Anti-cd28 nanobody and preparation method and application thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to an anti-CD28 nanobody and a preparation method and application thereof. The anti-CD28 nanobody provided by the application is prepared in the following manner: constructing a CD28 antigen to immunize a llama, constructing a phage library to the PBMC of the immunized llama, screening the phage library, selecting correct sequences to construct and express and purify, and finally obtaining the anti-CD28 nanobody through cell function detection screening. The nanobody provided by the application has specific recognition and binding capacity for CD28, has the advantages of high specificity, high sensitivity and the like, effectively reduces the development and production cost of the CD28 antibody, and shortens the expression time of the antibody.

Description

[0001] This application is a divisional application of patent application number "202311548011.9" with a filing date of "November 20, 2023" and an invention title of "Anti-CD28 Nanobody and Preparation Method Thereof and Application". TECHNICAL FIELD

[0002] The present application belongs to the technical field of molecular biology, and specifically relates to an anti-CD28 nanobody and a preparation method and application thereof. BACKGROUND

[0003] Leukocyte surface differentiation antigen 28 (CD28) molecules exist on the surface of most T cells and are considered a surface molecule unique to T cells. The reactivity of T cells to antigens is mainly mediated by the CD3-T cell antigen receptor (TCR) complex, but also relies on the cooperation of other molecules on the surface of T cells. CD28, as the first discovered costimulatory receptor, is the founder member of the costimulatory molecule subfamily characterized by extracellular variant immunoglobulin-like domains. CD28 mainly acts as a "second signal" (binding to B7-1 / B7-2 on the surface of target cells) to lower the threshold required for effective activation of T cells, strengthen the "first signal" (MHC-polypeptide recognition and binding of the T cell receptor (TCR)-CD3 complex to target cells) of activated T cells, and make T cells further develop and proliferate into cells with immune function. Based on the importance of CD28 costimulation for T cell activation, immunomodulation by activating or blocking the CD28 / B7-1 (CD80) / B7-2 (CD86) pathway is a promising approach: it can prevent inappropriate T cell activation rejection during transplantation, or treat T cell-mediated autoimmune diseases.

[0004] Studies have shown that CD28 mAb can also promote the proliferation of cytokine-induced killer cells (cytokine-induced killer cells, hereinafter referred to as CIK cells), promote the secretion of IFN-γ, TNF-α and IL-2 by CIK cells, and enhance the killing activity of CIK cells.

[0005] Cytokine-induced killer cells (cytokine-induced killer cells) are a group of immune cells with multiple cell types, with high proliferation capacity and strong cytotoxicity, and have great application value in the biological treatment of tumors. CIK cells are obtained by stimulating peripheral blood mononuclear cells with various cytokines such as IL-2, IFN-γ, etc. Current research on the clinical efficacy of CIK cells has shown that it has good efficacy on lung cancer, breast cancer, esophageal cancer, kidney cancer and other malignant tumors.

[0006] The nanobody is a heavy-chain single-domain antibody VHH (variable domain of heavy chain of heavy-chain antibody), the antibody only comprises a heavy chain variable region (VHH) and CH2 and CH3 regions, and the light chain is naturally lost compared to other antibodies. The nanobody has a crystal diameter of 2.5 nm and a length of 4 nm, is the smallest fragment that can bind to an antigen in nature, has an oval general structure, is very small in volume, has a molecular weight of 1 / 10 (15 kD) of a monoclonal antibody, is more active in chemical properties compared to a common antibody, has strong affinity, and can more effectively bind to an antigen gap. The secondary structure of the nanobody is two beta sheets forming a scaffold, and three hypervariable regions are gathered on one side to participate in antigen recognition. Compared to a common antibody, the nanobody has the following advantages: small molecular weight, easy penetration into dense tissues and even a blood-brain barrier; high affinity, strong specificity, good solubility and stability; weak immunogenicity in a human body, good biocompatibility; high expression in a prokaryotic or eukaryotic system, easy production; simple structure, easy engineering modification. Based on the characteristics of the nanobody, the nanobody has unique advantages in disease diagnosis and treatment. The small volume of the nanobody enables the nanobody to be firmly combined with a solid-phase carrier at a high density to capture trace antigens, and the nanobody-based immunodetection method can fully play a role in detecting and identifying target points and foreign pathogens or toxins that are difficult to detect in clinic. Therefore, the nanobody has great value in disease treatment and diagnosis, and has great development prospects in antibody-targeted diagnosis and treatment of tumors.

