Anti-ceacam5 antibody and use thereof

Abstract

The present invention relates to an anti-CEACAM5 antibody and the use thereof, particularly to an antibody that specifically binds to a CEACAM5 A3-B3 domain. The anti-CEACAM5 antibody exhibits a cross-reactivity with human and cynomolgus monkey CEACEA5, and specifically binds to the A3-B3 epitope of CEACAM5, thereby providing an antibody candidate molecule for the development of a CEACAM5-molecule-targeting tumor therapeutic or diagnostic reagent.

Description

Anti-ceacam5 antibodies and uses thereof

[0001] This international patent application claims priority to Chinese patent application No. 202411245210.7, filed September 5, 2024, Chinese patent application No. 202510192222.6, filed February 20, 2025, and Chinese patent application No. 202510898839.X, filed June 30, 2025, the entire contents of which are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present invention belongs to the field of biomedical or biopharmaceutical technology, and relates to an anti-CEACAM5 antibody. BACKGROUND

[0003] CEACAM5, also known as CEA or CD66e, is a carcinoembryonic antigen CEA-related cell adhesion molecule 5. This protein is bound to the membrane via glycosylphosphatidylinositol (GPI), and has seven immunoglobulin-like domains in the extracellular region, including one variable (lgV)-like domain and six lgC-like domains (A1-B3). There is a signal peptide of 34 amino acids before the lg domains.

[0004] CEACAM5 was first discovered as a tumor marker and can be used to assist in the diagnosis of various tumors. With further research, it has gradually been used as a tumor target for the development of new drugs. In the CEACAM family, in addition to CEACAM5, other family members mainly include CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19, CEACAM20, and CEACAM21.

[0005] CEACAM5 is widely expressed on the surface of endothelial cells, epithelial cells, immune cells and other cell types. CEACAM5 mainly acts as a cell adhesion molecule in normal tissues, mediates homotypic and heterotypic adhesion, and its high expression in most malignant tumors is related to tumor occurrence, invasion and metastasis. Its mechanism of action mainly includes the following aspects: 1) Anti-adhesion effect, cell separation is the first step of tumor invasion of surrounding tissues and distant metastasis, and plays a key role in the process of malignant tumor progression; 2) Extracellular matrix degradation is an important step in tumor cell migration, and excessive CEACAMs expression inhibits cancer cell anoikis and destroys cell structure; 3) Activation of tumor signaling pathways, CEACAMs have been shown to activate integrin signaling pathways, including ILK, PI3K and AKT. However, only GPI-anchored CEACAMs can bind to the alpha5beta1 integrin receptor to exert biological effects, and soluble CEACAMs that lose GPI anchoring have no binding capacity, which will block the transduction process of cell survival signals from the extracellular to the intracellular, and cause apoptosis.

[0006] Monoclonal antibodies have successfully applied in biological targeting therapy and cancer detection, and play an important role in biomedical and immunotherapy. Therefore, developing a therapeutic antibody or a diagnostic reagent for detecting the expression amount of CEACAM5 in cancer has broad prospects. SUMMARY

[0007] The present application adopts protein immunization of mice, and respectively detects the immune serum titer by ELISA and FACS; then the spleen of the mouse with better immune titer is selected to prepare a B cell single cell suspension, which is fused with non-secretory SP2 / 0 myeloma cells, and then the supernatant of the cultured cells after fusion is screened by ELISA and FACS to screen the clones that are combined with human CEACAM5, cynomolgus monkey CEACAM5 and CEACAM5A3-B3, weakly combined or not combined with human CEACAM-1, CEACAM-6, CEACAM-7 and CEACAM-8, and the positive clones are selected for multiple subcloning, and then the hybridoma is sequenced and analyzed, and finally the obtained sequence is constructed and verified to obtain 14 specific sequences. The antibody heavy chain and light chain variable region genes obtained by sequencing the hybridoma are synthesized and subcloned into an expression vector, the constructed light chain and heavy chain expression vectors are transiently co-transfected into 293F cells, expressed and purified, and then the purity is detected, and the binding activity of the antibody is detected by ELISA and FACS.

[0008] The inventors also humanized the obtained antibody clones and finally obtained 6 unique humanized sequences. The expression amount and affinity detection of the humanized antibodies showed that the humanized antibodies had high affinity. Endocytosis experiments showed that the humanized antibodies had good endocytosis activity, indicating that they were suitable for constructing antibody drug conjugates (ADC). The in vivo tumor killing data of the ADC obtained by coupling the humanized antibodies with linker-payload further confirmed the tumor inhibition effect of the humanized antibodies. The results showed that the humanized antibodies had good in vivo tumor inhibition effect.

[0009] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0010] (1) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 75, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 76; or

[0011] (2) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 102; or

[0012] (3) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 103; or

[0013] (4) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104; or

[0014] (5) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 105, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104; or

[0015] (6) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 106, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104; or

[0016] (7) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 107, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104.

[0017] In some embodiments, the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 18-20, respectively, and the VL comprises LCDR 1 having an amino acid sequence as set forth in SEQ ID NO: 21, LCDR2 having a SAS amino acid sequence, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 22.

[0018] In some embodiments, the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 95-97, respectively, and the VL comprises LCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 98-100, respectively.

[0019] In some embodiments, the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 95, 108, and 97, respectively, and the VL comprises LCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 98-100, respectively.

[0020] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0021] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 102.

[0022] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103.

[0023] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

[0024] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 105, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

[0025] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 106, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

[0026] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 107, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

[0027] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 75, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 76.

[0028] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 102.

[0029] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 103.

[0030] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104.

[0031] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 105, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104.

[0032] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 106, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104.

[0033] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 107, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104.

[0034] In some embodiments, the antibody comprises a heavy chain and a light chain, wherein:

[0035] (1) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 117; or

[0036] (2) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 118; or

[0037] (3) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119; or

[0038] (4) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 120, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119; or

[0039] (5) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 121, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119; or

[0040] (6) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 122, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119; or

[0041] (7) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 124.

[0042] In preferred embodiments, the antibody comprises a heavy chain and a light chain, wherein:

[0043] (1) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 116, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 117; or

[0044] (2) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 116, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 118; or

[0045] (3) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 116, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 119; or

[0046] (4) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 120, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 119; or

[0047] (5) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 121, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or

[0048] (6) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 122, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or

[0049] (7) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 123, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 124.

[0050] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0051] (1) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 71, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 72; or

[0052] (2) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 67, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 68; or

[0053] (3) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 69, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 70; or

[0054] (4) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 73, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 74; or

[0055] (5) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 77, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 78; or

[0056] (6) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 79, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 80; or

[0057] (7) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 81, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 82; or

[0058] (8) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 83, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 84; or

[0059] (9) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 85, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 86; or

[0060] (10) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 87, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 88; or

[0061] (11) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 89, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 90; or

[0062] (12) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 91, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 92; or

[0063] (13) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 93, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 94.

[0064] In some embodiments, the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 1, 11, and 3, respectively, and the VL comprises LCDR 1 having an amino acid sequence as set forth in SEQ ID NO: 4, LCDR2 having an AAS amino acid sequence, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 12.

[0065] In some embodiments, the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 1-3, respectively, and the VL comprises LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, LCDR2 having an AAS amino acid sequence, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 5.

[0066] In some embodiments, the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 6-8, respectively, and the VL comprises LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 9, LCDR2 having a WAS amino acid sequence, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 10.

[0067] In some embodiments, the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOs: 13-15, respectively, and the VL comprises LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, LCDR2 having a LAS amino acid sequence, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 17.

[0068] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 23-25, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 26, LCDR2 having the FAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 27.

[0069] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28-30, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 having the SAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 32.

[0070] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 33-35, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 36, LCDR2 having the AAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 37.

[0071] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 38-40, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 41, LCDR2 having the YAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42.

[0072] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28, 43, and 44, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 45, LCDR2 having the SAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 46.

[0073] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 47-49, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 50, LCDR2 having the NAK amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 51.

[0074] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 52-54, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 55, LCDR2 having the ATS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 56.

[0075] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 57-59, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 60, LCDR2 having the NAK amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 61.

[0076] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 62-64, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 65, LCDR2 having the ATS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 66.

[0077] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72.

[0078] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68.

[0079] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70.

[0080] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74.

[0081] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.

[0082] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80.

[0083] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.

[0084] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84.

[0085] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 85, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86.

[0086] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 87, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 88.

[0087] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 89, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 90.

[0088] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 91, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 92.

[0089] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 93, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 94.

[0090] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 71, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 72.

[0091] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 67, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 68.

[0092] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 69, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 70.

[0093] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 73, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 74.

[0094] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 77, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 78.

[0095] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 79, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 80.

[0096] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 81, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 82.

[0097] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 83, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 84.

[0098] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 85, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 86.

[0099] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 87, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 88.

[0100] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 89, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 90.

[0101] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 91, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 92.

[0102] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 93, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 94.

[0103] In some embodiments, the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0104] In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0105] In some embodiments, the antibody is of a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

[0106] In some embodiments, the antibody is a monovalent, bivalent or multivalent antibody.

[0107] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

[0108] In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv and ds-scFv.

[0109] In some embodiments, the antibody or antigen binding fragment thereof binds to CEACAM5 A3-B3, CEACAM5 A2-B2 and / or CEACAM5 Al-B l, preferably to CEACAM5 A3-B3.

[0110] In another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof as described herein.

[0111] In yet another aspect, the present disclosure provides a vector comprising a nucleic acid as disclosed herein.

[0112] In yet another aspect, the present disclosure provides a host cell comprising a nucleic acid or vector as disclosed herein.

[0113] In another aspect, the present disclosure provides a method of producing an antibody or antigen binding fragment thereof as described herein, the method comprising:

[0114] a) culturing the host cell of claim 14 under conditions suitable for expression of the antibody or antigen binding fragment thereof; and

[0115] b) isolating the antibody or antigen binding fragment thereof from the culture and / or culture supernatant of the host cell.

[0116] In yet another aspect, the present disclosure provides a conjugate comprising an antibody or antigen binding fragment thereof as described herein and a chemical moiety conjugated to the antibody or antigen binding fragment thereof, preferably the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety and an immunostimulatory molecule.

[0117] In some embodiments, the therapeutic agent is a topoisomerase inhibitor, preferably Dxd. In some embodiments, the antibody or antigen binding fragment thereof is conjugated to the chemical moiety via a linker, preferably the linker is MC-GGFG.

[0118] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an antibody or antigen binding fragment thereof as described herein.

[0119] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, a conjugate described herein, or a CAR described herein, and optionally a pharmaceutically acceptable carrier or excipient.

[0120] In yet another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, a conjugate described herein, or a CAR described herein, or a pharmaceutical composition described herein.

[0121] In yet another aspect, the present disclosure provides a method of treating a cancer in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, a conjugate described herein, or a CAR described herein, or a pharmaceutical composition described herein.

[0122] In some embodiments, the cancer is a cancer associated with CEACAM5 expression, preferably selected from a solid tumor, further preferably gastric cancer or colorectal cancer.

[0123] In another aspect, the present disclosure provides a method for detecting the presence or level of CEACAM5 in a sample, comprising:

[0124] (1) contacting the sample with an antibody or antigen-binding fragment thereof of any one of claims 1-11; and

[0125] (2) determining the presence or level of CEACAM5 in the sample by detecting binding of the antibody to the sample.

[0126] In another aspect, the present disclosure provides a method for diagnosing whether a subject has a cancer associated with CEACAM5 expression, comprising:

[0127] (1) contacting a sample from a subject with an antibody or antigen-binding fragment thereof described herein; and

[0128] (2) detecting binding of the antibody to the sample,

[0129] wherein increased binding of the antibody to the sample compared to binding to a healthy control indicates that the subject has a cancer associated with CEACAM5 expression.

