PD-1 and CTLA-4 dual-targeting inhibitory polypeptides or pharmaceutically acceptable salts thereof and uses thereof

Abstract

The application relates to the technical field of biopharmaceuticals, and discloses a PD-1 and CTLA-4 double-target inhibiting polypeptide or a pharmaceutically acceptable salt thereof and an application thereof, wherein the polypeptide has a structure as shown in formula (I): X1SPILYQMCDYKRX2 (I). The polypeptide or the pharmaceutically acceptable salt thereof can simultaneously produce high-affinity binding to PD-1 and CTLA-4, has excellent biological blocking activity, can significantly improve the proportion of effector T cell subgroups in tumor tissues, and effectively enhances the anti-tumor immune response of the body. Therefore, the polypeptide or the pharmaceutically acceptable salt thereof can be used for related detection of PD-1 and CTLA-4, and can be used as a new anti-tumor candidate drug, thereby providing a brand-new treatment scheme for cancer patients.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical technology, specifically relating to a dual-target inhibitory peptide of PD-1 and CTLA-4 or a pharmaceutically acceptable salt thereof and its use. Background Technology

[0002] In the field of tumor immunotherapy, programmed death receptor-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) are two of the most critical immune checkpoint proteins. Although both are involved in the regulation of tumor immune escape, their stages of action and molecular mechanisms differ significantly. PD-1 is mainly expressed on the surface of activated T cells. After binding to ligands (PD-L1 / PD-L2) expressed on the surface of tumor cells, it transmits inhibitory signals to T cells, leading to T cell exhaustion and thus mediating tumor cell 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 ligands, it restricts the initial activation and proliferation of T cells, indirectly weakening the immune system's ability to recognize tumor cells.

[0003] However, current research and development largely focuses on single-target antagonistic peptides targeting PD-1 or CTLA-4. Their core mechanism of action involves highly specific binding to PD-1 or CTLA-4, blocking their interaction with their corresponding ligands, thereby relieving the inhibitory effect on T cells and restoring the immune system's ability to recognize and kill tumor cells. However, tumor immune escape mechanisms are highly complex and compensatory—when the PD-1 pathway is blocked alone, tumor cells often achieve compensatory escape by activating other immune checkpoint pathways such as CTLA-4. As a result, existing single-target peptides, due to their singular mechanism of action, struggle to generate sufficient immune stimulation and fail to achieve the desired anti-tumor therapeutic effect.

[0004] More importantly, research on dual-target peptides capable of simultaneously and precisely binding to and blocking both PD-1 and CTLA-4 is extremely scarce in the field of peptide drugs. The design of such dual-target molecules demands extremely high precision in spatial configuration and screening techniques; existing technologies struggle to simultaneously balance the binding affinity of both targets with the stability of the molecule itself. Therefore, developing a novel dual-target inhibitor targeting PD-1 and CTLA-4 has significant clinical value and application prospects for the treatment of cancer and other related diseases. Summary of the Invention

[0005] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application provides a PD-1 and CTLA-4 dual-target antagonistic peptide, which aims to overcome the technical bottleneck of existing single-target peptides having limited efficacy and traditional PD-1 or CTLA-4 inhibitors being unable to simultaneously achieve dual-target binding efficacy and bioactivity, thus providing a more efficient and targeted new approach for cancer treatment.

[0006] This application utilizes precise molecular simulation calculations combined with iterative optimization using a deep learning model to design a novel dual-target antagonistic peptide for PD-1 and CTLA-4. Compared to traditional PD-1 and CTLA-4 inhibitors, this peptide possesses a unique and optimized amino acid sequence arrangement, overcoming the limitations of existing dual-target molecules that struggle to balance target binding specificity and molecular stability. It can simultaneously and specifically bind to both PD-1 and CTLA-4, effectively blocking their interaction with their corresponding ligands and inhibiting the PD-1 / CTLA-4-mediated immunosuppressive signaling pathway. Furthermore, the dual-target antagonistic peptide provided in this application exhibits excellent binding affinity and biological activity towards both PD-1 and CTLA-4. Compared to traditional single-target inhibitors or single antagonistic peptides, it can more efficiently relieve T-cell immunosuppression and potently activate T-cell proliferation and killing functions, blocking tumor immune escape through a dual pathway, thereby enhancing anti-tumor treatment efficacy and providing cancer patients with a novel and clinically promising treatment option.