[0007] The phage display technology is to insert an exogenous gene into a proper position of a phage coat protein structure gene, so that the exogenous gene is expressed along with the expression of the coat protein under the condition that the reading frame is normal and the normal function of the coat protein is not affected. The technology is to display a polypeptide or a protein on the surface of a phage, so as to screen the required polypeptide or protein in vitro. Different exogenous genes are respectively inserted into phage vectors, and the exogenous protein is displayed on the surface of the phage along with the passage of the phage, so as to form a phage library. Then, the phage library is screened by a specific protein, some phage clones are obtained through several rounds of affinity screening (such as bio-panning), the phage clones are amplified, and more rounds of screening are performed, so that the specific protein with strong specificity can be further obtained. SUMMARY

[0008] The application provides an anti-CD28 nanobody and a preparation method and application thereof, the anti-CD28 nanobody is screened by combining phage display and a high-throughput mammalian cell expression system, and further application of the anti-CD28 nanobody in preparation of a tumor treatment drug and tumor detection is provided.

[0009] Based on this, the technical scheme adopted by the application is that:

[0010] An anti-CD28 nanobody, comprising a framework region and a complementarity determining region, the complementarity determining region comprising CDR1, CDR2, CDR3, wherein the complementarity determining region CDR1 is SEQ ID NO. 4, the complementarity determining region CDR2 is SEQ ID NO. 5, and the complementarity determining region CDR3 is SEQ ID NO. 6.

[0011] Preferably, the anti-CD28 nanobody is one of MY-86 and MY-36, wherein the amino acid sequence of MY-86 is shown as SEQ ID NO. 13, and the amino acid sequence of MY-36 is shown as SEQ ID NO. 14.

[0012] Preferably, the nucleotide sequence encoding the amino acid sequence of the anti-CD28 nanobody MY-86 is shown as SEQ ID NO. 20, and the nucleotide sequence encoding the amino acid sequence of the anti-CD28 nanobody MY-36 is shown as SEQ ID NO. 21.

[0013] The application also provides a molecular expression vector, which comprises one of SEQ ID NO. 20-SEQ ID NO. 21.

[0014] The application also provides a host cell containing the molecular expression vector, wherein the host cell is a eukaryotic cell.

[0015] The application also provides a use of the anti-CD28 nanobody in the preparation of a CD28 tumor detection reagent.

[0016] The application also provides a preparation method of the anti-CD28 nanobody, and the preparation process is as follows:

[0017] S1, according to the protein sequence and gene sequence information of CD28, an immune antigen is analyzed and designed, and a His-tag is connected at the C terminal of the immune antigen to obtain a modified antigen;

[0018] S2, the antigen obtained in step S1 is used to immunize a llama, and the titer of the serum separated from the immunized llama is detected, then the effector B cells are extracted from the peripheral blood to obtain llama PBMC cells;

[0019] S3, the llama PBMC cells obtained in step S2 are used as raw materials to extract total RNA, and a cDNA fragment is obtained by reverse transcription PCR technology, and the target gene fragment is amplified as a template, then the target gene fragment is cloned into a phagemid vector, and the phagemid vector is transformed into a competent cell to construct a phage library;

[0020] S4, the phage library constructed in step S3 is subjected to phage packaging, enrichment by panning, and further picking of single clones for primary screening, and the positive clones are sent for detection, and correct antibody sequences are selected by sequencing analysis for eukaryotic expression;

[0021] S5, the antibody expressed in step S4 is subjected to cell function detection, and an antibody with good specificity and sensitivity is screened, and the antibody is obtained.

