Anti-CD28 nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to an anti-CD28 nano antibody as well as a preparation method and application thereof. The preparation process of the anti-CD28 nano antibody provided by the invention comprises the following steps: constructing a CD28 antigen to immunize alpaca, performing phage library construction on the immunized alpaca PBMC, screening the phage library, and selecting a correct sequence to construct, express and purify; and finally obtaining the anti-CD28 nano antibody through cell function detection and screening. The nano antibody provided by the invention has specific recognition and binding capacity to CD28, and has the advantages of strong specificity, high sensitivity and the like; meanwhile, the development and production cost of the CD28 antibody is effectively reduced, and the antibody expression time is shortened.

Description

[0001] This application is a divisional application with patent application number "202311548011.9", application date "November 20, 2023", and invention name "An anti-CD28 nanoantibody and its preparation method and application". Technical Field

[0002] The invention belongs to the technical field of molecular biology, and specifically relates to an anti-CD28 nano antibody and a preparation method and application thereof. Background Art

[0003] The leukocyte surface differentiation antigen 28 (CD28) molecule exists on the surface of most T cells and is considered to be a surface molecule unique to T cells. The responsiveness of T cells to antigens is mainly mediated by the CD3-T cell antigen receptor (TCR) complex, but it also depends on the cooperation of other molecules on the surface of T cells. As the first co-stimulatory receptor discovered, CD28 is the founding member of the co-stimulatory molecule subfamily characterized by an extracellular variant immunoglobulin-like domain. 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" for activating T cells (recognition and binding of the T cell receptor (TCR)-CD3 complex to the MHC-peptide of the target cell), and enable T cells to further develop and proliferate into cells with immune functions. Based on the importance of CD28 co-stimulation for T cell activation, immune regulation by activating or blocking the CD28 / B7-1 (CD80) / B7-2 (CD86) pathway is a promising approach: it can prevent inappropriate T cell activation and 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 (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 (CIK cells) are a group of immune cells with multiple cell types, which have the advantages of high proliferation ability and strong cytotoxicity, and have great application value in the biological treatment of tumors. CIK cells are obtained from mononuclear cells derived from peripheral blood and stimulated by multiple cytokines such as IL-2 and IFN-γ. The current research on the clinical efficacy of CIK cells has initially shown that it has a good effect on various malignant tumors such as lung cancer, breast cancer, esophageal cancer, and kidney cancer.

[0006] Nanobodies are heavy chain single-domain antibodies VHH (variable domain of heavy chain of heavy-chainantibody). This type of antibody contains only one heavy chain variable region (VHH) and CH2, CH3 regions. Compared with other antibodies, the light chain is naturally missing. The diameter of the nanoantibody crystal is 2.5nm and the length is 4nm. It is the smallest fragment that can bind to the antigen in nature. The general structure of nanoantibodies is elliptical, the volume is very small, and the molecular weight is 1 / 10 (15kD) of monoclonal antibodies. Compared with ordinary antibodies, the chemical properties are more active, with strong affinity, and can bind to the antigen gap more effectively. The secondary structure of nanoantibodies is that two β sheets form a scaffold, and three highly variable regions gather on one side to participate in antigen recognition. Compared with conventional antibodies, the advantages of nanoantibodies are: small molecular weight, easy to penetrate into dense tissues and even blood-brain barriers; high affinity, strong specificity, good solubility and stability; weak immunogenicity in the human body and good biocompatibility; high expression in prokaryotic or eukaryotic systems, easy to produce; nanoantibodies have a simple structure and are easy to engineer. Based on the characteristics of nano-antibodies, they have unique advantages in disease diagnosis and treatment. The small size of nano-antibodies enables them to firmly bind to solid phase carriers at high density to capture trace antigens, which can give full play to the role of immunoassays based on nano-antibodies to detect and identify targets that are difficult to detect clinically, as well as foreign pathogens or toxins. Therefore, nano-antibodies have great value in the treatment and diagnosis of diseases, and also have great development prospects in antibody-targeted diagnosis and treatment of tumors.

