An anti-human cd8 antibody and a preparation method and application thereof

Abstract

The present application relates to a kind of anti-human CD8 antibody and its preparation method and application.Belong to biological medicine technical field.The present application designs new type of anti-human CD8 antibody, the anti-human CD8 antibody has high affinity, and it is suitable for recombinant expression, can be carried out large-scale, high yield, high batch consistency production by genetic engineering method, solve the existing murine anti-human CD8 antibody heterogenicity is stronger, easy to trigger immune rejection, affinity decline after human source modification or natural antibody is difficult to realize scale stable production by genetic engineering, can provide excellent antibody material for immunodetection, immunotherapy field, with good development application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an anti-human CD8 antibody, its preparation method and application. Background Technology

[0002] CD8 is a leukocyte differentiation antigen and a signature glycoprotein on the surface of cytotoxic T lymphocytes (CTLs). As a co-receptor for the T cell receptor (TCR), it plays a crucial role in antigen recognition and the transduction of T cell activation signals. + T cells are the core effector cells in the body's anti-tumor and antiviral immune responses. Therefore, the CD8 molecule or CD8 + Specific detection, sorting, in vivo tracing, and in vivo delivery of T cells have significant clinical and research value for tumor immunotherapy, research on autoimmune diseases, and diagnosis of infectious diseases.

[0003] CD8 antibody is used for CD8 molecular detection, CD8 + T-cell isolation analysis is an indispensable tool in research and can also be used to develop targeted drugs for tumor immunotherapy. By conjugating drugs, toxins, or radionuclides, it can specifically eliminate abnormally activated CD8 cells in pathological states. + T cells, or mediators of CD8 + T-cell infiltration and targeted killing of tumors can improve patient prognosis.

[0004] Most existing CD8 antibodies are murine or murine-derived, which easily induce human anti-mouse antibody reactions (HAMA), reducing treatment efficacy and causing adverse immune reactions. Although there have been studies on anti-human CD8 antibodies, such as the human CD8 monoclonal antibody disclosed in CN117700550A, which can specifically recognize and bind to human CD8 protein and CD8+, the research has been ongoing. + Cells; CN111295394A discloses an anti-CD8 antibody that provides binding to human CD8 without stimulating or inhibiting CD8. + T-cell activated anti-CD8 antibody.

[0005] Currently, anti-human CD8 antibodies still have the following problems: First, insufficient affinity and specificity, and inability to efficiently enrich CD8. + T cells; second, poor conjugation performance, antibody shedding is likely to occur after conjugation with conjugates, leading to unstable targeted therapy effects; third, antibodies with insufficient humanization still pose certain immunogenic risks, and their safety in in vivo therapeutic applications still needs to be improved.

[0006] Therefore, there is still a need in this field to develop novel anti-human CD8 antibodies with high affinity and specificity to meet the needs of scientific research and clinical development. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides an anti-human CD8 antibody, its preparation method, and its application, with the aim of developing novel humanized CD8 antibodies with high affinity, specificity, and low immunogenicity.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an anti-human CD8 antibody, wherein the amino acid sequences of the heavy chain variable regions CDR1 to CDR3 of the anti-human CD8 antibody respectively include the sequences shown in SEQ ID NO.7 to SEQ ID NO.9, and the amino acid sequences of the light chain variable regions CDR1 to CDR3 respectively include the sequences shown in SEQ ID NO.10 to SEQ ID NO.12.

[0009] This invention designs and prepares novel anti-human CD8 antibodies. These antibodies exhibit high affinity for the human CD8 antigen and can be produced on a large scale with high yield and high batch-to-batch consistency through genetic engineering methods (such as stable expression in CHO cells). This invention enriches the variety of anti-human CD8 antibodies and provides more tool options for subsequent research and applications.

[0010] Optionally, the amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.4 or a sequence with at least 90% homology to its sequence, and the amino acid sequence of the light chain variable region includes any one of the sequences shown in SEQ ID NO.5 to SEQ ID NO.6 or a sequence with at least 90% homology to its sequence.

