Dual-targeting antagonistic polypeptides targeting pd-1 and ctla-4 or a pharmaceutically acceptable salt thereof and uses thereof

Abstract

The present application provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The polypeptide or the pharmaceutically acceptable salt thereof can bind to PD-1 and CTLA-4, and is used for effectively inhibiting PD-1 and CTLA-4. Thus, the polypeptide or 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 diseases mediated by PD-1 and / or CTLA-4 (such as tumors or cancers, for example, non-small cell lung cancer, melanoma, lymphoma and leukemia, etc.).

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology. Specifically, this invention relates to a dual-target antagonistic peptide targeting PD-1 and CTLA-4 or a pharmaceutically acceptable salt thereof and its uses. Background Technology

[0002] Programmed death receptor-1 (PD-1) is a key immune checkpoint receptor primarily expressed on the surface of activated T cells, B cells, and bone marrow cells. Under physiological conditions, PD-1 transmits inhibitory signals by binding to ligands (PD-L1 or PD-L2) expressed by tumor cells, inhibiting excessive T cell activation, thereby maintaining immune tolerance and preventing autoimmune diseases. However, in the tumor microenvironment, tumor cells often "hijack" this mechanism by overexpressing PD-L1, leading to T cell exhaustion and mediating tumor immune escape. Therefore, blocking the PD-1 pathway has become a core strategy in tumor immunotherapy, aiming to reactivate the ability of T cells to recognize and kill tumor cells.

[0003] Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is another transmembrane receptor primarily expressed on the surface of activated T cells. Belonging to the immunoglobulin superfamily, it is another key negative regulatory molecule in the immune system. CTLA-4 mainly functions in the early stages of the immune response (e.g., within lymph nodes), limiting the initial activation and proliferation of T cells by competitively binding to the B7 ligand (CD80 / CD86) with CD28. CTLA-4 plays a crucial role in regulating the strength and breadth of anti-tumor immune responses; inhibition of this target can significantly enhance the body's immune response against tumors.

[0004] In current technologies, the mainstream clinical approach targeting the aforementioned targets is monoclonal antibodies (mAbs). These antibodies bind to PD-1 or CTLA-4 with high affinity, blocking their interaction with their corresponding ligands and thus relieving T-cell suppression. In addition, some research has been conducted on peptide inhibitors targeting single targets of PD-1 or CTLA-4, but these are still in an immature stage regarding tissue penetration and efficacy.

[0005] However, these single-target inhibitors still have significant limitations in clinical application. Tumor immune escape mechanisms are highly complex and compensatory; blocking the PD-1 pathway alone often leads to compensatory escape by activating other immune checkpoints such as CTLA-4, resulting in treatment resistance or insufficient immune stimulation. Currently, although combination therapy regimens for monoclonal antibodies exist, research on dual-target peptides that can simultaneously and precisely bind to and block both PD-1 and CTLA-4 is extremely scarce in the peptide drug field. Designing such molecules requires extremely high spatial conformational precision, and current technologies struggle to balance the binding affinity of both targets with molecular stability. Therefore, developing a novel dual-target inhibitory peptide for PD-1 and CTLA-4, utilizing its synergistic blocking mechanism to enhance the immune system's tumor-killing efficacy, has significant clinical value and application potential in the field of tumor immunotherapy. Summary of the Invention

[0006] 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 dual-target inhibitory peptide for PD-1 and CTLA-4.

[0007] This application is based on the following discoveries of the inventors:

[0008] While current PD-1 or CTLA-4 single-target antagonistic peptides / inhibitors have shown some efficacy in anti-tumor immunotherapy, they still have some significant limitations:

[0009] (1) Stability and pharmacokinetic issues: Natural or existing polypeptide sequences usually have a short half-life in vivo and are easily degraded by proteases in vivo and rapidly cleared by the kidneys. This limits the effective accumulation of drugs in tumor tissues and the duration of efficacy, and increases the frequency of administration and the complexity of clinical application.

[0010] (2) Limitations and compensatory escape of single-target inhibition: Existing research and development focuses on single-target antagonism of PD-1 or CTLA-4. However, tumor immune escape mechanisms are highly complex and compensatory. Blocking the PD-1 pathway alone often leads to tumors escaping by activating other immune checkpoints such as CTLA-4. Existing single-target peptides, due to their singular mechanism of action, often fail to generate sufficient immune stimulation intensity and cannot achieve the expected depth of anti-tumor activity.

[0011] To address the aforementioned shortcomings of existing technologies, this application utilizes precise artificial intelligence calculations and deep learning iterative evolutionary models for optimization, resulting in novel dual-target inhibitory peptides for PD-1 and CTLA-4. Compared to traditional single-target inhibitors, these peptides possess a unique amino acid sequence, enabling them to simultaneously and precisely bind to and efficiently block the interaction between PD-1 and CTLA-4 target proteins and their corresponding ligands.

[0012] The peptides in this application, through combined blocking of two targets, can generate a complementary immune enhancement mechanism, effectively relieving the inhibitory signals of the tumor microenvironment on T cells, significantly enhancing the killing ability of effector T cells, and exhibiting excellent dual-target affinity and bioactivity. This invention not only overcomes the problems of drug resistance and escape associated with single-target drugs, but also improves molecular stability through sequence optimization (and optional modifications), providing an innovative treatment option for patients with malignant tumors (such as non-small cell lung cancer, melanoma, etc.).

[0013] In a first aspect of this application, a polypeptide or a pharmaceutically acceptable salt thereof is provided. According to embodiments of this application, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The polypeptide or a pharmaceutically acceptable salt thereof can bind to PD-1 and CTLA-4 to effectively inhibit PD-1 and CTLA-4. Therefore, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can be used to detect PD-1 and / or CTLA-4, and also for the treatment or prevention of PD-1 and / or CTLA-4 mediated diseases (such as tumors or cancer-related diseases, for example, diseases involving tumor immune escape, especially non-small cell lung cancer, melanoma, kidney cancer, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia, etc.).

