A nanobody against ceacam5 and related applications thereof

Abstract

The application discloses an anti-CEACAM5 nanobody and related application thereof, and relates to the field of antibodies. The anti-CEACAM5 nanobody comprises CDR1, CDR2 and CDR3 with amino acid sequences as shown in SEQ ID NO: 1-3 or 5-7 in sequence, can specifically bind to a CEACAM5 antigen, and has an affinity of 10 ‑9 KD or above, and belongs to a high-affinity nanobody. In addition, the application constructs a bispecific antibody by combining the nanobody targeting CEACAM5 and an anti-CD3 antibody OKT3, and experiments in vitro prove that the prepared specific antibody has strong killing activity on tumor cells expressing CEACAM5, thereby providing key materials for subsequent development of immunotherapy with high-efficiency and long-lasting anti-tumor activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular, to an anti-CEACAM5 nanobody and its related applications. BACKGROUND

[0002] Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) include a group of immunoglobulin-related vertebrate glycoproteins, containing 12 family members (CEACAM1, 3, 4, 5, 6, 7, 8, 16, 18, 19, 20 and 21), which play an important role in cell adhesion, intracellular and intercellular signal transduction, and complex biological processes such as cancer progression, metastasis, angiogenesis and inflammation. Among them, CEACAM1, CEACAM5 (CEA), CEACAM6 and CEACAM7 are more studied in terms of structure, expression and function under physiological and pathological conditions, and are closely related to tumor occurrence and development.

[0003] Human CEACAM5, also known as CEA (carcinoembryonic antigen) or CD66e, is the first member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAMs) family. It is composed of 642 amino acids, is bound to the membrane through glycosylphosphatidylinositol (GPI), and structurally contains seven immunoglobulin-like domains, one variable (lgV) -like domain (N domain) and six lgC-like domains (A1-B3). It was first discovered by Gold and Freeman in 1965, and is a glycoprotein related to various carcinogenic activities, involved in cell adhesion, differentiation, proliferation and survival, and often used as a tumor marker and cancer recurrence marker for colorectal cancer in clinical diagnosis and treatment.

[0004] At present, there is still a lack of antibodies targeting CEACAM5 with high specificity and high affinity on the market.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide an anti-CEACAM5 nanobody and its related applications.

[0007] The present application is implemented as follows:

[0008] In a first aspect, the present application provides an anti-CEACAM5 nanobody, which comprises a heavy chain variable region, the heavy chain variable region comprising the following complementarity determining regions:

[0009] the CDR1, CDR2 and CDR3 have amino acid sequences as shown in SEQ ID NO: 1-3 in order, or the CDR1, CDR2 and CDR3 have amino acid sequences as shown in SEQ ID NO: 5-7 in order.

[0010] In a second aspect, an antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region of the nanobody described in the preceding embodiments.

[0011] In a third aspect, an isolated nucleic acid or a recombinant vector comprising the isolated nucleic acid is provided, wherein the isolated nucleic acid encodes the nanobody described in the preceding embodiments or the antibody or the antigen-binding fragment thereof described in the preceding embodiments.

[0012] In a fourth aspect, a host cell comprising the recombinant vector described in the preceding embodiments is provided.

[0013] In a fifth aspect, a method for preparing the antibody or the antigen-binding fragment thereof is provided, comprising culturing the host cell described in the preceding embodiments.

[0014] In a sixth aspect, a conjugate is provided, comprising the nanobody described in the preceding embodiments or the antibody or the antigen-binding fragment thereof described in the preceding embodiments.

[0015] In a seventh aspect, a product is provided, comprising the nanobody described in the preceding embodiments or the antibody or the antigen-binding fragment thereof described in the preceding embodiments.

[0016] In a seventh aspect, the nanobody described in the preceding embodiments or the antibody or the antigen-binding fragment thereof described in the preceding embodiments are used in a product for detecting CEACAM5 protein or preparing a product for preventing, diagnosing, assisting diagnosis, treating or assisting treatment of a disease targeting CEACAM5 for a non-disease diagnosis or treatment purpose.