[0007] Therefore, in a first aspect of this application, a polypeptide or a pharmaceutically acceptable salt thereof is proposed. According to embodiments of this application, the polypeptide has a structure as shown in formula (I): X1SPILYQMCDYKRX2 (I); wherein X1 is TR or a first fragment, and X2 is a second or third fragment; the first fragment is PSCGMIRLHQEVYGLREKYGTA; the second fragment is KFNLEQAVNNLVYPVHEAYR; and the third fragment is CVGVFN. The polypeptide or a pharmaceutically acceptable salt thereof of this application can simultaneously bind with high affinity to PD-1 and CTLA-4, and possesses excellent biological blocking activity, significantly increasing the proportion of effector T cell subsets in tumor tissue and effectively enhancing the body's anti-tumor immune response. Therefore, the polypeptide or a pharmaceutically acceptable salt thereof of this application can be used for the detection of PD-1 and CTLA-4, and can also serve as a novel anti-tumor drug candidate, providing a new treatment option for cancer patients.

[0008] 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, and a modifying group, wherein the polypeptide or a pharmaceutically acceptable salt thereof is linked to the modifying group. As is previously known, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can simultaneously bind with high affinity to PD-1 and CTLA-4 and possesses excellent biological blocking activity. Therefore, polypeptide derivatives or pharmaceutically acceptable salts containing the aforementioned polypeptide can be used to treat or prevent diseases related to PD-1 and CTLA-4, including but not limited to cancer, thereby providing a novel treatment option for cancer patients.

[0009] In a third aspect, this application proposes a fusion protein. 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, the aforementioned polypeptide or its pharmaceutically acceptable salt can simultaneously bind with high affinity to PD-1 and CTLA-4 and possesses excellent biological blocking activity. Therefore, the fusion protein containing the aforementioned polypeptide can bind to PD-1 and CTLA-4 for the detection of PD-1 and / or CTLA-4 or for the inhibition of PD-1 and / or CTLA-4, and can also be used to treat or prevent PD-1 and / or CTLA-4 related diseases, including but not limited to cancer, thereby providing a novel treatment option for cancer patients.

[0010] In a fourth 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 third aspect. As is known prior art, the aforementioned polypeptides or pharmaceutically acceptable salts thereof can simultaneously bind with high affinity to PD-1 and CTLA-4 and possess excellent biological blocking activity. Therefore, the reagent or kit containing the aforementioned polypeptide can bind to PD-1 and CTLA-4 for the detection of PD-1 and / or CTLA-4.

[0011] In a fifth aspect, this application provides a pharmaceutical composition. 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 third aspect. As is known, the aforementioned polypeptides or pharmaceutically acceptable salts thereof can simultaneously bind with high affinity to PD-1 and CTLA-4 and possess excellent biological blocking activity. Therefore, the pharmaceutical composition containing the aforementioned polypeptide can be used to treat or prevent PD-1 and / or CTLA-4 related diseases, including but not limited to cancer, thereby providing a novel treatment option for cancer patients.

[0012] In a sixth 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 third aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament, wherein the medicament is a dual-target inhibitor of PD-1 and CTLA-4. As is known, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can simultaneously bind with high affinity to both PD-1 and CTLA-4 and possesses excellent biological blocking activity. Therefore, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, or a polypeptide derivative containing 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 related diseases, including but not limited to cancer, thereby providing a novel treatment option for cancer patients.

[0013] In a seventh 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 third aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament for treating or preventing PD-1 and / or CTLA-4 related diseases, inhibiting PD-1 and / or CTLA-4 activity, and / or promoting T cell proliferation.

[0014] In an eighth 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, a polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, a fusion protein described in the third aspect, or a reagent or kit described in the fourth 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 with high affinity to both PD-1 and CTLA-4 simultaneously. Therefore, the detection method based on this polypeptide can be effectively used for the detection of PD-1 and / or CTLA-4.

