WO2018192443A1

WO2018192443A1 - Polypeptide and application thereof

Info

Publication number: WO2018192443A1
Application number: PCT/CN2018/083206
Authority: WO
Inventors: 朱毅敏; 李春林; 赵梦雅; 崔雪原
Original assignee: 中国科学院苏州纳米技术与纳米仿生研究所
Priority date: 2017-04-17
Filing date: 2018-04-16
Publication date: 2018-10-25

Abstract

Provided are a polypeptide and an application thereof. The polypeptide has a conserved sequence of SEQ ID NO. 1, and the polypeptide sequence is any one of or a combination of at least two of amino acid sequences shown in SEQ ID NOs. 2-4. The polypeptide specifically binds to PD-L1 and blocks inhibition of T cells by PD-L1, thereby achieving the purpose of activating T cells. The polypeptide can be applied to the treatment of a tumor with high PD-L1 expression, such as lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer, and liver cancer.

Description

Polypeptide and application thereof

Technical field

The present application belongs to the field of biotechnology, and relates to a polypeptide and an application thereof, and particularly to a polypeptide targeting the programmed death ligand PD-L1 and an application thereof.

Background technique

Cancer has become one of the most dangerous diseases that destroy human health and deprive human life. Traditional tumor treatment methods have various drawbacks. For example, surgical treatment is only suitable for benign solid tumors with large volume and detectable imaging techniques. It is ineffective for smaller tumors or metastatic tumors; radiation therapy has less damage to local tissues. Large; chemotherapy requires systemic administration, poorly targeted, causing enormous toxic side effects to patients.

In view of the above defects in the treatment of tumors, researchers have been working hard to activate the body's immune system to achieve the purpose of treating tumors. In the case of other drug-treated metastatic melanoma failure, CTLA-4 (Cytotoxic T-lymphocyte-associated Protein 4) antibody significantly improved patient survival, a revolutionary result brought immunotherapy to treat tumors. new Hope. With the understanding of immune tolerance, immunosuppression, regulation of anti-tumor immunity and tumor-targeted therapy, people are increasingly convinced that activation of the immune system can provide long-term therapeutic effects in cancer patients.

Activation of tumor-specific T cells is critical for the regulation of the entire immune response system. In addition to TCR recognition, the TCR recognizes the MHC (Major Histocompatibility Complex)-antigen peptide complex signal on the surface of antigen-presenting cells (APC). In addition to stimulation, TCR-MHC-Peptide; a co-stimulatory signal between APC cells and T cells, the B7-CD28 molecule, is also often used as an immune checkpoint. Lack of costimulatory signals will result in incompetence of T cells, while overactivation is manifested as autoimmune disease.

Programmed Cell Death Protein 1 (PD-1/CD279, Programmed Cell Death Protein 1) is a newly discovered CD28 family member whose ligand PD-L1 (B7-H1/CD274, Programmed Death-ligand 1) is a B7 molecule. family members. PD-1/PD-L1 is a pair of co-stimulatory factors with negative regulatory functions, which inhibits peripheral T cell activity in inflammatory or autoimmune diseases; meanwhile, PD-1/PD-L1 signal suppression T cell proliferation, affecting survival and function (including killing and release of cellular molecules, promoting tumor-specific T cell apoptosis, promoting the conversion of CD4+ T cells into regulatory T cells (Tregs) expressing Foxp3+, and resisting toxic T cells ( Killing effect of CTL) PD-1 is widely expressed in activated T cells, B cells, DC (Dendritic Cell) cells, etc. PD-L1 is composed in many tissues and cells such as monocytes, endothelial cells, lungs and hepatocytes. Expression. So far, it has been expressed in many cell solid tumor cells such as lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer, liver cancer, etc. In addition, PD-1 is up-regulated in tumor invasive lymphocytes, which may It is the main cause of tumor cells causing T cell incompetence.

The PD-1/PD-L1 signaling pathway plays an important negative regulatory role as an important checkpoint for the T cell cell cycle. Drug blocking of this target is one of the important ways to interfere with the body's immune system and activate T cells to achieve anti-tumor immunotherapy, which has potential application value. PD-1 antibodies and PD-L1 antibodies have achieved clinically important breakthroughs in the treatment of tumors. However, the safety of heterologous antibody treatment and the cost of preparation hinder the development of antibody drugs.

