An anti-CD28 nanobody, its preparation method and application

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to an anti-CD28 nanobody, its preparation method, and its application. The preparation process of the anti-CD28 nanobody provided by this invention is as follows: CD28 antigen is constructed to immunize alpacas; a phage library is constructed from the immunized alpaca PBMCs; the phage library is screened, and correct sequences are selected for construction, expression, and purification; the anti-CD28 nanobody is finally obtained after screening by cell function detection. The nanobody provided by this invention has specific recognition and binding ability for CD28, exhibiting advantages such as high specificity and high sensitivity; simultaneously, it effectively reduces the development and production costs of CD28 antibodies and shortens antibody expression time.

Description

[0001] This application is a divisional application of patent application number "202311548011.9", filed on "November 20, 2023", and entitled "An anti-CD28 nanobody and its preparation method and application". Technical Field

[0002] This invention belongs to the field of molecular biology technology, specifically relating to an anti-CD28 nanobody, its preparation method, and its application. Background Technology

[0003] The leukocyte differentiation antigen 28 (CD28) molecule is present on the surface of most T cells and is considered a T cell-specific surface molecule. T cell responsiveness to antigens is primarily mediated by the CD3-T cell antigen receptor (TCR) complex, but also relies on the synergy of other molecules on the T cell surface. CD28, as the first discovered co-stimulatory receptor, is a founding member of a subfamily of co-stimulatory molecules characterized by extracellular variant immunoglobulin-like domains. CD28 primarily acts as a "second signal" (binding to B7-1 / B7-2 on the target cell surface) to lower the threshold required for effective T cell activation, strengthening the "first signal" for T cell activation (the T cell receptor (TCR)-CD3 complex's recognition and binding of MHC-peptides to target cells), thus enabling T cells to further develop and proliferate into immune-functional cells. Given the importance of CD28 co-stimulation 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 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 various cell types, possessing advantages such as high proliferative capacity and strong cytotoxicity, and have great application value in tumor biotherapy. CIK cells are obtained from peripheral blood mononuclear cells through stimulation with various cytokines such as IL-2 and IFN-γ. Preliminary studies on the clinical efficacy of CIK cells show that they have good therapeutic effects on various malignant tumors, including lung cancer, breast cancer, esophageal cancer, and kidney cancer.

[0006] Nanobodies, or variable domain of heavy chain antibody (VHH), contain only one heavy chain variable region (VHH) and CH2 and CH3 regions. Unlike other antibodies, they naturally lack the light chain. Nanobody crystals are 2.5 nm in diameter and 4 nm in length, representing the smallest naturally occurring fragments capable of binding to antigens. Nanobodies are generally elliptical in shape, very small in size, and have a molecular weight about 1 / 10 that of monoclonal antibodies (15 kDa). Compared to conventional antibodies, they are chemically more reactive, possess strong affinity, and can bind more effectively to antigen gaps. The secondary structure of nanobodies consists of two β-sheets forming a scaffold, with three hypervariable regions clustered on one side to participate in antigen recognition. Compared to conventional antibodies, nanobodies offer the following advantages: small molecular weight, facilitating penetration into dense tissues and even the blood-brain barrier; high affinity, strong specificity, good solubility and stability; weak immunogenicity in humans and good biocompatibility; high expression in prokaryotic or eukaryotic systems, facilitating production; and simple structure, making them easy to engineer. The unique properties of nanobodies give them a distinct advantage in disease diagnosis and treatment. Their small size allows them to bind securely and densely to solid-phase carriers to capture trace amounts of antigens, fully leveraging nanobody-based immunoassay methods to detect and identify clinically difficult-to-detect targets, as well as foreign pathogens or toxins. Therefore, nanobodies have significant value in disease treatment and diagnosis, and hold great promise for antibody-targeted diagnosis and treatment of tumors.

