PD-1 and CTLA-4 double-target inhibition polypeptide or derivative, pharmaceutically acceptable salt and application thereof

Abstract

The invention relates to the technical field of biological pharmacy, and particularly provides a PD-1 and CTLA-4 double-target inhibition polypeptide or a derivative thereof, a pharmaceutically acceptable salt and application thereof, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. The polypeptide or the derivative and the pharmaceutically acceptable salt thereof can be combined with PD-1 and CTLA-4 at the same time, combination between PD-1 and ligand and combination between CTLA-4 and ligand can be effectively blocked, the inhibition effect of immune checkpoint protein PD-1 and CTLA-4 on immune cells is relieved, and the immune response of a body is remarkably enhanced. The polypeptide or the derivative and the pharmaceutically acceptable salt thereof can be used for detecting PD-1 and / or CTLA-4, and can also be used for treating or preventing related diseases caused by PD-1 and / or CTLA-4.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical technology, specifically relating to dual-target inhibitory peptides of PD-1 and CTLA-4 or their derivatives, pharmaceutically acceptable salts and their applications. Background Technology

[0002] Programmed death receptor-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are two important immune checkpoint proteins. PD-1 is mainly expressed on the surface of activated T cells. By binding to ligands expressed by tumor cells (PD-L1 / PD-L2), it recruits the protein tyrosine phosphatase SHP-2 through the intracellular ITSM motif, leading to dephosphorylation of TCR-related signaling molecules, weakening TCR signaling, causing T cell exhaustion, and mediating tumor immune escape. CTLA-4, on the other hand, mainly functions in the early stages of the immune response (within lymph nodes). By competing with CD28 for the binding of B7 ligand, it limits the initial activation and proliferation of T cells.

[0003] Currently, the mainstream clinical approach targeting PD-1 and CTLA-4 is monoclonal antibodies (mAbs). These antibodies highly bind to PD-1 or CTLA-4, blocking their interaction with their corresponding ligands, thereby relieving T-cell suppression and restoring the immune system's ability to recognize and kill tumor cells. While some research has been conducted on peptides targeting PD-1 or CTLA-4, further research is needed, and most studies focus on single targets. Dual-target peptides targeting both PD-1 and CTLA-4 have not yet been reported. In the field of peptide drugs, research on dual-target peptides that can simultaneously and precisely bind to and block both PD-1 and CTLA-4 is extremely rare. Designing such molecules requires extremely high spatial configuration precision and screening techniques, and current technologies struggle to balance the binding affinity of both targets with molecular stability.

[0004] Therefore, developing a novel dual-target inhibitory peptide against PD-1 and CTLA-4 is of great significance for clinical treatment. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a PD-1 and CTLA-4 dual-target inhibitory peptide or a derivative thereof, a pharmaceutically acceptable salt, and its application.

[0006] In a first aspect, this application provides a polypeptide or a derivative thereof, or a pharmaceutically acceptable salt. According to embodiments of this application, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1. The polypeptide or its derivative thereof, or the pharmaceutically acceptable salt of this application, can simultaneously bind to PD-1 and CTLA-4, and block the binding between PD-1 and CTLA-4 and their respective ligands, thereby relieving the inhibitory effect of immune checkpoint proteins PD-1 and CTLA-4 on immune cells, significantly enhancing the body's immune response, and thus achieving the treatment of diseases related to PD-1 and / or CTLA-4. Furthermore, the aforementioned polypeptide or its derivative thereof, or the pharmaceutically acceptable salt, can be used for the qualitative and / or quantitative detection of PD-1 and / or CTLA-4.

[0007] In a second aspect, this application provides a nucleic acid molecule. According to embodiments of this application, the nucleic acid molecule encodes the aforementioned polypeptide or its derivative, or a pharmaceutically acceptable salt. The polypeptide or its derivative, or a pharmaceutically acceptable salt encoded by the nucleic acid molecule according to embodiments of this application can simultaneously bind to PD-1 and CTLA-4, and simultaneously block the binding between PD-1 and CTLA-4 and their respective ligands, thereby relieving the inhibitory effect of immune checkpoint proteins PD-1 and CTLA-4 on immune cells, significantly enhancing the body's immune response, and thus achieving the treatment of diseases related to PD-1 and / or CTLA-4. Furthermore, the aforementioned polypeptide or its derivative, or a pharmaceutically acceptable salt, can be used for the qualitative and / or quantitative detection of PD-1 and / or CTLA-4.

[0008] In a third aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the aforementioned nucleic acid molecule. This enables the effective expression of the aforementioned polypeptide or its derivatives, and pharmaceutically acceptable salts, thereby achieving the large-scale in vitro production of the polypeptide or its derivatives, and pharmaceutically acceptable salts.

[0009] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell comprises the aforementioned nucleic acid molecule or expression vector or expresses the aforementioned polypeptide or its derivative, or a pharmaceutically acceptable salt. Using this recombinant cell, under suitable conditions, the aforementioned polypeptide or its derivative, or a pharmaceutically acceptable salt, can be efficiently expressed intracellularly.

[0010] In a fifth aspect, this application provides a conjugate. According to embodiments of this application, the conjugate comprises: the aforementioned polypeptide or a derivative thereof, a pharmaceutically acceptable salt; and a conjugated portion thereto, the conjugated portion being selected from purified tags or labels. The conjugate of this invention can specifically recognize PD-1 and / or CTLA-4, detect PD-1 and / or CTLA-4 proteins, and can also effectively detect and diagnose diseases related to PD-1 and / or CTLA-4.

[0011] In a sixth aspect of this application, the use of the aforementioned polypeptide or its derivatives, pharmaceutically acceptable salts, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of a medicament, wherein the medicament is a dual-target inhibitor of PD-1 and CTLA-4. As is known from the foregoing, the aforementioned polypeptide or its derivatives, and pharmaceutically acceptable salts, can effectively inhibit both PD-1 and CTLA-4 simultaneously. Therefore, the aforementioned polypeptide or its derivatives, and pharmaceutically acceptable salts, can be formulated as dual-target inhibitors of PD-1 and CTLA-4 for the treatment or prevention of diseases related to PD-1 and / or CTLA-4.

[0012] In a seventh aspect of this application, the use of the aforementioned polypeptides or derivatives thereof, pharmaceutically acceptable salts, nucleic acid molecules, expression vectors or recombinant cells in the preparation of a medicament for the treatment or prevention of diseases associated with PD-1 and / or CTLA-4 is provided.

[0013] According to embodiments of this application, the diseases associated with PD-1 and / or CTLA-4 include tumors or cancer.

[0014] According to embodiments of this application, the tumor or cancer includes solid tumors or hematologic malignancies.

[0015] According to embodiments of this application, the tumor or cancer includes at least one of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, skin cancer, biliary tract cancer, mesothelioma, sarcoma, lymphoma, or leukemia.

