A preparation method and coding sequence of an anti-human PDL1 nanobody that blocks the binding of PD1 to PDL1

Abstract

The present invention discloses an anti-human PDL1 nano antibody and its VHH chain, including a framework region FR and a complementary determining region CDR region, wherein the framework region is selected from the amino acid sequence of FR1-FR4, and the CDR region is selected from the amino acid sequence of CDR1-CDR3. Also disclosed are a gene sequence encoding the nano antibody and a host cell expressing the nano antibody. The PDL1 nano antibody has a small molecular weight and strong specificity. The nano antibody can express soluble protein in Escherichia coli, has a simple preparation method, and is low in cost, and has good application prospects in the detection of human PDL1 and in anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine or biopharmaceutical technology, and relates to a preparation method of an anti-human PDL1 nano antibody for blocking the binding of PD1 and PDL1 and a coding sequence thereof. Background Art

[0002] At present, the immunotherapy of tumors is one of the more promising methods in tumor treatment. The main target of immunotherapy is the immune checkpoint molecules. T cell-mediated cellular immunity plays an important role in the recognition and killing of tumor cells. Due to the mutation of proteins expressed by cancer cells and the new antigens produced by abnormal processing, these specific antigens can be recognized by T cell surface receptors, thereby stimulating the immune system to eliminate cancer cells. However, the process of T cell surface receptors recognizing specific antigens is regulated by a series of immune checkpoint molecules. In the normal body, immune checkpoints are an inhibitory pathway for the immune system to maintain self-tolerance and prevent autoimmunity. However, cancer cells can use the mechanism of immune checkpoints to escape the immune response regulated by T cells by targeting immune checkpoints. When the body is attacked by tumors, immune checkpoints are activated, thereby suppressing autoimmunity and promoting tumor cell growth and escape.

[0003] At present, the research and development of immune checkpoint inhibitors has achieved good results in the field of tumor treatment, and the research on immune checkpoint inhibitors has attracted more and more attention. In particular, drugs targeting programmed death ligand 1 (PDL1, B7-H1, CD274) and programmed death receptor-1 (PD1, CD279) have significant effects on the treatment of various types of tumors. PD-1 is a co-stimulatory molecule induced on the surface of activated T cells. It is CD4 + and CD8 +Members of the CD28 immunoglobulin family expressed by T cells interact with ligands in the B7 family (PD-L1 and PD-L2). PD-L1 is more widely expressed than PD-L2. PD-L1 (B7-H1, CD274) is expressed on tumor cells and antigen-presenting cells (APCs) including dendritic cells, macrophages, B cells, and activated T cells. Therefore, PDL1 is often used as a target for anticancer drug design. In normal cells, the binding of PD-1 to its ligand limits the activity of T cells, thereby preventing overstimulation and maintaining immune tolerance to self-antigens. However, tumor cells upregulate the expression of PDL1 protein, PD-1 / PD-L1 interaction, weaken endogenous anti-tumor immune response, inhibit T lymphocyte proliferation, cytokine release and cytotoxicity, and lead to exhaustion and apoptosis of tumor-specific T cells. Therefore, blocking the PD-1 / PD-L1 interaction can enhance T cell activity, thereby achieving the effect of tumor immunotherapy. Immunotherapy with antibodies that block the PD-1 / PDL1 pathway has shown impressive clinical results: sustained tumor regression and improved patient survival. Currently, the FDA-approved monoclonal antibodies targeting PD1 include Nivolumab and Pembrolizumab, and the monoclonal antibodies targeting PDL1 include Atezolizumab, Avelumab, and Durvalumab.

[0004] Currently, most antibodies targeting PDL1 are monoclonal antibodies, but the preparation process of traditional monoclonal antibodies is relatively complicated and the production cost is high. In addition, monoclonal antibodies have a large molecular weight and poor tissue penetration ability, and cannot exert the maximum therapeutic effect in tumor tissue. Nanobodies are currently the smallest functional fragments that can bind to antigens, and are only 1 / 10 the size of monoclonal antibodies. Due to its unique structure that is different from monoclonal antibodies, it will not cause aggregation of red blood cells and platelets, and will not produce side effects such as bleeding and thrombocytopenia during treatment. In addition, nanobodies have good solubility, high stability, and can be expressed in prokaryotic expression systems, which overcomes the problems of long preparation cycles and high manufacturing costs of traditional antibodies. It is a new type of antibody molecule with great application prospects.

[0005] Currently, nanobodies targeting PDL1 targets have not been clinically applied, so there is an urgent need in this field to develop new and effective specific nanobodies targeting PDL1 targets. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide an anti-PDL1 nanobody that can block the binding of PD1 to its ligand PDL1, and at the same time provide the coding sequence of the nanobody and a method for preparing the nanobody.

