Nanometer antibody for resisting programmed death molecule 1 and application of nanometer antibody

Abstract

The invention relates to a nano antibody for resisting a programmed death molecule 1 and application of the nano antibody. The amino acid sequence of the nano antibody comprises CDR1 as shown in SEQ ID No. 1 and CDR3 as shown in SEQ ID No. 2; the amino acid sequence of the nano antibody further comprises a CDR2 as shown in SEQ ID No. 3 or SEQ ID No. 4. The nano antibody prepared by the invention is high in specificity, and can block the combination of PD-1 and PD-L1 and enhance the function of immune cells.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a nano antibody against programmed death molecule 1 and applications thereof. Background Art

[0002] In the immune system, T cell-mediated cellular immunity is the "main force" to eliminate tumors. T cells need to be activated to exert anti-tumor effects. The activation amplitude and quality of T cells depend on the balance between activation signals and inhibitory signals. Activating antibodies send activation signals like the accelerator of a car, and inhibitory antibodies send inhibitory signals like stepping on the brakes. Immune checkpoints can be considered the brake system of immune cells. They refer to a series of molecules expressed on immune cells that can regulate the degree of immune activation, so that the activation level of T cells remains within the normal range and prevents damage to the body's healthy tissues.

[0003] Programmed death 1 (PD-1) is widely expressed on the surface of immune cells and is an important immunosuppressive molecule. PD-1 belongs to the immunoglobulin superfamily CD28 / B7. It is a type I transmembrane glycoprotein composed of 288 amino acids and is an immunosuppressive receptor. It has at least two ligands, PD-L1 and PD-L2. Under normal circumstances, PD-1 on the surface of T cells can inhibit the function of T lymphocytes, thereby inhibiting autoimmune responses and preventing the occurrence of autoimmune diseases. However, in tumors, after PD-L1 expressed by tumor cells binds to PD-1 on the surface of T cells, it can inhibit T lymphocytes, lead to downregulation of T cell proliferation, and even induce T cell apoptosis, thereby promoting tumor immune escape. Blocking the PD-1 / PD-L1 negative regulatory pathway can activate the immune system function and kill tumor cells. There are many PD-1-targeted monoclonal antibodies on the market, including Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab, Cindilimab, etc. These PD-1 monoclonal antibodies have been successfully used in clinical practice. However, these antibodies are all common antibodies.

[0004] The research carriers of ordinary antibodies are mainly mice and rabbits. Compared with ordinary antibodies, alpaca antibodies have many advantages: First, they are small in size, one-tenth of ordinary antibodies, which allows them to penetrate into tissues and cells, especially the blood-brain barrier. Second, they are highly stable. Nano antibodies can maintain stability under extreme temperature and pH conditions, and can still maintain biological activity at high temperatures up to 90 degrees. Third, they are simple in structure. Nano antibodies are easier to transform, such as humanization and multivalent construction, and have low production costs, making them suitable for large-scale production. Fourth, they are highly specific. The alpaca's immune system can produce antibodies that are highly specific and affinity for specific antigens, and have weak immunogenicity to humans. Nano antibodies have a low risk of causing an immune response.

[0005] Therefore, how to prepare nano-antibodies against programmed death molecule 1 has become a problem that needs to be solved urgently. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a nano antibody against programmed death molecule 1 and its application. The alpaca antibody prepared by the present invention has high specificity, can block the binding of PD-1 and PD-L1, and enhance the function of immune cells.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a nanobody against programmed death molecule 1, wherein the amino acid sequence of the nanobody comprises CDR1 shown in SEQ ID No.1 and CDR3 shown in SEQ ID No.2.

[0009] The amino acid sequence of the Nanobody also includes CDR2 shown in SEQ ID No.3 or SEQ ID No.4.

[0010] SEQ ID No. 1: GTIFSMND.

[0011] SEQ ID No. 2: NREIRGSSGSWYPLHY.

[0012] SEQ ID No. 3: IDSGLSA.

[0013] SEQ ID No.4: IDSALSA.

[0014] In the present invention, nano antibodies are obtained by immunizing alpacas with PD-1, which are small in size and highly specific. The immune system of alpacas can produce antibodies with high specificity and affinity for specific antigens, and have weak immunogenicity to humans. After experimental detection, the antibody has high affinity and high specificity, and is used in the field of scientific research, for example, it can be used to explore theoretical research on immune checkpoints, and to screen more drugs for treating diseases with high expression of PD-1, etc.