[0016] In an eighth aspect of this application, the use of the aforementioned polypeptides or derivatives thereof, pharmaceutically acceptable salts, or conjugates in the preparation of reagents or kits for the detection of PD-1 and / or CTLA-4 is provided.

[0017] In a ninth aspect of this application, a medicament is proposed. According to embodiments of this application, the medicament comprises the aforementioned polypeptide or its derivative, a pharmaceutically acceptable salt, a nucleic acid molecule, an expression vector, or recombinant cells. As is known from the foregoing, the aforementioned polypeptide or its derivative, the pharmaceutically acceptable salt, and a series of products capable of obtaining this substance, such as nucleic acid molecules, expression vectors, or recombinant cells, can ultimately enable the polypeptide or its derivative, or the pharmaceutically acceptable salt, to bind to PD-1 and CTLA-4, effectively blocking the binding between PD-1 and CTLA-4 and their respective ligands. Therefore, a medicament containing the aforementioned polypeptide can be used to treat or prevent diseases related to PD-1 and / or CTLA-4.

[0018] According to embodiments of this application, the drug may further include pharmaceutically acceptable excipients.

[0019] In a tenth aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises the aforementioned polypeptide or derivative thereof, a pharmaceutically acceptable salt, or a conjugate. As is known from the foregoing, the aforementioned polypeptide or derivative thereof, pharmaceutically acceptable salt, or conjugate can bind to PD-1 and CTLA-4, and can be used to effectively block the binding between PD-1 and CTLA-4 and their respective ligands. Therefore, a reagent or kit containing the aforementioned polypeptide or derivative thereof, pharmaceutically acceptable salt, or conjugate can bind to PD-1 and / or CTLA-4 for the detection of PD-1 and / or CTLA-4.

[0020] In its eleventh aspect, this application discloses a method for detecting PD-1 and / or CTLA-4. According to embodiments of this application, the method includes: contacting a sample to be tested with the aforementioned polypeptide or its derivative, a pharmaceutically acceptable salt, a conjugate, or a reagent or kit; and determining whether the sample to be tested contains PD-1 and / or CTLA-4 based on the signal generated by the contact product. As is previously known, the aforementioned polypeptide or its derivative, and pharmaceutically acceptable salt, can bind to PD-1 and CTLA-4; therefore, the above method can be used for qualitative and / or quantitative detection of PD-1 and / or CTLA-4.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1This describes the generation and screening process of PD-1 & CTLA-4 targeting peptides in the embodiments of this application; Figure 2 The results of the detection of PD-1 & CTLA-4 targeting peptides inhibiting the binding of PD-1 and PD-L1 in the embodiments of this application are shown. Figure 3 The results of the detection of PD-1 & CTLA-4 targeting peptides inhibiting the binding of CTLA-4 and B7-1 in the embodiments of this application are shown. Figure 4 In the embodiments of this application, the PD-1 & CTLA-4 targeting peptides activate CD4. + T cell detection results; Figure 5 In the embodiments of this application, the PD-1 & CTLA-4 targeting peptides activate CD8. + T cell detection results; Figure 6 The above are the statistical analysis results of the effect of PD-1 & CTLA-4 targeting peptides on tumor volume in humanized mice in the embodiments of this application. The horizontal axis represents the number of days after injection of PD-1 & CTLA-4 targeting peptides. Figure 7 The statistical analysis results of the effect of PD-1 & CTLA-4 targeting peptides on tumor weight in humanized mice in the embodiments of this application are as follows; Figure 8 This is a graph showing the effect of PD-1 & CTLA-4 targeting peptides on tumor size in humanized mice in the embodiments of this application. Detailed Implementation

[0023] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0024] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0025] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0026] In this article, the term "PD-1" refers to Programmed Cell Death Protein 1 (PD-1), an important immune checkpoint receptor primarily expressed on the surface of T cells, B cells, and bone marrow cells. Under physiological conditions, PD-1 binds to its ligands (PD-L1 or PD-L2) to inhibit excessive T cell activation, thereby maintaining immune tolerance and preventing autoimmune diseases. However, many tumor cells "hijack" this mechanism by expressing high levels of PD-L1, causing the immune system to generate inhibitory signals, leading to tumor immune escape. Therefore, PD-1 inhibitors have become important substances in tumor immunotherapy, aiming to block this inhibitory pathway and reactivate the T cell's ability to kill tumor cells.

[0027] In this article, the term "CTLA-4" refers to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), a transmembrane receptor expressed on the surface of activated T cells. Belonging to the immunoglobulin superfamily, it is another key negative regulatory molecule in the immune system. It competitively binds to ligands (B7-1 / CD80 and B7-2 / CD86) on the surface of antigen-presenting cells, along with the co-stimulatory receptor CD28. Because CTLA-4 has a much higher affinity for these ligands than CD28, its binding blocks helper activation signals, thereby inhibiting T cell proliferation and activation in the early stages of the immune response. Clinically, blocking CTLA-4 with drugs can enhance the body's anti-tumor immune response.

[0028] In this article, the term "CD4" refers to Cluster of Differentiation 4, a transmembrane glycoprotein primarily expressed on the surface of helper T cells (Th cells) and an important regulatory molecule in the immune system. CD4 is a lineage marker for helper T cells (Th cells), while ICOS (inducible costimulatory molecules) are activation markers whose expression is upregulated only after stimulation of the T cell receptor (TCR). When CD4... + When T cells simultaneously express high levels of ICOS, it usually means that the cells are in a highly activated state.

[0029] In this article, the term "CD8" refers to Cluster of Differentiation 8, a key transmembrane glycoprotein primarily expressed on the surface of cytotoxic T lymphocytes (CTLs). CD8 is a characteristic surface marker of cytotoxic T cells. CD44, on the other hand, is a cell adhesion molecule whose expression level reflects the T cell's response to antigen stimulation. high (High expression) indicates that the T cell has been activated and is transforming into an effector T cell or has become a memory T cell.

[0030] In this article, the term "target protein" refers to a protein that plays a key role in an organism and is often the target of drug development. By binding to a target protein, drugs can regulate its biological activity, thereby achieving the goal of treating diseases.

[0031] In this article, the term "peptide" refers to a biological macromolecule composed of many amino acids, typically 50 or fewer amino acids linked together by peptide bonds. Peptides perform a variety of functions in living organisms, including participating in various physiological processes as enzymes, hormones, and antibodies. Furthermore, due to their excellent biocompatibility, selectivity, and high biological activity, peptides are widely studied for drug discovery and the treatment of various diseases, such as metabolic disorders.

[0032] In this article, the term "dual-target peptide" refers to a polypeptide molecule that can bind to two different targets simultaneously. This gives dual-target peptides a unique advantage in drug development, as they can enhance therapeutic effects by simultaneously regulating multiple biological pathways.

