ANTI-CEACAM5 MONOCLONAL ANTIBODY, PREPARATION METHOD THEREOF AND USE THEREOF

Abstract

Provided are a monoclonal antibody capable of specifically binding to human carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5) and an antigen binding fragment thereof. Further provided are a preparation method for and use of said antibody and antigen binding fragment thereof.

Claims

1.-25. (canceled)

26. An antibody or antigen-binding fragment thereof that specifically binds to CEACAM5 protein, wherein the antibody or antigen-binding fragment thereof comprises: 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 7; and/or, 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 8; wherein, the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined by Kabat, IMGT or Chothia numbering system.

27. The antibody or antigen-binding fragment thereof according to claim 26, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH) comprising the following 3 complementarity determining regions (CDRs): (i) VH CDR1, which consists of the following sequence: SEQ ID NO: 1, or a sequence having a substitution, deletion or addition of one or several amino acids as compared therewith, (ii) VH CDR2, which consists of the following sequence: SEQ ID NO: 2, or a sequence having a substitution, deletion or addition of one or several amino acids as compared therewith, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO: 3, or a sequence having a substitution, deletion or addition of one or several amino acids as compared therewith; and/or, (b) a light chain variable region (VL) comprising the following 3 complementarity determining regions (CDRs): (iv) VL CDR1, which consists of the following sequence: SEQ ID NO: 4, or a sequence having a substitution, deletion or addition of one or several amino acids as compared therewith, (v) VL CDR2, which consists of the following sequence: SEQ ID NO: 5, or a sequence having a substitution, deletion or addition of one or several amino acids as compared therewith, and (vi) VL CDR3, which consists of the following sequence: SEQ ID NO: 6, or a sequence having a substitution, deletion or addition of one or several amino acids as compared therewith.

28. The antibody or antigen-binding fragment thereof according to claim 27, wherein the antibody or antigen-binding fragment thereof is characterized by one or more of the following: (1) the substitution described in any one of (i) to (vi) is a conservative substitution; and, (2) the antibody or antigen-binding fragment thereof comprises the following three heavy chain CDRs: VH CDR1 having a sequence as shown in SEQ ID NO: 1, VH CDR2 having a sequence as shown in SEQ ID NO: 2, and VH CDR3 having a sequence as shown in SEQ ID NO: 3; and/or, the following three light chain CDRs: VL CDR1 having a sequence as shown in SEQ ID NO: 4, VL CDR2 having a sequence as shown in SEQ ID NO: 5, and VL CDR3 having a sequence as shown in SEQ ID NO: 6.

29. The antibody or antigen-binding fragment thereof according to claim 26, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region (VH), which comprises an amino acid sequence selected from the following: (i) the sequence as shown in SEQ ID NO: 7; (ii) a sequence having a substitution, deletion or addition of one or several amino acids as compared with the sequence shown in SEQ ID NO: 7; or (iii) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence shown in SEQ ID NO: 7; and/or (b) a light chain variable region (VL), which comprises an amino acid sequence selected from the following: (iv) the sequence as shown in SEQ ID NO: 8; (v) a sequence having a substitution, deletion or addition of one or several amino acids as compared with the sequence shown in SEQ ID NO: 8; or (vi) a sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% as compared with the sequence shown in SEQ ID NO: 8; optionally, the antibody or antigen-binding fragment thereof is characterized by one or more of the following: (1) the substitution described in (ii) or (v) is a conservative substitution; and, (2) the antibody or antigen-binding fragment thereof comprises: VH having the sequence shown in SEQ ID NO: 7 and VL having the sequence shown in SEQ ID NO: 8.

30. The antibody or antigen-binding fragment thereof according to claim 26, wherein the antibody or antigen-binding fragment thereof comprises a heave chain constant region (CH) of a human immunoglobulin and/or a light chain constant region (CL) of a human immunoglobulin; optionally, the antibody or antigen-binding fragment thereof is characterized by one or more of the following: (1) the heavy chain constant region is an IgG heavy chain constant region; (2) the heavy chain constant region has the sequence as shown in SEQ ID NO: 9; (3) the light chain constant region is a κ light chain constant region; (4) the light chain constant region has the sequence as shown in SEQ ID NO: 11; (5) the antigen-binding fragment is selected from the group consisting of Fab, Fab′, (Fab′).sub.2, Fv, disulfide-linked Fv, BsFv, DsFv, (dsFv).sub.2, dsFv-dsFv′, scFv, scFv dimer, camelized single chain domain antibody, diabody, ds diabody, nanobody, single domain antibody (sdAb), bivalent domain antibody; and/or, the antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody; and (6) the antibody or antigen-binding fragment thereof comprises a label.