[0015] 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

[0016] 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:

[0017] Figure 1 This is a schematic diagram of the peptide design and screening process in this application;

[0018] Figure 2 The detection results of the dual-target candidate peptides PD1CTLA4_8 and PD1CTLA4_9 inhibiting the binding of PD-1 and PD-L1, respectively, in Example 2 of this application;

[0019] Figure 3 The detection results of the dual-target candidate peptides PD1CTLA4_8 and PD1CTLA4_9 inhibiting the binding of CTLA-4 and B7-1, respectively, in Example 2 of this application;

[0020] Figure 4 The dual-target candidate peptides PD1CTLA4_8 and PD1CTLA4_9 in Example 2 of this application activate CD4, respectively. + T cell detection results;

[0021] Figure 5 The dual-target candidate peptides PD1CTLA4_8 and PD1CTLA4_9 in Example 2 of this application activate CD8, respectively. + Results of T cell testing. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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 anti-tumor, antiviral, antibacterial, and anti-inflammatory drugs.

[0027] 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.

[0028] 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 central to tumor immunotherapy, aiming to block this inhibitory pathway and reactivate the T cell's ability to kill tumor cells.

[0029] 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.

[0030] 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 a core regulatory molecule in the immune system. CD4 is a lineage marker for helper T cells (Th cells), while ICOS (inducible costimulatory molecule) is an activation marker whose expression is upregulated only after stimulation of the T cell receptor (TCR). When CD4+ T cells simultaneously express high levels of ICOS, it usually indicates that the cells are in a highly activated state.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In this article, the term "affinity maturation" refers to the process by which B cells, through mutation and selection mechanisms, increase the affinity of their antibodies for binding to antigens during an immune response. In this process, B cells undergo a high frequency of mutations in their germinal centers, producing a variety of variant antibodies, which are then selected based on their binding ability to antigens, ultimately resulting in antibodies with high affinity. This process is crucial for generating an effective immune response and for vaccine development.

[0035] 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.

[0036] 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.

[0037] In this paper, the structural formula for the term "αMeK" is: .

[0038] In this article, the structural formula for the term "HoK" is: .

[0039] In this article, the structural formula for the term "N-Me-K" is: .

[0040] In this article, the structural formula for the term "Orn" is: .

[0041] In this article, the structural formula for the term "Dab" is: .

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

[0043] 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.

[0044] 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.

[0045] In this document, "pharmaceutical composition" can refer to a drug 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 "pharmaceutical composition" 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 or revitalizer with a liquid excipient, a finely pulverized solid excipient, or both.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In this article, the term "inhibitor" refers to a substance (ligand) that inhibits the type of receptor.

[0050] 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 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 it; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any medication that administers a polypeptide or a pharmaceutically acceptable salt thereof, or a drug containing such polypeptide, to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administering a drug containing a polypeptide or a pharmaceutically acceptable salt thereof as described herein to an individual in need.

[0051] This application discloses a dual-target inhibitory peptide for PD-1 and CTLA-4, or a pharmaceutically acceptable salt thereof, and its uses, which will be described in detail below.

[0052] polypeptides or their pharmaceutically acceptable salts

[0053] In a first aspect of this application, this application provides a polypeptide or a pharmaceutically acceptable salt thereof. According to an embodiment of this application, the polypeptide has a structure as shown in formula (I): X1SPILYQMCDYKRX2 (I); wherein X1 is TR or a first fragment, X2 is a second fragment or a third fragment; the first fragment is PSCGMIRLHQEVYGLREKYGTA; the second fragment is KFNLEQAVNNLVYPVHEAYR; and the third fragment is CVGVFN.