[0011] Optionally, the amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.1 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.5 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.1 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.6 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.2 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.5 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.2 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.6 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.3 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.5 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.3 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.6 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.4 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.5 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.4 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.6 or a sequence with at least 90% homology to it.

[0012] It is understood that, based on the antibody designed in this invention, antibodies with similar functions obtained by using genetic modification methods in the art to substitute, delete, or add amino acids should all be within the scope of protection of this invention. The number of amino acids substituted, deleted, or added can be any value, such as 1, 5, 10, 15, or more, such that the sequence homology between the changed amino acid sequence and its corresponding original sequence can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. In the art, when amino acids with similar or comparable properties are conservatively substituted, the function of the protein is usually not changed, and the amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code; therefore, antibodies obtained by conservatively substituting amino acids with similar or comparable properties are also within the scope of protection of this invention.

[0013] In a second aspect, the present invention provides a nucleic acid molecule that encodes the anti-human CD8 antibody described in the first aspect.

[0014] In this invention, due to the degeneracy of the genetic code, a large number of nucleic acid molecules that can be used to encode the antibodies of this invention can be obtained. Therefore, given the identification of specific amino acid sequences, those skilled in the art can easily prepare any number of different nucleic acids by modifying the sequences of one or more codons without altering the amino acid sequence encoding the protein. More preferred polynucleotides can be selected through codon optimization based on the preferences of the host cells used in the actual preparation process. The nucleic acid molecules can be obtained using conventional methods, such as PCR amplification or artificial synthesis.

[0015] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecules described in the second aspect.

[0016] Fourthly, the present invention provides a recombinant cell containing the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0017] The recombinant cell expression of the anti-human CD8 antibody of the first aspect of the present invention may contain a nucleic acid molecule encoding the antibody or a recombinant vector containing the nucleic acid molecule. The host cell may be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell. Prokaryotic cells may be bacterial cells, lower eukaryotic cells may be yeast cells, and higher eukaryotic cells may be mammalian cells.

[0018] Fifthly, the present invention provides a method for preparing the anti-human CD8 antibody described in the first aspect, the method comprising: The nucleic acid molecule encoding the anti-human CD8 antibody described in the first aspect is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is transformed into host cells, cultured, and the product is purified to obtain the anti-human CD8 antibody.

[0019] Transformation of recombinant vectors into host cells can be performed using conventional methods well known to those skilled in the art. These include methods such as CaCl2 injection, electroporation, calcium phosphate co-precipitation, and conventional mechanical methods like microinjection, electroporation, and liposome packaging. The resulting transformants can be cultured using conventional methods well known to those skilled in the art, and the culture medium can be a standard culture medium. Antibodies produced by the transformants can be separated and purified using physical and chemical methods, such as salting out, centrifugation, cell disruption, and chromatography (e.g., affinity chromatography).

[0020] Optionally, the expression vector includes any one of pCDNA3.4, pTT5, pCMV, pMH, pCEP4, pComb3X, pPICZα, pGAPZα, or pFastBac.

[0021] Optionally, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0022] Optionally, the prokaryotic cells include any one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, lactic acid bacteria, lactococci, or Pseudomonas.

[0023] Optionally, the eukaryotic cells include any one of HEK293 cells, CHO cells, Pichia pastoris, Saccharomyces cerevisiae, Sf9 or Sf21 cells.

[0024] In a sixth aspect, the present invention provides an antibody conjugate comprising the anti-human CD8 antibody described in the first aspect and a conjugate substance thereto.

[0025] Optionally, the coupling substance includes at least one of cytotoxins, radioactive isotopes, luminescent substances, chromogenic substances, enzymes, or lipid nanoparticles.