[0007] Phage display technology involves inserting a foreign gene into an appropriate location within the structural gene of a phage coat protein. When the reading frame is normal and the normal function of the coat protein is not affected, the foreign gene will be expressed along with the coat protein. It is a technique that displays peptides or proteins on the surface of phages, allowing for in vitro screening of desired peptides or proteins. This technique involves inserting different foreign genes into phage vectors. As the phages are passaged, the foreign proteins are displayed on the phage surface, forming a phage library. The phage library is then screened using specific proteins. After several rounds of affinity screening (such as panning), some phage clones are obtained. These phage clones are then amplified and further screened to obtain more specific target proteins. Summary of the Invention

[0008] This invention provides an anti-CD28 nanobody, its preparation method, and its application. The anti-CD28 nanobody is obtained by screening through a combination of phage display and 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 as follows:

[0010] An anti-CD28 nanobody, the anti-CD28 nanobody comprising a framework region and a complementarity-determining region, the complementarity-determining region comprising CDR1, CDR2, and CDR3, wherein the complementarity-determining region CDR1 is SEQ ID NO.1, the complementarity-determining region CDR2 is SEQ ID NO.7, and the complementarity-determining region CDR3 is SEQ ID NO.8.

[0011] Preferably, the anti-CD28 nanobody is MY-127, with the amino acid sequence shown in SEQ ID NO.15.

[0012] Preferably, the nucleotide sequence encoding the amino acid sequence of the anti-CD28 nanobody MY-127 is shown in SEQ ID NO. 22.

[0013] The present invention also provides a molecular expression vector comprising the nucleotide sequence of SEQ ID NO.22.

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

[0015] The present invention also provides the application of the aforementioned anti-CD28 nanobody in the preparation of CD28-type tumor detection reagents.

[0016] The preparation method of the anti-CD28 nanobody is as follows:

[0017] S1. Based on the protein and gene sequence information of CD28, analyze and design an immune antigen, and link a His-tag to its C-terminus to obtain the modified antigen.

[0018] S2. Immunize alpacas with the antigen obtained in step S1, test the titer of the serum separated from the immunized alpacas, and then extract effector B cells from the peripheral blood to obtain alpaca PBMC cells.

[0019] S3. Using the alpaca PBMC cells obtained in step S2 as raw materials, total RNA was extracted, and cDNA fragments were obtained by reverse transcription PCR. The VHH gene fragment was amplified using this as a template. The target gene fragment was then cloned into a phage vector and transformed into competent cells to construct a phage library.

[0020] S4. Phage packaging is performed using the phage library constructed in step S3. Enrichment is achieved through panning, and single clones are further selected for initial screening. Positive clones are sent for testing, and sequencing analysis is used to select the correct antibody sequence for eukaryotic expression.

[0021] S5. Perform cell function tests using the antibody expressed in step S4, and screen for antibodies with good specificity and sensitivity to obtain the final product.

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

[0023] The present invention also provides the application of the aforementioned anti-CD28 nanobody in the preparation of drugs for the prevention and treatment of tumors.

[0024] Compared with existing technologies, the anti-CD28 nanobody provided by this invention has the following advantages:

[0025] (1) The present invention combines cell panning flow cytometry sorting technology, which can sort out target cells that are bound to bacteriophages and then wash them away to reduce non-specifically bound bacteriophages;

[0026] (2) The present invention utilizes a high-throughput mammalian cell expression system to express CD28 nanobodies, which effectively reduces the development and production costs of CD28 antibodies, shortens the antibody expression time, and increases throughput and improves efficiency. Attached Figure Description

[0027] Figure 1 The image shows the serum titer results obtained by ELISA.

[0028] Figure 2 This is a graph showing the results of agarose gel electrophoresis.

[0029] Figure 3 This is a graph showing the results of the first round of PCR testing;

[0030] Figure 4 This is a graph showing the results of the second round of PCR testing;

[0031] Figure 5 The image shows the detection results of the enzyme digestion reaction system;

[0032] Figure 6 This is a graph showing the results of flow cytometry analysis.