[0054] This application provides a dual-target antagonistic peptide or a pharmaceutically acceptable salt thereof targeting PD-1 and CTLA-4, aiming to offer an innovative treatment option for tumor immunotherapy. Through precise sequence design and structural optimization, this peptide exhibits high affinity and highly efficient biological blocking activity against both PD-1 and CTLA-4, significantly increasing the proportion of effector T cell subsets in tumor tissues and enhancing the body's anti-tumor immune response. Furthermore, the global incidence of malignant tumors continues to rise, while traditional monoclonal antibody drugs have inherent limitations in tissue penetration, production costs, and clinical application. In contrast, the dual-target peptide described in this application not only provides a novel treatment option for cancer patients but also holds the potential to overcome the application bottlenecks of existing immune checkpoint inhibitors, reshaping the research and development and market landscape of related drugs.

[0055] According to the embodiments of this application, the polypeptide satisfies one of the following: (1) X1 is TR and X2 is a second fragment; (2) X1 is a first fragment and X2 is a third fragment.

[0056] According to embodiments of this application, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0057] 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 the amino acid sequence shown in SEQ ID NO:A having a sequence similarity of 80% or more (e.g., 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more), 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 sequence similarity of the amino acid sequence shown in SEQ ID NO: A to 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%), 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 the receptor, and are all within the scope of protection of this application.

[0058] For example, "the amino acid sequence of the polypeptide is 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 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, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO:2" means that the polypeptide has an amino acid sequence shown in SEQ ID NO:2, an amino acid sequence in a conservative modified form of SEQ ID NO:2, or 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.

[0059] 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 80% of the total number, preferably not exceeding 90%. In this document, "amino acid sequences of conserved modification form" also includes naturally mutated amino acid modifications; "naturally mutated" refers to mutations caused by changes in alleles or other factors during the natural mutation process of a polypeptide.

[0060] 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.

[0061] .

[0062] 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 has been substituted, deleted, or added with 1-2 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.

[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 polypeptide's half-life in vivo.

[0066] According to embodiments of this application, the amino acid X is selected from K, k, αMeK, HoK, Dap, Dab, Orn, or N. Me K.

[0067] polypeptide derivatives or their pharmaceutically acceptable salts

[0068] 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, and a modifying group, wherein the polypeptide or a pharmaceutically acceptable salt thereof is linked to the modifying group. As is previously known, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can simultaneously bind with high affinity to PD-1 and CTLA-4 and possesses excellent biological blocking activity. Therefore, polypeptide derivatives or pharmaceutically acceptable salts containing the aforementioned polypeptide can be used to treat or prevent diseases related to PD-1 and CTLA-4, including but not limited to cancer, thereby providing a novel treatment option for cancer patients.

[0069] 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.

[0070] 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:

[0071] According to embodiments of this application, the modifying group is attached to -NH2, -SH, -OH or -COOH of the amino acid side chain in the polypeptide.

[0072] According to embodiments of this application, the modifying group is linked to the -NH2 of the amino acid side chain in the polypeptide.

[0073] According to embodiments of this application, the modifying group has at least one of the following structures:

[0074] ;

[0075] ;

[0076] ;

[0077] .

[0078] 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.

[0079] Fusion proteins, reagents or kits, pharmaceutical compositions

[0080] In a third aspect, this application proposes a fusion protein. 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, the aforementioned polypeptide or its pharmaceutically acceptable salt can simultaneously bind with high affinity to PD-1 and CTLA-4 and possesses excellent biological blocking activity. Therefore, the fusion protein containing the aforementioned polypeptide can bind to PD-1 and CTLA-4 for the detection of PD-1 and / or CTLA-4 or for the inhibition of PD-1 and / or CTLA-4, and can also be used to treat or prevent PD-1 and / or CTLA-4 related diseases, including but not limited to cancer, thereby providing a novel treatment option for cancer patients.

[0081] In an alternative embodiment of this application, the fusion protein further includes a functional fragment.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In a fourth 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 third aspect. As is known prior art, the aforementioned polypeptides or pharmaceutically acceptable salts thereof can simultaneously bind with high affinity to PD-1 and CTLA-4 and possess excellent biological blocking activity. Therefore, the reagent or kit containing the aforementioned polypeptide can bind to PD-1 and CTLA-4 for the detection of PD-1 and / or CTLA-4.

[0086] 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.

[0087] 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 is also contained in a container such as a test tube, microplate, or test strip.

