Non-antibody binding polypeptide or its derivatives for dual targeting of PD-1 receptor and its ligand PD-L1 and their applications

Abstract

The present invention discloses a non-antibody binding polypeptide or a derivative thereof for a PD-1 receptor, wherein the non-antibody binding polypeptide comprises a main chain and at least one branch chain connected to the main chain, the amino acid sequence of the branch chain is shown in SEQ ID NO.3, and the amino acid sequence of the main chain is shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.7. The present invention also provides a polynucleotide encoding the non-antibody binding polypeptide, a recombinant vector, and a host cell. The present invention also provides a preparation, which comprises the non-antibody binding polypeptide or a derivative thereof, and the preparation is a drug or a cell identification reagent. The present invention also provides the use of the non-antibody binding polypeptide or its derivative for the preparation of a tumor detection agent, an immunomodulatory drug, and an anti-tumor drug. The non-antibody binding polypeptide provided by the present invention has a small molecular weight, good permeability, can be artificially synthesized, has low cost, is not animal-derived, has high safety, has good binding to specific molecules, and has functions similar to or even superior to monoclonal antibodies.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a non-antibody binding polypeptide of a PD-1 receptor or a derivative thereof and an application thereof. Background Art

[0002] Programmed cell death receptor-1 (PD-1) is a membrane protein receptor with 288 amino acid residues, which is expressed in various immune cells such as T cells, B cells and myeloid cells. PD-1 has programmed cell death-ligand 1 (PD-L1) and programmed cell death-ligand 2 (PD-L2); the binding of PD-1 to its ligand upregulates E3-ubiquitin ligases CBL-b and c-CBL, downregulates T cell receptor expression, and inhibits T cell activation and cytokine release. Immune regulation targeting PD-1 is of great significance in anti-tumor, anti-infection, anti-autoimmune disease and organ transplant survival.

[0003] PD-1 and its ligands negatively regulate immune responses. Experiments have shown that PD-1 knockout mice are more susceptible to lupus-like glomerulonephritis and dilated cardiomyopathy. The combination of PD-1 and PD-L1 initiates programmed cell death of T cells and inhibits the secretion of interleukin-2 (IL-2) and interferon IFN-γ. Experimental studies on viral LCMV infection have shown that the interaction between PD-1 and PD-L1 inhibits the activation and proliferation of virus-specific CD8 cells, while antibody drugs block the binding of PD-L1 and PD-1, increase IL-2 secretion, and reverse the negative regulatory effect of CD8 cells.

[0004] PD-1's ligand PD-L1 is highly expressed in many cancer cells. PD-L1 / PD-1 is a key signaling pathway for cancer cells to escape immune responses. Inhibiting the interaction between PD-1 and PD-L1 can enhance the anti-tumor activity of T cells in vitro. Currently, nine antibody drugs are on the market. Nivolumab, Pembrolizumab, Cemiplimab, Toripalimab, Cindilimab, and Camrelizumab, which target PD-1, are all humanized or fully human immunoglobulin G (4IgG4) antibodies; Atezolizumab, Avelumab, and Durvalumab, which target PD-L1, are humanized or fully human IgG1 antibodies. No PD-1 and PD-L1 dual-targeting drugs are on the market.

[0005] Affinity is one of the important factors affecting the efficacy, dosage and toxic side effects of molecular targeted drugs. It is determined by the binding region, binding area, and the suitability of the three-dimensional configuration of the concave and convex binding. Affinity can be characterized by the dissociation constant (Kd). The smaller the Kd value, the greater the affinity. The main binding site of nivolumab is the "N-ring" structure of PD-1, with a binding area of ​​approximately 2, Kd is 1.45nmol / L; the main binding site of pembrolizumab is the C′D ring structure, and the binding area is about 2, Kd value is 27pmol / L; Kd value of simiprilimab is 6.11nmol / L; Teplilimab and sintilimab mainly bind to the FG ring, and the binding areas are 2 and 2, and the Kd values ​​were 0.324 nmol / L and 0.25 nmol / L, respectively; Carrelizumab mainly binds to the CC′ and FG loops, with a binding area of 2, with a Kd value of 3.31 nmol / L. Atezolizumab binds to the pre-β-fold of PD-L1 through three CDR loops from the heavy chain and one CDR loop from the light chain, i.e., binding is dominated by residues covering the BC, CC′, C′C″ and FG loops, with a binding area of 2, Kd value 400pmol / L; the binding epitope region of avelumab is mainly composed of the C chain, C' chain, F chain, G chain and CC' loop of PD-L1, with a binding area of 2, Kd value is 42.1pmol / L; the binding area of ​​durvalumab is the CC′ loop and N-terminal region on PD-L1, and the binding area is 2. Kd value is 667pmol / L.

[0006] Chinese patent ZL201710324664.7 discloses a non-antibody binding protein PD-1-nABP284 that binds to the PD-1 receptor, which can specifically bind to the PD-1 receptor. In vitro experiments show that nABP284 blocks the binding of the PD-L1 ligand to PD-1, enhances the IL-2 secretion of T lymphocytes and the killing effect of cancer cells, and inhibits tumor growth in mice. Compared with monoclonal antibodies, non-antibody binding peptides are easy to synthesize and control quality, rarely induce immunogenicity, have good tumor tissue targeting and penetration ability, and have low production costs. However, the affinity of nABP284 to the PD-1 receptor is relatively weak, and no experiments have shown that nABP284 has the ability to bind to the PD-1 ligand PD-L1. Summary of the invention

[0007] One object of the present invention is to provide a non-antibody binding polypeptide of the PD-1 receptor that can significantly improve the affinity with PD-1 and the blocking effect on PD-L1, improve the secretion of interleukin-2 of immune cells and the ability to kill cancer cells, in response to the above technical problems to be solved.

[0008] Another object of the present invention is to provide the use of the non-antibody binding polypeptide.