CN 103897036 A discloses an affinity peptide L8 of the extracellular domain of PD-1 protein and the use thereof, the amino acid sequence of the affinity peptide L8 is: Ser-Leu-Pro-Ser-Thr-Thr-Thr-Met-Arg -Leu-Thr-Ser, which has obvious inhibitory effects on tumors. High-affinity peptides have become an important adjuvant for anti-tumor immunotherapy because of its low preparation cost and small side effects, opening up for tumor immunotherapy. New approach.

In view of the advantages of targeting polypeptides as targeted drugs, it is important to screen for a polypeptide that targets PD-L1.

Summary of the invention

The purpose of the present application is to provide a polypeptide and a high-throughput screening affinity peptide targeting PD-L1 which utilizes bacterial surface display technology, and which has a broad therapeutic effect on antitumor drugs. Application prospects.

To achieve the object of the present invention, the present application adopts the following technical solutions:

In a first aspect, the application provides a polypeptide, wherein the polypeptide has a conserved sequence, and the amino acid sequence of the conserved sequence is the amino acid sequence set forth in SEQ ID NO.

SEQ ID NO. 1: CWCWR, ie Cys-Trp-Cys-Trp-Arg.

In the present application, the conserved sequence is obtained by screening a random bacterial polypeptide library for a polypeptide sequence which specifically binds to PD-L1. By analyzing these polypeptide sequences, the inventors unexpectedly found that they can specifically bind to PD-L1. The conjugated polypeptide sequences all have the amino acid sequence set forth in SEQ ID NO.

According to the present application, the polypeptide is any one or a combination of at least two of the amino acid sequences shown in SEQ ID NO. 2-4, preferably the amino acid sequence shown in SEQ ID NO.

The amino acid sequences shown in SEQ ID NO. 2-4 are as follows:

SEQ ID NO. 2: YASYHCWCWRDPGRS

SEQ ID NO. 3: YSAYQCWCWRQQGTS

SEQ ID NO. 4: YHQYSCWCWRPPGPY.

In the present application, the inventors combined these sequences with PD-L1 by ligating any five hydrophilic amino acids on both sides of the conserved sequence by constructing a bias library, and found the amino acids shown in SEQ ID NO. The sequence appeared frequently, and by further verifying the specificity of binding of these three amino acid sequences to PD-L1, it was found that all three sequences were able to bind to PD-L1, and the amino acid sequence shown in SEQ ID NO. PD-L1 binding works best.

In a second aspect, the application provides a DNA fragment comprising a nucleic acid sequence encoding a polypeptide as described in the first aspect.

In a third aspect, the application provides a recombinant vector comprising at least one copy of the DNA fragment of the second aspect.

In a fourth aspect, the application provides a recombinant cell comprising the expression vector of the third aspect.

In a fifth aspect, the present application provides a pharmaceutical composition, comprising the polypeptide of the first aspect, the nucleic acid sequence of the second aspect, the recombinant vector of the third aspect, or the like The recombinant cell described in the four aspects.

According to the application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In a sixth aspect, the application provides the use of a pharmaceutical composition according to the fifth aspect, the use of the pharmaceutical composition in the preparation of a tumor localization diagnostic reagent, an antitumor drug or a PD-L1 targeting preparation.

According to the present application, the tumor is feasible for tumors highly expressing PD-L1, and the tumor is selected from, but not limited to, high expression of PD-L1 such as lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer. Any one or combination of at least two of the tumors.

In the present application, the polypeptide is used as a drug to treat tumors on the premise that the tumor cells have high expression of PD-L1, and PD-L1 is in many tumor cells, such as melanoma, breast cancer, colorectal cancer, ovarian cancer and liver cancer. Both are constitutively high expression, so the polypeptide can also be used as a drug for treating other cancers.