[0007] Phage display technology is to insert a foreign gene into the appropriate position of the phage coat protein structural gene. When the reading frame is normal and does not affect the normal function of the coat protein, the foreign gene will be expressed along with the coat protein. It is a technology that displays polypeptides or proteins on the surface of the phage, thereby screening the desired polypeptides or proteins in vitro. This technology inserts different foreign genes into phage vectors respectively. As the phage is propagated, the foreign protein will be displayed on the phage surface to form a phage library. The phage library is then screened with a specific protein, and some phage clones are obtained after several rounds of affinity screening (such as bio-panning). These phage clones are then amplified and screened more rounds to further obtain target proteins with strong specificity. Summary of the invention

[0008] The present invention provides an anti-CD28 nano antibody and a preparation method and application thereof. The anti-CD28 nano antibody is screened and obtained by combining phage display with a high-throughput mammalian cell expression system, and further provides its application in the preparation of tumor therapeutic drugs and tumor detection.

[0009] Based on this, the technical solution adopted by the present invention is: An anti-CD28 nanobody, comprising a framework region and a complementary determining region, wherein the complementary determining region comprises CDR1, CDR2, and CDR3, wherein the complementary determining region CDR1 is SEQ ID NO.1, the complementary determining region CDR2 is SEQ ID NO.7, and the complementary determining region CDR3 is SEQ ID NO.8.

[0010] Preferably, the anti-CD28 nanobody is MY-127, and the amino acid sequence is SEQ ID NO.15.

[0011] Preferably, the nucleotide sequence encoding the amino acid sequence of the anti-CD28 nanobody MY-127 is shown as SEQ ID NO.22.

[0012] The present invention also provides a molecular expression vector, which contains the nucleotide sequence of SEQ ID NO.22.

[0013] The present invention also provides a host cell containing the molecular expression vector, wherein the host cell is a eukaryotic cell.

[0014] The present invention also provides an application of the anti-CD28 nano antibody in preparing a CD28 tumor detection reagent.

[0015] The preparation method of the anti-CD28 nanobody, the preparation process is as follows: S1. Analyze and design immune antigens based on the protein sequence and gene sequence information of CD28, and connect His-tag to its C-terminus to obtain modified antigens; S2, immunizing alpacas with the antigen obtained in step S1, performing titer detection on serum separated from the immunized alpacas, and then extracting effector B cells from peripheral blood to obtain alpaca PBMC cells; S3, using the alpaca PBMC cells obtained in step S2 as raw materials, extracting total RNA, obtaining cDNA fragments by reverse transcription PCR technology, amplifying VHH gene fragments using the cDNA fragments as templates, and then cloning the target gene fragments into phagemid vectors, and transforming them into competent cells to construct a phage library; S4, packaging phages using the phage library constructed in step S3, enriching by panning, and further selecting single clones for primary screening, screening positive clones for testing, and selecting the correct antibody sequence for eukaryotic expression by sequencing analysis; S5. Perform cell function detection using the antibodies expressed in step S4 to screen antibodies with good specificity and sensitivity.

[0016] Preferably, the eukaryotic expression vector in step S4 is a PcDNA3.4 vector.

[0017] The present invention also provides an application of the anti-CD28 nano antibody in preparing drugs for preventing and treating tumors.

[0018] Compared with the prior art, the anti-CD28 nanobody provided by the present invention has the following advantages: (1) The present invention combines the use of cell panning flow sorting technology to sort out the target cells bound to the phage and then wash out the infection, thereby reducing the non-specific binding of phage; (2) The present invention utilizes a high-throughput mammalian cell expression system to express CD28 nanoantibodies, which effectively reduces the development and production costs of CD28 antibodies, shortens the antibody expression time, and increases throughput and improves efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result diagram of serum titer detected by ELISA; Figure 2 It is the result of agarose gel electrophoresis detection; Figure 3 This is the result of the first round of PCR testing; Figure 4 This is the result of the second round of PCR testing; Figure 5 It is the result diagram of enzyme digestion reaction system detection; Figure 6 This is a graph of flow cytometry test results; Figure 7 , Figure 8 This is the result diagram of ELISA antigen-antibody test; Fig. 9 This is a graph showing the results of cell reporter gene detection. DETAILED DESCRIPTION