[0017] The present application has the following advantages:

[0018] The anti-CEACAM5 nanobody obtained by screening and selection in the embodiments of the present application can specifically bind to the CEACAM5 antigen, and the affinity is all above 10 -9 KD, which is a high-affinity nanobody. In addition, the nanobody targeting CEACAM5 and the anti-CD3 antibody OKT3 are constructed into a bispecific antibody, and experiments in vitro prove that the prepared specific antibody has strong killing activity on tumor cells expressing CEACAM5, which provides key materials for subsequent development of immunotherapy with high-efficiency and long-lasting anti-tumor activity. BRIEF DESCRIPTION OF DRAWINGS

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 SDS-PAGE of recombinant CEACAM5 protein;

[0021] Figure 2 This is a schematic diagram of the pcDNA3.1-hFc-His eukaryotic expression vector structure.

[0022] Figure 3 To identify the reactivity of recombinant nanobodies with antigens using indirect ELISA;

[0023] Figure 4 This is a diagram showing the binding effect of IFA on recombinant nanobodies and CEACAM5+-HeLa cells in an embodiment of the present invention.

[0024] Figure 5 This is a flow cytometry diagram showing the binding effect of recombinant nanobodies to tumor cell lines expressing CEACAM5 in an embodiment of the present invention.

[0025] Figure 6 This invention provides an in vitro cytotoxic activity assay for Nbs-OKT3 bispecific antibody (BiTE) against target cells. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] On one hand, embodiments of the present invention provide an anti-CEACAM5 nanobody, which includes a heavy chain variable region, wherein the heavy chain variable region includes the following complementarity-determining regions:

[0028] The amino acid sequences are as described in SEQ ID NO:1-3, namely CDR1, CDR2 and CDR3, or as described in SEQ ID NO:5-7, namely CDR1, CDR2 and CDR3.

[0029] In some embodiments, the nanobody has a KD ≤ 10 -9 M, 10 -10 M and 10-11 affinity binding to CEACAM5 protein.

[0030] In some embodiments, the heavy chain variable region further comprises a framework region. Specifically, the structure of the heavy chain variable region of the Nanobody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0031] In some embodiments, the Nanobody is at least one of a monovalent Nanobody, a multivalent Nanobody, a multispecific antibody, and a fusion Nanobody.

[0032] In some embodiments, when the Nanobody is a monovalent Nanobody, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 4 or 8.

[0033] In another aspect, embodiments of the present application provide an antibody or an antigen binding fragment thereof, comprising a heavy chain variable region of a Nanobody according to any of the preceding embodiments.

[0034] Nanobodies have a large number of hydrophilic residues on their surface, can maintain a strict monomer structure, and can specifically and highly bind to their antigens only in the form of monomers. Nanobodies are single gene encoded, easy to be genetically engineered, and can be aggregated by short connecting sequences to form multivalent or multispecific antibody structures.

[0035] In some embodiments, the antibody comprises any one of a full-length antibody, a heavy chain antibody, a chimeric antibody, a multivalent antibody (bivalent, trivalent, tetravalent, pentavalent, or hexavalent), a multispecific antibody, and a fusion antibody.

[0036] A "multivalent antibody" herein is a polymer of monovalent antibodies that recognize the same epitope, and has higher antigen affinity than the corresponding monovalent antibody.

[0037] A "multispecific antibody" herein is a polymer of monovalent antibodies that bind to different targets or different binding regions on the same target, and has stronger antigen recognition ability than the corresponding monovalent antibody. Optionally, the multispecific antibody comprises any one of a bispecific antibody, a trispecific antibody, and a tetraspecific antibody.

[0038] In some embodiments, when the antibody is a bispecific antibody, the bispecific antibody further comprises a CD3 binding domain. The CD3 binding domain can comprise an antibody or an antigen binding fragment thereof against CD3. The Nanobody against CEACAM5 is connected to the N-terminus or C-terminus of the CD3 binding domain.

[0039] Bispecific antibodies (BsAb) refer to artificial antibodies that can target two antigens or different epitopes of the same antigen simultaneously. Bispecific antibodies can be IgG-like, i.e. full-length bispecific antibodies, or non-IgG-like bispecific antibodies that are non-full-length antibody constructs. Full-length bispecific antibodies typically retain the traditional monoclonal antibody (mAb) structure with two Fab arms and one Fc region, except that the two Fab sites bind different antigens. Non-full-length bispecific antibodies can lack the entire Fc region. These include chemically linked Fabs, consist only of Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFv). There are also fusion proteins that mimic the variable domains of two antibodies.