[0033] In this paper, the term "Conditional Diffusion Model (Gong et al. 2022)" refers to the generation of data by introducing conditional signals (such as text, images, etc.) during the training process of the DDPM model. This type of model can control the output according to needs and generate high-quality data in a customized manner.

[0034] In this paper, the term "BERT (Bidirectional Encoder Representations from Transformers) (Devlin et al. 2018)" refers to a deep bidirectional Transformer encoder that is capable of capturing bidirectional contextual information in sequence data.

[0035] In this paper, the term "affinity maturation" refers to the process by which B cells increase the affinity of their antibodies (proteins) for binding to antigens through mutation and selection mechanisms during the immune response. In this process, B cells undergo a high frequency of mutations in the germinal centers, producing a variety of variant antibodies (proteins), which are then selected based on their binding ability to antigens, ultimately producing antibodies (proteins) with high affinity.

[0036] In this article, the term "peptide bioactivity" refers to the biological functions and activities of peptides in vivo. Peptides can regulate biological processes, such as signal transduction, immune responses, and cell proliferation, by binding to specific target proteins. The bioactivity of peptides depends on their amino acid sequence, structure, and interaction with their target. Due to their good biocompatibility and selectivity, peptides have significant potential in drug development, disease treatment, and biotechnological applications.

[0037] In this document, amino acids are referred to by their conventional single-letter and three-letter codes for natural amino acids, as well as the generally accepted three-letter codes for other α-amino acids. Unless otherwise specified, in this application, uppercase letters represent amino acids with the L-configuration and lowercase letters represent amino acids with the D-configuration.

[0038] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including: the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0039] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0040] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0041] In this paper, the structural formula for the term "αMeK" is: .

[0042] In this article, the structural formula for the term "HoK" is: .

[0043] In this article, the structural formula for the term "N-Me-K" is: .

[0044] In this article, the structural formula for the term "Orn" is: .

[0045] In this article, the structural formula for the term "Dab" is: .

[0046] In this article, the structural formula for the term "Dap" is: .

[0047] In this document, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components comprising a polypeptide or its derivatives and / or with the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0048] In this document, the term "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the polypeptides or their derivatives of this application. Pharmaceutically acceptable salts are well known to those skilled in the art. Salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts (such as hydrochlorides, hydrobromic acids, phosphates, sulfates, and perchlorates) formed by reaction with amino groups, and organic acid salts (such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates), or salts obtained by other methods described in the literature, such as ion exchange.

[0049] In this document, the term "drug" can refer to something used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "drug" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients that constitute one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining an active polypeptide or its derivative with a liquid excipient, a finely chopped solid excipient, or both.

[0050] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients is also within the scope of consideration for this application, except for any range of incompatibilities with the polypeptide or its derivatives, pharmaceutical compositions, or drugs containing them, such as any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition.

[0051] In addition to any conventional excipients, the use of any polypeptide or derivative thereof, drug or drug containing them that is incompatible with the present application, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition that occur in a harmful manner, is also within the scope of this application.

[0052] The pharmaceutical products disclosed herein include formulations suitable for parenteral administration. The formulations can be conveniently available in unit dosage forms and can be prepared by any method known in the pharmaceutical field. The amount of the active ingredient in a single-dose form, which can be prepared in combination with excipients, is generally the amount of the polypeptide or a derivative thereof that produces the therapeutic effect.

[0053] In this document, the term "reagent kit" refers to any container that carries raw materials or reagents, which may be in the form of a card, tube, box, strip, plate, etc., and may also include packaging units having one or more containers.

[0054] In this article, the term "inhibitor" refers to a substance (ligand) that inhibits the type of receptor.

[0055] In this document, the term "treatment" refers to the attainment of a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with it; (b) inhibition of disease, such as blocking disease progression; or (c) alleviation of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any medication that administers a polypeptide or its derivative, a pharmaceutically acceptable salt thereof, or a drug containing such polypeptide or its derivative to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to administering a drug containing the polypeptide or its derivative described herein, or a pharmaceutically acceptable salt thereof, to an individual in need.

[0056] This application discloses a dual-target inhibitory peptide for PD-1 and CTLA-4 or a derivative thereof, a pharmaceutically acceptable salt thereof, and their uses, which will be described in detail below.

[0057] polypeptides or their derivatives, pharmaceutically acceptable salts In one aspect of this application, a polypeptide or a derivative thereof, or a pharmaceutically acceptable salt, is provided. According to embodiments of this application, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

[0058] This application utilizes precise computation, deep learning models, and iterative evolutionary models to optimize and obtain novel dual-target peptides for PD-1 and CTLA-4. These peptides possess unique amino acid sequences, and the aforementioned peptides or their derivatives, as well as pharmaceutically acceptable salts, can simultaneously bind to both PD-1 and CTLA-4, exhibiting high affinity and bioactivity for both. They effectively inhibit or block the binding of PD-1 and CTLA-4 to their respective ligand proteins, thereby relieving the inhibitory effect of immune checkpoint protein molecules on the immune system. Therefore, the aforementioned peptides or their derivatives, as well as pharmaceutically acceptable salts, can be used to treat or prevent diseases related to PD-1 and / or CTLA-4, and can also be used for the qualitative or quantitative detection of PD-1 and / or CTLA-4.

[0059] In this document, the term "amino acid sequence of a polypeptide as shown in SEQ ID NO:A" includes the amino acid sequence of SEQ ID NO:A, the amino acid sequence of a conserved modified form of SEQ ID NO:A, or a sequence similarity of more than 80% to the amino acid sequence shown in SEQ ID NO:A (e.g., more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%), all of which are within the scope of protection of this application. Unless otherwise specified, the conserved modified amino acids or amino acids with different similarities in the amino acid sequence of the conserved modified form of SEQ ID NO: A, or the amino acid sequence shown in SEQ ID NO: A having a sequence similarity of more than 80% (e.g., more than 80%, more than 81%, more than 82%, more than 83%, more than 84%, more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) can be located at any position in the polypeptide of this application. Such amino acid modifications or differences in similarity do not significantly affect or change the structural stability of the polypeptide containing the amino acid and / or its binding activity with ligands, and are all within the scope of protection of this application.

[0060] In this document, "conservatively modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the properties of the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the peptides of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Exemplarily, the number of conserved modifications does not exceed 10% of the total number, preferably not exceeding 5%. In this document, "conserved amino acid sequences" also include naturally mutated amino acid modifications; "naturally mutated" refers to mutations caused by changes in alleles or other parameters during the natural mutation process of a polypeptide.

[0061] In this paper, the term "sequence similarity" is defined using a percentage similarity method, which is calculated by comparing the number of identical or similar amino acids in two protein or polypeptide sequences out of the total number of amino acids. .