[0020] The present invention is further explained below in conjunction with specific examples, but it should be noted that the following examples are only used to explain the present invention, and cannot be used to limit the present invention, and all technical solutions that are the same or similar to the present invention are within the scope of protection of the present invention. If no specific technology or conditions are specified in this embodiment, the operation is carried out according to the conventional technical methods and instrument instructions in the art; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0021] The present invention first constructs a CD28 antigen and immunizes alpacas three times to obtain alpaca PBMC cells; extracts RNA from the PBMC and performs reverse transcription, obtains antibody gene fragments by a nested PCR method, performs enzyme cutting and connection on a vector and the gene fragment, and transfers them into Escherichia coli for amplification to construct a phage library; screens antibodies that specifically bind to target antigens or target cells by solid phase panning and cell sorting, and then constructs a eukaryotic expression vector; and then induces high-throughput expression of antibodies by a mammalian cell high-throughput expression system to obtain an anti-CD28 nano antibody with high sensitivity and specificity. The primers involved in the present invention 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.

[0022] Example A method for preparing an anti-CD28 nanobody There are 7 types of anti-CD28 nano antibodies, namely MY-92, MY-86, MY-36, MY-127, MY-78, MY-91, and MY-119.

[0023] The method for preparing the anti-CD28 nanobody comprises the following steps: S1. Based on the protein sequence and gene sequence information of CD28, analyze and design an antigen that can effectively induce alpacas to produce specific antibodies against human CD28 (which can be the full sequence of CD28 protein), and connect a His-tag to its C-terminus to obtain a modified antigen; S2. Immunize the alpaca with the antigen obtained in step S1, perform titer detection on the serum separated from the immunized alpaca, and then extract effector B cells from the peripheral blood to obtain alpaca PBMC cells: immunize the alpaca three times with the mixture of the modified antigen obtained in step S1 and Freund's adjuvant, record the ear number of the blank alpaca and start the immunization experiment, observe for half an hour after each immunization to confirm that the alpaca is in good condition and has no discomfort symptoms. Immunize once every 2 weeks, collect blood, separate and save the upper serum for subsequent antibody titer detection.

[0024] The specific process is as follows: Immunization: Mix 200μg of CD28 / hFc protein with 200μL complete Freund's adjuvant, emulsify and inject subcutaneously at multiple points. On the 14th day, mix 200μg of CD28 / hFc with incomplete Freund's adjuvant in a 1:1 ratio, emulsify and inject subcutaneously at multiple points. On the 28th day, mix 200μg of CD28 / hFc with incomplete Freund's adjuvant in a 1:1 ratio, emulsify and inject subcutaneously at multiple points. One week after each immunization, blood was collected to detect the Anti-CD28 / His serum titer; one week after the third immunization, blood was collected to isolate alpaca PBMC.

[0025] Anti-CD28 / His serum titer was detected by ELISA. The ELISA plate was coated with CD28 / His protein at a concentration of 2μg / mL. 100μL of serum diluted 2 times in series was added to each well (the control was pre-immune alpaca serum). The plate was incubated at 25°C for 1 hour, washed 5 times, and a 1:10000 dilution of horseradish peroxidase-labeled Goat anti-Alpaca IgG (H+L) secondary antibody was added to each well. The plate was incubated at 25°C for 1 hour. After washing 5 times, 100μL of TMB substrate was added and the plate was incubated at 25°C. The reaction was terminated with 50μL 1M HCL and the OD 450nm was measured. The ELISA test for serum titer is defined as OD450 being more than 2 times that of the blank control. The specific results are as follows: Figure 1 As shown, the serum titer detected by ELISA is a dilution value at OD450 that is twice that of the blank control, and the results show that the antiserum titer after 3 immunizations is between 12800 and 25600. This shows that the antigen can induce alpacas to produce high-titer antiserum specific to CD28 protein.

[0026] Separation of lymphocytes: First add 15mL of cell separation solution (Ficoll-Raque PLUS, lymphocyte separation solution from GE, USA) to a 50mL centrifuge tube, and then slowly add 15mL of blood. Be careful and slow when adding blood to prevent mixing of blood and separation solution. After that, precool the centrifuge to 25℃, centrifuge at 400g for 30min, observe the blood separation in the centrifuge tube, store the upper serum in a new centrifuge tube at -80℃, and carefully pipette out the cotton-like upper immune cells in the middle into a new 50mL centrifuge tube. Add 10mL of PBS buffer at room temperature to each tube, centrifuge at 25℃, 400g for 20min. Remove the supernatant, add 5mL of PBS buffer at room temperature to each tube, mix gently, count the cells, and then centrifuge at 25℃, 400g for 20 minutes. The separated lymphocyte content is 107 / mL cell lysate, which is stored at -80℃.