[0015] Preferably, the amino acid sequence of the Nanobody comprises the sequence shown in SEQ ID No.5 or SEQ ID No.6.

[0016] SEQ ID No.5:

[0017] QVQLVESGGGLVQPGGSLRLSCAASGTIFSMNDMGWYRQAPGKQRELVARIDSGLSANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSP.

[0018] SEQ ID No.6:

[0019] EVQLVESGGGLVQPGGSLRLSCAASGTIFSMNDMGWYRQAPGKQRELVARIDSALSANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSS.

[0020] In a second aspect, the present invention provides a recombinant antibody, comprising a fusion sequence of the anti-programmed death molecule 1 nanobody described in the first aspect and a human Ig Fc fragment.

[0021] Preferably, the human Ig Fc includes any one of IgG1, IgG2, IgG3 or IgG4, or a combination of at least two of them.

[0022] Preferably, the sequence of the human Ig Fc fragment is as shown in SEQ ID No.7.

[0023] SEQ ID No.7:

[0024] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0025] Preferably, the sequence of the recombinant antibody is as shown in SEQ ID No.8 or SEQ ID No.9.

[0026] SEQ ID No.8:

[0027] QVQLVESGGGLVQPGGSLRLSCAASGTIFSMNDMGWYRQAPGKQRELVARIDSGLSANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSPAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0028] SEQ ID No.9:

[0029] EVQLVESGGGLVQPGGSLRLSCAASGTIFSMNDMGWYRQAPGKQRELVARIDSALSANYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNREIRGSSGSWYPLHYWGQGTQVTVSSAHHSEDPSSAAASGATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0030] In the present invention, the recombinant antibody constructed using nanoantibodies, human Ig Fc fragments and promoters has a strong ability to significantly block the binding of PD-1 to PD-L1, can effectively promote Jurkat-PD-1-Luciferase to express Luciferase, and still has a strong blocking ability at a lower concentration.

[0031] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-programmed death molecule 1 nanobody described in the first aspect or the recombinant antibody described in the second aspect.

[0032] In a fourth aspect, the present invention provides an expression vector, which contains the nucleic acid molecule described in the third aspect; and after the expression vector is transfected, transduced or transformed into a host cell, the host cell expresses the nano antibody against programmed death molecule 1 described in the first aspect or the recombinant antibody described in the second aspect.

[0033] Preferably, the expression vector further comprises a promoter.

[0034] Preferably, the promoter comprises any one or a combination of at least two of EF1a, PGK1, Ubc, human beta actin, CAG or SV40.

[0035] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the anti-programmed death molecule 1 nanobody described in the first aspect or the recombinant antibody described in the second aspect.

[0036] Preferably, the pharmaceutical composition further comprises immune cells.

[0037] In the seventh aspect, the present invention provides a use of the anti-programmed death molecule 1 nanobody described in the first aspect, the recombinant antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug that blocks PD-1.

[0038] Compared with the prior art, the present invention has at least the following beneficial effects:

[0039] 1. The nano-antibodies against programmed death molecule 1 of the present invention are small in size, highly stable and simple in structure. Tests have shown that they have high specificity. The immune system of alpacas can produce antibodies with high specificity and affinity for specific antigens, and have weak immunogenicity to humans. The risk of nano-antibodies causing immune responses is low.

[0040] 2. The recombinant antibody prepared in the present invention has high affinity and strong ability to block the binding of PD-1 and PD-L1, can effectively promote Jurkat-PD-1-Luciferase to express Luciferase, and can be used to develop immune-enhancing drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a flow cytometry test diagram for detecting the specificity of the recombinant single domain antibody 195-1-D08.

[0042] Figure 2 This is a flow cytometry analysis to detect the specificity of the recombinant single domain antibody 195-1-G06.

[0043] Figure 3 This is a graph showing the results of ELISA testing the EC50 of the recombinant single-domain antibody 195-1-D08.

[0044] Figure 4 This is a graph showing the results of ELISA testing the EC50 of the recombinant single-domain antibody 195-1-G06.