[0009] In order to achieve the above invention objectives, the present invention provides a non-antibody binding polypeptide or a derivative thereof for a PD-1 receptor, wherein the non-antibody binding polypeptide comprises a main chain and at least one branch chain connected to the main chain, the amino acid sequence of the branch chain is shown in SEQ ID NO.3, and the amino acid sequence of the main chain is shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.7. The amino acid L (Leu) of at least one (such as one, two, three, etc.) branch chain (SHHHRL) is connected to the amino acid K (Lys) on the main chain, thereby realizing the connection between the main chain and the branch chain. Preferably, the amino acid sequence of the main chain of the non-antibody binding polypeptide is as shown in SEQ ID NO.1, the amino acid sequence of the side chain is as shown in SEQ ID NO.3 (the non-antibody binding polypeptide can be named nABP386), and the amino acid L in the side chain is connected to the amino acid K at position 27 in the main chain; or, the amino acid sequence of the main chain of the non-antibody binding polypeptide is as shown in SEQ ID NO.2, the amino acid sequence of the side chain is as shown in SEQ ID NO.3 (the non-antibody binding polypeptide can be named nABP486), and the amino acid L in the side chain is connected to the amino acid K at position 7 in the main chain. Alternatively, the amino acid sequence of the main chain of the non-antibody binding polypeptide is as shown in SEQ ID NO.7, the amino acid sequence of the side chain is as shown in SEQ ID NO.3 (the non-antibody binding polypeptide can be named nABP786), and the amino acid L in the side chain is connected to the amino acid K at position 7 in the main chain.

[0010] The present invention also provides a polynucleotide encoding the non-antibody binding polypeptide.

[0011] Preferably, the nucleotide sequence encoding the main chain of the non-antibody binding polypeptide is shown as SEQ ID NO.4, and the nucleotide sequence encoding the side chain of the non-antibody binding polypeptide is shown as SEQ ID NO.6.

[0012] Preferably, the nucleotide sequence encoding the main chain of the non-antibody binding polypeptide is shown as SEQ ID NO.5, and the nucleotide sequence encoding the side chain of the non-antibody binding polypeptide is shown as SEQ ID NO.6.

[0013] Preferably, the nucleotide sequence encoding the main chain of the non-antibody binding polypeptide is shown as SEQ ID NO.8, and the nucleotide sequence encoding the side chain of the non-antibody binding polypeptide is shown as SEQ ID NO.6.

[0014] The present invention also provides a recombinant vector comprising the polynucleotide.

[0015] The present invention also provides a host cell, which comprises the recombinant vector.

[0016] The present invention also provides a preparation, which comprises the non-antibody binding polypeptide or a derivative thereof, and the preparation is a drug or a cell marking reagent.

[0017] Preferably, when the preparation is a drug, it also includes pharmaceutically acceptable excipients or carriers; when the preparation is a cell identification reagent, it also includes molecular identification acceptable carriers or excipients.

[0018] Preferably, the preparation comprises a compound formed by connecting a non-antibody binding polypeptide or its derivatives to a drug molecule and / or a detectable label; the amino acid sequence of the main chain of the non-antibody binding polypeptide is as shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.7, and the amino acid sequence of the side chain is as shown in SEQ ID NO.3.

[0019] Preferably, the drug molecule has the function of preventing and / or treating tumors and autoimmune diseases with activated PD-1 immune checkpoints. The tumor is preferably a tumor with upregulated PD-L1 expression, including but not limited to melanoma, non-small cell lung cancer, breast cancer, soft tissue sarcoma, and head and neck tumors.

[0020] Preferably, the detectable label comprises a combination of one or more of a fluorescent agent, a nuclear agent or a radioactive agent.

[0021] The present invention also provides the use of the non-antibody binding polypeptide or its derivatives for preparing tumor and immune disease detection agents. Preferably, the tumor and immune disease are diseases with abnormal expression of PD-L1 and PD-1.

[0022] The present invention also provides the use of the non-antibody binding polypeptide or its derivatives for binding to a marker or preparation of PD-1 molecules or PD-1 positive cells.

[0023] The present invention also provides the use of the non-antibody binding polypeptide or its derivative in the preparation of a preparation for marking, identifying, enriching, sorting or purifying PD-1 positive cells. Preferably, the PD-1 molecule is from total cell protein, cell secretory protein or living cell surface. The PD-1 positive cells include but are not limited to T cells, NK cells and leukemia cells.

[0024] The present invention also provides the use of the non-antibody binding polypeptide or its derivatives for preparing immunomodulatory drugs.

[0025] In particular, the present invention also provides the use of the non-antibody binding polypeptide in the preparation of an immunomodulatory drug for in vivo treatment of diseases with increased PD-L1 secretion; or the use of the non-antibody binding protein in the preparation of a preparation for enhancing the tumor killing effect of immune cells cultured in vitro.

[0026] The present invention also provides the use of the non-antibody binding polypeptide or its derivatives for preparing anti-tumor drugs. The tumor is preferably a tumor with upregulated PD-L1 expression, including but not limited to melanoma, non-small cell lung cancer, breast cancer, soft tissue sarcoma, head and neck tumors. Preferably, the present invention also provides the use of the non-antibody binding polypeptide or its derivatives as PD-1 and PD-L1 pathway blockers.

[0027] In vitro experiments have shown that the Kd value of nABP486's binding affinity to PD-1 is 11.9nmol / L; that of nABP386 is 75.1nM, which is significantly higher than the Kd value of nABP284 of 11.8μM.

[0028] Further experiments showed that the Kd value of the binding affinity between nABP486 and PD-L1 was 54.2nmol / L, indicating that nABP486 has dual targeting binding effects on PD-1 and PD-L1.

[0029] Further experiments showed that the Kd value of nABP786 binding affinity to PD-1 was 5.84 nmol / L, and the Kd value of nABP786 binding affinity to PD-L1 was 16.7 nmol / L, indicating that nABP786 has dual targeting binding to PD-1 and PD-L1, and has a higher affinity for targeting molecule binding than nABP486.