Compared with the prior art, the present application has the following beneficial effects:

(1) The inventors of the present application unexpectedly discovered a polypeptide sequence capable of specifically binding to PD-L1 by screening a random bacterial polypeptide library for a polypeptide sequence which specifically binds to PD-L1, and by analyzing these polypeptide sequences, the inventors unexpectedly discovered a polypeptide sequence which specifically binds to PD-L1. All have the amino acid sequence shown in SEQ ID NO. 1, and by further construction of the bias library, the inventors found the amino acid sequence shown in SEQ ID NO. 2-4, which has the best binding effect with PD-L1;

(2) The polypeptide of the present application and its products can specifically bind to PD-L1, the binding constant can reach ka=3192Ms ^-1 , and the dissociation equilibrium constant KD is 95×10 ^-9 M, and the binding is slow but not easy to fall off, and The polypeptide can specifically target and block the PD-1/PD-L1 signaling pathway, inhibit the T cell, achieve the purpose of activating T cells, and have antitumor activity;

(3) The polypeptide of the present application has broad application prospects in the treatment of PD-L1 high expression-related tumors such as lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer.

DRAWINGS

Figure 1 (A) is a screening of a PD-L1 targeting polypeptide from a random polypeptide library, and Figure 1 (B) is a polypeptide sequence analysis;

Figure 2 is a flow cytometric analysis of the conserved sequence CWCWR binding to MDA-MB-231 and MDA-MB-435, wherein Figure 2 (A) is the binding of MDA-MB-231 to the control polypeptide, Figure 2 (B) For the binding of MDA-MB-231 to the PD-L1 targeting polypeptide, Figure 2 (C) shows the binding of MDA-MB-435 to the control polypeptide, and Figure 2 (D) shows MDA-MB-435 and PD-L1. Targeting polypeptide binding;

Figure 3 (A) is a schematic diagram of the bias library construction, Figure 3 (B) is the result of screening the PD-L1 targeting polypeptide from the bias library, and Figure 3 (C) is the sequence analysis of the polypeptide screened by the bias library;

Figure 4 is a specific identification of the binding of the polypeptide to PD-L1, wherein Figure 4 (A) TPP-1 polypeptide and SPP-1 polypeptide binding to PD-L1, Figure 4 (B) using ELISA to verify the specificity of the polypeptide, Figure 4 (C) - Figure 4 (F) shows the specificity of binding of the polypeptide to the cell surface PD-L1 by fluorescence microscopy;

Figure 5 is a T cell activation experiment;

Figure 6 is a verification of the in vivo activity of the polypeptide, wherein Figure 6 (A) is the tumor volume in the TPP-1 group and the SPP-1 group, and Figure 6 (B) is the total bioluminescence intensity change;

Figure 7 shows the effect of TPP-1 polypeptide on T cell activation by modulating the expression of interferon-gamma and granzyme B to kill tumor cells.

detailed description

The technical solutions of the present application are further described below by way of specific embodiments. It should be understood by those skilled in the art that the present invention is only to be understood as an understanding of the present application and should not be construed as a limitation.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

material:

The polypeptides involved in the following examples were synthesized and purified by Shanghai Jill Biochemical Co., Ltd.;

The sequencing involved in the following examples was carried out by Suzhou Jinweizhi Biotechnology Co., Ltd.;

Example 1 Screening of a polypeptide sequence that specifically binds to PD-L1 from a library of random bacterial polypeptides

Screening for a polypeptide sequence that specifically binds to PD-L1, including the following steps:

(1) biotinylated PD-L1 protein using a Fluoreporter mini-biotin-xx protein labeling kit;

(2) The bacterial surface polypeptide display library used in the present application is integrated at the N-terminus of eCPX and consists of 15 random amino acids; at least 10 times the library capacity of the frozen bacterial library is inoculated to the LB containing chloramphenicol. In the medium, culture overnight;

(3) A certain amount of bacteria was taken from the overnight cultured bacteria at a ratio of 1:50 to 150 mL of chloramphenicol LB medium containing 34 μg/mL, and cultured at 37 ° C, 200 rpm for 2 h to an OD600 value of 0.5. Between -0.6;

(4) Adding arabinose in an amount of 200 μg/mL, and inducing culture at room temperature of about 25 ° C and 200 rpm for about 1 h, the OD600 value of which is 0.8-1.0, calculated according to an OD equal to 1 × 10 ⁹ bacteria, according to the bacteria a ratio of magnetic bead ratio of 100:1, and streptavidin-coated magnetic microbeads were added for negative separation of magnetic beads;

(5) Adsorption of bacteria not bound to streptavidin into the next round of positive selection of magnetic beads. In the positive screening, the concentration of PD-L1 protein in the mixture was 40 nM, mixed in silent rotation. Incubate for 45 minutes at 4 ° C;