[0007] Technical solution: To achieve the above objectives, the first part of the present invention is a VHH chain of an anti-human PDL1 nanobody, comprising a framework region (FR region) and an antigen complementarity determining region (CDR region). The framework region is selected from the following FR1-FR4 amino acid sequences:

[0008] FR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 3, FR4 shown in SEQ ID No. 4;

[0009] or FR1 shown in SEQ ID No. 5, FR2 shown in SEQ ID No. 6, FR3 shown in SEQ ID No. 7, FR4 shown in SEQ ID No. 8;

[0010] or FR1 shown in SEQ ID No. 9, FR2 shown in SEQ ID No. 10, FR3 shown in SEQ ID No. 11, FR4 shown in SEQ ID No. 12;

[0011] or FR1 shown in SEQ ID No. 13, FR2 shown in SEQ ID No. 14, FR3 shown in SEQ ID No. 15, FR4 shown in SEQ ID No. 16;

[0012] or FR1 shown in SEQ ID No. 17, FR2 shown in SEQ ID No. 18, FR3 shown in SEQ ID No. 19, FR4 shown in SEQ ID No. 20;

[0013] The antigen complementary determining region is selected from the following CDR1-CDR3 amino acid sequences:

[0014] CDR1 shown in SEQ ID No. 21, CDR2 shown in SEQ ID No. 22, and CDR3 shown in SEQ ID No. 23;

[0015] Or CDR1 shown in SEQ ID No. 24, CDR2 shown in SEQ ID No. 25, and CDR3 shown in SEQ ID No. 26;

[0016] Or CDR1 shown in SEQ ID No. 27, CDR2 shown in SEQ ID No. 28, and CDR3 shown in SEQ ID No. 29;

[0017] Or CDR1 shown in SEQ ID No. 30, CDR2 shown in SEQ ID No. 31, and CDR3 shown in SEQ ID No. 32;

[0018] Or CDR1 shown in SEQ ID No. 33, CDR2 shown in SEQ ID No. 34, and CDR3 shown in SEQ ID No. 35;

[0019] Preferably, it has the amino acid sequence shown in SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40.

[0020] The second part of the present invention is an anti-human PDL1 nanobody (Nb1, Nb2, Nb3, Nb4, Nb5), which is a nanobody against the human PDL1 molecular epitope, comprising a VHH chain having an amino acid sequence shown in SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQID No. 39, and SEQ ID No. 40.

[0021] The third part of the present invention provides a DNA molecule encoding a protein selected from the following group: the VHH chain of the anti-human PDL1 nanobody shown in claim 1 or 2, or the anti-human PDL1 nanobody shown in claim 3.

[0022] Preferably, the DNA molecule has a DNA sequence selected from the following group: SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45.

[0023] The fourth part of the present invention provides an expression vector, characterized in that it contains the nucleotide sequences shown in SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, and SEQ ID No. 45.

[0024] The fifth part of the present invention provides a host cell transformed with the recombinant expression vector or a host cell and its progeny cells having the nucleotide sequence encoding the anti-PDL1 nanobody integrated into its genome.

[0025] The host cells and their progeny cells refer to bacterial cells, fungal cells, animal cells or plant cells and the progeny of these host cells.

[0026] The sixth part of the present invention provides a method for preparing an anti-PDL1 antibody, comprising:

[0027] (1) Camels were immunized with human PDL1 eukaryotic protein to prepare a phage display library.

[0028] (2) Affinity screening of phage display library using human PDL1 protein; (3) Identification of positive clones by PE-ELISA; (4) Expression and purification of anti-PDL1 nanoantibodies; (5) Identification of the function of anti-PDL1 nanoantibodies.

[0029] The seventh part of the present invention provides the use of the human PDL1 nanobody of the present invention for detecting human PDL1.

[0030] The anti-PDL1 nanobody can be used to prepare a detection kit or a diagnostic kit for detecting PDL1 in a sample.

[0031] Application of the anti-PDL1 nanobody in drugs for treating cancer.

[0032] The anti-PDL1 nanobody can be used as an immune checkpoint inhibitor, as a cancer treatment drug alone, or in combination with other anti-cancer drugs.

[0033] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0034] The present invention uses eukaryotic human PDL1 eukaryotic protein to immunize Xinjiang Bactrian camels, separates camel peripheral blood lymphocytes to amplify VHH gene sequences, constructs a nano antibody phage display library, and uses a solid phase affinity panning method to screen the library, ultimately obtaining anti-human PDL1 specific nano antibody gene sequences.

[0035] The present invention does not subclone the positive clone sequences obtained by screening into other expression vectors, but directly uses the pMECS vector and the TG1 host bacteria to directly express the antibody protein, which greatly shortens the time and cost of obtaining the antibody.

[0036] The phage display library constructed by the present invention has a large library capacity, and the diversity of nano antibodies obtained by screening is high.