[0040] A "chimeric antibody" herein is an antibody that fuses the variable region of a non-human antibody with the constant region or the framework region of a human antibody, which can reduce the immune response reaction induced by the non-human antibody.

[0041] A "fusion antibody" herein includes fusion nanobodies, including but not limited to new fusion molecules formed by combining other structures (such as BSA, IgG-Fc, etc.) through genetic engineering technology, such as enzymes, antibacterial peptides or imaging substances that can prolong their half-life.

[0042] In some embodiments, the fusion antibody is fused from the constant region of an antibody and the nanobody of any of the preceding embodiments.

[0043] In some embodiments, the constant region of the antibody can be the heavy chain constant region of the antibody, which can be selected from the heavy chain constant region of a human antibody, a mouse heavy chain constant region, a rabbit heavy chain constant region, a sheep heavy chain constant region, or a monkey heavy chain constant region.

[0044] In some embodiments, the heavy chain constant region of the human antibody is selected from the heavy chain constant region of any one of hlgG1, hlgG2, hlgG3, hlgG4, or a mutation thereof. Taking the heavy chain constant region of IgG1 as the constant region of the nanobody against CLL1 has a very high inhibition rate on target cells and maintains good binding activity with the antigen.

[0045] In some embodiments, the antibody includes a mouse-derived antibody or a humanized antibody.

[0046] In some embodiments, the antigen-binding fragment includes any one of F(ab')2, Fab', Fab, Fv, and scFv, as long as they exhibit the desired antigen-binding activity.

[0047] The antigen-binding fragments of the above-mentioned antibodies are functional fragments of the antibodies, and generally have the same binding specificity as the antibodies from which they are derived. Those skilled in the art can easily understand from the content disclosed in the present application that the antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by methods of splitting disulfide bonds by chemical reduction. Those skilled in the art can easily obtain the antigen-binding fragments based on the structural basis of the antibodies disclosed in the present application.

[0048] The antigen-binding fragments can also be obtained by recombinant genetic engineering techniques, which are also known to those skilled in the art, or by synthesis, for example, using an automatic peptide synthesizer, such as an automatic peptide synthesizer sold by Applied BioSystems.

[0049] In another aspect, the present application provides an isolated nucleic acid or a recombinant vector containing the isolated nucleic acid, wherein the isolated nucleic acid encodes the nanobody according to any of the preceding embodiments or the antibody or antigen-binding fragment thereof according to any of the preceding embodiments. Considering the degeneracy of codons, the gene sequence encoding the antibody or antigen-binding fragment thereof can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody or antigen-binding fragment thereof; or the gene can be artificially synthesized and modified according to the codon bias of the host for expressing the antibody to improve the expression efficiency of the antibody.

[0050] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct that can be directly introduced into a host cell by transformation, transfection or transduction to express the target gene.

[0051] In another aspect, the present application provides a host cell containing the recombinant vector according to any of the preceding embodiments.

[0052] Specifically, the host cell includes 293 cells, 293T cells, 293FT cells, CHO cells, Per6 cells. The 293 series of cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those skilled in the art.

[0053] On the basis of the amino acid sequences of the antibody or the antigen-binding fragment thereof disclosed in the present application, it is easy for those skilled in the art to conceive that the antibody or the functional fragment thereof is prepared by using genetic engineering technology or other technologies (chemical synthesis, recombinant expression), for example, the nanobody or the antibody or the functional fragment thereof is isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the nanobody or the antibody or the functional fragment thereof according to any one of the preceding embodiments, which is easy for those skilled in the art to achieve. Therefore, no matter what technology is used to prepare the antibody or the functional fragment thereof of the present application, it belongs to the protection scope of the present application.

[0054] In another aspect, the present application provides a method for preparing the antibody or the antigen-binding fragment thereof, which comprises culturing the host cell according to any one of the preceding embodiments. Specifically, the culture conditions of the host cell are not specifically limited in the present application, and the culture conditions capable of making the host cell express the nanobody or the antibody or the antigen-binding fragment thereof can be obtained based on conventional technical knowledge.