[0062] According to embodiments of this application, the polypeptide has an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 80% sequence similarity to it; or, compared to the amino acid sequence shown in SEQ ID NO:1, the polypeptide has been substituted, deleted, or added with 1 to 3 amino acids and has PD-1 and CTLA-4 binding activity. For example, 1, 2, or 3 amino acids may be substituted, deleted, or added.

[0063] It should be noted that in this article, "substitution, deletion, or addition of one or more amino acids" means that the substitution, deletion, or addition of such amino acids does not significantly affect or alter the binding properties of the original amino acid sequence. Amino acid substitution refers to the replacement of amino acid residues in the original peptide chain with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0064] According to embodiments of this application, the amino acid used for substitution or addition is selected from amino acid X whose side chain contains -NH2, -SH, -OH or -COOH.

[0065] According to embodiments of this application, the amino acid used for substitution or addition is selected from amino acid X containing -NH2 in its side chain. This increases the number of modifiable sites in the original polypeptide, where the -NH2 side chain can bind to the modifying group, thereby effectively extending the half-life of the polypeptide or its derivatives, or pharmaceutically acceptable salts, in vivo.

[0066] In this document, the term "modifying group" should be interpreted broadly, and can refer to chemical groups or amino acid fragments. The specific type is not limited, and all are within the scope of protection of this application.

[0067] According to embodiments of this application, the modifying group is linked to the -NH2 group of the side chain of amino acid K in the polypeptide, or the substituted or added amino acid X; the modifying group has at least one of the following structures: .

[0068] In this article, the "" in the description of chemical groups "" is used to describe the position of a group substitution. That is, the above chemical group is substituted by... It is linked to the -NH2 group of an amino acid to form a -CO-NH- linkage.

[0069] According to embodiments of this application, the amino acid X is selected from K, k, αMeK, HoK, Dap, Dab, Orn, or N. Me K.

[0070] Nucleic acid molecules, expression vectors, recombinant cells, conjugates In one aspect of this application, a nucleic acid molecule is provided that encodes the aforementioned polypeptide or its derivative, or a pharmaceutically acceptable salt. According to embodiments of the present invention, the polypeptide or its derivative, or a pharmaceutically acceptable salt encoded by the nucleic acid molecule can simultaneously bind to PD-1 and CTLA-4, and simultaneously block the binding between PD-1 and CTLA-4 and their respective ligands, thereby relieving the inhibitory effect of immune checkpoint proteins PD-1 and CTLA-4 on immune cells, significantly enhancing the body's immune response, and thus achieving the treatment of diseases related to PD-1 and / or CTLA-4. Furthermore, the aforementioned polypeptide or its derivative, or a pharmaceutically acceptable salt, can be used for the qualitative and / or quantitative detection of PD-1 and / or CTLA-4.

[0071] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. Although in most cases only one strand is given, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0072] In another aspect, this application provides an expression vector carrying the aforementioned nucleic acid molecule. This effectively enables the expression of the aforementioned polypeptide or its derivatives, and pharmaceutically acceptable salts, thereby achieving the large-scale in vitro production of the polypeptide or its derivatives, and pharmaceutically acceptable salts.

[0073] When ligating the aforementioned nucleic acid molecules to an expression vector, the nucleic acid molecules can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. The key is that the nucleic acid molecules and control elements are operatively linked. In this context, "operatively linked" means linking a foreign gene to the vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids and bacteriophages.

[0074] According to embodiments of this application, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0075] According to an embodiment of this application, the expression vector is a plasmid expression vector.

[0076] In another aspect of the invention, this application provides a recombinant cell comprising the aforementioned nucleic acid molecule or expression vector or expressing the aforementioned polypeptide or its derivative, or a pharmaceutically acceptable salt. Using this recombinant cell, under suitable conditions, the aforementioned polypeptide or its derivative, or a pharmaceutically acceptable salt, can be efficiently expressed intracellularly.

[0077] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the polypeptide or its derivatives, or pharmaceutically acceptable salts, as described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the polypeptide or its derivatives, or pharmaceutically acceptable salts, include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the polypeptide or its derivatives, or pharmaceutically acceptable salts, based on the specific environment of the laboratory or specific needs.

[0078] According to embodiments of this application, the recombinant cells are eukaryotic cells or prokaryotic cells.

[0079] According to embodiments of this application, the recombinant cells are eukaryotic cells, preferably mammalian cells.

[0080] In another aspect, this application provides a conjugate comprising: the aforementioned polypeptide or a derivative thereof, a pharmaceutically acceptable salt; and a conjugated portion thereof, the conjugated portion being selected from purified tags or labels. The conjugate of this invention can specifically recognize PD-1 and CTLA-4, enabling the detection of PD-1 and CTLA-4 proteins, and can also effectively detect and diagnose diseases related to PD-1 and CTLA-4.

[0081] According to embodiments of this application, the coupling portion includes at least one selected from colloidal gold, radioactive labels, phosphorescent agents, chemiluminescent agents, fluorescein, enzymes, natural toxins, nucleic acids, and affinity markers.

[0082] According to an embodiment of this application, the coupling portion includes a radioactive isotope.

[0083] According to embodiments of this application, the coupling portion includes at least one selected from phycoerythrin, fluorophore, rhodamine, luciferase, luciferin isothiocyanate, green fluorescent protein, blue fluorescent protein, and red fluorescent protein.

[0084] According to embodiments of this application, the coupling portion includes at least one selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactosyloxidase, and glucose-6-phosphate dehydrogenase.

[0085] According to embodiments of this application, the coupling portion includes at least one selected from biotin and avidin.

[0086] According to embodiments of this application, the coupling portion includes at least one selected from magnetic beads, magnetic microspheres, plastic microspheres, plastic microparticles, microporous plates, nylon, and nitrocellulose membranes.

[0087] In this document, the coupling portion can be a substance capable of being suspended or dispersed in a liquid phase (e.g., solid-phase carriers such as particles or magnetic beads), or a solid phase capable of containing or carrying a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as well plates, microfluidic pathways, glass capillaries, nanopillars, and monolithic columns); it can also be a labeling carrier for labeling peptides or their derivatives, or pharmaceutically acceptable salts, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, β-phosphatase). Galactosidase), affinity substances (e.g., one of streptavidin and biotin, or one of complementary sense and antisense nucleic acids), fluorescent substances (e.g., luciferin, luciferin isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., insect luciferin, aequorin, acridinium ester, tri(2,2') Bipyridine, ruthenium, luminol, radioactive isotopes (e.g., 3H, 14C, 32P, 35S, 125I), and gold colloids, etc.

