Antibody for resisting cell adhesion molecule 5 related to human carcino-embryonic antigen, and preparation method and application thereof

Abstract

The invention provides an antibody for resisting a cell adhesion molecule 5 (CEACAM5) related to a human carcino-embryonic antigen as well as a preparation method and application of the antibody. The invention discloses a CEACAM5 specific antibody, which can be specifically combined with CEACAM5 and can be applied to inhibition of diseases related to CEACAM5 expression (including overexpression) or affected by CEACAM5 functions, such as tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine; more specifically, the present invention relates to an antibody against human carcinoembryonic antigen-related cell adhesion molecule 5, and a preparation method and application thereof. Background Art

[0002] Human carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), also known as CD66e, CEA (carcinoembryonic protein), is a member of the immunoglobulin (Ig) superfamily and CEA family, and is closely related to cell adhesion (BMC Cancer 7 (2007) 2). CEACAM5 protein has a total of 7 immunoglobulin-like domains in the extracellular space, with an Ig variable domain (lgV) at the N-terminus, followed by 6 domains homologous to the Ig constant domain (lgC). The protein binds to the membrane through glycosylphosphatidylinositol (GPI) and has 28 potential N-linked glycosylation sites, showing a high degree of glycosylation (Experimental Cell Research, 252 (2), 243–249). In normal adult tissues, CEACAM5 is mainly present in the colon, but also in the stomach, tongue, esophagus, cervix, sweat glands, and prostate. CEACAM5 is involved in cell adhesion, differentiation, proliferation and survival. Overexpression can change cell polarity and normal tissue structure, slow down cell differentiation and maturation, and ultimately lead to tumor formation (J. Int. Med. Res. 48 (9) (2020), 300060520959478). CEACAM5 can mediate metastatic signals through the DR5 receptor, and can also directly bind to the type I transforming growth factor-β receptor to promote liver metastasis of tumor cells (Cancer Research, 70 (20), 8159–8168). Studies have shown that CEACAM5 is overexpressed on the surface of a variety of tumor tissues, including 90% of gastrointestinal cancer, colorectal cancer and pancreatic cancer, and 50% of breast cancer (British Journal of Cancer, 26 (5), 356-360; J Clin Lab Anal 1991; 5: 344-366; Journal of the National Cancer Institute. 2011; 103: 689-97; J Int MedRes. 2020 Sep; 48 (9): 300060520959478; J Histochem Cytochem. 2015 Dec; 63 (12): 922-30). Therefore, CEACAM5 has always been a recognized tumor marker and cancer recurrence marker. Studies have found that CEACAM5 plays an important role in the differential diagnosis and prognosis of epithelial tumors such as the gastrointestinal tract, respiratory tract, and breast (Translational Oncology 24 (2022) 101491). In addition, researchers have found that high expression of CEACAM5 is associated with poor prognosis in patients with non-small cell lung cancer and is a potential therapeutic target for lung adenocarcinoma (Journal of International Medical Research, 48(9)1–15).CEACAM5 can be used as an independent prognostic indicator for gastric cancer and is associated with poor survival (Journal of Histochemistry & Cytochemistry 63.12 (2015): 922-930). In colorectal cancer, elevated preoperative serum CEA levels are an independent predictor of overall survival in patients with colon cancer (Journal of the National Cancer Institute, 103 (8), 689–697). For breast cancer, CEACAM5 in serum has been used to detect tumor metastasis and predict prognosis (Breast cancer research and treatment 174 (2019): 375-385). Therefore, drug research targeting CEACAM5 is of great significance for anti-tumor treatment.

[0003] Developing antibody-related drugs against CEACAM5 can achieve better therapeutic effects and significantly enhance the survival of patients by targeting tumor cells expressing CEACAM5, specifically killing cancer cells, and reducing damage to normal cells. Currently, CEACAM5 has become one of the targets of anticancer drugs. Some antibody-related drugs against CEACAM5, such as Tusamitamab ravtansine (Journal of Clinical Oncology. 2024 Volume 40, Number 16_suppl) and M9140 (Journal of Clinical Oncology. 2022 Volume 42, Number 16_suppl), are undergoing clinical studies. Nevertheless, this field still needs to further optimize antibody drugs against CEACAM5. Summary of the invention

[0004] The object of the present invention is to provide an antibody against human carcinoembryonic antigen-related cell adhesion molecule 5, and a preparation method and application thereof.

[0005] In a first aspect of the present invention, a CEACAM5-specific antibody is provided, wherein the antibody has heavy chain variable regions HCDR1, HCDR2 and HCDR3, comprising:

[0006] CA3, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively;

[0007] CA1, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively;

[0008] CA2, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively;

[0009] CA4, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively;

[0010] CA5, the amino acid sequences of its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0011] In one or more embodiments, the antibody comprises: a humanized antibody, a chimeric antibody or an alpaca antibody.

[0012] In one or more embodiments, the antibody comprises:

[0013] An antibody (humanized antibody) having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 23, or having 85% or more (such as 88%, 90%, 93%, 95%, 97% or 99% or more) identity with the sequence;

[0014] An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 3 or having 85% or more (e.g., 88%, 90%, 93%, 95%, 97% or 99% or more) identity with the sequence;

[0015] An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 1 or having 85% or more (e.g., 88%, 90%, 93%, 95%, 97% or 99% or more) identity with the sequence;

[0016] An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 2, or having 85% or more (e.g., 88%, 90%, 93%, 95%, 97% or 99% or more) identity with the sequence;

[0017] An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 4, or having 85% or more (e.g., 88%, 90%, 93%, 95%, 97% or 99% or more) identity with the sequence;

[0018] The heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 5, or an antibody having more than 85% (such as 88%, 90%, 93%, 95%, 97% or 99% or more) identity with the sequence.

[0019] In one or more embodiments, the chimeric antibody has a heavy chain variable region from an alpaca, but the heavy chain constant region of the antibody is replaced with a human antibody IgG1 Fc heavy chain constant region.

[0020] In one or more embodiments, the humanized antibody includes: for the antibody variable region, the alpaca CDR region is retained, but based on the alpaca variable region sequence, the FR region sequence of its heavy chain variable region is replaced with the sequence with the closest homology to the FR region of the human antibody germline gene.

[0021] In one or more embodiments, the heavy chain constant region of the humanized antibody is a human antibody IgG1 Fc heavy chain constant region.

[0022] In one or more embodiments, the heavy chain constant region of the humanized antibody is a mutant of the heavy chain constant region of human antibody IgG1 Fc; relative to human antibody IgG1 Fc, two pairs of disulfide bonds are added in the hinge region.

[0023] In another aspect of the present invention, a polynucleotide or a construct (such as an expression vector) containing the polynucleotide is provided, wherein the polynucleotide encodes any of the above-mentioned antibodies, and the construct comprises the polynucleotide.

[0024] In another aspect of the present invention, an antibody expression system is provided, wherein the expression system contains the construct or the exogenous polynucleotide is integrated into the genome.

