Anti-CEACAM5 antibody or antigen binding fragment thereof and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to an anti-CEACAM5 antibody or an antigen binding fragment thereof and application of the anti-CEACAM5 antibody or the antigen binding fragment. The invention also relates to an antibody coupling drug containing the anti-CEACAM5 antibody or the antigen binding fragment thereof and an application of the antibody coupling drug in tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-CEACAM5 antibody or its antigen-binding fragment and its applications. Background Technology

[0002] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is expressed in limited quantities in normal tissues, but is highly expressed on the surface of various epithelial tumor cells. CEACAM5, also known as CD66e, is located on human chromosome 19q13.2 and consists of 9 exons and 3 non-coding exons. CEACAM5 activates endothelial cell and tumor angiogenesis by participating in the regulation of cell differentiation, apoptosis, and cell polarity. High fucosylation of the 28 N-glycosylation sites on the CEACAM5 protein surface can directly promote epithelial-mesenchymal transition (EMT) and dedifferentiation. Studies have shown that the interaction between CEACAM5 on the surface of tumor cells and CEACAM1 on the surface of NK cells can inhibit NK cell-mediated anti-tumor immune effects. Therefore, CEACAM5 is one of the key driving factors in tumorigenesis and development.

[0003] CEACAM5 was initially discovered in the 1970s during studies of human embryonic development. Subsequent research has observed significantly increased expression levels of CEACAM5 protein in various tumors. Furthermore, CEACAM5 protein detaches from the surface of tumor cells to form soluble CEACAM5, making it an effective clinical biomarker and potential therapeutic target in melanoma, lung cancer, colorectal cancer, gastric cancer, and pancreatic cancer. In gastric cancer patients, over 90% of tumor tissues overexpress CEACAM5, and in colorectal cancer (CRC) patients, 98.8% show significantly higher expression levels than normal tissues. CEACAM5 is a biomarker for monitoring tumor recurrence in colorectal cancer patients; elevated expression levels are generally considered a sign of high tumor burden and poor prognosis. In CRC patients, 5-year survival is negatively correlated with tissue expression of CEACAM5, making it a current clinically used prognostic and monitoring biomarker for determining treatment strategies.

[0004] The present invention aims to develop a novel specific antibody targeting CEACAM5, which is of great significance for the diagnosis and treatment of tumors. Summary of the Invention

[0005] In this invention, to screen for antibodies specifically targeting the juxtamembranous domain of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mice were immunized with different fusion proteins, and multiple fusion proteins were designed for screening. Several mouse monoclonal antibodies specifically binding to the juxtamembranous domain of CEACAM5 were ultimately obtained. After chimeric modification, their specific binding ability and in vitro cytotoxic activity were evaluated at the protein and cellular levels, confirming excellent endocytosis activity and potential for further development into antibody-drug conjugates (ADCs). Further preparation of ADCs through site-specific conjugation of small molecule drugs showed significant killing effects on tumor cells. The specific scheme is as follows:

[0006] A first aspect of the present invention provides an anti-CEACAM5 antibody or an antigen-binding fragment thereof, wherein the anti-CEACAM5 antibody or the antigen-binding fragment thereof comprises VHCDR1-3 of the heavy chain variable region and / or VLCDR1-3 of the light chain variable region. Wherein:

[0007] The amino acid sequence of VHCDR1 comprises at least four consecutive amino acids from GYTFTNYGMN (SEQ ID NO: 55), preferably at least four, five, six, seven, eight, nine, or ten consecutive amino acid sequences. Alternatively, the amino acid sequence of VHCDR1 comprises at least four consecutive amino acids from GYIFTNYGMS (SEQ ID NO: 56), preferably at least four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, thirteen, eleven, thirteen, eleven, seventeen, or eighteen consecutive amino acid sequences. In one specific embodiment of the present invention, the amino acid sequence of VHCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, 22, 28, 34, 40 or 46, or comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more homology to the amino acid sequence shown in SEQ ID NO: 16, 22, 28, 34, 40 or 46, or comprises an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine or ten amino acids have been substituted, deleted or added.

[0008] The amino acid sequence of VHCDR2 includes at least four consecutive amino acids as specified in SEQ ID NO: 17, preferably at least four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or seventeen consecutive amino acid sequences. Alternatively, the amino acid sequence of VHCDR2 includes at least four consecutive amino acids as specified in SEQ ID NO: 35, preferably at least four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, thirteen, or seventeen consecutive amino acid sequences. In one specific embodiment of the present invention, the amino acid sequence of VHCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, 23, 29, 35, 41 or 47, or comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more homology to the amino acid sequence shown in SEQ ID NO: 17, 23, 29, 35, 41 or 47, or comprises an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine or ten amino acids have been substituted, deleted or added.

[0009] The amino acid sequence of VHCDR3 includes at least four consecutive amino acids as specified in SEQ ID NO: 30, preferably at least four, five, six, seven, eight, nine, or ten consecutive amino acid sequences. Alternatively, the amino acid sequence of VHCDR3 includes at least four consecutive amino acids as specified in SEQ ID NO: 48, preferably at least four, five, six, seven, or eight consecutive amino acid sequences. In one specific embodiment of the present invention, the amino acid sequence of VHCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18, 30, 36 or 48, or comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or more or 99% or more homology with the amino acid sequence shown in SEQ ID NO: 18, 30, 36 or 48, or comprises an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine or ten amino acids have been substituted, deleted or added.

[0010] The amino acid sequence of VLCDR1 includes at least four consecutive amino acids as in SEQ ID NO: 19, preferably at least four, five, six, seven, eight, nine, ten, eleven, thirteen, or eleven consecutive amino acid sequences. Alternatively, the amino acid sequence of VLCDR1 includes at least four consecutive amino acids as in SEQ ID NO: 37, preferably at least four, five, six, seven, eight, nine, ten, or eleven consecutive amino acid sequences. In one specific embodiment of the present invention, the amino acid sequence of VLCDR1 comprises the amino acid sequence shown in SEQ ID NO: 19, 31, 37 or 49, or comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or more or 99% or more homology with the amino acid sequence shown in SEQ ID NO: 19, 31, 37 or 49, or comprises an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine or ten amino acids have been substituted, deleted or added.

[0011] The amino acid sequence of VLCDR2 comprises at least two consecutive amino acids as shown in SEQ ID NO: 20, preferably at least two, three, four, five, six, or seven consecutive amino acid sequences. Alternatively, the amino acid sequence of VLCDR2 comprises at least two consecutive amino acids as shown in SEQ ID NO: 38, preferably at least two, three, four, five, six, or seven consecutive amino acid sequences. In one specific embodiment of the present invention, the amino acid sequence of VLCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, 32, 38 or 50, or comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or more or 99% or more homology with the amino acid sequence shown in SEQ ID NO: 20, 32, 38 or 50, or comprises an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine or ten amino acids have been substituted, deleted or added.

[0012] The amino acid sequence of VLCDR3 comprises at least two consecutive amino acids as in SEQ ID NO: 21, preferably at least two, three, four, five, six, seven, eight, or nine consecutive amino acid sequences. Alternatively, the amino acid sequence of VLCDR3 comprises at least two consecutive amino acids as in SEQ ID NO: 39, preferably at least two, three, four, five, six, seven, eight, or nine consecutive amino acid sequences. In one specific embodiment of the present invention, the amino acid sequence of VLCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21 or 39, or comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the amino acid sequence shown in SEQ ID NO: 21 or 39, or comprises an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine, or ten amino acids have been substituted, deleted, or added.

[0013] Preferably, in the anti-CEACAM5 antibody or its antigen-binding fragment, the amino acid sequence of VHCDR1 includes at least 4 consecutive amino acids from GYTFTNYGMN (SEQ ID NO: 55), preferably at least 4, 5, 6, 7, 8, 9, or 10 consecutive amino acid sequences; the amino acid sequence of VHCDR2 includes at least 4 consecutive amino acids from SEQ ID NO: 17, preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive amino acid sequences; the amino acid sequence of VHCDR3 includes at least 4 consecutive amino acids from SEQ ID NO: 30, preferably at least 4, 5, 6, 7, 8, 9, or 10 consecutive amino acid sequences; the amino acid sequence of VLCDR1 includes at least 4 consecutive amino acids from SEQ ID NO: 19, preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive amino acid sequences; the amino acid sequence of VLCDR2 includes SEQ ID NO: 55. NO: 20 contains at least two consecutive amino acids, preferably at least two, three, four, five, six or seven consecutive amino acid sequences; the amino acid sequence of VLCDR3 contains at least two consecutive amino acids, preferably at least two, three, four, five, six, seven, eight or nine consecutive amino acid sequences.

