the present invention may be a chimeric antigen receptor T cell (CAR-T). In such embodiments, the isolated nucleic acid molecule contained in the host cell may contain a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence encoding the chimeric antigen receptor further containing a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv). In some embodiments, the isolated nucleic acid molecule contained in the host cell encodes a chimeric antigen receptor containing an antigen-binding fragment of an antibody of the present invention (e.g., ScFv).

[0110] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, comprising culturing the host cell of the present invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0111] In some embodiments, the host cell used in the preparation method is a Chinese hamster ovary cell.

[0112] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that can be obtained by the aforementioned preparation methods.

[0113] Therapeutic applications

[0114] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule, a carrier, a host cell, a conjugate, a multispecific antibody, or a chimeric antigen receptor or a host cell expressing said chimeric antigen receptor, and a pharmaceutically acceptable carrier and / or excipient.

[0115] In some embodiments, the pharmaceutical compositions of the present invention comprise the antibody or antigen-binding fragment of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0116] In some embodiments, the pharmaceutical compositions of the present invention comprise the carrier or host cell of the present invention, and pharmaceutically acceptable carriers and / or excipients. In such embodiments, the isolated nucleic acid molecule comprised of the carrier comprises a nucleotide sequence encoding a chimeric antigen receptor, the nucleotide sequence encoding the chimeric antigen receptor further comprising a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof (e.g., ScFv); the host cell comprises the isolated nucleic acid molecule or carrier as described above. In some embodiments, the isolated nucleic acid molecule encodes a chimeric antigen receptor comprising an antigen-binding fragment of an antibody of the present invention (e.g., ScFv). In some embodiments, the host cell is an immune cell, such as a T cell. In some embodiments, the host cell is a chimeric antigen receptor T cell (CAR-T).

[0117] In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent. In some embodiments, the additional pharmaceutically active agent is a drug with antitumor activity. In some embodiments, the additional pharmaceutically active agent is selected from CEACAM5 inhibitors, CEACAM6 inhibitors, TROP2 inhibitors, B7H3 inhibitors, PTK7 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic agents, or any combination thereof. In some embodiments, the antibody or antigen-binding fragment of the present invention and the additional pharmaceutically active agent are provided as separate components or as mixed components. Therefore, the antibody or antigen-binding fragment of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0118] In some embodiments, the antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, conjugate, multispecific antibody, or chimeric antigen receptor or host cell expressing said chimeric antigen receptor in the pharmaceutical composition of the present invention is sufficient (e.g., in a subject):

[0119] (a) Inhibit cell (e.g., tumor cells) proliferation;

[0120] (b) Inhibits tumor growth;

[0121] (c) Inhibit CEACAM5-mediated signal transduction;

[0122] (d) Inducing CEACAM5 internalization;

[0123] (e) Treatment of CEACAM5-mediated diseases / conditions; or

[0124] Any combination of (f)(a)-(e).

[0125] In some embodiments, the CEACAM5-mediated disease / condition is a tumor, such as a tumor expressing CEACAM5. In some embodiments, the tumor is selected from colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, or skin cancer, or any combination thereof.

[0126] In another aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing said chimeric antigen receptor, or pharmaceutical composition thereof in the preparation of a medicament for the treatment and / or adjuvant treatment of tumors.

[0127] In another aspect, the present invention provides a method for inhibiting cell proliferation, comprising contacting the cells with an antibody or antigen-binding fragment thereof of the present invention, a nucleic acid molecule, a carrier, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor or a host cell expressing the chimeric antigen receptor, or a pharmaceutical composition. In some embodiments, the cells are cells expressing CEACAM5, such as tumor cells. In some embodiments, the method is performed in vitro.

[0128] In another aspect, the present invention provides a method for treating and / or adjuvantly treating tumors in a subject, the method comprising administering to a subject in need an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, a host cell, a conjugate, a multispecific antibody, a chimeric antigen receptor or a host cell expressing said chimeric antigen receptor, or a pharmaceutical composition thereof.

[0129] In some embodiments, the method further includes administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof. In some embodiments, the second therapy may be applied simultaneously, separately, or sequentially with the methods described above.

[0130] In any of the above embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention can be any tumor type. In some embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is a CEACAM5 positive tumor. In some embodiments, the tumor involved in the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, chimeric antigen receptor or host cell expressing the chimeric antigen receptor, or pharmaceutical composition of the present invention is selected from colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, or skin cancer, or any combination thereof.

[0131] The antibodies or antigen-binding fragments thereof of the present invention, and the pharmaceutical compositions of the present invention, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0132] Furthermore, the antibody or its antigen-binding fragment of the present invention may be present in the pharmaceutical composition in unit dose form for ease of administration.

[0133] The antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present invention are administered by intravenous infusion or injection.

[0134] The pharmaceutical compositions of the present invention may include a "therapeuticly effective amount" of the antibody or antigen-binding fragment thereof of the present invention. A "therapeuticly effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0135] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., therapeutic response). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0136] In this invention, the subject can be a mammal, such as a human.

[0137] Detection Application

[0138] The antibody or its antigen-binding fragment of the present invention can specifically bind to CEACAM5, thereby enabling it to be used to detect the presence or level of CEACAM5 in a sample.

[0139] Therefore, in another aspect, the present invention provides a kit comprising the antibody of the present invention or its antigen-binding fragment. In some embodiments, the antibody of the present invention or its antigen-binding fragment is labeled with a detectable marker. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody of the present invention or its antigen-binding fragment. Preferably, the second antibody further comprises a detectable marker.

[0140] In this invention, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Particularly preferred is that such labels are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acrid esters, and magnetic beads (e.g., The method includes: 1) thermometric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads; and 2) biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. In some embodiments, the detectable markers described above can be linked to the antibodies of the present invention via connectors of varying lengths to reduce potential steric hindrance.

[0141] In another aspect, the present invention provides a method for detecting the presence or level of CEACAM5 in a sample, comprising the step of using an antibody or antigen-binding fragment of the present invention. In a preferred embodiment, the antibody or antigen-binding fragment of the present invention is further labeled with a detectable marker. In another preferred embodiment, the method further comprises detecting the antibody or antigen-binding fragment of the present invention using a reagent labeled with a detectable marker. The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0142] In some embodiments, the method includes contacting the sample with the antibody or antigen-binding fragment of the present invention, and detecting the formation of the complex, under conditions that allow the antibody or its antigen-binding fragment to form a complex with CEACAM5.

[0143] Given that CEACAM5 is expressed at low or no levels in normal tissues, and expressed or expressed at high levels in some cancers, tumors can be diagnosed by detecting the presence or level of CEACAM5 in a sample. Therefore, in some embodiments, the method is used to diagnose tumors, such as CEACAM5-positive tumors, including colorectal cancer, gastric cancer, lung cancer, cervical cancer, pancreatic cancer, esophageal cancer, ovarian cancer, thyroid cancer, bladder cancer, endometrial cancer, breast cancer, liver cancer, prostate cancer, or skin cancer, or any combination thereof.

[0144] In some embodiments, the method includes detecting the expression level of CEACAM5 in a test sample from a subject and comparing the expression level with a reference value (e.g., a healthy control), wherein an increase in the expression level compared to the reference value is an indication of tumor.

[0145] In another aspect, the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a kit for detecting the presence or level of CEACAM5 in a sample and / or diagnosing tumors is provided.

[0146] In another aspect, the present invention provides diagnostic or therapeutic kits comprising the antibodies or antigen-binding fragments thereof described in this invention, nucleic acid molecules, vectors, host cells, conjugates, or multispecific antibodies, and instructions for use. The kits may also include a drug delivery device for local administration. The drug delivery device includes a drug-loaded syringe or a needleless device.