[0045] Figure 5 This is a graph showing the results of the blocking function test of the recombinant antibody 195-1-D08.

[0046] Figure 6 This is a graph showing the results of the blocking function test of the recombinant antibody 195-1-G06.

[0047] Figure 7 The graphs are the results of ELISA detection of IC50 of the recombinant single domain antibody 195-1-D08, wherein Figure A is the blocking curve of 194-1-D08, Figure B is the blocking curve of the negative control antibody, and Figure C is the blocking curve of the positive control antibody.

[0048] Figure 8 The graphs are the results of ELISA detection of IC50 of the recombinant single domain antibody 195-1-G06, wherein Figure A is the blocking curve of 194-1-D08, Figure B is the blocking curve of the negative control antibody, and Figure C is the blocking curve of the positive control antibody.

[0049] Reagents used in the following examples:

[0050] PE-anti-Human IgG (eBioscience, CAT# 12-4998-82);

[0051] PBS (bico, CAT# 14190-250);

[0052] Goat anti-Llama IgG (H+L)) Secondary Antibody [HRP] (Novus, AT#NB7242);

[0053] Nivolumab (MedChemExpress, HY-P9903);

[0054] Streptavidin PE (eBioscience, CAT# 12-4317-87);

[0055] Adjuvant (GERBU, CAT# 3030);

[0056] HRP-ProteinA (Boster, CAT# BA1080);

[0057] HRP-Streptavidin (Boster, CAT# BA1088);

[0058] PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, CAT# 6210B);

[0059] DNA fragment recovery kit (TakaRa, Cat# 9761);

[0060] THE™ V5 Tag Antibody [iFluor 647], mAb (GenScript, CAT# A01805). DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0062] Example 1

[0063] Alpaca immunization and antibody titer determination

[0064] Alpacas were immunized with PD-1 protein, and about 1000 μg of emulsified antigen was injected into both sides of the cervical lymph nodes. Immunization was performed once every 3 weeks for 4 times. 5 mL of peripheral blood was collected before immunization, the second, third and fourth immunizations, and centrifuged at 800 g for 10 min to collect the upper serum. 100 μl / well of 1 μg / mL PD-1-His protein was added to a 96-well plate and coated overnight at 4°C; then the supernatant was discarded, and the plate was washed 5 times with PBST buffer (PBS buffer containing 0.05% Tween-20); 200 μl / well of blocking buffer was added, and the plate was incubated at 37°C for 2 h; after discarding the blocking buffer, the plate was washed 5 times with PBST buffer; the serum collected above was gradiently diluted with PBS buffer, and 100 μL of the gradient diluted serum was added to the 96-well ELISA plate, and incubated at 25°C for 60 min. The control well was PBS buffer. The liquid in the well was discarded and washed 5 times with PBST buffer. Add 100 μl HRP anti-llama IgG (H+L) antibody (1:50000 dilution) and incubate at 25°C for 60 min. After discarding the liquid in the wells, wash the plate 5 times with PBST buffer; add 100 μl / well TMB colorimetric solution; incubate at 25°C in the dark for 15 min. Add 50 μL / well stop solution; read the OD450 value in the wells using a microplate reader.

[0065] "1:X" is used to represent the antibody titer (X refers to the maximum dilution multiple at which the antibody can be detected). The binding titer of the serum of the alpaca before immunization with the PD-1 protein was lower than 1:2000, indicating that the content of anti-PD-1 specific antibodies contained therein was extremely low. The binding titer of the serum after the fourth immunization with the PD-1 protein was significantly higher than 1:64,000, indicating that the content of anti-PD-1 specific antibodies in the alpaca serum was significantly increased after multiple immunizations, and can be used for the construction of yeast display libraries.

[0066] Example 2

[0067] Construction of a single domain antibody yeast display library

[0068] (1) Cloning of VHH antibody fragments: Collect 100 mL of peripheral blood from alpacas and separate PBMCs, extract RNA, and reverse transcribe using PrimeScript™ II 1st Strand cDNA Synthesis Kit to obtain cDNA. Use PBMC cDNA as a template to PCR amplify the alpaca heavy chain antibody sequence. The upstream primer binds to the signal peptide of the VHH antibody ORF, and the sequence is shown in SEQ ID NO.10; the downstream primer binds to the CH2 region, and the sequence is shown in SEQ ID NO.11. Use 1% agarose electrophoresis to recover the target fragment of about 750 bp.