[0025] In one or more embodiments, the antibody expression system is a cell expression system.

[0026] In another aspect of the present invention, a method for preparing any of the above-mentioned CEACAM5-specific antibodies is provided, comprising: expressing the antibody using the antibody expression system under conditions suitable for expressing the antibody, thereby obtaining the antibody.

[0027] In one or more embodiments, the preparation method further comprises: purifying and isolating the antibody.

[0028] In another aspect of the present invention, the use of the antibody is provided for: preparing anti-tumor drugs that specifically target tumor cells expressing CEACAM5; preparing antibody-drug conjugates or immunoconjugates; preparing bifunctional or multifunctional antibodies; preparing reagents for diagnosing tumors that express CEACAM5; or preparing immune cells modified with chimeric antigen receptors.

[0029] In another aspect of the present invention, an immunoconjugate is provided, which includes any of the antibodies described above; and a functional molecule connected thereto (including covalently linked, coupled, attached, and adsorbed).

[0030] In one or more embodiments, the functional molecules include (but are not limited to): tumor-suppressing molecules, molecules targeting tumor surface markers, cytotoxins, radioactive isotopes, biologically active proteins, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.

[0031] In one or more embodiments, the tumor-suppressing molecule is an anti-tumor toxin or an anti-tumor cytokine; preferably, the anti-tumor toxin includes (but is not limited to): a toxin that acts on DNA (or its derivatives), a toxin that acts on microtubules (or its derivatives), a compound that acts on intracellular metabolism, transcription, translation or signal transduction (or its derivatives); preferably, the toxin that acts on DNA includes: DXd (including its derivatives), Duocarmycin, Calicheamicin, Pyrrolobenzodiazepines (PBDs), SN-38 (including its derivatives); preferably, the toxin that acts on microtubules includes: Monomethyl auristatin (including its derivatives), maytansinoid (including its derivatives); preferably, the anti-tumor cytokines include (but are not limited to): IL-2, IL-12, IL-15, IFN-beta, TNF-alpha or its variants.

[0032] In one or more embodiments, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker; preferably, the antibody that binds to a tumor surface marker is an antibody that recognizes antigens other than CEACAM5, and the other antigens include: EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1 or CDH16.

[0033] In one or more embodiments, the molecule targeting the surface marker of immune cells is an antibody or ligand that binds to the surface marker of immune cells; preferably, the antibody that binds to the surface marker of immune cells, the surface marker includes: CD3, CD4, CD8, PD-1, PD-L1, LAG-3, TIM-3, 4-1BB, OX40, ICOS, B7-H3, CD16, CTLA-4, TIGIT, VISTA, GITR, CD27, SIRPα, CD32, CD64, ILT2, ILT4, NKG2A, NKG2D or KIR.

[0034] In one or more embodiments, the toxin also includes: related compounds and their derivatives that act on other functions in tumor cells such as metabolism, transcription, translation, signal transduction, etc.

[0035] In one or more embodiments, the anti-tumor toxin is connected to the antibody via a linker, which includes but is not limited to: maleimide-GGFG peptide linker, mc-Val-Cit-PAB (maleimidocaproyl valine-citrulline para-amino benzyloxycarbonyl), or mc-Val-Ala-PAB (maleimidocaproyl valine-alanine para-aminobenzyloxycarbonyl).

[0036] In one or more embodiments, the detectable markers include (but are not limited to): fluorescent markers and colorimetric markers.

[0037] In one or more embodiments, the intracellular signal region includes (but is not limited to): CD3ζ chain, FcεRIγ tyrosine activation motif, and intracellular signal regions of co-stimulatory signal molecules CD27, CD28, CD137, CD134, MyD88, and CD40.

[0038] In one or more embodiments, the transmembrane region includes (but is not limited to): the transmembrane region of CD8 or CD28.

[0039] In another aspect of the present invention, a pharmaceutical composition (including a diagnostic composition, such as a diagnostic reagent) is provided, which comprises: any of the above-mentioned antibodies or any of the above-mentioned immunoconjugates.

[0040] In one or more embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0041] In another aspect of the present invention, there is provided a use of any of the above antibodies, any of the above immunoconjugates or the above pharmaceutical compositions in the preparation of a preparation, a kit or a drug kit for diagnosing or treating a tumor; the tumor is a tumor expressing CEACAM5.

[0042] In one or more embodiments, the tumor includes (but is not limited to): colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, breast cancer.

[0043] In another aspect of the present invention, a kit or a medicine kit is provided, comprising any of the above-mentioned antibodies, any of the above-mentioned immunoconjugates or the above-mentioned pharmaceutical compositions.

[0044] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the titer test result of the present invention on the immunized alpaca serum.

[0046] Figure 2 The results are shown in Figure 1 and 2. The chimeric antibody of the present invention was tested by ELISA on human CEACAM5-ECD-His and monkey CEACAM5-ECD-His at the protein level.

[0047] Figure 3 The ELISA test results of the chimeric antibody of the present invention on human CEACAM1-ECD-His, human CEACAM6-ECD-His and human CEACAM8-ECD-His at the protein level.

[0048] Figure 4 The figure shows the flow cytometry detection result of the chimeric antibody of the present invention on human CEACAM5 and monkey CEACAM5 at the cellular level.

[0049] Figure 5 The chimeric antibody of the present invention is prepared into an antibody-drug killing test result at the cellular level.

[0050] Figure 6 The results are the test results of the anti-tumor effect of the chimeric antibody of the present invention prepared into an antibody-drug conjugate in the mouse LS174T tumor model.

[0051] Figure 7 The results are the affinity test results of the humanized antibody of the present invention to human CEACAM5 and monkey CEACAM5 by flow cytometry.

[0052] Figure 8 The humanized antibody of the present invention is prepared into an antibody-drug killing test result at the cellular level.

[0053] Fig. 9 The present invention is a humanized antibody prepared into an antibody-drug conjugate and the tumor inhibition test results in the mouse LS174T and GP2D tumor models. DETAILED DESCRIPTION

[0054] After in-depth research, the present inventors have revealed a CEACAM5-specific antibody that can specifically bind to CEACAM5 and can be used to inhibit diseases related to CEACAM5 expression (including overexpression) or affected by CEACAM5 function, such as tumors.

[0055] the term

[0056] As used herein, the term "antibody" or "immunoglobulin" is used as a general term herein, including full-length antibodies, single-chain antibodies, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments). In addition, the term "sequence" used herein (e.g., in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0057] The term "monoclonal antibody" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0058] The terms "heavy chain antibody", "single (structural) domain antibody (VHH)", and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, antibodies that naturally lack light chains and heavy chain constant regions 1 (CH1) are first obtained from alpaca immune serum, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0059] The term "heavy chain antibody" refers to an immunoglobulin domain comprising four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Therefore, the general structure or sequence of a single domain antibody (VHH) can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody (VHH) confers specificity to an antibody for an antigen by having an antigen binding site.