[0027] S3. Using the alpaca PBMC cells obtained in step S2 as raw materials, extract total RNA, obtain cDNA fragments by reverse transcription PCR technology, and use this as a template to amplify the VHH gene fragment, then clone the target gene fragment into a phagemid vector, and transform it into competent cells to construct a phage library: (1) RNA extraction process: A. Take out PBMC cells from -80℃ freezer, thaw on ice, take 500μL PBMC, add 500μL TrizoL, mix well, add 200μL chloroform, shake for 30s, let stand for 3min, then centrifuge at 4℃, 12000g for 15min; B. After centrifugation, separate the three layers. Take the upper aqueous phase, about 500 μL, add an equal volume of isopropanol, mix with a pipette tip, let stand at room temperature for 10 minutes, then centrifuge at 4°C, 12000g for 10 minutes, remove the supernatant, and collect the precipitate; C. Wash the precipitate with 1 mL of 75% ethanol, shake for 10 seconds, centrifuge at 12,000 g for 10 minutes at 4°C, remove the supernatant, and repeat the washing process; D. Dry on the clean bench for 5-10 minutes to evaporate the remaining alcohol; E. Preheat RNA Free H2O at 55℃ and add a certain amount of preheated RNA Free H2O to dissolve the RNA according to the amount of extracted RNA.

[0028] The RNA bands were identified by agarose gel and then stored at -80°C. Figure 2 shown.

[0029] After identification, RNA was reverse transcribed into cDNA using TAKARA's PrimeScript™ II Reverse Transcriptase Kit.

[0030] Then two rounds of PCR amplification were performed. The first round of PCR amplification system is shown in Table 2 below. The identification gel results are shown in Figure 2 below. Figure 3 shown.

[0031] Table 2 The first round of PCR amplification system

[0032] The amplification program was as follows: 98°C for 3 min; 98°C for 15 s, 56°C for 15 s, 72°C for 30 s, the whole process was cycled 20 times; 72°C for 2 min; and stored at 4°C.

[0033] Then the second round of PCR was performed, and the amplification system was shown in Table 3. The identification gel results were shown in Figure 3. Figure 4 shown.

[0034] Table 3 Second round PCR amplification system

[0035] The amplification program was as follows: 98°C for 3 min; 98°C for 10 s, 65°C for 15 s, 72°C for 30 s, with the whole process cycled 18 times; 72°C for 5 min; and stored at 4°C.

[0036] The second round PCR products were digested with enzymes. The digestion system is shown in Table 4. The gel results are shown in Figure 4. Figure 5 shown.

[0037] Table 4 Enzyme digestion reaction system

[0038] The identified correct fragment was connected to the vector, and the connection reaction system is shown in Table 5.

[0039] Table 5 Ligation reaction system

[0040] The ligation product is then introduced into cells by electroporation to construct a phage library: 1) Transform the ligation product into SS320 competent cells.

[0041] 2) DNA and competent cells were premixed, and the preset electroporation conditions were 2.5KV / 5.8ms.

[0042] 3) Add the competent cell and DNA mixture into the electroporation cuvette for electroporation, and then recover at 37°C for 1 hour.

[0043] 4) Titer plate dilution and plating: Centrifuge the electroporated library product at 3000 rpm for 5 min, discard most of the supernatant, and resuspend the remaining 300 μL of the supernatant in the bacterial suspension for plating.

[0044] 5) Incubation: Place the coated plate upside down in a 37°C incubator and incubate overnight for 15 hours.

[0045] 6) The next day, the bacteria were scraped and preserved, and single clones were picked for sequencing. The effective library capacity was 1.50E+08.

[0046] After the quality of the library construction is qualified through sequencing analysis, it will be stored in the library for subsequent screening.