[0022] Preferably, the eukaryotic expression vector in step S4 is a PcDNA3.4 vector.

[0023] The application also provides a use of the anti-CD28 nanobody in the preparation of a drug for preventing and treating tumors.

[0024] Compared with the prior art, the anti-CD28 nanobody provided by the application has the following advantages:

[0025] (1) The application combines the use of cell panning and flow sorting technology, and can sort out target cells combined with phage, and then elute and infect, thereby reducing non-specific binding phage;

[0026] (2) The application uses a high-throughput mammalian cell expression system to express the CD28 nanobody, effectively reduces the development and production cost of the CD28 antibody, shortens the antibody expression time, and at the same time increases the throughput and improves the efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is an ELISA detection serum titer result graph;

[0028] Figure 2 is an agarose gel electrophoresis detection result graph;

[0029] Figure 3 is a first round of PCR detection result graph;

[0030] Figure 4 is a second round of PCR detection result graph;

[0031] Figure 5 is an enzyme digestion reaction system detection result graph;

[0032] Figure 6 is a flow cytometry detection result graph;

[0033] Figure 7 、 Figure 8 is an ELISA antigen antibody detection result graph;

[0034] Figure 9 is a cell reporter gene detection result graph. DETAILED DESCRIPTION

[0035] The application will be further explained in connection with specific embodiments, but it should be noted that the following embodiments are only used to explain the application and cannot be used to limit the application, and all technical solutions same or similar to the application are within the protection scope of the application. The specific techniques or conditions not mentioned in the embodiments are operated according to the conventional technical methods and instrument instruction book contents; the reagents or instruments not mentioned by the manufacturer are all conventional products that can be obtained by market purchase.

[0036] The llama is first immunized three times with the CD28 antigen to obtain the PBMC cell of the llama; the RNA of the PBMC is extracted and reverse transcribed, the antibody gene fragment is obtained through the nest PCR method, the vector and the gene fragment are subjected to enzyme cutting and connection, and are transferred into the E. coli for amplification, the phage library is constructed, the antibodies specifically combined with the target antigen or the target cell are screened through the solid phase panning and cell sorting, and the eukaryotic expression vector is then constructed; then the high-throughput expression of the antibodies is induced through the mammalian cell high-throughput expression system, and the anti-CD28 nanobody with high sensitivity and specificity is obtained.

[0037] The primers involved in the application are as follows: DFL-01, DFL-02, DFL-03, DFL-04, DFL-05, DFL-06, DFL-07, DFL-08, DFL-09, DFL-10, DFL-11, DFL-12, DFL-13, DFL-14, DFL-15 and DFL-16.

[0038] The application relates to a preparation method of an anti-CD28 nanobody.

[0039] The anti-CD28 nanobody is 7 kinds, and is MY-92, MY-86, MY-36, MY-127, MY-78, MY-91 and MY-119.

[0040] The preparation method of the anti-CD28 nanobody comprises the following steps:

[0041] S1, according to the protein sequence and the gene sequence information of CD28, an antigen (which can be the whole sequence of CD28 protein) capable of effectively inducing the llama to produce a specific antibody against human CD28 is analyzed and designed, the His-tag is connected at the C end of the antigen, and the modified antigen is obtained;

[0042] S2, immunize the alpaca with the antigen obtained in step S1, detect the titer of the serum separated from the immunized alpaca, then extract the effector B cells from the peripheral blood to obtain alpaca PBMC cells: immunize the alpaca with the mixture of the modified antigen obtained in step S1 and Freund's adjuvant for 3 times, record the ear number of the blank alpaca and then start the immunization experiment, observe the alpaca for half an hour after each immunization to confirm that the alpaca is in good condition and has no symptoms of discomfort. Immunize every 2 weeks, collect blood to separate and store the upper serum for subsequent antibody titer detection.