[0060] The term "humanized antibody" refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin of a non-human species, wherein the rest of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only a complementarity determining region (CDR) transplanted to an appropriate framework region in a variable domain. The antigen binding site may be wild-type, or modified by one or more amino acid substitutions, such as modification to be closer to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences. Other forms have one or more CDRs that have been altered relative to the original antibody.

[0061] An "effective amount" of an agent is that amount necessary to effect a physiological change in the cell or tissue to which it is administered.

[0062] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.

[0063] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0064] The term "pharmaceutical composition" refers to a preparation which is in such form that the biological activity of the active ingredient contained therein is effective, and which contains no additional ingredients which would be unacceptably toxic to a subject to which the composition would be administered.

[0065] "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0066] The term "treatment / prevention" (and grammatical variations thereof) refers to an attempt to alter the natural course of a disease in a treated individual, and may be a clinical intervention performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a condition.

[0067] The term "detectable marker" refers to a marker that can be attached to an antibody and used to determine the presence or absence and amount of a specific target in a subject to be detected. The "detectable marker" can be, but is not limited to: an enzyme, a fluorescent marker, a nuclide, a quantum dot, colloidal gold, etc. More specifically, for example, it can be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0068] In the present invention, the tumor is a CEACAM5 tumor; preferably, the tumor includes but is not limited to: colorectal cancer, lung cancer (including small cell lung cancer), gastric cancer, pancreatic cancer, and breast cancer.

[0069] Antibodies and their preparation

[0070] The present invention provides an anti-CEACAM5 antibody, wherein the anti-CEACAM5 antibody has HCDR1-HCDR3 as listed in Table 3. In a preferred embodiment, the anti-CEACAM5 antibody has a heavy chain variable region as listed in SEQ ID NO: 23 or Table 2. The present invention also includes: an antibody having a heavy chain variable region amino acid sequence that is 85% or more (such as 88%, 90%, 93%, 95%, 97% or 99% or more) identical to the sequence listed in Table 6 or Table 2, an antibody having 85% or more (such as 88%, 90%, 93%, 95%, 97% or 99% or more) identical to the sequence listed in the examples, and an antibody having the same function or similar activity as the antibody described in the examples of the present invention.

[0071] The antigen binding properties of the antibody of the present invention are determined by three complementary determining regions CDR, which are arranged in order with the FR region, and the FR region does not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs therebetween are close to each other in spatial structure, forming the antigen binding site of the antibody. The CDR region is the sequence of a protein of interest in immunology, and the antibody of the present invention includes sequence modifications for the CDR region and the framework region.

[0072] The present invention also includes a fusion protein of an anti-CEACAM5 antibody, comprising the first domain of the antibody of the present invention and a second domain for prolonging the half-life in vivo and / or having a binding effect on effector cells. The fusion protein can be a binding molecule that can specifically bind to cells expressing CEACAM5.

[0073] In the second domain, the fragment used to extend the half-life in vivo may include serum albumin or its fragment, polyethylene glycol, a domain that binds serum albumin (such as an antibody against serum albumin), a polyethylene glycol-liposome complex, etc. In the second domain, the fragment that has a binding effect on effector cells may include an immunoglobulin Fc region, etc., preferably selected from a human immunoglobulin Fc region. The human immunoglobulin Fc region includes mutations for changing the effector function mediated by Fc, and the effector function includes a combination of one or more of CDC activity, ADCC activity, and ADCP activity. The immunoglobulin may be selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, IgM, etc., and the IgG may be specifically selected from a combination of one or more of IgG1, IgG2, IgG3 or IgG4 subtypes, etc. The immunoglobulin Fc region contained in the antibody fusion protein can cause the fusion protein to form a dimer, while extending the in vivo half-life of the fusion protein and increasing Fc-mediated related activities. In a specific embodiment of the present invention, the immunoglobulin Fc region can be the Fc region of human IgG1, more specifically, it can be a wild-type IgG1 Fc sequence, and the sequence can be introduced with mutations for changing Fc-mediated effector functions, for example, a) mutations that change Fc-mediated CDC activity; b) mutations that change Fc-mediated ADCC activity; or c) mutations that change Fc-mediated ADCP activity. Such mutations are described in the following literature: Leonard G Presta, Current Opinion in Immunology 2008, 20:460-470; Esohe E. Idusogie et al., J Immunol 2000, 164:4178-4184; RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4:443-446; Paul R. Hinton et al., J Immunol, 2006, 176:346-356.

[0074] In the fusion protein of the anti-CEACAM5 antibody provided by the present invention, a connecting peptide may be provided between the first domain and the second domain. The connecting peptide may be a flexible polypeptide chain composed of alanine (A) and / or serine (S) and / or glycine (G), and the length of the connecting peptide may be 3 to 30 amino acids, preferably 3-9, 9-12, 12-16, 16 to 20. In another specific embodiment of the present invention, the length of the connecting peptide may be 8 or 15.

[0075] The present invention also provides an isolated polynucleotide encoding the antibody of the present invention or encoding the fusion protein, and the polynucleotide may be RNA, DNA or cDNA, etc. The method of providing the isolated polynucleotide should be known to those skilled in the art, for example, it can be prepared by automatic DNA synthesis and / or recombinant DNA technology, or it can be isolated from a suitable natural source.

[0076] The present invention also provides a construct, which contains the isolated polynucleotide of the present invention. The construction method of the construct should be known to those skilled in the art. For example, the construct can be constructed by in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and other methods. More specifically, it can be constructed by inserting the isolated polynucleotide into the multiple cloning site of the expression vector. The expression vector in the present invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors. The vector may also include one or more regulatory sequences operably connected to the polynucleotide sequence, and the regulatory sequence may include a suitable promoter sequence. The promoter sequence is usually operably connected to the coding sequence of the amino acid sequence to be expressed. The promoter may be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequence may also include a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is linked to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice can be used in the present invention.

[0077] Generally speaking, suitable vectors may contain a replication origin, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers that function in at least one organism. For example, these promoters may include, but are not limited to, the lac or trp promoter of Escherichia coli; the λ phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. Marker genes can be used to provide phenotypic traits for selecting transformed host cells, for example, may include, but are not limited to, dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli, etc. When the polynucleotide is expressed, the expression vector may also include an enhancer sequence. If the enhancer sequence is inserted into the vector, transcription will be enhanced. The enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, which acts on the promoter to enhance the transcription of the gene.

[0078] The present invention also provides an antibody expression system, the expression system contains the construct of the present invention or the polynucleotide of the present invention integrated with an exogenous gene in the genome. Any cell suitable for expression by an expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, specifically including but not limited to Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS, HEK293 cells, or Bowes melanoma cells, etc. One or more combinations thereof. The method for constructing the expression system should be known to those skilled in the art, for example, it can be a combination of one or more of microinjection, gene gun method, electroporation method, virus-mediated transformation method, electron bombardment method, calcium phosphate precipitation method, etc.