[0014] In a second aspect of this application, a polypeptide derivative or a pharmaceutically acceptable salt thereof is proposed. According to embodiments of this application, the polypeptide derivative or a pharmaceutically acceptable salt thereof comprises the polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect of this application, and a modifying group, wherein the polypeptide or a pharmaceutically acceptable salt thereof and the modifying group are linked. The polypeptide derivative or a pharmaceutically acceptable salt thereof of this application can bind to PD-1 and CTLA-4 for effective inhibition of PD-1 and CTLA-4. Therefore, the aforementioned polypeptide derivative or a pharmaceutically acceptable salt thereof can be used to detect PD-1 and / or CTLA-4, and can also be used to treat or prevent PD-1 and / or CTLA-4 mediated diseases (such as tumors or cancer-related diseases, for example, diseases involving tumor immune escape, especially non-small cell lung cancer, melanoma, kidney cancer, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia, etc.).

[0015] In a third aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of this application can encode the polypeptide of the first aspect, which can bind to PD-1 and CTLA-4 for effective inhibition of PD-1 and CTLA-4.

[0016] In a fourth aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the third aspect. The expression vector of this application, carrying the nucleic acid molecule described in the third aspect, can express the polypeptide of the first aspect, which can bind to PD-1 and CTLA-4 for effective inhibition of PD-1 and CTLA-4.

[0017] In a fifth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect, or the recombinant cell expresses the polypeptide described in the first aspect. The recombinant cell of this application can express the polypeptide of the first aspect, which can bind to PD-1 and CTLA-4 for effective inhibition of PD-1 and CTLA-4.

[0018] In a sixth aspect of this application, the use of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, or the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, in the preparation of a dual-target inhibitor of PD-1 and CTLA-4 is disclosed. As is known prior art, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can effectively inhibit PD-1 and CTLA-4. Therefore, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can be formulated as a dual-target inhibitor of PD-1 and CTLA-4 for the treatment or prevention of PD-1 and / or CTLA-4 mediated diseases, such as tumors or cancers, for example, diseases involving tumor immune escape, particularly non-small cell lung cancer, melanoma, renal cell carcinoma, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia.

[0019] In a seventh aspect of this application, a fusion protein is proposed. According to embodiments of this application, the fusion protein comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, or the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof. As is known prior, the aforementioned polypeptide or its pharmaceutically acceptable salt can bind to PD-1 and CTLA-4, and can be used to effectively inhibit PD-1 and CTLA-4. Thus, the fusion protein containing the aforementioned polypeptide can bind to PD-1 and CTLA-4, and can be used to detect PD-1 and / or CTLA-4 or to inhibit PD-1 and CTLA-4. It can also be used to treat or prevent PD-1 and / or CTLA-4 mediated diseases, such as tumors or cancers, for example, diseases involving tumor immune escape, especially non-small cell lung cancer, melanoma, kidney cancer, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia.

[0020] In an eighth aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, or the fusion protein described in the seventh aspect. As is known prior, the aforementioned polypeptides can bind to PD-1 and CTLA-4 and can be used to effectively inhibit PD-1 and CTLA-4. Therefore, a reagent or kit containing the aforementioned polypeptides can bind to PD-1 and CTLA-4 for the detection of PD-1 and / or CTLA-4.

[0021] In a ninth aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, or the fusion protein described in the seventh aspect. As is known prior art, the aforementioned polypeptides can bind to PD-1 and CTLA-4 and can be used to effectively inhibit PD-1 and CTLA-4. Therefore, the pharmaceutical composition containing the aforementioned polypeptides can be used to treat or prevent PD-1 and / or CTLA-4 mediated diseases, such as tumors or cancers, for example, diseases involving tumor immune escape, especially non-small cell lung cancer, melanoma, kidney cancer, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia.

[0022] In a tenth aspect of this application, the use of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the seventh aspect, or the pharmaceutical composition described in the ninth aspect in the preparation of a medicament for the treatment or prevention of PD-1 and / or CTLA-4 mediated diseases, such as tumors or cancer.

[0023] In the eleventh aspect of this application, a method for detecting PD-1 and / or CTLA-4 is provided. According to embodiments of this application, the method includes: contacting a sample to be tested with the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the seventh aspect, or the reagent or kit described in the eighth aspect; 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 pharmaceutically acceptable salt can bind to PD-1 and CTLA-4, and can be used to effectively inhibit PD-1 and CTLA-4. Therefore, the above method can be used to bind to PD-1 and CTLA-4 for detection.

[0024] Additional aspects and advantages of the invention 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 the invention. Attached Figure Description

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0026] Figure 1 This is a schematic diagram of the process for generating and screening PD-1 and CTLA-4 dual-target inhibitory peptides based on artificial intelligence in Example 1 of this application.

[0027] Figure 2 This is a graph showing the in vitro detection results of the candidate dual-target peptides PD1CTLA4_6 and PD1CTLA4_7 inhibiting the binding of PD-1 and PD-L1 in Example 2 of this application.

[0028] Figure 3 This is an in vitro detection result of the candidate dual-target peptides PD1CTLA4_6 and PD1CTLA4_7 inhibiting the binding of CTLA-4 to B7-1 (CD80) in Example 2 of this application. Detailed Implementation

[0029] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0030] 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 indicated technical features. 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 invention, unless otherwise stated, "a plurality of" means two or more.

[0031] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0032] 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.

[0033] 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.

[0034] In this paper, 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 (such as the PD-1 and CTLA-4 pathways in this application).

[0035] 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 experience with antigen stimulation. CD44high (high expression) indicates that the T cell has been activated, is transforming into an effector T cell, or has become a memory T cell.