[0130] In some embodiments, the cancer is selected from a solid tumor, preferably gastric cancer or colorectal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0131] Figure 1 shows the results of FACS detection of serum titer after three immunizations of mice (IgG antibody);

[0132] Figure 2 shows the FACS binding of positive well hybridoma clones to cells expressing CEACAM5 and MKN45 cells;

[0133] Figure 3 shows the FACS binding of positive well hybridoma clones to cells expressing CEACAM1 / 6 / 7 / 8;

[0134] Figure 4 shows the results of endocytosis activity detection of positive well hybridoma clones;

[0135] Figure 5 shows the results of ELISA detection of chimeric antibodies;

[0136] Figure 6 shows the results of FACS detection of chimeric antibodies;

[0137] Figure 7 shows the results of specificity detection of chimeric antibodies;

[0138] Figure 8 shows the results of epitope recognition detection of chimeric antibodies.

[0139] Figure 9A shows the results of endocytosis activity detection of antibodies of the present application in MKN45 cells.

[0140] Figure 9B shows the results of endocytosis activity detection of antibodies of the present application in LS180 cells.

[0141] Figure 10 shows the results of tumor inhibition of antibody drug conjugates in a human colon cancer xenograft subcutaneous tumor PDX model. DETAILED DESCRIPTION

[0142] The above features and advantages of the present application, and additional features and advantages thereof, will be more clearly understood from consideration of the following detailed description when taken in conjunction with the accompanying drawings.

[0143] The embodiments described herein with reference to the accompanying drawings are explanatory, illustrative, and serve the purpose of general understanding of the present application. The embodiments should not be construed as limiting the scope of the present application. Identical or similar elements and elements having identical or similar functions are denoted by the same reference numerals throughout the specification.

[0144] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operation steps used herein are terms and conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of the related terms are provided below.

[0145] Definitions

[0146] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and reference to "an antibody" in some embodiments includes a plurality of antibodies, and so forth.

[0147] The term "comprising" and variations thereof as used herein are intended to mean including, but not excluding, any additional element or step. Thus, for example, a composition that comprises A and B also includes the possibility of comprising A, B, or both A and B.

[0148] As used herein, "and / or" means and, or, both, or any combination thereof, and includes any and all possible combinations of one or more of the associated listed items. For example, a composition comprising A and / or B can be interpreted to mean that the composition comprises A, the composition comprises B, or the composition comprises A and B.

[0149] As used herein, the term "about" generally means within 0.5%-10% above or below the specified value, e.g., within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0150] CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5), also known as Carcinoembryonic antigen (CEA) or CD66e, is a membrane protein widely used for tumor marker monitoring. CEACAM5 is composed of a large N-terminal ectoplasmic region, seven extracellular immunoglobulin-like domains, a transmembrane region and a short intracellular region. The extracellular immunoglobulin-like domains can be divided into N, A, B domains, i.e., N, A1-A3, B1-B3, a total of seven domains. This unique structure enables CEACAM5 to participate in intercellular adhesion processes and regulate various biological functions such as cell proliferation, differentiation and apoptosis.

[0151] The amino acid sequence of human CEACAM5 is shown below:

[0152] wherein the amino acids at positions 35-144 are an N domain, the amino acids at positions 145-232 are an Al domain, the amino acids at positions 240-315 are a Bl domain, the amino acids at positions 323-410 are an A2 domain, the amino acids at positions 418-495 are a B2 domain, the amino acids at positions 501-588 are an A3 domain, and the amino acids at positions 593-675 are a B3 domain.

[0153] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind a particular antigen. Such molecules typically comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the antibody heavy and light chains contain the binding sites for the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq (the first component in the classical pathway of complement activation).

[0154] The heavy chain of an immunoglobulin can be divided into three functional regions: an Fd region, a hinge region, and an Fc region (fragment, crystallizable). The Fd region comprises the VH and CH1 domains and, together with the light chain, forms the Fab (fragment, antigen binding) portion of an immunoglobulin. The Fc fragment is responsible for the effector functions of an immunoglobulin, including, for example, complement binding and binding to Fc receptors on effector cells. The hinge region, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer, allowing the Fab portions to move freely in space relative to the Fc region. Hinge domains are structurally diverse, differing in sequence and length between immunoglobulin classes and subclasses.

[0155] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions based on structure and function: the upper hinge, the core hinge, and the lower hinge. The upper hinge includes the amino acids from the carboxy-terminal end of CH1 to the first residue in the hinge that restricts movement, usually the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the fragment flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bonds. The lower hinge region connects the amino-terminal end of the CH2 domain and includes residues in the CH2 domain. Structural and flexibility-allowed conformational changes in the immunoglobulin hinge region polypeptide sequence can affect the effector functions of the Fc portion of an antibody.

[0156] A "heavy chain variable region" (VH) or "light chain variable region" (VL) is comprised of "framework" regions interrupted by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that specifically bind to an epitope of an antigen. The CDRs include the amino acid residues in an antibody that are primarily responsible for antigen binding. Both the VL and VH domains comprise, from amino-terminus to carboxyl-terminus, the following framework regions (FRs) and CDRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of a VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; and the CDR1, CDR2, and CDR3 of a VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0157] The amino acid arrangement of each VH and VL domain is consistent with any conventional definition of CDRs. Conventional definitions include, but are not limited to, the IMGT definition ((Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); the composite of Chothia and Kabat CDRs, in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). The disclosure can use CDRs defined according to any of these numbering systems, i.e., the Kabat definition, the Chothia definition, the Combined definition rule incorporating both the Kabat and Chothia definitions, the AbM definition, the CONTACT definition, the IMGT definition, etc., but preferred embodiments use CDRs defined by the IMGT definition or the Combined definition. CDR sequences determined according to the CDR definition rules commonly used in the art of antibodies are shown in the following table.

[0158] Based on the amino acid sequence of the heavy chain constant region, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subclasses, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of an antibody can be assigned to either a lambda (l) chain or a kappa (K) chain.

[0159] As used herein, the term "antibody" is to be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), antibody fragments, and multi-specific antibodies (e.g., bi-specific antibodies) containing at least two antigen binding regions. Antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0160] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, each antibody in the population is identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced through hybridoma technology, nor is it to be construed that antibodies are necessarily produced by any particular method.

[0161] The term "bi-specific antibody" is to be understood in the context of the present application as an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of one target. The term "bi-specific antibody" as used herein is to be understood in its broadest sense, including full-length bi-specific antibodies and antigen binding fragments thereof. Bi-specific antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Bi-specific antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modification to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0162] The term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody elicits a reduced likelihood of adverse immune reactions in a human individual as compared to the parental (e.g., mouse-derived) antibody.

[0163] The term "humanized antibody" generally refers to an antibody in which some or all amino acids of non-human origin have been replaced by corresponding amino acids of human origin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids can also be permissible, as long as they still retain the ability of the antibody to bind to a particular antigen. A humanized antibody can optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to that of the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies can minimally contain a chimeric antibody that contains sequences derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be replaced by CDR region residues of a non-human species (donor antibody) such as a mouse, rat, rabbit or non-human primate that have the desired properties, affinities and / or capabilities. In some cases, FR region residues of a human immunoglobulin can be replaced by corresponding non-human residues. Furthermore, a humanized antibody can comprise amino acid modifications not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity.

[0164] The term "fully human antibody" generally refers to an antibody that is expressed by a genetically engineered antibody knockout animal into which human antibody-encoding genes have been transferred, so that the animal expresses the antibody. All parts of the antibody, including the variable region and the constant region of the antibody, are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side reactions caused by heterologous antibodies in humans. Methods for obtaining fully human antibodies in the art can include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-polypeptide technology, etc.

[0165] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

[0166] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is simply a Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment consisting of the VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); (ix) a Nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Furthermore, the term also includes "linear antibodies", which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) with dual variable domain (D3) antibodies, which form a pair of antigen binding regions with a complementary light chain polypeptide, as well as modifications of any of the foregoing structures.

[0167] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of the fragments screened in the same manner as for whole antibodies.

[0168] As used herein, the term "binds" or "binds specifically" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity represents the equilibrium constant for dissociation of an antigen from an antibody (KD), a measure of the strength of binding between an antigenic determinant and an antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD.

[0169] Avidity is a measure of the strength of binding between an antibody and the relevant antigen. Avidity involves both the affinity between the antigenic determinant and the antigen binding site of an antibody and the number of relevant binding sites present on the antibody. Typically, an antibody will bind an antigen with a dissociation constant (KD) of 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 12 M or less, and more preferably 10-8 M to 10- 12 M, and / or have a binding affinity of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , such as at least 10 12 M -1 . It is generally accepted that any K -4 M value greater than 10 D M indicates non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, as well as different variants thereof known in the art.

[0170] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein or proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes usually comprise at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Epitopes define the minimum binding site of an antibody and are thus the specific targets of an antibody or antigen binding fragment thereof.

[0171] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acid) have identical residues in the same positions after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y and is set forth as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0172] Furthermore, in determining the extent of sequence identity between two amino acid sequences, the skilled person can take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions of an amino acid residue for another amino acid residue having a similar chemical structure that has little or essentially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0173] Such conservative substitutions are preferably substitutions of one amino acid for another within the following groups (a) to (e): (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0174] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; He to Leu or to Val; Leu to He or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to He; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to He, or to Leu.

[0175] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and that replicates in a suitable host cell into which it is introduced, and / or transfers into a host cell and / or between host cells.

[0176] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0177] The term "pharmaceutically acceptable" means that which the carrier or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof, and / or such carrier or excipient is approved or approvable by a regulatory agency of the Federal or a state government of the United States of America or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for inclusion in a drug product for administration to humans.

[0178] As used herein, the terms "treat," "treatment," "therapy," and the like, refer to the application of an agent or performance of a procedure for the purpose of effecting an outcome. The outcomes can be prophylactic, in terms of completely or partially preventing a disease or symptom thereof, and / or therapeutic, in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or condition (e.g., an inflammatory disease) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treatment can refer to any successful indication in the treatment or amelioration or prevention of cancer, including any objective or subjective parameters, such as reduction of symptoms; alleviation of disease symptoms or making the disease condition more tolerable to a patient; slowing in rate of decline or degeneration; or lessening of the disease's last node of deterioration. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including results of a physician examination. Thus, the term "treatment" includes the administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or ameliorate a symptom or condition associated with a disease (e.g., an inflammatory disease). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease or a side effect of a disease in a subject.

[0179] As used herein, the term "effective amount" refers to an amount of an agent administered to a subject to treat a disease sufficient to effect treatment of the disease.

[0180] As used herein, the term "subject" refers to any mammalian subject in which diagnosis, treatment or therapy is desired. A "mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory and sports or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0181] Anti-CEACAM5 antibodies

[0182] The present disclosure provides an antibody or antigen-binding fragment thereof that binds to CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0183] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 75, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 76.

[0184] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 102.

[0185] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 103.

[0186] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104.

[0187] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 105, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104.

[0188] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 106, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104.

[0189] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 107, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104.

[0190] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 71, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 72.

[0191] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 67, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 68.

[0192] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 69, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 70.

[0193] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 73, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 74.

[0194] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 77, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 78.

[0195] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 79, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 80.

[0196] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 81, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 82.

[0197] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 83, and the VL comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 84.

[0198] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 85, and the VL comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 86.

[0199] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 87, and the VL comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 88.

[0200] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 89, and the VL comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 90.

[0201] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 91, and the VL comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 92.

[0202] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 93, and the VL comprises LCDR1, LCDR2, and LCDR3 in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 94.

[0203] In this document, CDR sequences can be defined according to any CDR definition rule known in the art. In some embodiments, CDR sequences are defined according to IMGT, Kabat, Chothia, Combined, AbM, or CONTACT definition rules. In a preferred embodiment, CDR sequences are defined according to IMGT definition rules.

[0204] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 18-20, respectively, and the VL comprises LCDR 1 having the amino acid sequence set forth in SEQ ID NO: 21, LCDR2 having the SAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 22.

