CN119306838B

CN119306838B - Shark nanobody for resisting human PD-1 and application thereof

Info

Publication number: CN119306838B
Application number: CN202411582891.6A
Authority: CN
Inventors: 向阳; 张迎娜; 丁丽; 王玉芳
Original assignee: Jiangsu Baiying Biotechnology Co ltd
Current assignee: Jiangsu Baiying Biotechnology Co ltd
Priority date: 2024-11-07
Filing date: 2024-11-07
Publication date: 2025-09-09
Anticipated expiration: 2044-11-07
Also published as: CN119306838A

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

Shark nanobody for resisting human PD-1 and application thereof

Technical Field

The invention relates to a shark nano-antibody for resisting human PD-1 and application thereof, belonging to the technical field of biology.

Background

Programmed death receptor-1 (PD-1) is an important immunosuppressive molecule, and can be expressed on the surfaces of B cells and mature T cells, and the combination of the PD-1 and the PD-L1 can generate co-suppression signals, so that the T cells lose the capability of attacking cancer cells, in tumor cells, the tumor cells can regulate the expression of the PD-L1, and the expressed PD-L1 is combined with the PD-1 on the surfaces of the T cells, so that the T cells lose the killing function, and the growth of the tumor cells is not limited. There is therefore a need for an antibody that specifically binds to PD-1 and thus blocks the interaction of PD-1 and PD-L1, e.g. by using an anti-PD-1 antibody to specifically bind to PD-1 and thus block the interaction of PD-1 with PD-L1.

In the 90 s of the 20 th century, foreign students found in nurse shark antibodies in which the light chain portion of the naturally deleted antibody contained only the heavy chain portion, the heavy chain variable region of which specifically bound antigen was designated vNAR. Because of its molecular weight of only 12-15kDa, it is also known as nanobody. Compared with the traditional monoclonal antibody, the shark nanobody has the advantages of small relative molecular mass, high stability, strong drug property, less complex modification, simple preparation process and the like, and has good development prospect in the fields of drug development, in-vitro diagnosis, immunodetection and the like.

Structurally, vNAR is the antigen binding unit with the smallest molecular weight (< 11 kDa) in nature, and is stable with higher thermal stability and acid and alkali resistance, maintains better stability under different temperature and pH conditions, and in addition, the longer CDR3 region of vNAR makes it show high affinity and specificity for antigen binding.

Based on the above, the invention provides a novel shark nano-antibody with the function of blocking the combination of PD-1 and PD-L1, and has potential of being applied to the field of tumor treatment and diagnosis.

Disclosure of Invention

The main content of the invention is to provide a shark nanobody for resisting human PD-1 and application thereof, which can specifically recognize PD-1 and block the combination of PD-1 and PD-L1.

The technical scheme for solving the technical problems is as follows:

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

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

In a second aspect of the invention, there is provided a nucleotide sequence encoding a shark nanobody against human PD-1 as described in the first aspect.

Preferably, the nucleotide sequence is shown as SEQ ID NO. 1.

In a third aspect of the invention there is provided a vector having a nucleotide sequence as described in the second aspect.

Preferably, the vector is pcDNA3.4.

In a fourth aspect of the invention there is provided a host cell comprising a vector as described in the third aspect.

Preferably, the host cell is CHO-K1.

In a fifth aspect of the invention there is provided the use of a shark nanobody against human PD-1 as described in the first aspect for the preparation of a PD-1 detection reagent.

In a sixth aspect of the invention there is provided the use of a shark nanobody against human PD-1 as described in the first aspect for the preparation of a PD-1 and PD-L1 binding blocker.

Compared with the prior art, the invention has the following technical effects:

1) The invention selects the striped zebra shark as a model animal for antibody preparation to prepare the vNAR, which does not belong to endangered shark species, has small size and easy artificial breeding, and is suitable for antibody development.

2) The invention obtains the shark nano antibody targeting PD-1 through phage display technology, and the antibody can specifically recognize PD-1 and block the combination with PD-L1, thereby having application prospects of tumor therapeutic drug preparation and tumor detection.

Drawings

FIG. 1 shows 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.