[0043] The specific process is as follows: immunization: mix 200 μg of CD28 / hFc protein with 200 μL of complete Freund's adjuvant, emulsify, and then subcutaneously inject multiple points, on the 14th day, mix 200 μg of CD28 / hFc with incomplete Freund's adjuvant at a ratio of 1:1, emulsify, and then subcutaneously inject multiple points, on the 28th day, mix 200 μg of CD28 / hFc with incomplete Freund's adjuvant at a ratio of 1:1, emulsify, and then subcutaneously inject multiple points, collect blood to detect Anti-CD28 / His serum titer 1 week after each immunization; collect blood to separate alpaca PBMC 1 week after the third immunization.

[0044] Anti-CD28 / His serum titer is detected by ELISA, the enzyme-labeled plate is coated with CD28 / His protein at a concentration of 2 μg / mL, 100 μL of 2-fold gradient diluted serum is added to each well (the control is pre-immune alpaca serum), incubated at 25°C for 1 h, washed 5 times, 1:10,000 diluted horseradish peroxidase-labeled Goat anti-Alpaca IgG (H+L) secondary antibody is added to each well, incubated at 25°C for 1 h, washed 5 times, then 100 μL of TMB substrate is added, incubated at 25°C, the reaction is terminated by adding 50 μL of 1M HCL, and the OD 450nm is measured. The serum titer detected by ELISA is defined as being more than 2 times the blank control at OD450, and the specific results are shown in Figure 1 The serum titer detected by ELISA is the dilution value at which OD450 is 2 times the blank control, and the results show that the antiserum titer after the third immunization is between 12800-25600. It can be seen that the antigen can induce alpacas to produce high-titer antisera specific to CD28 protein.

[0045] Separation of lymphocytes: first add 15 mL cell separation solution (lymphocyte separation medium Ficoll-Raque PLUS of GE, USA) in a 50 mL centrifuge tube, then slowly add 15 mL blood. Be careful and slow when adding blood to prevent mixing of blood and separation solution. Then pre-cool the centrifuge to 25°C, centrifuge at 400g for 30 min, observe the blood separation in the centrifuge tube, save the upper serum in a new centrifuge tube, and store at -80°C. Carefully aspirate the intermediate cotton-like upper immune cells into a new 50 mL centrifuge tube with a pipette. Add 10 mL PBS buffer at room temperature to each tube, centrifuge at 400g for 20 min at 25°C. Remove the supernatant, add 5 mL PBS buffer at room temperature to each tube, mix gently, then centrifuge at 400g for 20 min at 25°C. The lymphocyte content is 107 / mL cell lysate, which is stored at -80°C.

[0046] S3, using the alpaca PBMC cells obtained in step S2 as raw material, extracting total RNA, obtaining cDNA fragments by reverse transcription PCR technology, amplifying VHH gene fragments with the template, then cloning the target gene fragments into phagemid vectors, transforming into competent cells, and constructing a phage library:

[0047] (1) RNA extraction process:

[0048] A. Take out the PBMC cells from the -80°C refrigerator, thaw on ice, then add 500 μL of PBMC to 500 μL of TrizoL, mix, add 200 μL of chloroform (chloroform), shake for 30 s, stand for 3 min, then centrifuge at 4°C, 12000g for 15 min;

[0049] B. After centrifugation, divide into three layers, take about 500 μL of the upper aqueous phase, add an equal volume of isopropanol, mix with a syringe, stand at room temperature for 10 min, then centrifuge at 4°C, 12000g for 10 min, remove the supernatant, and collect the precipitate;

[0050] C. Wash the precipitate with 1 mL of 75% ethanol, shake for 10 s, centrifuge at 4°C, 12000g for 10 min, remove the supernatant, and repeat the washing once;

[0051] D. Dry on a clean bench for 5-10 min to evaporate the residual alcohol;

[0052] E. Preheat RNA Free H2O to 55°C, add a certain amount of preheated RNA Free H2O to dissolve the RNA according to the amount of extracted RNA.