[0030] Through research, it was found that nABP386 or nABP486 can specifically bind to PD-1 on the surface of lymphocytes. At the same time, compared with nABP284 in the prior art, the affinity of nABP386 and nABP486 in the present invention for PD-1 is significantly improved.

[0031] Through research, it was found that nABP386 or nABP486 competitively binds to the PD-1 receptor on the surface of the tumor cell line Jurkat T cells with the PD-L1 ligand, exerting a competitive binding effect similar to that of PD-1 targeted monoclonal antibody drugs, and compared with nABP284 in the prior art, nABP386 and nABP486 in the present invention have better blocking effects.

[0032] Through research, it was found that nABP386 or nABP486 binds to the PD-1 receptor on the surface of lymphocytes, which can reverse the inhibitory effect of PD-L1 on the surface of tongue cancer cells on lymphocytes and restore the secretion of interleukin-2 by lymphocytes, indicating that nABP386 and nABP486 play a humoral immune regulatory role similar to that of PD-1 targeted monoclonal antibody drugs, and compared with nABP284 in the prior art, nABP386 and nABP486 in the present invention have better effects.

[0033] Through research, it was found that nABP386 or nABP486 had no toxic effect on peripheral blood-derived lymphocytes, tongue cancer cells and Jurkat cells. When nABP386 or nABP486 was added to the culture medium of co-cultured lymphocytes and tongue cancer cell line CAL27, the killing effect of lymphocytes on cancer cells was increased, which was similar to the anti-tumor effect of PD-1 targeted monoclonal antibody drugs. Compared with nABP284 in the prior art, nABP386 and nABP486 in the present invention have better effects.

[0034] Compared with therapeutic antibodies, the non-antibody binding polypeptides nABP486 and nABP786 of the present invention have dual targeting binding effects of PD-1 and PD-L1, and the non-antibody binding polypeptides nABP386 or nABP486 or nABP786 are artificially synthesized and have lower immunogenicity, higher in vivo stability, better tumor penetration, enhanced tumor accumulation and lower manufacturing cost.

[0035] In summary, the non-antibody binding polypeptide nABP386 or nABP486 or nABP786 provided by the present invention has many advantages. First, the non-antibody binding polypeptide has a small molecular weight and good permeability; second, it can be artificially synthesized, with low cost, no animal origin, and high safety; it has good binding to specific molecules and can play a similar or even better function than monoclonal antibodies; third, the non-antibody binding polypeptide nABP386 or nABP486 or nABP786 provided by the present invention has a PD-1 affinity close to that of a monoclonal antibody, and compared with the non-antibody binding polypeptide nABP284 of the invention patent (ZL201710324664.7) PD-1 receptor, it improves its weak affinity; importantly, the non-antibody binding polypeptide nABP486 or nABP786 has a dual-targeted binding effect of PD-1 and PD-L1, which is better than the unidirectional inhibition effect of monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1This is a schematic diagram of the main binding regions of human PD-1 and PD-1, as well as the amino acid sequences of human PD-L1, nABP284, nABP38, and nABP486 and their homology comparison: Figure A shows the two main binding regions of human PD-1 and human PD-L1, region 1 includes amino acid residues such as A121 K124 R125, and region 2 includes amino acid residues such as Q66; Figure B is a computer simulation of the simulated binding site of the histidine short chain that can enhance the affinity of nABP284 and human PD-1; Figure C shows the amino acid sequences of human PD-L1, nABP284, nABP38, and nABP486 and their homology comparison.

[0037] Figure 2 These are the affinity test results of nABP284, nABP386, nABP486 or nABP786 with recombinant human PD-1 protein, as well as the affinity test results of nABP486 or nABP786 with recombinant human PD-L1: A is the affinity of nABP284, B is nABP386, C is nABP486, and E is the result of determination of the affinity of nABP786 for PD-1 using surface plasmon resonance (SPR) technology; D is the affinity of nABP486, and F is the result of determination of the affinity of nABP786 for PD-L1 using SPR technology.

[0038] Figure 3 These are the immunofluorescence results of nABP284, nABP386, and nABP486 specifically binding to PD-1 on the surface of Juekat T cells.

[0039] Figure 4 These are the binding rates of different concentrations of nABP284, nABP386, and nABP486 with Jurkat T cells expressing PD-1.

[0040] Figure 5 These are the experimental results of the competition among nABP284, nABP386, and nABP486 for their ability to bind to cell surface PD-1.

[0041] Figure 6 These are the results of the neutralization ability of nABP284, nABP386, and nABP486 against recombinant human PD-L1 protein.

[0042] Figure 7The effects of nABP284, nABP386, and nABP486 on various cellular functions are shown. Among them, Figure A shows the method of inducing human peripheral blood mononuclear cells (PBMCs) by in vitro culture (PBMCs induced by in vitro culture are called cytokine-induced killer cells (iCIKs)), as well as the expression ratio of PD-1 on the surface of PBMCs and iCIKs; Figure B shows the expression level of PD-L1 on the surface of Cal27 cells after 48 hours of stimulation with different concentrations of IFN-γ; Figure C shows the cytotoxicity test results of nABP284, nABP386, and nABP486 on Juekat T cells, Cal27 cells, and iCIKs; Figure D shows the test results of nABP284, nABP386, and nABP486 blocking PD-1 / PD-L1 binding to increase cellular interleukin secretion function; Figure E shows the test results of nABP486 blocking PD-1 / PD-L1 binding to enhance lymphocyte tumor killing. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention.

[0044] The main research methods used in the examples include AutoDock and Pymol software simulation, non-antibody binding peptide and antibody immunofluorescence co-labeling confocal microscopy observation, surface plasmon resonance technology, flow cytometry, cytotoxicity experiments, immunoblotting, enzyme-linked immunosorbent assay, etc. The experimental operations are all routine experimental operations mastered by those skilled in the art unless otherwise specified. The reagents used are all purchased from commercial channels unless otherwise specified.