[0026] The anti-human CD8 antibody designed based on this invention can be further used to construct antibody conjugates and develop therapeutic or diagnostic reagents.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects: This invention designs a novel anti-human CD8 antibody, which possesses high affinity, with the highest affinity being for EC2. 50 The value can reach 10 -11 The M-level antibody exhibits a very strong binding affinity to the target CD8 molecule. Furthermore, this antibody is suitable for recombinant expression systems and can be industrially produced on a large scale with high yield and high batch-to-batch consistency through mature genetic engineering methods (e.g., stable expression using CHO cells). This design effectively solves a series of key problems in existing technologies, such as the high heterologousness of murine anti-human CD8 antibodies, which easily induces immune rejection; the significant decrease in affinity after humanization; and the difficulty in achieving large-scale, stable production of naturally derived antibodies through genetic engineering. Therefore, the anti-human CD8 antibody provided by this invention can provide high-performance, stable, and reliable antibody materials for related fields such as immunoassay (e.g., flow cytometry, immunohistochemistry) and immunotherapy (e.g., antibody-drug conjugates, bispecific antibody development), demonstrating broad market potential and promising development and application prospects. Attached Figure Description

[0028] Figure 1 The image shows the SDS-PAGE (polyacrylamide gel electrophoresis) results of different anti-human CD8 antibodies in Example 1.

[0029] Figure 2 This is a statistical graph showing the flow cytometry results of different anti-human CD8 antibodies binding to T cells in Example 2.

[0030] Figure 3 This is a sensor diagram showing the interaction between antigen and antibody in the SPR affinity test of different anti-human CD8 antibodies in Example 3. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0032] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0033] This invention provides a novel anti-human CD8 antibody that, while maintaining high affinity and specific binding ability to the CD8 molecule, significantly reduces the risk of immunogenicity in human applications and avoids triggering human anti-mouse antibody reactions (HAMA). Furthermore, this antibody holds promise for developing targeted conjugates, such as targeted drugs and targeted delivery vectors.

[0034] Example 1 This embodiment designs and prepares anti-human CD8 antibodies.

[0035] As shown in Table 1, four heavy chain variable regions (VH) were designed: heavy chain variable region 1 to heavy chain variable region 4 (VH1 to VH4). The amino acid sequences of each heavy chain variable region are shown in SEQ ID NO.1 to SEQ ID NO.4, and the CDR1 to CDR3 amino acid sequences of each heavy chain variable region are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively. Two light chain variable regions (VL) were designed: light chain variable region 1 to light chain variable region 2 (VL1 to VL2). The amino acid sequences of each light chain variable region are shown in SEQ ID NO.5 to SEQ ID NO.6, and the CDR1 to CDR3 amino acid sequences of each heavy chain variable region are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively.

[0036] By further utilizing the heavy chain variable region and light chain variable region designed above, eight different anti-human CD8 antibodies were constructed. The specific combinations include VH1 and VL1, VH1 and VL2, VH2 and VL1, VH2 and VL2, VH3 and VL1, VH3 and VL2, VH4 and VL1, and VH4 and VL2.

[0037] Table 1 Based on the designed amino acid sequences of the heavy chain and light chain variable regions, codon optimization was performed to adapt them for mammalian cell expression, and the corresponding gene sequences (SEQ ID NO.13~SEQ ID NO.18) were synthesized. The synthesized heavy chain variable region gene was fused with the IgG1 heavy chain constant region gene (GenBank: AH007035.2), and the light chain variable region gene was fused with the Kappa light chain constant region gene (GenBank: AH002839.2). A signal peptide, which guides the secretion of intracellularly synthesized antibodies extracellularly, was inserted into the 5' end of the fused heavy and light chain genes, and then cloned into the pCDNA3.4 expression vector to obtain heavy chain expression plasmids and light chain expression plasmids, respectively.

[0038] Heavy and light chain expression plasmids (at a 1:1 molar ratio) were co-transfected into CHO-S cells in suspension growth. After transfection, cells were cultured in a shaker at 37°C and 8% CO2 for 6–7 days. The cell culture supernatant was collected.