[0079] Immunoconjugates

[0080] The present invention provides an immunoconjugate, the immunoconjugate comprising the antibody of the present invention, or the fusion protein of the present invention. The immunoconjugate generally further comprises a functional molecule connected to the antibody or fusion protein (including but not limited to covalent connection, coupling, attachment, adsorption), the functional molecule may be but not limited to a detectable marker, a cytotoxin, a radioactive isotope, a biologically active protein, a molecule targeting a tumor surface marker, a molecule that inhibits tumors, a molecule targeting a surface marker of an immune cell, etc., or a combination thereof.

[0081] The method for preparing the immunoconjugate should be known to those skilled in the art. For example, the antibody and / or fusion protein can be connected to the functional molecule directly or through a spacer of appropriate length. The connection method can be chemical cross-linking or genetic engineering fusion expression to obtain the immunoconjugate.

[0082] For therapeutic purposes, it may be appropriate to combine with therapeutic effector groups such as radioactive groups, i.e., groups composed of radioisotopes or radionuclides (e.g. 3 H. 14 C. 15 N. 33 P. 35 S. 90 Y. 99 Tc, 111 ln, 123 1. 125 1. 131 1. 201 TI, 213 Bi), a toxin or a cytotoxic group such as a cytostatic group or a group consisting of or including it.

[0083] The immunoconjugate may comprise an antibody or fusion protein of the invention and a detectable marker. The detectable marker includes, but is not limited to, a fluorescent marker, a chromogenic marker, a protein tag; such as an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron emitting metal, and a non-radioactive paramagnetic metal ion. More than one marker may also be included. The label used to label the antibody for detection and / or analysis and / or diagnostic purposes depends on the specific detection / analysis / diagnosis technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels for detection / analysis / diagnosis techniques and / or methods known in the art are well known to those skilled in the art.

[0084] The antibody or fusion protein of the present invention can be coupled with a labeling group (labeled polypeptide), which can then be used, for example, for diagnostic purposes. Suitable labeling groups can be selected from radioisotopes (such as those mentioned above) or groups containing radioisotopes, radionuclides, fluorescent groups (such as fluorescent proteins such as GFP, RFP, etc., dyes, rhodamine, fluorescein and derivatives thereof such as FITC, cyanine dyes), enzyme groups (such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase), chemiluminescent groups, biotin groups, metal particles (such as gold particles), magnetic particles (such as having a core containing magnetite (Fe3O4) and / or hematite (Fe2O3)), predetermined polypeptide groups, etc.

[0085] The immunoconjugate may comprise the antibody or fusion protein of the present invention and a molecule targeting a surface marker of an immune cell. The molecule targeting a surface marker of an immune cell can recognize an immune cell, and it carries the antibody of the present invention to reach the immune cell, and the antibody of the present invention can target the immune cell to the tumor cell, thereby utilizing the killing effect of the antibody of the present invention while also inducing the immune cell to specifically kill the tumor.

[0086] The immunoconjugate may comprise an antibody or fusion protein of the present invention and at least one molecule targeting a tumor surface marker or a tumor-suppressing molecule. The tumor-suppressing molecule may be an anti-tumor cytokine or an anti-tumor toxin. For example, the cytokine may be, but is not limited to, IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, etc. The molecule targeting a tumor surface marker may, for example, synergize with the antibody of the present invention to more accurately target tumor cells.

[0087] The immunoconjugate can also be a chimeric antigen receptor (CAR), which can be expressed in immune cells. The "immune cells" and "immune effector cells" are used interchangeably, including: T lymphocytes, NK cells or NKT cells, etc., preferably, NK cells and T lymphocytes. The chimeric antigen receptor generally comprises an extracellular binding region, a transmembrane region and an intracellular signaling region connected in sequence, wherein the extracellular binding region comprises an antibody or fusion protein of the present invention. The design of the transmembrane region and the intracellular signaling region based on CAR technology is well known in the art: for example, the transmembrane region adopts the transmembrane region of molecules such as CD8 and CD28, and the intracellular signaling region adopts the intracellular signaling region of the immunoreceptor tyrosine activation motif (ITAM) CD3ζ chain or FcεRIγ tyrosine activation continuation and co-stimulatory signal molecules CD28, CD27, CD137, CD134, MyD88, CD40, etc. More specifically, for T lymphocytes, the first generation of CAR T lymphocytes contains only ITAM in the intracellular signaling region, in which the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-ITAM, which can stimulate anti-tumor cytotoxic effects; the second generation of CAR T lymphocytes adds the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), in which the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-ITAM or scFv-TM- / CD137-ITAM; the B7 / CD28 or 4-1BBL / CD137 co-stimulation in the intracellular signaling region causes the continuous proliferation of T lymphocytes, and can increase the level of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, while improving the survival period and anti-tumor effect of CAR T in vivo; the third generation of CAR T lymphocytes, in which the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further improving the survival period of CAR T in vivo and its anti-tumor effect.

[0088] According to the above, the chimeric antigen receptor prepared using the antibody or fusion protein of the present invention can be composed of the following sequentially connected extracellular binding regions, transmembrane regions and intracellular signaling regions: the antibody or fusion protein of the present invention, CD8 and CD3ζ; the antibody or fusion protein of the present invention, CD8, CD137 and CD3ζ; the antibody or fusion protein of the present invention, the transmembrane region (CD28a) of the CD28 molecule, the intracellular signaling region (CD28b) of the CD28 molecule and CD3ζ; or the antibody or fusion protein of the present invention, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, CD137 and CD3ζ.

[0089] The chimeric antigen receptor is expressed on the surface of immune effector cells, and while utilizing the killing effect of the antibody of the present invention, the immune effector cells can also have a highly specific cytotoxic effect on tumor cells expressing CEACAM5.

[0090] As a preferred embodiment of the present invention, the antibody of the present invention can be linked to a tumor suppressor molecule, preferably the tumor suppressor molecule is an anti-tumor toxin, including toxins acting on DNA, such as DXd, duocarmycin, calicheamicin, pyrrolobenzodiazepines (PBDs), SN-38 and other related compounds and their derivatives; toxins acting on tubulin, such as monomethyl auristatin related compounds and their derivatives, maytansinoid related compounds and their derivatives; and related compounds and their derivatives acting on other functions in cells such as metabolism, transcription, translation, signal transduction, etc. The present invention also includes analogs, isomers, precursors, etc. of these small molecule compounds as toxins.

[0091] Pharmaceutical composition

[0092] The present invention also provides a pharmaceutical composition comprising the anti-CEACAM5 antibody of the present invention, or the fusion protein of the anti-CEACAM5 antibody of the present invention, or the immunoconjugate of the present invention.

[0093] The pharmaceutical composition may also include various pharmaceutically acceptable carriers in the art. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used, and may include, but are not limited to: buffers, such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as blood albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; tonicity modifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactants such as polysorbates; salt-forming counterions such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants such as Or polyethylene glycol (PEG). Pharmaceutical preparations for in vivo administration are generally sterile, and methods for achieving sterility of pharmaceutical preparations should be known to those skilled in the art, for example, it can be achieved by filtering with a sterile filter membrane or the like. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the dosage form required for the pharmaceutical composition to prepare it into different dosage forms, for example, the pharmaceutical composition of the present invention can be in various dosage forms including but not limited to tablets, injections, lyophilized agents, etc.

