CN113234152B - Programmed death receptor-ligand 1 (PD-L1) specific binding polypeptide and application thereof - Google Patents

Abstract

The invention relates to a specific binding polypeptide aiming at a programmed death receptor-Ligand 1 (programmed cell death-Ligand 1, PD-L1) and application thereof, belonging to the technical field of biology. Wherein the polypeptide is obtained by mutating 1-20 amino acid residues from a Staphylococcal Protein A (SPA) immunoglobulin binding region (the polypeptide sequence is shown in SEQ ID: 2-5), and is capable of specifically binding to PD-L1 and blocking PD-1/PD-L1 interaction. The invention obtains the PD-L1 specific binding polypeptide coding sequence from the full-synthetic affinity small body polypeptide molecular library through four rounds of enrichment, and prepares a large amount of the obtained polypeptide by utilizing a prokaryotic expression system, wherein the polypeptide is specifically bound with PD-L1, has no obvious affinity with BSA protein, and can be used for PD-L1 detection. The polypeptide has PD-1/PD-L1 interaction blocking capacity, and the PD-1/PD-L1 interaction blocking capacity is enhanced with the increase of concentration.

Description

Programmed death receptor-ligand 1 (PD-L1) specific binding polypeptide and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a polypeptide specifically binding to a programmed death receptor-Ligand 1 (programmed cell death-Ligand 1, PD-L1), or a variant or a functional fragment thereof, and application of the polypeptide, or the variant or the functional fragment thereof.

Background

Immune checkpoints (immune checkpoint) are signaling pathways in the immune system that regulate the intensity of T cell immune responses by co-signaling (co-stimulatory or co-inhibitory) molecules, critical to maintaining autoimmune tolerance under normal physiological conditions. Apoptosis protein 1 (programmed cell death, PD-1) is an important negative regulatory molecule for immune cell activation that can exert immunosuppressive effects by binding to its ligand. PD-1 is predominantly expressed on the surface of activated effector T cells, regulatory T cells, B cells, monocytes, and natural killer T cells, but not on resting T cells, but can be detected within hours of T cell activation (Keir ME, button MJ, et al PD-1 and its ligands in tolerance and immunity.Annual Review of Immunology,2008, 26:677-704.). PD-1 has two ligands, namely PD-L1 and PD-L2. Among them, PD-L1 is widely expressed in T cells, B cells, monocytes, macrophages, various tumor cells, etc. The binding of PD-1 to PD-L1 inhibits proliferation, survival and effector functions of CD8+ cytotoxic lymphocytes (CTL) and promotes apoptosis of tumor-infiltrating lymphocytes (TIL) (Kim JW, nam KH, et al Prognosptic implications of immunosuppressive protein expression in tumors as well as immune cell infiltration within the tumor microenvironment in gastric Cancer,2016, 19 (1): 42-52). Many tumor cell lines and tumor cells attenuate the tumor immune response of the body by high expression of PD-L1, thereby causing tumor immune escape to occur.

In recent years, immune checkpoint blocking therapy targeting the PD-1/PD-L1 immune checkpoint has been extremely successful in the field of tumor immunotherapy, wherein PD-1/PD-L1 monoclonal antibody inhibitors have significant efficacy in tumor clinical therapy (Liu B, song Y, et al, receptor development in clinical applications of PD-1 and PD-L1 antibodies for cancer immunology. Journal of therapeutics & Oncology,2017, 10 (1): 174), including the observation of a significant tumor shrinkage, the generation of a durable immune response in tumor patients, etc., but as a four polypeptide chain large protein (molecular weight of-150 kDa), monoclonal antibodies have inherent disadvantages associated with their complex molecular structure: (1) complicated structure, difficult preparation, increased production cost, high treatment cost and limited wide application. (2) The stability is poor, the antibody is highly sensitive to external conditions, the activity of the antibody is lost due to some common external interference, and special transportation and storage conditions are required to ensure the activity of the antibody. (3) The molecules are larger, the capability of penetrating through tumor tissues is poor, the molecules cannot be effectively transported to the inside of the tumor tissues, the concentration of the medicine at the tumor site is very low, and the treatment effect on the solid tumor is poor.