[0014] Further preferably, in the anti-CEACAM5 antibody or its antigen-binding fragment, the amino acid sequence of VHCDR1 includes SEQ ID NO: 16, 22, 28; the amino acid sequence of VHCDR2 includes SEQ ID NO: 17, 23, 29; the amino acid sequence of VHCDR3 includes SEQ ID NO: 18, 30; the amino acid sequence of VLCDR1 includes SEQ ID NO: 19, 31; the amino acid sequence of VLCDR2 includes SEQ ID NO: 20, 32; and the amino acid sequence of VLCDR3 includes SEQ ID NO: 21.

[0015] More preferably, in the anti-CEACAM5 antibody or its antigen-binding fragment, the amino acid sequence of VHCDR1 includes SEQ ID NO: 16, 22; the amino acid sequence of VHCDR2 includes SEQ ID NO: 23; the amino acid sequence of VHCDR3 includes SEQ ID NO: 18; the amino acid sequence of VLCDR1 includes SEQ ID NO: 31; the amino acid sequence of VLCDR2 includes SEQ ID NO: 32; and the amino acid sequence of VLCDR3 includes SEQ ID NO: 21.

[0016] In one specific embodiment of the present invention, the amino acid sequences of VHCDR1-3 and VLCDR1-3 include:

[0017]

[0018]

[0019] Preferably, in the anti-CEACAM5 antibody or its antigen-binding fragment, the amino acid sequence of VHCDR1 includes at least 4 consecutive amino acids from GYIFTNYGMS (SEQ ID NO: 56), preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 consecutive amino acid sequences; the amino acid sequence of VHCDR2 includes at least 4 consecutive amino acids from SEQ ID NO: 35, preferably at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 consecutive amino acid sequences; the amino acid sequence of VHCDR3 includes at least 4 consecutive amino acids from SEQ ID NO: 48, preferably at least 4, 5, 6, 7 or 8 consecutive amino acid sequences; the amino acid sequence of VLCDR1 includes at least 4 consecutive amino acids from SEQ ID NO: 37, preferably at least 4, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acid sequences; the amino acid sequence of VLCDR2 includes SEQ ID NO: 56. The amino acid sequence of NO: 38 includes at least two consecutive amino acids, preferably at least two, three, four, five, six or seven consecutive amino acid sequences; the amino acid sequence of VLCDR3 includes at least two consecutive amino acids, preferably at least two, three, four, five, six, seven, eight or nine consecutive amino acid sequences.

[0020] Further preferably, in the anti-CEACAM5 antibody or its antigen-binding fragment, the amino acid sequence of VHCDR1 includes SEQ ID NO: 34, 40 or 46; the amino acid sequence of VHCDR2 includes SEQ ID NO: 35, 41 or 47; the amino acid sequence of VHCDR3 includes SEQ ID NO: 36 or 48; the amino acid sequence of VLCDR1 includes SEQ ID NO: 37 or 49; the amino acid sequence of VLCDR2 includes SEQ ID NO: 38 or 50; and the amino acid sequence of VLCDR3 includes SEQ ID NO: 39.

[0021] More preferably, in the anti-CEACAM5 antibody or its antigen-binding fragment, the amino acid sequence of VHCDR1 includes SEQ ID NO: 34, 40; the amino acid sequence of VHCDR2 includes SEQ ID NO: 41; the amino acid sequence of VHCDR3 includes SEQ ID NO: 36; the amino acid sequence of VLCDR1 includes SEQ ID NO: 49; the amino acid sequence of VLCDR2 includes SEQ ID NO: 50; and the amino acid sequence of VLCDR3 includes SEQ ID NO: 39.

[0022] In one specific embodiment of the present invention, the amino acid sequences of VHCDR1-3 and VLCDR1-3 include:

[0023]

[0024]

[0025] In one specific embodiment of the present invention, the anti-CEACAM5 antibody or its antigen-binding fragment comprises VHCDR1-3 of the heavy chain variable region and / or VLCDR1-3 of the light chain variable region.

[0026] Preferably, the amino acid sequences of VHCDR1-3 and VLCDR1-3 include:

[0027]

[0028] The anti-CEACAM5 antibody or its antigen-binding fragment further includes FR regions, preferably including VHFR1-4 of the heavy chain variable region and / or VLFR1-4 of the light chain variable region.

[0029] Preferably, the VHFR1-4 are each independently derived from humans or non-human animals.

[0030] Preferably, the heavy chain variable region includes VHFR1, VHCDR1, VHFR2, VHCDR2, VHFR3, VHCDR3, and VHFR4 sequentially from the N end to the C end.

[0031] Further preferably, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, 6, or 8, or comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence shown in SEQ ID NO: 4, 6, or 8, or comprises an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids have been substituted, deleted, or added.

[0032] Preferably, the VLFR1-4 are each independently derived from humans or non-human animals.

[0033] Preferably, the variable region of the light chain includes VLFR1, VLCDR1, VLFR2, VLCDR2, VLFR3, VLCDR3, and VLFR4 sequentially from the N end to the C end.

[0034] Further preferably, the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5, 7, or 9, or comprises an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the amino acid sequence shown in SEQ ID NO: 5, 7, or 9, or comprises an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids have been substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO: 5, 7, or 9.

[0035] In one specific embodiment of the present invention, the heavy chain variable region and the light chain variable region of the anti-CEACAM5 antibody or its antigen-binding fragment are selected from any one of the following groups:

[0036] i) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 4, or contains an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to SEQ ID NO: 4, or contains one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO: 4; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 5, or contains an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to SEQ ID NO: 5, or contains SEQ ID NO: 4. The amino acid sequence shown in ID NO: 5 is an amino acid sequence in which one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids have been substituted, deleted, or added; or,

[0037] ii) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 6, or contains an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the amino acid sequence shown in SEQ ID NO: 6, or contains one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO: 6; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 7, or contains an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology ... NO: 7 has an amino acid sequence with 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology, or contains one, two, three, four, five, six, seven, eight, nine or more amino acids that have been substituted, deleted or added to the amino acid sequence shown in SEQ ID NO: 7.

[0038] Preferably, the anti-CEACAM5 antibody or its antigen-binding fragment includes Fab, Fd, Fab', Fab'-SH, Fv, scFv, F(ab')2 or a double antibody (dAb).

[0039] The anti-CEACAM5 antibody or its antigen-binding fragment contains a heavy chain constant region and / or a light chain constant region.

[0040] Preferably, the heavy chain constant region is derived from humans or non-human animals.

[0041] In one specific embodiment of the present invention, the heavy chain constant region is derived from humans.

[0042] Preferably, the heavy chain constant region is selected from the constant region of any one of the heavy chains IgG, IgD, IgA, IgM or IgE.

[0043] More preferably, the IgG heavy chain includes IgG1 heavy chain, IgG2 heavy chain, IgG2b heavy chain, IgG3 heavy chain or IgG4 heavy chain; the IgA heavy chain includes IgA1 or IgA2.

[0044] In one specific embodiment of the present invention, the amino acid sequence of the heavy chain constant region comprises SEQ ID NO: 10, or comprises an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the amino acid sequence shown in SEQ ID NO: 10, or comprises one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids that have been substituted, deleted, or added.

[0045] Preferably, the light chain constant region is derived from humans or non-human animals.

[0046] In one specific embodiment of the present invention, the light chain constant region is derived from humans.

[0047] The constant region of the light chain is selected from the constant region of the λ-type light chain or the κ-type light chain.

[0048] In one specific embodiment of the present invention, the amino acid sequence of the light chain constant region includes SEQ ID NO: 11 or 12, or contains an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with SEQ ID NO: 11 or 12, or contains one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids that have been substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO: 11 or 12.

[0049] In one specific embodiment of the present invention, the heavy chain constant region is the constant region of the IgG1 heavy chain, and the light chain constant region is the constant region of the κ-type light chain.

[0050] In one specific embodiment of the present invention, the heavy chain constant region is the constant region of the IgG1 heavy chain, and the light chain constant region is the constant region of the λ-type light chain.

[0051] Preferably, the anti-CEACAM5 antibody or its antigen-binding fragment targets or specifically binds to human or non-human CEACAM5 protein or fragments thereof. More preferably, it targets or specifically binds to human CEACAM5 protein or fragments thereof.

[0052] Preferably, the anti-CEACAM5 antibody or its antigen-binding fragment targets or specifically binds to any one or more of the following: the IgV-like N-terminal domain, the IgC2-like domain (e.g., A1, B1, A2, B2, A3, and B3), or the membrane-anchored hydrophobic C-terminal domain of the CEACAM5 protein. More preferably, the anti-CEACAM5 antibody or its antigen-binding fragment targets or specifically binds to A3 and / or B3 within the IgC2-like domain.