[0037] Compared with conventional antibodies, the anti-PDL1 nanoantibody of the present invention has a small molecular weight (about 18 kDa), high affinity and good specificity. It has the potential to be developed as an immune checkpoint inhibitor, providing an alternative for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The total RNA was extracted after separating lymphocytes from camel peripheral blood (TRIZOL method);

[0039] Figure 2 This is the agarose gel electrophoresis image of the 750 bp DNA fragment recovered from the first round of PCR amplification products;

[0040] Figure 3 This is the agarose gel electrophoresis diagram of the DNA fragment of about 450 bp recovered from the gel excision of the second round of PCR amplification products;

[0041] Figure 4 This is a Coomassie Brilliant Blue staining image after SDS-PAGE electrophoresis after nanobody expression. The protein was obtained by high and low osmotic methods. From left to right, lane 1 is the total protein of the bacteria without induction, lane 2 is the periplasmic protein obtained by high and low osmotic methods, lane 3 is the antibody protein purified by HIS affinity chromatography, lane 5 is protein ultrafiltration, and lane 6 is the protein molecular weight marker.

[0042] Figure 5 This is a graph showing the purification results of five anti-PDL1 nanobodies: Nb1, Nb2, Nb3, Nb4, and Nb5;

[0043] Figure 6 This is a graph showing the specific binding results of anti-PDL1 nanoantibodies detected by indirect ELISA;

[0044] Figure 7 This is one of the anti-PDL1 nanobody affinity determination curves; DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with embodiments and drawings, but these specific embodiments should not be interpreted in any way as limiting the scope of application of the present invention.

[0046] The present invention first immunizes a female Xinjiang Bactrian camel with eukaryotic human PDL1 protein, and after six consecutive immunizations, extracts peripheral blood from the camel to separate and obtain peripheral blood lymphocytes of the camel, and constructs a PDL1-specific single-domain heavy chain antibody immune library. Then, a solid phase affinity panning method is used to screen the immune library with eukaryotic human PDL1 protein to obtain single-domain heavy chain antibodies against human PDL1, thereby obtaining a nano antibody strain that is efficiently expressed in Escherichia coli.

[0047] Case study 1: Construction of a single domain heavy chain antibody immune library targeting human PDL1:

[0048] (1) Immunize Xinjiang Bactrian camels with eukaryotic human PDL1 protein once every two weeks for a total of six times. (2) After the six immunizations, the serum titer was tested using indirect ELISA. Lymphocytes were isolated from the peripheral blood of camels and total RNA was extracted (TRIZOL method). (3) According to Primescript from TAKARA TmRT reagent kit with gDNAEraser reagent instructions will extract the total RNA, such as Figure 1 As shown. The RNA is then reverse transcribed into cDNA, and then the VHH chain is amplified using the PCR method, which includes two rounds of PCR. The first round of PCR:

[0049] Upstream primer CALL01-Leader: 5'-GTCCTGGCTGCTCTTCTACAAGG-3'

[0050] Downstream primer CALL02-CH2: 5'-GGTACGTGCTGTTGAACTGTTCC-3'

[0051] The first round of PCR reaction conditions: 95°C pre-denaturation for 5 min, 95°C, 10 s, 56°C, 30 s, 72°C, 1 min, 20 cycles, 95°C, 10 s, 68°C, 1 min, 72°C extension for 10 min.

[0052] The first round of PCR amplification products were separated by 1.5% agarose gel electrophoresis, and the DNA fragments with a size of 750 bp were recovered by gel excision. Figure 2 shown.

[0053] The second round of PCR uses the product recovered from the first round of gel excision as the template required for amplification to perform the second round of PCR:

[0054] Upstream primer VHH-Back: 5'-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3'

[0055] Downstream primer PMCF primer: 5'-CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3'

[0056] The reaction conditions for the second round of PCR were as follows: pre-denaturation at 95°C for 5 min, 28 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 40 s, and extension at 72°C for 10 min.

[0057] The second round of PCR products were separated by 2% agarose gel electrophoresis, and the gel was cut to recover DNA fragments of about 450 bp in size. Figure 3As shown; (4) The phagemid pMECS and the second round of PCR product VHH were double-digested with restriction endonucleases (purchased from TAKARA) QuickCut™ Not I and QuickCut™ PstI, respectively, and recovered by agarose gel electrophoresis. After nucleic acid quantification, the vector and gene were connected with T4 ligase at a mass ratio of 3:1 and connected at 16°C overnight. (5) The ligation product was purified and concentrated, and the ligation product was electrotransformed into competent TG1 cells to construct a PDL1 nanobody phage display library and calculate the library capacity. The library capacity is 4×10 8 , the library was collected and stored in aliquots at -80°C.