(6) After the end of the incubation, centrifuge at 3000 g and 4 ° C, remove the supernatant, and resuspend the pellet in 1 mL of PBS buffer solution, and magnetically according to the ratio of magnetic bead to bacteria 1:100. The beads were mixed with the bacteria resuspended in PBS buffer, and the pellet was washed 3 times with PBS buffer and resuspended in 1 mL LB;

(7) 10 μL of each sample was diluted 100 times during the cleaning process, coated on a solid LB plate, and cultured overnight at 37 ° C. The next day, the bacterial library capacity after magnetic bead sorting was calculated according to the number of bacterial clones grown;

(8) The enriched bacteria were resuspended in 5 mL of SOC medium, and cultured overnight at 37 ° C and 200 rpm for flow sorting;

(9) 100 μL of the overnight cultured bacteria were inoculated into 5 mL of LB medium and successfully induced the expression of the polypeptide. The 10-fold number of bacteria were mixed with the biotinylated PD-L1 protein bacteria to control the protein concentration below 40 nM. Incubate for 45 minutes under gentle rotation at °C and 20 rpm, and then centrifuge at 5 °C and 3000 g for 5 minutes to discard the supernatant;

(10) The pellet was resuspended in 100 μL of SAPE staining solution for 30 minutes, and the concentration of SAPE was 4 nM. After the incubation, centrifugation was carried out for 5 minutes at 4 ° C and 3000 g, and the supernatant was discarded;

(11) The pellet was resuspended in 600 μL of PBS buffer, and the bacteria resuspended in PBS buffer were sorted by flow cytometry, and the sorted bacterial-protein complex was inoculated into LB containing glucose. Culture in the medium overnight.

After 1 round of MACS screening and 9 rounds of FACS screening, bacteria in the bacterial pool that bind to PD-L1 were significantly enriched. The peak images of the first 9 rounds of the screening results were combined and summarized. As shown in Fig. 1(A), it can be clearly seen that the fluorescence intensity of PE is significantly enhanced. 40 clones were selected for sequencing, and the sequencing results were followed by extraction and analysis of the polypeptide sequence. As shown in Fig. 1(B), the conserved sequence SEQ ID NO. 1: CWCWR was found.

Example 2 Specificity verification of conserved sequences

PD-L1 highly expressed cells MAD-MB-231 cells and PD-L1 non-expressing cells MDA-MB-435 cells were digested to about 80% confluency, and the PBS adjusted cell concentration was 10 ⁶ /mL, 100 μL/tube cells. . The PD-L1 targeting polypeptide and the control polypeptide were separately added to a final concentration of 2 μM, and incubated at 4 ° C for 30 min. After the incubation, the cells were centrifuged at 1000 rpm for 5 min, and the precipitate was washed twice with 1 mL of PBS, and the binding rate was measured by flow cytometry. As shown in Figure 2 (A) - Figure 2 (D).

Figure 2 (A) - Figure 2 (D) shows that the conserved sequence has a higher binding rate to MAD-MB-231 cells than to MDA-MB-435 cells, indicating that conserved sequences and cells with high expression of PD-L1 have Certain binding specificity.

Embodiment 3 Construction method of biased library

The amino acid sequence of SEQ ID NO.: 1 is flanked by any five hydrophilic amino acids, respectively, and ligated to the N-terminus of eCPX. The sequence encoding the above polypeptide was amplified by a PCR method. The template for the PCR reaction was plasmid pBAD33-eCPX. In order to improve the hydrophilicity of the obtained polypeptide, it is preferred that the random amino acid is more hydrophilic, and thus a degenerate primer having a sequence of NVS is designed.

Forward primer (SEQ ID NO. 5): CGTAGCTGGCCAGTCTGGCCAGNVSNVSNVSNVSNVSTGTTGGTGTTGGAGGNVSNVSNVSNVSNVSGGCGGTTCTGGTGGCAGCGG, wherein N represents any nucleotide in A, T, C, G, V represents A, C or G, and S represents C or G;

Reverse primer (SEQ ID NO. 6): GGCTGAAAATCTTCTCTC.