[0088] In a fifth aspect, this application provides a pharmaceutical composition. 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 third aspect. As is known, the aforementioned polypeptides or pharmaceutically acceptable salts thereof can simultaneously bind with high affinity to PD-1 and CTLA-4 and possess excellent biological blocking activity. Therefore, the pharmaceutical composition containing the aforementioned polypeptide can be used to treat or prevent PD-1 and / or CTLA-4 related diseases, including but not limited to cancer, thereby providing a novel treatment option for cancer patients.

[0089] According to embodiments of this application, the pharmaceutical composition may further include pharmaceutically acceptable excipients.

[0090] In one alternative 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.

[0091] In one alternative embodiment of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field, such as a protectant.

[0092] In one alternative embodiment of this application, pharmaceutically acceptable mediators refer to pharmaceutical mediators conventional in the pharmaceutical field, such as solutions (e.g., water) and liposomes.

[0093] In one alternative embodiment 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.

[0094] In some optional embodiments of this application, the pharmaceutical composition may be an oral dosage form, such as a solid oral dosage form or a liquid oral dosage form, and the specific type is not limited, all of which are within the protection scope of this application.

[0095] In some alternative embodiments of this application, the pharmaceutical composition of this application may also contain other active ingredients for treatment.

[0096] The pharmaceutical composition 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 pharmaceutical composition 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.

[0097] use

[0098] In a sixth 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 third aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament, wherein the medicament is a dual-target inhibitor of PD-1 and CTLA-4. As is known, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can simultaneously bind with high affinity to both PD-1 and CTLA-4 and possesses excellent biological blocking activity. Therefore, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, or a polypeptide derivative containing 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 related diseases, including but not limited to cancer, thereby providing a novel treatment option for cancer patients.

[0099] In a seventh 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 third aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament for treating or preventing PD-1 and / or CTLA-4 related diseases, inhibiting PD-1 and / or CTLA-4 activity, and / or promoting T cell proliferation.

[0100] This application discloses 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 third aspect, or the pharmaceutical composition described in the fifth aspect in the treatment or prevention of PD-1 and / or CTLA-4 related diseases, the inhibition of PD-1 and / or CTLA-4 activity, and / or the promotion of T cell proliferation.

[0101] 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 third aspect or the pharmaceutical composition of the fifth aspect for the treatment or prevention of PD-1 and / or CTLA-4 related diseases, the inhibition of PD-1 and / or CTLA-4 activity and / or the promotion of T cell proliferation.

[0102] According to embodiments of this application, the above-described uses may further include at least one of the following technical features:

[0103] According to embodiments of this application, the PD-1 and / or CTLA-4 related diseases include cancer.

[0104] According to embodiments of this application, the 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, and hematologic malignancies.

[0105] method

[0106] In an eighth 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, a polypeptide derivative described in the second aspect or a pharmaceutically acceptable salt thereof, a fusion protein described in the third aspect, or a reagent or kit described in the fourth 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 with high affinity to both PD-1 and CTLA-4 simultaneously. Therefore, the detection method based on this polypeptide can be effectively used for the detection of PD-1 and / or CTLA-4.

[0107] According to a specific embodiment of this application, the signal includes a fluorescence signal.

[0108] According to a specific embodiment of this application, it 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.

[0109] In a ninth aspect of this application, a method for treating or preventing cancer is proposed. According to an embodiment of this application, the method includes administering to a subject a pharmaceutically acceptable dose 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 third aspect, or the pharmaceutical composition described in the fifth aspect.

[0110] In one alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0111] 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, 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.

[0112] 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.

[0113] According to embodiments of this application, the 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, and hematologic malignancies.

[0114] 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.

[0115] Example 1: Obtaining a dual-target protein inhibitory peptide for PD-1 and CTLA-4

[0116] The schematic diagram of the peptide design and screening process in this application is shown below. Figure 1This method encompasses a series of steps, from generating dual-target peptide sequences for PD-1 and CTLA-4 to affinity optimization and iterative evolution. The entire framework references the method proposed in patent 2025102889738. The method consists of multiple iterative evolutionary processes, after which the model-designed peptides achieve effective enhancements in biological activity. Each iterative evolution includes three parts: generation and optimization of novel peptides based on artificial intelligence, wet experimental determination of peptide biological activity, and feedback to strengthen the model's perception of biological activity.

