CN117700550A - Humanized CD8 monoclonal antibody and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of antibodies, in particular to a humanized CD8 monoclonal antibody, a preparation method and application thereof. The amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID NO. 1-3, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 4-6. The human CD8 antibody can specifically recognize and bind human CD8 protein and CD8 ⁺ Cells with high affinity for CD8 ⁺ Flow cytometry detection of cells, in human CD8 protein and CD8 ⁺ Has wide application prospect in cell detection.

Description

Humanized CD8 monoclonal antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to a humanized CD8 monoclonal antibody, a preparation method and application thereof.

Background

CD8 molecule is a leukocyte differentiation antigen, a glycoprotein on the surface of partial T cells, is an auxiliary receptor for MHCI-class restricted T cells to recognize antigen, can bind to MHCI-class molecules to assist TCR in recognizing antigen presented by the MHCI-class molecules, and enhances interaction between T cells and Antigen Presenting Cells (APCs) or target cells, so that the antigen is called a co-receptor of TCR.

Under natural conditions, CD8 typically forms dimers, which may have two structures. The first is a heterodimer (CD 8. Alpha. Beta.) consisting of an alpha-chain and a beta-chain, both of which are encoded by the leu-2a gene and the leu-2b gene, respectively. The second is a homodimer (CD 8. Alpha.) consisting of two alpha chains, both encoded by the leu-2a gene. Wherein the alpha chain is about 34-37kD in size and the beta chain is 32kD. Both peptide chains are transmembrane proteins, with extracellular portions linked by disulfide bonds.

About 30% to 35% of T cells are capable of expressing CD8, and these cells recognize antigenic peptides consisting of 8 to 10 amino acid residues (limited by self mhc class i molecules) and differentiate into cytotoxic T Cells (CTLs) upon activation, killing cells or tumor cells that are parasitized by the pathogen.

Monoclonal antibody technology (monoclonal antibody technique) was invented in 1975 by the uk scientist Milstein and Kohler on the principle that: b lymphocytes are capable of producing antibodies, but are unable to divide indefinitely in vitro; while tumor cells can be passaged indefinitely in vitro, they are unable to produce antibodies. The hybridoma obtained by fusing the two cells has the characteristics of the two parent cells. The monoclonal antibody obtaining process comprises animal immunization, cell fusion, cell screening, cloning, characteristic identification and the like, can recognize specific single antigen epitope and has high specificity.

For human CD8 (hCD 8), there is still a lack of human CD8 antibodies capable of binding thereto with high efficiency and suitable for detection by flow cytometry and the like.

Disclosure of Invention

The invention aims to provide a novel humanized CD8 monoclonal antibody, and a preparation method and application thereof.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a human CD8 monoclonal antibody or antigen binding fragment thereof, wherein the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID NO:1-3, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO:4-6, respectively.

The antibody or antigen binding fragment thereof is capable of specifically recognizing and binding to human CD8 protein and cells expressing human CD8 protein (CD 8) ⁺ Cells) with a higher affinity.

Further, the heavy chain variable region of the antibody is: an amino acid sequence shown in SEQ ID NO. 7; or polypeptide with the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 7; preferably, the polypeptide has at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 7 and has the same function.

Further, the light chain variable region of the antibody is: an amino acid sequence shown in SEQ ID NO. 8; or polypeptide with the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 8; preferably, the polypeptide has at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 8 and has the same function.

In one embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

In the case of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 and light chain complementarity determining regions CDR1, CDR2, CDR3, the amino acid sequence of the heavy chain variable region is an amino acid sequence corresponding to an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence similarity to the amino acid sequence shown as SEQ ID NO:7, and the amino acid sequence of the light chain variable region is an antibody or antigen binding fragment thereof corresponding to an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 99.5% sequence similarity to the amino acid sequence shown as SEQ ID NO: 8.

The antibody or antigen-binding fragment thereof is any one selected from monoclonal antibodies, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragments, single chain antibodies, and the like.

Among them, monoclonal antibodies include animal-derived antibodies (e.g., murine antibodies), chimeric antibodies, humanized antibodies, and the like.

In a second aspect, the invention provides a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.

In a third aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.

Based on the amino acid sequence and codon regularity of the antibody or antigen-binding fragment thereof, one skilled in the art can obtain the nucleotide sequence of a nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the antibodies or antigen binding fragments thereof are within the scope of the invention.