[0069] SEQ ID NO. 10: GTCCTGGCTGCTCTTCTACAAGG.

[0070] SEQ ID NO. 11: GGTACGTGCTGTTGAACTGTTCC.

[0071] The first round of PCR products were used as templates to perform the second round of PCR amplification of the heavy chain antibody VHH fragment. The upstream primer binds to the antibody FR1 region, and the 5' end contains the SfiI restriction site GGCCCAGCCGGCC, and the sequence is shown in SEQ ID NO.12; the downstream primer binds to the hinge region and FR4 region, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC, and the sequence is shown in SEQ ID NO.13. 1% agarose electrophoresis was used to recover the target fragment with a molecular weight of about 450 bp to obtain the VHH fragment library.

[0072] SEQ ID NO. 12: ACTACATGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCTGG.

[0073] SEQ ID NO. 13: GGCCCAGCCGGCCGATCACTAGTGGGGTCTTCGCTGTGGTGCG.

[0074] (2) Detection of electroporation of library vectors and library capacity and diversity: The yeast surface display vector pYDisplay and the VHH fragment library were digested with SfiI. The 5000 bp pYDisplay vector fragment was recovered by electrophoresis. The yeast competent strain frozen at -80°C was streaked onto a YPD solid culture medium plate and cultured at 30°C for 5 days. The yeast competent strain was inoculated into 50 mL YPD culture medium and cultured at 30°C for 2 days. The linearized vector fragment and PCR product were mixed and transformed by electroporation; the yeast competent strain after electroporation was transferred to a culture bottle and cultured at 30°C for 1 h. Take 20 μl of the resuspension, dilute 5000 times with SDCAA culture medium, aspirate 100 μL, apply it to the SDCAA plate, calculate the library capacity after 3 days of culture, and continue to culture the rest of the bacterial solution for 1 day. The remaining bacterial liquid was collected by centrifugation at 3000 g for 5 min, and the supernatant was discarded; 10 mL of SDCAA medium was added to resuspend, and 50% glycerol: resuspended liquid was mixed in a ratio of 1:1, and frozen at -80°C. The results showed that the library capacity of the single-domain antibody yeast display library obtained by alpaca PBMC was about 1.2×10 9 .

[0075] Example 3

[0076] Panning of yeast display libraries

[0077] (1) Pretreatment of streptavidin magnetic beads and yeast cells: To screen for antibodies with high affinity for PD-1, take 10 mL of yeast library (about 2×10 8 Yeast cells), centrifuge at 3000 g for 5 min, and discard the supernatant. Resuspend the yeast library with 0.5% PBSA (PBS + 0.5% BSA), transfer to a 1.5 mL centrifuge tube, centrifuge at 3000 g for 5 min, discard the supernatant, and repeat the wash once. Add 1 mL of 0.5% PBSA to three 1.5 mL centrifuge tubes, and then add 10 μL of streptavidin magnetic beads. Put the centrifuge tubes in a bag, fix them on a rotating mixer, and rotate and incubate at 4°C for 5 min. Place the centrifuge tubes on a magnetic stand for 5 min and discard the supernatant. Then add 1 mL of 0.5% PBSA again, rotate and incubate at 4°C for another 5 min, and discard the supernatant.

[0078] (2) Empty magnetic beads negative panning yeast display library: transfer the washed yeast solution to an empty magnetic bead tube, place it in a bag, and rotate and incubate at 4°C for 60 min; place the empty magnetic bead tube with yeast on a magnetic rack for 10 min, transfer the yeast solution to a new empty magnetic bead tube, and rotate and incubate at 4°C for 30 min. After the incubation, place it on a magnetic rack for 15 min, and transfer the yeast solution to a new empty magnetic bead tube.