[0058] Case study 2: Screening of anti-PDL1 nanobodies, identification and sequencing of positive clones

[0059] 1. Amplification of large-capacity PDL1 phage nanobody library

[0060] Inoculate the library in 10 mL of 2×YT liquid culture medium (containing 2% glucose, 100 μg / mL ampicillin) to an OD600 of 0.1, and culture at 37°C, 200 r / min until the OD600 reaches 0.5. Add helper phage M13K07 at a multiplicity of infection of 20:1, incubate at 37°C, incubate for 30 min, and then culture at 37°C, 100 r / min for 30 min. The culture was centrifuged and the precipitate was resuspended in 50 mL of 2×YT (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin). After overnight culture at 30°C and 220 r / min, the supernatant was centrifuged at 4°C and 8000 rpm, 5×PEG / NaCl solution was added and placed on ice for 2 h, centrifuged at 12000 rpm for 30 min, and the precipitate was resuspended in phosphate buffer (PBS, 0.01 M, pH 7.4) to obtain the anti-PDL1 single domain heavy chain antibody immune library. 10 μL was taken to determine the titer of the amplified library.

[0061] 2. Screening of anti-PDL1 nanobodies

[0062] Using the solid phase affinity panning method, the eukaryotic human PDL1 protein was diluted to 1-100ug / mL with coating buffer and coated on a high affinity ELISA plate at 4°C overnight. The next day, the plate was washed three times with PBS, and 200uL of PBS containing 3% BSA was added to each well and blocked at 37°C for 2 h. The phage titer was diluted to 1×10 12CFU (titer decreases in each subsequent round), mix the diluted phagemid with an equal volume of 3% skim milk powder, and rotate and mix at low speed at room temperature for 30 minutes to neutralize the phagemid bound to BSA. Wash the plate 5 times with PBS, add 100uL of pre-incubated phagemid to each well, and incubate at 37°C for 2 hours. Aspirate the unbound phagemid in the well, add PBST to wash 10-20 times (increase the number of washes with each round), then wash three times with PBS, add 100ul of glycine-HCl elution buffer with pH = 2.2, and shake at 37°C for 10 minutes. Gently blow the well to wash off the adsorbed phage and transfer it to a new EP tube, add an appropriate amount of neutralizing solution to make the pH value of the solution between 7.0-7.4. Take 10uL to determine the titer, and the rest will be used for the next round of panning after amplification. A total of three rounds of affinity screening were performed.

[0063] 3. PE-ELISA screening of PDL1-specific single positive clones

[0064] Add 200uL 2×YT-AG liquid culture medium (glucose content is 2%, ampicillin is 100ug / mL) to the 96-well deep-well plate, pick the monoclones on the output plate after the third round of screening and put them into each well, and culture them in a 37°C incubator overnight. The next day, add 500ul 2×YT-A (ampicillin is 100ug / mL) to the 96-well deep-well plate, inoculate 20uL / well of the cultured bacteria the night before, culture at 37°C 220rpm for 2h, add IPTG to each well to a final concentration of 1mM, and culture at 30°C 200rpm overnight. Centrifuge the 96-well deep-well plate and discard the supernatant, add 200uLTES hypertonic buffer to each well to resuspend the cells, shake at 4°C for 2h, then add 300μLTES / 4 hypotonic solution to each well, shake at 4°C for 2h, and centrifuge to obtain the supernatant. Coat two 96-well ELISA plates, one with human PDL1 antigen and the other with negative control BSA, overnight at 4°C. The next day, wash the plates twice with 0.05% PBST and block with 3% skim milk at room temperature for 2h. Aspirate the blocking solution and wash the plates twice with 0.05% PBST. Add 100uL of the corresponding deep-well plate supernatant to the antigen-coated wells and negative control wells, respectively, and incubate at 37°C for 2h. Wash the plates with PBST, add 100uL of 1:3000 dilution of anti-HA mouse monoclonal antibody to each well, and incubate at 37°C for 1h. Wash the plates three times with 0.05% PBST. Add 100uL of goat anti-mouse-HRP antibody to each well, and incubate at 37°C for 1h; aspirate the antibody dilution, wash the plates, add TMB for color development, and measure OD 450nm with an enzyme reader. The absorbance value was greater than 1, and the positive OD450 / negative OD450>2.5 was identified as a positive clone, and all positive clones were sequenced.

[0065] The DNA sequence was obtained by sequencing the positive clones, and the DNA was translated into an amino acid sequence using Vbase software. The framework region (FR region) of the protein sequence and the antibody recognition region (CDR region) were distinguished according to the International Immunogenetics Information System (IMGT). Clones with the same CDR1, CDR2, and CDR3 sequences were considered to be the same antibody strain, and clones with different CDR sequences were considered to be different antibody strains. Finally, 5 different nanoantibody sequences were obtained.

