A shark nanobody against human PD-1 and its application

Abstract

The present invention relates to a shark nanobody against human PD-1 and its application, and belongs to the field of biotechnology. The shark nanobody against human PD-1 of the present invention is composed only of a heavy chain, and the heavy chain includes a heavy chain variable region, and the antibody heavy chain variable region vNAR includes CDR1 and CDR3; CDR1 is the amino acid residues 25-32 in the amino acid sequence shown in SEQ ID NO: 2; CDR3 is the amino acid residues 84-102 in the amino acid sequence shown in SEQ ID NO: 2. The present invention also provides an encoding nucleic acid, an expression vector and a host cell for a shark nanobody against human PD-1, and also provides a method for producing the nanobody of the present invention and its application. The nanobody of the present invention has the advantages of high affinity and high specificity, can effectively block the PD-1 / PD-L1 pathway, and has the potential to be applied in the treatment and diagnosis of tumors.

Description

Technical Field

[0001] The present invention relates to a shark nanobody against human PD-1 and an application thereof, belonging to the field of biotechnology. Background Art

[0002] Programmed death receptor-1 (PD-1) is an important immunosuppressive molecule expressed on the surface of B cells and mature T cells. The binding of PD-1 and PD-L1 produces a co-inhibitory signal, causing T cells to lose their ability to attack cancer cells. In tumor cells, tumor cells can regulate the expression of PD-L1. The expressed PD-L1 binds to PD-1 on the surface of T cells, causing T cells to lose their killing function and allowing tumor cells to grow unrestricted. Therefore, an antibody that can specifically bind to PD-1 and thus block the interaction between PD-1 and PD-L1 is needed. For example, an anti-PD-1 antibody can be used to specifically bind to PD-1, thereby blocking the interaction between PD-1 and PD-L1.

[0003] In the 1990s, foreign researchers discovered antibodies in the nurse shark that naturally lack the light chain portion of an antibody and contain only the heavy chain. These heavy chain variable regions, which specifically bind to antigens, are called vNARs. With a molecular weight of only 12-15 kDa, they are also called nanobodies. Compared to traditional monoclonal antibodies, shark nanobodies offer advantages such as smaller molecular weight, higher stability, stronger drugability, less complex modifications, and simpler preparation processes. They hold great promise for development in drug development, in vitro diagnostics, and immunoassays.

[0004] Structurally, vNAR has a compact structure and is the smallest antigen-binding unit in nature (<11kDa). In terms of stability, vNAR has high thermal stability and acid and alkali resistance, and maintains good stability under different temperature and pH conditions. In addition, the longer CDR3 region of vNAR enables it to exhibit high affinity and specificity in binding to antigens.

[0005] Based on this, the present invention proposes a new type of shark nanoantibody with the function of blocking the binding of PD-1 and PD-L1, which has the potential to be used in the treatment and diagnosis of tumors. Summary of the Invention

[0006] The main content of the present invention is to provide an anti-human PD-1 shark nanobody and its application, which can specifically recognize PD-1 and block the binding of PD-1 and PD-L1.

[0007] The technical solution of the present invention to solve the technical problem is as follows:

[0008] In the first aspect of the present invention, a shark nanobody against human PD-1 is provided, wherein the shark nanobody is composed only of a heavy chain, wherein the heavy chain includes a heavy chain variable region, and the heavy chain variable region vNAR contains CDR1 and CDR3; the amino acid sequence of CDR1 is shown in SEQ ID NO: 3, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 4, the sequence of SEQ ID NO: 3 is the amino acid residues 25-32 in the amino acid sequence shown in SEQ ID NO: 2; the sequence of SEQ ID NO: 4 is the amino acid residues 84-102 in the amino acid sequence shown in SEQ ID NO: 2.

[0009] Preferably, the amino acid sequence of the antibody heavy chain variable region vNAR is shown in SEQ ID NO: 2.

