CN111548408A

CN111548408A - Improved tumor inhibiting peptide capable of being specifically combined with PD-1 and application thereof

Info

Publication number: CN111548408A
Application number: CN202010539221.1A
Authority: CN
Inventors: 王振; 张勇
Original assignee: Suzhou Lihao Biotechnology Co ltd
Current assignee: Deqi (Zhejiang) Pharmaceutical Technology Co.,Ltd.
Priority date: 2018-10-30
Filing date: 2018-10-30
Publication date: 2020-08-18
Anticipated expiration: 2038-10-30
Also published as: CN111548407B; CN111548408B; CN111647064A; CN111548407A; CN109265533A; CN111647064B; CN109265533B; CN111647065A; CN111647065B

Abstract

The invention relates to an improved tumor-inhibiting peptide mutant capable of being specifically combined with PD-1 and application thereof, wherein the peptide mutant can be used for tumor immunotherapy alone or combined with anti-PD-1 monoclonal antibodies or other anti-tumor drugs and the diagnosis and screening of PD-1 positive tumor patients, namely can be applied to the preparation of drugs for treating tumors, autoimmune diseases and the like.

Description

Improved tumor inhibiting peptide capable of being specifically combined with PD-1 and application thereof

Technical Field

The invention relates to the technical field of disease treatment, in particular to a tumor suppressor peptide mutant capable of being specifically combined with PD-1 and application thereof.

Background

Programmed death protein-1 (PD-1) and ligand (PD-L1) inhibitors are immune sentinel monoclonal antibody drugs, the response breadth, depth and persistence of the drugs are very rare, and the drugs are hot spots for tumor immunotherapy research in recent years. The marketed nivolumab (nivolumab) and palivizumab (pembrolizumab) belong to PD-1 inhibitors, are mainly used for treating melanoma and non-small cell lung cancer, and have better curative effects on renal cell carcinoma, bladder cancer, Hodgkin lymphoma and the like.

Currently, the main inhibitor of apoptosis protein-1 is monoclonal antibody, the earliest anti-PD-1 antibody was curetech, who promoted the anti-PD-1 antibody to the second stage clinical stage in combination with teva, but clinical trials of this drug have been discontinued due to failure of strategic cooperation, etc. Subsequently, Nivolumab, an anti-PD-1 antibody, was developed by combining Japanese minimus and BMS, which has been marketed in Japan as a first monoclonal antibody drug targeting Pd-1 in the world. BMS is currently being applied for BLA in the united states and is expected to be recently marketed. Furthermore, BMS has been well established in the field of tumor immunity, and the first anti-immune checkpoint antibody, anti-CTLA-4 antibody, is the product of this company. In addition, Roche and GSK focus on PD-1 as a drug target, and anti-PD-1 antibodies thereof enter a clinical research stage. BMS has been well established in the field of tumor immunotherapy and its anti-CTLA-4 antibodies are marketed in good quantity. However, Keytruda in September was the first to be approved without thinking that Merck's company came late. Based on the huge market prospect of the PD-1 inhibitor, all large pharmaceutical enterprises are killed in a dispute and great effort at present.

Designing polypeptides based on the spatial structure of proteins to inhibit the function of proteins in three-dimensional structure is a hot spot of current research, and a number of products have been marketed. Based on the high investment and uncertainty of the PD-1 antibody study, the study of specific inhibitory peptides against PD-1 is also a new direction of research.

CN104098651A discloses a PD-L1 IgV affinity peptide with antitumor activity and application thereof, but the binding site of the peptide is different from that of the invention, the sequence of the peptide is also different from that of the invention, and the effect is different.

The applicant discloses in CN107383174A of the previous application a tumor inhibiting peptide capable of being specifically combined with PD-1 and the application thereof, wherein the inhibiting peptide has better in vivo inhibiting effect on tumor growth.

Based on the needs of the prior art, the search for better inhibitory peptides that bind to PD-1 is an urgent need in the art.