The PCR amplification product was digested with Sfi1 and ligated into the same digested pBAD33-eCPX vector. The transformed pBAD33-eCPX plasmid was transformed into E. coli MC1061 strain, coated with LB plates, and the number of monoclonals was calculated. Twenty monoclonal clones were randomly selected and sequenced using pBAD-forward primers to make basic judgments on biased library diversity and amino acid bias. Figure 3 (A) shows the construction principle of the bias library. The X5CWCWRX5 mode polypeptide was inserted into the N-terminus of pBAD33-eCPX, and the number of clones on the LB plate was calculated, indicating that the bias library contained 5 × 10 ⁷ clones. The successful construction of the biased bacterial display library used the same method as the random display library for the screening of PD-L1 binding polypeptides. Figure 3 (B) shows that after one round of MACS screening and nine rounds of FACS screening, the bacterial library and The binding efficiency of PD-L1 is significantly improved. Twenty clones were selected for sequencing, and the sequencing results were followed by extraction and analysis of the polypeptide sequence. The results are shown in Figure 3 (C), SEQ ID NO. 2 (C1), SEQ ID NO. 3 (C2) and SEQ ID NO. The frequency of occurrence of the amino acid sequence shown in .4 (C3) is high.

SEQ ID NO. 2 (C1): YASYHCWCWRDPGRS

SEQ ID NO. 3 (C2): YSAYQCWCWRQQGTS

SEQ ID NO. 4 (C3): YHQYSCWCWRPPGPY

Since the above SEQ ID NO. 2-4 all have the property of specifically binding to PD-L1, and the amino acid sequence shown by SEQ ID NO. 2 has the best effect, the subsequent experiments employ the amino acid shown in SEQ ID NO. The sequence proceeds.

Example 4 ELISA and fluorescence microscopy methods to characterize the specificity of polypeptide binding to PD-L1

Fluorescent labels are all coupled to the N-terminus of the polypeptide to a FITC fluorescent molecule. In this example, the sequence of SEQ ID No. 3: YASYHCWCWRDPGRS was designated as TPP-1, and the control polypeptide SPP-1 of SEQ ID No. 3 was designed. PD-L1, mPD-L1 and hPD-L2 were each diluted to 1 μg/mL with PBS, and added to a black ELISA plate in a volume of 100 μL/well. The plate was incubated overnight at 4 °C. On the next day, the solution in the well was taken on the nonwoven fabric, and the coated well was washed with PBST containing 0.01% Tween-20 for 1 minute each time, and washed a total of 3 times. To the well-coated wells, 100 μL of a 2% (m/v) BSA blocking solution prepared using PBST was added, and the plate was incubated at room temperature for 1 h with shaking at 80 rpm. After the end, it was washed 3 times with PBST for 1 min each time. 100 μL of 10 μM FITC-labeled polypeptide solution was added to the blocked well-labeled well, and incubated at room temperature for 8 to 2 h with shaking at 80 rpm. After the end of the incubation, each well was washed 3 times with PBST for 3 min each. The FITC fluorescence value of each well solution was measured on a microplate reader.

CHO-K1 cells and CHO-K1/PD-L1 cells were plated in 24-well plates one day in advance, and the fusion degree was about 80%. After overnight culture, the medium was aspirated, and 2 mL of PBS was washed twice, respectively, and the final concentration was 5 μM. 1 mL of the fluorescently labeled polypeptide was incubated at 70 rpm for 30 min at room temperature with shaking, the supernatant was discarded, 2 mL of PBS was added to each well, and the chamber was washed at room temperature on a shaker at 70 rpm for 2 times. 1 mL of 4% paraformaldehyde solution was added to each well and fixed at room temperature for 10 min. After washing twice with PBS, 0.5 mL of Hoechst33342 with a final concentration of 2 μg/mL was added to each well, and stained for 10 min in the dark. The binding of the polypeptide to different cells was observed by fluorescence microscopy and photographed.

Figure 4 (A) shows that the binding of the TPP-1 polypeptide to PD-L1 increases with increasing polypeptide concentration, as is the case with the control polypeptide SPP-1. Figure 4 (B) shows that TPP-1 has better specificity for hPD-L1 and hPD-L2 than PD-L1, and Figure 4(C)-4(F) shows TPP-1 under fluorescence microscope. The polypeptide binds to CHO-K1 cells that specifically express PD-L1.