[0039] The antibody was purified from the supernatant using a Protein A affinity chromatography column (e.g., MabSelect SuRe). The column was equilibrated with PBS (pH 7.4), loaded with the sample, washed with PBS, and then eluted with 0.1 M glycine (pH 3.0), followed immediately by neutralization with 1 M Tris-HCl (pH 8.5). The eluted antibody was transferred to PBS buffer, and purity was assessed by SDS-PAGE gel electrophoresis, with concentration determined using Nanodrop.

[0040] The results are as follows Figure 1As shown in the figure, lane M represents the protein molecular weight standard; lane R represents the electrophoresis result of the purified antibody under reducing conditions (with 50 mM dithiothreitol added); and lane NR represents the electrophoresis result of the purified antibody under non-reducing conditions. The gel used was a 10% gradient polyacrylamide gel, stained with Coomassie Brilliant Blue R-250. As shown in the figure, the non-reducing lanes (lane NR) of the eight CD8 antibodies (VH1 and VL1 combination, VH1 and VL2 combination, VH2 and VL1 combination, VH2 and VL2 combination, VH3 and VL1 combination, VH3 and VL2 combination, VH4 and VL1 combination, and VH4 and VL2 combination) showed a single main band at approximately 150 kDa, indicating that the expressed heavy and light chains had been correctly assembled into complete IgG molecules via interchain disulfide bonds, and no obvious high-molecular-weight aggregates or degradation fragments were observed. In the reduced lane (lane R), clear heavy chain and light chain bands appeared at approximately 50 kDa and 25 kDa, respectively, indicating that the antibody of the present invention is composed of heavy chain and light chain subunits of the expected size, with an intact intrachain structure.

[0041] Example 2 This embodiment utilizes flow cytometry to detect the interaction between antibodies and CD8. + The binding capacity of T cells.

[0042] 1. Cell preparation: Human peripheral blood mononuclear cells (PBMCs) were collected and activated for 3 days with CD3 / CD28 beads (Thermo Fisher, catalog number: A56996-1). After removing the CD3 / CD28 beads, the cells were cultured for 11 days, cryopreserved, and then thawed. The cells were resuspended in DPBS buffer and the cell density was adjusted to 2 × 10⁻⁶ cells / day. 6 cells / mL.

[0043] 2. Antibody incubation: Take 100 μL of cell suspension and add serially diluted antibodies purified in Example 1 (starting concentration 2 μg / mL, 5-fold serial dilution). Incubate at room temperature in the dark for 30 minutes. Wash twice with DPBS buffer.

[0044] 3. Secondary antibody incubation: Add YSFluor™ 647-labeled goat anti-human IgG secondary antibody and incubate at room temperature in the dark for 30 minutes. Wash twice with DPBS buffer.

[0045] 4. Detection: Resuspend cells in 200 μL DPBS buffer and detect using a flow cytometer (e.g., Beckman CytoFLEX). Evaluate antibody binding ability to cells using mean fluorescence intensity (MFI) and percentage of positive cells.

[0046] Flow cytometry results of different anti-human CD8 antibodies binding to T cells are as follows: Figure 2 As shown in Table 2, all eight antibodies can bind to CD8 with high affinity. + T cells. Among them, the VH1+VL1 combination of EC 50 The smallest value indicates the highest affinity.

[0047] Table 2 Example 3 This embodiment utilizes surface plasmon resonance (SPR) technology to detect antibody affinity.

[0048] 1. Antigen fixation: Recombinant human CD8 protein (such as His-tagged protein) is coupled to the surface of the CM5 chip via amino coupling, with a coupling amount of approximately 100-200 RU.

[0049] 2. Affinity assay: The eight antibodies prepared in Example 1 were used as analytes and serially diluted with HBS-EP+ buffer (400 nM, 200 nM, 100 nM, 50 nM, and 25 nM). The solutions were flowed through the chip surface at a flow rate of 30 μL / min, with a binding time of 60 seconds and a dissociation time of 180 seconds.

