Method for screening polypeptides that specifically target t cells and use thereof

Abstract

Provided is a method for screening polypeptides that specifically target T cells, comprising: providing T cells having a surface antigen marker selected from at least one of CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD152 (CTLA-4), CD153, CD154 (CD40L), CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7; and contacting the T cells with a polypeptide display library, and screening therefrom target polypeptides that specifically bind to the T cells.

Description

Methods and applications for screening peptides that specifically target T cells Technical Field

[0001] This invention belongs to the field of polypeptide technology and relates to a method for screening polypeptides that specifically target T cells and its application. Background Technology

[0002] Chimeric antigen receptor-T cell (CAR-T) therapy and engineered T cell receptor-T cell (TCR-T) therapy are currently the two most effective methods of adoptive cell transfer therapy (ACT), demonstrating significant efficacy in cancer treatment and becoming a major approach. Several CAR-T cell therapies are now available globally, showing significant efficacy in treating B-cell malignancies, while TCR-T therapy has also achieved satisfactory results in treating solid tumors. For example, in August of this year, Adaptimmune Therapeutics' TCR-T therapy TECELRA received FDA approval for the treatment of adult patients with unresectable or metastatic synovial sarcoma, making it the world's first approved T-cell therapy for solid tumors. A bispecific TCR-T therapy for melanoma has also received FDA approval. The commercialization of TCR therapy marks a new chapter in tumor immunotherapy.

[0003] Although CAR-T and TCR-T therapies differ in their antigen recognition mechanisms, both utilize genetically engineered T cells. Currently, CAR-T and TCR-T therapies face numerous challenges in clinical application, including long preparation cycles, complex processes, and high costs, making repeated use difficult. Furthermore, traditional viral vector preparation carries potential safety risks, such as insertion mutations and oncogenic risks, limiting their widespread application.

[0004] In vivo induction of CAR-T or TCR-T cells can reduce systemic toxicity, providing new avenues for tumor cell therapy. However, current in vivo drug delivery mainly relies on carrier formulations and conjugated targeted delivery, which suffers from low delivery efficiency, poor targeting, and significant side effects. Although several molecules targeting CD3 or other T cell surface antigens have been commercialized, research has revealed that monoclonal antibodies have large molecular weights, long half-lives, high nuclear toxicity, and difficulty in penetrating certain barriers, hindering targeted drug delivery. Existing technology discloses a cyclic heptapeptide that specifically binds to T cell surface antigens, its encoding gene, and its applications. However, current methods utilize purchased purified T cell surface antigen proteins and employ solid-phase screening methods. Purchased antigen proteins often have a single structure, and solid-phase screening methods cannot obtain antibodies that specifically bind to antigens with stereoconformities. Specific targeted in vivo delivery remains an obstacle in the development of therapeutic agents, especially CAR-T or TCR-T immunotherapies. Summary of the Invention

[0005] To address one of the aforementioned technical problems in existing technologies, this invention utilizes peptide display libraries, such as phage display technology (PDT), to screen for peptides that specifically target T cells, providing targeted delivery molecules for the internal production of CAR-T or TCR-T. This invention establishes a standardized peptide screening process, aiming to solve current challenges in the field of cell therapy, and possesses commercial potential and broad application prospects.

[0006] In a first aspect, the present invention provides a method for screening peptides that specifically target T cells, comprising:

[0007] Provide T cells, said T cells having a subset selected from CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD152 (CTLA-4), CD153, CD154 (CTLA-4), ... At least one of the following surface antigen markers: CD40L, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7;

[0008] The T cells are brought into contact with a polypeptide display library, and target polypeptides that specifically bind to the T cells are screened from it.

[0009] In some embodiments, the T cells are obtained by purifying spleen mononuclear cells.

[0010] In some embodiments, the purification of the T cells is performed using a flow cytometer, a magnetic cell sorter, or a microfluidic chip, with a magnetic cell sorter being preferred.

[0011] In some embodiments, the polypeptide display library is selected from phage display libraries, yeast display libraries, or engineered cell surface display libraries, preferably phage display libraries.

[0012] In some embodiments, the peptide display library includes, but is not limited to, hexapeptide libraries, heptapeptide libraries, octapeptide libraries, dodecapeptide libraries, cyclic heptapeptide libraries, and 15-peptide libraries, with dodecapeptide libraries being preferred.

[0013] In some embodiments, the polypeptide display library contains 10 9 The above are polypeptide sequences.

[0014] In some embodiments, the method further includes: obtaining a candidate peptide display library that binds to the T cells, extracting DNA from the candidate peptide display library, and

[0015] Optionally, the DNA is amplified using primer pairs as shown in SEQ ID NO:1 and SEQ ID NO:2, and the amplification products are sequenced to obtain the sequence of the target polypeptide.

[0016] In some embodiments, the method further includes performing bioinformatics analysis on the sequencing results.

[0017] In some embodiments, the bioinformatics analysis includes calculating the frequency of occurrence of peptide sequences or specific amino acids using multiple sequence alignment and / or cluster analysis, and predicting the secondary and tertiary structures of peptides.

[0018] In some implementations, ClustalW or MUSCLE is used for multiple sequence alignment.

[0019] In some implementations, cluster analysis is performed using UPGMA or Neighbor-Joining.

[0020] Secondly, the present invention provides a polypeptide that specifically targets T cells.

[0021] In some embodiments, the polypeptide is obtained by the screening method described in the first aspect.

[0022] In some embodiments, the positive percentage of the peptide binding to T cells is greater than 15%. As shown in Figure 3, after the peptide is contacted with T cells, the percentage of T cells containing the peptide-displaying cells is measured as the positive percentage. In some embodiments, flow cytometry is used to determine the positive percentage. In some embodiments, a magnetic cell sorter or a microfluidic chip is used to determine the positive percentage.

[0023] In some embodiments, the polypeptide comprises the amino acid sequence shown in formula (I) WX1LX2X3GY.

[0024] Where X1, X2, or X3 represents any one of the amino acids selected from glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), tryptophan (W), serine (S), tyrosine (Y), cysteine ​​(C), phenylalanine (F), asparagine (N), glutamine (Q), threonine (T), aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), and histidine (H).

[0025] In some embodiments, the polypeptide comprises an amino acid sequence having at least 60% sequence identity with the amino acid sequence shown in Formula (I), for example at least 75% or at least 90% sequence identity.

[0026] In some implementations, X1 is selected from S.

[0027] In some implementations, X2 or X3 is independently selected from S, L, E, A or R.

[0028] In some implementations, X1 is selected from S, and X2 is selected from S, L, or E.

[0029] In some implementations, X1 is selected from S, and X3 is selected from S, A, or R.

[0030] In some implementations, X1 is selected from S, X2 is selected from S, L or E, and X3 is selected from S, A or R.

[0031] In some embodiments, the polypeptide is 7-25 amino acids long, preferably 10-18 amino acids long, and more preferably 12 amino acids long.

[0032] In some embodiments, the polypeptide further comprises no more than nine (e.g., nine, eight, seven, six, five, four, three, two, or one) amino acids at the C-terminus of the amino acid sequence shown in Formula (I).

[0033] In some embodiments, the polypeptide further comprises no more than nine (e.g., nine, eight, seven, six, five, four, three, two, or one) amino acids at the N-terminus of the amino acid sequence shown in formula (I).

[0034] In some embodiments, the polypeptide comprises an amino acid sequence selected from the following:

[0035] WSWSLSSGYADV(SEQ ID NO:3), VTYNWSLLAGYV(SEQ ID NO:4), YTWTLERGYSVN(SEQ ID NO:5), TSGTMQTNPLPV(SEQ ID NO:6), STWSLYAGYTHN(SEQ ID NO:7), NSVHVYHKSFLF(SEQ ID NO:8), TERTMESMTRFA(SEQ ID NO:9), FSVPSTPRTVVV (SEQ ID NO:10) and EWKVLEGHTTRD (SEQ ID NO:11).