[0147] The antibody of this invention binds to CEACAM5 with high affinity and exhibits extremely high specificity, refraining from binding to its family members CEACAM1, CEACAM3, CEACAM7, and CEACAM8. Specifically, the antibody of this invention specifically binds to membrane-bound CEACAM5 while avoiding binding to soluble native CEACAM5 protein in plasma, thereby potentially preventing interference with antibody function from plasma proteins. The antibody of this invention also possesses good endocytic activity. Therefore, the antibody of this invention has the potential for treating tumors and has significant clinical value.

[0148] abbreviations

[0149] Complementation-determining region in the CDR immunoglobulin variable region

[0150] FR (Antibody Framework Region): Amino acid residues in the antibody variable region other than CDR residues.

[0151] VH antibody heavy chain variable region

[0152] VL antibody light chain variable region

[0153] IgG Immunoglobulin G

[0154] IMGT is based on the international Immunogenetics information system initiated by Lefranc et al. For the numbering system of (IMGT), see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0155] Kabat is an immunoglobulin matching and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0156] The Chothia immunoglobulin numbering system, proposed by Chothia et al., is a classic rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883).

[0157] The AbM CDR definition method is derived from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272).

[0158] mAb monoclonal antibody

[0159] EC50 produces 50% efficacy or a binding concentration.

[0160] IC50 produces a concentration that inhibits 50% of the concentration.

[0161] ELISA (Enzyme-Linked Immunosorbent Assay)

[0162] PCR Polymerase Chain Reaction

[0163] HRP (Hordeum peroxidase)

[0164] K D Equilibrium dissociation constant

[0165] Ka binding rate constant

[0166] Kd dissociation rate constant

[0167] ADCC antibody-dependent cytotoxicity

[0168] FACS flow cytometry technology

[0169] Complementarity-determining region 1 in the variable region of CDR-H1 immunoglobulin heavy chain

[0170] Complementarity-determining region 2 in the variable region of CDR-H2 immunoglobulin heavy chain

[0171] Complementarity-determining region 3 in the variable region of CDR-H3 immunoglobulin heavy chain

[0172] Complementarity-determining region 1 in the variable region of CDR-L1 immunoglobulin light chain

[0173] Complementarity-determining region 2 in the variable region of CDR-L2 immunoglobulin light chain

[0174] Complementarity-determining region 3 in the variable region of CDR-L3 immunoglobulin light chain

[0175] definition

[0176] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0177] As used herein, the term "antibody" is used in the broadest sense to encompass a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can consist of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0178] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0179] The term "antibody" also includes embodiments in which the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or a C-terminal glycine-lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.

[0180] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0181] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the IMGT, Kabat, Chothia, or AbM numbering system.

[0182] The following general rules (published at www.bioinf.org.uk: Professor Andrew C. Martin's research group) can be used to define CDRs in antibody sequences, which include amino acids that specifically interact with the amino acids that form the antigenic epitope that the antibody binds to. In rare cases, these generally constant features may not appear; however, Cys residues are the most conserved feature.

[0183]

[0184]

[0185] V H The complete amino acid sequence is typically numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems. In some embodiments, V H The amino acid sites in the sequence can be numbered sequentially starting from amino acid site 1 until the end of the sequence, or they can be numbered according to Kabat. Unless otherwise stated, the V mentioned herein... H and V L The amino acid sites in the sequence are defined according to their sequential numbering.

[0186] The amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu. The amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330. The corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.

[0187] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0188] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0189] As used herein, the term “antigen-binding fragment” of an antibody refers to a molecule other than the full-length antibody, which includes a portion of the full-length antibody that binds to the antigen bound to the full-length antibody. For example, an antigen-binding fragment may be a polypeptide fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound to the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; this is also referred to as an “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, dAb and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody, linear antibody, nanobody (technology from Domantis), domain antibody (technology from Ablynx), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0190] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains". A "full-length heavy chain" or "heavy chain" refers to a polypeptide chain that, in the N-terminal to C-terminal direction, comprises a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. Preferably, the "full-length heavy chain" is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" or "light chain" is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0191] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains; the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region; the term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds of the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains); and the term "Fab'-SH" refers to a Fab fragment containing free thiol groups.

[0192] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0193] As used herein, the term "Fc fragment" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0194] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between VH and VL of scFv.

[0195] As used herein, the term “diabody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).

[0196] Each of the aforementioned antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen. In this document, those skilled in the art can use known conventional techniques to obtain antigen-binding fragments (e.g., the antibody fragments described above) from a given antibody (e.g., the antibody fragments provided in this invention), and specifically screen for antigen-binding fragments in the same manner as for intact antibodies.

[0197] As used herein, the term "multispecific antibody" refers to an antibody that has multiple different antigen-binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. A "bispecific antibody" is an antibody with two different antigen-binding specificities, formed by a conjugate of a first antibody (or a fragment thereof) and a second antibody (or a fragment thereof) or antibody analogue through a conjugate arm, the conjugation being including, but not limited to, chemical reactions, gene fusion, and enzymatic reactions. "Multispecific antibodies" include, for example, trispecific antibodies and tetraspecific antibodies; a trispecific antibody is an antibody with three different antigen-binding specificities, and a tetraspecific antibody is an antibody with four different antigen-binding specificities.

[0198] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0199] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case. For example, the term "chimeric antibody" may include an antibody whose heavy chain and light chain variable regions are derived from a first antibody (e.g., murine), while the heavy chain and light chain constant regions are derived from a second antibody (e.g., human).

[0200] As used herein, the term "humanized antibody" refers to an antibody that can be prepared by replacing a portion of a human antibody with a portion of a non-human antibody prepared through immunization against mammals other than humans. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). For example, humanized antibodies can be prepared by grafting a CDR sequence derived from a lineage of another mammalian species onto a human frame sequence.

[0201] As used herein, the term "variant," in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide containing an amino acid sequence altered by the introduction of amino acid residue substitutions, deletions, or additions. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, polypeptides can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, variants have similar, identical, or improved functions to the polypeptide or peptide from which they are derived.

[0202] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (KD) or half-maximal effective concentration (EC50) of the interaction. 50 )express.

[0203] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of KD, kon, and kdis can be measured using any effective method. In some embodiments, the dissociation constant can be measured using bioluminescent interferometry (e.g., the ForteBio Octet method). Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0204] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0205] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is the one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. As used herein, the term "expression vector" refers to a vector containing recombinant polynucleotides that include expression regulatory sequences effectively linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as entrapments, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0206] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 or CHO-EBNA cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0207] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0208] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0209] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0210] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

[0211] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0212] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) has a tumor, or is at risk of having the aforementioned disease.

[0213] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0214] As used herein, the term "effector function" refers to the biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region of an amino acid sequence variant), which vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, and the half-life / clearance of antibodies and antigen-antibody complexes. Methods for altering antibody effector functions are known in the art, for example, by introducing mutations into the Fc region.

[0215] As used herein, the term “antibody-dependent cell-mediated cytotoxicity (ADCC)” refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on cytotoxic cells (such as natural killer (NK) cells, neutrophils, or macrophages), enabling these cytotoxic effector cells to specifically bind to target cells to which the antigen is attached, and then kill the target cells by secreting cytotoxins.

[0216] As used in this article, the term "complement-dependent cytotoxicity (CDC)" refers to complement-mediated cytotoxicity, which involves the activation of the classical complement pathway by the binding of specific antibodies to corresponding antigens on the cell membrane surface to form a complex. The resulting membrane attack complex exerts a lytic effect on the target cell.