[0036] In this article, the term "Programmed Cell Death Protein 1 (PD-1)" refers to 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 and leading to tumor immune escape. Therefore, PD-1 inhibitors have become central to tumor immunotherapy, aiming to block this inhibitory pathway and reactivate the T cell's ability to kill tumor cells.

[0037] In this article, the term "Cytotoxic T-Lymphocyte-Associated Antigen 4 (CTLA-4)" refers to a transmembrane receptor expressed on the surface of activated T cells, belonging to the immunoglobulin superfamily, and another key negative regulatory molecule of 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.

[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 paper, the term "at least 80% identity" refers to an identity of at least 80% with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 89.5%, 89.9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0040] In this paper, the term "at least 90% identity" means at least 90% identity with each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0041] 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.

[0042] In this paper, the structural formula for the term "αMeK" is: .

[0043] In this article, the structural formula for the term "HoK" is: .

[0044] In this article, the structural formula for the term "N-Me-K" is: .

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

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

[0047] In this article, the structural formula for the term "Dap" is: .

[0048] 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.

[0049] 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 in the field. 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.

[0050] In this document, the term "pharmaceutical composition" generally refers to a unit dosage 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 a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0051] 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, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0052] In addition to any conventional excipients, the use of polypeptides or their derivatives, pharmaceutical compositions or pharmaceuticals containing them that are incompatible with the present application, such as any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition, is also within the scope of this application.

[0053] The pharmaceutical compositions 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 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.

[0054] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

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

[0056] In this paper, the term "Denoising Diffusion Probabilistic Model (DDPM)" refers to a deep generative model based on a diffusion process that learns the distribution of the training data to generate new samples that conform to the training data. The training process of DDPM mainly consists of two parts: 1) a forward diffusion process, which corrupts the original data by gradually adding Gaussian noise until it becomes completely random noise; 2) a backward diffusion process, which restores the corrupted data by estimating and removing noise at each time step through a series of Markov chains.

[0057] In this paper, the term "Conditional Diffusion Model (Gong et al. 2022)" refers to a model that introduces conditional signals (such as text, images, etc.) to generate data during the training process of a denoising diffusion probability model (DDPM). This type of model can control the output according to needs, customizing the generation of high-quality data. The Transformer model (Vaswani et al. 2017), a sequence-to-sequence model for natural language processing (NLP), is based on an encoder-decoder structure and learns the relationships between words in a sequence through a self-attention mechanism. Transformer models can better preserve long-term semantic information when processing long texts, showing significant performance improvements compared to previous recurrent neural networks and convolutional neural networks. Specifically, it refers to a deep generative model that introduces specific conditional signals (such as target protein sequences) to generate data during the training process of a denoising diffusion probability model (DDPM). In this application, it refers to the TPDiffusion method, which achieves specific generation of peptide sequences for specific targets by learning the relationship rules between target proteins and peptide sequences.

[0058] In this paper, the term "BERT (Bidirectional Encoder Representations from Transformers)" refers to a deep bidirectional Transformer encoder that can capture bidirectional contextual information in sequence data.

[0059] In this paper, the term "docking score" refers to the assessment of the stability and affinity of molecular binding. It includes a combination of various interaction energies, such as Coulomb interaction energy, van der Waals interaction energy, and hydrogen bond interaction energy, which can be used to evaluate the stability and affinity of molecular binding. A lower docking score generally indicates a more stable binding between the molecular ligand and the target protein, and a higher affinity.

[0060] In this article, the term "AlphaFold" refers to a tool released by DeepMind that uses multiple sequence alignment to accurately predict the three-dimensional structure of proteins based on physical and biological knowledge of protein structure. AlphaFold-Multimer, developed based on AlphaFold2, is primarily used for protein complex structure prediction, with a particular focus on predicting complex binding interfaces. AlphaFold-Multimer scores the interactions between chain residues of the complex using ipTM (interfacepredictedTM-score), which serves as a confidence level to assess the accuracy of interface predictions.

[0061] In this paper, the term "affinity maturation" refers to the process by which B cells enhance the affinity of their antibodies for antigens through mutation and selection mechanisms during the immune response. In this process, B cells undergo high-frequency mutations in the germinal centers, producing a variety of variant antibodies, which are then selected based on their binding affinity to antigens, ultimately resulting in antibodies with high affinity. This process is crucial for generating effective immune responses and vaccine development. Specifically, it involves using an artificial intelligence iterative evolutionary framework (such as the reinforcement learning reward model PepAF) to mutate and screen initially generated peptide sequences to improve the binding affinity and stability between peptides and target proteins (PD-1 and CTLA-4).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] In this document, the term "antagonism" refers to a substance (specifically, the polypeptide molecules PD1CTLA4_6 and PD1CTLA4_7 of this application) that can bind to receptors on the cell surface (such as PD-1 and CTLA-4) but does not activate the receptor. Instead, it inhibits or weakens the binding of natural ligands (such as PD-L1 or B7) to the receptor by occupying the receptor's binding site, thereby inhibiting or blocking the receptor's original biological function.

[0066] In this paper, the term "antagonist" refers to a class of molecules that can bind to receptors (such as cell surface receptors) but produce little or no biological effect themselves. Their key role is to competitively prevent or attenuate the binding of natural ligands (agonists) to the receptor by occupying its binding site, thereby blocking or downregulating the biological signaling pathway mediated by that receptor. Specifically, in this paper, an antagonist refers to a polypeptide that can simultaneously bind to both PD-1 and CTLA-4 immune checkpoint receptors on the surface of T cells. Through this binding, the polypeptide can effectively block the interaction between PD-1 and its ligands (PD-L1 / PD-L2) and CTLA-4 and its ligands (B7-1 / B7-2), thereby relieving the inhibition of T cell activity by these two signaling pathways and ultimately restoring and enhancing the anti-tumor immune response of T cells. In this paper, the term "antagonist" is synonymous with the term "inhibitor."