31. An isolated nucleic acid molecule, which encodes the antibody or antigen-binding fragment thereof according to claim 26, or its heavy chain variable region and/or light chain variable region.

32. A vector, which comprises the isolated nucleic acid molecule according to claim 31.

33. A host cell, which comprises the isolated nucleic acid molecule according to claim 31 or a vector comprising the isolated nucleic acid molecule.

34. A method for preparing the antibody or antigen-binding fragment thereof according to claim 26, which comprises: (i) culturing a host cell under a condition allowing expression of the antibody or antigen-binding fragment thereof, wherein the host cell comprises a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof; and (ii) recovering the antibody or antigen-binding fragment thereof from a culture of the host cell.

35. A bispecific or multispecific molecule, which comprises the antibody or antigen-binding fragment thereof according to claim 26; optionally, the bispecific or multispecific molecule is characterized by one or more of the following: (1) the bispecific or multispecific molecule specifically binds to CEACAM5, and additionally specifically binds to one or several other targets; (2) the bispecific or multispecific molecule further comprises at least one molecule having a second binding specificity for a second target; and (3) the bispecific or multispecific molecule further comprises a second antibody.

36. An immunoconjugate, which comprises the antibody or antigen-binding fragment thereof according to claim 26 and a therapeutic agent linked to the antibody or antigen-binding fragment thereof; optionally, the immunoconjugate is characterized by one or more of the following: (1) the therapeutic agent is a cytotoxic agent; (2) the therapeutic agent is selected from the group consisting of alkylating agent, mitotic inhibitor, anti-tumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide agent, and any combination thereof, and (3) the immunoconjugate is an antibody-drug conjugate (ADC).

37. A pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof according to claim 26, or a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof, or an immunoconjugate comprising the antibody or antigen-binding fragment thereof, with a pharmaceutically acceptable carrier and/or excipient; and optionally, comprises an additional pharmaceutically active agent; optionally, the pharmaceutical composition is characterized by one or more of the following: (1) the additional pharmaceutically active agent is a drug with an anti-tumor activity; (2) the additional pharmaceutically active agent is an alkylating agent, mitotic inhibitor, anti-tumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide, radiosensitizer, anti-angiogenesis agent, cytokine, molecular targeted drug, immune checkpoint inhibitor or oncolytic virus; and (3) the antibody or antigen-binding fragment thereof, bispecific or multispecific molecule or immunoconjugate and the additional pharmaceutically active agent are provided as separate components or as components of the same composition.

38. A kit, which comprises the antibody or antigen-binding fragment thereof according to claim 26; and optionally, a second antibody; wherein the antibody or antigen-binding fragment thereof and/or the second antibody optionally comprises a detectable label.

39. A chimeric antigen receptor, which comprises an antigen-binding domain of the antibody or antigen-binding fragment thereof according to claim 26; optionally, the chimeric antigen receptor is characterized by one or more of the following: (1) the antigen-binding domain comprises a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof according to claim 26; and (2) the antigen-binding domain is a scFv.

40. An isolated nucleic acid molecule, which encodes the chimeric antigen receptor according to claim 39.

41. A vector, which comprises the isolated nucleic acid molecule according to claim 40.

42. A host cell, which comprises the isolated nucleic acid molecule according to claim 40 or a vector comprising the isolated nucleic acid molecule; optionally, the host cell is characterized by one or more of the following: (1) the host cell is an immune effector cell; (2) the host cell is T cell or NK cell; and (3) the host cell is a chimeric antigen receptor T cell (CAR-T).