[0066] Case study 3: Expression and purification of PDL1 nanobody

[0067] 1. Expression of PDL1 Nanobody

[0068] This patent did not subclone the 5 positive clones identified after sequencing into other expression vectors, but used the original vector pMECS and the original host bacteria TG1 for protein expression, and the clone was named TG1-pMECS-VHH. The original strain was streaked onto a 2YT-AG solid culture medium plate, placed upright for 10 minutes, and then inverted for overnight culture. The next day, a single clone on the solid plate was picked and placed into 10mL 2YT-AG liquid culture medium containing 100ug / mL ampicillin antibiotics, and cultured at 37°C, 220rpm for 8 hours as a bacterial seed liquid.

[0069] Inoculate 1% of the bacterial seed liquid into 2YT-A liquid medium, shake and culture at 37℃, 220rpm until OD600=0.8, add IPTG to a final concentration of 1mM, and culture overnight at 30℃, 150rpm. The next day, centrifuge the bacterial liquid and discard the supernatant. Wash the bacteria twice with 1×PBS, and freeze-thaw the bacterial pellet twice at -80℃. After thawing, add an appropriate amount of TES to resuspend the cells, shake at 4℃, 200rpm for 4h. Add TES / 4 twice the volume of TES, shake at 4℃, 200rpm overnight. Centrifuge at 4℃, 12000rpm for 30min, and take the supernatant.

[0070] 2. Purification of PDL1 Nanobody

[0071] The antibody protein was purified by nickel column ion affinity chromatography, and PBS buffer was added to the obtained nano antibody solution, and centrifuged and ultrafiltered at 4°C, 4000rpm to the required volume. The molecular weight and purity of the PDL1 nano antibody were detected by 12% SDS-PAGE protein electrophoresis: Figure 4 The figure shows the protein expression of one of the nanobodies from protein induction to purification ultrafiltration. The SDS-PAGE electrophoresis results show an obvious band near 18kDa, which is consistent with expectations. The bands in the sample lane after ultrafiltration are relatively single, indicating that the antibody purity is relatively high.

[0072] Case study 4: Physical and chemical properties testing of PDL1 nanobody

[0073] 1. Specificity of PDL1 Nanobody Detected by Indirect ELISA

[0074] The human PD1, human PDL1, human SRIPa proteins, human CD47, human CD47-Fc, and CD47-HIS proteins used in this embodiment were all purchased from Beijing Sino Biological Biotech Co., Ltd.

[0075] Human PD1 protein, human PDL1 protein, human SRIPa protein, human CD47 protein, human CD47-Fc protein, and CD47-HIS protein were diluted to 1ug / mL using coating buffer, and 100uL per well was coated on a 96-well ELISA plate and coated overnight at 4°C. The next day, the plate was washed twice with 0.05% PBST and blocked with 5% skim milk at 37°C for 2h. After washing the plate three times, 100uL of a certain concentration (1ug / mL) of anti-PDL1 nanoantibodies (Nb1, Nb2, Nb3, Nb4, Nb5) was added to each antigen well and incubated at 37°C for 2h. After washing the plate three times, 100uL of secondary antibody (Mouse anti-HA tag antibody, purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added to each well for reaction and incubated at 37°C for 1h. After washing the plate three times, the tertiary antibody (HRP-coupled Goat anti-mouse IgG mAb, purchased from Kangwei Century) was added and incubated at 37°C for 1 hour. After washing the plate five times, TMB was added for color development, and OD 450nm was measured by a microplate reader after termination.

[0076] The absorbance value reflects the binding ability of anti-PDL1 nanobody to human PDL1, human PD1, human SRIPa, human CD47, human CD47-Fc, and CD47-HIS antigens. Figure 5 As shown, the results showed that the binding values ​​of anti-PDL1 nanoantibodies (Nb1, Nb2, Nb3, Nb4, Nb5) to the antigen PDL1 were high, but they basically did not bind to other control antigens, indicating that the prepared anti-PDL1 nanoantibodies had strong specific binding ability.

[0077] 2. Determination of affinity constants of anti-PDL1 nanobodies

[0078] The affinity constant of PDL1 nanobody was determined by non-competitive ELISA method.

[0079] Four different concentrations of human PDL1 protein solutions were prepared at 1ug / mL, 0.5ug / mL, 0.25ug / mL, and 0.125ug / mL, and 100uL was coated in each well of a 96-well ELISA plate at 4°C overnight. The next day, the plate was washed twice with 0.05% PBST and blocked with 5% skim milk at 37°C for 2h. The anti-PDL1 nanoantibody was diluted from 4ug / mL to 12 concentration gradients by a doubling dilution method. After washing the plate twice, the sample was added to a 96-well ELISA plate at 100μL / well and incubated at 37°C for 2h. After washing the plate three times, 100uL of secondary antibody (Mouse anti-HA tag antibody, purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added to each well for reaction and incubated at 37°C for 1h. After washing the plate three times, the tertiary antibody (HRP-coupled Goatanti-mouse IgG mAb, purchased from Kangwei Century) was added and incubated at 37°C for 1 hour. After washing the plate five times, TMB was added for color development. After termination, the OD 450nm was measured by microplate reader. The "S" curve was fitted to obtain the PDL1 nanoantibody concentration (EC50) corresponding to the four half-absorbance values. The results are shown in Figure 6 , as shown in 7.