[0045] Some reagents in the examples are described as follows:

[0046] Human PD-1 recombinant protein (PD1-H5221, Acro Biosystems), human PD-L1 recombinant protein (PD1-H82F3, Acro Biosystems), human PD-1 antibody (ab52587, abcam), human PD-1 flow cytometry antibody (130-120-389, Miltenyi Biotec), APC Streptavidin (405207, Biolegend), goat anti-mouse IgG secondary antibody (ab150115, abcam), RPMI1640 (R6504, Sigma-Aldrich), DMEM / F12 (D0697, Sigma-Aldrich), fetal bovine serum (FBS, 10099-141, Gibco), recombinant human interleukin-2 (200-02, PEPROTECH), human recombinant interferon-γ (300-02, PEPROTECH), Phorbol 12-myristate 13-acetate (1652981, PeproTech), Ionomycin Calcium Salt (5608212, PeproTech), PD-1 functional monoclonal antibody (16-9989-82, eBioscience | Thermo Fisher Scientific), Anti-CD3 monoclonal antibody (T210, TAKARA).

[0047] 1. Cell acquisition and culture

[0048] Human acute T lymphoblastic leukemia cell line (Jurkat T cells) and human tongue squamous cell carcinoma cell line (Cal27 cells) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Jurkat T cells were cultured in RPMI1640 medium containing 10% FBS; Cal27 cells were cultured in DMEM / F12 medium containing 10% FBS; human peripheral blood mononuclear cells (PBMCs) were obtained from the peripheral blood of healthy adult donors by density gradient centrifugation and cultured in RPMI1640 medium containing 10% FBS, 2000 U / ml IFN-γ, and 10 ng / ml IL-2.

[0049] 2. Design and synthesis of new peptides

[0050] The key region for human PD-L1 to bind to human PD-1 was searched, and the amino acid residue sequence of the key region of PD-L1 was compared with the amino acid sequence of nABP284 (SEQ ID NO.9, SRLKEIANSPTQFWRMVARNTLGGAKQSLNIEHARL) screened by phage display technology. 2RLKEIA7 and 28Q in nABP284 were considered to be the key regions for specific binding. Based on the fact that the cleft between Y68 and E136 near the key binding region of PD-1 is just enough to accommodate the ring structure on the amino acid side chain, and considering that histidine has an imidazole ring, it is assumed that the addition of histidine can enhance the binding affinity of nABP284 to PD1. Therefore, histidine was added near the key binding region of nABP284 to form a branched chain to obtain nABP386 and nABP486. For example, for nABP386, the leucine L in the side chain is linked to the lysine K at position 27 in the main chain; for nABP486 and nABP786, the leucine L in the side chain is linked to the lysine K at position 7 in the main chain.

[0051] The new peptide was commissioned to Qiangyao Biotechnology to be synthesized through solid phase synthesis, and the quality of the new peptide was tested through high performance liquid chromatography and electrospray mass spectrometry.

[0052] 3. Affinity analysis of new peptides

[0053] The affinity of the peptide was detected by surface plasmon resonance (SPR) technology using Biocore T100. The surface of the chip (CM5) was esterified using the crosslinker EDC / NHS at pH 4.5; human recombinant PD-1 protein or PD-L1 protein was coupled to the chip at a concentration of 5ug / ml; excess active carboxyl groups on the chip were blocked with 1M ethanolamine hydrochloride (pH 8.5); the channel not coated with human recombinant PD-1 protein was set as the reference channel; all SPR signals were calibrated by subtracting the corresponding value of the reference channel; the running buffer (pH 7.4, 100mMTris, 150nM The peptide stock solution was diluted with 1% aCl, 0.005% Tween-20) for a series of concentrations; for the affinity analysis of nABP284, one round of SPR measurement used seven concentrations (20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 uM), and for the affinity analysis of nABP386, nABP486 and nABP786, one round of SPR measurement used seven concentrations (2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 uM). After each round of binding and dissociation, the CM5 chip was regenerated with 10 mM glycine-HCl (pH 2.5). The affinity (Kd value) of the peptide was finally calculated by fitting the sensorgram curve on the Biacore software.

[0054] 4. Immunofluorescence and flow cytometry analysis of novel peptide-specific binding

[0055] The following experiments were divided into experimental and control groups. In the experimental group, Jurkat T cells were stimulated with 50ng / ml Phorbol 12-myristate13-acetate (PMA) and 1ug / ml Ionomycin Calcium Salt (Ionomycin) for 24 hours to express PD-1, and unstimulated Jurkat T cells that hardly expressed PD-1 were used as the control group. For immunofluorescence analysis, 1x106 Jurkat T cells were collected and blocked with 2% BSA for 1 hour at room temperature to reduce nonspecific binding. After blocking, the cells were collected and incubated with 10uM peptide with FITC (green fluorescence) at 37°C for 1 hour. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde. After washing three times with PBS, the cell pellet was resuspended with 1:100 diluted PD-1 antibody (ab52587) and incubated overnight at 4°C. Wash three times with PBS, stain the cells with goat anti-mouse IgG polyclonal antibody (ab150115), and incubate at 4°C in the dark for 1 hour. Wash three times with PBS, stain the nuclei with 1:1000 4',6-diamidino-2-phenylindole (DAPI), wash three times with PBS, and seal the slides with anti-fluorescence quenching sealing agent. In addition, fluorescence confocal microscopy images were analyzed with Image J software to evaluate the ability of peptides to compete for PD-1 sites. For flow cytometric analysis, 2x105 Jurkat T cells were collected and blocked with 2% BSA for 1 hour at room temperature to reduce nonspecific binding. Then incubate with 10 or 40uM peptide with FITC (green fluorescence) at 4°C for 1 hour. Wash three times with PBS, and finally resuspend the cells with 500ul PBS and transfer to flow tubes for analysis with a flow cytometer (CytoFLEX, BECK MANCOULTER, USA).