[0010] In the second aspect of the present invention, a nucleotide sequence encoding the anti-human PD-1 shark nanobody as described in the first aspect is provided.

[0011] Preferably, the nucleotide sequence is shown in SEQ ID NO: 1.

[0012] In the third aspect of the present invention, a vector having the nucleotide sequence as described in the second aspect is provided.

[0013] Preferably, the vector is pcDNA3.4.

[0014] In the fourth aspect of the present invention, a host cell containing the vector according to the third aspect is provided.

[0015] Preferably, the host cell is CHO-K1.

[0016] In the fifth aspect of the present invention, there is provided the use of the anti-human PD-1 shark nanobody as described in the first aspect in the preparation of a PD-1 detection reagent.

[0017] In the sixth aspect of the present invention, there is provided the use of the anti-human PD-1 shark nanobody as described in the first aspect in the preparation of a PD-1 and PD-L1 binding inhibitor.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] 1) The present invention selects the striped bamboo shark as a model animal for antibody preparation to prepare vNAR. It is not an endangered shark species, is small in size, and is easy to artificially breed, making it suitable for antibody development.

[0020] 2) The present invention obtains shark nanoantibodies targeting PD-1 through phage display technology. This antibody can specifically recognize PD-1 and block its binding to PD-L1, and has application prospects in the preparation of tumor therapeutic drugs and tumor detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Agarose gel electrophoresis of shark spleen RNA and nested PCR in Example 2. A is RNA, B is the first round of PCR, and C is the second round of PCR.

[0022] Figure 2 The results of monoclonal screening in Example 3, A is the first round of panning, B is the second round of panning, and C is the third round of panning.

[0023] Figure 3 The statistical results of sequencing of positive clones in Example 3 are shown.

[0024] Figure 4 This is a map of the antibody mammalian expression vector in Example 4.

[0025] Figure 5 The SDS-PAGE results of the purified antibody in Example 4, wherein R represents reducing conditions, NR represents non-reducing conditions, and M represents a marker.

[0026] Figure 6 This is the SEC-HPLC result of the purified antibody in Example 4, where A is 214 nM and B is 280 nM.

[0027] Figure 7 This is the binding of the purified antibody in Example 5 to the hPD-1 / His recombinant protein.

[0028] Figure 8 These are the SPR affinity test results of the purified antibodies in Example 6, where A is ANb-17-CD28-3LP-3, B is the positive control antibody Sintilimab, and C is the negative control antibody Anti-HEL IgG1 hFc.

[0029] Figure 9 This is the result of the purified antibody in Example 7 blocking the binding of PD-1 to PD-L1. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the examples. However, the present invention is not limited to the examples given. The methods used are conventional methods unless otherwise specified, and the reagents and materials used are commercially available unless otherwise specified.

[0031] In the present invention, the steps of the antibody preparation method are as follows: the present invention first immunizes the striped bamboo shark with hPD-1 / His recombinant protein for 6 times, and then takes the spleen of the striped bamboo shark; RNA is extracted and reverse transcribed from the spleen cells, and the heavy chain antibody variable region vNAR gene fragment is obtained by nested PCR method, the phagemid vector and the gene fragment are enzyme-cut and ligated, and transferred into Escherichia coli for amplification, and a phage library is constructed. Antibodies that specifically bind to the target antigen are screened by liquid phase panning, and then a mammalian system expression vector is constructed. The antibody is then expressed and purified through a mammalian cell expression system, and finally, a shark nanobody with high sensitivity and specificity against human PD-1 is obtained.

[0032] Example 1: Immunization of the striped bamboo shark

[0033] Anesthetic seawater was prepared by adding 500 μL of anesthetic to 10 L of water. The shark was placed in the seawater. When the shark became unresponsive or motionless, it was removed and its gills were wrapped with moist gauze. The immunization site was the left and right ventral fins. Immunization was performed once every 15 days for a total of 6 times. Each immunization was 100 μg of hPD-1 / His recombinant protein (Biointron, B2097) mixed with 100 μL of biphasic adjuvant. Whole blood and spleen were collected at an interval of two weeks after the sixth immunization.