[0053] Use agarose gel to identify the RNA band, then store at -80°C, and the identification gel diagram is shown in Figure 2 .

[0054] After identification, the RNA was reverse transcribed into cDNA by using PrimeScript™ II Reverse Transcriptase Kit of TAKARA.

[0055] Then, two rounds of PCR amplification were performed. The first round of PCR amplification system was shown in Table 2, and the identification gel result was shown in Figure 3 .

[0056] Table 2 The first round of PCR amplification system

[0057]

[0058] The amplification program was: 98℃ 3min; 98℃ 15s, 56℃ 15s, 72℃ 30s, the whole process was repeated for 20 times; 72℃ 2min; 4℃ preservation.

[0059] Then, the second round of PCR was performed, and the amplification system was shown in Table 3, and the identification gel result was shown in Figure 4 .

[0060] Table 3 The second round of PCR amplification system

[0061]

[0062] The amplification program was: 98℃ 3min; 98℃ 10s, 65℃ 15s, 72℃ 30s, the whole process was repeated for 18 times; 72℃ 5min; 4℃ preservation.

[0063] The second round of PCR product was digested, and the digestion system was shown in Table 4, and the identification gel result was shown in Figure 5 .

[0064] Table 4 Digestion reaction system

[0065]

[0066] The correct fragment after identification was connected with the vector, and the connection reaction system was shown in Table 5.

[0067] Table 5 Connection reaction system

[0068]

[0069] Then, the connection product was introduced into cells by electroporation to construct a phage library:

[0070] 1) The connection product was introduced into SS320 competent cells.

[0071] 2) DNA and competent pre-mixed, and the preset electroporation condition was 2.5KV / 5.8ms.

[0072] 3) The competent and DNA mixture is added to the electroporation cuvette for electroporation, and then recovered at 37°C for 1 hour.

[0073] 4) Titer plate dilution and plating: the electroporated library product is centrifuged at 3000 rpm for 5 min, most of the supernatant is discarded, and the remaining 300 μL or so of supernatant is resuspended and plated.

[0074] 5) Culture: the plated plate is inverted and placed in a 37°C incubator for overnight culture for 15 h.

[0075] 6) The next day, the bacteria are scraped and stored, and single colonies are picked for sequencing. The effective library capacity is 1.50E+08.

[0076] After sequencing analysis and passing the library quality, the library is stored for subsequent screening.

[0077] S4, the phage library constructed in S3 is subjected to panning, the first two rounds are subjected to solid-phase panning, the third round is subjected to cell panning and uses flow cytometry sorting technology to sort out the cells combined with the phage, and then eluted and infected with E. coli for amplification, and through picking single colonies for primary screening, the correct llama VHH gene is cloned into the eukaryotic expression vector pCDNA3.4, and high-throughput expression is carried out through the mammalian expression system, and a large amount of expressed antibody is obtained.

[0078] The specific steps of screening are as follows:

[0079] 1, Phage packaging:

[0080] 1) The constructed phage library is taken out from -80°C and thawed on ice;

[0081] 2) Inoculate into 2YT medium (12 μg / mL tetracycline, 50 μg / mL ampicillin) and shake at 37°C, 220 rpm for 2 h to make the OD value reach 0.3-0.4, and then add the thawed helper phage M13KO7 (add according to 100 times the amount of inoculation, to ensure that the helper phage is in the superabundant logarithmic growth phase) to each bottle of bacterial solution, and incubate at 37°C, 80 rpm for 0.5 h;

[0082] 3) Add Kana+ (kanamycin) with a final concentration of 50 μg / mL, and then add 100 μL of 0.1 M IPTG for induction, and incubate at 30°C, 220 rpm for 14-16 h overnight;

[0083] 4) The next day, centrifuge at 12000 rpm for 10 min at 4°C, and use a 0.45 μm filter to filter the supernatant into a pre-cooled 50 mL centrifuge tube. Add 1 / 4 volume of PEG6000 to the supernatant, mix by inverting, and then place the centrifuge tube flat in an ice box on a shaker at 80 rpm for 1 h.