[0053] More preferably, the anti-CEACAM5 antibody or its antigen-binding fragment targets or specifically binds to the vicinity of the A3 domain and / or B3 domain of the near-membrane end of the human CEACAM5 protein.

[0054] In one specific embodiment of the present invention, the anti-CEACAM5 antibody or its antigen-binding fragment targets or specifically binds to the vicinity of the B3 domain of the near-membrane end of the human CEACAM5 protein.

[0055] In one specific embodiment of the present invention, the anti-CEACAM5 antibody or its antigen-binding fragment targets or specifically binds to the vicinity of the A3 and B3 domains of the near-membrane end of the human CEACAM5 protein.

[0056] Preferably, the anti-CEACAM5 antibody or its antigen-binding fragment targets or specifically binds to the CEACAM5 protein or its fragment on the cell surface.

[0057] More preferably, the anti-CEACAM5 antibody or its antigen-binding fragment targets or specifically binds to the CEACAM5 protein or fragment thereof on the surface of tumor cells or NK cells.

[0058] In a second aspect, the present invention provides a VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3, a heavy chain variable region, a light chain variable region, a heavy chain, or a light chain.

[0059] A third aspect of the present invention provides a method for preparing the above-mentioned anti-CEACAM5 antibody or its antigen-binding fragment, the method comprising:

[0060] 1) Obtain the nucleic acid encoding the CEACAM5 antibody or its antigen-binding fragment;

[0061] 2) The nucleic acid obtained in step 1) is transformed into the host cell and then induced to express.

[0062] In a fourth aspect, the present invention provides an antigen receptor comprising the above-described anti-CEACAM5 antibody or its antigen-binding fragment.

[0063] The antigen receptor is selected from T cell antigen receptor (TCR), synthetic T cell receptor antigen receptor (STAR) or chimeric antigen receptor (CAR).

[0064] The STAR comprises the α chain and β chain of the TCR, wherein the variable regions of the α chain and β chain are replaced with the light chain variable region and heavy chain variable region described in the first aspect, or are replaced with the heavy chain variable region and light chain variable region described in the first aspect.

[0065] The STAR comprises an α-chain constant region and a β-chain constant region, preferably, the α-chain constant region and the β-chain constant region are connected by disulfide bonds.

[0066] The extracellular antigen-binding domain of the CAR includes the aforementioned anti-CEACAM5 antibody or its antigen-binding fragment, the aforementioned heavy chain variable region, the aforementioned light chain variable region, the aforementioned heavy chain, or the aforementioned light chain.

[0067] Preferably, the CAR has one or more identical or partially identical or different extracellular antigen-binding domains.

[0068] Preferably, the CAR further includes a transmembrane domain, an intracellular domain, and / or a hinge region.

[0069] More preferably, the hinge region connects the extracellular antigen-binding domain and the transmembrane domain.

[0070] Preferably, the transmembrane domain is selected from one or more of the following peptides: CD3ζ peptide, CD4 peptide, CD8 peptide, CD28 peptide, CD28-41BB peptide, OX40 peptide, ICOS peptide, CTLA-4 peptide, PD-1 peptide, LAG-3 peptide, 2B4 peptide, or BTLA peptide.

[0071] Preferably, the intracellular domain includes, but is not limited to, a signal transduction domain; more preferably, the intracellular domain further includes a co-stimulatory domain.

[0072] Preferably, the intracellular domains include, but are not limited to, one or more combinations of CD28, ICOS, 4-1BB, OX-40, CD27, or CD3ζ.

[0073] Preferably, the hinge region includes, but is not limited to, the extracellular hinge region of CD8, CD8α, CD28 or IgG.

[0074] In a fifth aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the aforementioned anti-CEACAM5 antibody or an antigen-binding fragment thereof, or comprising a nucleotide sequence encoding the aforementioned antigen receptor.

[0075] In a sixth aspect, the present invention provides a carrier comprising the above-described nucleic acid.

[0076] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0077] More preferably, the prokaryotic expression vector includes, but is not limited to, any one of the following: Escherichia coli expression system vectors, Bacillus subtilis expression system vectors, or streptomycin expression system vectors.

[0078] More preferably, the eukaryotic expression vectors include, but are not limited to, yeast expression system vectors, fungal expression system vectors, insect cell expression system vectors, or mammalian cell expression system vectors.

[0079] In one specific embodiment of the present invention, the expression vector is the PTT5 expression vector.

[0080] A seventh aspect of the present invention provides a host cell comprising the above-described nucleic acid and / or the above-described vector.

[0081] Preferably, the host cell can be a eukaryotic cell or a prokaryotic cell.

[0082] In an eighth aspect, the present invention provides an immune cell that expresses the above-described anti-CEACAM5 antibody or its antigen-binding fragment or the above-described antigen receptor.

[0083] The immune cells mentioned include one or more of the following: lymphocytes, dendritic cells, peripheral blood mononuclear cells, macrophages, granulocytes, or mast cells.

[0084] Preferably, the lymphocytes are selected from T cells, TIL cells, NK cells, or B cells.

[0085] In a ninth aspect, the present invention provides a method for preparing the above-described immune cells, the method comprising transfecting the above-described nucleic acid into immune cells for expression.

[0086] In a tenth aspect, the present invention provides the use of the above-described anti-CEACAM5 antibody or its antigen-binding fragment, the above-described nucleic acid, the above-described vector, or the above-described host cell in the preparation of an antibody-drug conjugate.

[0087] In an eleventh aspect, the present invention provides an antibody-drug conjugate comprising the above-described anti-CEACAM5 antibody or its antigen-binding fragment and a small molecule drug.

[0088] The theoretical average drug-to-antibody ratio (DAR) of the antibody-drug conjugate is 2.

[0089] The small molecule drugs mentioned include cytotoxins such as hydroxycamptothecin and its derivatives (e.g., 7-ethyl-10-hydroxycamptothecin (SN-38), Dxd), cyclophosphamide, nitrogen mustard, maytansine, monomethylauristatin E (MMAE), methotrexate, mitomycin C, cytarabine, paclitaxel, vinorelbine, or docetaxel.

[0090] The anti-CEACAM5 antibody or its antigen-binding fragment is linked to the small molecule drug via a linker. Preferably, the linker is a cleavable linker.

[0091] Preferably, the cleavable linker is a cathepsin B recognition site.

[0092] Preferably, an antibody-drug conjugate is obtained by site-specific conjugation of an anti-CEACAM5 antibody or its antigen-binding fragment with a small molecule drug.

[0093] Preferably, the small molecule drug is conjugated to a constant region of the anti-CEACAM5 antibody or its antigen-binding fragment, preferably the CH2 domain, more preferably the N-glycosylation site of the CH2 domain, and even more preferably one or more of positions 170-190 of SEQ ID NO: 10, such as positions 170, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185 or 190.

[0094] The site-specific coupling can be cysteine ​​coupling, non-natural amino acid coupling, enzymatic coupling, or glycoengineering, etc.

[0095] The cysteine ​​coupling described herein is achieved by introducing specific cysteine ​​residues into the antibody and then reacting them with a drug linker containing maleimide to achieve site-specific coupling.

[0096] The aforementioned non-natural amino acid conjugation is achieved by introducing non-natural amino acids (such as acetylphenylalanine or azidolysine) into the antibody, which then react specifically with the drug linker to achieve site-specific conjugation.

[0097] The aforementioned enzymatic coupling utilizes enzymes (such as transglutaminase) to transfer drug linkers or reactive spacers to specific locations on the antibody, achieving site-specific coupling.

[0098] The aforementioned sugar engineering involves introducing specific sugar molecules onto the sugar chain of an antibody and then coupling them with a drug linker to achieve site-specific coupling.

[0099] In a twelfth aspect of the present invention, a method for preparing the above-described antibody-drug conjugate is provided, wherein the anti-CEACAM5 antibody or its antigen-binding fragment is obtained by site-specific conjugation of a small molecule drug.

[0100] Preferably, the preparation method includes introducing an azide group onto the anti-CEACAM5 antibody or its antigen-binding fragment, and reacting it with a small molecule drug linked with DBCO (Dibenzocyclooctyne).

[0101] In one specific embodiment of the present invention, the preparation method includes:

[0102] 1) Remove the Fc fragment of the anti-CEACAM5 antibody or its antigen-binding fragment to expose N-acetylglucosamine (GlcNAc);

[0103] 2) Introduce an azide group into the Fc segment of the anti-CEACAM5 antibody or its antigen-binding fragment;

[0104] 3) The anti-CEACAM5 antibody or its antigen-binding fragment obtained in step 2) after the introduction of the azide group is reacted with a small molecule drug linked to DBCO in a click chemical reaction.