[0217] In this document, "combination" includes therapies that can be administered separately, such as those formulated separately for individual administration (e.g., those provided in kits), and therapies that can be administered together as a single formulation (i.e., "co-formulation"). In some embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment of the present invention may be administered sequentially. In other embodiments, the anti-CEACAM5 antibody or its antigen-binding fragment may be administered simultaneously. The anti-antibody or its antigen-binding fragment of the present invention may be used in combination with at least one other (active) agent in any manner.

[0218] In such combination therapies, the various active agents often have different complementary mechanisms of action, and the combination therapy may lead to a synergistic effect. Combination therapies include therapeutic agents that affect the immune response (e.g., enhance or activate the response) and therapeutic agents that affect (e.g., inhibit or kill) tumor / cancer cells. Combination therapies can reduce the likelihood of drug-resistant cancer cells developing. Combination therapies may allow for a reduction in the dosage of one or more agents in the regimen to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies may have a synergistic therapeutic or preventative effect on underlying diseases, conditions, or symptoms.

[0219] In this study, CEACAM5 positivity was obtained by professional clinical pathologists through immunohistochemistry and staining intensity evaluation.

[0220] The terms "cancer" and "tumor" are used interchangeably to refer to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

[0221] The embodiments of the present invention will now be described in detail with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the accompanying drawings and preferred embodiments. Attached Figure Description

[0222] Figure 1A MKN45 cell affinity of anti-human CEACAM5 antibody.

[0223] Figure 1B LS1714T cell affinity of anti-human CEACAM5 antibody.

[0224] Figure 2 Antibodies against human CEACAM5 bind to different domains of CEACAM5.

[0225] Sequence information

[0226] Information about the sequences involved in this invention is described in the table below:

[0227]

[0228]

[0229]

[0230]

[0231] Detailed Implementation

[0232] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0233] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0234] Example 1: Preparation of CEACAM5 and its family antigens, control antibody proteins

[0235] 1.1 Preparation of CEACAM5 antigen and related antigens by proteins and cell lines

[0236] The full-length sequences of human CEACAM1 (Uniprot:P13688-1), human CEACAM3 (Uniprot:P40198-1), human CEACAM5 (Uniprot:P06731-1), human CEACAM7 (Uniprot:Q14002-1), human CEACAM8 (Uniprot:P31997), and cynomolgus monkey CEACAM5 (NCBI:XP_005589491.1) were synthesized at GenScript and constructed into the pLVX vector. After plasmid extraction, the virus was packaged and used to construct 293T and CHOS overexpression cell lines. The extracellular nucleic acid sequences of human and monkey antigens were constructed into the pTT5 vector. Six histidine tags were introduced into the C-terminus of the extracellular segment. The recombinant plasmid was extracted and transiently transfected into HEK293-EBNA cells for expression. Cell supernatant was collected after 6 days and purified to obtain human CEACAM5-ECD-His and monkey CEACAM5-ECD-His proteins, respectively.

[0237] 1.2 Expression of CEACAM5 control antibody

[0238] According to database reports, the CEACAM5 control antibody tusamitamab sequence was codon optimized by GenScript. The heavy and light chain nucleotide sequences of the antibody were synthesized and cloned into the pTT5 vector. After plasmid extraction, the pTT5 plasmids corresponding to the heavy and light chains of the antibody were simultaneously transfected into CHOS-EBNA cells. After centrifugation and collection of cell supernatants, the supernatants were purified using Protein A (MabSelect SuRe, GE) to obtain the internally prepared control antibody protein tusamitamab.

[0239] 1.3 Mouse Immunization

[0240] Female BALB / c mice aged 6-8 weeks were alternately immunized with human CEACAM5-ECD-his and cynomolgus monkey CEACAM5-ECD-his proteins, with alternating immunizations every 7 days and four rounds of immunization for each protein. Immunization was performed via footpad, subcutaneous, tail root, and intraperitoneal sites using Freund's complete adjuvant system (Sigma) or Titermax Gold Adjuvant (Sigma) and Impect Alum (Thermo). Serum titers of anti-CEACAM5-ECD-his were monitored every two weeks using ELISA during immunization. Mouse hybridomas with optimal titers were generated using the following protocol.

[0241] 1.4 Hybridoma Fusion Screening

[0242] 1.4.1 Screening for CEACAM5-ECD His and Native CEACAM5 protein binding activities

[0243] Human CEACAM5-ECD-His, cynomolgus monkey CEACAM5-ECD-His protein, and native CEACAM5 protein (Abcam, Ab742) were used as antigens in an enzyme-linked immunosorbent assay (ELISA). In this example, the native CEACAM5 protein was purified from the plasma of cancer patients. For ELISA screening: 1 μg / ml of human CEACAM5-ECD-His, cynomolgus monkey CEACAM5-ECD-His, and native CEACAM5 protein were diluted with CBS buffer, 100 μl / well, and coated onto a bio-labeled plate (BIOFIL) overnight at 4°C. The plate was washed once with 300 μl PBST (0.05% Tween-20), and 100 μl of 2% BSA in PBS was added to each well. The plate was incubated at 37°C for 1 hour. 20 μl of hybridoma supernatant was then added directly to the plate and incubated at 37°C for 2 hours. Discard the solution. Wash the ELISA plate three times with PBST (0.05% Tween-20), 320 μl per well. Dry the ELISA plate and add 100 μl (1:10000) of HRP-conjugated Goat anti-Mouse IgG (Thermo Fisher) diluted in PBST to each well. Incubate at 37°C for 1 hour. Discard the solution. Wash the ELISA plate five times with PBST (0.05% Tween-20), 320 μl per well. Dry the ELISA plate and add 100 μl of TMB to each well and incubate in the dark. Then add 50 μl of 2M H2SO4 to stop the color development reaction. Read the absorbance at 450 nm using an ELISA reader. Select hybridoma clones that bind to human CEACAM5-ECD-his, bind to monkey CEACAM5-ECD-his, but weakly bind or do not bind to native CEACAM5.

[0244] 1.4.2 Screening for affinity binding activity of CEACAM5 antigen-positive cells

[0245] Human gastric cancer cells MKN45 (obtained from Nanjing Kebai Biotechnology Co., Ltd.), human colorectal adenocarcinoma cells LS174T (obtained from Nanjing Kebai Biotechnology Co., Ltd.), and 293T-hCEACAM5 (human) and 293T-cCEACAM5 (monkey) overexpressing cells were screened by flow cytometry. CEACAM5 overexpressing cells were prepared as follows: the full-length sequence of human CEACAM5 (Uniprot: P06731-1) or cynomolgus monkey CEACAM5 (NCBI: XP_005589491.1) was constructed into a lentiviral vector, then the virus was packaged and used to infect 293T cells (obtained from ATCC). Cells were then screened under pressure and flow cytometry validation was performed to obtain stably expressing human or monkey CEACAM5 overexpressing cells. For flow cytometry screening: cells were diluted to a density of 2 x 10^6 / ml with PBS + 2% BSA, and 50 μl was seeded into 96-well PCR conical plates (1 x 10^6 / ml). 5 Cells were collected per well. 50 μl of hybridoma supernatant was mixed with cells in the well plate and incubated at 4°C for 1 h. After washing three times with PBS + 2% BSA, 50 μl / well of diluted PE Goat Anti-Mouse IgG secondary antibody (Biolegend) was added at 4°C and the cells were resuspended. The cells were incubated at 4°C for 30 min and washed three times with PBS + 2% BSA. After centrifugation, 300 μl / well of PBS + 2% BSA was added to resuspend the cells. The affinity of the antibody for cell binding was assessed by flow cytometry (Beckman, Cytoflex). Positive clones that bound human MKN45 cells, LS174T cells, and 293T-hCEACAM5 and 293T-cCEACAM5 were selected.