[0079] (3) Biotin-PD-1-His magnetic panning yeast display library: Add 100 μL of 50 μg / mL biotin-PD-1-His protein solution (diluted with 0.5% PBSA buffer) to the centrifuge tube containing streptavidin magnetic beads, place the centrifuge tube in a bag, and rotate and incubate at 4°C for 60 min. Then add 1 mL of 0.5% PBSA buffer, let it stand for 5 min, keep the centrifuge tube on the magnetic rack, discard the supernatant, and add 1 mL of 0.5% PBSA buffer. Remove the centrifuge tube from the magnetic rack, blow and mix, place the centrifuge tube on the magnetic rack again, let it stand for 5 min, keep the centrifuge tube on the magnetic rack, and discard the supernatant. After repeating the above washing steps once more, the positive panning magnetic beads coated with biotin-PD-1-His are obtained. Add the positive selection magnetic beads bound to biotin-PD-1-His to the yeast cells that have completed negative selection, and incubate at 4°C with rotation for 60 min. After the incubation, place it on a magnetic stand and let it stand at 25°C for 15 min; keep the centrifuge tube on the magnetic stand and discard the yeast solution. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 1 mL of 0.5% PBSA buffer to the centrifuge tube, gently blow the magnetic beads, and transfer them to a sterile 1.5 mL centrifuge tube. Place the centrifuge tube on a magnetic stand, let it stand at 25°C for 5 min, discard the supernatant; repeat the wash twice. After the wash, resuspend the magnetic beads and adhered yeast cells with 1 mL SDCAA medium, pipette 20 μl of the resuspension into 180 μL SDCAA medium and apply it to two plates, each with a volume of 100 μL, and then pipette 5 μL of the resuspension into 95 μL SDCAA medium and apply it to one plate.

[0080] (4) Flow cytometry of yeast after selection: The yeast after the above two steps of selection were incubated with the PD-1 protein containing the His tag at 4°C for 60 min. The supernatant was then removed by centrifugation, and anti-His tag and anti-V5 tag flow cytometry antibodies were added (the yeast expression vector contains a V5 tag to indicate whether the expression vector has been successfully transferred into the yeast), and the cells were incubated at 4°C for 60 min. The supernatant was then removed by centrifugation, and 1 mL of PBS buffer was added to resuspend the cells. The supernatant was then removed by centrifugation, and 500 μL of PBS buffer was added to resuspend the cells for flow cytometry analysis. The results showed that compared with before selection, the number of yeast that could bind to the PD-1 protein in the yeast antibody display library after selection increased by 35%.

[0081] (5) Selection of monoclonal clones: Pick a yeast monoclonal clone and inoculate it into the culture medium to induce expression. After 48 hours, the bacterial solution was incubated with the PD-1 protein containing the His tag at 4°C for 60 min. Then centrifuge and discard the supernatant, resuspend the precipitate with a solution containing a biotin-conjugated anti-His tag flow cytometry antibody, and incubate at 4°C for 60 min. Then centrifuge and discard the supernatant, resuspend the precipitate with a solution containing PE-streptavidin, and incubate at 4°C for 60 min. Then centrifuge and discard the supernatant, resuspend the cells in 500 μl PBS, and perform flow cytometry analysis. Finally, two monoclonal clones with high positive rates were selected and named 195-1-D08 and 195-1-G06. According to Sanger sequencing, the CDR1 sequences of 195-1-D08 and 195-1-G06 are both SEQ ID No.1, and the CDR3 sequences are both SEQ ID No.2. The CDR2 sequence of 195-1-D08 is SEQ ID No.3, and the CDR2 sequence of 195-1-G06 is SEQ ID No.4. The amino acid sequence of 195-1-D08 is SEQ ID No.5, and the amino acid sequence of 195-1-G06 is SEQ ID No.6.

[0082] Example 4

[0083] Expression of recombinant single domain antibodies and detection of binding to target proteins

[0084] In order to express recombinant single domain antibodies 195-1-D08 and 195-1-G06, the VHH sequence, CMV promoter sequence and IgG1 Fc sequence of candidate 195-1-D08 and 195-1-G06 were amplified by PCR instrument, 50 μL of PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. The band size of CMV was 750 bp, the band size of Fc was 1400 bp, and the band size of VHH of 195-1-D08 and 195-1-G06 was 500 bp and 450 bp, respectively. The target band was cut from the gel and the PCR product was purified. The 195-1-D08 and 195-1-G06 VHH sequences were connected to the MV promoter and IgG1 Fc sequence respectively using a PCR instrument, and then 50 μL of the PCR product was taken, 1 / 10 volume of 10× loading buffer was added, and 1% agarose was used for electrophoresis analysis. The target band was cut out from the gel, and the PCR product was purified. After the obtained PCR product was transiently transfected into HEK293 cells, the harvested cell culture supernatant contained recombinant single domain antibodies 195-1-D08 and 195-1-G06, respectively.