In a fourth aspect, the invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria or host cells.

The expression cassette may be obtained by operably linking the nucleic acid molecule to regulatory elements such as promoters, terminators, and the like.

Such vectors include, but are not limited to, plasmid vectors, viral vectors, and the like.

The host cell includes a microbial cell or an animal cell. Wherein the microbial cells include, but are not limited to, E.coli, yeast, etc., and the animal cells include, but are not limited to, CHO cells, 293T cells, etc.

In a fifth aspect, the invention provides an antibody conjugate obtained by coupling the antibody or antigen-binding fragment thereof or the bispecific or multispecific antibody with a label or protein.

Preferably, the label may be selected from one or more of chemiluminescent dye labels, enzyme labels, biotin labels, fluorescent dye labels, colloidal gold labels, radioactive labels, and the like.

The antibodies or antigen-binding fragments thereof provided by the present invention can be prepared by methods conventional in the art, including chemical synthesis, host expression, and the like.

In a sixth aspect, the invention provides a method of preparing the antibody or antigen binding fragment thereof, the method comprising: culturing a host cell capable of expressing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

In a seventh aspect, the invention provides any one of the following uses of said antibody or antigen binding fragment thereof or said bispecific or multispecific antibody or said nucleic acid molecule or said biological material or said antibody conjugate:

(1) Use in the preparation of a product for detecting the presence or level of a human CD8 protein or a cell expressing a human CD8 protein in a sample;

(2) Use in detecting the presence or level of a CD8 protein of human origin in a sample;

(3) Use in detecting the presence or level of cells expressing a human CD8 protein in a sample.

In the application (1), the product may be a detection reagent or a kit.

In the applications (1) to (3), the sample may be a sample derived from a living human or animal (including blood, etc.), or may be a sample derived from a non-living human or animal such as cells or cell culture medium cultured in vitro.

The applications (2) and (3) are preferably assays for non-disease diagnosis and therapeutic purposes.

In the applications (1) to (3), the method for detection using the antibody or the antigen-binding fragment thereof provided by the present invention is preferably flow cytometry.

In an eighth aspect, the invention provides a biological product comprising said antibody or antigen binding fragment thereof, or comprising said bispecific or multispecific antibody, or comprising said antibody conjugate.

The biological product may be a detection reagent or a pharmaceutical composition.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the humanized CD8 antibody provided by the invention can specifically recognize and bind to humanized CD8 protein and CD8 ⁺ Cells with high affinity for CD8 ⁺ The detection of cell flow cytometry and the like has higher sensitivity and specificity, and can be used for detecting human CD8 protein and CD8 ⁺ Has wide application prospect in cell detection.

Drawings

FIG. 1 shows the results of the use of hCD8 monoclonal antibodies in flow cytometry in example 2 of the present invention, wherein A, B, C, D is the result of the detection of A, B, C, D four tubes, respectively.

Detailed Description

The invention provides a human CD8 (hCD 8) monoclonal antibody, which is obtained by animal immunization (taking recombinant hCD8 protein as antigen), cell fusion, screening and cloning of positive hybridoma cells, and ascites purification and flow cytometry verification. The humanized CD8 monoclonal antibody provided by the invention can effectively bind hCD8 and CD8 ⁺ The cells have stable performance and can be used for detecting CD8 by flow cytometry ⁺ And (3) cells.

In the invention, positive hybridoma cells are screened by recombinant hCD8 protein and then verified by flow cytometry, so that the monoclonal antibody can effectively identify natural hCD8 protein, the problem of later large-scale screening is avoided, the research and development period of the monoclonal antibody is shortened, and the operation is simple.

The reagents and formulations used in the following examples are shown in table 1.

TABLE 1

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

EXAMPLE 1 acquisition of human CD8 monoclonal antibodies

The hCD8 monoclonal antibody provided by the invention is obtained by the following steps:

1. immunization of animals

The purified recombinant hCD8 was diluted to 0.4mg/mL, and mixed with water-soluble rapid adjuvant (Boolon Quick antibody mouse w) in equal volume, and 6 female Balb/c mice 6 weeks old were inoculated in multiple points on leg muscle, with an antigen dose of 20 μg/mouse. After 21d, the injection was again performed, and the amount of immunity was the same as that of the first time. 3d before cell fusion, the recombinant hCD4 without adjuvant is injected into the abdominal cavity for immune impact, and then cell fusion is carried out.