[0105] The DBCO is linked to the small molecule drug via a cathepsin B recognition site. Preferably, the DBCO is linked to the small molecule drug via a linker peptide (e.g., Val-Ser). More preferably, the linker peptide and... connect.

[0106] In a thirteenth aspect, the present invention provides the use of the above-described anti-CEACAM5 antibody or its antigen-binding fragment, the above-described antigen receptor, the above-described nucleic acid, the above-described carrier, the above-described host cell, the above-described immune cell, or the above-described antibody-drug conjugate in the preparation of products for diagnosing and / or treating tumors.

[0107] The tumor includes tumor cells expressing CEACAM5 or NK cells expressing CEACAM5.

[0108] The tumor in question is one in which targeting or reducing CEACAM5 expression is beneficial for treatment.

[0109] Preferably, the tumors include lung cancer, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colorectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, or sarcoma.

[0110] In one specific embodiment of the present invention, the tumor is melanoma, lung cancer, pancreatic cancer, colorectal cancer, or gastric cancer.

[0111] In a fourteenth aspect, the present invention provides a drug or diagnostic kit comprising the above-described anti-CEACAM5 antibody or its antigen-binding fragment, the above-described antigen receptor, the above-described nucleic acid, the above-described carrier, the above-described host cell, the above-described immune cell, or the above-described antibody-drug conjugate.

[0112] Preferably, the drug targets or binds to cells expressing CEACAM5. The cells expressing CEACAM5 can be tumor cells or NK cells.

[0113] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0114] The pharmaceutically acceptable excipients are selected from one or more combinations of excipients, diluents, lubricants, wetting agents, emulsifiers, preservatives, antioxidants, buffers, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0115] Preferably, the drug can be formulated as a syrup, elixir, suspension, powder, granules, tablet, capsule, lozenge, aqueous solution, cream, ointment, lotion, gel, or emulsion. The various dosage forms of the drug can be prepared according to conventional pharmaceutical manufacturing methods.

[0116] Preferably, the pharmaceutical preparation is a unit dose preparation.

[0117] Preferably, the active ingredient contained in the drug (such as any of the anti-CEACAM5 antibodies or their antigen-binding fragments, antibody-drug conjugates, nucleic acids, carriers, antigen receptors, or immune cells, etc.) comprises any value from 0.001% to 99.9% by volume or mass ratio, for example, 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.1, 99.5, 99.9%, etc.

[0118] Depending on specific needs, the medication may also contain other suitable therapeutic agents.

[0119] The drug can be administered via any suitable route of administration, such as gastrointestinal (e.g., oral) or non-gastrointestinal (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, intravenous, intracerebral, intrathecal, transdermal, rectal, etc.).

[0120] Preferably, the drug can be used alone or in combination with other therapeutic agents.

[0121] Preferably, the drug is for human or veterinary use.

[0122] In a fifteenth aspect, the present invention provides a method for detecting CEACAM5, the method comprising contacting the above-described anti-CEACAM5 antibody or its antigen-binding fragment, the above-described antigen receptor, the above-described nucleic acid, the above-described carrier, the above-described host cell, the above-described immune cell, or the above-described antibody-drug conjugate or the above-described drug with a sample to be tested.

[0123] In a sixteenth aspect, the present invention provides a method for diagnosing or treating a tumor, the method comprising administering to a subject in need an effective amount of the aforementioned anti-CEACAM5 antibody or its antigen-binding fragment, the aforementioned antigen receptor, the aforementioned nucleic acid, the aforementioned carrier, the aforementioned host cell, the aforementioned immune cell, or the aforementioned antibody-drug conjugate, or the aforementioned drug.

[0124] In this invention, the term "tumor" can refer to any undesirable cell proliferation (or any disease that manifests as undesirable cell proliferation), vegetation, or an increased tendency or risk of undesirable cell proliferation, vegetation, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). A vegetation can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include the adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, mammary glands, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, and leukocytes. More preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (e.g., neuroglioma), colorectal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyosarcoma, squamous cell carcinoma of the tongue, nasopharyngeal carcinoma, ovarian cancer, placental choriocarcinoma, lymphoma (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic carcinoma, hematologic malignancy, head and neck cancer, or oropharyngeal carcinoma.

[0125] The term "nucleic acid" in this invention includes natural or modified ribonucleotide sequences and deoxyribonucleotide sequences. Preferably, it includes DNA, cDNA, pre-mRNA, mRNA, rRNA, hnRNA, miRNAs, scRNA, snRNA, siRNA, sgRNA, and tRNA.

[0126] In this invention, the term "CDR" refers to the complementarity-determining region within the variable sequence of an antibody. For each variable region, there are three CDRs in each variable region of the heavy chain and / or light chain. The exact boundaries of these CDRs are defined differently depending on the system. Such systems include Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)), Chothia (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)), IMGT, AbM, or the Contact system, etc., and the CDRs herein can be defined according to any of these systems. Of course, depending on the specific implementation requirements, the CDRs can also be defined using two or more combinations of the above systems.

[0127] The term "antigen-binding fragment" in this invention refers to a portion of an antibody that retains the specific binding activity of the antibody, meaning that any part of the antibody is capable of specifically binding to an epitope on the antibody's target molecule. It includes, for example, Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, scFv, and variants of these fragments. For example, the heavy and / or light chains of an antibody, the variable regions of the heavy and / or light chains of an antibody, or a single or more CDRs from the heavy or light chains of an antibody. Wherein, Fab is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. F(ab')2 is a divalent fragment comprising two Fab fragments connected by disulfide bridges in the hinge region. Fd is an Fd fragment consisting of VH and CH1 domains. Fv is an Fv fragment consisting of the VL and VH domains of a single arm of the antibody. Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH is a Fab' in which the cysteine ​​residues of the constant domain have at least one free thiol group. scFv refers to a single-chain antibody containing only the variable regions of the heavy chain and light chain, which are directly linked or linked through a short peptide (linker). Here, VH represents the heavy chain variable region, VL represents the light chain variable region, CL represents the light chain, and CH1 is the CH1 component that makes up the heavy chain constant region.

[0128] In this invention, the term "antibody variable region" refers to the portion of the amino acid sequence in the light and heavy chains of an antibody molecule that includes the complementarity-determining regions (CDRs, namely CDR1, CDR2, and CDR3) and the framework region (French, FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0129] In this invention, the term "double antibody" refers to an antibody containing two specific antigen-binding sites.

[0130] The term "small molecule drug" in this invention can be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The stereoisomers include geometric isomers (e.g., cis, trans structures) and optical isomers (e.g., enantiomers), as therapeutics consisting of monomers, racemates, racemic mixtures, and pharmaceutically acceptable salts thereof. The compounds containing asymmetric carbon atoms of this invention can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of this application. The small molecule drugs described in this invention also include tautomeric forms. Tautomeric forms arise from the exchange of a single bond with an adjacent double bond, accompanied by the migration of a proton.

[0131] The terms "comprising" or "including" in this invention are open-ended, encompassing the specified components or steps described, as well as other specified components or steps that do not substantially affect them. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the same or similar activity as the original sequence.

[0132] The term "and / or" in this invention encompasses all combinations of the items connected by the term, and should be regarded as each combination having been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0133] The term "treatment" in this invention means slowing, interrupting, preventing, controlling, stopping, reducing, mitigating, or reversing a sign, symptom, disorder, condition, or progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0134] The term "diagnosis" in this invention refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to determining the progression of a disease or its possible future progression.

[0135] The term "pharmaceutically acceptable" in this invention refers to the biological activity and properties of the active substance in the applied product that neither significantly irritates the organism nor inhibits it.

[0136] The term "effective amount" in this invention refers to the amount or dose of the product of this invention (e.g., a drug, antibody or its antigen-binding fragment, antigen receptor, immune cell or antibody-drug conjugate) that provides the desired treatment or prevention after being administered to a subject or organ in one or more doses.

[0137] The term "subject" in this invention can refer to a human or a non-human animal.

[0138] The term "non-human animal" in this invention can refer to non-human mammals, including wild animals, zoo animals, economically owned animals, pets, laboratory animals, etc. Preferably, the non-human mammals include, but are not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0139] The term "homology" in this invention refers to the ability of a person skilled in the art to adjust a protein or nucleotide sequence according to actual work needs, so that the sequence used has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 4% homology compared to sequences obtained by prior art. 1%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the sameness, and retain the same or similar activity or function. Attached Figure Description

[0140] Figure 1 SDS-PAGE electrophoresis image of the fusion protein reduction, M: 180kDa protein molecular weight standard (26616), band 1: CEACAM5-hFc; band 2: A3B3-hFc; band 3: B3-hFc.