[0084] 5) Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, and invert the centrifuge tube to remove the liquid. Take 1 mL of pre-cooled PBS, and resuspend the phage by gently blowing with a Pasteur pipette. Centrifuge at 12000 rpm for 5 min, and transfer the supernatant to a 1.5 mL centrifuge tube for subsequent experiments.

[0085] 6) Infection: In a 96-well dilution plate, perform 10-fold gradient dilution of the phage suspension using logarithmic phase SS320 (dilute 12 gradients), and allow the plate to stand at 37°C for 30 min for infection. This facilitates calculation of the phage titer the next day.

[0086] 7) Counting: The next day, observe the plate for contamination. If there is none, calculate the phage titer, and calculate the number of phages in 1 mL of solution.

[0087] 2. Elutriation: There are two methods, one is 3 rounds of solid-phase elutriation, and the other is 2 rounds of solid-phase elutriation and 1 round of cell elutriation.

[0088] 1) Solid-phase elutriation coating: Dilute CD28-his antigen (diluted to 10 μg / mL) in 5 mL immunization tubes with PBS, and rotate overnight at 4°C for coating;

[0089] 2) Blocking of phage and blank immunization tubes: Rotate blank immunization tubes and the desired phage sample at room temperature for 1 h with 1% BSA for blocking;

[0090] 3) Blocking of CD28-his coated immunization tubes: Remove the coating solution, wash the immunization tubes with PBST, and then rotate for 1 h at room temperature with 1% BSA for blocking;

[0091] 4) Incubation: After blocking, take the supernatant from the phage tube, add it to the CD28-his immunization tube, rotate for 1 h at room temperature for incubation, remove the supernatant, wash the cells with PBST for 8 times, wash with PBS for 2 times, and then add 1 mL of trypsin containing 0.25% EDTA to elute the bound phage;

[0092] 5) Infection: In 96-well dilution plate, dilute the eluted phage with the log phase SS320 bacteria, total 8 dilutions, 10 μL phage in the first well, 10 times dilution, 37°C for 30 min. Meanwhile, mix 500 μL eluted solution with 5 mL log phase SS320 bacteria, 37°C for 30 min, 3000 rpm, 5 min, resuspend the bacteria with the rest 300 μL medium, spread on solid plate (resistance: ampicillin / tetracycline hydrochloride / 2% glucose), invert the plate, 37°C overnight;

[0093] 6) Output phage library storage: about 0.5 mL output phage library eluted solution, mix with 15% glycerol, store in freezer;

[0094] 7) Counting: the next day, observe the plate for contamination. If not, count the phage titer, calculate the number of phage in 1 mL solution, scrape the plate and prepare the second round of phage preparation;

[0095] 8) After 3 rounds of adsorption-elution-amplification, the phage binding to the target protein CD28-his can be enriched.