[0088] According to some specific embodiments of this application, the coupling portion may be a protein tag, which includes, but is not limited to, His tags, Flag tags, GST tags, MBP tags, SUMO tags, and C-Myc tags.

[0089] It should be noted that the binding of the coupling moiety to the polypeptide or its derivative, or a pharmaceutically acceptable salt, can be achieved using methods known in the art. Examples include physical adsorption, covalent bonding, methods utilizing affinity substances (e.g., biotin, streptavidin), and ion bonding.

[0090] use In one aspect of this application, the use of the aforementioned polypeptide or its derivatives, pharmaceutically acceptable salts, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of a medicament, wherein the medicament is a dual-target inhibitor of PD-1 and CTLA-4. As is known from the foregoing, the aforementioned polypeptide or its derivatives, and pharmaceutically acceptable salts, can effectively inhibit both PD-1 and CTLA-4 simultaneously. Therefore, the aforementioned polypeptide or its derivatives, and pharmaceutically acceptable salts, can be formulated as dual-target inhibitors of PD-1 and CTLA-4 for the treatment or prevention of diseases related to PD-1 and / or CTLA-4.

[0091] In another aspect of this application, the use of the aforementioned polypeptides or derivatives thereof, pharmaceutically acceptable salts, nucleic acid molecules, expression vectors or recombinant cells in the preparation of medicaments for the treatment or prevention of diseases associated with PD-1 and / or CTLA-4 is proposed.

[0092] According to embodiments of this application, the diseases associated with PD-1 and / or CTLA-4 include tumors or cancer.

[0093] According to embodiments of this application, the tumor or cancer includes solid tumors or hematologic malignancies.

[0094] According to embodiments of this application, the tumor or cancer includes at least one of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, skin cancer, biliary tract cancer, mesothelioma, sarcoma, lymphoma, or leukemia.

[0095] In another aspect of this application, the use of the aforementioned polypeptides or derivatives thereof, pharmaceutically acceptable salts, or conjugates in the preparation of reagents or kits for the detection of PD-1 and / or CTLA-4 is provided. As is known from the foregoing, the aforementioned polypeptides or derivatives thereof, pharmaceutically acceptable salts, or conjugates thereof can specifically bind to PD-1 and CTLA-4. Furthermore, kits containing the above substances can also effectively bind to PD-1 and CTLA-4 and can be used for the effective detection of PD-1 and / or CTLA-4.

[0096] It should be noted that the kit can be used for scientific research, such as for qualitative or quantitative detection of PD-1 and / or CTLA-4 levels in biological samples (such as samples from laboratory animals such as rats and mice). It can also be used to assess the status of a subject, such as determining whether the PD-1 and / or CTLA-4 levels in biological samples from the subject are too high or too low after obtaining the PD-1 and / or CTLA-4 levels based on the reagent or kit. The biological samples can be saliva, urine, sweat, feces, skin, cells, tissues, blood, plasma, or serum, etc.

[0097] Drugs, reagents or kits In one aspect of this application, a drug is proposed comprising the aforementioned polypeptide or derivative thereof, a pharmaceutically acceptable salt, a nucleic acid molecule, an expression vector, or recombinant cells. As is known to the public, the aforementioned polypeptide or derivative thereof, the pharmaceutically acceptable salt, and a series of products capable of obtaining this substance, such as nucleic acid molecules, expression vectors, or recombinant cells, can ultimately enable the polypeptide or derivative thereof, or the pharmaceutically acceptable salt, to bind to PD-1 and CTLA-4, effectively blocking the binding between PD-1 and CTLA-4 and their respective ligands. Therefore, a drug containing the aforementioned polypeptide can be used to treat or prevent diseases related to PD-1 and / or CTLA-4.

[0098] According to embodiments of this application, the drug may further include pharmaceutically acceptable excipients.

[0099] According to embodiments of this application, the medicament may further include a pharmaceutically acceptable carrier.

[0100] According to embodiments of this application, the medicament may further include a pharmaceutically acceptable mediator.

[0101] According to an optional embodiment of this application, pharmaceutically acceptable excipients refer to pharmaceutical excipients that are conventional in the pharmaceutical field, such as diluents, buffer solutions, osmotic pressure regulators, pH regulators, etc.

[0102] According to an optional embodiment of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field, such as a protectant.

[0103] According to an optional embodiment of this application, pharmaceutically acceptable mediators refer to pharmaceutical mediators conventional in the pharmaceutical field, such as solutions (e.g., water) and liposomes.

[0104] According to optional embodiments of this application, examples of suitable pharmaceutically acceptable carriers, excipients, and mediators are well known in the art. Drugs comprising such carriers, excipients, and mediators can be formulated using known conventional methods.

[0105] According to optional embodiments of this application, the drug may be an oral preparation, such as a solid oral preparation or a liquid oral preparation, and the specific type is not limited, all of which are within the protection scope of this application.

[0106] According to optional embodiments of this application, the medicament of this application may also contain other active ingredients for treatment.

[0107] The medicament of this application can be administered via various routes, such as enterally, orally (e.g., liquid solution), or by injection (e.g., intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally). Preferably, the medicament of this application is in the form of a lyophilized preparation or an aqueous solution. The clinical dosing regimen will be determined by the attending physician and clinical factors. As is known in the medical field, the dosage for any given patient depends on many factors, including the patient's physique, body surface area, age, the drug to be administered, sex, time and route of administration, general health, and other concurrently administered drugs. The pharmaceutical composition of this application can be administered topically or systemically. Preferably, it can be administered intravenously or subcutaneously.

[0108] In another aspect of this application, a reagent or kit is provided, comprising the aforementioned polypeptide or derivative thereof, a pharmaceutically acceptable salt, or the aforementioned conjugate. As is known prior, the aforementioned polypeptide or derivative thereof, and the pharmaceutically acceptable salt, possess high affinity for PD-1 and CTLA-4. Therefore, a reagent or kit containing the aforementioned polypeptide or derivative thereof, or a pharmaceutically acceptable salt, can bind to PD-1 and CTLA-4 for the detection of PD-1 and / or CTLA-4.

[0109] In this paper, kits or reagents do not need to have a box structure, but only need to be relatively independent and have suitable loading or containers, such as tubes, boxes, bottles, and cards; some components are separated into different containers, and if permitted, some components can be combined into one container.

[0110] According to embodiments of this application, the kit includes reagents suitable for detection. In some embodiments, the kit may include instructions for use in the detection. In some embodiments, the kit may include calibrators or controls, such as standards or control samples. In some embodiments, the kit also includes containers such as test tubes, microplates, or test strips.

[0111] use In one aspect of this application, the use of the aforementioned polypeptides or derivatives thereof, pharmaceutically acceptable salts, nucleic acid molecules, expression vectors, recombinant cells or drugs in the treatment or prevention of diseases related to PD-1 and / or CTLA-4 is proposed.