Affinity body (affibody) molecules are a novel class of synthetic affinity ligands functionally similar to antibodies, derived from segment B of the immunoglobulin binding region of staphylococcal protein A (Staphylococcal protein A, SPA) (Nilsson B, moks T et al A synthetic IgG-binding domain based on staphylococcal protein A. Protein Engineering,1987,1 (2): 107-113). The Affibody molecule is a single-chain structure and consists of 58 amino acid residues, the relative molecular mass is about 6.5kDa, the structure contains 3 alpha-helices, the receptor binding site of the Affibody molecule contains 13 amino acid residues, Q9 (glutamine), Q10 (glutamine), N11 (asparagine), F13 (phenylalanine), Y14 (tyrosine), L17 (leucine), H18 (histidine) and E24 (glutamic acid), E25 (glutamic acid), R27 (arginine), N28 (asparagine), Q32 (glutamine) and K35 (lysine) of the second alpha-helix, the 13 amino acid residues are subjected to saturation mutation, the formed mutant forms a molecular library of the Affibody, and different Affibody molecules respectively have specific binding activity and high affinity (Nord K, nilsson J et al A combinatorial library of an alpha-helical bacterial receptor protein Engineering,1995,8 (6): 601-608) to different target molecules. Specific binding affibody molecules for a variety of important proteins have been successfully screened, including insulin, fibrin (fibrinogen), transferrin (TRF), tumor necrosis factor alpha (TNF-a), human serum albumin, interleukin-8 (IL-8), gpl20, CD28, igA, igE, igM, human epidermal growth factor receptor 2 (HER 2), and Epidermal Growth Factor Receptor (EGFR), among others. Compared with antibodies, the affinity small affibody has the advantages of small relative molecular weight, high stability, flexible application, easy preparation and the like, and is widely applied in a plurality of fields of bioscience. Therefore, developing PD-L1 specific binding affinity small body polypeptide lays an important material foundation and necessary data support for developing PD-1/PD-L1 immune checkpoint polypeptide blocking drugs and tumor PD-L1 non-invasive accurate detection.

Disclosure of Invention

The invention utilizes a full-synthetic affinity small-body polypeptide phage display technology platform to obtain a series of PD-L1 specific binding polypeptides through four rounds of enrichment and panning, and the polypeptide sequence is SEQ ID No:2-5. And preparing the PD-L1 specific binding polypeptide by using a prokaryotic escherichia coli expression system, and verifying the binding capacity and the binding specificity of the elutriated affinity small body polypeptide and the PD-L1 through ELISA binding experiments. The result shows that the obtained 4 affinity small polypeptides can be specifically combined with PD-L1, can effectively block the interaction of PD-1/PD-L1, and can be used for inhibiting the PD-1/PD-L1 immunosuppression signal path to strengthen immunity or detect PD-L1.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the polypeptide sequence specific to PD-L1 is obtained by mutating an immunoglobulin binding region of Staphylococcal Protein A (SPA) by 1-20 amino acid residues, and the polypeptide sequence is shown in SEQ ID No:2-5, has high PD-L1 specific affinity activity.

The PD-L1 specific binding polypeptide sequence comprises the polypeptide, or a variant or a functional fragment thereof, and any adjustment and modification to the polypeptide sequence by taking the polypeptide, or the variant or the functional fragment thereof as a core; modification materials include, but are not limited to, specific proteins, enzymes, fluorescent proteins, fluorescent luminescent groups, biotin, radiation-related groups, nanomaterials.

The invention also provides a preparation method of the PD-L1 specific binding polypeptide or a variant or a functional fragment thereof. One skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to the sequences of the invention to obtain variants of the sequences of the polypeptides or functional fragments thereof without substantially affecting the affinity properties of PD-L1. They are all considered to be included within the scope of the present invention.

The PD-L1 specific binding polypeptide sequences are useful in the detection of PD-L1, including but not limited to molecular imaging at the living, tissue water, cellular and subcellular levels.

An application of the polypeptide in PD-L1 detection.

Use of said polypeptide for inhibiting the PD-1/PD-L1 immunosuppressive signaling pathway to enhance immunity.

The invention has the advantages and positive effects that:

1. the PD-L1 specific binding polypeptide is selected from the full-synthetic affinity small body polypeptide molecular library through four rounds of enrichment based on phage display technology, and has the obvious advantages of low preparation cost, good stability and the like compared with a monoclonal antibody, and the specific affinity of the polypeptide is good.