2. Establishment of recombinant hCD8 indirect ELISA method

(1) Recombinant hCD8 was diluted to 1, 5, 10, 15, 20. Mu.g/mL, 100. Mu.L/well with carbonate buffer (pH 9.6), respectively, and incubated at 37℃for 1h at 4℃overnight.

(2) Spin-drying the coating liquid and washing the PBST for 3 times.

(3) 5% skim milk blocking solution, 250. Mu.L/well, was added and incubated at 37℃for 1h.

(4) The same PBST was washed 3 times.

(5) Negative and positive serum were diluted 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000, respectively, with 5% skim milk, 100 μl/well and incubated at 37 ℃ for 1h.

(6) The same PBST was washed 3 times.

(7) Goat anti-mouse IgG-HRP was diluted 1:10000 times with skim milk, 100. Mu.L/well, and incubated at 37℃for 0.5h.

(8) PBST was washed 4 times.

(9) Each well was added with a freshly prepared TMB developing solution at 100. Mu.L/well and developed for 10min at room temperature in the dark.

(10) Add 1M H ₂ SO ₄ The reaction was stopped at 50. Mu.L/well.

(11) Measuring OD of each well by enzyme labeling instrument ₄₅₀ 。

The optimal coating concentration of recombinant hCD8 protein, as well as the optimal dilutions of negative and positive serum, were determined by the above experiments.

3. Preparation of sp2/0 myeloma cells

Taking out sp2/0 cells in liquid nitrogen tank, thawing in 37deg.C water, centrifuging at 1000rpm for 5min, discarding supernatant, re-suspending with DMEM complete culture solution, transferring into cell bottle, thawing at 37deg.C, and concentrating with 5% CO ₂ Culturing in a saturated humidity incubator. The sp2/0 cells with uniform cell morphology, clear boundary and good growth state are taken in the day of fusion, the culture solution is discarded, the culture solution is washed for 2 times, then 20mL of the DMEM culture solution is used for resuspension of the cells, and then the cells are counted and placed at 4 ℃ for standby.

4. Preparation of feeder cells

Before fusion, selecting 2 non-immunized 10-week-old Kunming female mice, killing cervical dislocation, soaking in 75% alcohol for 5min, sterilizing, cutting off abdominal skin under aseptic condition, and fully exposing abdomen; lifting the peritoneum with forceps, injecting 8mL of HAT culture solution into the abdominal cavity with a syringe, gently pressing the abdominal cavity with an alcohol cotton ball, finally sucking the culture solution in the abdominal cavity with the syringe, counting cells, and re-suspending with HAT culture solution to regulate cell number to 2×10 ⁵ Each 100. Mu.L/well of 96-well plates were incubated at 37℃in 5% CO ₂ The mixture was placed in a saturated humidity incubator overnight, and whether contamination was observed.

5. Preparation of spleen cells

The titers of the serum of immunized mice were determined by indirect ELISA, and when the titers of hCD8 antibodies in the serum reached 16000, the recombinant hCD8 was boosted once. After 3d, the mice were collected from their eyeballs, and the blood was centrifuged at 37℃for 0.5h,4℃for 1h,4000rpm for 5min, and the separated serum was used as positive serum. Mice were sacrificed, and after 5min of sterilization by 75% alcohol, spleens were removed under aseptic conditions and ground on a 70 μm cell sieve. The obtained spleen cells were washed 2 times with DMEM medium (centrifugation at 1000rpm for 8 min), and then the cells were suspended in 20mL of DMEM medium and counted.

6. Cell fusion

1) The prepared sp2/0 myeloma cells and spleen cells are mixed in a ratio of 1:5-1:10, placed in a 50mL centrifuge tube, washed 3 times with DMEM culture solution, centrifuged at 1000rpm for 8min, and the supernatant is sucked dry after the last centrifugation, and the bottom of the centrifuge tube is tapped with fingers to loosen the cells.

2) The centrifuge tube was placed in a 37℃water bath, 1mL of 37℃preheated PEG1450 (Sigma) was pipetted into the centrifuge tube at constant speed, gently stirred while adding, completed within 1min, and allowed to stand for 1min.

3) 30mL of DMEM medium is slowly added, 1mL is added, 2 mL is added, 3min is added, 5mL is added, 4min is added, 7mL is added, and all 5min is added.