[0033] Figure 7 , Figure 8 This is a graph showing the results of ELISA antigen-antibody detection.

[0034] Figure 9 This is a graph showing the results of a cell reporter gene assay. Detailed Implementation

[0035] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Where specific techniques or conditions are not specified in this embodiment, they shall be operated in accordance with conventional technical methods and instrument manuals in the art; where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0036] This invention first constructs CD28 antigen and immunizes alpacas three times to obtain alpaca PBMC cells; RNA is extracted from PBMCs and reverse transcribed, and antibody gene fragments are obtained by nested PCR; the vector and gene fragments are digested and ligated with enzymes and transformed into E. coli for amplification, and a phage library is constructed; antibodies that specifically bind to the target antigen or target cells are screened by solid-phase panning and cell sorting, and then a eukaryotic expression vector is constructed; then, high-throughput expression of the antibody is induced by a mammalian cell high-throughput expression system, resulting in anti-CD28 nanobodies with high sensitivity and specificity.

[0037] The primers involved in this 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.

[0038] Example: A method for preparing an anti-CD28 nanobody

[0039] There are seven types of anti-CD28 nanobodies: MY-92, MY-86, MY-36, MY-127, MY-78, MY-91, and MY-119.

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

[0041] S1. Based on the protein and gene sequence information of CD28, analyze and design an antigen (which can be the full sequence of CD28 protein) that can effectively induce alpacas to produce specific antibodies against human CD28, and attach a His-tag to its C-terminus to obtain the modified antigen.

[0042] S2. Immunize alpacas with the antigen obtained in step S1. Analyze the titer of the serum isolated from the immunized alpacas. Then, extract effector B cells from the peripheral blood to obtain alpaca PBMCs. Immunize alpacas three times with a mixture of the modified antigen and Freund's adjuvant obtained in step S1. Record the ear tags of blank alpacas before starting the immunization experiment. Observe the alpacas for half an hour after each immunization to confirm they are in good condition and show no signs of discomfort. Immunize every two weeks, collect blood, separate and preserve the supernatant serum for subsequent antibody titer testing.

[0043] The specific process is as follows: Immunization: 200 μg of CD28 / hFc protein was mixed with 200 μL of complete Freund's adjuvant, emulsified, and injected subcutaneously at multiple sites. On day 14, 200 μg of CD28 / hFc was mixed with incomplete Freund's adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously at multiple sites. On day 28, 200 μg of CD28 / hFc was mixed with incomplete Freund's adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously at multiple sites. One week after each immunization, blood was collected to detect the serum titer of Anti-CD28 / His. One week after the third immunization, blood was collected to separate alpaca PBMCs.

[0044] Anti-CD28 / His serum titer was detected by ELISA. The plate was coated with CD28 / His protein at a concentration of 2 μg / mL. 100 μL of serially diluted serum (control: pre-immunized alpaca serum) was added to each well. The plate was incubated at 25°C for 1 h, washed 5 times, and then 1:10000 diluted 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 h, washed 5 times, and then 100 μL of TMB substrate was added. The plate was incubated at 25°C, and the reaction was terminated with 50 μL of 1M HCl. The OD450 nm was measured. The ELISA standard for serum titer is defined as an OD450 at least twice that of the blank control. Specific results are shown below. Figure 1 As shown, the serum titer detected by ELISA was the value at a dilution of 2 times that of the blank control at OD450. The results showed that the antiserum titer after 3 immunizations was between 12800 and 25600. This indicates that the antigen can induce alpacas to produce high-titer antiserum specifically targeting the CD28 protein.