[0205] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 1, 11, and 3, respectively, and the VL comprises LCDR 1 having the amino acid sequence set forth in SEQ ID NO: 4, LCDR2 having the AAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 12.

[0206] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 1-3, respectively, and the VL comprises LCDR 1 having the amino acid sequence set forth in SEQ ID NO: 4, LCDR2 having the AAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 5.

[0207] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 6-8, respectively, and the VL comprises LCDR 1 having the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 having the WAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 10.

[0208] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 13-15, respectively, and the VL comprises LCDR 1 having the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 having the LAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 17.

[0209] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 23-25, respectively, and the VL comprises LCDR 1 having the amino acid sequence set forth in SEQ ID NO: 26, LCDR2 having the FAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 27.

[0210] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28-30, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 having the SAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 32.

[0211] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 33-35, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 36, LCDR2 having the AAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 37.

[0212] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 38-40, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 41, LCDR2 having the YAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42.

[0213] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28, 43, and 44, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 45, LCDR2 having the SAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 46.

[0214] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 47-49, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 50, LCDR2 having the NAK amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 51.

[0215] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 52-54, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 55, LCDR2 having the ATS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 56.

[0216] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 57-59, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 60, LCDR2 having the NAK amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 61.

[0217] In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 62-64, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 65, LCDR2 having the ATS amino acid sequence, and LCDR 3 having the amino acid sequence set forth in SEQ ID NO: 66.

[0218] In other preferred embodiments, the CDR sequences are defined according to the Combined definition rules. In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 95-97, respectively, and the VL comprises LCDR1-3 having the amino acid sequences set forth in SEQ ID NOs: 98-100, respectively. In some embodiments, the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 95, 108, and 97, respectively, and the VL comprises LCDR1-3 having the amino acid sequences set forth in SEQ ID NOs: 98-100, respectively.

[0219] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0220] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 102.

[0221] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 103.

[0222] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

[0223] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 105, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

[0224] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 106, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

[0225] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 107, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104.

[0226] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72.

[0227] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68.

[0228] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70.

[0229] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74.

[0230] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.

[0231] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80.

[0232] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.

[0233] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84.

[0234] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 85, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86.

[0235] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 87, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 88.

[0236] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 89, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 90.

[0237] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 91 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 92.

[0238] In some embodiments, the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 93 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 94.

[0239] In some embodiments, the VH comprises a functional variant of an amino acid sequence as disclosed above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to CEACAM5. In some embodiments, the VL comprises a functional variant of an amino acid sequence as disclosed above by insertion, deletion and / or substitution of one or more amino acids thereof, provided that the functional variant retains the ability to bind to CEACAM5.

[0240] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0241] In the context of a functional variant, the number of inserted, deleted and / or substituted amino acids preferably does not exceed 40%, more preferably does not exceed 35%, more preferably is between 1 and 33%, more preferably is between 5 and 30%, more preferably is between 10 and 25%, more preferably is between 15 and 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and / or substituted amino acids can be between 1 and 20, preferably between 1 and 10, more preferably between 1 and 7, still more preferably between 1 and 5, most preferably between 1 and 2. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6 or 7.

[0242] In some embodiments, the insertion, deletion, and / or substitution can be made in a framework (FR) region, e.g., in FR1, FR2, FR3, and / or FR4.

[0243] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions preferably are substitutions of one amino acid for another within a group (a) to (e) below: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0244] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; He to Leu or to Val; Leu to He or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to He; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to He, or to Leu.

[0245] In preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 75, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 76.

[0246] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 102.

[0247] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 103.

[0248] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104.

[0249] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 105, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104.

[0250] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 106, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104.

[0251] In some embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 107, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104.

[0252] In another preferred embodiment, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 71, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 72.

[0253] In another preferred embodiment, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 67, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 68.

[0254] In another preferred embodiment, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 69, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 70.

[0255] In another preferred embodiment, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 73, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 74.

[0256] In another preferred embodiment, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 77, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 78.

[0257] In another preferred embodiment, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 79, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 80.

[0258] In another preferred embodiment, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 81, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 82.

[0259] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 83, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 84.

[0260] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 85, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 86.

[0261] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 87, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 88.

[0262] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 89, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 90.

[0263] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 91, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 92.

[0264] In other preferred embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO: 93, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 94.

[0265] In some embodiments, the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. In preferred embodiments, the antibody is a chimeric antibody.

[0266] Based on the amino acid sequences of the constant regions of the heavy chains, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. Based on the amino acid sequences of the light chains, the light chains of antibodies can be assigned to either lambda (l) chain or kappa (K) chain. The antibodies disclosed herein can be of any of the above classes or subclasses.

[0267] In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is of a subclass selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgGl antibody.

[0268] In some embodiments, the antibody is a monovalent, bivalent, or multivalent antibody.

[0269] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0270] In some embodiments, the antibody is a monoclonal antibody and comprises a heavy chain and a light chain. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 117. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 118. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 119. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 120, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 119. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 121, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 119.In some embodiments, the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 122, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119. In some embodiments, the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 124.

[0271] In preferred embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 116, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 117. In other preferred embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 116, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 118. In other preferred embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 116, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119. In other preferred embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 120, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119. In other preferred embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 121, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119. In other preferred embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 122, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119. In other preferred embodiments, the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 123, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 124.

[0272] In some embodiments, the antibody is a bispecific antibody further comprising a second antigen binding region that binds to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen or an immune cell antigen.

[0273] A number of tumor-associated antigens have been identified in the art that are associated with particular cancers. In some embodiments, the tumor-associated antigen is an antigen that can elicit a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are normally silent (i.e., not expressed) in normal cells, antigens that are expressed only at certain stages of differentiation, and antigens that are expressed over time such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried by RNA and DNA tumor viruses. A number of other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be prepared by those skilled in the art.

[0274] Examples of tumor-associated antigens include, but are not limited to, 5T4, alphafetoprotein, CA-125, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.

[0275] In some embodiments, the second antigen is a T cell antigen. In some embodiments, the T cell antigen is selected from the group consisting of a T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137), and NKG2D, or any combination thereof. In some embodiments, the T cell antigen is CD3, and the second antigen binding region binds to any one of the gamma, delta, epsilon, zeta, and eta chains of CD3.

[0276] The antibodies disclosed herein can be intact antibodies or antigen-binding fragments thereof. The antigen-binding fragment can be any fragment of an antibody that retains the ability to specifically bind to CEACAM5. Examples of antigen-binding fragments include, but are not limited to: Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fd segments (VH-CH1-VH-CH1), and modified forms of any of the foregoing fragments that retain antigen binding activity.

[0277] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In a preferred embodiment, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In another preferred embodiment, the antigen-binding fragment is scFv.

[0278] In some embodiments, the antibodies disclosed herein, or antigen-binding fragments thereof, bind to CEACAM5 A3-B3, CEACAM5 A2-B2, and / or CEACAM5 A1-B1, preferably to CEACAM5 A3-B3.

[0279] The antibodies disclosed herein can comprise an Fc region. The Fc region can be of any isotype, including but not limited to IgGl, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is of, or is derived from, an IgGl or IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgGl Fc.

[0280] In some embodiments, the Fc region has reduced effector function, e.g., reduced ADCC, ADCP, CDC, and / or Clq, FcyRI, FcyRII, or FcyRIIIA binding. For example, the Fc region can be of an IgGl isotype, or of a non-IgGl type, e.g., IgG2, IgG3, or IgG4, that has been mutated such that the ability to mediate effector function is reduced or even eliminated. Such mutations have been described, e.g., in Dall'Acqua WF, et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001). For example, the Fc region can comprise an amino acid sequence with one or more of the following amino acid substitutions compared to the wild-type sequence: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G.

[0281] In one embodiment, the Fc region comprises a mutation that removes the Asn-linked glycosylation acceptor site or is otherwise manipulated to alter the glycosylation properties. For example, in an IgGl Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Thus, in a specific embodiment, the Fc region comprises an IgGl sequence with the N297Q mutation.

[0282] In further embodiments, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, the method described in Shields et al. (1999) Nature Biotech 17:176 can be used to optimize ADCC. In another embodiment, the Fc region is engineered to enhance complement activation, as described in Natsume et al. (2009) Cancer Sci. 100:2411. In another embodiment, the Fc region is engineered to enhance complement activation, as described in Natsume et al. (2009) Cancer Sci. 100:2411.

[0283] In other embodiments, the Fc region has an extended serum half-life. For examples of altering (e.g., decreasing or increasing) the in vivo half-life of an antibody, see, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631; and U.S. Patent Nos. 5,869,046; 6,121,022; 6,277,375; and 6,165,745, all of which are incorporated herein by reference in their entireties. In some embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to decrease the in vivo half-life of the antibody. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, deletions, or insertions) are introduced into an IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to increase the in vivo half-life of the antibody. In a specific embodiment, the antibody can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgGl) and / or the third constant (CH3) domain (residues 341-447 of human IgGl) (numbering according to the EU numbering system).

[0284] Nucleic acid

[0285] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof disclosed herein.

[0286] The term "nucleic acid" includes single- and double-stranded nucleotide polymers. The nucleic acid can be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phenylphosphonothioate, phenylphosphonodithioate, and phosphoramidate.

[0287] In some embodiments, the present application provides a nucleic acid molecule encoding any of the heavy chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the heavy chain variable region sequences disclosed herein. In some embodiments, the present application provides a nucleic acid molecule encoding any of the light chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of the light chain variable region sequences disclosed herein.

[0288] In some embodiments, the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In some embodiments, the present application provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present application provides a deoxyribonucleic acid (DNA) comprising a deoxyribonucleotide sequence encoding an antibody disclosed herein.

[0289] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into a human cell in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is comprised in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is integrated into the genome of a cell.

[0290] In some embodiments, the ribonucleic acid (RNA) can be introduced into a human cell in vivo. In some embodiments, the ribonucleic acid (RNA) of the present application is comprised in a vector or delivery agent.

[0291] Vectors

[0292] The present disclosure provides vectors comprising a nucleic acid disclosed herein.

[0293] In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising a heavy chain or light chain variable region of an antibody. For example, the present application provides an expression vector comprising any of the nucleic acid molecules described above.

[0294] Any vector can be suitable for use in the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo large T cDNA, pBABE-hygro-hTERT, pMKO.lGFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0295] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, La Jolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as lambda GT10, lambda GT11, lambda Zap II (Stratagene), lambda EMBL4, and lambda NM1149, can also be used. Examples of plant expression vectors useful in the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0296] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain replication systems functional in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from COLEL, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0297] For example, the vector can be an adenoviral vector comprising a nucleotide sequence encoding an antibody disclosed herein. The vector can be administered into a subject, then in vivo into a cell of the subject, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the genome of the cell, and subsequently the cell expresses the antibody disclosed herein.

[0298] Host cells

[0299] The present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0300] Any cell can be used as a host cell for the nucleic acids or vectors of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobactehaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0301] The host cells of the present application are prepared by introducing the vectors disclosed herein or the nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present application can be administered to a subject in vivo, and the host cells express the antibodies disclosed herein in vivo.

[0302] Methods of manufacture

[0303] The present disclosure provides methods of making the antibodies or antigen-binding fragments thereof disclosed herein, comprising culturing the cells provided by the present application under conditions suitable for expression of the antibodies or antigen-binding fragments thereof disclosed herein; and isolating the antibodies or antigen-binding fragments thereof from the culture and / or culture supernatant of the cells.

[0304] Any method suitable for producing antibodies can be used to produce the antibodies of the present application. For example, the antibodies can be prepared using the hybridoma method, such as described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0305] The immunizing agent typically includes a protein antigen, a fragment thereof, or a fusion protein thereof. In this context, any suitable form of CEACAM5 can be used as an immunizing agent (antigen) for the production of non-human antibodies specific for CEACAM5, screening of the biological activity of the antibodies. For example, antigens suitable for use in the present application include, but are not limited to, full-length human CEACAM5, full-length cynomolgus CEACAM5, or human CEACAM5 A3-B3 peptide.