43. A method for reducing expression level of CEACAM5 on the surface of a cell, which comprises contacting a cell expressing CEACAM5 on the surface thereof with the following: (1) the antibody or antigen-binding fragment thereof according to claim 26, or (2) a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof, or (3) an immunoconjugate comprising the antibody or antigen-binding fragment thereof, or (4) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or (5) a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or (6) a host cell comprising the isolated nucleic acid molecule encoding the chimeric antigen receptor, or (7) any combination thereof, so as to reduce expression of CEACAM5 on the surface of the cell; optionally, the cell is a tumor cell expressing CEACAM5.

44. A method for inhibiting growth of a tumor cell expressing CEACAM5 and/or killing the tumor cell, which comprises contacting the tumor cell with an effective amount of the following: (1) the antibody or antigen-binding fragment thereof according to claim 26, or (2) a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof, or (3) an immunoconjugate comprising the antibody or antigen-binding fragment thereof, or (4) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or (5) a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or (6) a host cell comprising the isolated nucleic acid molecule encoding the chimeric antigen receptor, or (7) any combination thereof.

45. A method for preventing and/or treating a tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the following: (1) the antibody or antigen-binding fragment thereof according to claim 26, or (2) a bispecific or multispecific molecule comprising the antibody or antigen-binding fragment thereof, or (3) an immunoconjugate comprising the antibody or antigen-binding fragment thereof, or (4) a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or (5) a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or (6) a host cell comprising the isolated nucleic acid molecule encoding the chimeric antigen receptor; (7) any combination thereof, optionally, the method is characterized by one or more of the following: (1) the tumor involves a tumor cell expressing CEACAM5; (2) the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cell carcinoma hematological malignancies, primary mediastinal large B-cell lymphoma, T cell/histiocytic B-cell-rich lymphoma, EBV-positive and negative PTLD, EBV-related diffuse large B cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell Lymphoma, nasopharyngeal carcinoma HHV8-related primary exudative lymphoma, Hodgkin's lymphoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal axis tumor, and brainstem glioma; (3) the subject is a mammal; (4) the subject is a human; (5) the method further comprises administering an additional drug with an anti-tumor activity; (6) the method further comprises administering alkylating agent, mitotic inhibitor, anti-tumor antibiotic, antimetabolite, topoisomerase inhibitor, tyrosine kinase inhibitor, radionuclide, radiosensitizer, anti-angiogenesis agent, cytokine, molecular targeted drug, immune checkpoint inhibitor or oncolytic virus; (7) the method further comprises administering an additional an anti-tumor therapy; and (8) the method further comprises administering a surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative therapy.

46. A method for detecting the presence or amount of CEACAM5 in a sample, which comprises the following steps: (1) contacting the sample with the antibody or antigen-binding fragment thereof according to claim 26; (2) detecting formation of a complex comprising the antibody or antigen-binding fragment thereof and CEACAM5, or detecting the amount of the complex; optionally, the CEACAM5 is human CEACAM5.

47. A method for determining whether a tumor is capable of being treated by an anti-tumor therapy targeting CEACAM5, which comprises the following steps: (1) contacting a sample containing a cell of the tumor with the antibody or antigen-binding fragment thereof according to claim 26; (2) detecting formation of a complex comprising the antibody or antigen-binding fragment thereof and CEACAM5; optionally, the method is characterized by one or more of the following: (1) the CEACAM5 is human CEACAM5; and (2) the tumor is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoids, Merkel cell carcinoma hematological malignancies, primary mediastinal large B-cell lymphoma, T cell/histiocytic B-cell-rich lymphoma, EBV-positive and negative PTLD, EBV-related diffuse large B cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell Lymphoma, nasopharyngeal carcinoma HHV8-related primary exudative lymphoma, Hodgkin's lymphoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal axis tumor, and brainstem glioma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0214] FIG. 1A to 1B show the overexpression level of CEACAM5 in the CHO cell line constructed in the present application; as compared with conventional CHO cells, the overexpression level of CEACAM5 in the CHO cell line constructed in the present application is up to 904 times.

[0215] FIG. 2 shows the results of the ELISA experiment of the binding of antibody UM05-6L to CEACAM5 protein.

[0216] FIG. 3 shows the detection results of the binding ability of antibody UM05-6L to LOVO cells.