[0034] Example 2: Construction of Shark Nanobody Immune Library

[0035] Splenic RNA was extracted by TRIZOL (thermofisher, 15596018), its integrity was checked by agarose gel electrophoresis, and reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 2690A). Primers were designed based on the conserved sequence of the vNAR of the striped bamboo shark, with SfiI restriction sites at both ends for cloning into a phagemid vector. The vNAR region encoding gene was amplified by nested PCR. The results are shown in Figure 2. Figure 1 The constructed plasmid was transformed into Escherichia coli SS320, and the phage M13K07 system was used for amplification and packaging, and the vNAR was displayed on the surface of the phage to form a shark nanobody library.

[0036] Example 3: Screening of shark nanobodies against PD-1

[0037] Using liquid-phase panning, three 30 μL aliquots of Dynabeads™ M-280 Streptavidin magnetic beads (Thermo Fisher Scientific, 11206D) were transferred to 1.5 mL EP tubes. The beads were washed once with 1 mL of 0.05% PBST, and then blocked with 1 mL of 5% non-fat powdered milk (Sangon Biotech, A600669-0250) at 25°C for 1 hour. 2E+12 CFU of phage were added to 1 mL of 5% non-fat powdered milk and blocked at 25°C for 1 hour. Another 250 μL aliquot of magnetic beads was transferred to a 1.5 mL EP tube, and 1 mL of PBS and 15 μg of biotinylated hPD-1 / His recombinant protein were added. The beads were incubated at 25°C for 1 hour, washed five times with 1 mL of 0.05% PBST, and blocked with 1 mL of 5% non-fat powdered milk at 25°C for 1 hour. Remove the blocking buffer from the 30 μL magnetic beads and incubate the blocked phage with three 30 μL aliquots of blank magnetic beads for 0.5 hours each to remove background interference. Add the background-removed phage supernatant to the blocked 250 μL magnetic beads and incubate at 25°C for 1 hour. Remove any unbound phage supernatant and wash the remaining beads 15 times with 1 mL of 0.05% PBST. Resuspend and add to a logarithmic-phase SS320 culture for amplification and use in the next round of panning.

[0038] After three rounds of pressure panning, one plate was selected from each round, for a total of 264 single clones from three plates for screening. The sample preparation process was as follows: 600 μL of 2×YT dual-antibody culture medium containing tetracycline and carbenicillin was added to a 96-well deep-well plate, and a single clone was inoculated. The culture was incubated at 220 rpm and 37°C for 3 hours. 100 μL of the bacterial solution was aspirated from each well and transferred to a new 96-well plate for future use. 100 μL of 2×YT dual-antibody culture medium and IPTG were then added to the original 96-well plate, and expression was induced overnight at 30°C. The next day, the supernatant was discarded by centrifugation, and the cells were disrupted with TES buffer (30 mM Tris-HCl, 0.5 M sucrose, 0.5 mM EDTA, pH = 8.0) to release the test fusion protein in the periplasmic space.

[0039] The monoclonal detection process is as follows: 1 μg / mL hPD-1 / His recombinant protein and 1 μg / mL BSA were coated on ELISA plates (Corning, 3590), respectively, and incubated at 4°C overnight. The next day, the plates were washed three times with 0.1% PBST, blocked with 3% BSA (Sangon, A500023) for 1 hour, and washed three times with 0.1% PBST. The prepared samples were added at 100 μL / well and incubated at 25°C for 1 hour. The plates were washed five times with 0.1% PBST, and mouse anti-flag, HRP, and mAb (Sigma, A8592) were added at a dilution of 1:10,000 and incubated at 25°C for 1 hour. 10μg / mL Anti-PD1 / hFc antibody Sintilimab (Biointron, B682101) was used as a positive control, Anti-HELIgG1 hFc (Biointron, B117901) was used as a negative control, and Goat Anti-Human IgG-Fc, HRP (Sigma, A0170) was used as the control secondary antibody at a dilution of 1:10000. After washing 5 times with 0.1% PBST, TMB (Makewonderbio, 1001) was added and color was developed at room temperature in the dark. Finally, the color development was stopped with stop solution (Beyotime, P0215). The absorbance value at a wavelength of 450nm was read on a microplate reader. We defined the clones with 3×blank (PD-1) < OD450 < 2×blank (BSA) as positive clones. The test results are as follows Figure 2 shown.

