Anti-human PD-1 shark nano antibody and application thereof

Abstract

The invention relates to an anti-human PD-1 shark nano antibody and application thereof, and belongs to the technical field of biology. The anti-human PD-1 shark nano antibody provided by the invention is only composed of a heavy chain, the heavy chain comprises a heavy chain variable region, and the antibody heavy chain variable region vNAR comprises CDR1 and CDR3; cDR1 is amino acid residues 25-32 in the amino acid sequence as shown in SEQ ID NO: 2; cDR3 is amino acid residues 84-102 in the amino acid sequence as shown in SEQ ID NO: 2. The invention also provides a coding nucleic acid, an expression vector, a host cell and the like of the anti-human PD-1 shark nano antibody, and also provides a production method and application of the nano antibody. The nano antibody has the advantages of high affinity, high specificity and the like, can effectively block a PD-1 / PD-L1 pathway, and has the potential of being applied to the field of tumor treatment and diagnosis.

Description

Technical Field

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

[0002] Programmed death receptor-1 (PD-1) is an important immunosuppressive molecule that can be expressed on the surface of B cells and mature T cells. The combination of PD-1 and PD-L1 will produce a co-inhibitory signal, causing T cells to lose the 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, making the growth of tumor cells unrestricted. Therefore, an antibody that can specifically bind to PD-1 to block the interaction between PD-1 and PD-L1 is needed, such as using anti-PD-1 antibodies to specifically bind to PD-1, thereby blocking the interaction between PD-1 and PD-L1.

[0003] In the 1990s, foreign scholars discovered antibodies in nurse sharks that naturally lacked the light chain part of antibodies and only contained the heavy chain part. The heavy chain variable region that specifically binds to the antigen is called vNAR. Because its molecular weight is only 12-15kDa, it is also called nanoantibody. Compared with traditional monoclonal antibodies, shark nanoantibodies have the advantages of small relative molecular mass, high stability, strong drugability, less complex modification, and simple preparation process. They show good development prospects in the fields of drug development, in vitro diagnosis, and immunoassay.

[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 for antigen binding.

[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 applied 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, wherein the sequence of SEQ ID NO: 3 is amino acid residues 25-32 in the amino acid sequence shown in SEQ ID NO: 2; and the sequence of SEQ ID NO: 4 is 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 as 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 as 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 blocker.

[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. The bamboo shark is not an endangered shark species, is small in size, and is easy to breed artificially, making it suitable for antibody development.

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

[0021] Figure 1 This is the 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 This is the statistical result of sequencing of positive clones in Example 3.

[0024] Figure 4 This is a map of the antibody mammalian system 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 The SEC-HPLC results of the purified antibody in Example 4, wherein 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, wherein 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] Fig. 9 This is the result of the purified antibody in Example 7 blocking the binding of PD-1 and 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 after the immunization; extracts RNA from the spleen cells and reverse transcribes it, obtains the heavy chain antibody variable region vNAR gene fragment by the nested PCR method, enzyme-cuts and connects the phagemid vector and the gene fragment, and transfers them into Escherichia coli for amplification, constructs a phage library, screens antibodies that specifically bind to the target antigen by liquid phase panning, constructs a mammalian system expression vector, and then expresses and purifies the antibody by a mammalian cell expression system, and finally obtains a shark nanoantibody with high sensitivity and specificity against human PD-1.

[0032] Example 1: Immunization of 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 until it became unresponsive or motionless, then taken out and its gills were wrapped with moist gauze. The immunization site was the left and right pelvic fins. Immunization was performed once every 15 days, for a total of 6 times. Each time, 100 μg of hPD-1 / His recombinant protein (Biointron, B2097) was mixed with 100 μL of biphasic adjuvant. Whole blood was collected and the spleen was taken at intervals of two weeks after the sixth immunization.

[0034] Example 2: Construction of shark nanoantibody immune library

[0035] Spleen RNA was extracted by TRIZOL (thermofisher, 15596018) and its integrity was checked by agarose gel electrophoresis. It was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 2690A). Primers were designed based on the conservative sequence of vNAR of bamboo shark. Both ends of the primers contained SfiI restriction sites for cloning into phagemid vectors. The vNAR region encoding gene was amplified by nested PCR. The results are shown in Figure 1 The constructed plasmid was transformed into Escherichia coli SS320, assisted by the bacteriophage M13K07 system for amplification and packaging, and vNAR was displayed on the surface of the phage to form a shark nanoantibody library.

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

[0037] Using the liquid phase panning method, three 30 μL DynabeadsTM M-280 streptavidin magnetic beads (Thermo Fisher Scientific, 11206D) were taken into a 1.5 mL EP tube, washed once with 1 mL 0.05% PBST, and then 1 mL of 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250) was added, and blocked at 25°C for 1 hour. Take 2E+12C.FU of phage, add 1 mL 5% NON-fat Powdered Milk, and block at 25°C for 1 hour. Take another 250 μL magnetic beads into a 1.5 mL EP tube, add 1 mL PBS, 15 μg biotin-labeled hPD-1 / His recombinant protein, incubate at 25°C for 1 hour, wash 5 times with 1 mL 0.05% PBST, add 1 mL 5% NON-fat Powdered Milk, and block at 25°C for 1 hour. Remove the blocking solution from the 30 μL magnetic beads, and incubate the blocked phage with three portions of 30 μL blank magnetic beads for 0.5 hours 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 the phage supernatant that is not bound to the magnetic beads, wash the remaining magnetic beads 15 times with 1 mL of 0.05% PBST, resuspend them, and add them to the logarithmic phase bacterial solution of SS320 for amplification for the next round of panning.