[0217] FIG. 4A to 4B show the ADCC reporter gene activity of antibody UM05-6L; in which, FIG. 4A shows the ADCC reporter gene activity of the positive control (Erbitux antibody) and the negative control, and FIG. 4B shows the ADCC reporter gene activity of UM05-6L.

[0218] FIG. 5A to 5B show the results of flow cytometry; in which FIG. 5A shows the expression efficiency of control T cells (Mock T), and FIG. 5B shows the expression efficiency of UM05-6L-CAR.

[0219] FIG. 6A to 6B show the results of the secretion of cytokines IFNγ (left) and IL-2 (right) after UM05-6L-CAR-T is mixed with tumor cells.

[0220] FIG. 7 shows the killing results of UM05-6L-CAR-T cells on tumor cell KATO3.

[0221] FIG. 8 shows the expansion results of UM05-6L-CAR-T cells stimulated by tumor cell LS174T.

[0222] FIG. 9 shows the results of UM05-6L-CAR-T inhibiting tumors in mice.

[0223] FIG. 10 shows the release of IFNγ in mice receiving UM05-6L-CAR-T.

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0224] The present application will now be described with reference to the following examples which are intended to illustrate the present application (not limit the present application).

[0225] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present application basically referred to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F M Ausubel et al., Compiled Molecular Biology Experiment Guide, 3rd edition, John Wiley & Sons, Inc., 1995; the restriction enzymes were used in accordance with the conditions recommended by the product manufacturer. If specific conditions were not indicated in the examples, it should be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication were all conventional products that were purchased commercially. Those skilled in the art know that the examples describe the present application by way of example, and are not intended to limit the scope of protection claimed by the present application.

Example 1: Acquisition of Monoclonal Antibody Against Human CEACAM5 Protein

[0226] In the present application, a CHO cell line overexpressing CEACAM5 protein was constructed. In short, the CEACAM5 plasmid (Beijing Yiqiao Shenzhou Technology Co., Ltd., HG11077-UT) was transfected into a CHO cell line (ATCC) and expressed. The plasmid was resistant to hygromycin (Hygromycin), so that a cell stably transfected with this plasmid could be stably passaged in a medium containing hygromycin. The cells were picked and the expression of CEACAM was measured. As shown in FIGS. 1A and 1B, the CHO-CEACAM5 cell detected by FACS had a CEACAM overexpression factor of 904 times.

[0227] The CHO-CEACAM5 cells and the CEACAM5 protein (L2C01001) purified from tumor patients were used to immunize 6 Balb/c mice aged 5-8 weeks (Shanghai Slack) according to the immunization schedule in Table 2.

TABLE-US-00002 TABLE 2 Immunization schedule in mice Date Operation Antigen Adjuvant Day 1 Immunization by CHO-CEACAM5 cells CFA intraperitoneal injection Day 26 Immunization by CHO-CEACAM5 cells IFA intraperitoneal injection Day 49 Immunization by CHO-CEACAM5cells IFA intraperitoneal injection Day 91 Immunization by CEACAM5 protein Gerbu(MM3001) intraperitoneal injection Day 104 Immunization by CEACAM5 protein Gerbu(MM3001) intraperitoneal injection Day 105 Immunization by CEACAM5 protein Gerbu(MM3001) intraperitoneal injection Day 106 Immunization by CEACAM5 protein Gerbu(MM3001) intraperitoneal injection

[0228] On the second day after the immunization, the spleen cells of the immunized mice were taken, and fused with SP2/0-AG14 cells (ATCC) to prepare hybridoma cells, and an appropriate amount of the fused cells were plated on a 96-well plate. About 10 days after the fusion, the supernatant of each well was taken, and the binding activity of the mouse antibody secreted by the hybridoma cells to human CEACAM5 was detected by ELISA.

[0229] Furthermore, the supernatant of the well with strong positivity detected by ELISA was taken, its binding activity to LOVO cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) was detected by flow cytometry, and hybridoma cells with high binding activity to LOVO cells were obtained. These cells were subcloned to obtain monoclonal cells. Table 3 showed the detection data of some hybridoma cells.