[0067] In this article, the term "inhibitor" refers to a substance (ligand) that inhibits the type of receptor.

[0068] In this document, the term "dual-target antagonistic peptide" refers to a biomolecule composed of amino acids linked by peptide bonds, characterized by its ability to specifically bind to and antagonize two different biological targets. More specifically, the "dual-target antagonistic peptide" can bind simultaneously or independently to two specific targets referred to in this application—the PD-1 receptor and the CTLA-4 receptor. By binding to these two targets, the peptide exerts an antagonistic effect, i.e., it does not activate or only weakly activates the PD-1 and CTLA-4 receptors, but effectively prevents their respective natural ligands (PD-L1 / PD-L2 for PD-1, B7-1 / B7-2 for CTLA-4) from binding to the receptors. This dual antagonistic effect leads to the blocking or significant attenuation of the PD-1 and CTLA-4-mediated immunosuppressive signaling pathways, thereby synergistically deactivating T cell activity. Therefore, the "dual-target antagonistic peptide" of this invention is an active ingredient with multiple targeting capabilities designed through molecular engineering, aiming to achieve more effective and sustained immune activation by simultaneously regulating multiple key immune checkpoint pathways.

[0069] This application discloses a dual-target antagonistic peptide targeting PD-1 and CTLA-4, or a pharmaceutically acceptable salt thereof, and its uses, which will be described in detail below.

[0070] polypeptides or their pharmaceutically acceptable salts

[0071] In one aspect of this application, a polypeptide or a pharmaceutically acceptable salt thereof is provided. According to embodiments of this application, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0072] The inventors of this application have obtained a novel dual-target peptide for PD-1 and CTLA-4 through precise calculations, deep learning models, and iterative evolutionary models. Compared with traditional PD-1 and CTLA-4 inhibitors (such as monoclonal antibodies or single-target antagonistic peptides), this peptide has a unique amino acid sequence. Furthermore, the aforementioned peptide or its pharmaceutically acceptable salt can bind to PD-1 and CTLA-4, effectively inhibiting both PD-1 and CTLA-4, exhibiting high affinity and bioblocking activity for both PD-1 and CTLA-4 simultaneously. Therefore, the aforementioned peptide or its pharmaceutically acceptable salt can be used to detect PD-1 and / or CTLA-4, and can also be used to treat or prevent PD-1 and / or CTLA-4 mediated diseases (such as tumors or cancers, for example, diseases involving tumor immune escape, especially non-small cell lung cancer, melanoma, kidney cancer, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia).

[0073] In particular, with the rising global incidence of malignant tumors and the limitations of existing monoclonal antibody therapies in terms of tissue penetration and cost, the peptides or pharmaceutically acceptable salts of this application can not only provide new treatment options for cancer patients, but also are expected to reshape the market landscape of immune checkpoint inhibitors.

[0074] According to embodiments of this application, the above-mentioned polypeptide or its pharmaceutically acceptable salt may further include at least one of the following technical features:

[0075] WQDFENWVLNEVYVGKASPILYQMCCDYKVFN (SEQ ID NO: 1).

[0076] NYVSLNYNAYPSWHWHTAASPILYQMCCDYKRVFN (SEQ ID NO: 2).

[0077] 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 conservatively modified form of SEQ ID NO:A, or a sequence similarity of more than 90% (e.g., 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%) to the amino acid sequence shown in SEQ ID NO:A, all of which are within the scope of protection of this application. Unless otherwise specified, the amino acids in the conserved modified form of the amino acid sequence of SEQ ID NO:A, or the amino acid sequence shown in SEQ ID NO:A having a sequence similarity of more than 90% (e.g., 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, or at any position in the N-terminal hypervariable region of the polypeptide of this application (e.g., positions 1-16 of SEQ ID NO:1, or positions 1-17 of SEQ ID NO:2), or at any position in positions 19-20 (CC), 22 (D), or 24 (K) (based on SEQ ID NO:1) of the middle conserved region. Such conserved modifications or differences in similarity of amino acids do not significantly affect or change the structural stability of the polypeptide containing the amino acid and / or its binding activity with the receptor (i.e., PD-1 and / or CTLA-4), and are all within the scope of protection of this application.

[0078] For example, "a polypeptide having an amino acid sequence as shown in SEQ ID NO:1" means that the polypeptide has an amino acid sequence as shown in SEQ ID NO:1, an amino acid sequence in a conservative modified form of SEQ ID NO:1, or an amino acid sequence with a sequence similarity of more than 80% (e.g., more than 85%, 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. For example, "a polypeptide having an amino acid sequence as shown in SEQ ID NO:2" means that the polypeptide has an amino acid sequence as shown in SEQ ID NO:2, an amino acid sequence in a conservative modified form of SEQ ID NO:2, or an amino acid sequence with a sequence similarity of more than 80% (e.g., more than 85%, 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.

[0079] In this document, "conservatively modified amino acids" or "similarly different amino acids" refers to amino acids that do not significantly affect or alter the properties of the sequence containing that amino acid. Such modifications or differences include amino acid substitutions, additions, and deletions. Modifications or differences 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 or similarly different 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 or differences in similarity does not exceed 80% of the total number, preferably not exceeding 90%. In this document, "amino acids with conserved modifications" or "amino acids with differences in similarity" also includes 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.

[0080] 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.

[0081] .

[0082] According to embodiments of this application, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO: 1-2, or an amino acid sequence having at least 80% sequence similarity to it; or, compared to the amino acid sequence shown in any one of SEQ ID NO: 1-2, the polypeptide is substituted, deleted, or added with 1-5 amino acids and has PD-1 and CTLA-4 binding activity. For example, 1, 2, 3, 4, or 5 amino acids may be substituted, deleted, or added.

[0083] 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).

[0084] 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.

[0085] 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 polypeptide's half-life in vivo.

[0086] According to embodiments of this application, the amino acid X is selected from K, k, αMeK, HoK, Dap, Dab, Orn, or N. Me K.