Example 5 Determination of the Affinity of the Polypeptide of the Present Application with PD-L1

The peptide was analyzed by surface plasmon resonance instrument Biacore T200 for affinity between the polypeptide and PD-L1. The XanTEX chip was used in the experiment to perform related experiments. The specific process is as follows: First, 50 μL NHS/EDC mixture (0.05 mol/L NHS and 0.2 mol/L EDC, mixed at a volume ratio of 1:1 before use) was injected at a flow rate of 10 μL/min to activate the surface glucose of the chip. Carboxyl group; after completion of the activation, the PD-L1 protein diluted with sodium acetate was injected, and the sample was injected at a flow rate of 10 μL/min for 5 minutes. After the fixation signal reached 2000 RU or more, it was blocked with 20 μL of 1 M ethanolamine hydrochloride for 7 minutes. The target polypeptide to be detected is then dissolved in HBS buffer (pH=7.4) and formulated into a concentration gradient of 0.625 μg/mL, 1.25 μg/mL, 2.5 μg/mL, 5.0 μg/mL, and 10.0 μg/mL. At a flow rate of 25 μL/min, approximately 15 min was injected.

SPR used a single cycle to determine the binding properties of TPP-1 polypeptide to PD-L1. As the concentration of peptide increased, the response of TPP-1 and PD-L1 increased. The data was analyzed by Biacore T200 software to obtain a dissociation equilibrium constant KD of 95 x 10 ^-9 M. The dissociation constant kd=3.022×10 ^-4 , and the binding constant ka=3192Ms ^-1 . The equilibrium dissociation constant shows that TPP-1 binds to PD-L1 as a slower reaction process, and at the same time, dissociation is more difficult once bound.

Example 6 Effect of the polypeptide of the present application on T cell activation

10 mL of normal human peripheral blood was taken, and mononuclear cells were separated by lymphocyte separation solution. CD4+ T cells were sorted using CD4 antibody and flow cytometry. The sorted cells were cultured in an IMDM complete medium containing 200 ng/mL of CD3/CD28 antibody and 20 ng/mL of IL2, and placed at 37 ° C in a 5% CO 2 atmosphere. The fresh medium was replaced according to the color of the medium for 2-3 days, and the activated CD4+ T cells were extensively expanded and frozen after 7-10 days.

In the T cell activation experiment, the CD3 antibody concentration was adjusted to 0.5 μg/mL, Corning 96-well plate was 100 μL per well, and incubated overnight at 4 °C. CD4+ T cells were resuscitated and cultured overnight using IMDM medium supplemented with 10% FBS and 20 ng/ml IL2. PD-L1 protein was diluted to 75 μg/mL with PBS, 20 μL per well was added, and the control antibody MEDI4736, TPP-1 polypeptide and control polypeptide SPP-1 were added, incubated at 37 ° C for 3-4 h, and T cells were added, 150 μL per well. The cells contained 3-4 million cells, and cultured at 37 ° C for 48 hours. 50 μL of the cell culture supernatant was taken, and the IFN-gamma content was determined by ELISA.

Figure 5 shows that 10 μg/mL of MEDI 4736 antibody showed complete inhibition of PD-L1. The inhibitory effect of 10μg/mL TPP-1 polypeptide on PD-L1 was small. When the polypeptide concentration was increased to 50μg/mL, TPP-1 polypeptide showed significant inhibition on PD-L1. Secretion of the cytokine IFN-gamma is returned to the CD3 antibody activation state. The SPP-1 polypeptide still failed to exhibit inhibition of PD-L1 at 50 μg/mL. It is indicated that TPP-1 polypeptide can block the inhibitory effect of PD-L1 on T cells and achieve the purpose of activating T cells.

Example 7 Method for detecting antitumor activity of the polypeptide of the present application

H460-specific CD8+ T cells are capable of specifically killing H460 cells and releasing the cytokine IFN-gamma. When tumor cells overexpress PD-L1, the activity of T cells will be inhibited. Blocking the PD-1/PD-L1 pathway will help to increase T cell activity for the purpose of treating tumors.

The luciferase gene-expressing H460 cells (H460-luc) used in the present application were obtained by transfecting viral particles of the luciferase gene and under pressure screening of 1 μg/mL puromycin.