[0141] Figure 2 ELISA screening results of hybridoma supernatant.

[0142] Figure 3 Flow cytometry results of the binding of positive hybridoma cells (mab186, mab192, or mab211) to cells.

[0143] Figures 4A-4C Results of the binding activity of candidate chimeric antibodies to cynomolgus monkey CEACAM5, human CEACAM1, and human CEACAM6 proteins.

[0144] Figure 5 : Schematic diagram of the structure of the fusion protein.

[0145] Figure 6 Results of the binding activity assay of candidate chimeric antibodies to fusion proteins of different domains of CEACAM5.

[0146] Figure 7 Results of the binding activity of chimeric antibodies to LoVo cells.

[0147] Figure 8 Results of the binding activity of chimeric antibodies to MKN45 cells.

[0148] Figure 9 Results of the binding activity of chimeric antibodies to HEK293-CEACAM5 cells.

[0149] Figure 10 Results of CEACAM5 expression levels on different cell surfaces (ch186 antibody concentration 11.1 μg / ml).

[0150] Figure 11 Results of DT3C method for detecting the endocytic activity of chimeric antibodies.

[0151] Figure 12 : ADC fabrication flowchart.

[0152] Figure 13A Results of the binding activity assay of ch186 / ch186-MMAE with A3B3-mFc.

[0153] Figure 13B Results of the binding activity assay of ch192 / ch192-MMAE with A3B3-mFc.

[0154] Figure 14A Flow cytometry results of ch186 / ch186-MMAE binding activity with MKN45 cells.

[0155] Figure 14B Results of flow cytometry binding activity of ch186 / ch186-MMAE with LoVo cells.

[0156] Figure 14CFlow cytometry results of the binding activity of ch192 / ch192-MMAE to MKN45 cells.

[0157] Figure 14D Results of flow cytometry binding activity of ch192 / ch192-MMAE with LoVo cells.

[0158] Figure 15 The in vitro killing effect of ADC on HEK293 (CEACAM5 negative) cells.

[0159] Figure 16 The in vitro killing activity of ADC against HEK293 cells overexpressing human CEACAM5.

[0160] Figure 17 : The in vitro killing effect of ADC on human gastric cancer cells MKN45.

[0161] Figure 18 The in vitro killing effect of ADC on human colorectal cancer cells LoVo. Detailed Implementation

[0162] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0163] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0164] Unless otherwise specified, all reagents, materials, and instruments used in the following examples are commercially available.

[0165] Example 1: Preparation of CEACAM5 fusion protein

[0166] The human CEACAM5 amino acid sequence (P06731) was retrieved from the Uniport protein database. The full-length extracellular domain, A3B3 domain, and B3 domain were sent to GenScript for whole-genome synthesis and cloned into a pcDNA3.1 vector containing human IgG1 Fc. The fusion protein particle was transformed into *E. coli* DH5α competent cells for amplification. After large-scale plasmid extraction, the plasmid DNA was transfected into suspension Expi-293F cells at a PEI concentration of 1 μg / mL, with a cell density of 3 × 10⁶ cells at transfection. 6The plasmid DNA was collected at a concentration of 1:4 (plastid DNA to PEI) and cultured in a shaker at 37°C, 8% CO2, and 115 rpm. After 6 days of culture, the supernatant was collected by centrifugation, and the Fc fusion protein was purified using a Protein A affinity chromatography column. The purity was then determined by SDS-PAGE electrophoresis after replacing the buffer with PBS.

[0167] The fusion proteins were expressed by transfection with Expi293F and purified by Protein A affinity to successfully obtain CEACAM5-hFc (SEQ ID NO: 1), A3B3-hFc (SEQ ID NO: 2), and B3-hFc (SEQ ID NO: 3) fusion proteins. Purity analysis was performed using reducing SDS-PAGE, and the major band sizes of the purified fusion proteins were as expected, at 180 kDa, 70 kDa, and 55 kDa, respectively. Figure 1 This meets the experimental needs for subsequent animal immunization and antibody screening.

[0168] SEQ ID NO: 1: CEACAM5-hFc amino acid sequence

[0169] MESPSAPPHRWCIPWQRLLLTASLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWY

[0170] KGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQF

[0171] RVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVT

[0172] RNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVN

[0173] GTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALT

[0174] CEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVL

[0175] YGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANN

[0176] SASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQL

[0177] SNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSA

[0178] SNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGGGGEPKSCD

[0179] KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

[0180] TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

[0181] DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0182] SEQ ID NO:2:A3B3-hFc amino acid sequence

[0183] MEFGLSWVFLVAILKGVQCELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPR

[0184] LQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSC

[0185] HSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGGGGEPK

[0186] SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0187] NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

[0188] PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0189] SEQ ID NO: 3: Amino acid sequence of B3-hFc

[0190] MEFGLSWVFLVAILKGVQCDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIA

[0191] KITPNNNGTYACFVSNLATGRNNSIVKSITVSASGGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

[0192] PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ

[0193] DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

[0194] EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0195] Preparation of the hybridoma in Example 2

[0196] The purified fusion protein from Example 1 was diluted to 1 mg / ml, mixed with an equal volume of Freund's adjuvant, and emulsified to immunize BALB / c mice. For the first immunization, 200 μL of Freund's complete adjuvant emulsion was injected intraperitoneally. A second immunization was performed 14 days later using 200 μL of Freund's incomplete adjuvant via subcutaneous injection. The third to fifth immunizations used 200 μL of Freund's incomplete adjuvant via intraperitoneal injection. Blood was collected from the tail vein after five immunizations, and serum titers were determined using ELISA. A booster immunization was administered three days prior to fusion via intraperitoneal injection of 20 μg of recombinant protein.

[0197] Hybridomas were prepared by fusing mouse spleen cells with SP2 / 0 using an electrofusion method. After 14 days of static culture at 37°C and 5% CO2, the supernatant of the hybridomas was collected for antigen binding ELISA detection.

[0198] Example 3: ELISA screening for positive hybridomas

[0199] Positive hybridomas were screened using an indirect ELISA method. In 96-well ELISA plates, CEACAM5-hFc, A3B3-hFc, or B3-hFc fusion proteins were diluted to 1 μg / mL with PBS, and 100 μL was added to each well. The plates were incubated overnight at 4°C, washed three times with PBST, and blocked overnight at 4°C with 200 μL of 10% skim milk powder. 100 μL of hybridoma supernatant was added to each well, and the plates were incubated at 37°C for 45 minutes. After washing three times with PBST, 100 μL of Goatanti Mouse IgG(H+L)-HRP secondary antibody diluted 1:8000 with 5% skim milk powder was added to each well. The plates were incubated at 37°C for 30 minutes, washed three more times with PBST, and developed with 100 μL / well TMB chromogenic buffer for 15 minutes. Finally, 50 μL / well 2M sulfuric acid was added to stop the reaction. OD values ​​were read from the microplate reader. 450nm Numerical value.

[0200] The results are as follows Figure 2 As shown, after ELISA screening of CEACAM5-hFc, A3B3-hFc, and B3-hFc fusion proteins, three hybridoma cell lines with strong binding activity to all three fusion proteins were obtained and named mab186, mab192, and mab211, respectively.

[0201] Example 4: Screening of hybridomas by flow cytometry

[0202] To detect the binding activity between the positive hybridoma cells obtained in Example 3 and cells expressing CEACAM5, the supernatant of the hybridoma cells that tested positive by ELISA was further screened by flow cytometry. Specifically, LoVo cells (purchased from the Cell Bank of the Chinese Academy of Sciences, TCU82) with low CEACAM5 expression in logarithmic growth phase, MKN45 cells with high CEACAM5 expression, and HEK293-CEACAM5 cell line (purchased from ACRO) were taken, digested with trypsin, and their density was adjusted to 5 × 10⁻⁶. 6 Cells / mL. Add 100 μL to each well of a 2 mL deep-well plate, resuspend and wash with 300 μL of flow cytometry buffer (PBS + 2% FBS), centrifuge at 1800 rpm / min for 5 min, and repeat this step 3 times. Add 100 μL of hybridoma supernatant to each well, mix well, and incubate on ice for 1 hour. After washing three times with 300 μL of flow cytometry buffer, dilute PE-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend, 405307) 1:500 with flow cytometry buffer, add 100 μL to each well, mix well, and incubate on ice for 45 min. Wash three times to ensure thorough washing and remove nonspecific bindings. Resuspend with 200 μL of flow cytometry buffer and perform flow cytometry analysis.