[0096] Cell panning:

[0097] 1) Digest the uninfected and human-CD28 infected CHOK1 cells, count respectively, the density of human-CD28 infected cells is 5.00E+06 / mL, CFSE staining with a final concentration of 1 μM, 37°C, 7 min, avoid light, resuspend in 1 mL PBS, 4°C for standby;

[0098] 2) The density of uninfected CHOK1 cells is 6.00E+06 / mL, wash with pre-cooled PBS for 3 times, remove the supernatant;

[0099] 3) Use 6.00E+06 uninfected CHOK1 cells and the required phage to block with 1% BSA, 4°C, 1 h rotation incubation;

[0100] 4) After blocking, 400 g, 5 min, remove the supernatant, add to the mixture of 2.50E+06 infected human-CD28 cells and 2.50E+06 uninfected cells, 4°C, 1 h rotation incubation, remove the supernatant, wash the cells with PBST for 3 times, centrifuge to remove the supernatant;

[0101] 5) Wash the cells with PBS for 3 times, centrifuge to remove the supernatant, resuspend the cells with 1 mL PBS containing 1 mM EDTA, and sort the positive cells with flow cytometry;

[0102] 6) After sorting, 800g, 5min, discard supernatant, add 500ul 20mM citric acid PH2.3 buffer, incubate at room temperature for 6min;

[0103] 7) Elute the bound phage, 800g, 5min, transfer the supernatant to 500ul 1M Tris-HCL PH8.0 buffer, infect the cells, 500ul to 5ml of SS320 bacteria liquid, mix well, and place in a 37°C constant temperature incubator for 30min;

[0104] 8) Centrifuge the 15ml centrifuge tube from the previous step at 3000rpm for 5min, enrich the bacteria, resuspend the bacteria in the remaining 300ul medium, and plate. Invert the plate in a 37°C constant temperature incubator overnight.

[0105] Monoclonal screening:

[0106] 1) Coating: the day before, add 100ul of 1ug / mL CD28-his to each well of a 96-well ELISA enzyme plate and coat at 4°C overnight;

[0107] 2) Wash the plate: the next day, remove the supernatant and wash the plate 3 times with 200ul PBST per well;

[0108] 3) Blocking: 1% BSA 200ul / well, room temperature for 1h.

[0109] 4) Sample preparation: the day before, add 600ul of 2YT medium (12ug / mL tetracycline, 50ug / mL ampicillin) to a 96-well deep well plate, select the third round of solid-phase panning monoclonal and the third round of cell panning monoclonal, incubate overnight at 220rpm, 37°C, centrifuge the overnight bacteria at 4000rpm for 5min, and add 100ul / well of the supernatant sample to the corresponding wells using a multi-channel pipette, incubate at room temperature for 1h.

[0110] 5) Add secondary antibody: dilute enough secondary antibody, add 100ul / well to the corresponding 96-well enzyme plate, incubate at room temperature for 1h, wash the plate 5 times with PBST, then add 100ul of TMB at room temperature, develop at room temperature for 5min;

[0111] 6) Stop the reaction: add 100ul of TMB color development termination solution (450nm, without sulfuric acid) to stop the reaction;

[0112] 7) Data acquisition and analysis: read the data on the enzyme marker at OD450, and process the data as shown in Tables 6-12, where A12-D12 wells are CD28 positive control antibodies, a total of 129 positive clones are screened, and 35 unique sequences are analyzed for construction of expression;

[0113] 8) The positive clones screened by the picking screen are sent for sequencing, and the correct sequences obtained by sequencing analysis are constructed and expressed in large quantities.

[0114] Table 6 Flow sorting results 1

[0115]

[0116] Table 7 Solid phase panning results 1

[0117]

[0118] Table 8 Flow sorting results 2

[0119]

[0120] Table 9 Flow sorting results 3

[0121]

[0122] Table 10 Flow sorting results 4

[0123]

[0124] Table 11 Flow sorting results 5

[0125]

[0126] Table 12 Flow sorting results 6

[0127]

[0128] S5, the antibody expressed in step S4 is subjected to cell function detection, and the binding of the antibody to the cells is detected by flow cytometry. The results show that 7 antibodies have good binding activity with the cells for subsequent experiments. The specific test steps are as follows:

[0129] FACS detection:

[0130] 1) Cell suspension preparation: adjust the cell number to 4.00E+06 / mL in pre-cooled MACS buffer (PBS, 2% FBS, 2mM EDTA), and add 50μL / well of cell suspension to a 96-well plate.