[0246] 1.4.3 Evaluation of the non-specific screening effect of anti-CEACAM5 antibody on CEACAM family members

[0247] Overexpression of other members of the same family (including human CEACAM1, human CEACAM3, human CEACAM7, and human CEACAM8) in CHOS cells (obtained from Thermo Fisher Scientific) was used to obtain stably overexpressing cells. The binding of these overexpressing cells to hybridoma supernatant was screened by flow cytometry to examine the non-specific binding of antibodies. For flow cytometry binding screening: cells were diluted to a density of 2 x 10^6 / ml with PBS + 2% BSA, and 50 μl was seeded into 96-well PCR conical plates (1 x 10^5 cells / well). 50 μl of hybridoma supernatant was mixed with the cells in the wells and incubated at 4°C for 1 h. After washing three times with PBS + 2% BSA, 50 μl / well of PE Goat Anti-Mouse IgG secondary antibody (Biolegend) diluted with PBS + 2% BSA was added at 4°C, and the cells were resuspended. The cells were incubated at 4°C for 30 min, and washed three times with PBS + 2% BSA. The cells were then centrifuged and resuspended with 200 μl / well of PBS + 2% BSA. The specificity of the antibodies binding to CEACAM family members was assessed by flow cytometry (Beckman, Cytoflex). Hybridoma clones that did not bind to CEACAM1, CEACAM3, CEACAM7, and CEACAM8 were selected.

[0248] In summary, subcloning was performed using parent clones that bind to human CEACAM5-ECD-His, cynomolgus monkey CEACAM5-ECD-His protein, and human gastric cancer cell line MKN45 that weakly bind to native CEACAM5 protein but do not bind to CEACAM5 family members. Subclones were screened using a similar method, and the best single clones were selected for scale-up culture.

[0249] Example 2: Evaluation of anti-human CEACAM5 murine antibodies

[0250] 2.1 Evaluation of cell-binding activity of anti-human CEACAM5 murine antibody

[0251] All selected monoclonal antibodies were amplified and cultured in serum-free medium. 5-10 ml of culture supernatant was purified by affinity using Protein-A beads. The antibody protein concentration was quantified using a UV spectrophotometer and then used for candidate evaluation.

[0252] Affinity screening was performed using human gastric cancer cell line MKN45 and CHOS-Cynodon dactylus CEACAM5 overexpressing cells via flow cytometry. Cells were diluted to a density of 2 x 10^6 / ml with PBS + 2% BSA, and 50 μl of each was seeded into 96-well PCR conical plates (1 x 10^5 cells / well). Serially diluted monoclonal antibodies or tusamitamab were mixed with the cells and incubated at 4°C for 1 h. After washing three times with PBS + 2% BSA, 50 μl / well of diluted PE Goat Anti-Mouse IgG secondary antibody (Biolegend) or PE Goat Anti-human IgG secondary antibody (Biolegend) was added at 4°C, and the cells were resuspended by pipetting. The cells were incubated at 4°C for 30 min, and washed three times with PBS + 2% BSA. After centrifugation, 200 μl / well of PBS + 2% BSA was added to resuspend the cells. The affinity of the antibodies for cell binding was assessed by flow cytometry. Data processing: The median fluorescence intensity was exported and then imported into software for nonlinear curve fitting to calculate EC50. The results showed that the affinity of the Ab-C8 murine antibody of this invention for monkey CEACAM5 was approximately 6 times stronger than that of the tusamitamab antibody (as shown in Table 1). The EC50 value of the Ab-C8 murine antibody of this invention binding to MKN45 cells was 2516 ng / ml.

[0253] Table 1: Binding affinity of murine antibodies to CHOS-cCEACAM5 overexpressing cells

[0254]

[0255] 2.2 Evaluation of cell-binding activity of murine anti-human CEACAM5 antibody

[0256] Screening was performed using flow cytometry on stable cells overexpressing family members human CEACAM1, human CEACAM3, human CEACAM7, and human CEACAM8. The remaining procedures were the same as in section 2.1. The results are shown in Table 2. These results indicate that the antibody of this invention does not bind to human CEACAM1, human CEACAM3, human CEACAM7, or human CEACAM8. In contrast, the control antibody tusamitamab shows some non-specific binding to family member human CEACAM8.

[0257] Table 2: Cross-binding results of anti-human CEACAM5 hybridoma clone antibodies within the same family

[0258] Antibody / Cell hCEACAM1 hCEACAM3 hCEACAM7 hCEACAM8 Ab-C8 Do not combine Do not combine Do not combine Do not combine tusamitamab Do not combine Do not combine Do not combine weak binding

[0259] 2.3 Evaluation of the protein ELISA binding activity of anti-human CEACAM5 mouse antibody

[0260] The protein binding ability of the antibody was detected using human and monkey CEACAM5-ECD-his proteins. The procedure was described in section 1.4.1. The results are shown in Table 3, indicating that the candidate antibody Ab-C8 could bind to both human and monkey CEACAM5-ECD-His proteins.

[0261] Table 3: ELISA affinity assay for anti-CEACAM5 antibody binding to CEACAM5 protein;

[0262]

[0263]

[0264] 2.4 Sequencing and Chimeric Antibody Construction of Anti-Human CEACAM5 Mouse-Derived Antibody

[0265] Hybridoma cells of Ab-C8 were cultured to approximately 8000 cells, then lysed, and first-strand cDNA was synthesized using a cDNA reverse transcription kit (Thermo Fisher). The VH and VL genes were amplified from the cDNA by PCR using primers. The PCR products were purified using a DNA purification kit (MACHEREY-NAGEL) and homologously recombinated into a pTT5 vector expressing the human heavy chain constant region IgG1 and the light chain constant region CL to construct a chimeric antibody expression vector. Positive clones were selected for sequencing after PCR verification. The sequences were analyzed using IMGT and Abysis. The variable region sequence and CDR sequence of the anti-human CEACAM5 antibody are shown in Table 4.

[0266] Example 3: Humanization and Evaluation of Anti-human CEACAM5 Antibody

[0267] 3.1 Humanization and Expression of Anti-human CEACAM5 Antibody

[0268] The mouse antibody Ab-C8 was humanized using a CDR transplantation antibody humanization method. In short, the humanization process involves the following steps: The amino acid sequence of the mouse monoclonal antibody was compared with the amino acid sequence of the human embryonic antibody to identify sequences with high homology and superior physicochemical properties, which were then used as human embryonic framework sequences; HLA-DR affinity was analyzed and examined to select human embryonic framework sequences with low affinity; and the six CDRs of the mouse antibody were then transplanted onto the selected heavy and light chain framework sequences, respectively.

[0269] Further utilizing computer simulation techniques, molecular docking analysis was applied to the variable region and its surrounding framework amino acid sequences to examine their spatial binding mechanism. By calculating electrostatic forces, van der Waals forces, hydrophilicity / hydrophobicity, and entropy, key amino acids in the murine antibody's amino acid sequence that interact with the CEACAM5 protein and maintain its spatial structure were analyzed, and these murine amino acids were retained in the transplanted antibody. Specifically, a series of reversion mutations were performed on the FR region amino acid residues of the aforementioned humanized template to ensure that the humanized antibody retained as much of the murine antibody's antigen-binding ability as possible. Furthermore, to reduce the risks of deamidation, isomerization, and breakage, the NS, DS, and DP sites in the sequence were modified to obtain the final variable region sequence of the humanized antibody.