[0056] 5. Neutralization experiment of new peptide

[0057] Jurkat T cells were divided into two groups. The experimental group was stimulated with 50ng / ml Phorbol 12-myristate13-acetate (PMA) and 1ug / ml Ionomycin Calcium Salt (Ionomycin) 24 hours in advance, and unstimulated Jurkat T cells served as the control group. 5X105 cells were collected in each tube, washed once with FACS buffer (PBS containing 2% BSA), human PD-L1 recombinant protein (PD1-H82F3, Acro Biosystems) was diluted to 4ug / ml with FACS buffer, and PD-1 binding peptide was diluted to a series of concentrations (0.4, 1.33, 4, 13.3, 40uM) with FACS buffer. Subsequently, the two working solutions (PD-L1 protein diluent and peptide diluent) were mixed with equal volumes, and 100ul of the mixed solution of different concentrations was added to the centrifuge tube together with the cells and incubated at 4°C for 1 hour. The cells were washed three times with FACS buffer, 0.12ug / ml APC Streptavidin (405207, Biolegend) was added to each tube, incubated at 4°C for 1 hour, washed three times with FACS buffer, and the cells were resuspended with 500ul PBS. The cell suspension was transferred to a flow tube and analyzed by flow cytometry.

[0058] 6. Cytotoxicity detection of new peptides

[0059] Cytotoxicity was determined by cell counting kit-8 (CCK8, Dujindo, Japan). Jurkat T cells (4000 cells per well), PBMCs (4000 cells per well) or Cal27 cells (2000 cells per well) were added to 96-well plates and cultured for 24 hours. A series of concentrations (1, 2, 3, 4, 8, 16, 32, 64uM) of nABP284, nABP386, and nABP486 were added to the culture medium. After the cells were cultured in a cell culture incubator for 24 hours, 10ul of CCK-8 solution was added to each well and the cells were cultured in a cell culture incubator for another 2 hours. The absorbance of each well at 450nm was measured using a VictorX5 multilayer plate reader (PerkinElmer, Singapore). Cell viability (%) was calculated by the following formula: Cell viability (%) = [(A sample-A baseline) / (A control-A blank) x 100%. Asample represents the absorbance of the experimental wells (containing cells, CCK-8 solution, culture medium and peptide diluent); Abaseline represents the absorbance of the baseline wells (without cells, containing CCK-8 solution, culture medium and peptide diluent); Acontrol represents the absorbance of the control wells (containing cells, CCK-8 solution, culture medium, and no peptide diluent); Ablank represents the absorbance of the blank wells (without cells, containing CCK-8 solution, culture medium and no peptide diluent).

[0060] 7. Activated Jurkat T cells were co-cultured with stimulated cal27 cells to analyze the activity of Jurkat T cells

[0061] Jurkat T cells were stimulated with 50ng / ml Phorbol 12-myristate 13-acetate (PMA) and 1ug / ml Ionomycin Calcium Salt (Ionomycin) for 24 hours, and CAL27 cells were stimulated with 500U / ml IFN-γ for 48 hours. The stimulated Jurkat T cells were incubated with nABP284, nABP386, nABP486 (10uM) or PD-1 functional monoclonal antibody (2ug / ml, J116, eBioscience | ThermoFisher Science) at 37°C for 1 hour in advance. The stimulated CAL27 cells were added to a 96-well plate at 1x104 per well. After the CAL27 cells adhered, the supernatant was discarded, and Jurkat T cells and 200ul culture medium were added to each well at a ratio of 4:1. The supernatant was collected after 24 h of co-culture, and the IL-2 content in the supernatant was detected using an ELISA kit (88-7025-86, Thermo Fisher Scientific).

[0062] 8. Effect of the new peptide on the tumor killing efficiency of improving cytokine-induced killer cells (CIK)

[0063] Human peripheral blood was mixed with PBS at a ratio of 1:1, and the mixture was added to the same volume of Ficoll liquid surface, the liquid surface of the mixture and Ficoll was kept layered, and the human peripheral blood components were separated by density gradient centrifugation (400g, 20min). The cell culture dish was coated with 10ug / ml Anti-CD3 monoclonal antibody (T210, TAKARA) and kept at 4℃ overnight. The separated PBMCs were added to the coated culture dish, and RPMI1640 containing 10% fetal bovine serum, 2000U / ml IFN-γ, and 10ng / ml IL-2 was added as the culture medium. The culture medium was changed every three days. Culture for 10 days. This activated PBMCs is called cytokine-induced killer cells (iCIKs).

[0064] For iCIKs-mediated cell killing analysis. Cal27 cells were stimulated with 500U / ml IFN-γ for 48 hours, and iCIKs were pre-incubated with nABP284, nABP386, nABP486 (10uM) or PD-1 functional monoclonal antibody (2ug / ml, J116, eBioscience | ThermoFisher Science) at 37°C for 1 hour. Subsequently, the stimulated CAL27 cells were added to 96-well plates at a quantity of 1x104 per well. After CAL27 cells adhered, the supernatant was discarded, and iCIKs cells and 100ul culture medium were added to each well at a ratio of 10:1. The 96-well plate was centrifuged at 250g for 4 minutes to ensure that iCIKs were fully in contact with cal27 cells. After 6 h of culture, the lactate dehydrogenase (LDH) level in the culture medium was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit, and the absorbance was measured at 490 nm using a VictorX5 multilayer plate reader (PerkinElmer, Singapore).

[0065] 9. Statistical analysis

[0066] Statistical analysis was performed using GraphPad Prism 8 software, and all data were analyzed using t-test or one-way ANOVA (*p<0.05, *p<0.01, *p<0.001; ns, no significant difference). Experimental data are the mean of three independent experiments.

[0067] result:

[0068] 1. Optimize nABP284 and synthesize new peptides

[0069] 1.1n Characteristics of ABP284.