[0112] According to embodiments of this application, the above-described uses may further include at least one of the following technical features: According to embodiments of this application, the diseases associated with PD-1 and / or CTLA-4 include tumors or cancer. According to embodiments of this application, the tumor or cancer includes at least one of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, skin cancer, biliary tract cancer, mesothelioma, sarcoma, or hematologic malignancies.

[0113] In another aspect of this application, this application proposes the aforementioned polypeptides or derivatives thereof, pharmaceutically acceptable salts, nucleic acid molecules, expression vectors, recombinant cells, or drugs for the treatment or prevention of diseases related to PD-1 and / or CTLA-4.

[0114] According to embodiments of this application, the above-described uses may further include at least one of the following technical features: According to embodiments of this application, the diseases associated with PD-1 and / or CTLA-4 include tumors or cancer. According to embodiments of this application, the tumor or cancer includes at least one of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, skin cancer, biliary tract cancer, mesothelioma, sarcoma, or hematologic malignancies.

[0115] method In one aspect of this application, a method for detecting PD-1 and / or CTLA-4 is provided. The method includes: contacting a sample to be tested with the aforementioned polypeptide or its derivative, a pharmaceutically acceptable salt, a conjugate, or a reagent or kit; and determining whether the sample to be tested contains PD-1 and / or CTLA-4 based on the signal generated by the contact product. As is known, the aforementioned polypeptide or its derivative, and pharmaceutically acceptable salt, can bind efficiently to PD-1 and CTLA-4; therefore, the above method can be used to detect PD-1 and / or CTLA-4.

[0116] According to a specific embodiment of this application, the signal includes a fluorescence signal.

[0117] According to a specific embodiment of this application, the method further includes determining the content values ​​of PD-1 and / or CTLA-4 in the sample to be tested based on the signal generated by the contact product. That is, PD-1 and / or CTLA-4 can be qualitatively detected or quantitatively detected.

[0118] In another aspect of this application, a method for treating or preventing diseases associated with PD-1 and / or CTLA-4 is provided, the method comprising: administering to a subject a pharmaceutically acceptable dose of the aforementioned polypeptide or a derivative thereof, a pharmaceutically acceptable salt, a nucleic acid molecule, an expression vector, a recombinant cell, or a drug.

[0119] According to an alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0120] The effective amount of the polypeptide or its derivatives, or pharmaceutically acceptable salts, may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0121] The polypeptides or derivatives thereof, pharmaceutically acceptable salts, polypeptide derivatives or pharmaceutically acceptable salts thereof, or pharmaceutical compositions of this application may be incorporated into suitable pharmaceuticals, which may be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, including but not limited to solid dosage forms, semi-solid dosage forms, liquid dosage forms, and gaseous dosage forms. Various routes of administration of the polypeptides or derivatives thereof, pharmaceutically acceptable salts, polypeptide derivatives or pharmaceutically acceptable salts thereof, pharmaceutical compositions, or pharmaceuticals of this application are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, topical, nasal, pulmonary, rectal, and topical administration; however, this application is not limited to these exemplified routes of administration.

[0122] According to embodiments of this application, the diseases associated with PD-1 and / or CTLA-4 include tumors or cancer.

[0123] According to embodiments of this application, tumors or cancers include solid tumors or hematologic malignancies.

[0124] According to embodiments of this application, the tumor or cancer includes at least one of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, skin cancer, biliary tract cancer, mesothelioma, sarcoma, lymphoma, or leukemia.

[0125] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0126] Example 1: Obtaining a dual-target protein inhibitory peptide for PD-1 and CTLA-4 The schematic diagram of the peptide design and screening process in this application is shown below. Figure 1 This method encompasses a series of steps, from generating dual-target peptide sequences for PD-1 and CTLA-4 to screening and optimization. The entire approach consists of multiple iterative evolutionary processes, with each iteration resulting in a significant improvement in the bioactivity of the model-designed peptides. Each iteration comprises three parts: the generation and optimization of novel peptides based on artificial intelligence, wet experimental determination of peptide bioactivity, and feedback to enhance the model's perception of bioactivity.

[0127] First, an innovative deep learning method, TPDiffusion, can generate peptide sequences that can bind to target proteins based on their amino acid sequences. This method, which transforms target protein sequences into peptide sequences, can be viewed as providing a specific answer to a specific problem. By training the conditional diffusion model TPDiffusion, the relationship rules between target proteins and peptide sequences are learned, enabling the generation of specific peptides (i.e., PD-1 and CTLA-4 dual-target peptides) for specific target proteins (PD-1: uniprot ID: Q15116, and CTLA-4: uniprot ID: P16410). The training of the peptide sequence generation model TPDiffusion mainly includes forward diffusion and reverse diffusion processes. The forward diffusion process includes the following steps: 1. Encoding the amino acid sequence: The target protein and polypeptide sequences are spliced ​​together as a whole, and an embedding transformation function is introduced to map each discrete amino acid word or character to a continuous vector encoding space to model the joint feature space of the protein-peptide.

[0128] 2. Gradually add noise to the peptide sequence: Gaussian noise is gradually added to the peptide part of the vector encoding based on the Markov chain until the peptide sequence is completely destroyed.

[0129] After the forward diffusion process is completed, a clean set of target protein and noise peptide sequences is obtained. Then, the peptide sequence is restored through the reverse diffusion process, which mainly includes the following steps: 1. Stepwise denoising of peptide sequences: The distribution of noise added during forward diffusion is estimated by constructing a denoising network, and the peptide part is denoised stepwise until the peptide sequence is completely restored.

[0130] 2. Calculate the loss function: The model outputs the predicted probability distribution of the peptide sequence. The mean squared error loss function is used to calculate the difference between the model prediction and the actual result, and backpropagation is performed to update the model parameters.

[0131] The denoising network in TPDiffusion employs the BERT model (see Devlin, Jacob, et al. "Bert: Pre-training of deep bidirectional transformers for language understanding." Proceedings of the 2019 conference of the North American chapter of the association for computational linguistics: human language technologies, volume 1 (long and short papers). 2019). The BERT model architecture primarily consists of multi-layer Transformer encoders, with each Transformer block containing both self-attention and a feed-forward neural network. Through the self-attention mechanism, TPDiffusion introduces target sequence information during the backdiffusion process to recover the peptide sequence, implicitly modeling the relationship between the target protein and the peptide sequence, thus achieving a mapping from the target protein to the peptide sequence. During generation, given an arbitrary target protein sequence, the model first randomly samples from Gaussian noise and then performs a backdiffusion process. Guided by the target protein sequence, noise is gradually eliminated over a fixed time step, ultimately generating a binding peptide sequence targeting a given target. Using a trained TPDiffusion, the protein sequences of the aforementioned two targets, PD-1 and CTLA-4, were used as input to generate a batch of candidate peptide sequences that may have high affinity for both PD-1 and CTLA-4.