[0040] The results showed that 118 of the 264 monoclonal clones recognized PD-1. Sequencing analysis was performed on these 118 positive clones, and 5 unique sequences were obtained, among which Unique 1 was the dominant enriched clone. Figure 3 shown.

[0041] The dominant monoclone corresponding to Unique1 is Shark-PD1-6M-3LP-72, whose heavy chain variable region DNA sequence is SEQ ID NO: 1 and heavy chain variable region amino acid sequence is SEQ ID NO: 2.

[0042] In the amino acid sequence, amino acid residues 25-32 (ie, SEQ ID NO: 3) are vNAR CDR1, and amino acid residues 84-102 (ie, SEQ ID NO: 4) are vNAR CDR3.

[0043] Example 4: Mammalian expression and purification of anti-PD-1 shark nanobody (vNAR-hFc)

[0044] The mammalian expression vector pcDNA3.4 of the Shark-PD1-6M-3LP-72 nanobody in Example 3 was constructed as follows: Figure 4 , and then use this to prepare the plasmid. CHO-K1 cells were selected as the host cells for antibody expression, and the expression volume was 40 mL. The supernatant after expression was purified using a Protein A affinity chromatography column. The specific operation was as follows: 5.5 mL of CHO-K1 cells with a density of 7.2E+6 cells / mL and 4 generations of passages were taken, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded; 1.4 mL of electroporation solution was added to the centrifuge tube, mixed well, and then the plasmid was added; 1.0 mL of the mixture in the previous step was added to the electroporation cup, and then placed in the electroporator at 540V-580V for electroporation; two shaking flasks were prepared in advance, each containing 20 mL DMEM culture medium, the electroporated cells are evenly divided into the two shake flasks, and allowed to stand at room temperature for 30 minutes; the shake flasks are placed in a CO2 incubator at 37°C, a rotation speed of 250 rpm and a controlled CO2 concentration of 5.0% for culture. After 24 hours, 2 mL of feed is added and the culture is continued for 4 days; the Protein A affinity chromatography column is equilibrated with 1×PBS, and after the equilibration is completed, the sample (i.e., cell culture supernatant) is prepared, and the sample flow rate is set to 1.0 mL / min. After the sample is loaded, impurities are washed with 1×PBS, and then the antibodies bound to the affinity column are eluted with sodium acetate buffer at pH = 3.4. Finally, the high-concentration protein is aspirated into a dialysis bag and dialyzed in a beaker filled with 1×PBS to obtain high-purity expressed antibodies.

[0045] The purity was determined by SDS-PAGE and SEC-HPLC. Figure 5 and Figure 6 As shown in the figure, the purity of the antibodies reached more than 95%, which is relatively high.