TABLE-US-00003 TABLE 3 Binding results of hybridoma cells to CEACAM Hybridoma cells ELISA OD value LOVO FACS binding 1-2C5 1.0690 12.1 1-9G3 1.0390 7.01 3-2F3 1.4480 11 3-6F5 1.3520 6.51 3-8C10 2.3030 137 3-8G8 1.9270 82.9 2A10A5 2.392 / 2A10C4 2.434 /

[0230] The hybridoma cell 3-8G8 with better binding activity was selected for sequencing. After sequencing, an antibody was obtained, named UM05-6, and the CDR sequences of the UM05-6 antibody were determined by using the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991), pages 647-669), and shown in Table 4.

TABLE-US-00004 TABLE 4 UM05-6 variable region sequences SEQ ID NO: Monoclonal Numbering antibody VH VL system HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 UM05-6 7 8 Kabat 1 2 3 4 5 6

Example 2: Preparation of Human-Mouse Chimeric Antibody

[0231] According to the sequence of UM05-6 in Table 4, a human-mouse chimeric antibody was designed and expressed, and named as UM05-6L. In short, the sequences encoding the mouse antibody VH and VL were respectively linked to the sequence encoding the human IgG1 heavy chain constant region (its amino acid sequence was shown in SEQ ID NO: 9, and its nucleotide sequence was shown in SEQ ID NO: 10) and the κ chain constant region sequence (its amino acid sequence was shown in SEQ ID NO: 11, and its nucleotide sequence was shown in SEQ ID NO: 12) to obtain the human-mouse chimeric antibody UM05-6L.

[0232] The nucleotide sequences encoding the heavy chain and light chain of the antibody were cloned into the mammalian cell expression vector pcDNA3.4, respectively. The heavy chain expression vector and light chain expression vector were transfected into HEK293 cells with Lipofectamine 2000 transfection reagent (Invitrogen) at a molar ratio of 2:1, and cultured at 37° C. and 5% carbon dioxide for 7 days. The culture medium supernatant was collected, and the antibody in the supernatant was purified by Protein A affinity chromatography. After the purification, the antibody was dialyzed with PBS solution, freeze-dried and concentrated, and stored at −20° C.

Example 3: Binding of Antibody to CEACAM5 Protein

[0233] A 96-well high-affinity plate was coated with a human CEACAM5 protein solution with a concentration of 1 μg/mL, 100 μL/well at 4° C., and shaken overnight. On the next day, it was first washed with 300 μL of PBST (Tween20: 0.5‰) for 3 times, then blocked with 100 μL/well of 5% BSA/PBS for 1 hour, and shaken at room temperature. Washing was performed 3 times with 300 μL of PBST. A series of dilution solutions of antibody samples were prepared with PBS, and added at 100 μL/well to the 96-well plate and shaken at room temperature for 1 hour. Washing was performed 3 times with 300 μL of PBST. A secondary antibody goat anti-human IgG HRP solution was prepared, added at 100 μL/well to the 96-well plate, and shaken at room temperature for 30 minutes. Washing was performed 4 times with 300 μL of PBST. 100 μL/well TMB was added to perform color development for 20 min. 100 μL/well of 0.6N H.sub.2SO.sub.4 was added to stop the color development, and OD450 nm was detected. After detection, the results were shown in FIG. 2. The EC50 of the binding of the chimeric antibody UM05-6L to CEACAM5 protein was 47.47 ng/mL.

Example 4: Binding of Antibody to Cells Expressing CEACAM5

[0234] Flow cytometry (FACS) was used to detect the binding of antibody to LOVO tumor cells that naturally expressed human CEACAM5 protein or cells that overexpressed different CEACAM proteins. In short, cells were first collected, washed with PBS, counted, and diluted to obtain 2*10.sup.6/ml cell suspension; 10 μl of an antibody working solution was added to 100 μl of the cell suspension, and incubated at 4° C. for 30 min in the dark; after washing twice with PBS, a corresponding fluorescent-labeled secondary antibody Goat-anti-Human IgG (H+L) (Invitrogen) was added, incubated at 4° C. for 30 min in the dark, after washing twice with PBS, it was suspended in 400 μl of FACS buffer, and the binding situations of the antibody to the cells were detected by FACS.