[0085] The binding specificity of 195-1-D08 and 195-1-G06 was tested. The culture supernatants containing 195-1-D08 and 195-1-G06 were mixed with 3×10 5 CHO-K1 or CHO-K1-PD-1 cells were incubated at room temperature for 1 h. After centrifugation at 800g for 5 min at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. 100 μL of PE-labeled Anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature for 45 min in the dark. After centrifugation at 800g for 5 min at room temperature, the supernatant was discarded and the cells were washed 3 times with PBS. The cells were resuspended in 500 μL of PBS for flow cytometry analysis.

[0086] The results of 195-1-D08 are as follows Figure 1 As shown, the expression supernatant of the negative control group did not significantly bind to CHO-K1 or CHO-K1-PD-1; the positive control antibody significantly bound to CHO-K1-PD-1 but not to CHO-K1 cells, indicating that CHO-K1 cells do not express PD-1 protein, while CHO-K1-PD-1 cells express PD-1 protein and can be used as detection cells for alpaca antibodies. 195-1-D08 did not bind to CHO-K1 cells, but significantly bound to CHO-K1-PD-1, and was able to detect that most CHO-K1-PD-1 cells expressed PD-1 protein, indicating that 195-1-D08 had good affinity and specificity.

[0087] The results of 195-1-G06 are as follows Figure 2 As shown, 195-1-G06 did not bind to CHO-K1 cells, but significantly bound to CHO-K1-PD-1, and most CHO-K1-PD-1 cells could be detected to express PD-1 protein, indicating that 195-1-G06 had good affinity and specificity.

[0088] Example 5

[0089] Purification of recombinant antibodies and determination of half effective concentration (EC50)

[0090] To determine the EC50 of 195-1-D08 and 195-1-G06 antibodies, expression plasmids expressing recombinant heavy chain alpaca antibodies were transiently transfected into 293F cells, respectively, and shaken flasks were cultured to express and purify the antibodies. Since the target recombinant antibodies contain human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30 mL of PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20 mg IgG / mL Protein A magnetic beads) to the 293F cell shake flask according to the required amount of the sample. Incubate at room temperature for 2 h in a shaking incubator at 120 rpm. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50 mL centrifuge tube. Wash twice with 30 mL of PBS buffer and deionized water, respectively, and resuspend with 1 mL of elution buffer. After incubation at room temperature for 5 min, collect the magnetic beads with a magnetic separation rack and transfer them to a 15 mL centrifuge tube. After eluting the Protein A magnetic beads twice, the eluates were combined and the neutralization buffer was added to adjust the pH of the solution. The eluted sample was dialyzed with PBS at least 100 times the volume of the sample, first at 20°C for 2 h, then the solution was changed once and then dialyzed at 6°C for 15 h. Finally, the protein concentration was determined and the sample was filtered with a 0.22 μm sterile filter membrane and aliquoted, and stored in a -80°C refrigerator for use.

[0091] Use coating solution to dilute PD-1 protein to 2 μg / mL, pipette 100 μL / well into 96-well ELISA plate, and coat overnight at 4°C. Wash 5 times with PBST, add 200 μL / well of blocking solution, and block at room temperature for 2 h. Wash 5 times with PBST, add different concentrations of 195-1-D08 candidate antibody, which are 10 μg / mL, 3.3 μg / mL, 1.1 μg / mL, 0.37 μg / mL, 0.12 μg / mL, 0.04 μg / mL, 0.014 μg / mL, 0 μg / mL, and incubate at room temperature for 60 min. Wash 5 times with PBST, dilute HRP-Protein A at 1:50000, add 100 μL / well into ELISA plate, and incubate at room temperature for 45 min. Wash 5 times with PBST, add 100 μL of TMB colorimetric solution to each well, and color for 10 min at room temperature in the dark. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm on a microplate reader.

[0092] The results are shown in Table 1 and Figure 3 As shown, the EC50 value of 195-1-D08 for CHO-K1-PD-1 was 0.018 μg / mL, indicating that 195-1-D08 had a high affinity.