FIG. 2 shows the results of the monoclonal screening in example 3, A for the first round of panning, B for the second round of panning, and C for the third round of panning.

FIG. 3 shows the sequencing statistics of positive clones in example 3.

FIG. 4 is a map of an expression vector of the antibody nursing system in example 4.

FIG. 5 shows SDS-PAGE results of purified antibodies of example 4, wherein R is a reducing condition, NR is a non-reducing condition, and M is a marker.

FIG. 6 is a SEC-HPLC result of the purified antibody of example 4, wherein A is 214nM and B is 280nM.

FIG. 7 shows the binding of purified antibodies of example 5 to hPD-1/His recombinant proteins.

FIG. 8 shows the SPR affinity assay results for the purified antibodies of example 6, wherein A is ANb-17-CD28-3LP-3, B is the positive control antibody Sintillimab, and C is the negative control antibody Anti-HEL IgG1 hFc.

FIG. 9 is the result of blocking the binding of PD-1 to PD-L1 by the purified antibody of example 7.

Detailed Description

The present invention will be described in further detail with reference to examples. The 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 products unless otherwise specified.

The preparation method of the antibody comprises the steps of immunizing stripe bamboo sharks with hPD-1/His recombinant protein for 6 times, taking spleen of the stripe bamboo sharks after the immunization is finished, extracting RNA from spleen cells, carrying out reverse transcription, obtaining a heavy chain antibody variable region vNAR gene fragment by a nested PCR method, carrying out enzyme digestion connection on a phagemid vector and the gene fragment, transferring the phagemid vector and the gene fragment into escherichia coli for amplification, constructing a phage library, screening the antibody specifically combined with a target antigen by a liquid phase panning mode, constructing a mammalian expression vector, carrying out expression and purification of the antibody by a mammalian cell expression system, and finally obtaining the anti-human PD-1 shark nanobody with high sensitivity and specificity.

Example 1 immunization of striped Spot shark

The anesthetic seawater was prepared in a proportion of 500. Mu.L of anesthetic added to 10L of water, the shark was put into the anesthetic solution, taken out and covered with wet gauze after the shark became unresponsive or stationary, the immunized part was left and right ventral fins, immunized once every 15 days for 6 times, 100. Mu. g hPD-1/His recombinant protein (Biointron, B2097) each time was mixed with 100. Mu.L of biphasic adjuvant, whole blood was collected at two weeks after the sixth immunization, and spleen was taken.

EXAMPLE 2 construction of shark nanobody immune repertoire

Spleen RNA is extracted by a TRIZOL (thermofisher, 15596018) method, the integrity of the spleen RNA is detected by agarose gel electrophoresis, a reverse transcription kit (TaKaRa, 2690A) is adopted to carry out reverse transcription to obtain cDNA, a primer is designed according to a stripe bamboo shark vNAR conserved sequence, sfiI enzyme cutting sites cloned into phagemid vectors are arranged at two ends of the primer, and a coding gene of a vNAR region is obtained by nested PCR amplification, and the result is shown in figure 1. And (3) transforming the constructed plasmid into escherichia coli SS320, amplifying and packaging an auxiliary phage M13K07 system, and displaying the vNAR on the surface of phage to obtain the shark nano antibody library.

Example 3 screening of shark nanobody against PD-1

Three 30. Mu.L Dynabeads M-280 streptavidin beads (Thermo FisherScientific, 11206D) were pipetted into a 1.5mL EP tube using liquid phase panning, 1mL of 5% NON-fat Powdered Milk (Sangon Biotech, A600669-0250) were added after one wash with 1mL of 0.05% PBST, and blocked at 25℃for 1 hour. 2E+12C.F.U.phage was taken and blocked for 1 hour at 25℃with 1mL of 5% NON-fat Powdered Milk. Another 250. Mu.L of magnetic beads was placed in a 1.5mL EP tube, 1mL of LPBS, 15. Mu.g of biotin-labeled hPD-1/His recombinant protein was added, incubated at 25℃for 1 hour, 1mL of 0.05% PBST was washed 5 times, 1mL of 5% NON-fat Powdered Milk was added, and blocked at 25℃for 1 hour. The blocking solution in 30. Mu.L of magnetic beads was removed, and the blocked phage were incubated with 3 parts of 30. Mu.L of blank magnetic beads for 0.5 hour, respectively, in order to remove background interference. The phage supernatant from which the background was removed was added to the blocked 250. Mu.L magnetic beads and incubated at 25℃for 1 hour. Phage supernatant not bound to beads was removed and the remaining beads were washed 15 times with 1mL of 0.05% PBST, resuspended and added to the log phase bacterial fluid of SS320 for amplification for the next round of panning.