Disclosure of Invention

Aiming at the defects of the prior art, the inventor conducts a large number of experiments to optimize the activity of the inhibitory peptide proposed in the prior application CN107383174A, and obtains a mutant short peptide inhibitor which has a remarkably improved inhibitory effect on PD-1 and can be used as a pharmaceutical active ingredient for treating and/or preventing diseases related to PD-1. Therefore, the invention aims to provide a mutant improved short peptide inhibitor and application thereof.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

in one aspect, the invention provides a short peptide inhibitor, which has an amino acid sequence shown as SEQ ID NO. 1.

In another aspect, the invention provides an improved mutant short peptide inhibitor, the sequence of which is mutated based on the amino acid sequence shown in SEQ ID NO. 1. 5 cases after mutation of the invention: (1) SEQ ID NO:1 polypeptide which is mutated to 10H/Q; (2) SEQ ID NO:1 polypeptide mutated to 40G/R and 42L/S; (3) SEQ ID NO:1 polypeptide having a mutation of 44R/Q; (4) SEQ ID NO:1 polypeptide, which is mutated to 45K/N; (5) SEQ ID NO:1 polypeptide, wherein the mutation is 97H/I.

Preferably, the sequence of the short peptide inhibitor adopts amidation modification at the N segment, and C end is connected with cholesterol modification.

In another aspect of the present invention, there is provided a peptide-nanoparticle composite system prepared and modified by the following steps:

(1) the inhibitory peptide is prepared by Fmoc/tert-butyl strategy and HOBt/TBTU/NMM coupling method through chemical synthesis (or directly synthesized by biological company);

(2) synthesizing 18nm gold nano-microspheres, namely synthesizing 18nm gold particles by one step by using a sodium citrate reduction method;

(3) synthesizing gold nanorods by using a seed growth method;

(4) surface polypeptide modification of the gold nanoparticles;

(5) characterization of the modified gold nanoparticles the physicochemical properties of the modified gold particles can be characterized by measuring their spectra and Zeta-potentials.

In yet another aspect, the present invention provides a pharmaceutical composition comprising a mutated anti-PD-1 inhibitor of the present invention, and a pharmaceutically acceptable carrier.

In yet another aspect, the present invention provides a method for treating or preventing cancer or an infectious disease in a subject in need thereof, comprising administering to the subject a mutated polypeptide and/or a pharmaceutical composition of the present invention.

In yet another aspect, the present invention provides a method for enhancing a T cell immune response in a subject in need thereof, comprising administering to the subject a mutated polypeptide and/or a pharmaceutical composition of the present invention. In some embodiments, the enhancing a T cell immune response comprises enhancing cytokine production by a T cell, preferably the cytokine comprises IL-2 and/or IFN- γ. In some preferred embodiments, the enhancing cytokine production by T cells comprises anti-CD 3 antibody-stimulated cytokine production by T cells. In other preferred embodiments, the subject is a cancer patient, e.g., a PD-L1 positive cancer patient, preferably a lung cancer and melanoma patient.

In yet another aspect, the present invention provides a method for promoting T cell activation in a subject in need thereof, comprising administering to the subject a mutated polypeptide and/or a pharmaceutical composition of the present invention. Preferably, the method further comprises administering to the subject an anti-CD 3 antibody.

In yet another aspect, the present invention provides a method for abrogating the inhibition of T cell activation by PD-L1 in a subject in need thereof, comprising administering to the subject a mutated polypeptide and/or a pharmaceutical composition of the present invention. Preferably, the method further comprises administering to the subject an anti-CD 3 antibody.

In yet another aspect, the invention provides a method of promoting T cell activation (preferably in vitro) comprising contacting a mutant polypeptide or pharmaceutical composition of the invention with a T cell. Preferably, the method further comprises contacting the anti-CD 3 antibody with a T cell.

In yet another aspect, the invention provides a method (preferably in vitro) of abrogating the inhibition of T cell activation by PD-L1, comprising contacting a mutant polypeptide and/or pharmaceutical composition of the invention with a T cell. Preferably, the method further comprises contacting the anti-CD 3 antibody with a T cell.