[0270] Based on the above methods, a humanized antibody, named Ab-hzDP, was constructed using the CDR of the murine antibody Ab-C8 as a basis. The heavy chain constant region of each antibody was the human IgG1 heavy chain constant region (SEQ ID NO:5), and the light chain constant region of each antibody was the human Kappa light chain constant region (SEQ ID NO:6). Furthermore, following the above methods, the human IgG1 heavy chain constant region (SEQ ID NO:5) of Ab-hzDP was replaced with the mutant human heavy chain constant region IgG1m (SEQ ID NO:7), and the expressed antibody was named Ab-hzDPmut. Its variable region and CDR sequence are shown in Table 4.

[0271] The aforementioned chimeric and humanized antibodies underwent codon optimization at Nanjing GenScript Biotech Co., Ltd., and cDNA was synthesized and ligated into the expression plasmid pTT5. The heavy and light chain expression plasmids of the humanized antibody were simultaneously transfected into CHO-S cells. After 7 days of expression, the supernatant was collected by centrifugation. The recombinant antibody in the supernatant was purified using Protein A (MabSelect SuRe, GE) to obtain the anti-human CEACAM5 chimeric and humanized antibody.

[0272] Table 4: Variable region and CDR amino acid sequence of anti-human CEACAM5 antibody

[0273]

[0274] 3.2 Detection of anti-CEACAM5 antibody affinity for tumor cells

[0275] Screening was performed using flow cytometry with human gastric cancer cells MKN45 and human colorectal adenocarcinoma cells LS174T overexpressing cells. Cells were diluted to a density of 2 x 10^6 / ml with PBS + 2% BSA, and 50 μl of each cell was seeded into 96-well PCR conical plates (1 x 10^5 cells / well). Serially diluted antibodies were mixed with the cells in the plates and incubated at 4°C for 1 h. Cells were washed twice with PBS, and then 50 μl of diluted secondary antibody was added to each well, mixed, and incubated at 4°C for 30 min. Cells were washed twice with PBS, and then resuspended in 200 μl of PBS for flow cytometry analysis. Data processing: The median fluorescence intensity was exported and imported into data analysis software to calculate EC50. The results are shown in Table 5. Figure 1A As shown in 1B.

[0276] Table 5: Cell affinity results of anti-CEACAM5 humanized antibody

[0277]

[0278] 3.3 Affinity detection of anti-CEACAM5 antibody binding to CEA family members

[0279] Because there are many members in the CEACAM5 family, but based on sequence similarity, sequences with high similarity to CEACAM5 include CEACAM1, CEACAM3, CEACAM7, and CEACAM8. Therefore, it is necessary to detect the binding activity of antibodies to members of the same family.

[0280] The specificity of the antibody was detected by flow cytometry using CHOS-overexpressing cells containing genes CEA1, CEA3, CEA7, and CEA8. Cells were diluted to a density of 2 x 10^6 / ml with PBS + 2% BSA, and 50 μl of each cell was seeded into 96-well PCR conical plates (1 x 10^5 cells / well). 10 μg / ml antibody was mixed with the cells and incubated at 4°C for 1 h. Cells were washed twice with PBS + 2% BSA, and then 50 μl of diluted secondary antibody was added to each well, mixed, and incubated at 4°C for 30 min. Cells were washed twice with PBS + 2% BSA, and then resuspended in 200 μl of PBS for flow cytometry analysis. Data processing: The median fluorescence intensity and positive rate were derived, and the results are shown in Table 6. The results indicate that the Ab-hzDPmut antibody of this invention does not bind to hCEA1, hCEA3, hCEA7, or hCEA8, while the tusamitamab antibody exhibits some non-specific binding activity to CEA8.

[0281] Table 6: Affinity test of anti-CEACAM5 antibody binding to CEA family members

[0282]

[0283] 3.4 CEACAM5 protein binding of anti-human CEACAM5 antibody, species cross-detection

[0284] The dynamic affinity of anti-human CEACAM5 antibody with human CEACAM5-ECD-his, monkey CEACAM5-ECD-his, and mouse CEACAM5-ECD(ACRO) was detected using a ForteBio (Pall Life Sciences) instrument. The specific method is as follows: The antibody to be tested was diluted to 5 μg / ml with PBST (0.02% Tween-20). Each antigen protein was serially diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM. Then, the antibody to be tested was captured for 60 s in PBST (0.02% Tween-20) solution using a Protein A Sensor (Pall Life Sciences). After equilibration in buffer for 30 s, the antibody was bound to the above proteins for 60 s, followed by dissociation for 180 s. The results were opened in Data Analysis 11.0 software, using 1:1 mode and global fitting, to analyze the results and obtain the affinity constant. The results are shown in Table 7. The results show that both Ab-hzDPmut and Tusamitamab bind to human and monkey CEACAM5-ECD-His, and Ab-hzDPmut has stronger binding activity to monkey protein. Neither antibody binds to mouse CEACAM5-ECD-his.

[0285] Table 7: Dynamic affinity results of antigen proteins of humanized anti-human CEACAM5 antibodies

[0286]

[0287] 3.5 Binding of anti-human CEACAM5 antibody to native CEACAM5 protein: detection

[0288] The plasma of cancer patients contains high concentrations of native CEACAM5 protein. If antibodies can bind to native CEACAM5 in plasma, it may affect the binding of antibodies to tumor target cells. Therefore, it is necessary to detect the binding effect of antibodies on free native CEACAM5 present in plasma.

[0289] The dynamic affinity of anti-human CEACAM5 antibody to human Native CEACAM5 protein (Abcam, Ab742) was detected using a ForteBio (Pall Life Sciences) instrument. The specific method is as follows: The antibody to be tested was diluted to 5 μg / ml with PBST (0.02% Tween-20). The antigen protein was serially diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, and 0 nM. The antibody was then captured for 60 seconds in PBST (0.02% Tween-20) solution using a Protein A Sensor (Pall Life Sciences). After equilibration in buffer for 30 seconds, the antibody was bound to the aforementioned protein for 60 seconds, followed by dissociation for 180 seconds. The results were opened in Data Analysis 11.0 software, using 1:1 mode and global fitting, to analyze the results and obtain the affinity constant. The results are shown in Table 8. The results indicate that Ab-hzDPmut does not bind to the native CEACAM5 protein, but the TUSAMITAMAB antibody shows significant binding to native CEACAM5, with a dynamic affinity of 3.9 nM. These results demonstrate that the Ab-hzDPmut antibody can avoid binding to soluble native CEACAM5 protein in plasma, thereby preventing the influence of plasma proteins on antibody binding to target cells.

[0290] Table 8: Dynamic affinity results of anti-human CEACAM5 antibody antigen protein

[0291]

[0292] 3.6 Detection of endocytic activity of anti-human CEACAM5 antibody

[0293] To evaluate the endocytic activity of anti-CEACAM5 antibody in human gastric cancer cells MKN45, the cell density was adjusted to 1×10⁶ cells / year using complete culture medium. 5Cells were cultured at a density of 100 μl / ml in a 96-well plate, and incubated at 37°C in a CO2 incubator for 24 h. The culture medium was discarded, and 50 μl of fresh complete culture medium was added. The test antibody and negative control antibody hIgG1 were diluted with complete culture medium to a starting concentration of 4.8 μg / ml, followed by 3-fold serial dilutions to 8 concentration points. pHrodo reagent (Thermo, Cat#Z25612) was diluted with complete culture medium to a concentration of 12 μg / ml. The test antibody and pHrodo reagent were mixed 1:1 (30 μl:30 μl) and incubated at room temperature in the dark for 30 min. 50 μl of the test antibody and pHrodo reagent mixture was added to the cells and incubated at 37°C in 5% CO2 for 24 h. The cells were then analyzed using a flow cytometer (Thermo, Attune NxT). Data processing: The median fluorescence intensity was exported and imported into data analysis software to calculate EC50. The results are shown in Table 9. The above results demonstrate that the Ab-hzDPmut antibody of the present invention exhibits superior EC50 endocytic activity compared to the control antibody tusamitamab.