[0093] The results are shown in Table 2 and Figure 4 As shown, the EC value of 195-1-G06 for CHO-K1-PD-1 50 The value was 0.083 μg / mL, indicating that 195-1-G06 had a higher affinity.

[0094] Table 1 Absorbance values ​​of 195-1-D08 at different dilution concentrations at OD450nm

[0095]

[0096] Table 2 Absorbance values ​​of 195-1-G06 at OD450nm at different dilution concentrations

[0097]

[0098] Example 6

[0099] Recombinant antibody blocking function test

[0100] In order to detect the function of 195-1-D08 and 195-1-G06 in blocking the binding of PD-1 to PD-L1, 100 μL of cell suspension was added to a 96-well plate, including 2×10 4 Effector cells Jurkat-PD-1-Luciferase (expressing PD-1 molecules, which can inhibit the expression of Luciferase fluorescent protein after binding to PD-L1) and 8×10 4 Target cells CHO-K1-PD-L1 (expressing PD-L1 molecules, which can bind to PD-1 and inhibit the expression of Luciferase fluorescent protein in Jurkat-PD-1-Luciferase cells). Then, 100 μL of different concentrations of 195-1-D08 and 195-1-G06 were added to the corresponding wells, respectively, so that the final concentrations in the corresponding wells were 120 μg / mL, 40 μg / mL, 13.3 μg / mL, 4.4 μg / mL, 1.6 μg / mL, 0.5 μg / mL, 0.16 μg / mL, 0.05 μg / mL, 0.02 μg / mL, and 0 μg / mL. After 18 h of co-culture, 20 μL of One-Glo reagent was added to each well, and the Luciferase fluorescence value was read.

[0101] result Figure 5As shown, the maximum induction effect value of 195-1-D08 is 2.2 (the difference between the maximum Luciferase fluorescence value and the 0μg / mL Luciferase fluorescence value), indicating that 195-1-D08 has a strong ability to block the binding of PD-1 and PD-L1, can effectively promote Jurkat-PD-1-Luciferase to express Luciferase, and can be used to develop immune-enhancing drugs.

[0102] result Figure 6 As shown, the maximum induction effect value of 195-1-G06 is 2.0 (the difference between the maximum fluorescence value and the fluorescence value of 0 μg / mL), indicating that 195-1-G06 has a strong ability to block the binding of PD-1 and PD-L1, can effectively promote Jurkat-PD-1-luciferase to express luciferase, and can be used to develop immune-enhancing drugs.

[0103] Example 7

[0104] Recombinant antibody half inhibitory concentration (IC50) experiment

[0105] In order to detect the half-inhibitory concentration of 195-1-D08 and 195-1-G06 blocking the binding of PD-1 to PD-L1, the PD-1 protein was diluted to 2 μg / mL with the coating solution, and 100 μL / well was pipetted into a 96-well ELISA plate and coated overnight at 4°C. Washed 5 times with PBST, 200 μL / well was added with blocking solution, and blocked for 2 h at room temperature. Washed 5 times with PBST, and different concentrations of 195-1-D08 and 195-1-G06 (25μg / mL, 8.3μg / mL, 2.8μg / mL, 0.9μg / mL, 0.3 μg / mL, 0μg / mL) and anti-PD-1 positive control antibody Nivolumab were added separately, and incubated at room temperature for 15 min. Biotin-PD-L1 protein (4 μg / mL) was added and incubated at room temperature for 45 min. Wash 5 times with PBST, dilute the secondary antibody (Streptavidin-HRP) at 1:10000, add 100 μL / well to the ELISA plate, and incubate at room temperature for 45 min. Wash 5 times with PBST, add 100 μL of TMB colorimetric solution to each well and color for 10 min at room temperature in the dark. Add 50 μL of stop solution to each well, and read the absorbance at 450 nm on an ELISA reader.

[0106] The results are shown in Table 3 and Figure 7As shown in the figure, the IC50 value of 195-1-D08 is 0.24 μg / mL, and the IC50 value of the positive control antibody is 0.3 μg / mL. The IC50 value of 195-1-D08 is lower than that of the positive control antibody Nivolumab. At the same time, when the antibody concentration is only 0.034 μg / ml, the absorbance values ​​of the 195-1-D08 treatment group (2.207 and 2.168) are still significantly lower than those of the control group without the addition of the antibody (3.079 and 3.092); on the contrary, the absorbance values ​​of the positive control antibody at this concentration (1.626 and 1.669) are no longer significantly different from those of the control group without the addition of the antibody (1.703 and 1.602). This result shows that the blocking ability of 195-1-D08 is stronger than that of the positive control antibody Nivolumab.