[0132] Second, affinity maturation is performed on the high-affinity candidate peptide sequences obtained in the first step. This method is an innovative deep reinforcement learning scheme specifically designed to optimize peptide sequences, guiding the evolution of candidate peptide sequences by simulating the environment and defining actions. This deep reinforcement learning framework can incorporate reward models as prior knowledge to guide peptide evolution. These reward models can be affinity prediction models, solubility prediction models, or toxicity prediction models, etc.

[0133] The reward model, PepAF, is an innovative technical solution that effectively predicts the binding affinity of target proteins and peptides by comprehensively utilizing structural information, flexibility, and advanced pre-training strategies. PepAF first learns on two pre-training tasks: 1) estimating the binding free energy of protein (PD-1 and CTLA-4)-peptide complexes; and 2) predicting the affinity of protein (PD-1 and CTLA-4)-peptide complexes. The first task enables the model to learn the complex interactions between proteins (PD-1 and CTLA-4) and peptides, as well as their structural and physicochemical properties, providing a foundation for understanding the binding patterns and key features of protein (PD-1 and CTLA-4)-peptide interactions. The second task enables the model to capture a wide range of molecular interactions at the atomic scale. PepAF also improves prediction accuracy by modeling the target protein structure and the peptide flexibility. The core technology PepAF from this patent is used as a reward model in a deep reinforcement learning method to guide the mutation of candidate peptide sequences.

[0134] After obtaining the affinity-matured peptide, its dual-target bioactivity was evaluated, and the results were fed back to the model to continue the next round of peptide generation and optimization. After multiple rounds of iteration, a peptide that simultaneously inhibited both PD-1 and CTLA-4 target proteins was designed and named PD-1-CTLA4-2, whose amino acid sequence is shown in SEQ ID NO:1 in Table 1.

[0135] Table 1

[0136] Example 2: Inhibitory ability of candidate peptides against dual-target proteins PD-1 and CTLA-4 This example aims to evaluate the inhibitory ability of the candidate peptide (PD-1-CTLA4-2) obtained in Example 1 against the dual-target proteins PD-1 and CTLA-4. The specific details are as follows: Peptide Synthesis and Purification: Candidate peptides were synthesized using solid-phase peptide synthesis (Fmoc-SPPS) technology based on the designed amino acid sequence shown in SEQ ID NO:1. High-performance liquid chromatography (HPLC) was employed for purification to ensure high purity and the absence of significant impurities in the synthesized peptides. Furthermore, the precise molecular weight of the peptides was verified by mass spectrometry (MS).

[0137] 2.1 In vitro PD-1 / PD-L1 binding blockade detection based on TR-FRET technology The purified candidate peptide PD-1-CTLA4-2 was dissolved in water to prepare a 1 mg / mL stock solution for subsequent experiments.

[0138] This experiment was conducted using the TR-FRET kit (purchased from BPS Bioscience, San Diego, USA, catalog number #72038). The specific experimental procedures were strictly performed in accordance with the manufacturer's instructions, and all reagent components were prepared and used at the recommended concentrations.

[0139] The experiments were conducted in 384-well white, non-binding microtiter plates. Different concentrations (0.125 μg / ml, 0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 6 μg / ml, 8 μg / ml) of the peptide inhibitor of this invention were added to the wells, with three triplicates for each concentration gradient. The specific concentrations of PD-1 and PD-L1 proteins were determined according to the kit instructions to ensure the reliability of the experimental data.

[0140] Signal detection: Fluorescence intensity was detected using a Spark Multi-Plate Analyzer (TECAN). The instrument was configured with two consecutive measurement channels: first, the emission signal at 620 nm was detected, followed by the emission signal at 665 nm.

[0141] Data processing and analysis: Experimental results were evaluated by calculating the TR-FRET ratio.

[0142] Figure 2 The results showed the effect of a dual-target peptide inhibitor concentration of 8 μg / ml on the binding between PD-1 and PD-L1, and the dual-target peptide inhibitor could effectively block the binding between PD-1 and PD-L1.

[0143] 2.2 In vitro detection of CTLA-4 and B7-1 (CD80) binding blockade based on TR-FRET technology The purified candidate peptide PD-1-CTLA4-2 was dissolved in water to prepare a 1 mg / mL stock solution for subsequent experiments.

[0144] This experiment used the TR-FRET kit (purchased from BPSBioscience, San Diego, USA, catalog number #72120) specifically for CTLA-4:B7-1 binding detection. The entire experimental process strictly followed the official operating procedures provided by the manufacturer, and all reagent components were prepared and used at their recommended concentrations.

[0145] The experiments were conducted in 384-well white, non-binding microtiter plates. To ensure the statistical significance of the experimental data, different concentrations (0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.4 μg / ml, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 3 μg / ml) of the target peptide inhibitor PD-1-CTLA4-2 were added to the corresponding wells, with three triplicates for each concentration gradient.

[0146] Fluorescence intensity readings were performed using a Spark Multi-Functional Microplate Analyzer (TECAN). The detection program was set to two consecutive time-resolved measurement channels: first, the emission signal at 620 nm was measured, followed by the emission signal at 665 nm.

[0147] The experimental data were quantitatively analyzed using the TR-FRET ratio.

[0148] Figure 3 The results showed the effect of a dual-target peptide inhibitor concentration of 3 μg / ml on the binding of CTLA-4 and B7-1, demonstrating that the dual-target peptide inhibitor could effectively block the binding between CTLA-4 and B7-1.

[0149] 2.3 Detection of PD-1-CTLA4-2-activated T cells in vitro This experiment aims to evaluate the enhancing effect of the dual-target peptide inhibitor PD-1-CTLA4-2 described in this invention on the function of human primary T cells by detecting the expression levels of cell surface activation markers. The specific experimental procedure is as follows: (1) Isolation and culture of T cells CD8 was isolated and purified from human peripheral blood using fluorescence activated cell sorting (FACS) technology. + T cells and CD4 +T cell transplantation was performed, a routine procedure in this field. The experiment was conducted in 96-well plates. The isolated T cells were co-cultured with A549 tumor cells (purchased from American Type Culture Collection (ATCC)) in RPMI 1640 medium containing 10% FBS, with 1 × 10⁶ cells seeded per well. 4 A549 tumor cells and 5×10 4 T cells.

[0150] (2) Cell activation and peptide stimulation Activation signal: T cells were initially activated and stimulated using plate-bound anti-human CD3 antibody (1 μg / ml, purchased from Sinocare, catalog number 10997-H001) and soluble anti-human CD28 antibody (5 μg / ml, purchased from Sinocare, catalog number 11524-H001).