4) Centrifuge at 1000rpm for 8min and discard supernatant.

5) The HAT culture solution is used for suspending the fused cells, the action is gentle, the cells which are just fused are prevented from being blown away, 60-100mL of HAT culture solution is added, 96-well cell culture plates are paved, and 6-10 cells can be paved.

6) The cell culture plate was placed at 37℃in 5% CO ₂ Culturing in saturated humidity incubator, mixing for 3d, and changing with HAT culture solution for half amount, and changing into HT culture solution for 10 d.

7. Establishment of hCD8 hybridoma cell line

1) Screening of positive hybridoma cells

And 10d, HT culture solution is used instead, and detection is carried out when the hybridoma cells grow to 1/4 of the bottom of the hole. Coating an ELISA plate with recombinant hCD8 according to the optimal coating concentration, taking hybridoma cell culture supernatant after 2d of liquid exchange, reacting with the recombinant hCD8, taking sp2/0 cell culture supernatant as negative control, taking positive mouse serum as positive control, and selecting hybridoma cell holes reacting with the recombinant hCD8 as positive cell strains for next subcloning.

2) Subcloning cells

Preparing feeder cells according to the fourth step, and plating 96-well plates with 100 μl/well of feeder cells obtained by washing abdominal cavity of mice with HT nutrient solution, placing 96-well plates at 37deg.C and 5% CO ₂ Culturing overnight in a saturated humidity incubator. The cells in the hybridoma cell wells detected as positive were blown up and mixed uniformly, 10. Mu.L of cells were taken, the cells were counted after 10-fold dilution, 100 cells were calculated from the corresponding wells and added to 10mL of HT medium, and the cells were inoculated into 96-well cell culture plates plated with feeder cells at 100. Mu.L/well, i.e., about 1 cell per well. The cell plates were exposed to 5% CO at 37 ℃ ₂ Culturing in a saturated humidity incubator. After 4d, observing the clone number of each hole, when 7-10d cells grow to 1/4 hole bottom, performing ELISA detection, and taking 2 times of monoclonal holes positive in detection for subcloning. After 3 subcloning until the detection positive rate of all cloned cell holes is 100%, the hybridoma cell strain capable of stably secreting monoclonal antibodies can be determined.

3) Cryopreservation and resuscitation of hCD8 hybridoma cells

And (3) performing amplification culture on the final clone which is positive and is observed to be a single clone hole under a microscope, blowing off cells in the hole into a 6-hole plate, and transferring the cells into a 50mL cell bottle for culture after the cells in the 6-hole plate are full. After the cells grow to 80% -90%, subculturing, and simultaneously freezing hybridoma cell strains, wherein each strain is frozen for 3 tubes. Taking cells in good state in growth log phase, blowing off, mixing, centrifuging at 1000rpm for 8min, discarding supernatant, and re-suspending cells with frozen stock solution to reach cell number of 1×10 ⁶ Transferring the mixture into a freezing tube, placing 1 mL/tube into a program cooling box at-70 ℃ overnight, and transferring the mixture into liquid nitrogen for preservation the next day.

4) Ascites preparation

10-week-old Balb/c female mice were intraperitoneally injected with 0.5mL of liquid paraffin. After 7d, the abdominal cavity was inoculated with 1X 10 ⁶ Individual/hybridoma cells. Before inoculation, hybridoma cells were washed twice with DMEM medium at a concentration of 1X 10 ⁷ Each mL was taken as 0.1mL of the cell suspension, and injected into the abdominal cavity of the mouse. After 7d, canAscites is produced and collected by aspiration with a syringe. The collected ascites was centrifuged at 3000rpm for 5min, and the supernatant was collected.

8. hCD8 monoclonal antibody purification

1) The ascites is centrifuged at 12000rpm for 5min, the supernatant is collected and the precipitate is discarded.

2) The centrifuged ascites was filtered with a 0.22 μm filter.

3) The Protein A column was rinsed with 10 volumes of PBS of 1mL/min flow rate.

4) The ascites after filtration is added into a chromatographic column with the flow rate of 1mL/min.

5) The Protein A column was rinsed with 10 volumes of PBS of 1mL/min flow rate.

6) Eluting with glycine-hydrochloric acid (pH 3.0, 0.1M), collecting eluate with 1.5mL centrifuge tube at a flow rate of 1mL/min, collecting 0.5mL each tube, continuously collecting 12 tubes, adding 1M Tris-HCl (pH 8.8) 20 μl each tube after collection, and neutralizing, and performing PAGE-SDS electrophoresis.