[0036] In some embodiments, the polypeptide comprises an amino acid sequence selected from the following: WWSLSSGYADV (SEQ ID NO:3), VTYNWSLLAGYV (SEQ ID NO:4), YTWTLERGYSVN (SEQ ID NO:5), and STWSLYAGYTHN (SEQ ID NO:7).

[0037] In some embodiments, the polypeptide comprises an amino acid sequence selected from the following: YTWTLERGYSVN (SEQ ID NO:5).

[0038] In some preferred embodiments, the T cells are CD2+, CD3+, CD4+, CD5+, CD7+, CD8+, CD25+, CD28+, CD45+, CD69+, CD152+, CD154+ and / or PD-1+ T cells.

[0039] In some embodiments, the T cells may be human or non-human mammal T cells. Non-human mammals include, for example, livestock and pets, such as mammals belonging to the families Sheep, Bovidae, Suidae, Canidae, Felidae, and Muridae. Preferably, the T cells are human T cells.

[0040] In some embodiments, the polypeptide includes modified peptides, including but not limited to N-terminal modifications, C-terminal modifications, backbone modifications, side chain modifications, and amino acid modifications. The modifications are intentionally drug-induced and performed while preserving the polypeptide's activity. The purposes of these modifications include prolonging half-life, increasing water solubility, and reducing or eliminating toxic side effects.

[0041] Thirdly, the present invention provides a delivery system specifically targeting T cells, comprising the polypeptide described in the second aspect.

[0042] In some embodiments, the delivery system is selected from liposomes, lipid nanoparticles, micelles, exosomes, or any combination thereof that exhibit the polypeptide on their surface.

[0043] In some embodiments, the delivery system is selected from lipid nanoparticles.

[0044] In some embodiments, the delivery system further comprises at least one reagent encapsulated in the peptide-modified liposomes, lipid nanoparticles, micelles, exosomes, or combinations thereof.

[0045] In some embodiments, the delivery system further includes a connector for linking the polypeptide to the at least one reagent.

[0046] In some embodiments, the at least one reagent is selected from drugs for preventing or treating diseases, imaging agents, diagnostic agents, markers, and detection agents.

[0047] In some embodiments, the drug for disease prevention or treatment is selected from small molecule drugs, polypeptide drugs, nucleic acid drugs, or any combination thereof.

[0048] In some embodiments, the nucleic acid drug is selected from one or more of siRNA, mRNA, shRNA, microRNA, ASO, DNA, or plasmid.

[0049] Fourthly, the present invention provides a fusion protein comprising one or more polypeptides as described in the second aspect.

[0050] In some embodiments, the fusion protein further includes one or more other targeting moieties.

[0051] In some implementations, the other targeting portion includes a tumor antigen-binding region.

[0052] In some embodiments, the fusion protein optionally further comprises one or more domains selected from the following: hinge region, transmembrane region, at least one co-stimulatory region, and intracellular signal transduction region.

[0053] In some embodiments, the polypeptide is fused to the C-terminus or N-terminus of the other targeting moiety.

[0054] In some embodiments, the polypeptide is fused to the C-terminus or N-terminus of the other targeting moiety via a linker peptide or adapter.

[0055] In some embodiments, the linker peptide or adapter can be any amino acid sequence, preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.

[0056] In some embodiments, the linker peptide or adapter is less than 12 amino acids in length, for example, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2. Examples of suitable linkers include, but are not limited to: GGGSGGGG (SEQ ID NO:12), GGGGS (SEQ ID NO:13), GGGSG (SEQ ID NO:14), GGSGG (SEQ ID NO:15), GSGGG (SEQ ID NO:16), GSGGGP (SEQ ID NO:17), GGEPS (SEQ ID NO:18), GGEGGGP (SEQ ID NO:19), and GGEGGGSEGGGS (SEQ ID NO:20) (as described in WO2010 / 133828). Preferred linkers or adapters comprise sequences having the formula (GGGGS)n, optionally with other amino acid sequences.

[0057] In some implementations, n in the formula (GGGGS)n is 1, 2, 3 or 4.

[0058] In some embodiments, the hinge region is selected from the hinge regions of the following molecules: CD8α, 4-1BB, OX40, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or any combination thereof.

[0059] In some embodiments, the transmembrane region is selected from the transmembrane regions of the following molecules: CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or any combination thereof.

[0060] In some embodiments, the co-stimulatory region or intracellular signal transduction region is selected from the co-stimulatory regions or intracellular signal transduction regions of the following molecules: 4-1BB (CD137), CD27, CD19, CD4, CD28, ICOS (CD278), CD8α, CD8β, BAFFR, HVEM, LIGHT, PD-1, KIRDS2, SLAMF7, NKG2D, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TLR2, CD2, CD7, CD70, CD134, CD226, or any combination thereof.

[0061] Fifthly, the present invention provides a nucleic acid molecule encoding the polypeptide described in the second aspect or the fusion protein described in the fourth aspect.

[0062] In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0063] In a sixth aspect, the present invention provides a carrier comprising the nucleic acid molecules described in the fifth aspect.

[0064] In some embodiments, the vector includes, but is not limited to, DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0065] In some preferred embodiments, the viral vector is selected from lentiviruses, adenoviruses, and retroviral vectors.

[0066] In a seventh aspect, the present invention provides an engineered cell comprising the nucleic acid molecule described in the fifth aspect or the carrier described in the sixth aspect.

[0067] In some embodiments, engineered cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia coli cells), and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0068] In some embodiments, the engineered cells are immune cells selected from T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and / or peripheral blood mononuclear cells.

[0069] In an eighth aspect, the present invention provides a conjugate comprising the polypeptide described in the second aspect or the fusion protein described in the fourth aspect.

[0070] In some embodiments, the conjugate further comprises modifications to the polypeptide or fusion protein, including detectable labels and therapeutic agents.

[0071] In some embodiments, the detectable markers include enzymes, radionuclides, fluorescent dyes, luminescent substances, and biotin.

[0072] In a ninth aspect, the present invention provides a composition.

[0073] In some embodiments, the composition comprises the polypeptide described in the second aspect, the delivery system described in the third aspect, the fusion protein described in the fourth aspect, the nucleic acid molecule described in the fifth aspect, the carrier described in the sixth aspect, the engineered cell described in the seventh aspect, or the conjugate described in the eighth aspect.

[0074] In some embodiments, the composition further includes a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0075] In a tenth aspect, the present invention provides the use of peptides obtained by the screening method described in the first aspect, peptides described in the second aspect, delivery systems described in the third aspect, fusion proteins described in the fourth aspect, nucleic acid molecules described in the fifth aspect, vectors described in the sixth aspect, engineered cells described in the seventh aspect, conjugates described in the eighth aspect, or compositions described in the ninth aspect in the preparation of preventive, diagnostic, and / or therapeutic drugs targeting T cells.

[0076] In one aspect, the present invention provides a method for preventing, diagnosing and / or treating a disease, comprising administering to a subject in need the polypeptide described in the second aspect, the delivery system described in the third aspect, the fusion protein described in the fourth aspect, the nucleic acid molecule described in the fifth aspect, the carrier described in the sixth aspect, the engineered cell described in the seventh aspect, the conjugate described in the eighth aspect or the composition described in the ninth aspect.

[0077] In some implementations, the disease is an autoimmune disease.

[0078] In some implementations, the disease is cancer or a tumor.

[0079] In some embodiments, the tumor is selected from hematologic malignancies, solid tumors, or any combination thereof.

[0080] In some embodiments, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, neuroblastoma, Ewing sarcoma, myelodysplastic syndrome, or any combination thereof.

[0081] In some embodiments, the solid tumor is selected from gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer, glioma, endometrial cancer, testicular cancer, urinary tract tumor, thyroid cancer, or any combination thereof.