[0306] The immunizing agent can be used alone or in combination with one or more immunogenicity enhancers known in the art, such as Freund's adjuvant. Typically, if human cells are desired, peripheral blood lymphocytes are used; if non-human mammalian sources are desired, spleen cells or lymph node cells are used. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevents the growth of HGPRT-deficient cells.

[0307] The culture medium in which the hybridoma cells are cultured can then be assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeutic applications, it is important to identify antibodies that have high specificity for the target antigen and high binding affinity.

[0308] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purposed include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in mammals. The monoclonal antibodies secreted by the subclones can be separated from the culture medium or the ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0309] Monoclonal antibodies can also be made by recombinant DNA methods, such as described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the application can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the application serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulins, to obtain the synthesis of recombinant antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or a portion of the coding sequence for a polypeptide which is not an immunoglobulin, such as a tissue factor or a portion thereof. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the application or can be substituted for the variable domains of one antigen-combining site of an antibody of the application to create a chimeric bivalent antibody.

[0310] The sequences of the antibodies of the present application can be obtained using conventional techniques, such as by PCR amplification or screening of genomic libraries, among others. In addition, the coding sequences for the light and heavy chains can be fused together to form a single chain antibody.

[0311] Once the relevant sequences are obtained, the antibodies can be obtained in large quantities using recombinant methods. This is typically done by cloning the sequences into vectors, which are then transferred into cells, and the antibodies are then isolated from the propagated host cells using conventional methods. In addition, the relevant sequences can be synthesized using artificial synthesis, particularly for shorter fragments. Typically, longer fragments are obtained by first synthesizing smaller fragments, which are then ligated together. The nucleic acid molecule can then be introduced into various existing vectors known in the art, which can be used to transform appropriate host cells to enable expression of the protein, thereby obtaining the antibodies of the present application.

[0312] Conjugates

[0313] The present disclosure provides conjugates comprising an antibody or antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

[0314] In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates." When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC."

[0315] Accordingly, the present disclosure provides antibody drug conjugates comprising an antibody or antigen binding fragment thereof disclosed herein and a chemical moiety conjugated to the antibody or antigen binding fragment thereof via a linker, wherein the chemical moiety is a therapeutic agent.

[0316] The term "conjugate" or "linkage" can refer to the joining of two polypeptides into one continuous polypeptide molecule. In one embodiment, the antibody is linked to a chemical moiety. In another embodiment, the antibody linked to a chemical moiety is further linked to a lipid or other molecule to the protein or peptide to increase its half-life in vivo. The linkage can be by chemical or recombinant means. In one embodiment, the linkage is chemical, wherein a reaction between the antibody moiety and the chemical moiety results in a covalent bond formed between the two molecules to form one molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the chemical moiety.

[0317] Chemical moieties can be attached to the antibodies of the application using any number of means known to those of skill in the art. Both covalent and non-covalent modes of attachment can be used. The procedure for attaching a chemical moiety to an antibody varies depending on the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2) or thiol (-SH) moieties, which can be used to react with appropriate functional groups on the antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any of a number of known linker molecules. A linker can be any molecule used to attach an antibody to a chemical moiety. The linker is capable of forming a covalent bond to both the antibody and the chemical moiety. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the chemical moiety are polypeptides, the linker can be attached to a constituent amino acid through a side group (such as through a disulfide bond to a cysteine) or to the alpha carbon amino and carboxyl groups of terminal amino acids.

[0318] The linker can be susceptible to cleavage (cleavable linker), such as acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide cleavage, under conditions in which the compound or antibody remains active. Alternatively, the linker can be substantially resistant to cleavage (e.g., stable or noncleavable linker).

[0319] In some embodiments, the linker comprises a cleavable linker or a noncleavable linker. For example, the linker can be selected from, for example, an acid-labile linker, a light-labile linker, a protease-cleavable linker, a hydrazone linker, an esterase-cleavable linker, a dimethyl linker, a disulfide-containing linker, a hydrophilic linker, a charged linker, and an acid group linker. In preferred embodiments, the linker is a cathepsin-cleavable linker, preferably a cathepsin B-cleavable linker.

[0320] In some embodiments, the linker can comprise an amino acid unit. In one such embodiment, the amino acid unit allows for protease cleavage of the linker, thereby facilitating release of the drug from the antibody drug conjugate upon exposure to intracellular proteases, e.g., lysosomal enzymes. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys): or N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker comprises a vc (valine-citrulline) unit. The amino acid units can be designed and optimized to be selectively cleaved by a particular enzyme, e.g., a tumor-associated protease, cathepsins B, C, and D, or plasmin protease.

[0321] In some embodiments, the linker is selected from MC (6-maleimidocaproyl), Val-Cit (valine-citrulline), PABC (p-amino-benzyloxycarbonyl), DMEA (dimethyl ethylamine), Val-Cit-PABC, MC-Val-Cit-PABC, MC-Val-Cit-PABC-DMEA, GGFG (glycine-glycine-phenylalanine-glycine), MC-GGFG, AcBut (4-(4-acetylphenoxy)-butyric acid), and AcBut-dimethyl hydrazide. In a preferred embodiment, the linker is MC-GGFG.

[0322] In certain cases, it is desirable to release the chemical moiety from the antibody when the immunoconjugate reaches its target site. Thus, in these cases, the immunoconjugate will comprise a linkage that is cleavable in the vicinity of the target site.

[0323] The conditions experienced by the enzymatic activity or immunoconjugate within the target cell or in the vicinity of the target site can prompt cleavage of the linker to release the chemical moiety from the antibody.

[0324] In view of the large number of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, markers (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one of skill in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0325] The antibodies disclosed herein can be derivatized or linked to another molecule, such as another peptide or protein. Typically, the antibody or portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association with another molecule, such as a streptavidin core region or a polyhistidine tag.

[0326] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same or different type). Suitable cross-linking agents include heterobifunctional or homobifunctional cross-linking agents having two distinctly reactive moieties separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or a sulfosuccinimidyl propionate. Such linkers are commercially available.

[0327] In some embodiments of the conjugates disclosed herein, the chemical moiety is selected from a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0328] In some embodiments, the therapeutic agent includes, but is not limited to, an immunomodulatory agent, a radioactive compound, an enzyme (e.g., a perforin), a chemotherapeutic agent (e.g., cisplatin), or a toxin. In some embodiments, the therapeutic agent is selected from a topoisomerase inhibitor, a microtubule inhibitor, an antibiotic, a DNA synthesis inhibitor, an RNA polymerase II inhibitor, and an RNA spliceosome inhibitor. In some embodiments, the therapeutic agent can be, for example, a maytansinoid, a geldanamycin, a tubulin inhibitor such as a tubulin binding agent (e.g., an auristatin) or a minor groove binder such as a calicheamicin. In some embodiments, the therapeutic agent can be, for example, MMAE, Dxd, or a derivative thereof. In some embodiments, the therapeutic agent is a topoisomerase inhibitor, preferably a topoisomerase I inhibitor, such as Dxd and SN-38. In preferred embodiments, the therapeutic agent is Dxd.

[0329] In some embodiments, the linker-load moiety conjugated to the antibody comprises MC-GGFG-Dxd, SPDB-DM4, vc-MMAD, vc-MMAE, vc-calicheamicin, or PEG2-vc-MMAD. In preferred embodiments, the linker-load moiety conjugated to the antibody is MC-GGFG-Dxd. MC-GGFG-Dxd has the following structure:

[0330] In some embodiments, the detectable moiety can be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

[0331] The antibody can be conjugated to a detectable label; for example, a detectable label that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy. Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent labels such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) can also be used.

[0332] The antibody or antigen-binding fragment can also be conjugated to an enzyme that can be used for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When the antibody or antigen-binding fragment is conjugated to a detectable enzyme, detection can be accomplished by the addition of an additional reagent with which the enzyme acts to produce a recognizable reaction product. For example, when horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product, which can be detected visually. The antibody or antigen-binding fragment can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0333] The antibody can be fused to a self-labeling protein tag, such as a HaloTag. For example, the protein tag can be cloned to the end of the constant region. HaloTag is a self-labeling protein tag derived from a bacterial enzyme (haloalkane dehalogenase) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

[0334] The antibody can be labeled with a magnetic agent, such as gadolinium. The antibody can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0335] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with predetermined polypeptide epitopes recognized by a second reporter molecule (e.g., a leucine zipper pair sequence, a binding site for a second antibody, a metal binding domain, an epitope tag). In some embodiments, the tag is attached with spacers of various lengths to reduce potential steric hindrance.

[0336] Antibodies can also be labeled with radiolabeled amino acids. Radiolabeling can be used for both diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect expression of a target antigen by X-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H、 14 C、 15 N、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I.

[0337] In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule can be a cytokine such as IL-2 and IFN-gamma, a chemokine such as IL-8, platelet factor 4, melanoma growth stimulatory protein, a complement activator; a viral / bacterial protein domain, or a viral / bacterial peptide.

[0338] Chimeric antigen receptor

[0339] The present disclosure provides a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof disclosed herein. The present disclosure also provides a genetically modified cell comprising a chimeric antigen receptor disclosed herein.

[0340] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule engineered to contain an antigen binding domain that targets a specific antigen and, when bound to that antigen, activates an immune cell (e.g., a T cell or NK cell, such as a naive T cell, central memory T cell, effector memory T cell, or a combination thereof) to attack and destroy cells bearing that antigen. When these antigens are present on tumor cells, the CAR-expressing immune cells can target and kill the tumor cells.

[0341] A classic chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that links an extracellular antigen binding domain to an intracellular signaling domain. The antigen binding domain is usually an antigen binding fragment derived from a monoclonal antibody (mAb), for example a scFv, but it can be based on other formats containing antibody-like antigen binding sites or antigen binding domains derived from natural ligands of the antigen. A hinge domain is usually required to separate the antigen binding domain from the membrane and allow its proper orientation. A common hinge domain used is the Fc of IgGl. Depending on the antigen, a more compact spacer can suffice, for example the stalk from CD8a, and even just the IgGl hinge alone. The transmembrane domain anchors the protein in the cell membrane and links the hinge domain to the intracellular domain (endodomain).

[0342] According to at least one non-limiting view, there have been at least three“generations” of CAR molecules. In the first generation CAR, it was designed to have an intracellular domain derived from the intracellular portion of the gamma chain of Fc epsilon Rl or CD3 zeta. Thus, these first generation CARs transmit immune signal 1, which is sufficient to trigger T cell killing of cognate target cells, but is not able to fully activate T cell proliferation and survival. To overcome this limitation, second generation CARs have been constructed, which have a composite endodomain generated by fusing the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3 zeta, so that both an activation signal and a costimulatory signal can be transmitted upon antigen recognition. The most commonly used costimulatory domain is that of CD28. This provides the most potent costimulatory signal, immune signal 2, which triggers T cell proliferation. Some CARs have also been described that include TNF receptor family endodomains, such as the closely related OX40 and 41BB, which transmit survival signals. Even more potent third generation CARs have now been described, which have endodomains capable of transmitting activation, proliferation, and survival signals.

[0343] Thus, a CAR typically comprises: (i) an antigen binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain comprising a signaling domain and one or more costimulatory domains.

[0344] By“antigen binding domain” is meant the portion of the chimeric antigen receptor that recognizes an antigen. In classic CARs, the antigen binding domain comprises a single chain variable fragment (scFv) derived from a monoclonal antibody. CARs have also been produced with domain antibodies (dAbs), VHH antigen binding domains, or antigen binding domains derived from natural ligands of the antigen. In the present application, the antigen binding domain can be an antibody or antigen binding fragment thereof as described herein.