After three rounds of pressurized panning, 3 plates were picked up for each round of selection of 264 monoclonal antibodies, and sample preparation was performed by adding 600. Mu.L of 2 XYT dual-antibody medium containing tetracycline and carbenicillin to a 96-well deep-well plate, inoculating the monoclonal antibodies, culturing at 220rpm and 37℃for 3 hours, sucking 100. Mu.L of bacterial liquid from each well to a new 96-well plate for storage, and supplementing 100. Mu.L of 2 XYT dual-antibody medium and IPTG to the original 96-well plate for induction expression at 30℃overnight. The supernatant was discarded by the next day centrifugation, and the cells were disrupted with TES buffer (30 mM Tris-HCl,0.5M sucrose, 0.5mM EDTA, pH=8.0) to release the fusion protein to be assayed in the periplasmic space.

The monoclonal assay was performed by coating 1. Mu.g/mLhPD-1/His recombinant protein and 1. Mu.g/mLBSA, respectively, on an ELISA plate (Corning, 3590), overnight at 4 ℃, and then washing 3 times with 0.1% PBST, blocking 3% BSA (Sangon, A500023) for 1 hour, washing 3 times with 0.1% PBST, adding a sample prepared in advance, incubating at 100. Mu.L/well, 25℃for 1 hour, washing 5 times with 0.1% PBST, adding a Mouse-flag, HRP, mAb (Sigma, A8592), diluting 1:10000, and incubating at 25℃for 1 hour. 10 μg/mLAnti-PD1/hFc antibody Sintilimab (Biointron, B682101) served as positive control, anti-HEL IgG1 hFc (Biointron, B117901) served as negative control, and control antibody secondary antibody was diluted 1:10000 using GoatAnti-Human IgG-Fc, HRP (Sigma, A0170). 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 at 450nm was read by a microplate reader, and 3 Xblank (PD-1) < OD450<2 Xblank (BSA) clones were defined as positive clones. The detection results are shown in FIG. 2.

The results indicated that 118 of the 264 monoclonal clones recognized positive PD-1. Sequencing analysis was performed on these 118 positive clones to yield 5 Unique sequences, of which Unique 1 was the dominant rich clone, as shown in FIG. 3.

The dominant monoclonal corresponding to Unique1 is sharp-PD 1-6M-3LP-72, the 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.

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

Example 4 expression and purification of shark nanobody (vNAR-hFc) against PD-1 in a mammalian system

A mammalian expression vector pcDNA3.4 for the Shark-PD1-6M-3LP-72 nanobody of example 3 was constructed as shown in FIG. 4, and plasmids were prepared therefrom. The antibody expression host cell is prepared by selecting CHO-K1 cells with an expression volume of 40mL, purifying the expressed supernatant by using protein A affinity chromatography columns, and the specific operation is that 5.5mL of CHO-K1 cells with a density of 7.2E+6cells/mL and transmitted for 4 generations are taken, centrifuged at 1000rpm for 5min, the supernatant is discarded, 1.4mL of electrotransfer liquid is added into a centrifuge tube, the plasmid is added after mixing uniformly, 1.0mL of the mixed liquid in the last step is added into an electric shock cup, then the electric shock cup is placed into an electrotransfer instrument for electrotransfer with 540V-580V, two shake flasks are prepared in advance, each shake flask is filled with 20mL of DMEM culture medium, the shocked cells are evenly distributed into the two shake flasks, the shake flasks are kept at room temperature for 30min, the shake flasks are placed into a CO ₂ ℃ incubator for culture at a speed of 250rpm, the concentration of 5.0% CO ₂ is controlled, 2mL of the supernatant is added after the centrifugation is continued to be cultured for 4 days, the buffer solution is used for balancing the protein A by 1X, the buffer solution is added into PBS (PBS is added after the buffer solution is added into the buffer solution for eluting the buffer solution after the buffer solution is filled into the buffer solution for 1X 1.0mL, and the buffer solution is washed by the buffer solution is filled into the buffer solution for eluting the buffer solution after the buffer solution is filled into the buffer solution for 1.1.1X 1.3 min, and the buffer solution is washed by the buffer solution.