[0082] Beneficial effects of the present invention

[0083] 1. Phage display technology is innovative in screening T-cell-targeting peptides using 12-peptide libraries because the highly diverse and complex 12-peptide libraries can cover a wide range of potential T-cell receptor (TCR) binding sites. Peptides obtained by combining high-throughput sequencing with comprehensive screening methods ensure high specificity and efficacy, and stronger affinity. The method of this invention has the advantages of simple operation, low cost, and easy preparation, modification, and high biosafety of the peptides screened by this method.

[0084] 2. The screened peptides were validated in vitro and in animal models, demonstrating their specificity in targeting T cells and their ability to effectively activate or regulate T cell function, proliferation, and effector activity. These peptides can serve as candidate molecules for novel immunotherapeutic drugs. This technology facilitates the development of novel immunotherapeutic strategies for cancer treatment or immune disease therapy, and has broad potential for clinical application. Attached Figure Description

[0085] Figure 1 shows a flowchart of peptide-targeted T-cell screening.

[0086] Figure 2 shows the purity results of T cells sorted by flow cytometry.

[0087] Figure 3 shows the in vitro validation results of the screened peptides targeting T cells.

[0088] Figure 4 shows the specific distribution of the screened peptides in mice and the results of targeting T cells.

[0089] Figure 5 shows the validation results of the targeted T cell effect of the selected peptide-modified lipid nanoparticles in Cre-loxP mice. Detailed Implementation

[0090] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0091] The methods used in this article to represent polypeptides, amino acids, and chemical groups are all recognized in the relevant fields. The abbreviations for amino acids can be found in Table 1. Unless otherwise specified, amino acids in this article generally refer to L-type amino acids.

[0092] Table 1

[0093] definition

[0094] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply.

[0095] Unless the context clearly indicates otherwise, references to a specific number herein include their plural forms. For example, references to “cell” include one or more such cells and equivalents known to those skilled in the art, etc.

[0096] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0097] The term "TCR" as used in this article refers to the T-cell receptor, a transmembrane protein located on T cells. Belonging to the immunoglobulin superfamily of cell surface molecules, it specifically recognizes a peptide major histocompatibility complex (pMHC) complex on antigen-presenting cells (APCs). The interaction between T cells and other cell membrane surface molecules of APCs triggers a series of subsequent cell signaling and other physiological responses, enabling T cells specific to different antigens to exert immune effects on their target cells. The TCR is a heterodimeric protein complex composed of α / β or γ / δ chains, with approximately 95% of T cells being αβ-type T cells composed of α / β chains. The TCR lacks any signaling domains but is non-covalently coupled to the CD3 complex (composed of CD3εγ, CD3εδ, and CD3ζζ dimers), which is responsible for the TCR signaling pathway.

[0098] The term "Chimeric Antigen Receptor (CAR)" as used in this article refers to the core component of chimeric antigen receptor T cells (CAR-T), which may include an antigen-binding domain (e.g., T cell-specific antigens and / or tumor-associated antigens), a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain. Genetically modified T cells expressing CAR can specifically recognize and eliminate malignant cells expressing target antigens in a non-MHC-dependent manner, thus T cell activation is no longer dependent on MHC presentation of antigens. First-generation CARs contain an extracellular antigen-binding domain, a transmembrane domain, and a single intracellular signal transduction domain; second-generation CARs introduce a co-stimulatory molecule, enhancing tumor-killing efficacy; third-generation CARs incorporate multiple co-stimulatory factors, such as CD28, OX40 (CD134), and 4-1BB (CD137); and fourth-generation CARs add selective markers and promoters encoding CAR amplification.

[0099] The term "tumor antigen binding region" as used in this article refers to polypeptides that are selective for tumor-associated antigens. These tumor-associated antigens include, but are not limited to: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, liver glycoside B2, IGF-I receptor, CAIX, LMP2, gp100, and bcr-ab. l, Tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / Galectin8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGFR1, AFP and IGLL1.

[0100] The terms "specific targeting" or "specific binding" as used herein can be used when referring to the interaction between an antibody, protein, or peptide and a second chemical species. This means that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, a peptide may recognize and bind to a specific protein structure while generally recognizing and binding proteins. In some embodiments, the specifically binding peptide is a peptide that recognizes a specific antigen but does not substantially recognize or bind to other molecules in the sample. For example, a peptide that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, this cross-species reactivity itself does not alter the antibody's classification as specific. In some embodiments, a peptide that specifically binds to an antigen may also bind to different allelic forms of that antigen.

[0101] The "splenic mononuclear cells (SMCs)" used in this article include T lymphocytes, B lymphocytes, and monocytes. Their volume, morphology, and density differ from other cells. Red blood cells and polymorphonuclear leukocytes have a higher density, around 1.090, while lymphocytes and monocytes have a density of 1.075–1.090, and platelets have a density of 1.030–1.035. Density gradient centrifugation was performed using a near-isotonic solution (layering solution) with a density between 1.075 and 1.092 to separate cells according to their density gradient.

[0102] In some embodiments, the method for purifying specific antigen-marked T cells from splenic mononuclear cells mainly employs a direct screening method, in which target cells are directly captured, enriched, and separated using antibodies carrying identifiable markers. In other embodiments, the method for purifying specific antigen-marked T cells from splenic mononuclear cells employs an indirect screening method, in which target cells are first captured using biotin-labeled antibodies, and then enriched and separated using streptavidin.

[0103] The term "CD3+ T cells" as used in this article generally refers to T cells that express CD3 on their cell surface. CD3 is an important marker protein on the surface of T lymphocytes and is expressed in all mature T lymphocytes. The CD3 molecule includes εδ, εγ, and ζζ dimers. The γ, ε, and δ chains each contain an immune receptor tyrosine-based activation motif (ITAM). CD3 can bind to the TCR to form the TCR-CD3 complex. When the MHC on the surface of antigen-presenting cells (APCs) binds to the TCR on the surface of T lymphocytes, the TCR-CD3 complex transmits the stimulus signal into the cell, activating tyrosine kinases (PTKs) and causing ITAM phosphorylation. This triggers a cascade of kinase activation, thereby mediating the T lymphocyte's functions of antigen recognition, signal transduction, differentiation, proliferation, and effector activity. CD3 is one of the best markers of mature T lymphocytes in peripheral blood. Measuring CD3+ T lymphocytes is of great significance for evaluating the subtyping and diagnosis of immunodeficiency diseases (T-lymphocytopenia), leukemia, and lymphoma (T-lymphocytic type). Anti-CD3 antibodies can be used for immunosuppressive therapy during organ or bone marrow transplantation, and also for immunomodulatory therapy in severe autoimmune diseases to eliminate T lymphocytes.

[0104] In one embodiment of the present invention, phage display technology (PDT) is used to screen for peptides that specifically target T cells. Phage display technology is a relatively new technology developed in recent years. In 1985, George Smith first inserted a foreign DNA fragment encoding a peptide sequence into gene III of filamentous phage f1, producing a fusion protein, thereby displaying the foreign peptide on the phage surface. Phage conjugates are screened using specific affinity for target molecules. As the virus infects and infects host bacteria, it simulates the natural evolutionary screening process, rapidly identifying peptide sequences that specifically bind to and are enriched with the target. The greatest advantage of PDT technology is that it directly links genotype and phenotype, and the displayed peptides or proteins can maintain relatively independent spatial structures and biological activities, which is beneficial for the recognition and binding of target molecules, allowing for the screening of desired proteins. Phage surface display technology achieves genotype and phenotype conversion, making it a highly efficient screening system.