[0087] polypeptide derivatives or their pharmaceutically acceptable salts

[0088] In a second aspect of this application, a polypeptide derivative or a pharmaceutically acceptable salt thereof is proposed. According to embodiments of this application, the polypeptide derivative or a pharmaceutically acceptable salt thereof comprises the polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect of this application, and a modifying group, wherein the polypeptide or a pharmaceutically acceptable salt thereof and the modifying group are linked. The polypeptide derivative or a pharmaceutically acceptable salt thereof of this application can bind to PD-1 and CTLA-4 for effective inhibition of PD-1 and CTLA-4. Therefore, the aforementioned polypeptide derivative or a pharmaceutically acceptable salt thereof can be used to detect PD-1 and / or CTLA-4, and can also be used to treat or prevent PD-1 and / or CTLA-4 mediated diseases (such as tumors or cancer-related diseases, for example, diseases involving tumor immune escape, especially non-small cell lung cancer, melanoma, kidney cancer, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia, etc.).

[0089] According to embodiments of this application, the above-mentioned polypeptide derivatives or pharmaceutically acceptable salts thereof may further include at least one of the following technical features:

[0090] According to embodiments of this application, the modifying group is attached to an active group on the side chain or end of an amino acid in the polypeptide; the active group is selected from at least one of -NH2, -SH, -OH and -COOH.

[0091] According to embodiments of this application, the modifying group is linked to the -NH2 of the amino acid side chain in the polypeptide.

[0092] According to embodiments of this application, the modifying group has at least one of the following structures:

[0093] ;

[0094] ;

[0095] ;

[0096] .

[0097] 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.

[0098] According to embodiments of this application, the modifying group further includes at least one selected from polyethylene glycol and its derivatives, fatty acid chains, protein tags, Fc fragments, signal peptides, and targeting ligands.

[0099] 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.

[0100] Nucleic acid molecules, expression vectors and recombinant cells

[0101] In a third aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of this application can encode the polypeptide of the first aspect, which can bind to PD-1 and CTLA-4 for effective inhibition of PD-1 and CTLA-4.

[0102] According to an embodiment of this application, the nucleic acid molecule is DNA.

[0103] 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. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the nucleic acid molecule sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0104] In a fourth aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the third aspect. The expression vector of this application, carrying the nucleic acid molecule described in the third aspect, can express the polypeptide of the first aspect, which can bind to PD-1 and CTLA-4 for effective inhibition of PD-1 and CTLA-4.

[0105] 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 expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can be derived directly from the expression vector itself or be exogenous, i.e., not originating from the expression vector itself. The key is to ensure that the nucleic acid molecules and control elements are operatively linked.

[0106] In this document, "operably ligated" refers to ligating a foreign gene to an expression vector, enabling the control elements within the expression vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the expression vector is introduced into suitable recipient cells, the aforementioned polypeptide can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro production of the polypeptide.

[0107] According to embodiments of this application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0108] According to an embodiment of this application, the expression vector is a lentiviral vector.

[0109] According to an embodiment of this application, the expression vector is a plasmid expression vector.

[0110] In a fifth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect, or the recombinant cell expresses the polypeptide described in the first aspect. The recombinant cell of this application can express the polypeptide of the first aspect, which can bind to PD-1 and CTLA-4 for effective inhibition of PD-1 and CTLA-4.

[0111] According to embodiments of this application, the recombinant cells are obtained by introducing the expression vector described in the fourth aspect into the host cell.

[0112] It should be noted that the host cell in this application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. The prokaryotic cell can be *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*, etc. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*, insect cells such as armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0113] According to an embodiment of this application, the host cell is a eukaryotic cell.

[0114] According to embodiments of this application, the host cell is a mammalian cell, including but not limited to BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0115] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the peptide described in this application. Those skilled in the art will readily understand that suitable conditions for peptide expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable 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 peptide expression based on the specific environment of their laboratory.

[0116] use

[0117] In a sixth aspect of this application, the use of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, or the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, in the preparation of a dual-target inhibitor of PD-1 and CTLA-4 is disclosed. As is known prior art, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can effectively inhibit PD-1 and CTLA-4. Therefore, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can be formulated as a dual-target inhibitor of PD-1 and CTLA-4 for the treatment or prevention of PD-1 and / or CTLA-4 mediated diseases, such as tumors or cancers, for example, diseases involving tumor immune escape, particularly non-small cell lung cancer, melanoma, renal cell carcinoma, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia.

[0118] Fusion proteins, reagents or kits, pharmaceutical compositions

[0119] In a seventh aspect of this application, a fusion protein is proposed. According to embodiments of this application, the fusion protein comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, or the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof. As is known prior, the aforementioned polypeptide or its pharmaceutically acceptable salt can bind to PD-1 and CTLA-4, and can be used to effectively inhibit PD-1 and CTLA-4. Thus, the fusion protein containing the aforementioned polypeptide can bind to PD-1 and CTLA-4, and can be used to detect PD-1 and / or CTLA-4 or to inhibit PD-1 and CTLA-4. It can also be used to treat or prevent PD-1 and / or CTLA-4 mediated diseases, such as tumors or cancers, for example, diseases involving tumor immune escape, especially non-small cell lung cancer, melanoma, kidney cancer, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia.

[0120] According to embodiments of this application, the fusion protein further includes a functional fragment.

[0121] In this document, the term "functional fragment" refers to an amino acid fragment, which may be a functionally active fragment or a protein tag. The specific type is not limited and is within the scope of protection of this application.

[0122] It should be noted that the aforementioned functionally active fragments can be used to exert their effects in animals or in vitro. For example, when used in animals, the functionally active fragment can be used to prevent and / or treat diseases; when used in vitro, it can be used to specifically bind to a substance, detect that substance, or diagnose diseases in vitro.