[0094] In the pharmaceutical composition, the content of the fusion protein and the immunoconjugate is usually an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined according to the object to be treated and the specific administration method. For example, based on the total mass of the pharmaceutical composition, the content of the fusion protein and the immunoconjugate can range from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99%.

[0095] The fusion protein, immunoconjugate and pharmaceutical composition of the present invention can be administered as a single active ingredient, or can be administered in a combination therapy, i.e., in combination with other agents. For example, the combination therapy can be the fusion protein, immunoconjugate, pharmaceutical composition combined with at least one other anti-tumor drug. For another example, the combination therapy can be the fusion protein, immunoconjugate, pharmaceutical composition combined with antibodies targeting other tumor-specific antigens, such as but not limited to: anti-EGFR antibodies, anti-VEGF antibodies, anti-HER2 antibodies, or anti-Claudin18A2 antibodies, etc. One or more combinations, the inhibitor can preferably be a monoclonal antibody.

[0096] The present invention also provides a detection kit, comprising the antibody, fusion protein or immunoconjugate of the present invention. The kit may also include: a container, a control (negative or positive control), a buffer, an adjuvant, etc. as required, and those skilled in the art may select them according to specific circumstances. The kit may also include instructions for use to facilitate operation by those skilled in the art.

[0097] The present invention further provides a detection method for detecting CEACAM5 protein using the antibody, including but not limited to qualitative detection, quantitative detection and localization detection. Specifically, the detection method includes but is not limited to immunofluorescence assay, immunohistochemistry and radioimmunoassay.

[0098] A method for detecting whether CEACAM5 protein is present in a sample may include: contacting the sample with the antibody of the present invention; observing whether an antibody complex is formed, and the formation of the antibody complex indicates that CEACAM5 protein is present in the sample. The sample may be a cell and / or tissue sample; the sample may be fixed or dissolved in a medium; and the level of CEACAM5 protein in the fixed or dissolved sample is detected. In some embodiments, the detection object may be a cell-containing sample present in a cell preservation solution. In other embodiments, the antibody is also conjugated with a fluorescent dye, chemical substance, polypeptide, enzyme, isotope, label, etc. that can be used for detection or can be detected by other reagents.

[0099] application

[0100] The present invention also provides use of the antibody, fusion protein, immunoconjugate or pharmaceutical composition of the present invention in the preparation of a drug for diagnosing, treating or preventing a disease associated with cells expressing (or overexpressing) CEACAM5.

[0101] The "therapeutically effective amount" of the fusion protein, immunoconjugate, and pharmaceutical composition provided by the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevents damage or disability caused by the pain of the disease. For example, for the treatment of CEACAM5-related tumors (including colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, breast cancer, etc.), relative to untreated subjects, the "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts the efficacy of human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, and such inhibition can be determined in vitro by tests known to those skilled in the art. A therapeutically effective amount of fusion protein, immunoconjugate, or pharmaceutical composition is generally capable of reducing tumor size or otherwise alleviating symptoms of the subject. Those skilled in the art can select an appropriate therapeutically effective amount based on actual conditions, for example, the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected. The prescription for treatment (e.g., determination of dosage, etc.) can be determined by a doctor, and factors generally considered include, but are not limited to, the disease being treated, the individual patient's condition, the delivery site, the method of administration, and other factors. A preventive effective amount refers to an amount that is effective in achieving the desired preventive effect at the necessary dose and time. Usually, but not necessarily, since a preventive dose is used for a subject before the onset of the disease or in the early stages of the disease, a "preventive effective amount" is usually lower than a "therapeutically effective amount."

[0102] The beneficial technical effects of the present invention include:

[0103] 1. Provides a novel anti-human CEACAM5 antibody. The antibody of the present invention has a unique sequence and very ideal binding specificity.

[0104] 2. Compared with the reported antibodies, the antibodies of the present invention only have the heavy chain part, which has a smaller molecular weight, a higher expression level, and better tissue penetration, and can be used for the unmet medical needs related to CEACAM5.

[0105] 3. The anti-human CEACAM5 antibody of the present invention has a significant killing effect on tumor cells and can significantly inhibit the growth of CEACAM5-positive tumors.

[0106] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, or according to the conditions recommended by the manufacturer.

[0107] Example 1: Preparation of antibodies specific for CEACAM5

[0108] A fusion protein of the extracellular region (ECD) of human CEACAM5 and a His tag (Human CEACAM5 ECD-His) was used as an immunogen and immunized at multiple points subcutaneously near the cervical lymph nodes of alpacas to stimulate the production of antibodies against the extracellular region of human CEACAM5 in alpacas.

[0109] ELISA was used to detect the titer of antibodies in alpaca serum after three immunizations. First, human CEACAM5-ECD-His protein was adsorbed on the ELISA plate, and then the gradient diluted serum was added. After incubation at room temperature for 1 hour, the ELISA plate was washed. Then, the HRP-labeled alpaca full-length secondary antibody (Jackson, 128-035-160) was added, and the ELISA plate was washed after incubation at room temperature for 1 hour. Finally, TMB was used for color development, and the absorbance value at 450nm was recorded with an ELISA reader. The results are shown in Figure 2. Figure 1 A. The antibody titer in the serum reaches above 1:145K.

[0110] Flow cytometry was used to detect the binding titer of antibodies in alpaca serum to cells expressing human CEACAM5. ExpiCHO stable cell lines expressing full-length human CEACAM5 were taken, counted and centrifuged, HBSS was added to resuspend the cells and added to a 96V-type well plate to centrifuge and remove the supernatant, and then the test serum of immune alpaca was added. After incubation at 4°C for 30 minutes, the cells were washed twice, and PE-labeled alpaca full-length secondary antibody (Jackson, 128-115-160) was added. After incubation at 4°C in the dark for 30 minutes, the cells were washed, resuspended with HBSS and detected by flow cytometry. The results are shown in Figure 1 B, The binding titer of the antibody in the serum to cells expressing human CEACAM5 reached above 1:48.6K.

[0111] The results of serum titer testing showed that the immunized alpacas produced antibodies specific to CEACAM5. PBMCs were collected and phage display technology was used to screen specific heavy chain antibodies against CEACAM5.

[0112] Example 2: Obtaining a monoclonal antibody that specifically binds to human CEACAM5

[0113] Peripheral blood of immune alpacas was collected, PBMC was separated and mRNA was extracted, and reverse transcribed into cDNA. VHH antibody fragments were amplified from reverse transcribed cDNA using PCR, and the amplified VHH antibody fragments were cloned into phage plasmids and transfected into competent cells TG1 to obtain phage libraries. VHH phages that can bind to human CEACAM5 were screened using phage display technology, and then the VHH phages that bind to human CEACAM5 were infected with Escherichia coli cells SS320, and finally SS320 was induced to express VHH antibodies.