[0345] A“hinge domain” positions the antigen binding domain away from the surface of an effector cell to enable proper cell / cell contact, antigen binding, and activation to function. A CAR optionally includes one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise an amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular region of type 1 membrane proteins such as CD8 (e.g., CD8a), CD4, CD28, 4-1BB, and CD7, which can be from the wild-type hinge region of these molecules or can be altered.

[0346] A“transmembrane domain” (TM domain) refers to the portion of the CAR that fuses the extracellular binding portion to the intracellular portion (e.g., costimulatory and intracellular signaling domains) and anchors the CAR to the plasma membrane of an immune effector cell, optionally via a hinge domain. The transmembrane domain is typically a hydrophobic region of the CAR that spans the plasma membrane. The TM domain can be a transmembrane region of a transmembrane protein (e.g., a type 1 transmembrane protein or other transmembrane protein) or a fragment thereof, an artificial hydrophobic sequence, or a combination thereof.

[0347] An“intracellular domain (endodomain)” is the signaling portion of a chimeric antigen receptor. It contains a signaling domain and one or more costimulatory domains. Upon antigen recognition, the receptor clusters naturally CD45 and CD148 away from the synapse, and signals to the cell, thereby activating one or more immune cell effector functions (e.g., a natural immune cell effector function). The most commonly used endodomain component is the endodomain component of CD3 zeta, which contains 3 ITAMs. Upon antigen binding, it transmits an activation signal to the T cell. CD3 zeta can not provide a fully sufficient activation signal, and additional costimulatory signaling can be needed. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and those belonging to the TNF family (OX40, 41BB, CD27, GITR, etc.).

[0348] An "intracellular signaling domain" refers to the portion of a CAR polypeptide that is involved in transducing the information of effective CAR binding to a target antigen into the interior of an immune effector cell to elicit effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors toward target cells to which the CAR binds, or other cellular responses elicited upon antigen binding to the extracellular CAR domain. Non-limiting examples of intracellular signaling domains containing immunoreceptor tyrosine-based activation motifs (ITAMs) include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 theta, CD3 delta, CD3 eta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0349] A "costimulatory domain" refers to an intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide co-stimulatory signals required for efficient activation and function of T lymphocytes upon antigen binding. The costimulatory domain can be, for example, a costimulatory domain of 4-1BB, CD27, CD28, or OX40.

[0350] Pharmaceutical compositions

[0351] The present disclosure provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid described herein, a vector described herein, a host cell described herein, a conjugate described herein, or a CAR described herein, and optionally a pharmaceutically acceptable carrier or excipient.

[0352] The antibodies or antigen-binding fragments thereof of the present application (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise an antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Such vehicles and agents for use in

[0353] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0354] The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended routes of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), mucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as tetraethylamine oxalate (EDTA); buffers, such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or dextran. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0355] Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid present in a manner easily injectable. It must be stable under preparation and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. The action of microorganisms can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. The absorption of injectable compositions can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0356] A sterile injectable solution can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of a sterile powder, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0357] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0358] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or a nebulizer or a dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide.

[0359] Systemic administration can also be by way of transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into a

[0360] The active compounds can also be prepared in the form of suppositories (e.g., medical delivery by use of conventional binding agents such as natural or hardened oils, waxes, fats, and semi-solid or liquid polyols such as polyethylene glycols) or in the form of retention enemas.

[0361] In one embodiment, the active compound is prepared with an agent that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are apparent to those skilled in the art.

[0362] The present application provides therapeutic compositions comprising the antibodies or antigen binding fragments thereof of the present application. The therapeutic compositions according to the present application will be administered with suitable carriers, excipients and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Many suitable formulations are found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions with various types of polyols, semi-solid gels, and semi-solid mixtures with polyols. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0363] Kit

[0364] The present disclosure provides kits comprising an antibody or antigen binding fragment thereof disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, a host cell described herein, a conjugate described herein, or a CAR described herein, or a pharmaceutical composition disclosed herein.

[0365] In certain instances, the kits can include instructions for use, including information about the antibody, nucleic acid, vector, host cell, conjugate, CAR, or pharmaceutical composition in the kit and how to use them. Typically, such information aids patients and physicians in the effective and safe use of the encapsulated antibody, nucleic acid, vector, host cell, conjugate, CAR, or pharmaceutical composition. Containers to be used in such kits are typically at least one vial, test tube, flask, bottle, syringe or other container suitable for use with the antibody, nucleic acid, vector, host cell, conjugate, CAR, or pharmaceutical composition.

[0366] In some embodiments, the present application provides a kit, which can comprise an antibody or antigen-binding fragment thereof disclosed herein, a nucleic acid described herein, a vector described herein, a host cell described herein, a conjugate described herein, or a CAR described herein, or a pharmaceutical composition described herein, instructions for administration of the foregoing, and an administration device for administration of the foregoing. The administration device can introduce one of the foregoing into a patient by a parenteral route, e.g., intramuscularly, subcutaneously, or intravenously. For example, the administration device can be a syringe (e.g., a pre-filled syringe with an antibody or pharmaceutical composition of the present application, such as an auto-injector), which can include a barrel for containing a fluid to be injected (e.g., an antibody or pharmaceutical composition of the present application) and a needle (which can be used to pierce the skin and / or a blood vessel). The mode of administration can vary. Routes of administration can include oral, intramuscular injection, subcutaneous injection, rectal administration, and the like.

[0367] Methods of treatment and uses

[0368] The present disclosure provides a method of treating a cancer in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, a conjugate described herein, or a CAR described herein, or a pharmaceutical composition described herein.

[0369] The present disclosure also provides the use of an antibody or antigen-binding fragment thereof described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, a conjugate described herein, or a CAR described herein, or a pharmaceutical composition described herein, in the manufacture of a medicament for treating a cancer in a subject.

[0370] The present disclosure also provides an antibody or antigen-binding fragment thereof described herein, a nucleic acid described herein, a vector described herein, a host cell described herein, a conjugate described herein, or a CAR described herein, or a pharmaceutical composition described herein, for use in treating a cancer in a subject.

[0371] In some embodiments, the cancer is a cancer associated with CEACAM5 expression or a CEACAM5-positive cancer. In some embodiments, the cancer is a solid tumor. In preferred embodiments, the cancer is gastric cancer. In another preferred embodiment, the cancer is colorectal cancer.

[0372] In some embodiments, the dosage administered to a subject can vary depending on the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the effective amount of administration to a human or other subject to treat a medical condition. The precise amount required can depend on many factors, such as the activity of the antibody and the route of administration.

[0373] A dose of an antibody, nucleic acid, vector, host cell, conjugate, CAR, or pharmaceutical composition described herein can be administered to a mammal in a suitable period of time in a single portion or in a series of subdoses, for example, daily, semi-weekly, weekly, biweekly, semimonthly, bimonthly, semiannual, or annual, as desired. Dose units comprising an effective amount of an antibody, composition, conjugate, or chimeric antigen receptor can be administered as a single daily dose, or the total daily dose can be administered in two, three, four, or more divided doses administered daily, as desired.

[0374] A suitable mode of administration can be selected by a physician. The route of administration can be parenteral administration, for example, administration by injection, nasal administration, pulmonary administration, or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition, conjugate, or chimeric antigen receptor is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, for example, orally. The dose and method of administration can vary depending on the weight, age, condition, etc. of the subject, and can be appropriately selected.

[0375] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, conjugate, CAR, or pharmaceutical composition disclosed herein is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug. In certain embodiments, an antibody, composition, conjugate, or chimeric antigen receptor disclosed herein is administered prior to, substantially simultaneously with, or after administration of the second therapeutic agent.

[0376] Detection / diagnostic methods and uses

[0377] The present disclosure provides a method of detecting CEACAM5 protein in vitro or in vivo, comprising contacting a sample with a CEACAM5 antibody disclosed herein to thereby detect the presence of CEACAM5 protein.

[0378] Provided herein is a method of detecting the presence or level of CEACAM5 in a sample, comprising:

[0379] (1) contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein; and

[0380] (2) determining the presence or level of CEACAM5 in the sample by detecting the binding of the antibody to the sample.

[0381] Also provided herein is a method of diagnosing whether a subject has a cancer associated with CEACAM5 expression, comprising:

[0382] (1) contacting a sample from a subject with an antibody or antigen-binding fragment thereof disclosed herein; and

[0383] (2) detecting binding of the antibody to the sample,

[0384] wherein increased binding of the antibody or antigen-binding fragment thereof disclosed herein to the sample from the subject as compared to binding of the antibody or antigen-binding fragment thereof disclosed herein to a healthy control indicates that the subject has a cancer associated with CEACAM5 expression.

[0385] Also provided herein is the use of an antibody or antigen-binding fragment thereof disclosed herein or a conjugate disclosed herein in the manufacture of a kit for diagnosing a cancer associated with CEACAM5 expression in a subject.

[0386] The present disclosure also provides an antibody or antigen-binding fragment thereof disclosed herein or a conjugate disclosed herein for use in diagnosing a cancer associated with CEACAM5 expression in a subject.

[0387] In some embodiments, the sample can be any sample, including but not limited to a blood sample, tissue from a biopsy, autopsy, and pathology specimens. Samples also include body fluids, such as blood, serum, plasma, sputum, spinal fluid, or urine. In some embodiments, the healthy control sample is a sample from a subject who does not have cancer. In particular embodiments, the sample is a blood or tissue sample.

[0388] In some embodiments, the cancer associated with CEACAM5 expression is a solid tumor, preferably gastric cancer or colorectal cancer.

[0389] In some embodiments of the diagnostic and detection methods, the anti-CEACAM5 antibody is directly labeled with a detectable label. In another embodiment, the anti-CEACAM5 antibody (first antibody) is unlabeled, while a second antibody or other molecule that can bind the first antibody is labeled. As is well known to those skilled in the art, a second antibody is chosen that is capable of specifically binding to the first antibody of a particular species and class. For example, if the first antibody is human IgG, the second antibody can be anti-human IgG. Other molecules that can bind to antibodies include, but are not limited to, protein A and protein G, both of which are commercially available.

[0390] Suitable labels for either the primary or secondary antibodies include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium; and non-limiting exemplary radioactive labels include 125 I, 131 I, 35 S or 3 H.

[0391] In alternative embodiments, CEACAM5 can be measured in a biological sample by a competitive immunoassay that utilizes a CEACAM5 protein standard labeled with a detectable substance and unlabeled anti-CEACAM5 antibody. In this assay, the biological sample, the labeled CEACAM5 protein standard, and the anti-CEACAM5 antibody are combined, and the amount of labeled CEACAM5 protein standard bound to the unlabeled antibody is determined. The amount of CEACAM5 in the biological sample is inversely proportional to the amount of labeled CEACAM5 protein standard bound to the anti-CEACAM5 antibody.

[0392] The immunoassays and detection methods disclosed herein can be used for a variety of purposes. In one embodiment, the anti-CEACAM5 antibody can be used to detect the production of CEACAM5 in cells in a cell culture. In another embodiment, the antibody can be used to detect the amount of CEACAM5 in a biological sample, such as a tissue sample or a blood or serum sample.

[0393] Examples

[0394] The following examples are given for the purpose of illustrating various embodiments of the application and are not meant to limit the present application in any way. This example, as well as the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the application. Changes therein and other uses will occur to those skilled in the art. The experimental methods in the following examples, where no specific conditions are indicated, are carried out under conventional conditions or under conditions as suggested by the manufacturer. Experimental materials and reagents used in the following examples are commercially available unless otherwise stated.

[0395] Example 1 Mouse Immunization

[0396] 1.1 Mouse Immunization

[0397] Take Balb / c mice (mouse marker A1-A5), use human CEACAM5 (hCEACAM5) full-length protein (Acro, CAT#CE5-H5226), cynomolgus CEACAM5 (cyno CEACAM5) full-length protein (Acro, CAT#CE5-C52H5), human CEACAM5 A3-B3 truncated protein (Ekonade preparation), add Freund's adjuvant, and immunize mice by subcutaneous multiple point injection, intraperitoneal injection and other ways.