[0070] The amino acid sequence of nABP284 is shown in SEQ ID NO. 7. In this study, nABP284 was synthesized by solid phase method, and nABP284 and its purity were confirmed by ESI-MS / HPLC. Figure 3 -A, immunofluorescence technique was used to determine whether nABP284 could specifically bind to PD-1 expressed on the surface of Jurkat T cells. In order to detect the binding affinity of nABP284 to recombinant human PD-1 protein, surface plasmon resonance (SPR) ( Figure 2 , A).

[0071] NABP284 specifically binds to PD-1 with moderate affinity. Flow cytometry showed that the binding rate of nABP284 to PD-1 on the surface of stimulated Jurkat T cells increased with increasing concentration.

[0072] 1.2 Design of nABP386, nABP486 and nABP786

[0073] By analyzing the structure of the complex of PD-1 and its ligand PD-L1 (4ZQK) in RCSBPDB and comparing the homology between human PD-L1 and nABP284, it was found that the two regions 2RLKEIA7 and 28Q on nABP284 have homology with 125RKYDA121 and 66Q in the extracellular domain of PD-L1, which are the key regions for the binding of PD-1 and PD-L1. In particular, the A121K124 and R125 residues of these sequences are the key determinants of the binding of PD-1 and PD-L1 ( Figure 1 , A and C). This region including A121K124 and R125 is called "Region 1". At the same time, the "SHHHRL" motif was designed for the gap between Y68 and E136 of PD-1. The imidazole ring on the histidine may have the potential to bind to this hotspot. Computer simulation structural analysis using Autodock and Pymol showed that the three histidine imidazole rings of "SHHHRL" can bind to the amino acid residues in PD-1 region 1 and form hydrogen bonds, enhancing the binding ( Figure 1 , B). In summary, by comparing the homology between nABP284 and PD-L1 and adding an amino acid chain containing "SHHHRL" in the region that may bind to PD-1, two new peptides, nABP386 and nABP486 ( Figure 1 , C).

[0074] In order to improve the binding affinity of nABP486 for dual targeting of PD-1 and PD-L1, the main chain amino acid sequence of nABP486 was further optimized to obtain the third new peptide nABP786 ( Figure 1 , C).

[0075] 1.3 Synthesis of nABP386, nABP486 and nABP786

[0076] China Peptide Company (Jiangsu) was commissioned to synthesize nABP386, nABP486 and nABP786 using Fmoc chemistry. The purity of the new peptides was confirmed by ESI-MS / HPLC analysis.

[0077] 2. Binding specificity and affinity of novel PD-1 binding peptides

[0078] Surface plasmon resonance (SPR) was used to detect the binding affinity (Kd value) of nABP386, nABP486 and nABP786 to PD-1. The human recombinant PD-1 protein was coated on a CM5 chip, and the Kd values ​​of nABP386, nABP486 and nABP786 were 75.1nmol / L, 11.9nmol / L and 5.84nmol / L, respectively. Figure 2 , B, C and E), which was significantly higher than the Kd value of nABP284 of 11.8 μmol / L ( Figure 2 , A).

[0079] The human recombinant PD-L1 protein was coated on the CM5 chip, and the Kd values ​​of nABP486 and nABP786 were detected to be 52.4nmol / L and 16.7nmol / L, respectively, indicating that the non-antibody binding peptides nABP486 and nABP786 have dual targeting binding to PD-1 and PD-L1 ( Figure 2 , D).

[0080] Jurkat cells not stimulated by PMA barely expressed PD-1, whereas Jurkat T cells stimulated by PMA highly expressed PD-1 ( Figure 3 , A). To demonstrate the binding specificity of nABP386 and nABP486 to PD-1, the new peptides were used to bind to Jurkat T cells before and after stimulation. Figure 3 As shown in Figure 1-A, nABP386 and nABP486 can bind to Jurkat T cells expressing PD-1 stimulated by PMA and ionomycin, while unstimulated Jurkat T cells have almost no binding. Compared with nABP284, the new peptide has a higher binding rate to Jurkat T cells expressing PD-1. Similarly, the binding rate is positively correlated with concentration ( Figure 4, A and B). These results indicate that the binding of nABP386 and nABP486 to Jurkat T cells expressing PD-1 is a specific binding mediated by PD-1.

[0081] 3. Blocking and neutralizing ability of new peptides

[0082] The purpose of these studies was to determine whether the enhanced binding capacity of the novel peptides compared with nABP284 was reflected in enhanced blocking and neutralizing capacity. Jurkat T cells were prestimulated with PMA and ionomycin for 24 h. After incubation with nABP284, nABP386, nABP486, or functional anti-PD-1 antibody for 1 h, the number of PD-1 sites that could bind to the surface of Jurkat T cells incubated with the peptides was significantly reduced compared with untreated Jurkat cells ( Figure 5 , A). Quantification of mean fluorescence intensity showed that nABP284, nABP286 and nABP486 all had the ability to block PD-1 on the surface of stimulated Jurkat T cells. At the same concentration, nABP486 had a stronger blocking ability than A and B, and there was no significant difference between nABP284 and nABP386 ( Figure 5 , B).

[0083] Next, the neutralizing ability of nABP284 and the new peptide on recombinant human PD-L1 protein was studied. As a ligand for PD-1, PD-L1 can specifically bind to PD-1. Before binding to Jurkat T cells expressing PD-1, nABP284, nABP386, or nABP486 was mixed with recombinant human PD-L1 protein at a series of concentrations. The binding rate of recombinant human PD-L1 protein was determined by flow cytometry, and the results showed that by increasing the concentration of nABP284, nABP386, or nABP486, the binding of human PD-L1 recombinant protein to Jurkat T cells overexpressing PD-1 after stimulation could be inhibited ( Figure 6 , D). In terms of neutralization ability, nABP386 and nABP486 are stronger than nABP284. Among the three peptides, nABP486 has the strongest neutralization ability ( Figure 6 , AD).