[0105] Phage display technology typically involves fusing exogenous peptides or proteins with a capsid protein of the phage. The fusion protein is displayed on the surface of the phage particle, allowing for the recognition of specific polypeptide chains displayed on the phage surface through interactions. This technology enables the rapid and efficient sorting of large libraries composed of selectively randomized protein variants (or randomly cloned cDNAs) to find sequences that bind with high affinity to target molecules. Phage display establishes a direct link between a large number of random peptides and their DNA coding sequences, allowing for the rapid identification of peptide ligands for various target molecules (antibodies, enzymes, cell surface receptors, etc.) through an in vitro selection procedure called panning. The simplest panning procedure involves co-incubating the phage-displayed peptide library with plates (or magnetic beads) coated with the target molecule, washing away unbound phages, and then eluting specifically bound phages. The eluted phages are amplified, and then another round of binding / amplification cycles is performed to enrich the binding sequences. After 3-4 rounds of panning, each binding clone is identified by DNA sequencing. The successful display of antibodies using phage display technology has enabled its application to other immune proteins, including TCR. The specific peptide library presented in this paper can be displayed using any suitable phage display system. For example, cyclic peptide libraries can be displayed using single-chain filamentous phage display systems, λ phage display systems, T4 phage display systems, or T7 phage display systems.

[0106] The term "phage vector" as used in this article refers to a double-stranded phage containing a heterologous gene and capable of replication. A phage vector possesses an origin of phage replication, thereby allowing phage replication and phage particle formation. Phages are mainly classified into two categories: double-stranded phages and single-stranded filamentous phages. Double-stranded phages are classified as λ-type phages, while single-stranded filamentous phages include M13, f1, and fd phages. The preferred phages are filamentous phages, such as M13 phage or its derivatives. λ-type phages include, but are not limited to, phi80, phage 21, 82, 424, 432, lambda.imm343, lambda.imm21, lambda.EMBL, or lambdab.gt, or all their derivatives, genetically engineered derivatives, and hybrids. In some preferred embodiments, M13 phage is used. Phage vectors are categorized based on several factors: expression system (phage-derived vector expression peptide libraries and phageparticle vector expression peptide libraries); fusion location of the peptide with the coat protein (gpI phage peptide libraries, gpV cyclic phage peptide libraries, etc.); peptide length (6-peptide libraries, 8-peptide libraries, 15-peptide libraries, etc.); peptide valence (monovalent phage peptide libraries and multivalent phage peptide libraries); conformation (linear phage peptide libraries and conformational phage peptide libraries); and sequence diversity (partially random peptide libraries and randomly random peptide libraries). In some implementations, a phage-displayed random cyclic heptapeptide library (Ph.D-C7C) is used. TM Phage Display Peptide Library Kit. In some implementations, a random dodecapeptide library (Ph.D-12) is displayed using phage. TM Phage Display Peptide Library Kit).

[0107] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein consisting of two or more amino acids linked together by peptide bonds. As used herein, it refers to both short chains (often referred to as, for example, peptides, oligopeptides, and oligomers) and longer chains (often referred to as proteins). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. In some embodiments, the conformation of each amino acid in a specific polypeptide is independently selected from either the D or L conformation. For example, in the polypeptide shown in SEQ ID NO: 3 (Trp-Ser-Trp-Ser-Leu-Ser-Ser-Gly-Tyr-Ala-Asp-Val, abbreviated as WWSLSSGYADV), each amino acid is either in the D configuration or the L configuration, preferably in the L configuration.

[0108] As used in this article, "a nucleic acid molecule encoding...", "a DNA sequence encoding...", or "DNA encoding..." refers to the sequence of deoxyribonucleotides along a deoxyribonucleic acid (DNA) chain. These deoxyribonucleotide sequences determine the sequence of amino acids along the polypeptide chain. Therefore, the DNA sequence encodes these amino acid sequences.

[0109] As used herein, “encoding” refers to the inherent properties of a specific nucleotide sequence in a polynucleotide such as a gene, complementary DNA (cDNA), or messenger RNA (mRNA) that serves as a template for other polymers and macromolecules having defined nucleotide (i.e., ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA)) sequences or defined amino acid sequences in biological processes, and the resulting biological properties. Thus, a gene encodes a protein—if the transcription and translation of the mRNA corresponding to that gene produces that protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand that serve as the transcription template for a gene or cDNA can be referred to as the protein or other product encoding that gene or cDNA.

[0110] In some embodiments, the polypeptides or fusion proteins of the present invention can be generated entirely or partially by chemical synthesis. They can be readily prepared according to standard liquid-phase peptide synthesis methods or preferred solid-phase peptide synthesis methods, which are well known in the art. Alternatively, they can be prepared in solution by liquid-phase methods, or by any combination of solid-phase, liquid-phase, and solution chemistry methods, for example by first completing the individual peptide moieties and then, as needed and as appropriate, introducing residue X by reacting with various carbonic or sulfonic acids or their reactive derivatives after removing any existing protecting groups. In some embodiments, the polypeptides or fusion proteins can be generated by expression in an expression system using nucleic acid molecules encoding the polypeptides or fusion proteins.

[0111] As used herein, "isolated" refers to the preferred state of the polypeptides, fusion proteins, or nucleic acids encoding the polypeptides, fusion proteins, or nucleic acids of the present invention. The polypeptides, fusion proteins, or nucleic acids will generally be free of or substantially free of materials naturally associated with them, such as other polypeptides or nucleic acids in their natural environment, or other polypeptides or nucleic acids found in the environment in which they are prepared (e.g., cell culture medium) when the preparation is carried out by recombinant DNA technology performed in vitro or in vivo. The polypeptides, fusion proteins, or nucleic acids may be formulated with diluents or adjuvants, but are still isolated for practical purposes—for example, polypeptides or fusion proteins are typically mixed with gelatin or other carriers if used to coat microtiter plates for immunoassay, or mixed with pharmaceutically acceptable carriers or diluents when used for diagnostic or therapeutic purposes. The polypeptides may be naturally glycosylated or glycosylated via a heterologous eukaryotic cell system, or they may be (e.g., if produced by expression in prokaryotic cells) unglycosylated.

[0112] The term "primer" as used in this article refers to a natural or synthetic nucleotide sequence that, when combined with a polynucleotide template to form a double helix, serves as the starting point for nucleic acid synthesis and extends from its 3' end along the template, thereby forming an extended double helix. The nucleotide sequence added during the extension process is determined by the sequence of the template polynucleotide.

[0113] The term "sequencing" as used in this article refers to determining the sequence of nucleotides (base sequences) in a nucleic acid sample (such as DNA or RNA). Compared to first-generation sequencing (Sanger sequencing), next-generation sequencing (NGS) or high-throughput sequencing can sequence hundreds of thousands to millions of DNA molecules simultaneously. NGS sequencing includes second-generation sequencing (SGS) and third-generation sequencing (TGS). SGS technology is suitable for obtaining short read lengths, while third-generation sequencing (TGS) can obtain longer read lengths. Methods used for second-generation sequencing include sequencing-by-synthesis (SBS) and sequencing by ligation (SBL). SBS methods include pyrosequencing, sequencing by reversible terminators, and sequencing by the detection of hydrogen ions. Various commercial next-generation sequencing platforms, including but not limited to, such as the GS FLX sequencing platform (454Life Sciences / Roche Diagnostics), HiSeq, MiSeq, and NextSeq sequencing platforms (Illumina), SOLiD sequencing platform (ABI), and Ion Torrent PGM. TM and Ion Torrent Proton TM Sequencing platforms (Thermo Fisher); various commercial sequencing platforms available for third-generation sequencing, Helicos TM Genetic Analysis System (SeqLL, LLC), SMRT Sequencing (Pacific Biosciences), Nanopore Sequencing Platform (Oxford Nanopore).

[0114] In some implementations, prior to sequencing, the mutant region of the specific polypeptide is amplified from the autophage, preferably using polymerase chain reaction (PCR). The sequence and length to be amplified are determined based on the sequencing method and the mutant region to be analyzed.