The purity of the antibody is measured by SDS-PAGE and SEC-HPLC, and the results are shown in FIG. 5 and FIG. 6 respectively, the purity of the antibody is over 95%, and the purity is higher.

Example 5 binding of antibodies to hPD-1/His recombinant proteins

1 Μg/mLhPD-1/His recombinant protein was coated onto an ELISA plate, 4℃overnight, 0.1% PBST plate washed 3 times, 3% BSA blocked for 1 hour, 0.1% PBST plate washed 3 times, sharp-PD 1-6M-3LP-72 nanobody and control antibody diluted to 100nM as first Kong Nongdu, 4-fold gradient diluted, end wells blank, 25℃incubation for 1 hour, 0.1% PBST plate washed 5 times, secondary antibodies were HRP-labeled Goat anti-human IgG-Fc,1:10000 dilution, 25℃incubation for 0.5 hours, 0.1% PBST plate washed 5 times. After 100 mu LTMB is added to each hole and reacts at room temperature for 3-5 minutes in a dark place, the color development is stopped, and the absorbance at the wavelength of 450nm is read by an enzyme-labeled instrument. Wherein the positive control is Anti-Human PD-1 (Sintilimab), and the negative control is Anti-HEL, human IgG1 Fc.

ELISA results are shown in FIG. 7. Shark-PD1-6M-3LP-72 binds hPD-1/His with an EC50 of 0.30nM.

Example 6 SPR affinity detection of antibodies with hPD-1/His

Purified antibodies of 1. Mu.g/mL were injected into the experimental channel at a flow rate of 10. Mu.L/min, respectively, with a capture amount of about 257-510RU. hPD-1/His recombinant protein was diluted 2-fold with HBS-EP+buffer running from 200 nM. The diluted hPD-1/His recombinant protein was injected into the experimental channel and the reference channel at a flow rate of 30. Mu.L/min, and the binding and dissociation steps were all performed in running buffer. The ProteinA Chip (Cytiva, 29127556) was regenerated with 10mM Gly-HCl pH=1.5 at a flow rate of 30. Mu.L/min for 30s, washing away undissociated analytes. The KD values of the samples were calculated using Biacore 8K analysis software. The reference channel was used for background subtraction, patterning model 1:1.

As a result, the affinity of the sharp-PD 1-6M-3LP-72 was 4.80E-08M, as shown in FIG. 8.

Example 7 detection of antibody blocking function

1. Mu.g/mLhPD-1/His recombinant protein was coated onto an ELISA plate, 4℃overnight, 0.1% PBST plate 3 times, 3% BSA blocked for 1 hour, 0.1% PBST plate 3 times, shark-PD1-6M-3LP-72 nanobody and control antibody diluted to 200nM as initial well concentration, 3-fold gradient dilution, end wells blank, 50. Mu.L/well added to the ELISA plate, hPD-L1/mFc (Biointron, B1576) at a final concentration of 0.13. Mu.g/mL, 50. Mu.L/well, 25℃incubation for 1 hour after mixing, 0.1% PBST plate 5 times, secondary antibodies incubated with HRP-labeled GoatAnti-MoIgG (Jackson, 115-035-164), 1:10000 dilution used, 25℃incubation for 1 hour, 0.1% PBST plate 5 times. After 100 mu LTMB is added to each hole and reacts at room temperature for 3-5 minutes in a dark place, the color development is stopped, and the absorbance at the wavelength of 450nm is read by an enzyme-labeled instrument.

As a result, as shown in FIG. 9, sharp-PD 1-6M-3LP-72 can block the binding of PD-1 to PD-L1.

The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in other related technical fields are included in the 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.

The vector according to claim 3 , wherein the vector is pcDNA3.4.

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

The host cell according to claim 5 , wherein 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.