[0294] Table 9: Antibody MKN45 internalizes EC50 against human CEACAM5

[0295]

[0296] 3.7 Analysis of Antigen-Binding Epitopes of Humanized Anti-human CEACAM5 Antibodies

[0297] To determine the binding sites of Ab-hzDPmut antibody and tusamitamab to CEACAM5 antigen, the binding domains of anti-CEACAM5 antibody to CEACAM5 protein were measured using ELISA. Using the same coating conditions as previously described, 96-well plates were coated with the N-A1-B1 (35-315), A1-B1 (145-315), A2-B2 (323-495), and A3-B3 (501-675) domains of human CEACAM5 protein and incubated overnight at 4°C. After washing once with 300 μl PBST (0.05% Tween-20), 300 μl of 2% BSA blocking buffer was added to each well, and the plates were incubated at 37°C for 1 hour. The plates were then dried, and 100 μl each of the humanized antibody and control antibody were diluted with 2% BSA to 10 μg / ml and added to the plates, followed by incubation at 37°C for 1 hour. Discard the solution. Wash the ELISA plate three times with PBST (0.05% Tween-20), 320 μl per well. Dry the ELISA plate and add 100 μl (1:10000) of HRP-conjugated Goat anti-human IgG (H+L) (Jackson) diluted in PBST to each well. Incubate at 37°C for 1 hour. Discard the solution. Wash the ELISA plate five times with PBST (0.05% Tween-20), 320 μl per well. Dry the ELISA plate and add 100 μl of TMB to each well for light-protected color development. Then add 50 μl of 2M H2SO4 to stop the color development reaction. Read the absorbance at 450 nm using an ELISA reader. Import the raw data into data analysis software for analysis, such as... Figure 2 As shown, the humanized anti-human CEACAM5 antibody Ab-hzDPmut specifically binds to the A1-B1 domain of the CEACAM5 protein. In contrast, the tusamitamab antibody primarily binds to the A3-B3-his region of the CEACAM5 protein.

[0298] 3.8 Hydrophilicity Detection of Humanized Anti-CEACAM5 Antibody

[0299] The hydrophilicity of antibodies was detected using an Agilent 1260 TSKgel Butyl-NPR analytical column; column temperature 30℃, detection wavelength 280nm, flow rate 0.5ml / min; mobile phase A: 1.5mol / L (NH4)2SO4; mobile phase B: 25mmol / L Na2HPO4, pH=7.0, 25% IPA. An appropriate amount of the test sample was diluted with diluent (0.75mol / L (NH4)2SO4) to prepare a 1.0mg / ml solution as the test solution. Control antibodies (hydrophilic control: tetilimab; hydrophobic control: sacituzumab, both manufactured by Sichuan Kelun Biotech Co., Ltd.) were diluted with diluent to prepare a 1mg / ml solution as the system suitability solution. Approximately 40 μg of sample was injected, and gradient elution was performed: 0-3 min, maintaining mobile phase A at 95% and mobile phase B at 5%; 3-40 min, mobile phase B was increased from 5% to 100%; 40-45 min, maintaining mobile phase A at 95% and mobile phase B at 5%. After detection, the hydrophobicity value of the test sample was calculated based on the control sample using the formula: (Retention time of test sample - Retention time of hydrophilic control) / (Retention time of hydrophobic control - Retention time of hydrophilic control). The smaller the retention time and hydrophobicity value, the better the hydrophilicity of the antibody. The results are shown in Table 10. The candidate derived antibody Ab-hzDPmut has better hydrophilicity than the control tusamitamab. Good hydrophilicity will be beneficial for antibody production, quality control, or use in small molecule conjugation, while also improving in vivo efficacy and drug metabolism.

[0300] Table 10: Hydrophilicity assay of anti-human CEACAM5 humanized antibody

[0301] Antibody Retention time Hydrophobicity Hydrophilic comparison 11.8 0.00 Hydrophobicity comparison 17.7 1.00 Ab-hzDPmut 13.0 0.2 Tusamitamab 16.4 0.8

[0302] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CEACAM5, wherein, The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2 and CDR-H3 contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 3 or 1; and / or, CDR-L1, CDR-L2 and CDR-L3 contained in a light chain variable region (VL) as set forth in SEQ ID NO: 4 or 2; or (b) CDR-H1, CDR-H2 and CDR-H3 contained in a heavy chain variable region (VH) and / or CDR-L1, CDR-L2 and CDR-L3 contained in a light chain variable region (VL), wherein the heavy chain variable region (VH) and / or light chain variable region (VL) comprises at least one mutation in at least one CDR as compared to the heavy chain variable region and / or light chain variable region of (a), which mutation is a substitution, deletion or addition of one or several amino acids (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids); preferably, the substitution is a conservative substitution; Preferably, the CDRs are defined according to the IMGT, Kabat, Chothia or AbM numbering system.