[0115] In some implementations, after amplifying a nucleic acid molecular library from the bacteriophage, sequencing can be performed using methods known in the art. For example, bridge PCR next-generation sequencing can be used. Alternatively, commercially available deep sequencing platforms, such as the Illumina MiSeq sequencing platform, can be employed.

[0116] After sequencing, multiple sequence alignment and sequence clustering analysis can be performed to determine the enrichment level of the sorted population and the abundance of different sequences in the population.

[0117] The term "alignment" as used in this article refers to the comparison of two or more nucleotide sequences based on the presence of short or long sequences with the same or similar nucleotides. Multiple sequence alignment (MSA) is a method that aligns two or more character sequences and compares their characters column by column to make each column as consistent as possible in order to discover their common structural features. The MSA software used in this article includes, but is not limited to, Muscle, MAFFT, ClustalW, and T-coffee.

[0118] The term "cluster analysis" as used in this article refers to a statistical method for classifying research objects corresponding to data. It involves dividing a set of individuals into clusters according to certain criteria, aiming for samples within a cluster to be as similar as possible, while clusters should be as dissimilar as possible. In some implementations, cluster analysis includes converting the nucleic acid sequences obtained from sequencing into amino acid sequences and calculating the frequency of each polypeptide sequence or specific amino acid. Commonly used cluster analysis methods include, but are not limited to, hierarchical clustering, K-means clustering, fuzzy clustering, graph-based clustering, dynamic clustering, spectral clustering, and self-organizing map neural networks. In some implementations, hierarchical clustering is used. In some implementations, UPGMA (Unweighted Pair-group Method with Arithmetic Means) is used to construct a UPGMA phylogenetic tree, or Neighbor Joining (NJ) is used to construct an NJ phylogenetic tree, and / or the Bayesian clustering method in STRUCTURE software is used for cluster analysis.

[0119] As used in this article, “identity” refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing sequences. Percentage identity is determined by the number of identical amino acid residues or nucleotides in the compared sequences (i.e., % identity = number of identical positions / total number of positions × 100).

[0120] As used in this article, "modification" refers to structural alterations to peptides or proteins through chemical or enzymatic methods to enhance their stability, biological activity, solubility, or other properties. These modifications can include chemical modifications of amino acid side chains, terminal modifications, cyclization, PEGylation (polyethylene glycolization), alkylation, biotinylation, D-amino acid substitution, etc.

[0121] C-terminal modifications, including amidation and esterification, can alter the charge state of a polypeptide, thereby affecting its interaction with other molecules.

[0122] N-terminal modifications, including acetylation and methylation, can enhance the stability of peptides, prevent their degradation, and alter their biological activity.

[0123] Intermediate residue modifications include glycosylation, phosphorylation, fatty acid modification, and other labeling. Glycosylation involves introducing sugar molecules into the polypeptide chain via enzymatic reactions, increasing its immunogenicity and pharmacological activity. For example, adding glycosyl groups to amino acid residues such as Ser, Tyr, and Thr can alter protein stability, activity, and localization. Phosphorylation involves adding phosphate groups to hydroxyl-containing amino acid residues such as Ser, Tyr, and Thr. Phosphorylation can change the charge properties of proteins, thus affecting their interactions with other molecules and regulating protein activity. Fatty acid modification involves adding fatty acid chains to the side chains of certain amino acids (such as lysine), which can alter the lipophilicity and cell penetration ability of polypeptides. Labeling is well known to those skilled in the art and can be any desired structure. Preferably, such labels can be selected from any known detectable groups, such as dyes, luminescent labeling groups such as chemiluminescent groups, or fluorescent dyes such as fluorescein, coumarin, and their derivatives.

[0124] Cyclization includes head-and-tail cyclization and side-chain cyclization. Head-and-tail cyclization refers to forming a cyclic structure by connecting the N-terminus and C-terminus of a polypeptide, which can improve the stability and biological activity of the polypeptide. Side-chain cyclization refers to forming a cyclic structure by connecting functional groups on two side chains (such as the ε-amino group of lysine and the γ-carboxyl group of glutamic acid), which can enhance the rigidity and selectivity of the polypeptide.

[0125] PEGylation refers to the addition of polyethylene glycol (PEG) molecules to one or more sites on a peptide. By reacting activated PEG derivatives (such as maleimide-PEG) with specific amino acids (such as cysteine) in the peptide, the half-life of the peptide can be prolonged, immunogenicity reduced, and solubility improved.

[0126] As used in this article, “delivery” means the act or manner of delivering a compound, substance, entity, part, carrier, or payload.

[0127] As used in this article, "liposome" refers to a typically spherical aggregate of amphiphilic compounds, usually existing as one or more concentric layers, such as a bilayer. Liposomes have been extensively studied for drug delivery. Hydrophilic drugs can be encapsulated in the aqueous interior region of the liposome, while hydrophobic drugs can be encapsulated in the hydrocarbon chain region of the lipid bilayer. The characteristics and behavior of liposomes in vivo can be modified by adding a hydrophilic polymer coating, such as polyethylene glycol (PEG), to the surface of the liposome to impart steric stability. Furthermore, liposomes can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or the ends of the attached PEG chains.

[0128] In some embodiments, lipid nanoparticles (LNPs) refer to particles having at least one nanometer (nm) size (e.g., 1 to 1,000 nm) containing one or more types of lipid molecules (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids).

[0129] Cationic lipids can be, for example, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium chloride propane (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleoyloxy-3-trimethylaminopropane chloride salts), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dilinoleoyloxy-N,N-dimethylamino1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl... 3-Dimethylaminopropane (DLinDAP), 1,2-Dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoxy-3-trimethylaminopropane chloride (DLin-TMA.CL), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.CL), 1,2-dilinoleoyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), or 3-(N,N-dilinoleoamino)-1,2-propanediol (DLinAP), 3-(N,N-dilinoleoamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM) A) 2,2-Dilinyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadec-9,12 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazinediyl)bisdodecane-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyldimethylaminomethyl-[1,3]-dioxolane (DLin-DMA), (6Z,9Z,28Z,31Z)-heptantriane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-M-C3-DMA).Other cationic lipids include, but are not limited to, N,N-distearate-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1-(2,3-dioleoyloxy)propyl)-N(sperminecarbamoyl)ethyl)-N,N-dimethyltrifluoroacetate ammonium (DOSPA), bis(octadecylaminoglycylcarboxyspermine) (DOGS), 1,2-dioleoyl-sn-3-phosphate ethanolamine (DOPE), and 1,2-dioleoyl-3-dimethylpropane ammonium (DODAP). Additionally, commercial formulations of cationic lipids, such as Lipofectin, can be used. TM (Contains DOTMA and DOPE, available from Thermo Fisher Scientific) and Lipofectamine TM (Includes DOSPA and DOPE, available from Thermo Fisher Scientific).

[0130] In some embodiments, non-cationic lipids, such as neutral lipids, anionic lipids, or amphiphilic lipids, may be used. Neutral lipids can be any of many lipid species present at physiological pH in an uncharged or neutral zwitterionic form. Such lipids include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for the particles described herein is generally guided by considerations such as LNP size and LNP stability in blood flow. Preferably, neutral lipids are lipids having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine). In some embodiments, neutral lipids contain saturated fatty acids with carbon chain lengths in the C10 to C20 range. In other embodiments, neutral lipids having monounsaturated or diunsaturated fatty acids with carbon chain lengths in the C10 to C20 range are used. Alternatively, neutral lipids having a mixture of saturated and unsaturated fatty acid chains may be used. Suitable neutral lipids include, but are not limited to, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), and dimyristoylphosphatidylethanolamine (DMPE). Anionic lipids suitable for LNP include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups linked to neutral lipids.