[0398] 1.2 titer detection

[0399] After three immunizations, the serum titer of the mice was detected, including ELISA and FACS, and the data was counted from the beginning of the three immunizations.

[0400] 1) ELISA screening

[0401] Coat 96-well enzyme-labeled plates with 100 μL of human CEACAM5 full-length protein (Acro, CAT#CE5-H5226), cynomolgus CEACAM5 full-length protein (Acro, CAT#CE5-C52H5), human / cynomolgus CEACAM5 A3-B3 truncated protein (Ekonade preparation), CEACAM1 (Acro, CAT#CE1-H5220), CEACAM6 (Acro, CAT#CE6-H5223), CEACAM7 (Ekonade preparation), CEACAM8 (Acro, CAT#CE8-H5224) (final concentration 0.5 μg / mL) at 4°C overnight. After washing 3 times with PBST (containing 0.05% Tween), add blocking buffer (3% BSA) at 37°C for 1h, wash the plate 3 times with PBST. Add mouse immune serum with 5-fold serial dilution starting from 1:1000, incubate at 37°C for 1h, wash 3 times with PBST, add 100 μL 0.1 μg / mL HRP Goat Anti-Mouse IgG H&L (Biolegend, CAT#BF03001) per well, incubate at 37°C for 0.5h, wash 3 times with PBST, add 100 μL / well TMB developing solution, incubate at room temperature for 10 min in the dark, add 50 μL stop solution to stop the reaction, and read the OD450nm value in the well with an enzyme-labeled instrument. The ELISA results are shown below.

[0402] The above results show that the immune titer of the mice is high, the titer of human CEACAM5 is 1:625K, the titer of cynomolgus CEACAM5 is 1:125K, the titer of human / cynomolgus A3-B3 is 1:125K, the titer of CEACAM1, 6, 8 is 1:125K, and the titer of CEACAM7 is 1:25K.

[0403] 2) FACS screening

[0404] CHO (provided by Akonadi), CHO-human CEACAM5 (constructed by Akonadi), CHO-cynomolgus CEACAM5 (constructed by Akonadi), CHO-hCEACAM5-A3-B3 (constructed by Akonadi), MKN45 (CEACAM5 positive cell line, Basic Medical College of Peking Union Medical College) cell strains were divided into several parts, and the number of cells in each part was 3x10 5 Each part of cells was incubated with 50 μL of mouse immune serum, mixed well, and incubated at room temperature for 1 h. Anti-CEACAM5 antibody Tusamitamab was used as a positive control. After centrifugation, the cells were washed with PBS 3 times, and PE-labeled anti-mouse IgG (Abeam, CAT# ab97024) was added. After mixing well, the cells were incubated at room temperature for 30 min in the dark. After centrifugation, the cells were washed with PBS 3 times, resuspended with PBS, and detected by flow cytometry.

[0405] The results are shown in Figure 1. The serum of mice immunized for 3 times had good FACS activity, indicating that good antibodies against CEACAM5 protein were produced.

[0406] Example 2 Hybridoma fusion and screening

[0407] 2.1 Cell fusion

[0408] After 3 days of impact immunization, the mouse with good immune titer detection was sacrificed by cervical dislocation, and the mouse spleen was obtained. B cell single cell suspension was prepared, mixed with non-secretory SP2 / 0 myeloma cells at a ratio of 1:1, and cell fusion was performed using a cell electrofusion instrument. After electrofusion, all cells were immediately suspended in complete culture medium (DMEM, 20% FBS and HAT) and inoculated into 96-well plates; about 10 days after fusion, HT medium was used for culture for two days, and then the supernatant was detected for specific antibodies.

[0409] 2.2 Hybridoma screening

[0410] Take 100 μL of culture supernatant from each well of the 96-well plate, and replenish fresh HT medium into each well. Perform ELISA binding test on the removed culture supernatant and cynomolgus CEACAM5-His. Select the clones with strong ELISA binding, and perform ELISA retest using human CEACAM5-His, human CEACAM-1-His, CEACAM-6-His, CEACAM-7-His, CEACAM-8-His, and CEACAM5 A3-B3-His. Select the clones that bind to human CEACAM5 and CEACAM5 A3-B3, and do not bind or weakly bind to human CEACAM-1, CEACAM-6, CEACAM-7, and CEACAM-8, and perform flow retest by incubating CHO-hCEACAM5, CHO-cyno-CEACAM5, MKN45, and CHO cells. After each round of subcloning, perform ELISA and FACS identification until single clones are formed.

[0411] 1) ELISA test

[0412] Dilute the antigen proteins (cynomolgus CEACAM5-His, human CEACAM5-His, human CEACAM-1-His, CEACAM-6-His, CEACAM-7-His, CEACAM-8-His, and CEACAM5 A3-B3-His) using PBS to a final concentration of 0.5 μg / mL, take a new 96-well enzyme plate, add 100 μL per well, and coat overnight at 4°C. Remove the antigen coating solution, and wash the plate 3 times using PBST (containing 0.05% Tween). Add 200 μL / well of 3% BSA, and block at 37°C for 2 hours. After removing the blocking buffer, wash the plate 3 times using PBST. Add 100 μl of hybridoma supernatant, and incubate at room temperature for 1 hour. The control wells are PBS. Remove the liquid in the wells, and wash 3 times using PBST. Add 100 μL of HRP Anti-Mouse IgG H&L (Biolegend, CAT#BF03001, diluted 1:10000), and incubate at room temperature for 1 hour. After removing the liquid in the wells, wash the plate 3 times using PBST. Add 100 μL / well of TMB developing solution. Incubate at room temperature for 10 minutes in the dark. Add 50 μL / well of stop solution. Read the OD value in the wells using an enzyme labeler. The ELISA test results are shown in Tables 1 and 2.

[0413] Table 1. Positive wells binding to cynomolgus CEACAM5

[0414] Table 2. Binding of positive cynomolgus CEACAM5 binding well hybridoma clones to CEACAM family proteins

[0415] The results of Table 1 and Table 2 show that there are more clones that bind to cynomolgus CEACAM5, human CEACAM5, CEACAM5 A3-B3, weakly bind to or do not bind to human CEACAM-1, CEACAM-6, CEACAM-7, CEACAM-8. The clones that bind to human CEACAM5, CEACAM5 A3-B3, weakly bind to human CEACAM-1, CEACAM-6, CEACAM-7, CEACAM-8 are selected for cell recheck.

[0416] 2) FACS detection

[0417] The CHO-hCEACAM5, CHO-cyno-CEACAM5, MKN45 and CHO cell strains were recovered from liquid nitrogen, and the cell state was adjusted to the logarithmic growth phase using complete culture medium. The cells were divided into several portions, and the number of cells in each portion was 3x10 5 The target cells were incubated with 50 μL of hybridoma supernatant, and then mixed well. After incubation at room temperature for 1 hour, the cells were centrifuged at room temperature for 5 minutes, and the supernatant containing the antibody was removed. The cells were washed with PBS for 3 times. PE-labeled Anti-mouse IgG H&L (Acro, CAT#CE5-C52H5, 1:1000 dilution) was added, and then mixed well. After incubation at room temperature for 30 minutes in the dark, the cells were centrifuged at room temperature for 5 minutes, and the supernatant containing the secondary antibody was removed. The cells were washed with PBS for 3 times. The cells were resuspended with 200 μL of PBS, and then the fusion clone titer was detected by flow cytometry.

[0418] The FACS recheck results are shown in Figures 2-3. The clones that cross-bind to human CEACAM5, cynomolgus CEACAM5 and MKN45, and do not bind to CEACAM1 / 6 / 7 / 8 were selected for endocytosis activity detection.

[0419] 3) Endocytosis activity detection (MKN45 cells)

[0420] The MKN45 cells in the logarithmic growth phase were taken, and the number of cells in each well was 5x10 5Cell group. Add 200 μL hybridoma supernatant, incubate at 4°C for 30 min. 4°C, centrifuge for 5 min, remove the supernatant containing the antibody, and wash the cells with pre-cooled PBS 3 times. Divide the above cells equally into 2 parts, and incubate at 4°C and 37°C for 240 min, respectively. After incubation, immediately add pre-cooled PBS to terminate the endocytosis experiment, centrifuge at 4°C for 5 min, resuspend the cells with 100 μL pre-cooled PBS, and immediately add PE-labeled Anti-mouse IgG H&L (Abeam, CAT# ab97024, 1:1000 dilution), incubate at 4°C for 30 min. Then centrifuge at 4°C for 5 min, remove the supernatant containing the antibody, and wash the cells with pre-cooled PBS 3 times. Resuspend the cells with 200 μL pre-cooled PBS, and immediately perform flow cytometry analysis. The results of FACS endocytosis activity detection are shown in Figure 4 and Table 3.

[0421] Table 3. Results of FACS endocytosis activity detection

[0422] The results show that the clones that cross-bind with human CEACAM5, cynomolgus CEACAM5 and MKN45, and do not bind with CEACAM1 / 6 / 7 / 8, basically all have endocytosis activity.

[0423] 2.3 Hybridoma clone subcloning

[0424] Collect well-grown hybridoma cell strains to make a single cell suspension. Count using a hemocytometer, and take 100 cells, dilute to 10 mL. Take a 96-well plate, add 100 μL cell suspension to each well, finally supplement the volume to 200 μL, place in a 37°C incubator, and label. After continuous culture for 3 days, select single clones under a microscope. Replace fresh culture medium on the 7th day of culture. On the 8th day of culture, perform ELISA and FACS detection (methods same as ELISA and FACS detection in Example 2.2). Select clones that specifically bind by ELISA and FACS, and perform subcloning again, a total of two times. After subcloning, select clones that cross-bind with human CEACAM5, cynomolgus CEACAM5 and MKN45, and do not bind with CEACAM1 / 6 / 7 / 8, and have endocytosis activity, and sequence.

[0425] Example 3 Hybridoma sequencing

[0426] Take 1 x 10 6The hybridoma cells obtained from the final screening were lysed using Trizol, and total RNA was extracted from the hybridoma cells. The total RNA was reverse transcribed into cDNA using a reverse transcription kit. Then, the heavy and light chain variable regions of the antibody were amplified by PCR using hybridoma sequencing primers. TA cloning was then performed, and the PCR-obtained fragments were subcloned into a T vector. Clones were selected for sequencing, and the final antibody sequences were analyzed. A total of 14 unique sequences were obtained. The CDR sequences of the heavy chain variable region (VH) and light chain variable region (VL), as well as the VH and VL sequences, are shown in Tables 4 and 5, respectively.

[0427] Table 4. Antibody CDR Sequences

[0428] Table 5. Antibody VH and VL sequences

[0429] Example 4: Construction and Validation of Chimeric Antibody Expression Vector

[0430] The variable region of the antibody heavy chain, obtained from hybridoma sequencing, was subcloned into the pcDNA3.4-IgG1 expression vector (provided by Aikon), and the variable region of the light chain was subcloned into the pcDNA3.4-IgKc expression vector (provided by Aikon). The constructed light and heavy chain expression vectors were transiently co-transfected into 293F cells. Five days after transfection, the supernatant was collected, and the antibody was purified by Protein A affinity chromatography and then analyzed by FACS.

[0431] 4.1 Expression of chimeric antibodies

[0432] Remove the LVTransm transfection reagent (prepared by Aikon) and antibody expression vector from the freezer. After thawing at room temperature, mix thoroughly by pipetting. Remove the PBS buffer and warm to room temperature. Add 2 mL of PBS to one well of a 6-well plate, and add 50 μg of heavy chain expression vector and 50 μg of light chain expression vector respectively. Mix thoroughly by pipetting, then add 360 μL of LVTransm transfection reagent. Immediately mix by pipetting and incubate at room temperature for 10 minutes. Add the above DNA / LVTransm complex to 100 mL of 293F cells (1 cell per well). 6 Mix the cells thoroughly by gently agitating them at a density of 1 / mL. Incubate the cells at 37°C and 5% CO2 for 130 rpm. After 7 days of continuous culture, collect the supernatant by centrifugation, filter through a 0.45 μm filter membrane, transfer the filtrate to a sterile centrifuge tube, and purify the chimeric antibody using a Protein A affinity column.