The positive progress effects of the invention are as follows: the invention optimizes the mutation of the polypeptide in the previous stage to obtain mutant peptide with better activity than the original polypeptide, and the mutant peptide also has high affinity with PD-1 protein, and the polypeptide has good biological activity. The polypeptide can be used in tumor immunotherapy alone or in combination with anti-PD-1 monoclonal antibodies or other anti-tumor drugs and diagnosis and screening of PD-1 positive tumor patients, namely can be used in preparation of drugs for treating tumors, autoimmune diseases and the like.

Detailed Description

The present invention is described in detail below with reference to examples, but they are not intended to limit the present invention further.

EXAMPLE I preparation of inhibitory peptide mutants

According to previous research of the applicant, the preparation method of the inhibitory peptide is disclosed in CN107383174A, and the specific polypeptide sequence is as follows:

KWQEEGQAIHSTNLTQYPHRSGLRVGCHDWRTWPHNYPCGALPRKNYSLFPQDHTPCYIWYYSPDLQHMPNNVKHNSSRSPDLLASKPPVTESPWWHIDTQMSGYMA. The underlined three are extracellular regions of protein receptors that specifically bind to PD-1, forming disulfide bonds between amino acids 11 and 77.

According to the binding characteristics of the inhibitory peptide and the extracellular region, 400 simulation structures are respectively designed at three binding sites, according to protein binding isoelectric points and three-dimensional structure simulation data, 20 optimized mutation combination forms are finally obtained through optimization, and only 6 mutations have remarkably improved activity through later verification, wherein 3 optimized forms are used as conventional controls. The specific mutant forms are respectively: the 6 mutations with better effect are: 10H/Q, 40G/R, 42L/S, 44R/Q, 45K/N or 97H/I combinations thereof; 3 differences of 42L/G, 45K/R, 97H/T as controls. Wherein H/Q represents the replacement of the original amino acid H with Q.

EXAMPLE 2 determination of the affinity of inhibitory peptides for PD-1

(1) The polypeptide was prepared in a 1mg/mL solution using 1 XPBS and the anti-PD-1 polypeptide was prepared in a 100. mu.g/mL solution, 2.5. mu.L of the solution was dropped on a carboxylated SPR chip (available from Plexera, Kx5 SPR standard substrate), and the polypeptide was immobilized on the SPR chip by a specific reaction between streptavidin and biotin. Then PD-1 solutions (diluted by 1 XPBS) with the concentrations of 0.1. mu.g/mL, 1. mu.g/mL, 10. mu.g/mL and 50. mu.g/mL are used as flowing phases to pass through the surface of the chip, the combination time is 150s, the dissociation time is 130s, and the regeneration time is 200 s. 1 XPBS was used as a dissociation solution, and 0.5% phosphoric acid was used as a regeneration solution. The binding dissociation curves of the polypeptides with TNF-. alpha.were recorded on a K.times.5 model SPR instrument (Plexara).

(2) The SPR curve of the polypeptide to PD-1 was fitted with the Langmuir equation. Equilibrium association/dissociation constants were calculated. The results are as follows:

compared with the original sequence 1, the equilibrium dissociation constant KD value of the five mutated polypeptides is remarkably reduced, which indicates that the binding capacity of the polypeptide and PD-1 is further enhanced.

Example 3 detection of biological Activity of mutant polypeptide inhibitors

(1) Jurkat/PD-1/NFAT cells deposited at the general microbiological center of China Committee for culture Collection of microorganisms under the accession number 10298 were selected as target cells, and DMEM/F12+ 10% FBS medium was used as 2.5 × 10⁴The density of each well, 100 mu 1/well, was uniformly plated in 96-well plates (corning) at 37 ℃ with 5% CO₂Incubating in an incubator overnight;

(2) preparing mutant polypeptide solutions with different concentrations by using a 1640+ 10% fetal bovine serum culture medium, diluting to 1 mu g/mL, then downwards diluting 7 concentration points by a 10-fold ratio, sucking out a DMEM/F12+ 10% FBS culture medium in the 96-well plate, and adding the prepared polypeptide inhibitor solution into the 96-well plate according to 50 mu 1/well;