[0131] 2) Primary antibody incubation: dilute the antibody to be tested by 4-fold gradient in the first hole 200nM with MACS buffer. After dilution, add 50μL of antibody dilution buffer to the cell suspension of 1), mix well, and incubate at 4℃ for 60min;

[0132] 3) Wash cells: Add 100 μL of MACS buffer to the 96-well, 400g centrifuge for 5 min, discard the supernatant, add 200 μL of MACS buffer to the well to wash the cells, 400g centrifuge for 5 min, discard the supernatant, wash twice;

[0133] 4) Secondary antibody incubation: Resuspend the cells with 100 μL of fluorescent secondary antibody (Goat anti-Human Fc, Alexa Fluor 647, 1:1000 dilution), incubate at 4°C for 30 min;

[0134] 5) Wash cells: Add 100 μL of MACS buffer to the 96-well, 400g centrifuge for 5 min, discard the supernatant, add 200 μL of MACS buffer to the well to wash the cells, 400g centrifuge for 5 min, discard the supernatant, wash twice;

[0135] 6) Detection and data analysis: Resuspend the cells with 200 μL of MACS buffer and detect them on a flow cytometer, and process the data as shown in Figure 6 Seven antibodies showed good binding to the cells, and the seven antibodies were then separately subjected to ELISA detection with cyno-CD28 antigen and mouse-CD28, and the results are shown in Figure 7 、 Figure 8 The results showed that the seven antibodies all showed good cross-reactivity with cyno-CD28, with an EC50 of about 0.3 nM, and two of them showed cross-reactivity with mouse-CD28. As shown in Figure 8 MY-36 and MY-86 can bind to cyno-CD28 and show weak binding to mouse-CD28.

[0136] 2. Cell reporter assay:

[0137] 1) Prepare 1-fold concentration of CD28 monoclonal antibody CD28 Monoclonal Antibody (CD28.2) and CD3 monoclonal antibody Anti-Human CD3 epsilon OKT-3 (muromonab). The concentration of OKT-3 is 0.01 μg / mL and the concentration of anti-CD28.2 is 100 nM;

[0138] 2) Antibody coating: Coat the 96-well plate with 100 μL / well of antibody, and coat at 4°C overnight;

[0139] 3) Cell preparation: Add 100 μL / well of prepared Jurkat-luc2p-IL2 cells to the coated 96-well cell culture plate, and incubate in a 37°C, 5% CO2 incubator for 6 hours;

[0140] 4) Add Bio-Glo luciferase for detection. Incubate at room temperature on a shaker at 400 rpm for 10 minutes, then detect the fluorescence signal using a microplate reader. Data processing is as follows: Figure 9 As shown. By Figure 9 It was found that when CD3 and CD28 antibodies were present simultaneously, CD28 nanobodies MY-92, MY-127, MY-91, and MY-119 exhibited activating activity in a concentration-dependent manner. Compared with the reference antibody, MY-92 and MY-127 showed significant activating activity.

[0141] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An anti-CD28 nanobody, characterized in that, The anti-CD28 nanobody comprises a framework region and a complementarity determining region, the complementarity determining region comprising CDR1, CDR2, CDR3, wherein the complementarity determining region CDR1 amino acid sequence is shown as SEQ ID NO. 4, the complementarity determining region CDR2 amino acid sequence is shown as SEQ ID NO. 5, and the complementarity determining region CDR3 amino acid sequence is shown as SEQ ID NO.

6.

2. Anti-CD28 Nanobody according to claim 1, characterized in that, The anti-CD28 nanobody has an amino acid sequence selected from any one of SEQ ID NO. 13, SEQ ID NO.

14.

3. Use of the anti-CD28 nanobody of claim 1 in the preparation of a CD28 tumor marker detection reagent.

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