[0117] First, referring to patent 202410192333.2, entitled "Training of Ligand Information Generation Model, Method and Apparatus for Generating Ligand Information," which discloses an innovative deep learning method, TPDiffusion, capable of generating polypeptide sequences that can bind to target proteins based on their amino acid sequences. This method, which converts target protein sequences into polypeptide sequences, 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 target proteins and polypeptide sequences are learned, enabling the generation of specific polypeptides (i.e., PD-1 and CTLA-4 dual-target peptides) for specific target proteins (i.e., PD-1 and CTLA-4 dual-target peptides). The training of the polypeptide sequence generation model TPDiffusion mainly includes forward diffusion and reverse diffusion processes, with the forward diffusion process comprising the following steps:

[0118] 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.

[0119] 2. Gradually add noise to the peptide sequence: Gaussian noise is gradually added to the peptide portion of the vector encoding based on the Markov chain until the peptide sequence is completely destroyed.

[0120] 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:

[0121] 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.

[0122] 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.

[0123] The denoising network in TPDiffusion employs the BERT model (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 (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 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, sequences of PD-1 and CTLA-4 as inputs were used to generate a batch of candidate peptide sequences that may have high affinity for both PD-1 and CTLA-4.

[0124] 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.

[0125] 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 in this patent is used as a reward model in a deep reinforcement learning method to guide the mutation of candidate peptide sequences.

[0126] After obtaining peptides with mature affinity, their bioactivity was evaluated, and the results were fed back to the model to continue the next round of peptide generation and optimization. After one round of iterations, peptides that effectively inhibited the two target proteins PD-1 and CTLA-4 were designed, with sequences shown in SEQ ID NO:1~2, respectively. The amino acid sequences of SEQ ID NO:1~2 are shown in Table 1.

[0127] Table 1

[0128]

[0129] Example 2: Evaluation of the inhibitory effect of candidate peptides on both PD-1 and CTLA-4 target proteins

[0130] This example aims to evaluate the inhibitory ability of the candidate peptides (PD1CTLA4_8, PD1CTLA4_9) obtained in Example 1 against the dual-target proteins of PD-1 and CTLA-4. The specific details are as follows:

[0131] 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 2. 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).

[0132] 1. In vitro experiment on PD-1 / PD-L1 binding blockade based on TR-FRET technology

[0133] This experiment aims to evaluate the in vitro inhibitory activity of the two candidate peptides (PD1CTLA4_8 and PD1CTLA4_9) obtained in Example 1 against the interaction between PD-1 and PD-L1. The specific experimental steps are as follows:

[0134] Reagent preparation: The purified candidate peptides PD1CTLA4_8 and PD1CTLA4_9 were dissolved in water to prepare a 1 mg / mL stock solution for subsequent experiments.

[0135] Experimental reagents and instruments: The TR-FRET kit (purchased from BPS Bioscience, San Diego, USA, catalog number #72038) was used, and the experiments were performed strictly in accordance with the manufacturer's instructions. All reagent components were prepared and used at the recommended concentrations.

[0136] Sample preparation and loading: The experiment was conducted in 384-well white, non-binding microtiter plates. Candidate peptides at 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) were added to the wells, with three triplicates for each concentration gradient to ensure the reliability of the experimental data.

[0137] 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.

[0138] Data processing and bioactivity analysis: Experimental results were evaluated by calculating the TR-FRET ratio.

[0139] Test results as follows Figure 2As shown, both synthesized candidate peptides were found to effectively inhibit the interaction between PD-1 and PD-L1, achieving an inhibition effect of about 50% at a concentration of 8 μg / ml. This indicates that both candidate peptides have good PD-1 inhibitory activity and high PD-1 inhibitory biological activity, and can effectively block the binding between PD-1 and PD-L1.