[0055] In another aspect, the present application provides a conjugate, which comprises the nanobody according to any one of the preceding embodiments or the antibody or the antigen-binding fragment thereof according to any one of the preceding embodiments.

[0056] In some embodiments, the conjugate further comprises a conjugated moiety combined with the nanobody or the antibody or the antigen-binding fragment thereof.

[0057] In some embodiments, the conjugated moiety comprises any one of a purification tag, a label for detection or tracking, and a solid carrier.

[0058] In some embodiments, the label is selected from at least one of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.

[0059] In another aspect, the present application provides a product, which comprises the nanobody according to any one of the preceding embodiments or the antibody or the antigen-binding fragment thereof according to any one of the preceding embodiments.

[0060] In some embodiments, the product is any one of a drug, a reagent, a detection plate, a detection chip, or a kit.

[0061] In addition, the present application also provides the use of the nanobody according to any one of the preceding embodiments or the antibody or the antigen-binding fragment thereof according to any one of the preceding embodiments in the preparation of a product for detecting CEACAM5 protein or preventing, diagnosing, assisting diagnosis, treating, or assisting treatment of a disease related to CEACAM5 as a target.

[0062] "Treatment" in the present disclosure includes preventing or reducing a condition, reducing the speed of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination thereof.

[0063] For cancer, "treatment" can mean inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cells, or some combination thereof. For a tumor, "treatment" includes eradicating all or part of a tumor, inhibiting or slowing the growth and metastasis of a tumor, preventing or delaying the development of a tumor, or some combination thereof.

[0064] In some embodiments, the related disease includes: a CEACAM5-positive tumor.

[0065] In some embodiments, the related disease includes: any one or more of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, medullary thyroid cancer, liver cancer, lung cancer, ovarian cancer, and urological tumors.

[0066] There are many detection cases for non-disease diagnosis or treatment purposes, such as when the sample to be detected is selected from an artificially prepared sample, a negative sample, and an environmental sample, the detection is for non-disease diagnosis or treatment purposes.

[0067] The features and performances of the present disclosure are further described in detail below in conjunction with the embodiments.

[0068] Example 1 Preparation of CEACAM5 recombinant protein

[0069] The nucleic acid sequence encoding human CEACAM5 (NM_004363.2) was synthesized by Anhui General Biologic Co. Ltd. The extracellular domain (ECD) (Met 1-Ala 685) of CEACAM5 was amplified by PCR and subcloned into pcDNA3.1 expression vector (Invitrogen) with a His tag at the C-terminus. Then, the recombinant CEACAM5 protein was purified by affinity chromatography using NTA-Ni column through transient transfection of 293FT, and the FreeStyle 293 Expression System (Life Technologies) was used to express the recombinant protein in serum-free medium (Life Technologies) in a shake flask for 5-7 days. The supernatant was collected and the recombinant CEACAM5 protein was purified to high purity. The SDS-PAGE result of the recombinant CEACAM5 protein is shown in FIG. 1. TM The supernatant was collected and the recombinant CEACAM5 protein was purified to high purity. The SDS-PAGE result of the recombinant CEACAM5 protein is shown in FIG. 1. Figure 1 .

[0070] Example 2 Preparation of anti-CEACAM5 protein specific nanobody

[0071] Firstly, the CEACAM5 protein (1 mg / time) and an equal volume of Freund's adjuvant were mixed and then the bactrian camel was immunized for 4 times successively, and the peripheral blood was collected to separate the lymphocytes. Then, the total RNA of the lymphocytes was extracted by using an RNA extraction kit, and the VHH gene was amplified by RT-PCR. The amplified VHH gene was cloned into the phage display vector pMECS to successfully construct the VHH phage antibody library. Two strains of anti-CEACAM5 nanobodies were obtained by screening 3 rounds of phage display technology, and the VHH gene was amplified by using the two strains as templates and was constructed into the eukaryotic expression vector pcDNA3.1-hFc-His by homologous recombination (see Figure 2 ). Then, the recombinant nanobodies were purified by affinity chromatography by using NTA-Ni column.