[0047] S4. The phage library constructed in S3 was used for panning. The first two rounds adopted solid phase panning method, and the third round adopted cell panning method and flow cytometry sorting technology to sort out the cells bound to the phage, and then eluted and infected Escherichia coli for amplification. Single clones were picked for initial screening, and the correct alpaca VHH gene was selected and cloned into the eukaryotic expression vector pCDNA3.4, and high-throughput expression was performed through the mammalian expression system to obtain large-scale expressed antibodies.

[0048] The specific steps of screening are as follows: 1. Phage packaging: 1) Take out the constructed phage library from -80℃ and thaw it on ice; 2) Inoculate into 2YT medium (12µg / mL tetracycline, 50µg / mL ampicillin), shake at 37°C, 220rpm for 2h, until the OD value reaches 0.3-0.4, add thawed helper phage M13KO7 to each bottle of bacterial solution (the amount added is 100 times the amount of inoculation, to ensure that the helper phage is a bacteria in the super logarithmic growth phase), and culture at 37°C, 80rpm for 0.5h; 3) Add Kana+ (kanaher penicillin) with a final concentration of 50 μg / mL, incubate at 37°C, 220 rpm for 0.5 h, then add 100 μL of 0.1 M IPTG for induction, incubate at 30°C, 220 rpm for 14-16 h overnight; 4) On the next day, centrifuge at 12000 rpm for 10 min at 4°C. Filter the supernatant with a 0.45 μm filter membrane into a pre-cooled 50 mL centrifuge tube. Add 1 / 4 volume of PEG6000 to the supernatant. Mix by inversion and place the centrifuge tube flat. Place the tube in an ice box and place the ice box on an oscillator at 80 rpm for 1 h.

[0049] 5) Centrifuge at 4°C, 12,000 rpm for 10 min, discard the supernatant and invert the centrifuge tube to drain the liquid; aspirate 1 mL of pre-cooled PBS and gently pipette to resuspend the phage; centrifuge at 12,000 rpm for 5 min, transfer the supernatant to a 1.5 mL centrifuge tube for subsequent experiments; 6) Infection: In a 96-well dilution plate, use logarithmic phase SS320 to make a 10-fold gradient dilution of the phage suspension (12 dilution gradients), and incubate the plate at 37°C for 30 minutes to facilitate calculation of the phage titer the next day; 7) Counting: The next day, take out the plate and check for contamination. If not, count the phage titer and calculate the number of phages in 1 mL of solution.

[0050] 2. Panning: There are two methods, one is three rounds of solid phase panning, and the other is the first two rounds of solid phase and the third round of cell panning strategy.

[0051] 1) Solid phase panning coating: dilute CD28-his antigen (diluted to 10 μg / mL) with PBS into a 5 mL immunotube and rotate overnight at 4°C for coating; 2) Block the phage and blank immunotubes: Block the blank immunotubes and the desired phage with 1% BSA at room temperature for 1 hour by rotating. 3) Block the immunotubes coated with CD28-his: remove the coating solution, wash the immunotubes with PBST, and block with 1% BSA at room temperature for 1 hour with rotation: 4) Incubation: After blocking, take the supernatant from the blocked phage tube and add it to the CD28-his immunotube. Incubate with rotation at room temperature for 1 hour. Remove the supernatant, add PBST to wash the cells 8 times, PBS to wash twice, and then add 1 mL of trypsin containing 0.25% EDTA to elute the bound phage. 5) Infection: In a 96-well dilution plate, dilute the eluted phage with shaking logarithmic phase SS320 bacteria, with a total of 8 dilution gradients. Add 10 μL of phage to the first well, dilute in 10-fold gradients, and stand at 37°C for 30 minutes. At the same time, take 500 μL of the eluate and mix with 5 mL of logarithmic phase SS320 cells. After standing and culturing at 37°C for 30 minutes, centrifuge at 3000 rpm for 5 minutes at room temperature. Resuspend the bacteria in the remaining 300 μL of culture medium and spread on a solid culture plate (resistance: ampicillin / tetracycline hydrochloride / final concentration of 2% glucose). Invert the plate and culture overnight in a 37°C incubator. 6) Storage of output phage library: Add glycerol to a final concentration of about 15% to the remaining 0.5 mL of output phage library eluate, mix well and freeze; 7) Counting: The next day, take out the plate and check for contamination. If not, count the phage titer, calculate the number of phages in 1 mL of solution, scrape the plate and prepare the second round of phage selection; 8) After three rounds of adsorption-elution-amplification, phages binding to the target protein CD28-his can be enriched.