2. The PD-L1 specific binding polypeptide or the variant or the functional fragment thereof obtained by the invention can be used as a specific recognition element of PD-L1 in molecular imaging detection of living body, tissue water, cells and subcellular level, and can also be used as a PD-1/PD-L1 immunosuppression signal path blocker for enhancing organism immunity.

Drawings

FIG. 1 shows the fold enrichment of each round during phage display screening for polypeptides that specifically bind to PD-L1.

FIG. 2 shows ELISA results of positive phage clones screened, and BSA as a negative control.

FIG. 3 shows the purification results of a polypeptide that specifically binds to PD-L1.

FIG. 4 shows the results of in vitro affinity and specificity verification of the specific binding of PD-L1 polypeptides to PD-L1.

FIG. 5 is a graph showing the result of blocking PD-1/PD-L1 interactions by specifically binding to a PD-L1 polypeptide.

FIG. 6 is a graph showing the results of blocking PD-1/PD-L1 interactions by binding specifically to PD-L1 polypeptides at different concentrations.

Detailed Description

The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way. The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like are all commercial products unless specified.

Example 1

Panning and identification of PD-L1 specific binding polypeptides.

1. Mutant construction and monoclonal selection

Amino acid residues 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32 and 35 of the immunoglobulin binding region polypeptide against staphylococcal protein A are designed with degenerate primers using NNK as codons, and the degenerate primers are used to obtain the encoding genes of the library of affinity small polypeptides by over-lap PCR. Cloning the polypeptide mutant encoding gene into phage display vector, and electrically transforming the recombinant plasmid into TG1 host cell to obtain 5X 10 library capacity ⁷ Is described. Affinity minibody Using helper phageAnd displaying the polypeptide mutant to obtain a polypeptide mutant phage display library.

Taking PD-L1 extracellular domain protein (purchased from Sino Biological, with the product number of 10084-H08H) as a target, adding an affinity small phage display library, performing four rounds of enrichment panning, enriching the phage captured in the fourth round by 200 times compared with the first round, collecting phage supernatant obtained by four rounds of enrichment, verifying the affinity of each round of phage to PD-L1 through an indirect ELISA experiment, and increasing the affinity of phage supernatant and PD-L1 round by round as shown in the attached figure 1.

2. Phage ELISA (enzyme Linked immunosorbent assay) for detecting binding capacity of polypeptide and PD-L1

Through four rounds of panning, single clone is selected, inoculated into 1mL 2 XYTAG culture medium (1.6 g tryptone, 1g yeast extract, 0.5g sodium chloride constant volume to 100mL, high pressure sterilization, and then added with 2% glucose and 100 mug/mL ampicillin for overnight culture at 37 ℃ and 180 rpm; the cultures were inoculated in 1:50 into 5mL of 2 XYTAG medium at 37℃and 180rpm to logarithmic phase; adding helper phage M13KO7 (purchased from New England Biolabs, cat#) according to the ratio of 1:20, standing at 37deg.C for 30min for infection, and shake culturing at 37deg.C at 180rpm for 30min; taking 1mL of bacteriophage culture, centrifuging at 7000rpm for 10min, and discarding the supernatant; the cells were resuspended in 5mL of 2 XYTAK medium (1.6 g tryptone, 1g yeast extract, 0.5g sodium chloride to 100mL, autoclaved; and then added with 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin) at a final concentration and incubated overnight at 30℃and 185 rpm. Centrifuging the culture at 10000rpm and 4 ℃ for 10min, and collecting the supernatant to obtain the phage monoclonal displaying the polypeptide mutant.

PD-L1 and BSA (negative control) were diluted to 5. Mu.g/mL with carbonate buffer at pH 9.6, and 96-well ELISA plates (available from Nunc, cat#) were coated overnight at 4 ℃; blocking with 3% skimmed milk powder-PBS, and standing at 37deg.C for 2 hr; adding the collected phage monoclonal supernatant, and incubating for 2 hours at room temperature; HRP-labeled anti-M13 antibody (ex Yiqiao Shenzhou, cat 11973-MM 05T-H) diluted 1:2000 was added and incubated for 2H at room temperature; adding TMB substrate for color development for 10min; add 2M H ₂ SO ₄ The reaction was terminated and OD450 was read on a microplate reader. In ELISA, each stepPlates were washed 4 times with PBST (PBS+0.5% Tween-20). For ELISA results, clones with positive values/negative values > 2.1 were selected as positive clones, and a total of 20 positive clones were selected, and specific data of each positive clone are shown in FIG. 2.