[0072] Example 3: ELISA identification of the reactivity of the recombinant nanobodies with the antigen

[0073] To identify the reactivity of the recombinant nanobody-hFc fusion protein with the antigen, 200 ng / well of the CEACAM5 recombinant protein was coated on an enzyme-labeled plate in advance, and the plate was blocked at 4°C overnight. Then, different amounts of the recombinant nanobodies (dilution: 10 -5 ~ 10 2 μg / mL) were added, and the secondary antibody was washed, developed, and terminated. The optical density (OD450) at 450 nm was measured by using an enzyme-labeled instrument, and the binding capacity was determined by using four-parameter nonlinear regression curve fitting. The results showed (see Figure 3 ) that the two recombinant nanobodies of CEACAM5 could specifically bind to the CEACAM5 recombinant protein.

[0074] Example 4: Affinity determination of the CEACAM5 nanobody

[0075] The two nanobodies of CEACAM5 were verified for the affinity of the recombinant nanobody by surface plasmon resonance, and the binding kinetic constant (Kd) was determined. The GE Biacore TM 8K instrument was used, and the anti-mouse IgG antibody was immobilized on the surface of the CM5 chip by using the coupling buffer in the amino coupling kit. Then, the CEACAM5 (ECD)-mFc was captured on the CM5 chip by 2-fold serial dilution. Then, the purified CEACAM5 nanobody was allowed to flow through the surface of the chip, and the machine read the Ka (1 / Ms), kd (1 / s), and KD (M), i.e., the affinity of the recombinant nanobody was measured. The affinity determination results showed that the two candidate nanobodies of CEACAM5 could specifically bind to the CEACAM5 (ECD)-mFc protein, and the affinity was 10 -9KD magnitude above, all belong to high affinity antibody, the specific data see table 1.

[0076] Table 1 affinity data summary table of nanobody

[0077] Antibody Ka (1 / Ms) kd (1 / s) KD (M) NbC40 5.06 x 10 4 ]] 3.51 x 10 -5 ]] 6.93 x 10 -10 ]] NbC74 6.08 x 10 4 ]] 4.69 x 10 -5 ]] 7.72 x 10 -9 ]]

[0078] Example 5 IFA detection of recombinant nanobody binding to CEACAM5+-HeLa cells

[0079] First, HeLa cells are infected with lentivirus containing the full-length gene of CEACAM5, and high-purity CEACAM5+-HeLa cells stably expressing the CEACAM5 gene are obtained by flow sorting. The recombinant nanobody prepared in Example 1 and the positive control antibody TUSA (Tusamitamab, 5 μg / mL) are incubated with CEACAM5+-HeLa cells at 37°C for 40 min, washed with PBS for 3 times, and then incubated with 594@goat anti-human secondary antibody. After washing with PBS for 3 times, imaging is performed by fluorescence microscopy, and the results are shown in Figure 4 As shown, the recombinant nanobodies NbC40 and NbC74 and the positive control antibody TUSA can bind to the above cells well.

[0080] Example 6 Flow detection of recombinant nanobody binding to tumor cell lines expressing CEACAM5

[0081] The recombinant nanobodies NbC40 and NbC74 prepared in Example 2 and the positive control antibody TUSA are incubated with colorectal cancer cells HT29, gastric cancer cells SNU16 and pancreatic cancer cells BXPC3, and non-small cell lung cancer cells A549 which do not express CEACAM5 at 37°C for 40 min, washed with PBS for 3 times, and then incubated with APC@goat anti-human IgG (H+L) secondary antibody. After washing with PBS for 3 times, analysis is performed by flow cytometry, and the results are shown in Figure 5 As shown, the recombinant nanobodies NbC40 and NbC74 and the positive control antibody TUSA can bind to the above cells well.

[0082] Example 7 Preparation and activity detection of anti-CEACAM5 and CD3 bispecific antibody (BiTE)

[0083] Two anti-CEACAM5 nanobodies NbC40 and NbC74 provided in Example 2 of the application are respectively connected in series with the antibody OKT3 against CD3 molecule using (G4S)3linker to construct a eukaryotic expression vector, transfected into HEK293T cells for expression, and purified by Ni column.