[0052] Cell panning: 1) Digest uninfected and human-CD28 infected CHOK1 cells, count them separately, the cell density of human-CD28 infected cells is 5.00E+06 / mL, stain with CFSE at a final concentration of 1μM, stain at 37℃ for 7min in the dark, resuspend in 1mL PBS at 4℃ for later use; 2) Uninfected CHOK1 cells were washed 3 times with pre-cooled PBS at a density of 6.00E+06 / mL and the supernatant was discarded; 3) Block the uninfected CHOK1 cells 6.00E+06 and the required loading Phage with 1% BSA and incubate with rotation at 4°C for 1 hour; 4) After blocking, take the supernatant at 400g for 5 min, add it to a mixture of 2.50E+06 infected human-CD28 cells and 2.50E+06 uninfected cells, incubate with rotation at 4°C for 1 h, remove the supernatant, wash the cells 3 times with PBST, and remove the supernatant by centrifugation; 5) Wash the cells three times with PBS, remove the supernatant by centrifugation, resuspend the cells in 1 mL of PBS containing 1 mM EDTA, and sort the positive cells by flow cytometer; 6) Centrifuge the sorted cells at 800g for 5 minutes, discard the supernatant, add 500 μL of 20 mM citric acid pH 2.3 buffer, and incubate at room temperature for 6 minutes; 7) Elute the bound phages, centrifuge at 800g for 5 min, transfer the supernatant to 500 μL of 1M Tris-HCL PH8.0 buffer, infect the cells, add 500 μL to 5 mL of shaken SS320 bacterial solution, mix well, and place in a 37°C constant temperature incubator for 30 min; 8) Centrifuge the 15 mL centrifuge tube at 3000 rpm for 5 min to enrich the cells, resuspend the cells in the remaining 300 μL culture medium, and apply to the plate. Place the plate upside down in a 37°C constant temperature incubator overnight.

[0053] Monoclonal screening: 1) Coating: The day before, add 100 μL of 1 μg / mL CD28-his to each well of a 96-well ELISA plate and coat overnight at 4°C; 2) Washing: On the second day, remove the supernatant and wash the plate three times with 200 μL of PBST per well; 3) Blocking: 1% BSA 200 μL / well, room temperature for 1 hour.

[0054] 4) Sample preparation: Add 600 μL 2YT medium (12 μg / mL tetracycline, 50 μg / mL ampicillin) to a 96-well deep-well plate the day before, select the single clones from the third round of solid phase panning and the third round of cell panning, culture overnight in a shaker at 220 rpm and 37°C, centrifuge the overnight bacteria at 4000 rpm for 5 min, add 100 μL / well of the supernatant sample to the corresponding well plate using a multichannel gun, and incubate at room temperature for 1 hour.

[0055] 5) Add secondary antibody: dilute enough secondary antibody, add 100µL / well to the corresponding 96-well ELISA plate, incubate at room temperature for 1h, wash the plate 5 times with PBST, then add 100µL TMB at room temperature, incubate at room temperature for 5min, and develop color; 6) Stop the reaction: add 100 µL of TMB colorimetric stop solution (450 nm, without sulfuric acid) to stop the reaction; 7) Data collection and analysis: The OD450 data was read by the microplate reader. The data processing is shown in Table 6-12. Wells A12-D12 are CD28 positive control antibodies. A total of 129 positive clones were screened and sent for testing. Sequencing analysis showed that 35 unique sequences were constructed and expressed. 8) Select the positive clones screened and send them for sequencing, and use the correct sequence obtained from sequencing analysis for construction and large-scale expression.

[0056] Table 6 Flow cytometry results 1

[0057] Table 7 Solid phase panning results 1

[0058] Table 8 Flow sorting results 2

[0059] Table 9 Flow cytometry results 3

[0060] Table 10 Flow cytometry results 4

[0061] Table 11 Flow sorting results 5

[0062] Table 12 Flow cytometry results 6

[0063] S5. Cell function test was performed using the antibodies expressed in step S4. The binding of antibodies to cells was detected by flow cytometry. The results showed that 7 antibodies had good binding activity with cells for subsequent experiments. The specific test steps are as follows: FACS assay: 1) Preparation of cell suspension: Adjust the cell number to 4.00E+06 / mL in pre-cooled MACS buffer (PBS, 2% FBS, 2mM EDTA) and add 50 μL / well of the cell suspension to a 96-well plate.