[0045] Lymphocyte isolation: Add 15 mL of cell separation medium (Ficoll-Raque PLUS lymphocyte separation medium from GE Healthcare) to a 50 mL centrifuge tube, then slowly add 15 mL of blood. Add the blood carefully and slowly to prevent mixing with the separation medium. After centrifuging to 25°C, centrifuge at 400 g for 30 min. Observe the blood separation in the centrifuge tube. Store the supernatant serum in a new centrifuge tube at -80°C. Carefully pipette the cotton-like supernatant immune cells into a new 50 mL centrifuge tube. Add 10 mL of room-temperature PBS buffer to each tube and centrifuge at 25°C, 400 g for 20 min. Remove the supernatant, add 5 mL of room-temperature PBS buffer to each tube, mix gently, count the cells, and then centrifuge at 25°C, 400 g for 20 min. The resulting cell lysate has a lymphocyte concentration of 10⁷ / mL and is stored at -80°C.

[0046] S3. Using the alpaca PBMC cells obtained in step S2 as raw materials, total RNA was extracted, and cDNA fragments were obtained by reverse transcription PCR. The VHH gene fragment was then amplified using this cDNA fragment as a template. The target gene fragment was then cloned into a phage vector and transformed into competent cells to construct a phage library.

[0047] (1) RNA extraction process:

[0048] A. Remove PBMC cells from the -80℃ freezer, thaw them on ice, take 500μL of PBMCs, add 500μL of TrizoL and mix well, add 200μL of chloroform, shake for 30s, let stand for 3min, and then centrifuge at 12000g at 4℃ for 15min.

[0049] B. After centrifugation, the mixture separates into 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 min, then centrifuge at 12000g for 10 min at 4℃, remove the supernatant, and collect the precipitate.

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

[0051] D. Allow the clean bench to air dry for 5-10 minutes to evaporate any residual alcohol;

[0052] 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 RNA extracted.

[0053] RNA bands were identified using agarose gel, and then stored at -80°C. The gel image is shown below. Figure 2 As shown.

[0054] After identification, RNA was reverse transcribed into cDNA using TAKARA's PrimeScript™ II Reverse Transcriptase kit.

[0055] Then, two rounds of PCR amplification were performed. The PCR amplification system for the first round is shown in Table 2 below, and the identification gel results are shown in the figure below. Figure 3 As shown.

[0056] Table 2 First-round PCR amplification system

[0057]

[0058] The amplification program was as follows: 98℃ for 3 min; 98℃ for 15 s, 56℃ for 15 s, 72℃ for 30 s, repeating the entire process 20 times; 72℃ for 2 min; and stored at 4℃.

[0059] Then a second round of PCR was performed. The amplification system is shown in Table 3, and the identification gel results are shown in the figure below. Figure 4 As shown.

[0060] Table 3 Second-round PCR amplification system

[0061]

[0062] The amplification program was as follows: 98℃ for 3 min; 98℃ for 10 s, 65℃ for 15 s, 72℃ for 30 s, repeating the process 18 times; 72℃ for 5 min; and stored at 4℃.

[0063] The second round of PCR products were digested with enzymes, and the digestion system is shown in Table 4. The identification gel results are shown in the figure below. Figure 5 As shown.

[0064] Table 4 Enzyme digestion reaction system

[0065]

[0066] The correctly identified fragments were ligated to the vector, and the ligation reaction system is shown in Table 5.

[0067] Table 5 Connection Reaction System

[0068]

[0069] The ligation products were then introduced into cells via electroporation to construct a phage library.

[0070] 1) Transform the ligation product into SS320 competent cells.

[0071] 2) DNA and competent cells were premixed, and the preset electroporation conditions were 2.5KV / 5.8ms.

[0072] 3) The mixture of competent cells and DNA was added to an electroporation vessel for electroconversion, and then the cells were thawed at 37°C for 1 hour.

[0073] 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 bacterial culture in the remaining 300 μL of supernatant for plating.

[0074] 5) Incubation: Invert the coated plate and place it in a 37℃ incubator for overnight incubation for 15 hours.

[0075] 6) On the second day, the bacteria were scraped, preserved, and single clones were picked and sent for sequencing. The effective library size was 1.50E+08.