3. DNA sequencing

DNA sequencing of good positive phage clones, wherein individual clones are identical sequences, the different sequences are designated Z _{PD-L1 A2} ，Z _{PD-L1 D3} 、Z _{PD-L1 E3} 、Z _{PD-L1 GA2} 、Z _{PD-L1 GA2} 、Z _{PD-L1 GE2} 、Z _{PD-L1 P2} 、Z _{PD-L1 P10} 、Z _{PD-L1 P3} 、Z _{PD-L1 P9} The amino acid sequence can be obtained according to the DNA sequencing result and codon table.

Example 2

Expression and purification of PD-L1 specific binding polypeptides.

The 10 Affibody polypeptides identified in example 1 that specifically bind PD-L1 were subject for further investigation and large amounts of the polypeptide were expressed using a prokaryotic expression system. 10 Affibody coding genes are cloned to an expression vector to select pET-21b (stored in a laboratory), and whether the recombinant expression vector is successfully constructed is identified by a double enzyme digestion combined DNA sequencing technology. E.coli BL21 (DE 3) is selected as an expression host (preserved in the laboratory), a recombinant expression vector with correct sequencing is transformed into E.coli BL21 (DE 3), a single clone is selected to 5mL of LB/A culture medium (1 g of tryptone, 0.5g of yeast extract, 1g of sodium chloride to a volume of 100mL, the culture medium is autoclaved, and then 100 mu g/mL of ampicillin with a final concentration is added for overnight culture at 37 ℃ and 180 rpm; cultures were inoculated 1:50 into 200mL LB/A medium, incubated at 37℃at 180rpm to logarithmic phase, added with 0.1mM IPTG at final concentration, and induced overnight at 20℃at 180 rpm. Centrifuging the culture at 4 ℃ and 4400rpm to collect thalli; with 20mL of precooled PBS (8 g NaCl, na ₂ HPO ₄ ·12H ₂ O，0.2g KCl，0.24gNaH ₂ PO ₄ ·2H ₂ O) re-suspending the cells, sonicating (3 s more, 10s more) for 20min, centrifuging at 12000rpm for 15min, and collecting the supernatant.

Purification of Affibody polypeptide Ni-TED column (available from Biotechnology Co., ltd.)Cargo number C600803). First, the Ni-TED column was equilibrated with 10 column volumes of phosphate buffer; filtering the collected protein broken supernatant with a 0.45 μm filter membrane, loading, carrying out flow-through for 4 times, and reserving flow-through liquid for SDS-PAGE analysis; washing the column with phosphate buffer (20 column volumes) containing 20mM imidazole to wash off the contaminating proteins; phosphate buffers containing 50mM, 100mM, 250mM and 400mM imidazole were prepared, and the target protein was eluted with 2 column volumes, respectively. Performing SDS-PAGE analysis on protein samples collected by imidazole at different concentrations; according to SDS-PAGE result, selecting high purity protein, dialyzing with phosphate buffer, performing SDS-PAGE analysis of dialyzed protein, and purifying the 10 kinds of affinity small polypeptides shown in figure 3, wherein Z _{PD-L1 J1} ，Z _{PD-L1 J2} Is a positive control (laboratory existing PD-L1 specific affinity Affibody).

Example 3

Analysis of binding characteristics of PD-L1 specific binding polypeptides.

In this experiment, 8 PD-L1 specific binding Affibody polypeptides purified in example 2 were used as further subjects, their affinity to PD-L1 was detected by ELISA, and BSA was selected as an irrelevant antigen negative control, specifically as follows:

diluting PD-L1/BSA to 10 mug/mL by using carbonate buffer with pH of 9.6, coating a 96-well ELISA plate, and standing at 4 ℃ overnight; blocking with 3% skimmed milk powder-PBS, and standing at 37deg.C for 2 hr; adding Z with HA tag _{PD-L1 A2} ，Z _{PD-L1 D3} 、Z _{PD-L1 E3} 、Z _{PD-L1 GA2} 、Z _{PD-L1 GA2} 、Z _{PD-L1 GE2} 、Z _{PD-L1 P2} 、Z _{PD-L1 P10} 、Z _{PD-L1 P9} Incubating the recombinant protein at 37 ℃ for 2 hours; diluted HA antibody (purchased from bioengineering company, inc., cat No. D191044) was added and incubated at 37 ℃ for 2h; diluted HRP-labeled rabbit anti-mouse secondary antibody (purchased from bioengineering company, inc., cat No. D110098) was added and incubated at 37 ℃ for 45min; adding TMB substrate for color development for 10min; add 2M H ₂ SO ₄ Terminating the reaction, and reading OD in an ELISA reader ₄₅₀ . During ELISA, the plates were washed 4 times with PBST (PBS+0.5% Tween-20) for each step. The results are shown in FIG. 4. It can be seen that 8 Afsfibody has the specific binding capacity of PD-L1.

