阅读说明：本技术 一种改进的能与pd-1特异性结合的肿瘤抑制肽及其用途 (Improved tumor inhibiting peptide capable of being specifically combined with PD-1 and application thereof ) 是由 王振 张勇 于 2018-10-30 设计创作，主要内容包括：本发明涉及一种改进的能与PD-1特异性结合的肿瘤抑制肽突变体及其用途,该肽突变体可以单独或与抗PD-1单克隆抗体或其它抗肿瘤药物联合的肿瘤免疫治疗和PD-1阳性肿瘤病人的诊断和筛查中,即能够运用于治疗肿瘤、自身免疫性疾病等药物的制备中。(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.)

1. A tumor suppressor peptide, comprising: the amino acid sequence of the inhibitory peptide is a single mutated SEQ ID NO:1, wherein the mutation is 44R/Q.

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.

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-L1IgV 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.