[0076] After sequencing analysis confirms the library construction quality is satisfactory, the libraries are stored for future screening.

[0077] S4. The phage library constructed in S3 was panned. The first two rounds used solid-phase panning, and the third round used cell panning and flow cytometry to separate the cells that were bound to the phage. The cells were then washed and infected with E. coli for amplification. Single clones were selected for initial screening, and the correct alpaca VHH gene was cloned into the eukaryotic expression vector pCDNA3.4. The gene was then expressed in high throughput using a mammalian expression system to obtain a large number of expressed antibodies.

[0078] The specific steps for filtering are as follows:

[0079] 1. Phage packaging:

[0080] 1) Remove the constructed phage library from -80℃ and thaw it on ice;

[0081] 2) Inoculate into 2YT medium (12µg / mL tetracycline, 50µg / mL ampicillin), shake at 37℃ and 220rpm for 2h to achieve an OD value of 0.3-0.4. Add thawed helper phage M13KO7 to each bottle of bacterial culture (add 100 times the amount of inoculum to ensure that the helper phage is in the superlogarithmic growth phase), and incubate at 37℃ and 80rpm for 0.5h.

[0082] 3) Add Kana+ (kanacillin) to a final concentration of 50 μg / mL, incubate at 37°C and 220 rpm for 0.5 h, then add 100 μL of 0.1 M IPTG for induction, incubate at 30°C and 220 rpm for 14-16 h overnight;

[0083] 4) On the second day, centrifuge at 12000rpm for 10min at 4℃. Filter the supernatant through a 0.45μm filter membrane into a pre-cooled 50mL centrifuge tube. Add 1 / 4 volume of PEG6000 to the supernatant, mix by inverting, lay the centrifuge tube flat, embed it in an ice box, and place the ice box on a shaker at 80rpm for 1h.

[0084] 5) Centrifuge at 4℃ and 12000rpm for 10 min, discard the supernatant and invert the centrifuge tube to drain the liquid; take 1mL of pre-cooled PBS and gently resuspend the phage by pipetting with a Pasteur pipette; centrifuge at 12000rpm for 5 min, and transfer the supernatant to a 1.5mL centrifuge tube for subsequent experiments;

[0085] 6) Infection: In a 96-well dilution plate, the phage suspension was serially diluted 10-fold with logarithmic SS320 (12 dilutions). The plate was then incubated at 37°C for 30 minutes to infect the plate for easy calculation of phage titers the next day.

[0086] 7) Counting: The next day, remove the plate and observe for contamination. If there is no contamination, count the phage titer and calculate the number of phages contained in 1 mL of solution.

[0087] 2. Panning: There are two methods: one is a 3-round solid-phase panning, and the other is a strategy of the first two rounds of solid-phase panning and the third round of cell panning.

[0088] 1) Solid-phase panning and coating: Dilute CD28-his antigen with PBS (to 10 μg / mL) into 5 mL immunotubes and rotate to coat overnight at 4°C;

[0089] 2) Blocking phage and blank immunotherapy tubes: The blank immunotherapy tubes and the required phage were blocked together with 1% BSA by rotation at room temperature for 1 hour.

[0090] 3) Blocking CD28-his-coated immunosorbent assay tubes: Remove the coating solution, wash the immunosorbent assay tubes with PBST, and then rotate-block them with 1% BSA at room temperature for 1 hour.

[0091] 4) Incubation: After blocking, take the supernatant from the blocked phage tube and add it to the CD28-his immunotherapy tube. Incubate at room temperature for 1 hour by rotation. Remove the supernatant, wash the cells 8 times with PBST, wash twice with PBS, and then add 1 mL of trypsin containing 0.25% EDTA to elute the bound phage.