[0114] First, ELISA was used to screen antibodies that specifically bind to CEACAM5 but not to CEACAM family proteins, CEACAM1, CEACAM6, and CEACAM8. Human CEACAM5-His, Human CEACAM1-His (Kactus, CEA-HM201), Human CEACAM6-His (Kactus, CAM-HM406), and Human CEACAM8-His (Kactus, CEA-HM108) proteins were adsorbed on the ELISA plate, incubated at room temperature for 1 hour with 5% skim milk, and then the ELISA plate was washed. Then, the Escherichia coli supernatant containing VHH was added, and the ELISA plate was washed after incubation at room temperature for 1 hour. Then, a secondary antibody with an HRP-labeled anti-myc tag (Bethyl, A190-104P) was added, and the ELISA plate was washed after incubation at room temperature for 1 hour. Finally, TMB was used for color development, and the absorbance value at 450nm was recorded with an ELISA reader. The antibodies were then screened by flow cytometry. ExpiCHO stable cells overexpressing the full length of Human CEACAM5 were centrifuged, resuspended in HBSS and counted, added to a 96 V-well plate, centrifuged to remove the supernatant, and then added with Escherichia coli supernatant containing VHH. After incubation at 4°C for 30 minutes, the cells were washed twice, and a PE-labeled anti-myc tag secondary antibody (CST, 3739S) was added. After incubation at 4°C in the dark for 30 minutes, the cells were washed twice, resuspended in HBSS and the fluorescence value was detected by flow cytometry.

[0115] As shown in Table 1, after extensive screening, 5 clones were finally obtained, which did not bind to human CEACAM1, CEACAM6, and CEACAM8.

[0116] Table 1

[0117]

[0118]

[0119] According to Table 1, these five clones can not only bind to human CEACAM5 at the protein level, but also bind to the ExpiCHO cell line overexpressing the full length of Human CEACAM5 at the cell level, indicating that these five clones can specifically bind to human CEACAM5.

[0120] Example 3: Analysis of antibody variable region structure

[0121] The E. coli obtained in Example 2 that specifically binds to human CEACAM5 was used to obtain the variable region amino acid sequence of the alpaca heavy chain antibody against human CEACAM5 by using sequencing primers of phage plasmid, as shown in Table 2.

[0122] Table 2

[0123]

[0124] The variable region amino acid sequence was analyzed using the IMGT method, and the CDR amino acid sequence that plays a decisive role in the binding of the alpaca heavy chain antibody to human CEACAM5 was obtained, as shown in Table 3.

[0125] Table 3

[0126]

[0127]

[0128] Example 4. Preparation of recombinant antibodies

[0129] By using PrimeSTAR@Max DNA Polymerase (TAKARA, R045B), PCR cloned the heavy chain antibody variable region nucleic acid sequence obtained in Example 3, and simultaneously synthesized and cloned the Fc gene of the human IgG1 antibody heavy chain, the gene and amino acid sequence of which are as follows:

[0130] The Fc gene sequence of human IgG1 antibody heavy chain (SEQ ID NO: 21):

[0131] gacaaaactcacacatgcccaccgtgcccagcaccagaactgctcggcggaccctccgtgttcctgtttccacccaagcctaaagacaccctcatgatatctcggactcccgaggtcacctgcgtggtcgtggacgtgagccacgaggaccccgaagtcaagttcaactggtacgtggatggagtcgaagtgcataatgctaagacaaaaccaagggaggaacagtacaacagcacttatagagtcgtgtccgtcctgaccgtgctccaccaggattggctgaacggaaaggagtataagtgcaaagtgagcaataaggccctgcccgctcctatcgagaaaaccatttccaaggccaaagggcagcctcgggaaccacaggtgtacacactgcctccatcccgcgaggaaatgaccaagaaccaggtctctctgacatgtctcgtgaaagggttctatccttcagacatcgctgtggagtgggaaagcaatggtcagccagagaacaattacaagaccacaccccctgtgctggacagtgatgggtcattctttctgtattctaagctcaccgtggataaaagtagatggcagcagggtaacgtcttttcctgttctgtgatgcacgaagcactccacaaccactacactcagaagtcactcagcctctctccagggaag

[0132] Fc amino acid sequence of human IgG1 heavy chain (SEQ ID NO: 22):

[0133] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0134] The heavy chain antibody variable region gene and the Fc gene of the heavy chain of the human IgG1 antibody were separated by agarose gel electrophoresis, and the target gene was obtained using a gel recovery kit (Omega, D2500-02), and finally recombined and connected to the expression vector using the ClonExpress UltraOne Step Cloning Kit (Vazyme, C115-02). After sequencing to verify that the expression vector is correct, it was transfected into ExpiCHO cells for expression and purification to obtain the chimeric antibody.

[0135] Example 5: ELISA to detect antibody affinity

[0136] ELISA was used to detect the affinity of the anti-human CEACAM5 chimeric antibody in Example 4 to human CEACAM5 and monkey CEACAM5. Human CEACAM5-His and cynomolgus CEACAM5-His proteins (Kactus, CAM-CM105) were first adsorbed on the ELISA plate, incubated with 5% skim milk at room temperature for 1 hour, and then the ELISA plate was washed. The gradient diluted antibodies were added, and the ELISA plate was washed after incubation at room temperature for 1 hour. HRP-labeled goat anti-human Fc secondary antibody (Jackson, 109-035-098) was added, and the ELISA plate was washed after incubation at room temperature for 1 hour. Finally, TMB was used for color development, and the absorbance at 450nm was recorded with an ELISA reader.

[0137] The results are as follows Figure 2 A and Figure 2 As shown in B, all chimeric antibodies have high affinity for Human CEACAM5 and cynomolgus CEACAM5, and the affinity is significantly higher than that of the control antibody Tusamitamab (the antibody amino acid sequence is derived from WO2014079886).

[0138] Example 6: ELISA method to detect antibody binding specificity

[0139] ELISA was used to detect the affinity of the anti-human CEACAM5 chimeric antibody in Example 4 for human CEACAM1, human CEACAM6, and human CEACAM8. First, human CEACAM1-His, human CEACAM6-His, and human CEACAM8-His proteins were adsorbed on the ELISA plate, respectively, incubated with 5% skim milk at room temperature for 1 hour, and then the ELISA plate was washed. The gradient diluted antibodies were added, and the ELISA plate was washed after incubation at room temperature for 1 hour. The HRP-labeled goat anti-human Fc secondary antibody was added, and the ELISA plate was washed after incubation at room temperature for 1 hour. Finally, TMB was used for color development, and the absorbance value at 450nm was recorded with an ELISA reader.

[0140] The results are as follows Figure 3 A, Figure 3 B, Figure 3 As shown in C, all chimeric antibodies do not bind to human CEACAM1, human CEACAM6, and human CEACAM8, indicating that the chimeric antibodies specifically bind to human CEACAM5.