(3) the target cell suspension was prepared using 1640+ 10% fetal bovine serum medium and was as per 5 × 10⁴The density of each well, 50. mu.l/well, was uniformly plated on the above 96-well plate at 37 ℃ with 5% CO₂Incubating in an incubator for 6 hours;

(4) relative chemiluminescent unit values were read using chemiluminescence on an M200 or Pherastar plate reader (rlu), biological activity of the polypeptide inhibitor was calculated by data processing, and half-effective concentration of the polypeptide inhibitor was calculated based on four-parameter regression. The results are as follows:

from the results, it can be seen that there are 5 cases after mutation in the present invention: (1) SEQ ID NO:1 polypeptide which is mutated to 10H/Q; (2) SEQ ID NO:1 polypeptide mutated to 40G/R and 42L/S; (3) SEQ ID NO:1 polypeptide having a mutation of 44R/Q; (4) SEQ ID NO:1 polypeptide, which is mutated to 45K/N; (5) SEQ ID NO:1 polypeptide, the mutation of which is 97H/I, has the EC50 value which is obviously reduced, and has better effect than the original peptide.

Example 4 treatment of in vivo tumor models with anti-inhibitor antibodies

The inhibitory peptides are respectively amidated and modified at N sections by adopting a conventional operation method in the field, and the C end is connected with cholesterol for modification, and the inhibitory peptides are named as PD-1-YZL-C;

in addition, the inhibitory peptide-nanoparticle composite system was prepared and modified by methods that are conventional in the art (1) inhibitory peptides were synthesized directly by bio-companies; (2) synthesizing 18nm gold nano-microspheres, namely synthesizing 18nm gold particles by one step by using a sodium citrate reduction method; (3) synthesizing gold nanorods by using a seed growth method; (4) and modifying the surface polypeptide of the gold nanoparticles. Is named as PD-1-YZL-NM.

Treatment of cancerous tumor implanted mice with inhibitory peptides to examine the in vivo effect of antibodies on tumor growth female AJ mice between 6-8 weeks of age were randomly divided into 6 groups based on body weight 2 × 10 dissolved in 200 μ l DMEM medium on day 0⁶Individual SA1/N fibrosarcoma cells were subcutaneously implanted in mice on the right side. Treatment with PBS control or 1mg/kg inhibitory peptideA mouse. Animals were dosed by intraperitoneal injection on days 1, 4, 8 and 11 with approximately 20 μ l of PBS containing inhibitory peptide or control. Each group contained 10 animals, each group consisting of PBS control group, 5 different inhibitory peptide types. The mice were monitored twice weekly for tumor growth for approximately 6 weeks. The tumors were measured in three dimensions (height X width X length) using an electronic caliper and tumor volume was calculated. When the tumor reached the tumor end point (1500 mm)³) Mice were sacrificed. The results are shown in table 1:

the results show that the mutated inhibitor polypeptide will reach the tumor end-point volume (1500 mm)³) The average time of (2) was increased from 25 days in the control group to 59 days in the longest period, and 55 days in the shortest period, which was significantly higher than the original 50 days. Thus, the mutant inhibitory peptide treatment had a significantly improved in vivo inhibitory effect on tumor growth.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.

Sequence listing

<110> Suzhou Lihao Biotechnology Limited

<120> an improved tumor suppressor peptide capable of specifically binding to PD-1 and use thereof

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Lys Trp Gln Glu Glu Gly Gln Ala Ile His Ser Thr Asn Leu Thr Gln

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Tyr Pro His Arg Ser Gly Leu Arg Val Gly Cys His Asp Trp Arg Thr

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Trp Pro His Asn Tyr Pro Cys Gly Ala Leu Pro Arg Lys Asn Tyr Ser

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Claims

1. A tumor suppressor peptide, comprising: the amino acid sequence of the inhibitory peptide is double-mutated SEQ ID NO:1 polypeptide, the specific mutation is 40G/R and 42L/S.

2. Use of the inhibitory peptide of claim 1 in the manufacture of a medicament for inhibiting PD-1 activity.

3. Use of the inhibitory peptide of claim 1 in the manufacture of a medicament for inhibiting cancer.

4. A method of treating cancer, comprising: administering to a patient a tumor suppressor peptide according to claim 1.