[0203] See results Figure 3 Flow cytometry analysis revealed that mab186, mab192, and mab211 specifically bind to LoVo, MKN45, and HEK293-CEACAM5 cells.

[0204] Example 5: Construction and expression of chimeric antibodies

[0205] Antibody subtypes of candidate antibodies were detected using the SBA Clonotyping System-HRP subtype detection kit. Total mRNA was extracted from hybridomas using the TakaRa MiniBEST Universal RNA Extraction Kit. The variable region sequence of the murine antibody was obtained using the SMARTer RACE 5' / 3' Kit PCR. Following the method described in "Recombinant Antibodies" (Shen Beifen, 2005), 5' and 3' specific primers were designed based on the cDNA sequence encoding the murine monoclonal antibody subtype. PCR of the murine monoclonal antibody variable region was performed according to the SMARTer RACE 5' / 3' Kit instructions. Homologous recombination primers were designed based on the variable region sequencing results, and the PCR-amplified variable region fragments were recovered using gel electrophoresis.

[0206] The CDR sequences of the antibodies obtained through screening are shown in Table 1-3.

[0207] Table 1: CDR sequence of mab186 antibody

[0208]

[0209]

[0210] SEQ ID NO: 4: mab186 heavy chain variable region

[0211] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWMKQAPGKGLKWMGWINTYTGEATYVDDF

[0212] KGRFAFSLETSASTAYLQINNIKNEDTATYLCVRSGYGHYDYWGQGTSVTVSS

[0213] SEQ ID NO: 5: mab186 light chain variable region

[0214] QAVVTQESALTTSPGETVTTLTCRSSTGAVTTSNYAHWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSL

[0215] IGDKAALTITGAQTEDEAIYFCALWYSNHLVFGGGTKLTVL

[0216] Table 2: CDR sequence of mab192 antibody

[0217]

[0218] SEQ ID NO: 6: mab192 heavy chain variable region

[0219] QIQLVQSGHELKKPGETVKISCKASGYIFTNYGMSWVTQAPGKGLKWMGWINTITEEPTYAEE FKGRFAFSLETSASTAYLQINNLKNEDTATYFCTTYGNAAYWGQGTLVTVSA

[0220] SEQ ID NO: 7: mab192 light chain variable region

[0221] DIQMTQSPASSLSASVGETVTITCRASENIYSYLAWFQQKQGKSPQLLVYNAKTLAEGVPSRFSG SGSGTQFSLKINNLQPEDFGTYYCQHHYDIPFTFGAGTKLELK

[0222] Table 3: CDR sequence of mab211 antibody

[0223]

[0224] SEQ ID NO: 8: mab 211 heavy chain variable region

[0225] QVQLQQSGAELARPGASVKMSCKASGYTFTTYSMHWVKQRPGQGLEWIGYINPNSGYTNYN QKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSMYYDNSRDFALDYWGQGTAVTVSS

[0226] SEQ ID NO: 9: mab 211 light chain variable region

[0227] QIVLTQSPAIMSVSPGEKVTITCSVSSSVTYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSG SGTSYSLTLSRMEAEDAATYYCQQRSSYPFTFGSGTKLEIN

[0228] The selected antibody variable region sequences were homologously recombinated with the expression vector PTT5 containing the enzyme-digested human IgG1 heavy chain constant region sequence (SEQ ID NO: 10) and the κ or λ light chain constant region sequence (SEQ ID NO: 11 or SEQ ID NO: 12) to obtain chimeric antibody heavy and light chain expression vectors. Plasmids were extracted using a commercially available plasmid extraction kit. The density of logarithmically growing Expi-293F cells was adjusted to 1.0–2.0 × 10⁶ cells / day before transfection. 6 Cells were cultured at a concentration of 1 μg / mL for 24 hours, followed by transfection with a total plasmid concentration of 1 μg / mL and a heavy chain plasmid:light chain plasmid:PEI (polyethyleneimine) ratio of 1:1:3. After 7 days, the cells were centrifuged to collect the culture supernatant, and the antibodies were purified using a Protein A affinity column to obtain chimeric antibodies ch186, ch192, and ch211.

[0229] SEQ ID NO: 10: Amino acid sequence of hIgG1 heavy chain constant region

[0230] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 11: Human kappa light chain constant region amino acid sequence

[0231] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0232] SEQ ID NO: 12: Amino acid sequence of the constant region of the human λ light chain

[0233] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNN KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0234] Example 6: Specific binding activity of candidate chimeric antibodies

[0235] To detect species cross-reactivity of the candidate antibodies, the binding activity of the candidate chimeric antibodies ch186, ch192, and ch211 constructed in Example 5 with cynomolgus monkey CEACAM5 and other family members human CEACAM1 and human CEACAM6 was detected by ELISA.

[0236] Cynomolgus monkey CEACAM5 (ACRO, CE5-C52H5), human CEACAM1 (ACRO, CE1-H5220), and human CEACAM6 (ACRO, CE6-H5223) were coated onto ELISA plates at 1 μg / mL, 100 μL per well, and blocked with 10% skim milk overnight at 4°C. Candidate chimeric antibodies were diluted 5-fold from 10 μg / mL in eight gradients, 100 μL / well, and added to the cell-coated ELISA plates. After incubation at 37°C for 1 hour, the plates were washed three times with PBST. Mouse anti-human IgG Fc-HRP diluted 1:2000 was added, and the plates were incubated at 37°C for 45 minutes. After washing three times with PBST, the plates were developed with TMB chromogenic buffer for 15 minutes, and the reaction was terminated with 2M H2SO4. OD450 values ​​were read, and EC50 was calculated using GraphPad Prism 9 software for plotting and graphing. 50 .

[0237] See results Figures 4A-4C The results indicate that ch186 and ch192 have no binding activity with cynomolgus monkey CEACAM5, human CEACAM1, and human CEACAM6 proteins; while ch211 has strong cross-binding activity with cynomolgus monkey CEACAM5, human CEACAM1, and human CEACAM6 proteins.

[0238] Example 7: Detection of the CEACAM5 binding domain of the candidate chimeric antibody

[0239] To detect the binding sites of candidate chimeric antibodies at the CEACAM5 domain sites, this embodiment designed several fusion proteins for ELISA detection. These included fusing the A3B3 domain and B3 domain near the membrane, the full-length extracellular truncated B3 domain, and the full-length extracellular truncated A3B3 domain with mFc, respectively named A3B3-mFc, B3-mFc, CEACAM5-DelB3-mFc (SEQ ID NO: 13), and CEACAM5-DelA3B3-mFc (SEQ ID NO: 14). Simultaneously, the B1 domain of the CEACAM1 extracellular domain was replaced with the B3 domain of CEACAM5 to construct and express the NAB3A-His protein (SEQ ID NO: 15). A schematic diagram of the fusion protein structure is shown below. Figure 5 Perform ELISA detection according to the method in Example 6.

[0240] CEACAM5-Del B3-mFc amino acid sequence (SEQ ID NO: 13):

[0241] MESPSAPPHRWCIPWQRLLLTASLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWY

[0242] KGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQF

[0243] RVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVT

[0244] RNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVN

[0245] GTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALT

[0246] CEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVL

[0247] YGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANN

[0248] SASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQL

[0249] SNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPGGGSPTIKPCPPCKCPAPNLLGGP

[0250] SVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSAL

[0251] PIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDF

[0252] MPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK*

[0253] CEACAM5-Del A3B3-mFc amino acid sequence (SEQ ID NO: 14):

[0254] MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWY

[0255] KGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQF

[0256] RVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVT

[0257] RNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVN

[0258] GTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALT

[0259] CEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVL

[0260] YGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANN

[0261] SASGHSRTTVKTITVSAGGGSPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSE

[0262] DDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER

[0263] TISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK*

[0264] NAB3A-His amino acid sequence (SEQ ID NO: 15):

[0265] MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQQLFGYSW

[0266] YKGERVDGNRQIVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEAT

[0267] GQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYLWWINNQSLPVSPRLQLSNGNRTLTLL

[0268] SVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTPTISPPDSSYLSGANLNLSCHSASNPSPQYSW

[0269] RINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSALSPVVAKPQIKASKTTVTGD

[0270] KDSVNLTCSTNDTGISIRWFFKNQSLPSSERMKLSQGNTTLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALPQENGSHHHHHHHH*

[0271] See results Figure 6 ELISA results showed that ch186 specifically binds to A3B3-mFc, B3-mFc, and NAB3A-His, but not to CEACAM5-Del B3-mFc or CEACAM5-Del A3B3-mFc; indicating that ch186 specifically binds near the B3 domain of the lipometa-terminus of CEACAM5.