[0084] 4. New peptide rescues the inhibitory effect of Cal27 cells on Jurkat T cells

[0085] After PMA and ionomycin stimulation of Jurkat T cells, PD-1 sites were highly expressed on the cell surface ( Figure 3 , A), and Jurkat T cells secrete a large amount of IL-2. Similarly, as the concentration of pre-stimulated IFN-γ increases, the number of PD-L1 sites on the surface of Cal27 cells increases ( Figure 7 , B). Cytokine secretion level is an important indicator for evaluating T cell function. After co-culture, Cal27 cells inhibited Jurkat T cells through the interaction between PD-1 and PD-L1, which was demonstrated by the decrease in IL-2 levels detected by ELISA ( Figure 7 , D). We examined whether the new peptides could more effectively block the interaction between PD-1 and PD-L1 than nABP284. After confirming that nABP284, nABP386, and nABP486 had no direct cytotoxicity to Cal27 and Jurkat T cells ( Figure 7 , C), nABP284, nABP386, nABP486 or anti-PD-1 functional antibody was added into the culture system. The results showed that compared with the control group, after adding nABP284, nABP386, nABP486 or anti-PD-1 antibody, the IL-2 level in the co-culture system increased significantly ( Figure 7 , D). Therefore, nABP284 and the new peptides can restore the suppression of Jurkat T cells by blocking the interaction between PD-1 and PD-L1, among which nABP486 has the best blocking effect among the three peptides.

[0086] 5. New peptides enhance the tumor cell killing efficiency of iCIKs

[0087] like Figure 7 As shown in Figure A, PBMCs were isolated from the peripheral blood of volunteers and stimulated by a series of cytokines, including OKT-3, IL-2, and IFN-γ. These activated PBMCs are called cytokine-induced killer cells (iCIKs). Flow cytometry showed that compared with freshly isolated PBMCs, cytokine-induced killer cells (iCIKs) significantly overexpressed PD-1 ( Figure 7 , A). IFN-γ stimulated cal27 cells were co-cultured with cytokine-induced killer cells (iCIKs), and the LDH concentration in the culture medium was detected by ELISA to evaluate the cytotoxicity of iCIKs to cal27 cells. Due to the presence of the PD-1 / PD-L1 pathway, the cytotoxicity of iCIKs to cal27 cells is relatively weak ( Figure 7 , E), as the most effective peptide in the above experiment, adding nABP486 to the co-culture system of iCIKs and cal27 cells significantly increased the killing ability of iCIKs compared with the control group ( Figure 7 , E). Considering that nABP486 has no direct killing ability on Cal27 cells ( Figure 7 , C), the above experimental results show that nABP486 can enhance iCIKs-mediated cytotoxicity by blocking the interaction between PD-1 and PD-L1 ( Figure 7, A and E).

[0088] In this study, it was demonstrated that two new peptides (nABP386 and nABP486) optimized from nABP284 found in previous studies had better effects on blocking the interaction between PD-1 and PD-L1. The affinity of nABP386 or nABP486 bound to PD-1 was significantly higher than that of nABP284. These peptides can block the binding of human recombinant PD-L1 to PD-1 on the surface of stimulated JurkatT cells. The inhibitory effect of cal27 cells on JurkatT cells and PBMCs can be reversed by adding these peptides to the co-culture system. In summary, the results show that the new peptides optimized from nABP284, especially nABP486, have good blocking ability on the interaction between PD-1 and PD-L1.

[0089] The surface plasmon resonance (SPR) in the experiment showed that K d The binding affinity of nABP386 to PD-1 is about 75.1nM and 11.9nm. The Kd value of nABP284 binding affinity to PD-1 is 11.8μM. In addition, the Kd value of nABP486 binding affinity to PD-1 is 11.9nM, and the Kd value of nABP486 binding affinity to PD-L1 is 54.2nM, with dual-targeted binding to PD-1 and PD-L1. This dual-targeted blocking effect of PD-1 and PD-L1 is expected to improve the therapeutic effect of tumor immune checkpoint inhibition.

[0090] These SPR results showed that after optimization of nABP284, two peptides evolved from nABP284 had higher binding affinity to PD-1 than to PD-L1. By comparing the PD-1 binding peptide sequences directly screened by phage display technology and the extracellular domain of PD-L1, the key binding region of nABP284 was inferred, and nANP284 was modified by adding histidine to the key region of nABP284, which is also the side chain formed by nABP284. Compared with nABP284, the affinity of the new peptides that bind to PD-1 is significantly improved, which may be a more effective way to optimize the target binding peptides directly screened by phage display technology.

[0091] The half-effective inhibitory concentration (IC50) of the peptide obtained by neutralization experiments showed that at low concentrations, the new peptide effectively blocked the binding of PD-L1 to PD-1. The co-culture system of Jurkat cells and cal27 cells was used as a model to simulate the in vitro inhibitory effect of PD-1-expressing tumor cells on activated lymphocytes, and the degree of inhibition was demonstrated by evaluating the concentration of IL-2 in the culture medium. After incubation of activated Jurkat cells with nABP386, nABP486 or functional PD-1 antibody, the interaction between PD-1 and PD-L1 could reverse the inhibition of activated Jurkat cells, and nABP486 showed a more significant effect than PD-1 antibody. PBMCs were isolated from human peripheral blood and cultured with anti-CD3 antibodies, IL-2, IFN-γ and other cytokines for 10 days to obtain iCIK cells. Compared with PBMCs, the expression level of PD-1 on the surface of ICIK cells was increased. When tongue cancer CAL27 cells were co-cultured with iCIK, the tongue cancer cells inhibited the tumor killing ability of iCIK cells. Pretreatment of stimulated iCIK with nABP486 or functional PD-1 antibody significantly enhanced its ability to reverse the immune escape of tongue cancer cells and enhance the tongue cancer cell killing ability of iCIK cells. At the same time, nABP486 performed better than PD-1 functional antibody. These in vitro experiments showed that the two new peptides (nABP386 and nABP486) optimized from the PD-1 binding peptide nABP284 have the ability to block the interaction between PD-1 and PD-L1. Compared with nABP284 directly screened by phage display technology, nABP386 and nABP486 have better blocking effects at the same concentration, and nABP486 is better than nABP386.