[0084] CEACAM5+-HeLa cells and HeLa cells were added into RTCA instrument special 96-well plate and common 96-well cell culture plate respectively at 5000 per well, 100 μL / well (for duplicate), the RTCA special 96-well plate was placed in the instrument and cultured until the Cell Index value was between 1.0-2.0, then the bispecific antibody prepared in Example Five of different concentrations (0.1 nM and 0.01 nM) and positive control antibody TUSA-OKT3 were respectively co-incubated with activated T cells from healthy donor peripheral blood at an effector-target ratio of 2:1, and the RTCA monitoring time was 48 h;

[0085] The results are shown in Figure 6 The above-mentioned bispecific antibody has no killing effect on wild-type HeLa cells but has strong killing activity on CEACAM5+-HeLa cells, and C40 shows relatively stronger killing activity at a low concentration of 10 pM.

[0086] The antibody sequences involved in the foregoing examples and the CDR sequences analyzed and aligned according to the IMGT database antibody sequence are shown in Table 2 below.

[0087] Table 2 Sequence information of antibodies

[0088]

[0089] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An anti-CEACAM5 Nanobody, characterized in that, It comprises a heavy chain variable region, which comprises the following complementarity determining regions: CDR1, CDR2 and CDR3 with the amino acid sequences shown in SEQ ID NO: 1~3 in order, or CDR1, CDR2 and CDR3 with the amino acid sequences shown in SEQ ID NO: 5~7 in order.

2. The Nanobody according to claim 1, characterized in that, The heavy chain variable region further comprises a framework region.

3. The Nanobody according to claim 1, characterized in that, The nanobody is at least one of a monovalent nanobody and a multivalent nanobody.

4. The Nanobody according to claim 3, characterized in that, When the nanobody is a monovalent nanobody, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4 or 8.

5. An antibody or antigen-binding fragment thereof, characterized in that, It comprises: The heavy chain variable region of the nanobody according to any one of claims 1~4; The antibody comprises any one of a heavy chain antibody, a bispecific antibody and a fusion antibody; When the antibody is a bispecific antibody, the bispecific antibody is constructed by connecting the heavy chain variable region of the nanobody according to any one of claims 1~4 to the N-terminus of anti-CD3 antibody OKT3; When the antibody is a fusion antibody, the fusion antibody is hFc connected to the C-terminus of the heavy chain variable region of the nanobody according to any one of claims 1~4.

6. An isolated nucleic acid or a recombinant vector comprising the isolated nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO:

2. The isolated nucleic acid encodes the nanobody according to any one of claims 1~4 or the antibody according to claim 5 or an antigen binding fragment thereof.

7. A host cell, characterized in that, It contains the recombinant vector according to claim 6.

8. A method of producing an antibody or antigen-binding fragment thereof, characterized in that, It comprises: The host cell according to claim 7 is cultured.

9. A conjugate, characterized in that, It consists of: the nanobody according to any one of claims 1~4 or the antibody according to claim 5 or an antigen binding fragment thereof; and a coupling moiety bound to the nanobody or the antibody or the antigen binding fragment thereof; The coupling moiety is selected from any one of a purification tag, a label for detection or tracking and a solid support.

10. A product characterized by, It comprises: The nanobody according to any one of claims 1~4 or the antibody according to claim 5 or an antigen binding fragment thereof.

11. The product of claim 10, wherein, The product is a reagent.

12. The product of claim 10, wherein, The product is a drug.

13. The product of claim 10, wherein, The product is a detection plate or a detection chip.

14. The product of claim 10, wherein, The product is a kit.

15. Use of the nanobody according to any one of claims 1~4 or the antibody according to claim 5 or an antigen binding fragment thereof in the preparation of a product for the diagnosis, auxiliary diagnosis, treatment or auxiliary treatment of a disease related to CEACAM5 as a target; The disease related to CEACAM5 as a target is a CEACAM5 positive tumor, and the disease diagnosed or diagnosed with CEACAM5 as a target is any one or more of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, medullary thyroid cancer, liver cancer, lung cancer, ovarian cancer and urological tumors; The treatment or adjuvant therapy targets diseases related to CEACAM5, including: Any one or more of colorectal cancer, pancreatic cancer and gastric cancer.

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