7) The purified monoclonal antibody was dialyzed against PBS and dialyzed overnight at 4℃and quantified using the BCA quantification kit, and the concentration was adjusted to 1mg/mL.

Sequencing the hCD8 monoclonal antibody, and the sequencing result shows that the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the hCD8 monoclonal antibody are shown as SEQ ID NO. 1-3, the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 4-6, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

8) hCD8 monoclonal antibody titer detection

(a) Recombinant hCD8 was diluted to 1. Mu.g/mL with carbonate buffer (pH 9.6), 100. Mu.L/well, incubated at 37℃for 1h, overnight at 4 ℃.

(b) Spin-drying the coating liquid and washing the PBST for 3 times.

(c) 5% skim milk blocking solution, 250. Mu.L/well, was added and incubated at 37℃for 1h.

(d) The same PBST was washed 3 times.

(e) 1mg/mL of hCD8 monoclonal antibody and 1mg/mL of hCD14 monoclonal antibody (purchased from Biolegend) were diluted with 5% skim milk, 100. Mu.L/well, respectively, and incubated at 37℃for 1h at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000.

(f) The same PBST was washed 3 times.

(g) Sheep anti-mouse IgG-HRP was diluted 1:10000 times with skim milk, 100. Mu.L/well and incubated at 37℃for 0.5h.

(h) PBST was washed 4 times.

(i) Each well was added with a freshly prepared TMB developing solution at 100. Mu.L/well and developed for 10min at room temperature in the dark.

(j) Add 1M H ₂ SO ₄ The reaction was stopped at 50. Mu.L/well.

(k) Measuring OD of each well by enzyme labeling instrument ₄₅₀ 。

The results are shown in Table 2, with hCD8 monoclonal antibody titers of 1:128000.

TABLE 2

EXAMPLE 2 humanized CD8 monoclonal antibody for flow cytometry detection

The humanized CD8 monoclonal antibody prepared in the example 1 is used for flow cytometry detection, and the specific method is as follows:

1. 4 centrifuge tubes (1.5 mL) were used, A, B, C, D labeled, and the PBMC concentration was adjusted to 1X 10 ⁶ Each tube was taken at 0.5mL, 1mL of PBS was added, and 350g was centrifuged for 5min, and the supernatant was discarded.

2. The cells were resuspended in 200. Mu.L PBS by addition to the A-tube.

3. The B tube was resuspended in 100. Mu.L PBS, then 2. Mu.L anti-mouse IgG-PE flow antibody (BioLegend), thoroughly mixed, incubated in the dark for 15min,1mL PBS stopped the reaction, 350g centrifuged for 5min, the supernatant discarded, 200. Mu.L PBS was added to resuspend the cells, then 2. Mu.L 7AAD staining solution (BioLegend) was added, thoroughly mixed, incubated in the dark for 15min.

4. 100. Mu.L of PBS was added to the C tube to resuspend the cells, then 2. Mu.L of anti-hCD8-PE flow antibody (BioLegend) was added, thoroughly mixed, incubated for 15min in the dark, 1mL of PBS stopped the reaction, 350g centrifuged for 5min, the supernatant was discarded, 200. Mu.L of PBS was added to resuspend the cells, then 2. Mu.L of 7AAD staining solution (BioLegend) was added, thoroughly mixed, incubated for 15min in the dark.

5. The D-tube was resuspended in 100. Mu.L PBS, 1. Mu.L of hCD8 monoclonal antibody was added, mixed well and incubated for 30min at room temperature. Adding 1mLPBS, mixing, centrifuging at 350g for 5min, discarding supernatant, and washing for 2 times. 100. Mu.L PBS was used to resuspend cells, 2. Mu.L anti-mouse IgG-PE flow antibody was added, mixed well, incubated for 15min in the dark, 1mL PBS was used to stop the reaction, 350g was centrifuged for 5min, the supernatant was discarded, 200. Mu.L PBS was added to resuspend cells, 2. Mu.L 7AAD staining solution (BioLegend) was added, mixed well, and incubated for 15min in the dark.

6. 4-tube cells were mechanically examined.