[0123] It should be noted that the aforementioned protein tag refers to a short peptide co-expressed with the target protein, which facilitates the expression, detection, tracing, or purification of the polypeptide of this application. For example, the protein tag includes at least one of the following: His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

[0124] In an eighth aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, or the fusion protein described in the seventh aspect. As is known prior, the aforementioned polypeptides can bind to PD-1 and CTLA-4 and can be used to effectively inhibit PD-1 and CTLA-4. Therefore, a reagent or kit containing the aforementioned polypeptides can bind to PD-1 and CTLA-4 for the detection of PD-1 and / or CTLA-4.

[0125] 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.

[0126] 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.

[0127] In a ninth aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, or the fusion protein described in the seventh aspect. As is known prior art, the aforementioned polypeptides can bind to PD-1 and CTLA-4 and can be used to effectively inhibit PD-1 and CTLA-4. Therefore, the pharmaceutical composition containing the aforementioned polypeptides can be used to treat or prevent PD-1 and / or CTLA-4 mediated diseases, such as tumors or cancers, for example, diseases involving tumor immune escape, especially non-small cell lung cancer, melanoma, kidney cancer, liver cancer, gastric cancer, colorectal cancer, lymphoma, and leukemia.

[0128] According to embodiments of this application, the pharmaceutical composition may further include pharmaceutically acceptable excipients.

[0129] According to embodiments of this application, pharmaceutically acceptable excipients refer to pharmaceutical excipients commonly used in the pharmaceutical field, such as diluents, buffer solutions, osmotic pressure regulators, pH regulators, etc.

[0130] According to embodiments of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field, such as a protective agent.

[0131] According to embodiments of this application, pharmaceutically acceptable mediators refer to conventional pharmaceutical mediators, such as solutions (e.g., water) and liposomes.

[0132] According to embodiments of this application, examples of suitable pharmaceutically acceptable carriers, excipients, and mediators are well known in the art. Pharmaceutical compositions comprising such carriers, excipients, and mediators can be formulated using known conventional methods.

[0133] According to embodiments of this application, the pharmaceutical composition 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.

[0134] According to embodiments of this application, the pharmaceutical composition may also contain other therapeutic active ingredients. According to embodiments of this application, the pharmaceutical composition may 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 pharmaceutical composition is in the form of a lyophilized formulation or an aqueous solution. Clinical dosing regimens are 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 patient size, 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 may be administered topically or systemically. Preferably, it may be administered intravenously or subcutaneously.

[0135] use

[0136] In a tenth aspect of this application, the use of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the seventh aspect, or the pharmaceutical composition described in the ninth aspect in the preparation of a medicament for the treatment or prevention of PD-1 and / or CTLA-4 mediated diseases is provided.

[0137] This application proposes the use of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the seventh aspect, or the pharmaceutical composition described in the ninth aspect for the treatment or prevention of PD-1 and / or CTLA-4 mediated diseases.

[0138] This application proposes the polypeptide of the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative of the second aspect or a pharmaceutically acceptable salt thereof, the fusion protein of the seventh aspect, or the pharmaceutical composition of the ninth aspect for the treatment or prevention of PD-1 and / or CTLA-4 mediated diseases.

[0139] According to embodiments of this application, the above-described uses may further include at least one of the following technical features:

[0140] According to embodiments of this application, the PD-1 and / or CTLA-4 mediated diseases include tumors or cancers.

[0141] According to embodiments of this application, the tumor or cancer includes solid tumors or hematologic malignancies.

[0142] According to embodiments of this application, the solid tumor or hematologic malignancy includes diseases involving tumor immune escape.

[0143] According to embodiments of this application, the disease is selected from 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, and leukemia.

[0144] According to an embodiment of this application, the lung cancer is selected from non-small cell lung cancer.

[0145] Methods for detecting PD-1 and / or CTLA-4

[0146] In the eleventh aspect of this application, a method for detecting PD-1 and / or CTLA-4 is provided. According to embodiments of this application, the method includes: contacting a sample to be tested with the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the seventh aspect, or the reagent or kit described in the eighth aspect; 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 pharmaceutically acceptable salt can bind to PD-1 and CTLA-4, and can be used to effectively inhibit PD-1 and CTLA-4. Therefore, the above method can be used to bind to PD-1 and CTLA-4 for detection.

[0147] According to embodiments of this application, the signal includes a fluorescence signal.

[0148] According to embodiments 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 signals generated by the contact product.

[0149] In a twelfth aspect of this application, a method for treating or preventing PD-1 and / or CTLA-4 mediated diseases is provided. According to embodiments of this application, the method comprises administering to a subject a pharmaceutically acceptable dose of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, a polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, a fusion protein described in the seventh aspect, or a pharmaceutical composition described in the ninth aspect.

[0150] In one alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0151] The effective amount of the polypeptide or its pharmaceutically acceptable salt 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 (e.g., tumor stage, size, and metastasis); 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.

[0152] The polypeptides, pharmaceutically acceptable salts thereof, polypeptide derivatives 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, pharmaceutically acceptable salts thereof, polypeptide derivatives thereof, pharmaceutical compositions, or pharmaceutical compositions 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.

[0153] According to embodiments of this application, the PD-1 and / or CTLA-4 mediated diseases include tumors or cancers.

[0154] According to embodiments of this application, the tumor or cancer includes solid tumors or hematologic malignancies; specifically, it includes at least one of lung cancer, melanoma, kidney cancer, liver cancer, stomach cancer, colorectal cancer, lymphoma, and leukemia.

[0155] According to an embodiment of this application, the lung cancer is selected from non-small cell lung cancer.