[0131] In some embodiments, the polyethylene glycol (PEG) modified lipids include, but are not limited to, PEG-diacylglycerol (DAG), PEG-dialkylglycerol, PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof (e.g., PEG-Cer14 or PEG-Cer20). PEG-DAA conjugates may be, for example, PEG-dilauoxypropyl (C12), PEG-dimyristoxypropyl (C14), PEG-dispalmitoxypropyl (C16), or PEG-distearateoxypropyl (C18). Other PEGylated lipids include, but are not limited to, polyethylene glycol-dimyristoylglycerol (C14-PEG or PEG-C14, wherein the PEG has an average molecular weight of 2000 Da) (PEG-DMG), (R)-2,3-bis(octadecoxy)propyl-1-(methoxy polyethylene glycol)2000)propylcarbamate (PEG-DSG), PEG-carbamoyl-1,2-dimyristoyloxypropylamine, wherein the PEG has an average molecular weight of 2000 Da (PEG-cDMA), N-acetylgalactosamine-(R)-2,3-bis(octadecoxy)propyl-1-(methoxy polyethylene glycol)2000)propylcarbamate (GalNAc-PEG-DSG), mPEG(Mw2000)-distearatephosphatidylethanolamine (PEG-DSPE), and polyethylene glycol-dipalmitoylglycerol (PEG-DPG).

[0132] The total amount of at least one reagent, particularly one or more preventative or therapeutic drugs, encapsulated in lipid nanoparticles varies and can be defined, for example, by the w / w ratio of nucleic acids to total lipids. In some embodiments of the invention, the nucleic acid to total lipid ratio is less than 0.06 w / w, preferably from 0.03 w / w to 0.04 w / w.

[0133] In some embodiments, the LNP has a median diameter size of about 50 nm to about 300 nm, such as about 50 nm to about 250 nm, for example, about 50 nm to about 200 nm. In some embodiments, smaller LNPs can be used. Such particles can include diameters from 0.1 pm to 100 nm. In other embodiments, smaller LNPs can be used to deliver at least one reagent, for example, with diameters of about 1 nm to about 100 nm, about 1 nm to about 20 nm, about 1 nm to about 40 nm, about 1 nm to about 60 nm, about 1 nm to about 80 nm, about 5 nm to about 100 nm, about 10 nm to about 50 nm, about 20 nm to about 50 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 40 nm to about 60 nm, about 20 nm to about 70 nm, about 50 nm to about 70 nm, about 60 nm to about 70 nm, about 30 nm to about 80 nm, or about 20 nm to about 90 nm. In some implementations, the LNP may have a diameter greater than 100 nm, greater than 200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, greater than 600 nm, greater than 700 nm, greater than 800 nm, greater than 900 nm, or greater than 1000 nm.

[0134] The term "micelle" as used in this article refers to a multifunctional drug carrier that is self-assembled from an amphiphilic copolymer. By controlling the structure and properties of the polymer, polymer micelles can load hydrophobic drugs through non-covalent interactions or through covalent bonding.

[0135] The term "exosomes" (Exo) used in this article refers to naturally occurring nanoscale microvesicles (or "nanoparticles") synthesized and secreted by living cells. They range in size from 30 to 150 nm and consist of a lipid bilayer containing transmembrane proteins, cytoplasmic proteins, and various nucleic acids. Exosomes can regulate the biological activity of recipient cells through the proteins, nucleic acids, lipids, etc., they carry.

[0136] In practical use, those skilled in the art can select appropriate markers based on detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0137] In some embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0138] In some embodiments, the radioactive markers include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186Re、211At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0139] In some embodiments, the "medicine for the prevention or treatment of disease" of the present invention is not limited to any specific drug, but covers any suitable drug that may be included in the delivery system. The drug includes, but is not limited to: antiviral agents, antibacterial agents, antioxidants, thrombolytic agents, chemotherapeutic agents, anti-inflammatory agents, immunogenic agents, anesthetics, analgesics, pharmaceutical reagents, small molecule drugs, peptides, nucleic acids, etc.

[0140] In some embodiments, the imaging agent is a material that allows the delivery system to image cells or tissues after exposure. Imaging includes imaging for the naked eye, as well as imaging that requires instrumental detection or the detection of information typically invisible to the eye, and includes imaging that requires the detection of photons, sound, or other energy quanta. Examples include, but are not limited to: staining agents, in vivo dyes, fluorescent markers, radiolabeled substances, enzymes, or plasmid constructs encoding markers or enzymes. Molecular imaging-based imaging typically involves detecting biological processes or biomolecules at the tissue, cellular, or molecular level. Molecular imaging can be used to evaluate specific targets for gene therapy, cell-based therapies, and as a diagnostic or research tool to image pathological conditions. Imaging agents capable of intracellular delivery are particularly useful because these agents can be used to evaluate intracellular activity or conditions. Imaging agents must effectively reach their targets. In some embodiments, the imaging agent is a magnetic resonance imaging contrast agent. In some embodiments, the imaging agent is an X-ray contrast agent.

[0141] As used herein, "pharmaceutically acceptable carriers, diluents, and / or excipients" refers to at least one component that may be included in a pharmaceutical composition in addition to the active molecule. These may be materials known to those skilled in the art, and should be non-toxic and not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral or injectable, such as intravenous. The composition may also be administered via delivery systems such as microspheres, liposomes, lipid nanoparticles, etc., as described herein, or as a sustained-release formulation placed in certain tissues (including blood). The composition may also be applied topically to the desired site or delivered in a manner that targets relevant cells.

[0142] The pharmaceutical compositions of the present invention can be administered in a manner suitable for treating (or preventing) the disease. The amount and frequency of administration are determined by the attending physician based on the patient's condition, the type and severity of the disease.

[0143] Depending on the disease to be treated, the pharmaceutical composition of the present invention can be administered alone or together with other treatments, and can be administered simultaneously or sequentially.

[0144] Experiments have confirmed that by using the method of this invention and phage surface display peptide library technology, a highly diverse and complex peptide library can be constructed, covering many potential T cell receptor (TCR) binding sites. Simultaneously, by utilizing deep sequencing and cluster analysis to select high-frequency sequences (i.e., dominant amino acid sequences) from the sorted population, and preferably further combining this with the detection of dominant amino acid residues, peptides that specifically bind to T cells can be accurately screened, reducing non-specific binding.

[0145] The inventors further validated the screened peptides comprehensively, confirming their ability to effectively activate or regulate the function, proliferation, and effector properties of T cells. Validation using in vitro and in vivo models yielded identification results for peptide sequences that met the desired binding properties in multiple aspects. Preclinical studies in animal models showed that these peptides possess high affinity and specific targeting, avoiding excessive immune responses in the body. The method of this invention is simple, easy to operate, and highly reproducible, and can be standardized into a peptide screening process, providing important support for advancements in the field of immunotherapy.

[0146] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0147] Example

[0148] The process for screening peptide-targeted T cells is shown in Figure 1. Animal experiments were conducted strictly in accordance with regulations governing laboratory animals, and the experimental procedures met ethical requirements.

[0149] Example 1. Sorting of mouse CD3+ T cells

[0150] (1) Isolation of mouse spleen mononuclear cells

[0151] Mice were humanely euthanized by cervical dislocation (purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.), and the spleen was quickly removed in a sterile laminar flow hood, with the surrounding connective tissue removed.

[0152] The spleen was chopped and ground, placed in a 40μm cell sieve, and 3mL of PBS was added to submerge the spleen.

[0153] Take the plunger of a 5mL sterile syringe and use the flat end of the plunger to gently grind the spleen tissue in a circular motion until no obvious red clumps remain.

[0154] Gently pipette the cell suspension to further disperse any remaining clumps and ensure a uniform single-cell suspension.

[0155] In a sterile test tube, first add an appropriate amount of Ficoll separation solution. Carefully tilt the test tube and slowly add the prepared cell suspension along the tube wall, being careful to maintain a clear interface.