[0092] 5) Infection: In a 96-well dilution plate, dilute the eluted phage with shaken logarithmic SS320 cells, making a total of 8 dilutions. Add 10 μL of phage to the first well, and then dilute 10-fold. Incubate at 37°C for 30 min. At the same time, mix 500 μL of elution buffer with 5 mL of logarithmic SS320 cells, incubate at 37°C for 30 min, centrifuge at 3000 rpm at room temperature for 5 min, and resuspend the cells in the remaining 300 μL of culture medium. Spread the culture on a solid culture plate (resistance: ampicillin / tetracycline hydrochloride / final concentration 2% glucose), invert the plate, and incubate overnight at 37°C.

[0093] 6) Preservation of the exported phage library: Add approximately 0.5 mL of the remaining phage library eluent to approximately 15% glycerol, mix well, and freeze.

[0094] 7) Counting: The next day, remove the plate and observe for contamination. If there is no contamination, count the phage titer, calculate the number of phages in 1 mL of solution, scrape the plate, and perform a second round of phage preparation.

[0095] 8) After three rounds of adsorption-elution-amplification, phages that bind to the target protein CD28-his can be enriched.

[0096] Cell selection:

[0097] 1) Digest uninfected and human-CD28-infected CHOK1 cells, count them separately. The density of human-CD28-infected cells was 5.00E+06 / mL. Stain with CFSE at a final concentration of 1μM, at 37℃ for 7 min in the dark, and resuspend in 1mL PBS at 4℃ for later use.

[0098] 2) The density of uninfected CHOK1 cells was 6.00E+06 / mL. After washing three times with pre-cooled PBS, the supernatant was discarded.

[0099] 3) Block uninfected CHOK1 cells 6.00E+06 and the required loading Phage with 1% BSA, and incubate at 4°C for 1 h by rotation.

[0100] 4) After blocking, take the supernatant at 400g for 5min and add it to a mixture of infected human-CD28 cells 2.50E+06 and uninfected cells 2.50E+06. Incubate at 4℃ for 1h by rotation, remove the supernatant, wash the cells 3 times with PBST, and centrifuge to remove the supernatant.

[0101] 5) Wash cells 3 times with PBS, centrifuge to remove supernatant, resuspend cells in 1 mL of PBS containing 1 mM EDTA, and sort positive cells using a flow cytometer.

[0102] 6) After sorting, centrifuge at 800g for 5 min, discard the supernatant, add 500μL of 20mM citric acid pH 2.3 buffer, and incubate at room temperature for 6 min;

[0103] 7) Elute the bound phage, centrifuge at 800g for 5 min, transfer the supernatant to 500 μL of 1M Tris-HCl pH 8.0 buffer, infect cells, add 500 μL to 5 mL of shaken SS320 bacterial culture, mix well, and incubate at 37℃ for 30 min.

[0104] 8) Centrifuge the 15mL centrifuge tube from the previous step at 3000rpm for 5min to enrich the bacterial cells. Resuspend the bacterial cells in the remaining 300μL of culture medium and plate them. Invert the plates and incubate overnight at 37℃.

[0105] Monoclonal screening:

[0106] 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.

[0107] 2) Washing: On the second day, remove the supernatant and wash the plate 3 times with 200 μL of PBST per well;

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

[0109] 4) Sample preparation: The day before, add 600 μL of 2YT medium (12 µg / mL tetracycline, 50 µg / mL ampicillin) to a 96-well deep-well plate. Select single clones from the third round of solid-phase panning and the third round of cell panning. Incubate overnight in a shaker at 220 rpm and 37°C. Centrifuge the overnight bacteria at 4000 rpm for 5 min. Add 100 µL of the supernatant sample to the corresponding well using a multichannel pipette and incubate at room temperature for 1 h.

[0110] 5) Add secondary antibody: Dilute sufficient secondary antibody, add 100µL / well to the corresponding 96-well microplate, incubate at room temperature for 1 hour, wash the plate 5 times with PBST, then add 100µL of TMB at room temperature, incubate at room temperature for 5 minutes, and perform color development.