Example 4

PD-L1 specific binding to Affibody polypeptide PD-1/PD-L1 interaction blocking characterization.

Identification of the ability of PD-L1 to specifically bind to Affibody polypeptide to block PD-1/PD-L1 interactions

In this experiment, 8 PD-L1 specific binding Affibody polypeptides with PD-L1 specific binding capacity in example 3 were used as further subjects, and blocking of PD-1/PD-L1 interaction was detected by competition ELISA, and the specific procedures were as follows:

100. Mu.L (1. Mu.g/mL) of PD-1-Fc-His (purchased from Sino Biological under the trade designation 10377-H03H)/Fc-His (laboratory self-made) protein was pre-coated in a 96-well plate, overnight coated at 4 ℃; blocking with 5% skimmed milk powder-PBST, and standing at 37deg.C for 2 hr; PD-L1-His-biotin (available from Sino Biological under the trade designation 10084-H08H-B) and PD-L1 specifically bind to the Affibody polypeptide (0.5. Mu.g) were added at 37℃for 2H; HRP-streptavidin was added at 37℃for 45min. Adding TMB substrate for color development for 10min; the reaction was stopped by adding 2M H2SO4 and the OD was read on an ELISA reader ₄₅₀ . During ELISA, the plates were washed 4 times with PBST (PBS+0.5% Tween-20) for each step. The results are shown in FIG. 5.Z is Z _{PD-L1 P9} ，Z _{PD-L1 D3} 、Z _{PD-L1 E3} 、Z _{PD-L1 P2} Has good PD-1/PD-L1 interaction blocking capability.

Identification of PD-1/PD-L1 interaction blocking concentration dependence of PD-L1 specific binding to Affibody polypeptide

In this experiment, Z is _{PD-L1 P9} ，Z _{PD-L1 D3} 、Z _{PD-L1 E3} 、Z _{PD-L1 P2} The polypeptide is taken as a further research object, the blocking of the interaction of the PD-1/PD-L1 by the Affibody polypeptides with different concentrations is detected by using a competition ELISA method, and the specific operation is as follows:

100. Mu.L (1. Mu.g/mL) of PD-1-Fc-His/Fc-His protein was pre-coated in 96-well plates overnight at 4 ℃; blocking with 5% skimmed milk powder-PBST, and standing at 37deg.C for 2 hr; adding PD-L1-His-biotin and PD-L1 specifically binding Affibody polypeptide (the addition amounts are 0.5, 1, 2, 4 and 8 mug respectively), and carrying out 2 hours at 37 ℃; HRP-streptavidin was added at 37℃for 45min. Display by adding TMB substrateColor for 10min; the reaction was stopped by adding 2M H2SO4 and the OD was read on an ELISA reader ₄₅₀ . During ELISA, the plates were washed 4 times with PBST (PBS+0.5% Tween-20) for each step. The results are shown in FIG. 6.Z is Z _{PD-L1 P9} ，Z _{PD-L1 D3} 、Z _{PD-L1 E3} 、Z _{PD-L1 P2} The blocking ability against PD-1/PD-L1 interactions increases with increasing concentration.

Claims (2)

1. A polypeptide that specifically binds to the extracellular domain of programmed death receptor-Ligand 1 (programmed cell death-Ligand 1, PD-L1) or a functional fragment thereof, said polypeptide that specifically binds to the extracellular domain of PD-L1 is obtained by mutating 13 amino acid residues of the immunoglobulin binding region of staphylococcal protein a (staphylococcal protein A, SPA), the amino acid sequence of which is set forth in SEQ ID NO: 4.

2. Use of the polypeptide of claim 1 for the preparation of a PD-L1 extracellular domain detection reagent or a medicament.

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