[0092] For immune checkpoint inhibitors, in vivo stability and half-life and binding affinity to the target site are important evaluation criteria. Adding a branch generally increases the stability of the peptide in vivo. Compared with nABP284, nABP386 and nABP486, they all have a branch (SHHHRL), which increases the stability of the two new peptides in vivo, thereby slowing down the rate of removal from the circulation.

[0093] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention. Sequence Listing <110> The Affiliated Stomatological Hospital of Sun Yat-sen University Guangzhou Yida Pharmaceutical Technology Co., Ltd. <120> Non-antibody binding polypeptide or its derivatives for dual targeting of PD-1 receptor and its ligand PD-L1 and their applications <160> 9 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 35 <212> PRT <213> Artificial Sequence <400> 1 Ser Arg Leu Lys Glu Ile Ala Asn Ser Pro Thr Gln Phe Trp Arg Met 1 5 10 15 Val Ala Arg Asn Thr Leu Gly Asn His Ala Lys Gln Ser Leu Asn Ile 20 25 30 Glu His Ala 35 <210> 2 <211> 40 <212> PRT <213> Artificial Sequence <400> 2 Ser His His His Arg Leu Lys Leu Lys Glu Ile Ala Asn Ser Pro Thr 1 5 10 15 Gln Phe Trp Arg Met Val Ala Arg Asn Thr Leu Gly Asn Gly Ala Lys 20 25 30 Gln Ser Leu Asn Ile Glu His Ala 35 40 <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <400> 3 Ser His His His Arg Leu 1 5 <210> 4 <211> 105 <212> DNA <213> Artificial Sequence <400> 4 tcaaggctga aggaaatagc aaattcacct acacagttct ggaggatggt tgcacgaaac 60 acactgggga atcacgcaaa acagtctttg aatatcgaac acgca 107 <210> 5 <211> 120 <212> DNA <213> Artificial Sequence <400> 5 tcacaccacc acaggctgaa gctgaaggaa atagcaaatt cacctacaca gttctggagg 60 atggttgcac gaaacacact ggggaatgga gcaaaacagt ctttgaatat cgaacacgca 122 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 tcacaccacc acaggctg 18 <210> 7 <211> 21 <212> PRT <213> Artificial Sequence <400> 7 Ser His His His Arg Leu Lys Leu Lys Glu Ile Ala Asn Cys Ser Pro 1 5 10 15 Thr Gln Phe Lys Cys 20 <210> 8 <211> 63 <212> DNA <213> Artificial Sequence <400> 8 tcacaccacc acaggctgaa gctgaaggaa atagcaaatt gctcacctac acagttcaag 60 tgc 63 <210> 9 <211> 36 <212> PRT <213> Artificial Sequence <400> 9 Ser Arg Leu Lys Glu Ile Ala Asn Ser Pro Thr Gln Phe Trp Arg Met 1 5 10 15 Val Ala Arg Asn Thr Leu Gly Gly Ala Lys Gln Ser Leu Asn Ile Glu 20 25 30 His Ala Arg Leu 35

Claims

1. A non-antibody binding polypeptide that dual-targets PD-1 receptor and its ligand PD-L1, characterized in that: The non-antibody binding polypeptide consists of a main chain and a branch chain connected to the main chain, the amino acid sequence of the branch chain is shown in SEQ ID NO.3, and the amino acid sequence of the main chain is shown in SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.7; When the amino acid sequence of the side chain is as shown in SEQ ID NO.3, and the amino acid sequence of the main chain is as shown in SEQ ID NO.1, the amino acid L in the side chain is connected to the amino acid K at position 27 in the main chain; When the amino acid sequence of the side chain is as shown in SEQ ID NO.3, and the amino acid sequence of the main chain is as shown in SEQ ID NO.2, the amino acid L in the side chain is connected to the amino acid K at position 7 in the main chain; When the amino acid sequence of the side chain is as shown in SEQ ID NO.3, and the amino acid sequence of the main chain is as shown in SEQ ID NO.7, the amino acid L in the side chain is connected to the amino acid K at position 7 in the main chain.

2. A polynucleotide, characterized in that It encodes the non-antibody binding polypeptide according to claim 1.

3. The polynucleotide according to claim 2, characterized in that The nucleotide sequence encoding the main chain of the non-antibody binding polypeptide is shown in SEQ ID NO.4, and the nucleotide sequence encoding the side chain of the non-antibody binding polypeptide is shown in SEQ ID NO.

6.

4. The polynucleotide according to claim 2, characterized in that The nucleotide sequence encoding the main chain of the non-antibody binding polypeptide is shown in SEQ ID NO.5, and the nucleotide sequence encoding the side chain of the non-antibody binding polypeptide is shown in SEQ ID NO.

6.

5. The polynucleotide according to claim 2, characterized in that The nucleotide sequence encoding the main chain of the non-antibody binding polypeptide is shown in SEQ ID NO.8, and the nucleotide sequence encoding the side chain of the non-antibody binding polypeptide is shown in SEQ ID NO.

6.

6. A recombinant vector, characterized in that: It comprises the polynucleotide according to any one of claims 2 to 5.

7. A host cell, characterized in that It comprises the recombinant vector according to claim 6.

8. A preparation, characterized in that The preparation comprises the non-antibody binding polypeptide according to claim 1, and the preparation is a drug or a cell marking agent.

9. Use of the non-antibody binding polypeptide according to claim 1 for preparing an immunomodulatory drug or an anti-tumor drug, wherein the tumor is tongue cancer.

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