[0080] Nanobodies are monovalent antibodies. The affinity constant is calculated according to the formula KA = (n-1) / (nAb '-Ab) (where Ab and Ab' represent the antibody concentration (mol / L) that produces half the absorbance value when the antigen concentration is Ag and Ag', respectively, and n = Ag / Ag'). Six KA values ​​are obtained, and the average value and standard deviation are calculated. The results are shown in Table 1. Taking the affinity constant determination of one of the anti-PDL1 (Nb2) nanobodies as an example, the calculated affinity constant is (0.93304) × 10 6 L·mol⁻ 1 , indicating that the prepared anti-PDL1 nanobody has a higher affinity.

[0081] Sequence Listing <110> Xinjiang Youmai Biotechnology Co., Ltd. <120> A preparation method and coding sequence of an anti-human PDL1 nanoantibody that blocks PD1-PDL1 binding <130> 1 <140> 20201217 <141> 2020-12-17 <160> 45 <170> SIPOSequenceListing 1.0 <210> 1 <211> 25 <212> PRT <213> Synthesis <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Val Ser 20 25 <210> 2 <211> 17 <212> PRT <213> Synthesis <400> 2 Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala 1 5 10 15 Gly <210> 3 <211> 38 <212> PRT <213> Synthesis <400> 3 Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala Phe Ser Arg Asp Asn 1 5 10 15 Gly Gln Asn Thr Val Tyr Leu Glu Met Ser Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 4 <211> 11 <212> PRT <213> Synthesis <400> 4 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 5 <211> 25 <212> PRT <213> Synthesis <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser 20 25 <210> 6 <211> 17 <212> PRT <213> Synthesis <400> 6 Asp Arg Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly 1 5 10 15 Val <210> 7 <211> 38 <212> PRT <213> Synthesis <400> 7 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile 1 5 10 15 Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Glu Pro Glu Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> 8 <211> 11 <212> PRT <213> Synthesis <400> 8 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 9 <211> 25 <212> PRT <213> Synthesis <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Val Ser 20 25 <210> 10 <211> 17 <212> PRT <213> Synthesis <400> 10 Leu Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val Ala 1 5 10 15 Gly <210> 11 <211> 38 <212> PRT <213> Synthesis <400> 11 Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala Leu Ser Arg Asp Asn 1 5 10 15 Gly Gln Asn Thr Val Tyr Leu Glu Met Ser Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 12 <211> 11 <212> PRT <213> Synthesis <400> 12 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 13 <211> 25 <212> PRT <213> Synthesis <400> 13 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser 20 25 <210> 14 <211> 17 <212> PRT <213> Synthesis <400> 14 Asp Arg Met Val Trp Phe Arg Pro Val Pro Glu Lys Glu Arg Glu Gly 1 5 10 15 Val <210> 15 <211> 38 <212> PRT <213> Synthesis <400> 15 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile 1 5 10 15 Gly Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> 16 <211> 11 <212> PRT <213> Synthesis <400> 16 Trp Gly Gln Gly Thr His Val Thr Val Ser Ser 1 5 10 <210> 17 <211> 25 <212> PRT <213> Synthetic <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Thr Ala Ser 20 25 <210> 18 <211> 17 <212> PRT <213> Synthetic <400> 18 Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val Gly 1 5 10 15 Cys <210> 19 <211> 38 <212> PRT <213> Synthetic <400> 19 Val Tyr Ala Val Pro Val Glu Gly Arg Phe Thr Met Ser Arg Glu Asn 1 5 10 15 Arg Gln Asn Thr Val Tyr Leu Gln Met Asp Ser Leu Lys Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 20 <211> 10 <212> PRT <213> Synthesis <400> 20 Trp Gly Gln Gly Thr Gln Val Thr Val Ser 1 5 10 <210> twenty one <211> 8 <212> PRT <213> Synthesis <400> twenty one Gly Asp Thr Ser Ser Leu Asn Ile 1 5 <210> twenty two <211> 10 <212> PRT <213> Synthesis <400> twenty two Ile Tyr Thr Asp Ala Gly Thr Pro Thr Tyr 1 5 10 <210> twenty three <211> 19 <212> PRT <213> Synthesis <400> twenty three Ala Ala Gly Thr Met Pro Val Arg Ala Ala Thr Leu Arg Pro Ala Arg 1 5 10 15 Tyr Ile Tyr <210> twenty four <211> 8 <212> PRT <213> Synthesis <400> twenty four Gly Val Asp Tyr Asn Ser Arg Thr 1 5 <210> 25 <211> 10 <212> PRT <213> Synthesis <400> 25 Ala Ala Ile Tyr Thr Tyr Asn Asn Arg Thr 1 5 10 <210> 26 <211> 18 <212> PRT <213> Synthesis <400> 26 Ala Arg Asp Trp Arg Phe Ser Ser Gly Met Leu Asp Lys Asn Met Tyr 1 5 10 15 Lys Tyr <210> 27 <211> 8 <212> PRT <213> Synthesis <400> 27 Gly Asp Thr Ser Thr Leu Asn Ile 1 5 <210> 28 <211> 10 <212> PRT <213> Synthesis <400> 28 Ile Tyr Phe Ala Ala Gly Thr Pro Thr Tyr 1 5 10 <210> 29 <211> 19 <212> PRT <213> Synthesis <400> 29 Ala Ala Gly Thr Met Pro Val Arg Ala Ala Thr Leu Arg Pro Ala Arg 1 5 10 15 Tyr Thr Tyr <210> 30 <211> 8 <212> PRT <213> Synthesis <400> 30 Gly Asp Asp Tyr Asn Leu Lys Thr 1 5 <210> 31 <211> 10 <212> PRT <213> Synthesis <400> 31 Ala Ser Ile Tyr Thr Tyr Asn Asn Thr Thr 1 5 10 <210> 32 <211> 18 <212> PRT <213> Synthesis <400> 32 Ala Arg Asp Trp Ser Phe Ser Ser Gly Met Leu His Lys Asn Met Tyr 1 5 10 15 Lys Tyr <210> 33 <211> 8 <212> PRT <213> Synthesis <400> 33 