[0111] 6) Termination of reaction: Add 100µL of TMB colorimetric stop solution (450nm, sulfuric acid-free) to terminate the reaction;

[0112] 7) Data acquisition and analysis: Data were read by the OD450 microplate reader. Data processing is shown in Table 6-12. Wells A12-D12 were CD28 positive control antibodies. A total of 129 positive clones were screened and sent for testing. Sequencing analysis revealed 35 unique sequences that were constructed and expressed.

[0113] 8) Select the positive clones from the screening, send them for sequencing, and use the obtained correct sequences for construction and large-scale expression.

[0114] Table 6 Flow Cyclic Sorting Results 1

[0115]

[0116] Table 7 Solid phase panning results 1

[0117]

[0118] Table 8 Flow Cyclic Sorting Results 2

[0119]

[0120] Table 9 Flow Cycling Sorting Results 3

[0121]

[0122] Table 10 Flow Cytometry Sorting Results 4

[0123]

[0124] Table 11 Flow Cytometry Sorting Results 5

[0125]

[0126] Table 12 Flow Cycling Sorting Results 6

[0127]

[0128] S5. Cell function was assessed using the antibodies expressed in step S4. Flow cytometry was used to detect the binding of the antibodies to the cells. The results showed that seven antibodies exhibited good binding activity to the cells and were used in subsequent experiments. The specific experimental steps are as follows:

[0129] FACS testing:

[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 of cell suspension per well to a 96-well plate.

[0131] 2) Primary antibody incubation: Dilute the antibody to be tested 4-fold with MACS buffer, starting at 200 nM in the first well. After dilution, add 50 μL of antibody dilution buffer to the cell suspension from step 1), mix well, and incubate at 4°C for 60 min.

[0132] 3) Washing cells: Add 100 μL of MACS buffer to each well, centrifuge at 400g for 5 min, discard the supernatant, add 200 μL of MACS buffer to each well to wash the cells, centrifuge at 400g for 5 min, discard the supernatant, and wash twice.

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

[0134] 5) Washing cells: Add 100 μL of MACS buffer to each well, centrifuge at 400g for 5 min, discard the supernatant, add 200 μL of MACS buffer to each well to wash the cells, centrifuge at 400g for 5 min, discard the supernatant, and wash twice.

[0135] 6) Detection and Data Analysis: Resuspend cells in 200 μL MACS buffer and perform flow cytometry analysis. Data processing is as follows: Figure 6 As shown, seven antibodies exhibited good binding efficacy to cells. These seven antibodies were then subjected to ELISA assays with cyno-CD28 antigen and mouse-CD28, respectively. The results are as follows. Figure 7 , Figure 8 As shown, the results indicated that all seven antibodies exhibited good cross-reactivity with cyno-CD28, with EC50 values ​​around 0.3 nM. Two of these antibodies also showed cross-reactivity with mouse-CD28. Figure 8 As shown, MY-36 and MY-86 can bind to cyno-CD28 and mouse-CD28, showing a weak binding.

[0136] 2. Cell reporter gene assay:

[0137] 1) Prepare a 1-fold concentration of CD28 monoclonal antibody CD28.2 and CD3 monoclonal antibody Anti-Human CD3 epsilon OKT-3 (muromonab). The concentrations of OKT-3 and anti-CD28.2 were 0.01 μg / mL and 100 nM, respectively.

[0138] 2) Coating antibody: 100 μL of antibody was used to coat 96-well plates overnight at 4°C;

[0139] 3) Cell preparation: Add 100 μL / well of Jurkat-luc2p-IL2 cells to a coated 96-well cell culture plate and incubate at 37°C in a 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 includes a framework region and a complementarity-determining region (CDR). The CDR includes CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

8.

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

15.

3. The use of the anti-CD28 nanobody according to claim 1 in the preparation of CD28-type tumor detection reagents.

Images