[0141] Example 7: Detection of antibody affinity for cell membrane CEACAM5 antigen by flow cytometry

[0142] Flow cytometry was used to detect the affinity of chimeric antibodies for human CEACAM5 and monkey CEACAM5 on the cell membrane. ExpiCHO cell lines overexpressing human CEACAM5 and monkey CEACAM5 were selected, and antibodies of different dilutions were added. After incubation at 4°C for 0.5 hours, unbound antibodies were washed away. Then, PE-labeled goat anti-human Fc secondary antibody (Jackson, 109-115-098) was added, and unbound secondary antibodies were washed away after incubation at 4°C for 0.5 hours. Finally, flow cytometry was used for detection.

[0143] Through analysis, such as Figure 4 As shown in A, when binding to cells expressing human CEACAM5, the chimeric antibodies CA1, CA2, CA3, CA4, and CA5 not only have better EC50 than the control antibody Tusamitamab, but also have higher maximum binding amounts than the control antibody Tusamitamab. In addition, the EC50 of each antibody is also better than the affinity of mAb1 (the antibody amino acid sequence is derived from WO2022048883A1). Figure 4 As shown in B, in binding to cells expressing monkey CEACAM5, not only were the EC50s of each antibody superior to the control antibody Tusamitamab, but the maximum binding amount was also higher than the control antibody Tusamitamab. In addition, although the EC50 of each antibody was comparable to mAb1, from the perspective of the maximum binding amount, CA2 and CA3 had more antibodies able to bind to cells than mAb1.

[0144] Example 8: Octet detection of affinity of antibody to human CEACAM5

[0145] Octet was used to detect the affinity of CA1, CA2, CA3, CA4 and CA5 to human CEACAM5.

[0146] The results are shown in Table 4. Antibodies CA1, CA2, CA3, CA4 and CA5 have higher affinity for CEACAM5 and are better than the control antibody Tusamitamab. In addition, the affinity of CA1, CA2, CA3 and CA4 for CEACAM5 is also better than the control antibody mAb1.

[0147] Table 4

[0148]

[0149]

[0150] Example 9. Preparation of antibody-drug conjugates and their killing effects

[0151] Five chimeric antibodies, CA1, CA2, CA3, CA4 and CA5, were prepared into antibody-drug conjugates to detect their killing effects on colorectal cancer tumor cells SK-CO-1, DiFi and LS174T expressing human CEACAM5. The chimeric antibody was first mixed with TCEP (sigma) at a molar ratio of 1:3 to open the disulfide bonds between the chains, and then 4 to 10 times the molar amount of maleimide-GGFG-DXd (MedChemExpress) was added for conjugation. Finally, the ultrafiltration tube (Merck, UFC801096) was used for centrifugation to remove excess small molecule drugs. The obtained antibody-drug conjugates were named CA1-DXd, CA2-DXd, CA3-DXd, CA4-DXd and CA5-DXd, and negative control IgG-DXd and positive control mAb1-DXd were prepared at the same time. Through hydrophobic interaction chromatography analysis, the ratio of antibody to DXd in all prepared antibody-drug conjugates was about 1:4.

[0152] Detect the cell-killing effect of the antibody-drug conjugate of the chimeric antibody on tumor cells expressing CEACAM5. SK-CO-1, DiFi and LS174T cells were digested and resuspended, and the density was adjusted to 5000 cells / 55μL with complete medium and plated on a 96-well plate (Corning, 3599). After adding equal volumes of antibody-drug conjugate samples of different dilution concentrations, the cells were placed in a 37°C incubator for 5 days before detection. On the day of detection, the cells were taken out and returned to room temperature, and the detection reagent (Novozyme, DD1101-1) was added to the cells to be tested at 100μL / well and mixed thoroughly, and allowed to stand for 5 minutes in the dark for full reaction. Take out 100μL of the cell reagent mixture from each well and transfer it to a 96-well plate with a white bottom, and use Cytation to detect the luminescence signal value.

[0153] Through analysis, the results are as follows Figure 5 A. Figure 5 B. Figure 5As shown in Figure 3, in SK-CO-1 and DiFi cells, the killing effects of CA1-DXd, CA2-DXd, CA3-DXd, CA4-DXd and CA5-DXd were comparable to those of mAb1-DXd; however, in LS174T cells, the killing effects of CA1-DXd, CA2-DXd, CA3-DXd, CA4-DXd and CA5-DXd were significantly better than those of mAb1-DXd.

[0154] Example 10: In vivo antitumor effect of CA3-DXd

[0155] CA3-DXd was selected to study its anti-tumor efficacy in mice. SCID female mice were selected and LS174T cells were inoculated subcutaneously on the back to construct a tumor-bearing mouse model. When the tumor size grew to 250mm 3 Afterwards, the mice were randomly divided into 4 groups, with 5 mice in each group. PBS, 3.0 mg / kg IgG-DXd, 1.6 mg / kg CA3-DXd, and 3.0 mg / kg mAb1-DXd were injected into the tail vein. After one administration, the body weight and tumor volume of mice in each group were measured.

[0156] The results are as follows Figure 6 As shown, compared with the PBS and IgG-DXd control groups, the experimental group CA3-DXd can significantly inhibit the growth of LS174T tumors, and after 19 days of tumor bearing, its anti-tumor effect is better than the positive control mAb1-DXd with the same molar amount.

[0157] Example 11. Preparation and affinity analysis of CA3 humanized antibody

[0158] The chimeric antibody CA3 was humanized, the CDR region binding to the antigen was retained, and the FR region was replaced with an amino acid sequence that was closest to the human antibody germline gene homology. At the same time, based on the predicted antibody structure, the amino acids at several key sites that maintained the antibody structure were retained, and finally the humanized antibody variable region was obtained. The amino acid sequence of the variable region (SEQ ID NO: 23) is as follows:

[0159] EVQLVESGGGLVQPGGSLRLSCAAS GDTLSNRA MSWFRQAPGKEREFVSA ITWSGGST HYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYC TATHPLGPVPITPDKHNA WGQGTLVTVSS

[0160] The variable region amino acid sequence of this antibody was converted into a nucleotide sequence, and gene synthesis was performed after codon optimization. In order to increase the number of small molecule drug couplings when preparing antibody-drug conjugates with heavy chain antibodies, a new Fc gene of the heavy chain of human IgG1 antibody with mutations in the hinge region was resynthesized, and two pairs of disulfide bonds were added to the hinge region. Its gene and amino acid sequences are as follows:

[0161] The Fc gene sequence of the human IgG1 antibody heavy chain with a mutated hinge region (SEQ ID NO: 24):

[0162] Gagcgcaaatgtgaggagtgtgtcgagtgcccaccgtgcccagcaccagaactgctcggcggaccctccgtgttcctgtttccacccaagcctaaagacaccctcatgatatctcggactcccgaggtcacctgcgtggtcgtggacgtgagccacgaggaccccgaagtcaa gttcaactggtacgtggatggagtcgaagtgcataatgctaagacaaaaccaagggaggaacagtacaacagcacttatagagtcgtgtccgtcctgaccgtgctccaccaggattggctgaacggaaaggagtataagtgcaaagtgagcaataaggccctgcccgctccta tcgagaaaaccatttccaaggccaaagggcagcctcgggaaccacaggtgtacacactgcctccatcccgcgaggaaatgaccaagaaccaggtctctctgacatgtctcgtgaaagggttctatccttcagacatcgctgtggagtgggaaagcaatggtcagccagagaac aattacaagaccacaccccctgtgctggacagtgatgggtcattctttctgtattctaagctcaccgtggataaaagtagatggcagcagggtaacgtcttttcctgttctgtgatgcacgaagcactccacaaccactacactcagaagtcactcagcctctctccagggaag

[0163] The amino acid sequence of the Fc gene of the heavy chain of a human IgG1 antibody with a mutated hinge region (SEQ ID NO: 25):

[0164] ERKCEECVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0165] The humanized CA3 variable region gene and the Fc gene of the heavy chain of the above-mentioned mutated human IgG1 antibody were recombinantly cloned into an expression vector. After the sequencing results were correct, the plasmid was extracted and transfected into ExpiCHO cells for expression. The humanized antibody protein hCA3 was obtained by Protein A purification.