[0151] Drug treatment: Add the dual-target peptide inhibitor PD-1-CTLA4-2 (final concentration of 4 μM, i.e. PD1CTLA4-2 group) designed in this invention to the aforementioned activated co-culture system, and incubate in a cell culture incubator at 5% CO2 and 37°C for 72 hours.

[0152] Reference settings: ① A group containing only activated T cells (not co-cultured with A549 cells) and without drug administration was used as a positive control, namely the activated T cell group; ② It contains activated T cells and A549 lung cancer cells, and does not include a drug-free group, to demonstrate the inhibitory effect on the tumor microenvironment, namely the activated T cell & A549 group.

[0153] (3) Flow cytometry detection CD8 + T-cell detection: CD8+ T-cell assay using flow cytometry. + Gated T cells were analyzed to identify those highly expressing CD44 (CD44) high The percentage of CD8 cells as a measure + Indicators of T cell activation and effector status.

[0154] CD4 + T-cell detection: CD4+ detection was performed using flow cytometry. + Analysis of gated T cells using ICOS + The percentage of cells used as a measure of CD4 + An indicator of the degree of activation of T cell helper function.

[0155] (4) Signal acquisition and data analysis: Cell surface antigens were labeled using fluorescent staining, and data were acquired using a BD FACS Canto flow cytometer (BDBiosciences). The acquired flow cytometry data were analyzed using FlowJo software (version 10.5.3).

[0156] Figure 4 and Figure 5 The results showed that the dual-target multi-inhibitor could effectively relieve the inhibitory effect of immune checkpoint molecules PD-1 and CTLA4 on T cells and effectively activate CD4 T cells and CD8 T cells.

[0157] Example 3: In vivo antitumor efficacy evaluation of humanized NOG mouse model This embodiment establishes a humanized mouse model loaded with human peripheral blood mononuclear cells (PBMCs) to evaluate the in vivo antitumor activity and tumor growth inhibitory ability of the dual-target peptide inhibitor PD-1-CTLA4-2 described in this invention. The specific experimental procedures are as follows: 3.1 Establishment of Experimental Model Humanization of the mouse immune system: Six 6-week-old female NOG-MHCI / II-2 KO-deficient mice (purchased from Charles River Laboratory Animal Technology Company) were selected from each group and transplanted with 5 × 10⁶ cells via intravenous injection (iv). 6 The PBMC cells were isolated from human blood. The method of PBMC cell isolation is the same as that in Example 2, which are all conventional methods in the art.

[0158] Tumor modeling: Three days after immune system reconstruction, each mouse was subcutaneously (sc) injected with 1×10⁻⁶ cells. 6 A subcutaneous xenograft model was established using personal lung cancer A549 cells (purchased from the American Type Culture Collection (ATCC)).

[0159] 3.2 Dosing regimen and grouping Experimental group (drug administration group, i.e. PD1CTLA4-2 group): The dual-target peptide inhibitor PD-1-CTLA4-2 was injected intraperitoneally (ip) once every 3 days, with an injection dose of 4 mg / kg body weight; Negative control group: PBS group (blank control, injection dose of 4 mg / kg body weight); The disordered peptide control group (i.e., the Scrambled (control) group, whose sequence is shown in SEQ ID NO:2, was used to establish a baseline for tumor growth in the presence of human immune cells, with an injection dose of 4 mg / kg body weight).

[0160] MQGPNLDKHTAMYWRIFKMQGELQNGYTCRTVP (SEQ ID NO: 2).

[0161] Both the experimental and control groups of mice were intraperitoneally injected (ip) with the reagents or drugs of each group every 3 days, and the observation continued for 26 days.

[0162] 3.3 Tumor Monitoring and Data Calculation During the experiment, the tumor's long diameter and small diameter were measured every other day using a digital caliper. Tumor volume was calculated using the following formula: V = [(minor axis)] 2 × (major diameter) × 0.5] 3.4 Experimental Evaluation Indicators By plotting tumor growth curves, the volume differences among the groups were compared over a 26-day experimental period.

[0163] The tumors were weighed after treatment to assess the differences in tumor weight among the groups.

[0164] 3.5 Experimental Results The results are as follows Figure 6 , Figure 7 and Figure 8 As shown, compared with the PBS and Scrambled control groups, the dual-target peptide inhibitor significantly reduced the tumor volume and weight of humanized tumor mice, demonstrating a significant tumor inhibition effect and potential as an anti-tumor drug.

[0165] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0166] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A polypeptide or a derivative thereof, or a pharmaceutically acceptable salt, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide or its derivative as described in claim 1, or a pharmaceutically acceptable salt.

3. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 2 or the expression vector of claim 3, or express the polypeptide or its derivative of claim 1, or a pharmaceutically acceptable salt.

5. A coupling agent, characterized in that, It includes the polypeptide or derivative thereof as described in claim 1, a pharmaceutically acceptable salt, and a coupling moiety thereto, the coupling moiety being selected from a purification tag or label.

6. Use of the polypeptide or derivative thereof of claim 1, a pharmaceutically acceptable salt, the nucleic acid molecule of claim 2, the expression vector of claim 3, or the recombinant cell of claim 4 in the preparation of a medicament, wherein the medicament is a dual-target inhibitor of PD-1 and CTLA-4.

7. Use of the polypeptide or derivative thereof of claim 1, a pharmaceutically acceptable salt, the nucleic acid molecule of claim 2, the expression vector of claim 3, or the recombinant cell of claim 4 in the preparation of a medicament for the treatment or prevention of diseases related to PD-1 and / or CTLA-4.

8. The use according to claim 7, characterized in that, The diseases associated with PD-1 and / or CTLA-4 include tumors or cancers.

9. The use according to claim 8, characterized in that, The tumor or cancer mentioned includes solid tumors or hematologic malignancies.

10. Use of the polypeptide of claim 1 or a derivative thereof, a pharmaceutically acceptable salt, or the conjugate of claim 5 in the preparation of a reagent or kit for the detection of PD-1 and / or CTLA-4.

11. A drug, characterized in that, It includes the polypeptide or its derivative as described in claim 1, a pharmaceutically acceptable salt, the nucleic acid molecule as described in claim 2, the expression vector as described in claim 3, or the recombinant cell as described in claim 4.

12. A reagent or kit, characterized in that, Includes the polypeptide or derivative thereof as described in claim 1, a pharmaceutically acceptable salt, or the conjugate as described in claim 5.

13. A method for detecting PD-1 and / or CTLA-4, characterized in that, include: The sample to be tested is contacted with the polypeptide or its derivative as described in claim 1, a pharmaceutically acceptable salt, the conjugate as described in claim 5, or the reagent or kit as described in claim 12. Based on the signal generated by the contact product, it is determined whether the sample to be tested contains PD-1 and / or CTLA-4.

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