[0156] The present invention will be explained below 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 the invention. 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 field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0157] Example 1: Design and screening of dual-target inhibitory peptides for PD-1 and CTLA-4

[0158] 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 PD-1 and CTLA-4 dual-target inhibitory peptide sequences to screening and optimization. The entire framework references the method proposed in patent number 202510288973.8, entitled "An Iterative Evolutionary Framework for Designing Highly Bioactive Peptides for Target Proteins." The entire method consists of multiple iterative evolutionary processes, after which the model-designed peptides achieve effective enhancements in bioactivity. Each iterative evolution includes three parts: generation and optimization of novel peptides based on artificial intelligence, wet experimental determination of peptide bioactivity, and feedback to strengthen the model's perception of bioactivity.

[0159] First, referring to patent 202410192333.2, entitled "Training of Ligand Information Generation Model, Method and Apparatus for Generating Ligand Information," an innovative deep learning method, TPDiffusion, is disclosed. This method can generate peptide sequences that can bind to the amino acid sequence of a target protein. Converting the target protein sequence into a peptide sequence can be seen as a process of providing a specific answer to a specific problem. By training the conditional diffusion model TPDiffusion, the relationship rules between the target protein and peptide sequences are learned, enabling the generation of specific peptides (i.e., PD-1 and CTLA-4 dual-target inhibitory 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:

[0160] (1) Encoding the amino acid sequence: In order to model the joint feature space of protein-peptide, the target protein and peptide sequences are spliced ​​into a whole, and an embedding transformation function is introduced to map each discrete amino acid word or character to a continuous vector encoding space.

[0161] (2) Gradually add noise to the polypeptide sequence: Gaussian noise is gradually added to the polypeptide part of the vector encoding based on the Markov chain until the polypeptide sequence is completely destroyed.

[0162] 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:

[0163] (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.

[0164] (2) Calculate the loss function: The model outputs the predicted probability distribution of the polypeptide sequence. The mean squared error loss function is used to calculate the difference between the model prediction result and the actual result, and backpropagation is performed to update the model parameters.

[0165] The denoising network in TPDiffusion employs the BERT (Devlin et al. 2018) model. The BERT model architecture primarily consists of multi-layer Transformer (Vaswani et al. 2017) encoders, with each Transformer block containing both self-attention and a feed-forward neural network. Through the self-attention mechanism, TPDiffusion incorporates target sequence information during the backdiffusion recovery of peptide sequences, implicitly modeling the relationship between target proteins and peptide sequences, thus achieving a mapping from target proteins to peptide sequences. During generation, given any target protein sequence, the model first randomly samples from Gaussian noise and then performs a backdiffusion process. Guided by the target protein sequence, the noise is gradually eliminated over fixed time steps, ultimately generating a binding peptide sequence targeting the given target. Using the trained TPDiffusion, sequences from the two targets PD-1 and CTLA-4 were used as input, generating a batch of candidate peptide sequences that may have high affinity for both targets.

[0166] Second, affinity maturation is performed on these high-affinity candidate peptide sequences. This step mainly references the deep reinforcement learning method for target protein-specific binding peptide generation and optimization proposed in patent 2025101234137. This method is an innovative deep reinforcement learning scheme specifically designed for optimizing 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.

[0167] The reward model references the affinity prediction method proposed in patent 2025101207356. This method is an innovative technical solution called PepAF, which effectively predicts the binding affinity of target proteins and peptides by comprehensively utilizing structural information, flexibility characteristics, 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; 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 structure and physicochemical properties, which provides 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 the accuracy of predictions by modeling the target protein structure and the peptide flexibility. The core technology PepAF is used as a reward model in deep reinforcement learning to guide candidate peptide sequences to mutate in the direction of higher affinity for both targets.

[0168] After obtaining the affinity-matured peptide, its dual-target bioactivity was calculated and evaluated. The results were then fed back into the model to continue the next round of peptide generation and optimization. After multiple iterations, peptides that effectively inhibit both PD-1 and CTLA-4 target proteins were designed. The sequence of peptide PD1CTLA4_6 is shown in SEQ ID NO: 1, and the sequence of PD1CTLA4_7 is shown in SEQ ID NO: 2. The amino acid sequences shown in SEQ ID NO: 1~2 are as follows:

[0169]

[0170] Example 2: In vitro activity assessment of candidate peptides in blocking PD-1 and CTLA-4 binding

[0171] This example aims to evaluate the inhibitory ability of the candidate peptides (PD1CTLA4_6, PD1CTLA4_7) obtained in Example 1 against the dual-target proteins of PD-1 and CTLA-4. The specific details are as follows:

[0172] Peptide Synthesis and Purification: Candidate peptides were synthesized using solid-phase peptide synthesis (Fmoc-SPPS) technology based on the designed amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2. High-performance liquid chromatography (HPLC) was used 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).

[0173] 1. This embodiment aims to evaluate the in vitro inhibitory activity of the two candidate peptides (PD1CTLA4_6 and PD1CTLA4_7) obtained in Example 1 against the interaction between PD-1 and PD-L1. By quantitatively measuring changes in TR-FRET signaling, this experiment can accurately assess the blocking effect of the candidate peptides on the PD-1 receptor, providing important bioactivity data for the development of tumor immunotherapy drugs. The experimental procedure is as follows:

[0174] The purified candidate peptides PD1CTLA4_6 and PD1CTLA4_7 were dissolved in water to prepare a 1 mg / mL stock solution for subsequent experiments.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] Data processing and analysis: Experimental results were evaluated by calculating the TR-FRET ratio.

[0179] 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.

[0180] 2. This example aims to evaluate the in vitro inhibitory activity of the candidate peptides PD1CTLA4_6 and PD1CTLA4_7 obtained in Example 1 on the interaction between CTLA-4 and B7-1 (CD80). The experimental procedure is as follows:

[0181] The purified candidate peptides PD1CTLA4_6 and PD1CTLA4_7 were dissolved in water to prepare a 1 mg / mL stock solution for subsequent experiments.

[0182] 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.

[0183] 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 inhibitors PD1CTLA4_6 and PD1CTLA4_7 were added to the corresponding wells, with three triplicates for each concentration gradient.