[0235] The binding situations of the supernatants of the chimeric antibody UM05-6L and the control antibody UM05-8 (anti-CEACAM5 antibody, prepared by the hybridoma method in our laboratory) to different cells at a concentration of 3 μg/mL were detected, in which SW620-CEACAM1 was a cell overexpressing CEACAM1 protein, SW620-CEACAM3 was a cell overexpressing CEACAM3 protein, CHO-CEACAM6 was a cell overexpressing CEACAM6 protein, CHO-CEACAM7 was a cell overexpressing CEACAM7 protein, and CHO-CEACAM8 was a cell overexpressing CEACAM8 protein. On the basis that the binding activity (that was, the detection value of FACS) of antibody to LOVO cells (which expressed CEACEM5) was 100%, the relative binding activity of the antibody to different cells (which expressed different CEACAM proteins) were calculated, and the detection results were shown in Table 5.

TABLE-US-00005 TABLE 5 Binding of antibodies to LOVO cells and cells overexpressing different CEACAM proteins Cell name UM05-6L UM05-8 LOVO 100.00% 100.00% SW620-CEACAM1 1.20% 120.47% SW620-CEACAM3 0.84% 91.56% CHO-CEACAM6 1.35% 69.61% CHO-CEACAM7 0.36% 13.71% CHO-CEACAM8 15.23% 61.60%

[0236] It could be seen from Table 5 that the chimeric antibody UM05-6L had particularly outstanding selectivity: it bound to CEACAM5 with high affinity, but did not obviously bind to CEACAM1, CEACAM3, CEACAM6, CEACAM7, and only weakly bound to CEACAM8. While the ordinary antibody (for example, the control antibody UM05-8) could bind to CEACAM5, and simultaneously bind to CEACAM1, CEACAM3, CEACAM6, CEACAM7 and CEACAM8 at a high level, and showed no selectivity.

[0237] Furthermore, we used FACS to detect the EC50 of the chimeric antibody UM05-6L binding to LOVO tumor cells that naturally expressed human CEACAM5. The results were shown in FIG. 3, in which the EC50 of the chimeric antibody UM05-6L binding to LOVO cells was 276.1 ng/mL. Based on the above results, the chimeric antibody UM05-6L had excellent binding affinity, specificity and selectivity to CEACAM5.

Example 5: ADCC Activity of Antibodies

[0238] Using LOVO cells as target cells, the LOVO cells were inoculated on a 96-well cell culture plate at a density of 20,000 cells/well, and incubated overnight at 37° C. and 5% CO.sub.2. On the second day, the antibody UM05-6L and the positive control antibody Erbitux were prepared at 20 μg/ml in the culture medium, and were diluted by 3 times to obtain 8 concentrations. The supernatant medium of the LOVO cells was removed by pipetting, and an antibody (positive control antibody Erbitux, chimeric antibody UM05-6L or negative control irrelevant antibody) with diluted to a specified concentration was added at 30 μL/well to the LOVO cells. Subsequently, effector cells Jurkat/ADC (Nanjing Nuoaixin Biotechnology Co., Ltd.) were plated into LOVO cell wells at 120,000 cells/30 μL/well, and incubated at 37° C. and 5% CO.sub.2 for 16 to 20 hours. After the incubation, Bright-Glo kit (Promega, cat. E2620) was used to detect the expression level of luciferase in the effector cells. The luciferase level represented the degree of ADCC activation of the effector cells. FIG. 4A showed the ADCC reporter gene activity of the positive control Erbitux antibody and the negative control, and FIG. 4B showed the ADCC reporter gene activity of UM05-6L. Based on the above results, it could be seen that UM05-6L had strong ADCC activity with an EC50 of 0.57 g/mL.

Example 6: Construction of CAR-T Cells Targeting CEACAM5

[0239] Based on the CAR-T structures of the second and third generations, we used anti-CEACAM5 single-chain antibody as CAR-T recognition antibody, and used one or several costimulatory components such as 41BB, CD28, OX40 and others to carry out the design of different CEA-CAR structures, and constructed corresponding lentiviral plasmids to generate lentivirus that could infect cells and express the corresponding CAR. The lentivirus could be used to infect T cells isolated from the peripheral blood of tumor patients, and produce CAR-T cells expressing corresponding CAR receptors on the cell membrane surface. This kind of CAR-T cells could effectively recognize and kill tumor cells expressing CEACAM5. Both in vitro and in vivo experiments, it was seen that this cellular immunotherapy had very good safety and effectiveness.