2. The antibody or antigen binding fragment thereof specifically binding to CEACAM5 of claim 1, wherein, The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): (a) CDR-H1, CDR-H2 and CDR-H3 contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 3; and, CDR-L1, CDR-L2 and CDR-L3 contained in a light chain variable region (VL) as set forth in SEQ ID NO: 4; or (b) CDR-H1, CDR-H2 and CDR-H3 contained in a heavy chain variable region (VH) as set forth in SEQ ID NO: 1; and, CDR-L1, CDR-L2 and CDR-L3 contained in a light chain variable region (VL) as set forth in SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system: (1a) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 of SEQ ID NO: 14 or a variant thereof; CDR-H2 of SEQ ID NO: 24 or a variant thereof; and CDR-H3 of SEQ ID NO: 16 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 of SEQ ID NO: 17 or a variant thereof; CDR-L2 of SEQ ID NO: 18 or a variant thereof; and CDR-L3 of SEQ ID NO: 27 or a variant thereof; or (1b) a heavy chain variable region (VH) comprising the following 3 CDRs: CDR-H1 of SEQ ID NO: 14 or a variant thereof; CDR-H2 of SEQ ID NO: 24 or a variant thereof; and CDR-H3 of SEQ ID NO: 16 or a variant thereof; and / or, a light chain variable region (VL) comprising the following 3 CDRs: CDR-L1 of SEQ ID NO: 17 or a variant thereof; CDR-L2 of SEQ ID NO: 18 or a variant thereof; and CDR-L3 of SEQ ID NO: 27 or a variant thereof. (1 b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 14 or a variant thereof; CDR-H2 of SEQ ID NO: 15 or a variant thereof; CDR-H3 of SEQ ID NO: 16 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 17 or a variant thereof; CDR-L2 of SEQ ID NO: 18 or a variant thereof; CDR-L3 of SEQ ID NO: 13 or a variant thereof; or, (2) a heavy chain variable region (VH) and / or a light chain variable region (VL) wherein the CDRs are defined according to the Chothia numbering system: (2a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 19 or a variant thereof; CDR-H2 of SEQ ID NO: 25 or a variant thereof; CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 11 or a variant thereof; CDR-L2 of SEQ ID NO: 12 or a variant thereof; CDR-L3 of SEQ ID NO: 27 or a variant thereof; or (2b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 19 or a variant thereof; CDR-H2 of SEQ ID NO: 20 or a variant thereof; CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 11 or a variant thereof; CDR-L2 of SEQ ID NO: 12 or a variant thereof; CDR-L3 of SEQ ID NO: 13 or a variant thereof; or, (3) a heavy chain variable region (VH) and / or a light chain variable region (VL) wherein the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 21 or a variant thereof; CDR-H2 of SEQ ID NO: 26 or a variant thereof; CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 11 or a variant thereof; CDR-L2 of SEQ ID NO: 12 or a variant thereof; CDR-L3 of SEQ ID NO: 27 or a variant thereof; or (3b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 21 or a variant thereof; CDR-H2 of SEQ ID NO: 22 or a variant thereof; CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 11 or a variant thereof; CDR-L2 of SEQ ID NO: 12 or a variant thereof; CDR-L3 of SEQ ID NO: 13 or a variant thereof; or, (4) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system: (4a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 8 or a variant thereof; CDR-H2 of SEQ ID NO: 23 or a variant thereof; CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 11 or a variant thereof; CDR-L2 of SEQ ID NO: 12 or a variant thereof; CDR-L3 of SEQ ID NO: 27 or a variant thereof; or (4b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 8 or a variant thereof; CDR-H2 of SEQ ID NO: 9 or a variant thereof; CDR-H3 of SEQ ID NO: 10 or a variant thereof; and / or, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 11 or a variant thereof; CDR-L2 of SEQ ID NO: 12 or a variant thereof; CDR-L3 of SEQ ID NO: 13 or a variant thereof; wherein the variant of any one of (1 a), (1 b), (2a), (2b), (3a), (3b), (4a), (4b) has one or several (for example 1, 2 or 3) amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably said substitutions are conservative substitutions.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, the antibody or antigen binding fragment thereof comprises: (1) a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system: (1a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 14; CDR-H2 of SEQ ID NO: 24; CDR-H3 of SEQ ID NO: 16; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 17; CDR-L2 of SEQ ID NO: 18; CDR-L3 of SEQ ID NO: 27; or (1b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 14; CDR-H2 of SEQ ID NO: 15; CDR-H3 of SEQ ID NO: 16; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 17; CDR-L2 of SEQ ID NO: 18; CDR-L3 of SEQ ID NO: 13; or, (2) a heavy chain variable region (VH) and / or a light chain variable region (VL) in which the CDRs are defined according to the Chothia numbering system: (2a) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 19; CDR-H2 of SEQ ID NO: 25; CDR-H3 of SEQ ID NO: 10; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 11; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO: 27; or (2b) a heavy chain variable region (VH) comprising 3 CDRs: CDR-H1 of SEQ ID NO: 19; CDR-H2 of SEQ ID NO: 20; CDR-H3 of SEQ ID NO: 10; and, a light chain variable region (VL) comprising 3 CDRs: CDR-L1 of SEQ ID NO: 11; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO: 13; or, (3) a heavy chain variable region (VH) and a light chain variable region (VL) in which the CDRs are defined according to the Kabat numbering system: (3a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 21 ; CDR-H2 of SEQ ID NO: 26; CDR-H3 of SEQ ID NO: 10; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 11 ; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO: 27; or (3b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 21 ; CDR-H2 of SEQ ID NO: 22; CDR-H3 of SEQ ID NO: 10; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 11 ; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO: 13; or, (4) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDRs are defined by the AbM numbering system: (4a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 8; CDR-H2 of SEQ ID NO: 23; CDR-H3 of SEQ ID NO: 10; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 11 ; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO: 27; or (4b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 of SEQ ID NO: 8; CDR-H2 of SEQ ID NO: 9; CDR-H3 of SEQ ID NO: 10; and, a light chain variable region (VL) comprising three CDRs: CDR-L1 of SEQ ID NO: 11 ; CDR-L2 of SEQ ID NO: 12; CDR-L3 of SEQ ID NO:

13.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein, the antibody or antigen binding fragment thereof comprises: (a) a VH comprising a sequence as shown in SEQ ID NO: 3 or a variant thereof and / or a VL comprising a sequence as shown in SEQ ID NO: 4 or a variant thereof; or (b) a VH comprising a sequence as shown in SEQ ID NO: 1 or a variant thereof and / or a VL comprising a sequence as shown in SEQ ID NO: 2 or a variant thereof; wherein the variant has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it is derived, or one or several substitutions, deletions, or additions of amino acids (e.g. 1, 2, 3, 4, or 5 substitutions, deletions, or additions of amino acids) compared to the sequence from which it is derived; preferably the substitutions are conservative substitutions.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein, The antibody or antigen-binding fragment thereof comprises: (a) a VH comprising a sequence as set forth in SEQ ID NO: 3 and a VL comprising a sequence as set forth in SEQ ID NO: 4; or (b) a VH comprising a sequence as set forth in SEQ ID NO: 1 and a VL comprising a sequence as set forth in SEQ ID NO:

2.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein, The antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein, The antibody or antigen-binding fragment thereof further comprises a constant region from or derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region from or derived from a human immunoglobulin (e.g. IgGl, IgG2, IgG3, or IgG4); preferably the antibody or antigen-binding fragment thereof comprises a wild-type Fc region, or a mutated or chemically modified Fc region having altered effector function compared to a wild-type Fc region; Preferably, the antibody or antigen-binding fragment thereof comprises a variant of the human IgGl heavy chain constant region having the following substitutions compared to the wild-type sequence from which it is derived: Leu234Ala, Leu235Ala, and Gly237Ala (positions according to the EU numbering system); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region from or derived from a human immunoglobulin (e.g. kappa or lambda); Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 5 or a variant thereof having up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 5 (e.g. up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g. 1, 2, 3, 4, or 5 conservative substitutions of amino acids); Preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 6 or a variant thereof having up to 20 conservative substitutions of amino acids compared to SEQ ID NO: 6 (e.g. up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g. 1, 2, 3, 4, or 5 conservative substitutions of amino acids); Preferably, the antibody or antigen-binding fragment thereof comprises a variant of the human IgGl heavy chain constant region as set forth in SEQ ID NO: 7; More preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 5 or 7 and a light chain constant region (CL) as set forth in SEQ ID NO:

6.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein, The heavy chain constant region (CH) as set forth in SEQ ID NO: 5 or 7 or a variant thereof lacks a C-terminal lysine.

10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, wherein, The antibody or antigen-binding fragment thereof comprises: (1) a heavy chain comprising a VH as set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 7, and, a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 6; (2) a heavy chain comprising a VH as set forth in SEQ ID NO: 3 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 5, and, a light chain comprising a VL as set forth in SEQ ID NO: 4 and a light chain constant region (CL) as set forth in SEQ ID NO: 6; (3) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 5, and, a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO: 6; or (4) a heavy chain comprising a VH as set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 7, and, a light chain comprising a VL as set forth in SEQ ID NO: 2 and a light chain constant region (CL) as set forth in SEQ ID NO:

6.

11. The antibody or antigen-binding fragment thereof of claim 5, 6, or 10, wherein, The N-terminal glutamine of the VH comprising a sequence as set forth in SEQ ID NO: 3 or 1 or a variant thereof is subjected to cyclization to form pyroglutamic acid or a pyroglutamate salt.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein, The antibody comprises a heavy chain having a sequence as set forth in SEQ ID NO: 28 and a light chain having a sequence as set forth in SEQ ID NO:

29.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein, The N-terminal glutamine of the heavy chain having a sequence as set forth in SEQ ID NO: 28 is subjected to cyclization to form pyroglutamic acid or a pyroglutamate salt; and / or The C-terminal lysine of the heavy chain having a sequence as set forth in SEQ ID NO: 28 is lacking.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein, The antibody comprises a heavy chain having a sequence as set forth in SEQ ID NO: 30 and a light chain having a sequence as set forth in SEQ ID NO:

29.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein, The antibody comprises a heavy chain having a sequence as set forth in SEQ ID NO: 31 and a light chain having a sequence as set forth in SEQ ID NO:

29.

16. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein, The antibody comprises a heavy chain having a sequence as set forth in SEQ ID NO: 32 and a light chain having a sequence as set forth in SEQ ID NO:

29.

17. The antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein, The antibody or antigen-binding fragment thereof is selected from the group consisting of a ScFv, a Fab, a Fab', a Fab'-SH, a F(ab')2, a Fv fragment, a disulfide linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

18. The antibody or antigen-binding fragment thereof of any one of claims 1-17, wherein, The antibody or antigen-binding fragment thereof carries a label; preferably, the antibody or antigen-binding fragment thereof carries a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

19. The antibody or antigen-binding fragment thereof of any one of claims 1-18, wherein the antibody or antigen-binding fragment thereof has one or more features selected from the group consisting of: (1) Specific binding membrane-bound CEACAM5 and / or the extracellular domain (ECD) of CEACAM5, for example by flow cytometry or biofilm interferometry (BLI) (e.g., ForteBio). ) Measurement; (2) does not bind, or does not substantially bind, native CEACAM5, especially soluble native CEACAM5, e.g., as determined by Bio-Layer Interferometry (BLI) (e.g., ForteBio Octet®); and ) does not bind, or does not substantially bind, native CEACAM5, especially soluble native CEACAM5, e.g., as determined by Bio-Layer Interferometry (BLI) (e.g., (3) does not bind or does not substantially bind to CEACAM1, CEACAM3, CEACAM7, and CEACAM8, e.g., as determined by flow cytometry; (4) has reduced or ablated ADCC activity; (5) induces internalization of CEACAM5, e.g., as determined by flow cytometry; (6) inhibits cell (e.g., tumor cell) proliferation; and / or (7) inhibits tumor growth.

20. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, the heavy chain and / or light chain thereof, or the heavy chain variable region and / or light chain variable region thereof, of any one of claims 1-19.

21. A vector comprising the nucleic acid molecule of claim 20; preferably, the vector is a cloning vector or an expression vector.

22. A host cell comprising the nucleic acid molecule of claim 20 or the vector of claim 21.

23. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-19, comprising culturing the host cell of claim 22 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

24. The method of claim 23, wherein the host cell is a Chinese hamster ovary cell.

25. The antibody or antigen-binding fragment thereof obtainable by the method of claim 23 or 24.

26. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-19 and a conjugating moiety linked thereto; Preferably, the conjugating moiety is selected from a detectable label (e.g., a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme) or a therapeutic agent (e.g., a cytotoxic agent, a cytokine, a toxin, or a radionuclide).

27. A multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-19; Preferably, the multispecific antibody comprises the antibody or antigen-binding fragment thereof of any one of claims 1-19 as a first antigen-binding domain, and further comprises at least one second antigen-binding domain directed to another target; Preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

28. A chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof (e.g., ScFv) of any one of claims 1-19, a transmembrane domain, and one or more intracellular T cell signaling domains.

29. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-19, or the isolated nucleic acid molecule of claim 20, or the vector of claim 21, or the host cell of claim 22, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing said chimeric antigen receptor, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from the group consisting of a CEACAM5 inhibitor, a CEACAM6 inhibitor, a TROP2 inhibitor, a B7H3 inhibitor, a PTK7 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof; Preferably, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as a mixed component.

30. A diagnostic or therapeutic kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-19, or the isolated nucleic acid molecule of claim 20, or the vector of claim 21, or the host cell of claim 22, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing said chimeric antigen receptor, or the pharmaceutical composition of claim 29, and optionally instructions for use and / or a device for administration.

31. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-19, or the isolated nucleic acid molecule of claim 20, or the vector of claim 21, or the host cell of claim 22, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing said chimeric antigen receptor, or the pharmaceutical composition of claim 29, for the manufacture of a medicament for the treatment and / or adjuvant treatment of a tumor; Preferably, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, conjugate, multispecific antibody, or pharmaceutical composition is administered in combination with, e.g. simultaneously, separately or sequentially, an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity; Preferably, the additional pharmaceutically active agent is selected from the group consisting of: a CEACAM5 inhibitor, an EGFR inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an IGFR-1 inhibitor, an mTOR inhibitor, a PI3 kinase inhibitor, a c-met or VEGF inhibitor, a chemotherapeutic drug, or any combination thereof.

32. The use of claim 31, wherein the tumor is a CEACAM5-positive tumor; Preferably, the tumor is selected from the group consisting of a colorectal cancer, a gastric cancer, a lung cancer, a cervical cancer, a pancreatic cancer, an esophageal cancer, an ovarian cancer, a thyroid cancer, a bladder cancer, an endometrial cancer, a breast cancer, a liver cancer, a prostate cancer, or a skin cancer.

33. A method of inhibiting cell proliferation, comprising contacting the cell with the antibody or antigen-binding fragment thereof of any one of claims 1-19, or the isolated nucleic acid molecule of claim 20, or the vector of claim 21, or the host cell of claim 22, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing the chimeric antigen receptor, or the pharmaceutical composition of claim 29; Preferably, the cell is a CEACAM5-expressing cell, such as a tumor cell; Preferably, the tumor cell overexpresses CEACAM5.

34. A method for treating and / or adjuvant treatment of a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-19, or the isolated nucleic acid molecule of claim 20, or the vector of claim 21, or the host cell of claim 22, or the conjugate of claim 26, or the multispecific antibody of claim 27, or the chimeric antigen receptor of claim 28 or a host cell expressing the chimeric antigen receptor, or the pharmaceutical composition of claim 29.

35. The method of claim 34, further comprising administering to the subject a second therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof; Optionally, the second therapy can be applied simultaneously, separately or sequentially with the method of claim 34.

36. The method of claim 34 or 35, wherein, the tumor is a CEACAM5-positive tumor; Preferably, the tumor is selected from the group consisting of a colorectal cancer, a gastric cancer, a lung cancer, a cervical cancer, a pancreatic cancer, an esophageal cancer, an ovarian cancer, a thyroid cancer, a bladder cancer, an endometrial cancer, a breast cancer, a liver cancer, a prostate cancer, or a skin cancer.

37. A method of detecting the presence or level of CEACAM5 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-19 under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment thereof and CEACAM5, and detecting the formation of the complex. Preferably, the method is for diagnosing a tumor, such as a CEACAM5-positive tumor, such as a colorectal cancer, a gastric cancer, a lung cancer, a cervical cancer, a pancreatic cancer, an esophageal cancer, an ovarian cancer, a thyroid cancer, a bladder cancer, an endometrial cancer, a breast cancer, a liver cancer, a prostate cancer or a skin cancer or any combination thereof. Preferably, the method comprises detecting the expression level of CEACAM5 in a test sample from a subject, and comparing the expression level to a reference value, wherein an increase in the expression level compared to the reference value is indicative of a tumor.

38. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-19, or the isolated nucleic acid molecule of claim 20, or the vector of claim 21, or the host cell of claim 22, or the conjugate of claim 26, or the multispecific antibody of claim 27, for the manufacture of a diagnostic test kit for detecting the presence or level of CEACAM5 in a sample and / or for diagnosing a tumor. Preferably, the tumor is a CEACAM5-positive tumor. Preferably, the tumor is selected from the group consisting of a colorectal cancer, a gastric cancer, a lung cancer, a cervical cancer, a pancreatic cancer, an esophageal cancer, an ovarian cancer, a thyroid cancer, a bladder cancer, an endometrial cancer, a breast cancer, a liver cancer, a prostate cancer or a skin cancer or any combination thereof.

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