[0166] The affinity of humanized antibody hCA3 to human CEACAM5 and monkey CEACAM5 was detected by flow cytometry using the same method as in Example 7.

[0167] The results are as follows Figure 7 A~ Figure 7 As shown in B, hCA3 has good affinity for human CEACAM5 and monkey CEACAM5, and the affinity is similar to that of the chimeric antibody CA3.

[0168] Example 12: Conjugated drugs based on humanized antibody hCA3 and their killing effects

[0169] The humanized antibody hCA3 was prepared into an antibody-drug conjugate, and the same detection method as in Example 9 was used to make the ratio of antibody to DXd in the antibody-drug conjugate to be about 1:6. The obtained antibody-drug conjugate was named hCA3-DXd. At the same time, a negative control IgG-DXd and a positive control mAb1-DXd were prepared, and the ratio of antibody to DXd was 1:6 and 1:8, respectively. The cell killing ability of the humanized antibody hCA3-DXd was examined by the same method as in Example 9.

[0170] The results are as follows Figure 8 A, Figure 8 B, Figure 8 As shown in C, hCA3-DXd has a significant killing effect on tumor cells in colorectal cancer cells DiFi, LS174T and small cell lung cancer cells SHP77, and the killing effect is better than that of the control mAb1-DXd.

[0171] Example 13: In vivo killing effect of conjugate drugs based on humanized antibody hCA3

[0172] The anti-tumor efficacy of hCA-3 in mice was studied using the same method as in Example 10. Two animal models of colorectal cancer cells, LS174T and GP2D cells, were selected to construct tumor-bearing mouse models, and the mice were randomly divided into two groups after modeling. PBS and 1.6 mg / kg hCA3-DXd were injected respectively. After one administration, the body weight and tumor volume of each group of mice were measured.

[0173] Fig. 9 A and Fig. 9 The results in B show that compared with the control group, hCA3-DXd significantly inhibited tumor growth in both tumor-bearing mouse models.

[0174] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A CEACAM5-specific antibody having heavy chain variable regions HCDR1, HCDR2 and HCDR3, comprising: CA3, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; CA1, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; CA2, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; CA4, the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; CA5, the amino acid sequences of its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

2. The antibody according to claim 1, characterized in that The antibodies include: humanized antibodies, chimeric antibodies or alpaca antibodies.

3. The antibody according to claim 1, characterized in that The antibodies include: An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 23 or having more than 85% identity thereto; An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 3 or having more than 85% identity with the sequence; An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 1 or having more than 85% identity with the sequence; An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 2 or having more than 85% identity with the sequence; An antibody having a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 4 or having more than 85% identity with the sequence; The heavy chain variable region amino acid sequence is as shown in SEQ ID NO: 5 or an antibody having 85% or more identity thereto.

4. A polynucleotide or a construct containing the polynucleotide, wherein the polynucleotide encodes the antibody according to any one of claims 1 to 3, and the construct comprises the polynucleotide according to claim 4.

5. An antibody expression system, comprising the construct according to claim 4 or the exogenous polynucleotide according to claim 4 integrated into the genome.

6. A method for preparing the CEACAM5-specific antibody according to any one of claims 1 to 3, comprising: The antibody is expressed using the antibody expression system of claim 5 under conditions suitable for expressing the antibody, thereby obtaining the antibody.

7. Use of the antibody according to any one of claims 1 to 3, for: preparing anti-tumor drugs that specifically target tumor cells expressing CEACAM5; preparing antibody drug conjugates or immunoconjugates; Preparation of bifunctional or multifunctional antibodies; preparing a reagent for diagnosing a tumor that expresses CEACAM5; or Preparation of chimeric antigen receptor modified immune cells.

8. An immunoconjugate, comprising the antibody according to any one of claims 1 to 3; and a functional molecule linked thereto.

9. The immunoconjugate according to claim 8, characterized in that The functional molecules include: tumor-suppressing molecules, molecules targeting tumor surface markers, cytotoxins, radioactive isotopes, biologically active proteins, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof.

10. The immunoconjugate according to claim 9, characterized in that The tumor-suppressing molecule is an antitumor toxin or an antitumor cytokine; preferably, the antitumor toxin includes: a toxin acting on DNA, a toxin acting on microtubules, and a compound acting on intracellular metabolism, transcription, translation or signal transduction; preferably, the toxin acting on DNA includes: DXd, Duocarmycin, Calicheamicin, Pyrrolobenzodiazepines, SN-38; preferably, the toxin acting on microtubules includes: monomethyl auristatin, maytansinoids; preferably, the antitumor cytokines include: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha or variants thereof; or The molecule targeting the tumor surface marker is an antibody or a ligand that binds to the tumor surface marker; preferably, the antibody that binds to the tumor surface marker is an antibody that recognizes other antigens other than CEACAM5, and the other antigens include: EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1 or CDH16; or The molecule targeting the surface marker of immune cells is an antibody or ligand that binds to the surface marker of immune cells; preferably, the antibody that binds to the surface marker of immune cells, the surface marker includes: CD3, CD4, CD8, PD-1, PD-L1, LAG-3, TIM-3, 4-1BB, OX40, ICOS, B7-H3, CD16, CTLA-4, TIGIT, VISTA, GITR, CD27, SIRPα, CD32, CD64, ILT2, ILT4, NKG2A, NKG2D or KIR.

11. A pharmaceutical composition comprising: Any of the above antibodies or any of the above immunoconjugates.

12. Use of the antibody according to any one of claims 1 to 3, the immunoconjugate according to any one of claims 8 to 10, or the pharmaceutical composition according to claim 11 in the preparation of a preparation, a kit or a drug kit for diagnosing or treating a tumor; the tumor is a tumor expressing CEACAM5.

13. The use according to claim 12, characterized in that The tumors include: colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, and breast cancer.

14. A kit or a medicine box comprising the antibody according to any one of claims 1 to 3, the immunoconjugate according to any one of claims 8 to 10, or the pharmaceutical composition according to claim 11.

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