[0046] Example 5 Binding of Antibodies to hPD-1 / His Recombinant Protein

[0047] 1 μg / mL hPD-1 / His recombinant protein was coated onto an ELISA plate overnight at 4°C. The plate was washed three times with 0.1% PBST, blocked with 3% BSA for 1 hour, and washed three times with 0.1% PBST. Shark-PD1-6M-3LP-72 nanoantibody and control antibody were diluted to 100 nM as the first well concentration, followed by a 4-fold serial dilution. The final well was blanked and incubated at 25°C for 1 hour. The plate was washed five times with 0.1% PBST. HRP-conjugated Goat anti-human IgG-Fc was used as the secondary antibody at a 1:10,000 dilution. The plate was incubated at 25°C for 0.5 hour, and washed five times with 0.1% PBST. 100 μL TMB was added to each well and the reaction was stopped at room temperature in the dark for 3-5 minutes. The absorbance was read at 450 nm using a microplate reader. The positive control was anti-human PD-1 (Sintilimab), and the negative control was anti-HEL, human IgG1Fc.

[0048] ELISA results Figure 7 As shown: Shark-PD1-6M-3LP-72 can bind to hPD-1 / His with an EC50 of 0.30 nM.

[0049] Example 6: SPR affinity detection of antibodies and hPD-1 / His

[0050] 1 μg / mL of purified antibody was injected into the experimental channel at a flow rate of 10 μL / min, and the capture capacity was approximately 257-510 RU. The hPD-1 / His recombinant protein was diluted 2-fold starting from 200 nM using HBS-EP+Buffer running buffer. The diluted hPD-1 / His recombinant protein was injected into the experimental channel and the reference channel at a flow rate of 30 μL / min. The binding and dissociation steps were all performed in running buffer. The ProteinA Chip (Cytiva, 29127556) needed to be regenerated with 10 mM Gly-HCl, pH = 1.5, at a flow rate of 30 μL / min for 30 seconds to wash away the undissociated analyte. The KD value of the sample was calculated using Biacore 8K analysis software. The reference channel was used for background subtraction, and the fitting model was 1:1.

[0051] The results are as follows Figure 8 As shown: The affinity of Shark-PD1-6M-3LP-72 is 4.80E-08M.

[0052] Example 7: Antibody blocking function detection

[0053] 1 μg / mL hPD-1 / His recombinant protein was coated onto an ELISA plate overnight at 4°C, washed three times with 0.1% PBST, blocked with 3% BSA for 1 hour, and washed three times with 0.1% PBST. Shark-PD1-6M-3LP-72 nanoantibody and control antibody were diluted to 200 nM as the first well concentration, and then diluted three-fold serially. The last well was blank and 50 μL / well was added to the ELISA plate. Then, hPD-L1 / mFc (Biointron, B1576) was added at a final concentration of 0.13 μg / mL at 50 μL / well. After mixing, the plate was incubated at 25°C for 1 hour and washed five times with 0.1% PBST. HRP-conjugated Goat Anti-Mouse IgG (Jackson, 115-035-164) was used as the secondary antibody at a dilution of 1:10,000. The plate was incubated at 25°C for 1 hour and washed five times with 0.1% PBST. Add 100 μL TMB to each well and react in the dark at room temperature for 3-5 minutes, then terminate the color development and read the absorbance at 450 nm using a microplate reader.

[0054] The results are as follows Figure 9 As shown, Shark-PD1-6M-3LP-72 can block the binding of PD-1 to PD-L1.

[0055] The above are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A shark nanobody against human PD-1, characterized in that: The shark nanobody is composed only of a heavy chain, wherein the heavy chain includes a heavy chain variable region, and the amino acid sequence of the heavy chain variable region vNAR is shown in SEQ ID NO:

2.

2. A polynucleotide encoding the shark nanobody against human PD-1 as claimed in claim 1.

3. A vector containing the polynucleotide according to claim 2.

4. The carrier according to claim 3, characterized in that The vector is pcDNA3.

4.

5. A host cell containing the vector according to claim 3.

6. The host cell according to claim 5, characterized in that The host cell is CHO-K1.

7. Use of the anti-human PD-1 shark nanobody according to claim 1 in the preparation of a PD-1 detection reagent.

8. Use of the anti-human PD-1 shark nanobody according to claim 1 in the preparation of therapeutic drugs or diagnostic reagents for tumors.

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