PBMC cells were freshly isolated and CD8+ T cells were sorted by flow cytometry. The cells were activated in vitro using IMDM complete medium containing 200 ng/mL CD3/CD28 antibody and 20 ng/mL IL2, depending on cell density and medium color. Fresh medium was added for 2-3 days. The mitomycin C was added to the H460-luc cell culture medium at a final concentration of 10 μg/mL, and the cells were further cultured at 37 ° C for 2 h. After washing twice with PBS, the T cells of the fifth day of culture were added to the H460-luc cells and continued. After cocultivation for 3 days, H460 cell-specific CD8+ T cells were harvested.

Female Balb/c nude mice aged 5-6 weeks were selected and cultured in SPF environment for 3 days. H460-luc and activated CD8+ T cells were injected into the right thigh, and pre-mixed and injected 4:1, each small Rats were injected 2.5x10 ⁶ . The polypeptide was administered in situ from day 2, once every 3 days, at a dose of 5 mg/kg for a total of 6 doses. After 7 days of injection of H460-luc cells, IVIS Lumina II was used for in vivo imaging to analyze tumor size, and in vivo imaging was performed once a week. When the tumor volume was measurable, the tumor volume was measured every 3 days and the mice were weighed. The fluorescence intensity, tumor volume and animal body weight of each group of animals were statistically analyzed, and the antitumor activity of the polypeptide was analyzed.

Figure 6 (A) - Figure 6 (B) shows that the volume of the tumor increases as the time of injection of H460-luc increases. There was no significant change in tumor volume between the TPP-1 polypeptide group and the SPP-1 polypeptide group 15 days earlier. In the 20 days or so, the tumor in the control group increased significantly, while the experimental group was inhibited to some extent. The expression of luciferase gene showed a similar trend to tumor volume.

Referring to Figure 7, after observing the tumor volume for 35 days, the mice were sacrificed, tumor tissues were taken for immunohistochemistry, and tissue sections were stained with antibodies of interferon-gamma (IFN-gamma) and granzyme B (Granzyme B). The results showed that the expression of interferon and granzyme B in the mouse group given TPP-1 polypeptide was significantly higher than that in the control group, confirming that the inhibition of TPP-1 polypeptide on tumor is activated T cells, and the tumor is activated by T cells. The cells are caused by killing.

In summary, the polypeptide of the present application and its products can specifically bind to PD-L1, the binding constant can reach ka=3192Ms ^-1 , and the dissociation equilibrium constant KD is 95×10 ^-9 M, which shows that the binding is slow but not easy to fall off. And the polypeptide is capable of specifically targeting and blocking the PD-1/PD-L1 signaling pathway, inhibiting T cells, achieving the purpose of activating T cells, and having antitumor activity; the polypeptide of the present application is in lung cancer It has broad application prospects in the treatment of diseases such as melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer.

The applicant claims that the present application describes the process of the present application by the above embodiments, but the present application is not limited to the above process steps, that is, it does not mean that the application must rely on the above process steps to be implemented. It should be apparent to those skilled in the art that any modifications of the present application, equivalent substitution of the materials selected for the present application, and the addition of the auxiliary components, the selection of the specific manners, and the like, are all within the scope of the present application.

Claims

A polypeptide having a conserved sequence, the amino acid sequence of the conserved sequence being the amino acid sequence set forth in SEQ ID NO.
The polypeptide according to claim 1, wherein the polypeptide is any one or a combination of at least two of the amino acid sequences shown in SEQ ID NO.
The polypeptide according to claim 2, wherein the polypeptide is the amino acid sequence shown in SEQ ID NO.
A DNA fragment comprising a nucleic acid sequence encoding the polypeptide of any of claims 1-3.
A recombinant vector comprising at least one copy of the DNA fragment of claim 4.
A recombinant cell comprising the expression vector of claim 5.
A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 3, the nucleic acid sequence of claim 4, the recombinant vector of claim 5 or the method of claim 6. Recombinant cells.
The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
Use of the pharmaceutical composition according to claim 7 or 8 for the preparation of a tumor localization diagnostic reagent, an antitumor drug or a PD-L1 targeting preparation.
The use according to claim 9, wherein the tumor is any one or a combination of at least two of PD-L1 highly expressed tumors such as lung cancer, melanoma, breast cancer, colorectal cancer, ovarian cancer or liver cancer.