[0433] 4.2 Chimeric antibody ELISA detection

[0434] Dilute human CEACAM5 and cynomolgus CEACAM5 to final concentration of 0.5 pg / mL with PBS, take a new 96-well enzyme plate, add 100 pL per well, and coat overnight at 4°C. Remove the antigen coating solution and wash 3 times with PBST (containing 0.05% Tween). Add 200 pL / well of 3% BSA, and block at 37°C for 2 hours. After removing the blocking buffer, wash the plate 3 times with PBST. Add 100 pL of gradient-diluted chimeric antibody (10 pg / mL, 3-fold dilution of 7 points), and incubate at room temperature for 1 hour. The control well is PBS. Use the anti-CEACAM5 antibody Tusamitamab as a positive control. Remove the liquid in the well, and wash 3 times with PBST. Add 100 pL of HRP-Protein A (Boster, CAT# BA1080, 1:10000 dilution), and incubate at room temperature for 1 hour; after removing the liquid in the well, wash the plate 3 times with PBST. Add 100 pL / well of TMB developing solution. Incubate at room temperature for 10 minutes in the dark. Add 50 pL / well of stop solution. Use a microplate reader to read the OD value in the well. Use PRISM GraphPad to process the data and draw a curve.

[0435] The results are shown in Figure 5, and the binding of 17-A12-G9-G8, 7-E2-F2-C7, 16-D7-E1-C11, 43-D11-F3-F5, 5-H3-G1 1, 4-G5-E2, 8-G12-B3, 24-C3-2-D6, 6-E6-D7, 19-D1-B1, 22-B2-B4 and 8-C3-C12 chimeric antibodies to human CEACAM5 is better than the positive control antibody, wherein 7-E2-F2-C7, 16-D7-E1-C11, 17-A12-G9-G8, 40-H5-F10-F2, 14-H4-D1-C10, 43-D11-F3-F5, 24-C3-2-D6 and 22-B2-B4 all cross-recognize cynomolgus CEACAM5 and have better binding activity than the positive control antibody.

[0436] 4.3 Chimeric antibody FACS detection

[0437] Recover CHO-human CEACAM5 and CHO-cynomolgus CEACAM5 cells from liquid nitrogen, and adjust the cell state to the logarithmic growth phase. Divide the cells into several portions, and the number of cells in each portion is 3x10 5Each well of the plate was added with 100 μL of the chimeric antibody (30 μg / mL, 3-fold dilution of 10 points), mixed well, and incubated at room temperature for 1 hour. Anti-CEACAM5 antibody Tusamitamab was used as a positive control. Centrifugation at room temperature for 5 minutes, remove the supernatant containing the antibody, and wash the cells with PBS for 3 times. Added 100 μL PE-anti-human IgG (Invitrogen, CAT#12-4998-82, 1:500 dilution), mixed well, and incubated at room temperature for 45 min. Centrifugation at room temperature for 5 minutes, remove the supernatant containing the secondary antibody, and wash the cells with PBS for 3 times. Resuspend the cells with 500 μL of PBS for flow cytometry analysis. The data were processed using PRISM GraphPad to draw the curve graph.

[0438] The FACS results are shown in Figure 6. The binding strength of 7-E2-F2-C7, 16-D7-E1-C11, 17-A12-G9-G8, 5-H3-G11, 4-G5-E2, 8-G12-B3, 24-C3-2-D6, 19-D1-B1, 22-B2-B4 and 8-C3-C12 to human CEACAM5 was better than the positive control antibody, and the binding strength of 40-H5-F10-F2, 14-H4-D1-C10 and 43-D11-F3-F5 to human CEACAM5 was comparable to the positive control antibody. Among them, 7-E2-F2-C7, 16-D7-E1-C11, 17-A12-G9-G8, 40-H5-F10-F2, 14-H4-D1-C10, 43-D11-F3-F5, 24-C3-2-D6 and 22-B2-B4 cross-recognized cynomolgus CEACAM5 and had better binding activity than the positive control antibody, which was consistent with the ELSIA results.

[0439] 4.4 Detection of specificity of chimeric antibodies

[0440] CHO-human CEACAM1, CHO-human CEACAM6, CHO-human CEACAM7 and CHO-human CEACAM8 cells were recovered from liquid nitrogen, and the cell state was adjusted to the logarithmic growth phase. The cells were divided into several portions, and the number of cells in each portion was 3 x 105 5Each well was added with 100 μL chimeric antibody (30 μg / mL, 3-fold dilution of 3 points), mixed well, and incubated at room temperature for 1 hour. Anti-CEACAM5 antibody Tusamitamab was used as a positive control. Centrifugation at room temperature for 5 minutes, remove the supernatant containing the antibody, and wash the cells with PBS for 3 times. Added 100 μL PE-Anti-human IgG (Invitrogen, CAT#12-4998-82, 1:500 dilution), mixed well, and incubated at room temperature for 45 min. Centrifugation at room temperature for 5 minutes, remove the supernatant containing the secondary antibody, wash the cells with PBS for 3 times, resuspend the cells with 500 μL PBS, and perform flow cytometry analysis.

[0441] The results are shown in Figure 7, and the specific results of 7-E2-F2-C7, 16-D7-E1-C11, 17-A12-G9-G8, 4-G5-E2, 8-G12-B3, 5-H3-G11, and 8-C3-C12 chimeric antibodies are good.

[0442] 4.5 Detection of chimeric antibody recognizing epitope

[0443] CHO-human CEACAM5, CHO-cynomolgus CEACAM5, CHO-hCEACAM5-A1, CHO-hCEACAM5-A1-B1, CHO-hCEACAM5-A2-B2, and CHO-hCEACAM5-A3-B3 cells were recovered from liquid nitrogen, and the cell state was adjusted to the logarithmic growth phase. The cells were divided into several portions, and the number of cells in each portion was 3 x 105 5 Each well was added with 100 μL chimeric antibody (30 μg / mL, 3-fold dilution of 3 points), mixed well, and incubated at room temperature for 1 hour. Anti-CEACAM5 antibody Tusamitamab was used as a positive control. Centrifugation at room temperature for 5 minutes, remove the supernatant containing the antibody, and wash the cells with PBS for 3 times. Added 100 μL PE-Anti-human IgG (Invitrogen, CAT#12-4998-82, 1:500 dilution), mixed well, and incubated at room temperature for 45 min. Centrifugation at room temperature for 5 minutes, remove the supernatant containing the secondary antibody, wash the cells with PBS for 3 times. Resuspend the cells with 500 μL PBS, and perform flow cytometry analysis.

[0444] Results are shown in Figure 8, 7-E2-F2-C7, 16-D7-E1-C11, 17-A12-G9-G8 specifically bind human CEACAM5, and cross-bind cynomolgus monkey CEACAM5, the binding epitope is at A3-B3, and the FACS binding intensity is better than the positive antibody. 5-H3-G11, 4-G5-E2, 8-G12-B3, 6-E6-D7, 19-D1-B1, 8-C3-C12 specifically bind human CEACAM5, and the binding epitope is at A3-B3.

[0445] Example 5 Humanization of Antibodies

[0446] The sequence of the 16-D7-E1-C11 chimeric antibody (the amino acid sequence of VH is SEQ ID NO: 75, and the amino acid sequence of VL is SEQ ID NO: 76) was selected for humanization modification. The original murine antibody sequence was aligned with the fully human antibody sequence present in the IMGT database, and a human germline sequence with the highest framework (Framework) homology was found as a template for humanization, and the CDR region was determined according to the amino acids before and after the antibody framework region CDR (see http: / / www.bioinf.org.uk / abs / index.html#cdrid), as shown in Table 6 below.

[0447] Table 6. CDR sequence of 16-D7-E1-C11 antibody determined according to amino acids before and after antibody framework region CDR

[0448] The murine antibody CDR region sequence was transplanted (CDR grafting) into the selected humanization template framework to obtain the initial humanized antibody, and the important sites of key amino acids that can maintain the CDR conformation and affect the structure and function of the antibody were selected from the framework region for back mutation, and the different back mutation amino acids of light and heavy chains were combined to obtain multiple humanization variants, and the specific mutation types are shown in Table 7 below.

[0449] Table 7. Types of humanization mutations in antibody heavy and light chains

[0450] Note: (1) GH / GL: germline VH / VL sequence; (2) p1: variant without PTM (CDR-H2 "NYS" glycosylation site replaced N059Q).

[0451] The corresponding humanized antibody sequence is shown in Table 8 below.

[0452] Table 8. VH and VL sequences of humanized antibodies

[0453] Note: CDR sequences of heavy and light chains are shown in bold underlined, where HCDR2 sequence of 6 humanized antibodies (YIFPSSGYIQYSQKFKD, SEQ ID NO. 108) is different from that of parental antibody 16-D7-E1-C11 listed in Table 6, and other CDRs are the same as parental antibody.

[0454] Example 6. Affinity detection of humanized antibodies

[0455] The humanized antibody light and heavy chains were constructed into expression vectors containing human IgK light chain constant region fragment and human IgG heavy chain CH1, hinge, CH2 and CH3 fragments, respectively (TransGen Biotech). The expression plasmids were transfected into Expi293F cells (Invitrogen, CAT# A14528) using ExpiFectamine 293 Transfection Kit (Invitrogen, CAT# A14524). The expressed supernatant was collected after 5 days of culture in Expi293 expression medium (Invitrogen, CAT# A1435101) at 120 rpm, 37°C, 5% CO2 shaker incubator, and the expression amount was detected. The collected supernatant was purified using Protein A column (Cytiva, CAT# GE17-5438-02).

[0456] The affinity of each humanized antibody to human CEACAM5 protein was detected by SPR assay using Biacore T200. The purified antibodies were captured on anti-human IgG Fc antibody (Jackson ImmunoResearch) immobilized CM5 sensor chip (Cytiva). Different concentrations of human CEACAM5 protein were injected into the sensor chip at a flow rate of 30 μL / min, with a binding time of 180 s and a dissociation time of 3600 s. After each binding cycle, the chip was regenerated with 10 mM glycine (pH 1.5). The dissociation constant (KD) of each humanized antibody was obtained by fitting the data using 1:1 Binding Model using Biacore Evaluation Software, as shown in Table 9 below.

[0457] Table 9. Expression amount and binding affinity of humanized antibodies

[0458] Example 7. Endocytosis activity detection of humanized antibodies in MKN45 and LS180 cells

[0459] This example tested the endocytosis activity of 16-D7-E1-C11 chimeric antibody and its humanized antibodies z1-p1, z2-p1, z3-p1, z4-p1, z5-p1, z6-p1, using Tusamitamab as a control. The antibody sequences are as follows: Full-length sequence of z1-p1 antibody:

[0460] Heavy chain:

[0461] Light chain:

[0462] Full-length sequence of z2-p1 antibody:

[0463] Heavy chain:

[0464] Light chain:

[0465] Full-length sequence of z3-p1 antibody:

[0466] Heavy chain:

[0467] Light chain:

[0468] Full-length sequence of z4-p1 antibody: Heavy chain:

[0469] Light chain:

[0470] Full-length sequence of z5-p1 antibody:

[0471] Heavy chain:

[0472] Light chain:

[0473] Full-length sequence of z6-p1 antibody:

[0474] Heavy chain:

[0475] Light chain:

[0476] Full-length sequence of 16-D7-E1-C11 chimeric antibody:

[0477] Heavy chain:

[0478] Light chain:

[0479] MKN45 cells (College of Basic Medical Sciences, Peking Union Medical College) or LS180 cells (Nanjing Kebai) in logarithmic growth phase were used, at a ratio of 6 x 10 cells per well. 5 Cells were divided into groups. 100 μL of antibody (10 μg / mL) was added to each well, and the cells were incubated at 4°C for 45 min. After incubation, the cells were centrifuged at 800 x g for 5 min at 4°C, and the supernatant containing the antibody was removed. The cells were washed three times with pre-chilled PBS. The cells were then divided into two groups and incubated at 4°C and 37°C for 30 min, 1 h, 2 h, and 4 h, respectively. After incubation, pre-chilled PBS was immediately added to terminate the endocytosis experiment. The cells were centrifuged at 800 x g for 5 min at 4°C, resuspended in 100 μL of pre-chilled PBS, and immediately incubated with PE-labeled anti-human IgG (Invitrogen, CAT#12-4998-82) at 4°C for 30 min. Afterward, the cells were centrifuged at 800 x g for 5 min at 4°C, and the supernatant containing the antibody was removed. The cells were washed three times with pre-chilled PBS, and then resuspended in 200 μL of pre-chilled PBS. Flow cytometry analysis was performed immediately. The endocytosis level of antibodies bound to the cell surface was calculated as follows: MFI% at different time points = MFI of samples incubated at 37℃ / MFI of control samples incubated at 4℃; endocytosis percentage at different time points = 100% - MFI at different time points.