The detection results are shown in fig. 1, and the results show that: the positive cell ratio of the D tube is 25.9 percent, which is approximately equal to the result of the C tube (26.3 percent), which shows that the hCD8 monoclonal antibody can effectively recognize CD8 ⁺ Cells, which can be used for flow cytometry detection.

Example 3 human CD8 monoclonal antibody affinity detection with CD8+ cells

1. Fresh anticoagulated human whole blood was taken and PBS was added at a 1:1 volume ratio to dilute the blood. A proper amount of Ficoll separating liquid (GE company) is added into a sterile centrifuge tube, and the diluted blood sample is paved above the liquid level of the separating liquid. 800g, and centrifuged at room temperature for 30min. After centrifugation, the plasma layer was aspirated, and the Peripheral Blood Mononuclear Cell (PBMC) layer (i.e., buffy coat) was carefully aspirated and transferred to a 15mL centrifuge tube.

2. Positive sorting of cd8+ cells in PBMC cells using human cd8+ cell sorting kit (meitian gentle) according to instructions provided by the manufacturer, first labeling cd8+ cells with biotinylated antibodies, and then binding the labeled cd8+ cells with Anti-Biotin superparamagnetic nanobeads; when the cell suspension is placed in a magnetic field, the cells bound by the magnetic beads are cd8+ cells.

The human CD8 monoclonal antibodies were biotin-labeled with a biotin-labeled antibody kit (Beijing auspicious industry technology Co., ltd.), and the biotin-labeled human CD8 monoclonal antibodies of 0nM,10nM,20nM,40nM,80nM,160nM,320nM,640nM,1280nM were incubated with 10million CD8+ cells in a binding buffer (DPBS+2 mM EDTA,0.5% BSA) at 4℃for 15min, respectively. After centrifugation at 1000rpm for 5min, the cells were counterstained with strepitavidin-PE (Western-A. Biotechnology Co., ltd.) by washing with binding buffer three times. Flow cytometry was performed after three PBS washes. As a result, it was found that as the concentration of CD8 monoclonal antibody increases, its binding force to target cells gradually increases, and eventually tends to saturate. When the binding rate of the CD8 monoclonal antibody and the cells reaches 50%, the concentration of the CD8 monoclonal antibody is the equilibrium dissociation constant Kd. The CD8 monoclonal antibody is detected to bind to CD8+ cells with a Kd of 134+ -15 nM.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The human CD8 monoclonal antibody or antigen binding fragment thereof is characterized in that the amino acid sequences of heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID NO. 1-3, and the amino acid sequences of light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO. 4-6, respectively.

2. The antibody of claim 1, wherein the heavy chain variable region of the antibody is: an amino acid sequence shown in SEQ ID NO. 7; or polypeptide with the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 7; preferably, a polypeptide having at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 7 and having the same function;

the light chain variable region of the antibody is: an amino acid sequence shown in SEQ ID NO. 8; or polypeptide with the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 8; preferably, the polypeptide has at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 8 and has the same function.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, fab ', F (ab') ₂ Fd, fv, dAb, a complementarity determining region fragment, a single chain antibody.

4. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.

5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3.

6. A biological material comprising the nucleic acid molecule of claim 5, wherein the biological material is recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineering bacterium.

7. An antibody conjugate, characterized in that it is obtained by coupling the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 or the bispecific or multispecific antibody according to claim 4 with a label or protein;

preferably, the label is selected from one or more of chemiluminescent dye label, enzyme label, biotin label, fluorescent dye label, colloidal gold label, and radioactive label.

8. A method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising: culturing a host cell capable of expressing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.

9. Use of the antibody or antigen binding fragment thereof of any one of claims 1-3 or the bispecific or multispecific antibody of claim 4 or the nucleic acid molecule of claim 5 or the biological material of claim 6 or the antibody conjugate of claim 7 for any one of the following:

(1) Use in the preparation of a product for detecting the presence or level of a human CD8 protein or a cell expressing a human CD8 protein in a sample;

(2) Use in detecting the presence or level of a CD8 protein of human origin in a sample;

(3) Use in detecting the presence or level of cells expressing a human CD8 protein in a sample;

the use is for non-disease diagnosis and treatment purposes.

10. A biologic comprising the antibody or antigen binding fragment thereof of any one of claims 1-3, or comprising the bispecific or multispecific antibody of claim 4, or comprising the antibody conjugate of claim 7;

the product is a detection reagent or a pharmaceutical composition.