[0272] ch192 specifically binds to A3B3-mFc and NAB3A-His, but not to B3-mFc, CEACAM5-Del B3-mFc, or CEACAM5-Del A3B3-mFc; indicating that ch192 specifically binds near the A3 and B3 domains of the lipometa-terminus of CEACAM5.

[0273] ch211 binds to A3B3-mFc, CEACAM5-Del B3-mFc, and CEACAM5-Del A3B3-mFc, but not to B3-mFc; indicating that ch211 may bind to the A3 domain of CEACAM5.

[0274] Example 8: Flow cytometry detection of the binding activity of chimeric antibodies to target cells

[0275] For the chimeric antibodies ch186 and ch192 that specifically bind to the proximal end of CEACAM5, flow cytometry was used to further detect the binding activity of the chimeric antibodies to cells. Specifically, LoVo cells (purchased from the Cell Bank of the Chinese Academy of Sciences, TCU82), MKN45 cells, and HEK293-CEACAM5 cell lines (purchased from ACRO) in logarithmic growth phase were digested with trypsin and then adjusted to a density of 5 × 10⁻⁶ cells / cells. 6 Cells / mL. Add 100 μL to each well of a 2 mL deep-well plate, resuspend and wash with 300 μL of flow cytometry buffer (PBS + 2% FBS), centrifuge at 1800 rpm / min for 5 min, and repeat this step 3 times. Dilute the chimeric antibody to 100 μg / mL and then serially dilute 3-fold to obtain 11 concentrations. Add 100 μL to each well, mix well, and incubate on ice for 1 hour. After washing three times with 300 μL of flow cytometry buffer, dilute APC-labeled mouse anti-human IgG fluorescent secondary antibody (Biolegend, 410714) 1:500 with flow cytometry buffer and add 100 μL to each well. Mix well and incubate on ice for 45 min. Wash three times to ensure thorough washing and remove nonspecific conjugates. Resuspend in 200 μL of flow cytometry buffer and perform flow cytometry analysis.

[0276] See results Figure 7 , Figure 8 , Figure 9 The results indicate that the chimeric antibodies ch186 and ch192 have strong binding activity with LoVo cells, MKN45 cells, and HEK293-CEACAM5 cell lines (see Table 4).

[0277] By comparing the average fluorescence intensity of different cells at the same antibody concentration, it was found that the expression level of CEACAM5 protein in the three cell types, from highest to lowest, was HEK293-CEACAM5 cells, MKN45 cells, and LoVo cells. The results are shown in the table below. Figure 10 .

[0278] Table 4: Results of the binding activity of chimeric antibodies to target cells

[0279]

[0280] Example 9: Detection of antibody internalization activity using the DT3C method

[0281] The DT3C protein is a fusion of the diphtheria toxin (DT) toxin portion (33-417aa, P00588) and the 3C fragment of group G streptococci (33-417aa, P00588). This protein exhibits high affinity for the Fc terminus of antibodies. DT3C molecules bound to the antibody enter the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, toxic DT is released. DT inhibits EF2-ADP ribosylation activity, blocking protein translation and ultimately leading to cell death. DT3C molecules that do not enter the cell do not possess cytotoxic activity. Therefore, the internalization efficiency of antibodies can be evaluated based on cell-killing activity.

[0282] Specifically, MKN45 cells in the logarithmic growth phase were harvested. After digestion and counting, the cells were resuspended in medium containing 20% ​​FBS to a density of 3000 cells / well, and 100 μL of the cell suspension was added to each well of a 96-well cell plate. After overnight incubation at 37°C and 5% CO2, DT3C (purchased from Jiman Biotechnology, GM-046001RP) and antibody were mixed in serum-free medium at a molar ratio of 2:1 (i.e., a mass ratio of 1:1) and incubated at 37°C for 30 min. The mixture was serially diluted 3-fold with serum-free medium, resulting in 9 dilutions. 100 μL of the serially diluted mixture was added to each well of the 96-well cell plate. After incubation at 37°C and 5% CO2 for 48-72 h, the cell plates were removed from the CO2 incubator, and after equilibration to room temperature for 10 minutes, the antibody was added to each well. Luminescent CellViability assay solution (purchased from Promega), shaken at 200 rpm for 2 min, incubated in the dark for 10 min, and read using an M5 microplate reader. Result processing formula:

[0283]

[0284] Then, using GraphPad Prism 9.0 software, a four-parameter fitting graph was performed with the logarithm of concentration as the x-axis and the lethality as the y-axis.

[0285] See results Figure 11 The endocytic activity of MKN45 cells against ch186 antibody was detected by the DT3C method. It was found that DT3C alone did not have cytotoxic activity against MKN45 cells, but ch186 and DT3C were co-incubated and had cytotoxic activity against MKN45 cells. This indicates that ch186 is endocytosed after binding to CEACAM5 on the surface of MKN45 cells and has a dose-response effect.

[0286] Example 10: Preparation of Antibody-Drug Conjugates (ADCs)

[0287] Antibody-conjugated drugs were prepared using glycosylation conjugation technology. The MMAE site of the toxin molecule was specifically conjugated to the N-glycosylation site of the CH2 domain of IgG using a GlyCLICK kit (Genovis, catalog number: L1-T02-200), employing a cleavable linker with a theoretical DAR value of 2. Specific steps are as follows: Figure 12 As shown, the process includes: 1) Deglycosylation: using an immobilized glycan-cleaving enzyme GlycINATOR (EndoS2) to remove the Fc glycan of the antibody, exposing the GlcNAc (N-acetylglucosamine) glycosyl group; 2) Azide introduction: using UDP-GalNAz and a mutant GalT (Y298L) to introduce the azide group into the antibody; 3) Click chemistry: achieving specific conjugation of the drug through the reaction of DBCO-drug with the azide group. The group connected to the N-glycosylation site at position 297 of the antibody Fc portion (corresponding to position 180 of SEQ ID NO: 10 in this application) is DBCO-Val-Ser(Glc-A)-PAB-MMAE, with the following structural formula:

[0288]

[0289] Wherein, Ab is the chimeric antibody prepared in Example 5, Val-Ser is the linker peptide, Glc-A represents glucuronic acid, and PAB has the following structural formula:

[0290] During the use of the obtained ADC drug, after the antibody specifically targets cells expressing CEACAM5, it can be cleaved by cathepsin B. The concentration of the ADC drug after conjugation was determined by the BCA method, and the concentration of ch186-MMAE was 2.63 mg / mL, and the concentration of ch192-MMAE was 1.99 mg / mL.

[0291] Example 11: Detection of the binding activity of antibody-drug conjugates to target antigens

[0292] Following the method described in Example 7, the binding activity of ch186-MMAE or ch192-MMAE prepared in Example 10 with A3B3-mFc and NAB3A proteins was detected by ELISA. The results are as follows: Figure 13A and Figure 13B As shown, the binding activity of the ADC drugs ch186-MMAE and ch192-MMAE formed by conjugating small molecules ch186 and ch192 to the target antigen did not change significantly.

[0293] Example 12: Binding activity of antibody-drug conjugates to target cells

[0294] The binding activity of the ADC to target cells was detected according to the method in Example 8. Ch186, Ch186-MMAE, Ch192, and Ch192-MMAE were serially diluted 3-fold, starting at 60 μg / mL, for a total of 8 concentrations. Flow cytometry was then performed according to the experimental method in Example 8. The mean fluorescence intensity (MFI) was fitted to the antibody concentration using GraphPad Prism9 software using four parameters. Results are shown below. Figure 14A , Figure 14B , Figure 14C as well as Figure 14D The results showed that the specific binding activity of the ADC drugs ch186-MMAE and ch192-MMAE formed by conjugating small molecules with ch186 and ch192 to target cells did not change significantly.

[0295] Example 13: Detection of the cytotoxic activity of antibody-drug conjugates against target cells

[0296] To detect the specific killing activity of ADC drugs on target cells, this embodiment used the CTG method to detect the killing activity of ADCs on CEACAM5-negative HEK293 cells, CEACAM5-stable cell line HEK293-CEACAM5 cells, CECAM5-highly expressing tumor cells MKN45 cells, and CECAM5-low expressing cells LoVo cells.