[0480] The results are shown in Figures 9A and 9B. The endocytic activity of the 16-D7-E1-C11 chimeric antibody and the humanized antibodies z1-p1, z2-p1, z3-p1, z4-p1, z5-p1, and z6-p1 in MKN45 and LS180 cells generally increased with increasing incubation time. After 4 hours of incubation, the endocytosis percentages in MKN45 and LS180 cells reached approximately 70% and 90%, respectively, while the endocytosis percentages of Tusamitamab were lower, at 19% and 44%, respectively. This indicates that the antibodies of the present invention have a higher level of endocytosis and stronger endocytosis activity.

[0481] Example 8. Evaluation of tumor suppression in a human colon cancer xenograft subcutaneous tumor PDX model using antibody-drug conjugates.

[0482] By chemical conjugation, MC-GGFG-DXD (MCE, CAT#HY-13631E) was used as a linker-payload to conjugate the full-length sequences of humanized antibodies z1-p1, z5-p1, and z6-p1 (the full-length antibody sequences are shown in Example 7) to obtain antibody-drug conjugates (DAR value of 8), which were used as in vivo efficacy testing drugs to verify the in vivo efficacy characteristics of different antibodies linked to toxin molecules.

[0483] The structure of MC-GGFG-DXD is shown below (hereinafter referred to as "DXD"):

[0484] The modeling specimen is derived from liver metastasis of colon cancer patients, and the specimen is collected when the patient is not treated. The tumor tissue is passaged in Balb / c Nude mice (Huafukang), and when the subcutaneous tumor of the PDX model mice grows to 500-1000mm 3 Afterwards, the mice are euthanized, the tumor is peeled off, the necrotic part is removed, and the tumor is cut into small pieces with a size of about 2mm x 2mm x 2mm. The tumor pieces are inoculated into the right flank of the experimental animals subcutaneously using a trocar, and each mouse is inoculated with one piece. When the average tumor volume reaches 100-150mm 3 The mice are randomly grouped for drug administration according to the tumor volume and body weight (the day is recorded as D0), and are divided into 4 groups: the solvent control group, the z1-p1-DXD group, the z5-p1-DXD group and the z6-p1-DXD group. The drug administration mode is tail vein injection (i.v.), the drug dose is 3mg / kg, the drug volume is 10μL / g, the drug administration is once every two weeks, and a total of 2 times, and the experiment ends on D49. The tumor volume of the tumor-bearing mice is measured during the experiment, the efficacy is evaluated according to the tumor volume, and the safety is evaluated according to the animal body weight change and death.

[0485] The results are shown in Figure 10, and the average tumor volume of the solvent control group on D49 is 2250mm 3 The z1-p1-DXD group, the z5-p1-DXD group and the z6-p1-DXD group all show obvious tumor inhibition effect on D49 compared with the control group. In addition, no animal died during the test period, and no obvious drug toxicity clinical symptoms were shown, indicating that the tested drug has good safety.

Claims

1. An antibody or antigen-binding fragment thereof that binds CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 75, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 76; or (2) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 102; or (3) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 103; or (4) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104; or (5) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 105, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104; or (6) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 106, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 104; or (7) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 107, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:

104.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein (1) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 18-20, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 21, LCDR2 having the SAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 22; or (2) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 95-97, respectively, and the VL comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 98-100, respectively; or (3) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 95, 108, and 97, respectively, and the VL comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 98-100, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76; or (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 102; or (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

103. (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 101, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104; or (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 105, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104; or (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 106, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 104; or (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 107, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

104.

4. The antibody or antigen binding fragment thereof of claim 3, wherein (1) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 75, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 76; or (2) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 102; or (3) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 103; or (4) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 101, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104; or (5) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 105, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104; or (6) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 106, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104; or (7) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 107, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:

104. (5) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 105, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104; or (6) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 106, and the VL comprises an amino acid sequence as set forth in SEQ ID NO: 104; or (7) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 107, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:

104.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the antibody comprises a heavy chain and a light chain, wherein: (1) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 117; or (2) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 118; or (3) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 116, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119; or (4) the heavy chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 120, and the light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119; or (5) the heavy chain comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 121 and the light chain comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119; or (6) the heavy chain comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 122 and the light chain comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 119; or (7) the heavy chain comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 123 and the light chain comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

124.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the antibody comprises a heavy chain and a light chain, wherein: (1) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 116 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 117; or (2) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 116 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 118; or (3) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 116 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or (4) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 120 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or (5) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 121 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or (6) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 122 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 119; or (7) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 123 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:

124. (7) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 123, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:

124.

7. An antibody or antigen-binding fragment thereof that binds CEACAM5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 71, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 72; or (2) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 67, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 68; or (3) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 69, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 70; or (4) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 73, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 74; or (5) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 77, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 78; or (6) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 79, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 80; or (7) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 81, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 82; or (8) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 83, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 84; or (9) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 85, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 86; or (10) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 87, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 88; or (11) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 89, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 90; or (12) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 91, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 92; or (13) the VH comprises HCDR1, HCDR2, and HCDR3 in a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 93, and the VL comprises LCDR1, LCDR2, and LCDR3 in a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:

94.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein (1) the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOS: 1, 11, and 3, respectively, and the VL comprises LCDR 1 having an amino acid sequence as set forth in SEQ ID NO: 4, LCDR2 having an AAS amino acid sequence, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 12; or (2) the VH comprises HCDR 1-3 having an amino acid sequence as set forth in SEQ ID NOS: 1-3, respectively, and the VL comprises LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, LCDR2 having an AAS amino acid sequence, and LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 5; or (3) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 6-8, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 9, LCDR2 having the WAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 10; or (4) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 13-15, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 16, LCDR2 having the LAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 17; or (5) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 23-25, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 26, LCDR2 having the FAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 27; or (6) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28-30, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 31, LCDR2 having the SAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 32; or (7) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 33-35, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 36, LCDR2 having the AAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 37; or (8) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 38-40, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 41, LCDR2 having the YAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; or (9) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 28, 43, and 44, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 45, LCDR2 having the SAS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 46; or (10) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 47-49, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 50, LCDR2 having the NAK amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 51; or (11) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 52-54, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 55, LCDR2 having the ATS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 56; or (12) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 57-59, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 60, LCDR2 having the NAK amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 61; or (13) the VH comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 62-64, respectively, and the VL comprises LCDR1 having the amino acid sequence set forth in SEQ ID NO: 65, LCDR2 having the ATS amino acid sequence, and LCDR3 having the amino acid sequence set forth in SEQ ID NO:

66.

9. The antibody or antigen-binding fragment thereof of claim 7 or 8, wherein (1) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72; or (2) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68; or (3) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70; or (4) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74; or (5) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78; or (6) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80; or (7) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82; or (8) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84; or (9) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 85 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86; or (10) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 87 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 88; or (11) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 89 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 90; or (12) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 91 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 92; or (13) the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 93, and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

94.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein (1) the VH comprises the amino acid sequence set forth in SEQ ID NO: 71, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 72; or (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 67, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 68; or (3) the VH comprises the amino acid sequence set forth in SEQ ID NO: 69, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 70; or (4) the VH comprises the amino acid sequence set forth in SEQ ID NO: 73, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 74; or (5) the VH comprises the amino acid sequence set forth in SEQ ID NO: 77, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 78; or (6) the VH comprises the amino acid sequence set forth in SEQ ID NO: 79, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 80; or (7) the VH comprises the amino acid sequence set forth in SEQ ID NO: 81, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 82; or (8) the VH comprises the amino acid sequence set forth in SEQ ID NO: 83, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 84; or (9) the VH comprises the amino acid sequence set forth in SEQ ID NO: 85, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 86; or (10) the VH comprises the amino acid sequence set forth in SEQ ID NO: 87, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 88; or (11) the VH comprises the amino acid sequence set forth in SEQ ID NO: 89, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 90; or (12) the VH comprises the amino acid sequence set forth in SEQ ID NO: 91, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 92; or (13) the VH comprises an amino acid sequence as set forth in SEQ ID NO: 93, and the VL comprises an amino acid sequence as set forth in SEQ ID NO:

94.

11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody is of a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody is a monovalent, bivalent, or multivalent antibody.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

16. The antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and ds-scFv.

17. The antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof binds to CEACAM5 A3-B3, CEACAM5 A2-B2, and / or CEACAM5 Al-B l, preferably to CEACAM5 A3-B3.

18. A nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-17.

19. A vector comprising the nucleic acid of claim 18.

20. A host cell comprising the nucleic acid of claim 18 or the vector of claim 19.

21. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-17, the method comprising: a) culturing the host cell of claim 20 under conditions suitable for expression of the antibody or antigen-binding fragment thereof; and b) isolating the antibody or antigen-binding fragment thereof from the culture and / or culture supernatant of the host cell.

22. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17 and a chemical moiety conjugated to the antibody or antigen-binding fragment thereof, preferably the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

23. The conjugate of claim 22, wherein the therapeutic agent is a topoisomerase inhibitor, preferably Dxd.

24. The conjugate of claim 22 or 23, wherein the antibody or antigen-binding fragment thereof is conjugated to the chemical moiety via a linker, preferably the linker is MC-GGFG.

25. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17, the nucleic acid of claim 18, the vector of claim 19, the host cell of claim 20, the conjugate of any one of claims 22-24, or the CAR of claim 25, and optionally a pharmaceutically acceptable carrier or excipient.

27. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-17, the nucleic acid of claim 18, the vector of claim 19, the host cell of claim 20, the conjugate of any one of claims 22-24, the CAR of claim 25, or the pharmaceutical composition of claim 26.

28. A method of treating cancer in a subject comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-17, the nucleic acid of claim 18, the vector of claim 19, the host cell of claim 20, the conjugate of any one of claims 22-24, the CAR of claim 25, or the pharmaceutical composition of claim 26.

29. The method of claim 28, wherein the cancer is a cancer associated with CEACAM5 expression, preferably a solid tumor, further preferably gastric cancer or colorectal cancer.

30. A method for detecting the presence or level of CEACAM5 in a sample comprising: (1) contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-17; and (2) determining the presence or level of CEACAM5 in the sample by detecting binding of the antibody to the sample.

31. A method for diagnosing whether a subject has a cancer associated with CEACAM5 expression comprising: (1) contacting a sample from the subject with the antibody or antigen-binding fragment thereof of any one of claims 1-17; and (2) detecting binding of the antibody to the sample, wherein increased binding of the antibody to the sample compared to binding to a healthy control indicates that the subject has a cancer associated with CEACAM5 expression.

32. The method of claim 31, wherein the cancer is selected from a solid tumor, preferably gastric cancer or colorectal cancer.

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