[0240] The single chain antibody UM05-6L scFv (in the order of VH-G4S3-VL) as shown in SEQ ID NO: 13 was ligated to the sequence CD8α-CD137-CD3ζ-p2A-tEGFR to construct a chimeric antigen receptor CAR, and then the nucleotide sequence encoding the CAR was cloned into a lentiviral vector (Genechem, GV401), and the vector was named UM05-6L-CAR. The expression cassette was EF1a promoter-CAR-2A-tEGFR-WPRE.

[0241] The UM05-6L-CAR lentiviral vector and packaging plasmid were transiently transfected into 293T cells for 16 hours, the medium was changed, and the culture was continued for 24 to 48 hours. The medium supernatant (containing the lentivirus) was collected and stored at −80° C.

[0242] PBMC cells derived from healthy people were activated with CD3/28 antibody for 24 hours; the lentivirus and T cells were mixed according to MOI=3:1 and cultured for 96 hours, and the expression of UM05-6L-CAR was detected with PE fluorescent-labeled Cetuximab. The results were shown in FIG. 5. As compared with the control (FIG. 5A), UM05-6L-CAR (FIG. 5B) could be well expressed on the surface of human T cells.

Example 7: Function Detection of CAR-T Cells Targeting CEACAM5

[0243] 1. Detection of Cytokine Release

[0244] UM05-6L-CAR-T cells and CEACAM5-expressing tumor cells (for example, KATO3 and LS174T) were mixed, and a medium control (that was, only UM05-6L-CAR-T cells were used) and a CEACAM5 negative cell control (that was, UM05-6L-CAR-T cells and cells not expressing CEACAM5 such as RKO cells were used) were set; the two kinds of cells were mixed at a ratio of 1:1 and cultured for 16 hours, and the released IFNγ and IL2 in the supernatant were detected. The results were shown in FIG. 6, in which UM05-6L-CAR-T could recognize the cell line expressing CEACAM5 and cause the release of cytokines IFNγ and IL-2.

[0245] 2. Detection of Killing Tumor Cells

[0246] UM05-6L-CAR-T and CEACAM5-expressing tumor cells (for example, KATO3) were mixed and cultured in 96-well culture plate at a specified E:T ratio (30:1, 10:1, 3:1, 1:1, 0.3:1). Promega LDH detection kit was used to detect the release record data of lactate dehydrogenase (LDH) in the culture supernatant. The calculation results of killing tumor cells were shown in FIG. 7. It could be seen from the figure that UM05-6L-CAR-T could kill tumor cells efficiently and dose-dependently.

[0247] 3. Proliferation Experiment Stimulated by Tumor Cells

[0248] UM05-6L-CAR-T cells and CEACAM5-expressing tumor cells (for example, LS174T) were mixed and cultured for 72 hours (without IL2 supplement) according to E:T=1:1, and their proliferation was detected by cell counting. The results were shown in FIG. 8. UM05-6L-CAR-T could be efficiently proliferated when stimulated by the tumor cells expressing CEACAM5.

Example 8: In Vivo Pharmaceutical Efficacy Experiment of LS174T Tumor Model

[0249] LS174T cells (ATCC CL-187) were inoculated subcutaneously into NSG mice (Biocytometer) at 1×10.sup.7/mouse. When the tumor volume reached 200 to 400 mm.sup.3, PBS control, unmodified T cell control (Mock T) or UM05-6L CAR-T were administered via tail vein at doses of 100 μl, 1×10.sup.7 cells, and 1×10.sup.7 tEGFR+ cells, respectively. The tumor volume changes were recorded and the IFNγ release in peripheral blood was analyzed. The results were shown in FIG. 9 and FIG. 10. The UM05-6L recognized LS174T tumor cells in mice and released a large amount of IFNγ, resulting in strong tumor suppression effect.