[0297] Target cells in the logarithmic growth phase were harvested, digested with trypsin, and then their density was adjusted to 2.0 × 10⁻⁶. 4Cells were seeded at a rate of 100 μL / well in 96-well opaque white-background culture plates, with the entire plate seeded. 200 μL of PBS was added to the outermost well to prevent evaporation. After incubation at 37°C and 5% CO2 for 24 hours until complete cell adhesion, small molecule MMAE, ADC drugs (ch186-MMAE, ch192-MMAE), and chimeric antibody controls (ch186, ch192) were diluted in the deep-well plates with analytical medium (RMPI 1640 + 0.1% BSA) at an initial concentration of 60 μg / mL. A three-fold serial dilution was performed for a total of nine dilutions, with three replicates for each dilution. The supernatant was discarded, and 100 μL of the drug was added per well. An analytical medium blank control was also added. After culturing for 4-6 days, the cell culture plates were removed from the CO2 incubator and allowed to equilibrate to room temperature for 15 minutes. Then, 100 μL of the drug was added to each well. Luminescent Cell Viability assay solution (purchased from Promega), shake for 2 minutes, incubate in the dark for 10 minutes, and read the values ​​using an M5 microplate reader.

[0298] Result processing formula:

[0299]

[0300] Then, using GraphPad Prism 9.0 software, a four-parameter fitting graph was performed with the logarithm of concentration as the x-axis and the lethality as the y-axis.

[0301] See results Figure 15 and Figure 16 As shown, ch186-MMAE and ch192-MMAE have significant in vitro killing effects on HEK293-CEACAM5 cells that highly express human CEACAM5, but no in vitro killing effect was observed on human CEACAM5-negative HEK293 cells. Similarly, naked anti-ch186 and anti-ch192 drugs did not show any killing effect on the above two cell types, indicating that the ADC drugs ch186-MMAE and ch192-MMAE can specifically kill CEACAM5-positive cells.

[0302] The results are as follows Figure 17 As shown, ch186-MMAE and ch192-MMAE have significant in vitro killing effects on the human gastric cancer cell line MKN45, which highly expresses CEACAM5.

[0303] The results are as follows Figure 18 As shown, ch186-MMAE and ch192-MMAE only exhibit significant killing effects on LoVo cells with low CEACAM5 expression in humans at high concentrations.

[0304] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0305] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An anti-CEACAM5 antibody or its antigen-binding fragment, characterized in that, The anti-CEACAM5 antibody or its antigen-binding fragment comprises the heavy chain variable region VHCDR1-3 and / or the light chain variable region VLCDR1-3, wherein: The amino acid sequence of VHCDR1 includes the amino acid sequence shown in SEQ ID NO: 16, 22, 28, 34, 40 or 46; The amino acid sequence of VHCDR2 includes the amino acid sequence shown in SEQ ID NO: 17, 23, 29, 35, 41 or 47; The amino acid sequence of VHCDR3 includes the amino acid sequence shown in SEQ ID NO: 18, 30, 36 or 48; The amino acid sequence of VLCDR1 includes the amino acid sequence shown in SEQ ID NO: 19, 31, 37 or 49; The amino acid sequence of VLCDR2 includes the amino acid sequence shown in SEQ ID NO: 20, 32, 38 or 50; The amino acid sequence of VLCDR3 includes the amino acid sequence shown in SEQ ID NO: 21 or 39.

2. The anti-CEACAM5 antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequences of VHCDR1-3 and VLCDR1-3 include:

3. The anti-CEACAM5 antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequences of VHCDR1-3 and VLCDR1-3 include:

4. The anti-CEACAM5 antibody or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The anti-CEACAM5 antibody or its antigen-binding fragment further includes FR regions, preferably including VHFR1-4 of the heavy chain variable region and / or VLFR1-4 of the light chain variable region; Preferably, the VHFR1-4 or VLFR1-4 is independently derived from humans or non-human animals.

5. The anti-CEACAM5 antibody or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The heavy chain variable region, from the N-terminus to the C-terminus, sequentially includes VHFR1, VHCDR1, VHFR2, VHCDR2, VHFR3, VHCDR3, and VHFR4; and / or, The light chain variable region includes VLFR1, VLCDR1, VLFR2, VLCDR2, VLFR3, VLCDR3, and VLFR4 sequentially from the N-end to the C-end.

6. The anti-CEACAM5 antibody or its antigen-binding fragment according to any one of claims 1-5, characterized in that, The amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4 or 6; The amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5 or 7.

7. The anti-CEACAM5 antibody or its antigen-binding fragment according to any one of claims 1-6, characterized in that, The anti-CEACAM5 antibody or its antigen-binding fragment includes Fab, Fd, Fab', Fab'-SH, Fv, scFv, F(ab')2 or a double antibody (dAb).

8. The anti-CEACAM5 antibody or its antigen-binding fragment according to any one of claims 1-7, characterized in that, The anti-CEACAM5 antibody or its antigen-binding fragment comprises a heavy chain constant region and / or a light chain constant region, preferably, the heavy chain constant region and / or light chain constant region are derived from humans or non-human animals.

9. The anti-CEACAM5 antibody or its antigen-binding fragment according to claim 8, characterized in that, The heavy chain constant region is selected from the constant region of any one of the heavy chains IgG, IgD, IgA, IgM or IgE. Preferably, the IgG heavy chain includes IgG1 heavy chain, IgG2 heavy chain, IgG2b heavy chain, IgG3 heavy chain or IgG4 heavy chain. Preferably, the amino acid sequence of the heavy chain constant region includes SEQ ID NO:

10.

10. The anti-CEACAM5 antibody or its antigen-binding fragment according to claim 8 or 9, characterized in that, The constant region of the light chain is selected from the constant region of either the λ-type light chain or the κ-type light chain; Preferably, the amino acid sequence of the light chain constant region includes SEQ ID NO: 11 or 12.

11. A method for preparing the anti-CEACAM5 antibody or its antigen-binding fragment according to any one of claims 1-10, characterized in that, The preparation method includes: 1) Obtain the nucleic acid encoding the CEACAM5 antibody or its antigen-binding fragment; 2) The nucleic acid obtained in step 1) is transformed into the host cell and then induced to express.

12. A nucleic acid, characterized in that, The nucleic acid comprises a nucleotide sequence encoding the anti-CEACAM5 antibody or its antigen-binding fragment as described in any one of claims 1-10.

13. A carrier, characterized in that, The vector comprises the nucleic acid as described in claim 12.

14. A host cell, characterized in that, The host cell comprises the nucleic acid of claim 12 and / or the vector of claim 13.

15. The use of an anti-CEACAM5 antibody or its antigen-binding fragment as described in any one of claims 1-10, the nucleic acid as described in claim 12, the vector as described in claim 13, or the host cell as described in claim 14 in the preparation of an antibody-drug conjugate.

16. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises the anti-CEACAM5 antibody or its antigen-binding fragment as described in any one of claims 1-10, and a small molecule drug.

17. The antibody-drug conjugate according to claim 16, characterized in that, The small molecule drugs mentioned include cytotoxins such as hydroxycamptothecin and its derivatives, cyclophosphamide, nitrogen mustard, maytansine, methylauratestatin E, methotrexate, mitomycin C, cytarabine, paclitaxel, vinorelbine, or docetaxel.

18. The antibody-drug conjugate according to claim 16 or 17, characterized in that, Anti-CEACAM5 antibodies or their antigen-binding fragments can be site-specifically conjugated with small molecule drugs to obtain antibody-drug conjugates.

19. A method for preparing an antibody-drug conjugate according to any one of claims 16-18, characterized in that, This includes introducing an azide group onto the anti-CEACAM5 antibody or its antigen-binding fragment as described in any one of claims 1-10, and reacting it with a small molecule drug linked to a DBCO.

20. The use of an anti-CEACAM5 antibody or its antigen-binding fragment as described in any one of claims 1-10, the nucleic acid as described in claim 12, the vector as described in claim 13, the host cell as described in claim 14, or the antibody-drug conjugate as described in any one of claims 16-18 in the preparation of products for the diagnosis and / or treatment of tumors.

21. The application according to claim 20, characterized in that, The tumor includes tumor cells expressing CEACAM5 or NK cells expressing CEACAM5.

22. The application according to claim 20 or 21, characterized in that, The tumors mentioned include lung cancer, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colorectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, or sarcoma.

23. A drug or diagnostic kit, characterized in that, The drug or diagnostic kit comprises the anti-CEACAM5 antibody or its antigen-binding fragment as described in any one of claims 1-10, the nucleic acid as described in claim 12, the vector as described in claim 13, the host cell as described in claim 14, or the antibody-drug conjugate as described in any one of claims 16-18.

24. A method for detecting CEACAM5, characterized in that, The method comprises contacting the anti-CEACAM5 antibody or its antigen-binding fragment according to any one of claims 1-10, the nucleic acid according to claim 12, the vector according to claim 13, the host cell according to claim 14, or the antibody-drug conjugate according to any one of claims 16-18 with the sample to be tested.

Images