[0184] 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.

[0185] The experimental data were quantitatively analyzed using the TR-FRET ratio.

[0186] The results showed that the synthesized candidate peptides PD1CTLA4_6 and PD1CTLA4_7 effectively blocked the binding of CTLA-4 to B7-1, indicating that both peptides PD1CTLA4_6 and PD1CTLA4_7 possessed high CTLA-4 inhibitory bioactivity. The test results can be found in [link to relevant documentation]. Figure 3 .

[0187] Example 3: In vitro T cell activation and in vivo antitumor efficacy evaluation of candidate peptides

[0188] This embodiment aims to evaluate the activation effect of candidate peptides PD1CTLA4_6 and PD1CTLA4_7 on T cell function in vitro, and their anti-tumor efficacy in a humanized mouse model in vivo. The specific steps are as follows:

[0189] 1. In vitro activation assay of T cells based on flow cytometry:

[0190] This experiment aims to evaluate the enhancing effect of the dual-target peptides of the present invention on the function of human primary T cells by detecting the expression levels of cell surface activation markers.

[0191] (1) Isolation and culture of T cells: CD8 cells were 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.

[0192] (2) Cell activation and peptide stimulation:

[0193] ① 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).

[0194] ② Drug treatment: Add the dual-target peptides PD1CTLA4_6 and PD1CTLA4_7 (final concentration 4 μM, i.e., PD1CTLA4_6 and PD1CTLA4_7 group) designed in this invention to the co-culture system, and incubate in a cell culture incubator at 5% CO2 and 37℃ for 72 hours. Control settings:

[0195] ① The group containing only activated T cells (not co-cultured with A549 cells) and without drug administration served as a positive control, namely the Stimulated T cells group;

[0196] ② The group containing activated T cells and A549 lung cancer cells, and without drug administration, was used to demonstrate the inhibitory effect on the tumor microenvironment, namely the Stimulated T cells & A549 group.

[0197] (3) Flow cytometry detection and data analysis: Cell surface antigens were labeled using fluorescent staining technology, and data were acquired using a BDFACS Canto flow cytometer (BD Biosciences). The acquired flow cytometry data were analyzed using FlowJo software (version 10.5.3).

[0198] ①CD8 + T-cell detection: CD8+ T-cell assay using flow cytometry. + The T cells within the gated region were analyzed, with the percentage of CD44 high cells used as a measure of CD8. + Indicators of T cell activation and effector status.

[0199] ②CD4 + 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.

[0200] 2. In vivo antitumor efficacy evaluation based on humanized NOG mouse model:

[0201] This experiment established a humanized mouse model loaded with human peripheral blood mononuclear cells (PBMCs) to evaluate the in vivo antitumor activity and tumor growth inhibition ability of the dual-target peptides PD1CTLA4_6 and PD1CTLA4_7 described in this invention.

[0202] (1) Establishment of experimental model

[0203] 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 isolation method of the PBMC cells was the same as that of the T cells in this embodiment, which are all conventional methods in the field.

[0204] 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)).

[0205] (2) Dosing regimen and grouping

[0206] Experimental group (drug administration group, i.e. PD1CTLA4_6 and PD1CTLA4_7 group): The dual-target peptide inhibitors PD1CTLA4_6 and PD1CTLA4_7 were injected intraperitoneally (ip) once every 3 days, with an injection dose of 4 mg / kg body weight.

[0207] Negative control group:

[0208] PBS group (blank control, injection dose of 4 mg / kg body weight);

[0209] Scrambled (control) group (disordered peptide control, the sequence of which is shown in SEQ ID NO:3, 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).

[0210] MQGPNLDKHTAMYWRIFKMQGELQNGYTCRTVP (SEQ ID NO: 3).

[0211] 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.

[0212] (3) Tumor monitoring and assessment indicators

[0213] During the experiment, the tumor's long diameter and short diameter were measured every other day using a digital caliper. Tumor volume was calculated using the following formula: [(short diameter)] 2 [× (long diameter)×0.5]. Tumor growth curves were plotted to compare the volume differences among the groups over a 26-day experimental period; tumors were weighed after treatment to assess the differences in tumor weight among the groups.

[0214] The results showed that the candidate peptides PD1CTLA4_6 and PD1CTLA4_7 in Example 1 had the following effects:

[0215] 1) In an in vitro co-culture system, it can effectively relieve target inhibition and significantly enhance CD8. +The percentage of CD44high cells within the T-cell-gated region, successfully activating CD8 + The killing and effector functions of T cells;

[0216] 2) Significantly improves CD4 + T-cell-gated intracellular ICOS + Percentage of cells that effectively activate CD4 + The immune support function of T cells;

[0217] 3) It showed a very good tumor inhibition effect in humanized tumor mice. During the 26-day experimental period, the tumor growth curve of the experimental group mice was flat and the tumor volume was effectively inhibited.

[0218] 4) After treatment, the tumor weight of mice in the experimental group was significantly lower than that in the PBS blank control group and the disordered peptide control group.

[0219] In summary, the above-mentioned candidate peptides PD1CTLA4_6 and PD1CTLA4_7 can effectively inhibit both PD-1 and CTLA-4 targets simultaneously in vivo and in vitro, successfully activate T cell immune responses, and exert significant tumor growth inhibition effects in humanized mouse models, demonstrating their great potential as anti-tumor drug candidates.

[0220] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. 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.

[0221] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

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

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1.

3. An expression carrier, characterized in that, The expression vector carries the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule of claim 2 or the expression vector of claim 3; or The recombinant cells express the polypeptide of claim 1.

5. A reagent or kit, characterized in that, Includes the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition, characterized in that, Includes the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.

7. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 6 in the preparation of a medicament for the treatment or prevention of at least one of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colorectal cancer, bladder cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, biliary tract cancer, mesothelioma, sarcoma, lymphoma, and leukemia.

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