[0107] The results are shown in Table 4 and Figure 8 As shown in the table, the IC50 value of 195-1-G06 is 0.29 μg / mL, and the IC50 value of the positive control antibody is 0.3 μg / mL. The IC50 value of 195-1-G06 is comparable to that of the positive control antibody Nivolumab, indicating that the blocking ability of 195-1-G06 is comparable to that of the positive control antibody Nivolumab. (The above results are from the same batch of experiments, so Tables 3 and Figure 7 The results of the control group in Table 4 and Figure 8 are the same)

[0108] Table 3 Blocking ability of 195-1-D08 at different concentrations

[0109]

[0110] Table 4 Blocking ability of 195-1-G06 at different concentrations

[0111]

[0112] In summary, the present invention uses PD-1 protein to immunize alpacas. After determining the titer of PD-1-specific antibodies in alpaca serum by ELISA, alpaca peripheral blood mononuclear cells (PBMCs) are separated, RNA is extracted and reverse transcribed to obtain cDNA. Alpaca single-domain antibody-specific primers are used to amplify the VHH sequence and clone it into a yeast expression plasmid to construct a yeast display library. The yeast display library is then positively and negatively screened with biotin-PD-1 protein-streptavidin magnetic beads or streptavidin magnetic beads to enrich yeast that binds to PD-1 protein. Monoclones are picked from the enriched products, and the specific binding of candidate antibodies to PD-1 is detected by ELISA. After the positive antibodies are expressed and purified, their affinity for binding to PD-1 protein is detected. Antibody clones with strong affinity to PD-1 are selected to detect the ability to block the binding of PD-1 and PD-L1. The results show that the alpaca antibody has high specificity, and the effect of blocking the binding of PD-1 and PD-L1 is stronger than the prior art, thereby enhancing the function of immune cells.

[0113] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. A nanobody against programmed death molecule 1, characterized in that: The amino acid sequence of the Nanobody includes CDR1 shown in SEQ ID No. 1 and CDR3 shown in SEQ ID No. 2; The amino acid sequence of the Nanobody also includes CDR2 shown in SEQ ID No.3 or SEQ ID No.

4.

2. The Nanobody according to claim 1, characterized in that The amino acid sequence of the nanobody includes the sequence shown in SEQ ID No.5 or SEQ ID No.

6.

3. A recombinant antibody, characterized in that: The recombinant antibody comprises a sequence obtained by fusing the anti-programmed death molecule 1 nanobody according to claim 1 or 2 with a human Ig Fc fragment.

4. The recombinant antibody according to claim 3, characterized in that The human Ig Fc includes any one or a combination of at least two of IgG1, IgG2, IgG3 or IgG4; The sequence of the human Ig Fc fragment is shown in SEQ ID No.

7.

5. The recombinant antibody according to claim 3, characterized in that The sequence of the recombinant antibody is shown as SEQ ID No.8 or SEQ ID No.

9.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-programmed death molecule 1 nanobody according to claim 1 or 2 or the recombinant antibody according to any one of claims 3-5.

7. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule of claim 6; and after transfecting, transducing or transforming the host cell, the expression vector causes the host cell to express the anti-programmed death molecule 1 nanobody of claim 1 or 2 or the recombinant antibody of any one of claims 3-5.

8. The expression vector according to claim 7, characterized in that The expression vector further comprises a promoter; The promoter includes any one or a combination of at least two of EF1a, PGK1, Ubc, human beta actin, CAG, CMV or SV40.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-programmed death molecule 1 nanobody according to claim 1 or 2 or the recombinant antibody according to any one of claims 3-5; The pharmaceutical composition also includes immune cells.

10. Use of an anti-programmed death molecule 1 nanobody according to claim 1 or 2, the recombinant antibody according to any one of claims 3-5, the nucleic acid molecule according to claim 6, the expression vector according to claim 7 or 8, or the pharmaceutical composition according to claim 9 in the preparation of a drug that blocks PD-1.

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