[0050] 3. Data Analysis: A 1:1 binding model was used to globally fit the sensor map, and the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) were calculated.

[0051] The results are as follows Figure 3 As shown in Table 3, Chi 2 It is the goodness-of-fit parameter, with units of RU. 2 The smaller the value of (resonance unit squared), the lower the noise level of the experiment and the higher the agreement between the model and the data. Rmax is the theoretical maximum binding response value, and the unit is RU (resonance unit). The larger the value, the stronger the maximum response signal that the instrument can detect when saturation binding is achieved on the chip surface. The results show that antibodies with different VH / VL combinations all show high affinity for human CD8 antigen.

[0052] Table 3 In summary, this invention designs a novel anti-human CD8 antibody, which possesses high affinity, with the highest affinity being EC21. 50 It can reach 10 -11M-level antibodies, suitable for recombinant expression, can be produced on a large scale, in high yield, and with high batch-to-batch consistency through genetic engineering methods (such as stable expression in CHO cells). This solves the problems of existing murine anti-human CD8 antibodies having strong heterogeneity that easily leads to immune rejection, decreased affinity after human modification, and the difficulty in achieving large-scale stable production of natural antibodies through genetic engineering. It can provide high-performance antibody materials for the fields of immune detection and immunotherapy, and has good prospects for development and application.

[0053] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An anti-human CD8 antibody, characterized in that, The amino acid sequences of the heavy chain variable regions CDR1 to CDR3 of the anti-human CD8 antibody include the sequences shown in SEQ ID NO.7 to SEQ ID NO.9, respectively, and the amino acid sequences of the light chain variable regions CDR1 to CDR3 include the sequences shown in SEQ ID NO.10 to SEQ ID NO.12, respectively.

2. The anti-human CD8 antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.4 or a sequence with at least 90% homology to its sequence, and the amino acid sequence of the light chain variable region includes any one of the sequences shown in SEQ ID NO.5 to SEQ ID NO.6 or a sequence with at least 90% homology to its sequence.

3. The anti-human CD8 antibody according to claim 1 or 2, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.1 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.5 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.1 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.6 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.2 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.5 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.2 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.6 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.3 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.5 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.3 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.6 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.4 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.5 or a sequence with at least 90% homology to it; or, The amino acid sequence of the heavy chain variable region of the anti-human CD8 antibody includes the sequence shown in SEQ ID NO.4 or a sequence with at least 90% homology to it, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.6 or a sequence with at least 90% homology to it.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-human CD8 antibody according to any one of claims 1-3.

5. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 4.

6. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecule of claim 4 or the recombinant vector of claim 5.

7. The method for preparing the anti-human CD8 antibody according to any one of claims 1-3, characterized in that, The preparation method includes: The nucleic acid molecule encoding the anti-human CD8 antibody according to any one of claims 1-3 is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is transformed into host cells, cultured, and the product is purified to obtain the anti-human CD8 antibody.

8. The method for preparing anti-human CD8 antibody according to claim 7, characterized in that, The expression vector includes any one of pCDNA3.4, pTT5, pCMV, pMH, pCEP4, pComb3X, pPICZα, pGAPZα, or pFastBac; Optionally, the host cell includes a prokaryotic cell or a eukaryotic cell; Optionally, the prokaryotic cells include any one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, lactic acid bacteria, lactococci, or Pseudomonas. Optionally, the eukaryotic cells include any one of HEK293 cells, CHO cells, Pichia pastoris, Saccharomyces cerevisiae, Sf9 or Sf21 cells.

9. An antibody conjugate, characterized in that, The antibody conjugate includes the anti-human CD8 antibody as described in any one of claims 1-3 and the conjugate substance therewith.

10. The antibody conjugate according to claim 9, characterized in that, The coupling substance includes at least one of cytotoxins, radioactive isotopes, luminescent substances, chromogenic substances, enzymes, or lipid nanoparticles.

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