[0140] 2. In vitro experiment on CTLA-4 and B7-1 (CD80) binding blockade based on TR-FRET technology

[0141] This experiment aimed to evaluate the in vitro inhibitory activity of the two candidate peptides (PD1CTLA4_8 and PD1CTLA4_9) obtained in Example 1 against the interaction between CTLA-4 and B7-1 (CD80). The specific experimental methods are as follows:

[0142] Reagent preparation: The purified candidate peptides PD1CTLA4_8 and PD1CTLA4_9 were dissolved in water to prepare a 1 mg / mL stock solution for subsequent experiments.

[0143] Experimental reagents and instruments: The TR-FRET kit (purchased from BPSBioscience, San Diego, USA, catalog number #72120) specifically designed for CTLA-4:B7-1 binding detection was used. The entire experiment strictly followed the manufacturer's official operating procedures, and all reagent components were prepared and used at their recommended concentrations.

[0144] Sample preparation and reaction system: The experiment was conducted in 384-well white, non-binding microtiter plates. To ensure the statistical significance of the experimental data, candidate peptides at 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) were added to the corresponding wells, and three parallel replicates were set up for each concentration gradient to ensure the reliability of the experimental data.

[0145] Signal acquisition and detection: Fluorescence intensity was read using a Spark multi-functional microplate analyzer (TECAN). The detection program was set to two consecutive time-resolved measurement channels: first, the emission signal value at 620 nm was detected, followed by the emission signal value at 665 nm.

[0146] Data processing and bioactivity analysis: Experimental data were quantitatively analyzed using the TR-FRET ratio.

[0147] Test results as follows Figure 3 As shown, both synthesized candidate peptides effectively blocked the interaction between CTLA-4 and B7-1, achieving an inhibition rate of 40%-90% at a concentration of 3 μg / ml, indicating that both possess good CTLA-4 inhibitory activity. Among them, peptide PD1CTLA4_8 showed better inhibitory effect on CTLA-4 than PD1CTLA4_9.

[0148] Based on this, it can be seen that the two candidate peptides (PD1CTLA4_8 and PD1CTLA4_9) of this application can serve as dual-target peptides, effectively inhibiting the dual targets of PD-1 and CTLA-4, and have the potential to be used as subsequent anti-tumor drugs.

[0149] Example 3: In vitro activation experiment of T cells based on flow cytometry

[0150] This experiment aimed to evaluate the enhancing effects of two candidate peptides (PD1CTLA4_8 and PD1CTLA4_9) obtained in Example 1 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:

[0151] (1) Isolation and culture of T cells

[0152] 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 the 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.

[0153] (2) Cell activation and peptide stimulation

[0154] 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).

[0155] Drug treatment: Add candidate peptides (final concentration of 4 μM, i.e. PD1CTLA4_8 group and PD1CTLA4_9 group) to the aforementioned activated co-culture system, and incubate in a cell culture incubator at 5% CO2 and 37°C for 72 hours.

[0156] Reference settings:

[0157] ① Containing only unactivated T cells (not co-cultured with A549 cells) and without drug administration, serving as a negative control;

[0158] ② 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;

[0159] ③ Contains activated T cells and A549 tumor cells, but without drug administration, to demonstrate the inhibitory effect on the tumor microenvironment, namely the Stimulated T cell s & A549 group.

[0160] (3) Flow cytometry detection:

[0161] CD8 + T-cell detection: CD8+ T-cell assay using flow cytometry. + The T cells within the gated region were analyzed, with the percentage of CD44high cells used as a measure of CD8. + Indicators of T cell activation and effector status.

[0162] 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.

[0163] (4) Signal acquisition and data analysis: Cell surface antigens were labeled using fluorescent staining technology, and data were acquired using a BD FACSCanto flow cytometer (BD Biosciences). The acquired flow cytometry data were analyzed using FlowJo software (version 10.5.3).

[0164] Experimental results are as follows Figure 4 and 5 As shown, the two candidate peptides (PD1CTLA4_8 and PD1CTLA4_9) of this application can effectively relieve the inhibitory effect of immune checkpoint molecules PD-1 and CTLA4 on T cells and can effectively activate CD4 T and CD8 T cells.

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

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

4. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 3, 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, and leukemia.

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