[0156] Perform horizontal centrifugation at room temperature, 2000 rpm, for 20 minutes, and carefully aspirate the central mononuclear cells (white membrane layer).

[0157] Transfer the collected mononuclear cells to a new test tube and add 10 mL of PBS and mix well.

[0158] Centrifuge at 300g for 5 minutes and discard the supernatant.

[0159] (2) Mouse T cell sorting

[0160] Resuspend the cell pellet with sorting buffer and adjust the cell density to 1×10⁻⁶. 8 cells / mL.

[0161] Transfer the cell suspension to the bottom of a sterile flow cytometry tube, add 2 μL of Biotin-antibody Mix to every 100 μL of cell suspension, and mix gently. Incubate at 4°C for 10 minutes.

[0162] After incubation, add 20 μL of Streptavidin beads to every 100 μL of cell suspension. Mix gently and continue incubation at 4°C for 10 minutes.

[0163] Add 2.5 mL of sorting buffer to the flow cytometry tube and gently pipette five times to ensure thorough mixing. Place the flow cytometry tube containing cells on a magnetic rack and let it stand for 5 minutes.

[0164] Carefully pour the cell suspension into a new sterile centrifuge tube, avoiding contact with the tube wall. Centrifuge at 500g for 5 minutes, discard the supernatant, and collect the purified T cells.

[0165] The purity of sorted T cells was determined using flow cytometry, and cell viability was assessed using trypan blue staining. Typically, purity should be above 93%, and viability above 90%. The purity test results are shown in Figure 2, which indicates a CD3 positivity rate of 97.41%. Trypan blue staining showed T cell viability greater than 90%.

[0166] Example 2.12 Screening of T cells targeting peptide libraries

[0167] High-purity T cells isolated in vitro were co-incubated with a 12-peptide display library (NEB, #E8210S) to allow the peptides displayed on the surface of the phage to bind to the T cells.

[0168] (1) Co-incubation of peptides with T cells

[0169] The purified T cells were resuspended in 300 μL of a 12-peptide display library (2 × 10⁻⁶). 10 Mix gently with pfu. Incubate at 4°C for 30 minutes.

[0170] Add 5 mL of elution buffer (PBS + 0.5% BSA + 0.05% Tween 20), centrifuge at 500 g for 5 minutes, and discard the supernatant. Repeat the washing process 3 times.

[0171] Resuspend the cells in 1 mL of L-lycine-HCl (0.2 M, pH 2.5) and gently shake for 10 minutes at room temperature. Centrifuge at 500 g for 5 minutes, collect the supernatant into a new centrifuge tube, add 110 μL of Tris-HCl (1 M, pH 9.0) to neutralize, and mix thoroughly. This is the elution buffer for phages bound to the surface of T cells.

[0172] (2) Amplification and purification of bacteriophages

[0173] Take 1 / 5 of the eluted specific polypeptide for amplification. Prepare host bacteria E. coli ER2738 plates from glycerol bacterial culture, and pick single colonies to inoculate into LB+Tet medium.

[0174] Culture at 37℃ and 250rpm for 12-16 hours until the logarithmic growth phase.

[0175] Take 200 μL of the overnight cultured host bacteria and inoculate it into 20 mL of LB medium (250 mL Erlenmeyer flask), and add phage elution buffer.

[0176] Amplification was performed by incubating at 37℃ and 250rpm for 4.5-5 hours with shaking.

[0177] Centrifuge at 10000g for 15 minutes at 4℃, collect the supernatant, and centrifuge again to remove the precipitate.

[0178] Take 80% of the supernatant from the top layer, add 1 / 5 volume of 20% PEG / 2.5M NaCL, mix well, and precipitate on ice for 2 hours or overnight.

[0179] Centrifuge at 12000g, 4℃ for 15 minutes, discard the supernatant, add 1mL TBS to resuspend the precipitate, and transfer to a 1.5mL centrifuge tube.

[0180] Centrifuge at 20000g, 4℃ for 1 minute, and collect the supernatant into a new centrifuge tube. Add 1 / 5 volume of 20% PEG / 2.5M NaCl, mix well, and precipitate on ice for 15 minutes.

[0181] Centrifuge at 20000g, 4℃ for 10 minutes, discard the supernatant, and resuspend the precipitate in 200μL TBS.

[0182] Centrifuge at 20000g, 4℃ for 1 minute, and collect the supernatant, which is the amplified phage fluid.

[0183] (3) Phage titer determination

[0184] Prepare the host bacterium E. coli ER2738 in advance during its logarithmic growth phase.

[0185] Preheat the LB / IPTG / X-Gal plate in a 37°C incubator for at least 30 minutes.

[0186] Prepare diluted elution buffer containing bacteriophage or amplified bacteriophage. The dilution range for the elution buffer is 10. 2 -10 5 The amplified phage was diluted in a range of 10. 9 -10 12 .

[0187] Add 200 μL of bacterial culture to each EP tube, and add 10 μL of phage fluid at different dilutions to each tube. Vortex to mix and let stand for 1-5 minutes.

[0188] Add the incubated phage solution to 3 mL of pre-warmed top-layer agar medium (42-50℃), vortex to mix, and quickly pour onto LB / IPTG / X-Gal plates. Let stand for 5 minutes, then invert the plates and incubate overnight at 37℃. Potency was assessed; the blue spot potency was approximately 10. 10PFU and glycerol are stored for the next round of screening. The above steps are repeated 2-3 times. Each round of phage is amplified using the elution buffer from the previous round, resulting in the enrichment of phages that bind to the target cells. After 2-3 rounds of "adsorption-elution-amplification," peptides that specifically bind to T cells are highly enriched.

[0189] Example 3. Analysis and identification of specific peptides

[0190] (1) Selection of monoclonal phage spots:

[0191] Titer determination was performed on the phage eluent obtained after the third round of screening. On plates with fewer than 100 plaques, single plaques that were well-separated, in good growth condition, and blue in color were randomly selected. Each selected plaque was added to a culture tube containing 1 mL of logarithmic-phase host bacterial culture and incubated at 37°C and 250 rpm with shaking for 4.5 hours.

[0192] The amplified phage suspension was centrifuged, and the supernatant was divided into two parts. One part was stored in a 4°C refrigerator for later use, and the other part was used to extract phage DNA.

[0193] (2) DNA extraction:

[0194] Take the phage supernatant from the DNA extraction, add 200 μL of 20% PEG / 2.5M NaCl solution, mix well, and let stand at room temperature for 15 minutes.

[0195] Centrifuge at 10000g for 10 minutes at 4℃, discarding the supernatant. After a brief second centrifugation, carefully remove any remaining supernatant.

[0196] Resuspend the precipitate in 100 μL of iodide buffer, then add 250 μL of anhydrous ethanol, mix well, and let stand at room temperature for 10 minutes.

[0197] Centrifuge 10000g at 4℃ for 10 minutes and discard the supernatant.

[0198] Add 500 μL of 70% anhydrous ethanol, centrifuge at 10000 g and 4 °C for 10 minutes, and discard the supernatant. After a brief centrifugation, carefully remove any remaining supernatant.

[0199] Let the sample stand at room temperature for 5 minutes to allow the ethanol to evaporate completely. Finally, add 30 μL of ddH2O to resuspend the DNA precipitate.

[0200] (3) PCR amplification of polypeptide sequences:

[0201] Design a pair of primers with the following sequences:

[0202] Upstream primer: 5′-GGCGATGGTTGTTGTCATTG-3′ (SEQ ID NO:1)

[0203] Downstream primer: 5′-CCCTCATAGTTAGCGTAACG-3′ (SEQ ID NO:2)

[0204] This primer pair can amplify a PCR product of about 300 bp, including the variable region of phage 12 peptide.

[0205] PCR amplification was performed using extracted monoclonal phage DNA as a template, and the PCR products were sent for sequencing.