Gly Tyr Thr Ser Gly Ile Leu Tyr 1 5 <210> 34 <211> 8 <212> PRT <213> Synthesis <400> 34 Ile Tyr Pro Ser Gly Arg Ser Thr 1 5 <210> 35 <211> 16 <212> PRT <213> Synthesis <400> 35 Gly Ala Arg Pro Gln Ser Cys Gly Ala Pro Val Ser Thr Val Asp Tyr 1 5 10 15 <210> 36 <211> 128 <212> PRT <213> Synthetic <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Ala Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Val Ser Gly Asp Thr Ser Ser Leu Asn 20 25 30 Ile Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Gly Ile Tyr Thr Asp Ala Gly Thr Pro Thr Tyr Thr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Ala Phe Ser Arg Asp Asn Gly Gln Asn Thr 65 70 75 80 Val Tyr Leu Glu Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Ala Gly Thr Met Pro Val Arg Ala Ala Thr Leu Arg Pro 100 105 110 Ala Arg Tyr Ile Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 37 <211> 127 <212> PRT <213> Synthetic <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Val Asp Tyr Asn Ser Arg 20 25 30 Thr Asp Arg Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu 35 40 45 Gly Val Ala Ala Ile Tyr Thr Tyr Asn Asn Arg Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile Gly Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg Asp Trp Arg Phe Ser Ser Gly Met Leu Asp Lys Asn 100 105 110 Met Tyr Lys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 38 <211> 127 <212> PRT <213> Synthetic <400> 38 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly Asp Asp Tyr Asn Leu Lys 20 25 30 Thr Asp Arg Met Val Trp Phe Arg Pro Val Pro Glu Lys Glu Arg Glu 35 40 45 Gly Val Ala Ser Ile Tyr Thr Tyr Asn Asn Thr Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile Gly Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg Asp Trp Ser Phe Ser Ser Gly Met Leu His Lys Asn 100 105 110 Met Tyr Lys Tyr Trp Gly Gln Gly Thr His Val Thr Val Ser Ser 115 120 125 <210> 39 <211> 123 <212> PRT <213> Synthetic <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Tyr Thr Ser Gly Ile Leu 20 25 30 Tyr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Gly Cys Ile Tyr Pro Ser Gly Arg Ser Thr Val Tyr Ala Val Pro Val 50 55 60 Glu Gly Arg Phe Thr Met Ser Arg Glu Asn Arg Gln Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Ala Arg Pro Gln Ser Cys Gly Ala Pro Val Ser Thr Val Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 40 <211> 128 <212> PRT <213> Synthetic <400> 40 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Val Ser Gly Asp Thr Ser Thr Leu Asn 20 25 30 Ile Leu Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Gly Ile Tyr Phe Ala Ala Gly Thr Pro Thr Tyr Thr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Ala Leu Ser Arg Asp Asn Gly Gln Asn Thr 65 70 75 80 Val Tyr Leu Glu Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Ala Ala Gly Thr Met Pro Val Arg Ala Ala Thr Leu Arg Pro 100 105 110 Ala Arg Tyr Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 41 <211> 385 <212> DNA <213> Synthetic <400> 41 caggtgcagc tgcaggagtc tggaggaggc tcggcgcagg ctggagggtc tctgaggctc 60 tcctgtgaag tctctggaga cacctccagt ctgaacatcc tgggttggtt ccgccaggct 120 ccagggaagg agcgcgaggg ggtggcaggc atttatactg acgctggtac cccaacctat 180 acctatgccg actccgtgaa gggccgattc gccttctccc gtgacaacgg ccagaacacg 240 gtgtatcttg aaatgagcag cctgaagcct gaggacactg ccatgtacta ctgcgcggcg 300 ggtacgatgc ccgttcgggc ggccactctt cgccccgctc gttatattta ttggggccag 360 gggacccagg tcaccgtctc ctcag 385 <210> 42 <211> 382 <212> DNA <213> Synthetic <400> 42 caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtatag cttctggagt tgattacaat agtaggaccg atcgcatggc ttggttccgc 120 caggttccag ggaaggagcg cgagggagtg gcagccattt atacttataa taatagaaca 180 tattatgccg actccgtgaa gggccgattc accatctccc aagacattgg caagaatacg 240 gtatatctgc agatgaatag cctggaacct gaggacactg ccacatacta ctgtgcgcga 300 gattggcgtt tctcgagtgg gatgttggat aaaaatatgt ataagtactg gggccagggg 360 acccaggtca ccgtctcctc ag 382 <210> 43 <211> 215 <212> DNA <213> Synthetic <400> 43 aggtgcagct gcaggagtct gggggaggct cggtgcagcc tggagggtct ctgaggctct 60 cctgtgaagt ctctggagac acctccactc tgaacatcct gggttggttc cgccagactc 120 cagggaagga gcgcgagggg gtggcaggca tttattttgc cgctggtacc ccaacctata 180 cctatgccga ctccgtgaag ggccgattcg ccctc 215 <210> 44 <211> 381 <212> DNA <213> Synthetic <400> 44 caggtgcagc tgcaggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtatag cttctggaga tgattacaat cttaagaccg atcgcatggt ttggttccgt 120 ccggttccag agaaggagcg cgagggagtg gcatccattt atacttataa caatacaaca 180 tattatgccg actccgtgaa gggccgattc accatctccc aagacattgg caagaatacg 240 gtatatctgc aaatgaatag cctgaaacct gaggacactg ccacatacta ctgtgcgcga 300 gattggagtt tctcgagtgg gatgttgcat aaaaatatgt ataagtactg gggccagggg 360 acccacgtca ccgtctcctc a 381 <210> 45 <211> 370 <212> DNA <213> Synthetic <400> 45 caggtgcagc tgcaggagtc tggaggaggc tcggtgcagg ctggagggtc tctgaaactc 60 tcctgtacag cctctggata caccagcggt atcttgtaca tggcctggtt ccgccaggct 120 ccggggaagg agcgcgaggc ggtcggctgc atttatccca gtggacgtag cacagtctat 180 gccgtccccg tggagggccg attcaccatg tccagggaga accgccagaa cacggtgtat 240 ctgcagatgg acagcctgaa agctgaggac actgccgtgt actattgtgg ggcaaggccg 300 cagtcctgtg gagcccctgt ttctacggtt gactactggg gccaggggac ccaggtcacc 360 gtctcctcag 370