[0064] 2) Primary antibody incubation: Use MACS buffer to dilute the antibody to be tested 4 times in a gradient of 200nM in the first well. After dilution, add 50μL of antibody dilution buffer to the cell suspension in 1), mix well, and incubate at 4℃ for 60min; 3) Wash cells: add 100 μL of MACS buffer to the 96-well plate, centrifuge at 400 g for 5 min, discard the supernatant, add 200 μL of MACS buffer to the well to wash the cells, centrifuge at 400 g for 5 min, discard the supernatant, and wash twice; 4) Secondary antibody incubation: Resuspend the cells with 100 μL fluorescent secondary antibody (Goat anti-Human Fc, Alexa Fluor 647, 1:1000 dilution) and incubate at 4°C for 30 min; 5) Wash cells: add 100 μL of MACS buffer to the 96-well plate, centrifuge at 400 g for 5 min, discard the supernatant, add 200 μL of MACS buffer to the well to wash the cells, centrifuge at 400 g for 5 min, discard the supernatant, and wash twice; 6) Detection and data analysis: Resuspend the cells in 200 μL MACS buffer and analyze on a flow cytometer. Data processing is as follows: Figure 6 As shown in Figure 2, 7 antibodies have a good binding effect with cells. These 7 antibodies were then tested with cyno-CD28 antigen and mouse-CD28 by ELISA. The results are as follows: Figure 7 , Figure 8 As shown in the results, all seven antibodies showed good cross-reactivity with cyno-CD28, with EC50 around 0.3nM, and two of them showed cross-reactivity with mouse-CD28. Figure 8 As shown, MY-36 and MY-86 can bind to cyno-CD28 and show weak binding to mouse-CD28.

[0065] 2. Cell reporter gene assay: 1) Prepare 1x concentration of CD28 monoclonal antibody CD28 Monoclonal Antibody (CD28.2) and CD3 monoclonal antibody Anti-Human CD3 epsilon OKT-3 (muromonab). The concentrations are 0.01 μg / mL OKT-3 and 100 nM anti-CD28.2; 2) Antibody coating: 100 μL / well of the antibody was coated on a 96-well plate at 4°C overnight; 3) Cell preparation: Add 100 μL / well of prepared Jurkat-luc2p-IL2 cells to the coated 96-well cell culture plate and culture in a 37°C, 5% CO2 incubator for 6 hours; 4) Add Bio-Glo luciferase assay, shake at 400 rpm at room temperature on a shaker, incubate for 10 minutes, and detect the fluorescence signal on a microplate reader. Data processing is as follows Fig. 9 As shown. Fig. 9 It can be seen that when CD3 antibodies and CD28 antibodies are present at the same time, CD28 nanoantibodies MY-92, MY-127, MY-91, and MY-119 show activation activity, and there is a concentration-dependent effect. Compared with the reference antibody, MY-92 and MY-127 have significant activation activity. Finally, it should be noted that the above embodiments are only illustrative of the principles, performance and efficacy of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by 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 includes a framework region and a complementary determining region, and the complementary determining region includes CDR1, CDR2, and CDR3, wherein the amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO.

8.

2. The anti-CD28 nanobody according to claim 1, characterized in that The amino acid sequence of the anti-CD28 nanobody is shown in SEQ ID NO.

15.

3. The anti-CD28 nanobody according to claim 2, characterized in that The nucleotide sequence encoding the anti-CD28 nanobody amino acid sequence is shown in SEQ ID NO.

22.

4. A molecular expression vector, characterized in that: The vector comprises a sequence of SEQ ID NO.22 as described in claim 3.

5. A host cell containing the molecular expression vector according to claim 4, characterized in that: The host cell is a eukaryotic cell or a prokaryotic cell.

6. Use of the anti-CD28 nanobody according to claim 1 in the preparation of tumor treatment or detection reagents.

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