[0038] After three rounds of pressure selection, 1 plate was selected in each round, and a total of 264 monoclones were selected from 3 plates for screening. The sample preparation process was as follows: 600 μL 2×YT dual-antibody culture medium containing tetracycline and carbenicillin was added to a 96-well deep-well plate, and a monoclonal clone was inoculated. The culture was cultured at 220 rpm and 37°C for 3 hours. 100 μL of bacterial solution was aspirated from each well to a new 96-well plate for storage, and then 100 μL 2×YT dual-antibody culture medium and IPTG were 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 bacteria were broken with TESbuffer (30 mM Tris-HCl, 0.5 M sucrose, 0.5 mM EDTA, pH = 8.0) to release the fusion protein to be tested in the periplasmic space.

[0039] The detection process of monoclonal was 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, washed three times with 0.1% PBST, and the prepared samples were added at 100 μL / well, incubated at 25°C for 1 hour, washed five times with 0.1% PBST, and Mouse anti-flag, HRP, mAb (Sigma, A8592) were added, diluted 1:10000, 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 a control antibody secondary antibody, diluted 1:10000. After washing 5 times with 0.1% PBST, TMB (Makewonderbio, 1001) was added to develop color at room temperature in the dark, and finally the color development was stopped with stop solution (Beyotime, P0215). The absorbance value at a wavelength of 450nm was read with an enzyme 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 there were 118 positive clones that recognized PD-1 among the 264 monoclonal clones. These 118 positive clones were sequenced and analyzed to obtain 5 unique sequences, 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 the 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: Expression and purification of anti-PD-1 shark nanobody (vNAR-hFc) in mammalian system

[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 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 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 mixed solution in the previous step was added to the electroporation cup, and then placed in the electroporator at 540V-580V for electroporation; two shake flasks were prepared in advance, each containing 20 mL of DMEM culture medium, and the electroporated cells were evenly divided into the two shake flasks, and left to stand at room temperature for 30 minutes; the shake flasks were placed in CO 2 The incubator was set at 37°C, 250 rpm, and 5.0% CO 2 The cells were cultured at a concentration of 1.5447 W / m2 and 2 mL of feed was added after 24 h, and the culture was continued for 4 days. The Protein A affinity chromatography column was equilibrated with 1×PBS, and the sample (i.e., cell culture supernatant) was prepared after the equilibration. The sample loading flow rate was set to 1.0 mL / min. After the sample loading, impurities were washed with 1×PBS, and the antibodies bound to the affinity column were eluted with sodium acetate buffer at pH=3.4. Finally, the high-concentration protein was aspirated into a dialysis bag and dialyzed in a beaker filled with 1×PBS to obtain a highly pure expressed antibody.

[0045] The purity was measured by SDS-PAGE and SEC-HPLC, and the results were as follows: Figure 5 and Figure 6 As shown, 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] 1μg / mL hPD-1 / His recombinant protein was coated on the ELISA plate, overnight at 4℃, washed 3 times with 0.1% PBST, blocked with 3% BSA for 1 hour, washed 3 times with 0.1% PBST, diluted Shark-PD1-6M-3LP-72 nanoantibody and control antibody to 100nM as the first well concentration, 4-fold gradient dilution, the last well was blank, incubated at 25℃ for 1 hour, washed 5 times with 0.1% PBST, and the secondary antibody was HRP-labeled Goat anti-human IgG-Fc, diluted 1:10000, incubated at 25℃ for 0.5 hour, and washed 5 times with 0.1% PBST. Add 100μLTMB to each well and react at room temperature in the dark for 3-5 minutes to terminate the color development, and read the absorbance at 450nm wavelength with an ELISA reader. The positive control is Anti-Human PD-1 (Sintilimab) and the negative control is 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 amount 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, and the binding and dissociation steps were all performed in the running buffer. ProteinA Chip (Cytiva, 29127556) needs to be regenerated with 10 mM Gly-HCl at pH = 1.5 at a flow rate of 30 μL / min for 30 s 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, Fitting model 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 on the ELISA plate, incubated overnight at 4°C, washed 3 times with 0.1% PBST, blocked for 1 hour with 3% BSA, and washed 3 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 3-fold gradient dilution was performed. The last well was blank and 50 μL / well was added to the ELISA plate. Then hPD-L1 / mFc (Biointron, B1576) with a final concentration of 0.13 μg / mL was added at 50 μL / well. After mixing, the plate was incubated at 25°C for 1 hour, and the plate was washed 5 times with 0.1% PBST. The secondary antibody was HRP-labeled Goat Anti-Mouse IgG (Jackson, 115-035-164), diluted 1:10000, incubated at 25°C for 1 hour, and washed 5 times with 0.1% PBST. Add 100 μL TMB to each well and react at room temperature in the dark for 3-5 minutes to terminate the color development, and read the absorbance at 450 nm using an enzyme reader.

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

[0055] The above are only 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 directly or indirectly applied 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 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.

2. The anti-human PD-1 shark nanobody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region vNAR is shown in SEQ ID NO:

2.

3. A nucleotide sequence encoding the shark nanobody against human PD-1 as claimed in claim 1 or 2.

4. A vector containing the nucleotide sequence as claimed in claim 3.

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

4.

6. A host cell containing the vector according to claim 4.

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

8. Use of the anti-human PD-1 shark nanobody as claimed in claim 1 or 2 in the preparation of a PD-1 detection reagent.

9. Use of the anti-human PD-1 shark nanobody as claimed in claim 1 or 2 in the preparation of a PD-1 and PD-L1 binding inhibitor.

10. Use of the anti-human PD-1 shark nanobody according to claim 1 or 2 in the preparation of therapeutic drugs or diagnostic reagents for tumors.

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