Claims

1. A VHH chain of a human PDL1 nanobody, comprising a framework region FR region and an antigen complementary determining region CDR region, characterized in that: The framework region FR region of the VHH chain of the human PDL1 Nanobody is derived from the amino acid sequence of its FR1-FR4: FR1 shown in SEQ ID No. 1, FR2 shown in SEQ ID No. 2, FR3 shown in SEQ ID No. 3, FR4 shown in SEQ ID No. 4; The antigen complementary determining region CDR region of the VHH chain of the human PDL1 nanobody is selected from the following CDR1-CDR3 amino acid sequences: CDR1 shown in SEQ ID No. 21, CDR2 shown in SEQ ID No. 22, and CDR3 shown in SEQ ID No. 23; The human PDL1 nanobody VHH chain has the amino acid sequence shown in SEQ ID No.

36.

2. A human PDL1 nanobody, characterized in that The human PDL1 nanobody is a nanobody targeting a human PDL1 molecular epitope and has a VHH chain with an amino acid sequence shown in SEQ ID No.

36.

3. A DNA molecule, characterized in that The DNA molecule encodes a protein selected from the following group: the VHH chain of a human PDL1 nanobody according to claim 1 or a human PDL1 nanobody according to claim 2.

4. The DNA molecule according to claim 3, characterized in that The DNA molecule has a DNA sequence selected from the following group: SEQ ID No.

41.

5. An expression vector, characterized in that: The expression vector contains the nucleotide sequence shown in SEQ ID No.

41.

6. A host cell, characterized in that The host cell comprises the expression vector according to claim 5.

7. Use of a human PDL1 nanobody, wherein the human PDL1 nanobody according to claim 2 is used to prepare a diagnostic kit for detecting PDL1 or a cancer therapeutic drug.

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