[0206] After obtaining the sequencing results, bioinformatics tools were used for multiple sequence alignment and cluster analysis, frequency statistics, and structure prediction to screen for high-frequency peptide sequences with potential functions. In addition to using multiple sequence alignment methods such as ClustalW or MUSCLE, cluster analysis methods such as UPGMA or Neighbor-Joining were performed to calculate the frequency of each peptide sequence or specific amino acid, and to predict the secondary and tertiary structures of the peptides, identifying potential functional regions of the peptides and providing important evidence for subsequent experimental validation and application research.

[0207] Sequencing results of 114 DNA sequences showed that the polypeptide sequences with high repetition frequencies were 5## (VTYNWSLLAGYV, SEQ ID NO:4), 4## (WSWSLSSGYADV, SEQ ID NO:3), 14## (TSGTMQTNPLPV, SEQ ID NO:6), 25## (STWSLYAGYTHN, SEQ ID NO:7), 43## (NSVHVYHKSFLF, SEQ ID NO:8), 10## (TERTMESMTRFA, SEQ ID NO:9), 41## (YTWTLERGYSVN, SEQ ID NO:5), 56## (FSVPSTPRTVVV, SEQ ID NO:10), and 83## (EWKVLEGHTTRD, SEQ ID NO:8). NO:11) appeared at frequencies of 33.33%, 14.91%, 9.65%, 6.14%, 3.51%, 1.75%, 1.75%, 1.75%, and 1.75% respectively (as shown in Figure 1).

[0208] Example 4. In vitro validation of specific peptide targeting T cells

[0209] The phage supernatant of the candidate peptide was further amplified and purified to achieve a titer of 2 × 10⁻⁶. 10 pfu / mL or higher.

[0210] Mice were humanely euthanized via cervical dislocation, and spleen mononuclear cells and CD3+ T cells were isolated in vitro under sterile conditions. T cells were then isolated using an immunomagnetic bead negative sorting kit.

[0211] Splenic mononuclear cells and CD3+ T cells were divided into several portions, each containing 1×102 cells. 6 Cells were collected, and the supernatant was removed after centrifugation.

[0212] Each cell pellet was treated with 100 μL of peptide phage suspension (2 × 10⁻⁶). 10 Resuspend the pfu and incubate on ice for 30 minutes.

[0213] Add 1 mL of washing buffer, centrifuge at 500 g and 4 °C for 5 minutes, and remove the supernatant. Repeat washing 3 times to remove unbound phages.

[0214] Resuspend the cells in 30 μL of PBS buffer containing antibodies such as anti-M13-FITC and anti-CD3-PE, and incubate at 4°C in the dark for 15 minutes.

[0215] Add 1 mL of washing buffer, centrifuge at 500 g and 4 °C for 5 minutes, and remove the supernatant. Repeat the washing process 3 times.

[0216] Cells were resuspended in 300 μL PBS + 0.5% BSA and analyzed by flow cytometry to determine the percentage of M13-positive T cells. This percentage was used as the positive percentage of peptide binding to T cells. The results are shown in Figure 3. As can be seen from Figure 3, compared with the empty vector phage and non-targeted peptide phage groups, peptide sequences 4##, 5##, and 41## could specifically bind to mouse T cells, with binding rates of 18.6%, 31.3%, and 45.6%, respectively. These three peptides will be further validated in vivo. The binding rates of the remaining peptides to T cells were approximately 1-5% (results not shown).

[0217] Example 5. In vivo validation of specific peptide targeting T cells

[0218] Based on the results of in vitro validation, positive phage peptides targeting T cells were screened, further amplified and purified to achieve a titer of 2 × 10⁻⁶. 10 pfu / mL or higher. Its distribution, targeting, and safety were validated in mice.

[0219] 200 μL of the corresponding phage suspension (2 × 10⁻⁶) was injected via tail vein. 10 PFU was injected into C57BL / 6 mice (purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.) to allow the phage to circulate in vivo for 2 hours, with non-targeting peptide phage as a negative control (Ctrl).

[0220] After removing the eyeballs to collect blood, the mice were euthanized humanely by cervical dislocation. The spleen and bone marrow were removed, the surrounding tissues were removed, and the surface blood was washed away with PBS.

[0221] Various organizational processes:

[0222] Spleen: Grind the spleen using the flat end of a syringe in a gentle circular motion, then filter it through a 40μm cell sieve to obtain a single-cell suspension;

[0223] Bone marrow: Cut off both ends of the femur with a scalpel or sharp scissors, ensuring the incision is clean. Use a 2mL syringe to aspirate complete culture medium to flush out the bone marrow, and then filter it through a 40μm cell sieve to obtain a single-cell suspension.

[0224] Resuspend the blood in PBS to 10 mL.

[0225] Mononuclear cells were isolated from the cell suspension using Ficoll separation solution.

[0226] The mononuclear cells of each tissue were divided into 1×10 portions. 6 After centrifuging and removing the supernatant, the cells were resuspended in 30 μL of PBS buffer containing antibodies such as anti-M13-FITC and anti-CD3-PE, and incubated at 4°C in the dark for 15 minutes.

[0227] Add 1 mL of washing buffer, centrifuge at 500 g and 4 °C for 5 minutes, and discard the supernatant. Repeat the washing process 3 times.

[0228] Cells were resuspended in 300 μL PBS + 0.5% BSA and analyzed by flow cytometry to detect the percentage of M13-positive T cells. The proportion of M13 cells in different tissues was compared to further evaluate the specific distribution of the phages in vivo and their targeting ability to T cells. The results are shown in Figure 4. Figure 4 shows that, compared with the negative control group (Ctrl), peptides 4##, 5##, and 41## specifically bound to CD3+ positive T cells in mouse bone marrow, spleen, and blood, while peptides 4##, 5##, and 41## bound less to CD3+ positive T cells in other tissues.

[0229] Example 6. Validation of specific peptide-modified lipid nanoparticles in Cre-loxP mice

[0230] Based on the results of in vitro and in vivo validation of phage peptides, candidate specific peptide-modified lipid nanoparticles were prepared.

[0231] 15 μg of specific peptide-modified lipid nanoparticles were injected into Cre / loxP mice (purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.) via tail vein for 72 hours of in vivo expression. Non-targeted peptide-modified lipid nanoparticles were used as a negative control (Ctrl). When the Cre enzyme in Cre / loxP mice was cleaved by the targeted mRNA, the expression of tdTomato red fluorescent protein was activated, which facilitated detection.

[0232] Mice were euthanized by cervical dislocation, and major organs such as the spleen and kidneys were removed. AniView Pro small animal live imaging was used to take pictures and observe the expression of tdTomato red fluorescence in each tissue.

[0233] Each tissue was ground to prepare a single-cell suspension, and mononuclear cells were separated from each cell suspension using Ficoll separation solution.

[0234] The mononuclear cells of each tissue were divided into 1×10 portions. 6 After centrifuging and removing the supernatant, the cells were resuspended in 30 μL of PBS buffer containing antibodies such as anti-CD3-AF647 and incubated at 4°C in the dark for 15 minutes.

[0235] Add 1 mL of washing buffer, centrifuge at 500 g and 4 °C for 5 minutes, and discard the supernatant. Repeat the washing process twice.

[0236] Cells were resuspended in 300 μL PBS + 0.5% BSA and analyzed by flow cytometry to detect the percentage of CD3+ positive cells in tdTomato+ cells. The results are shown in Figure 5. The in vitro tissue fluorescence imaging results of small animals in the figure show that LNP particles conjugated with peptides 4##, 5##, and 41## were mainly delivered to the spleen, with peptide 41## showing the strongest fluorescence signal (A). Flow cytometry analysis of spleen single-cell suspensions showed that the CD3+ positivity rate targeted by peptides 4##, 5##, and 41## was greater than 20%, proving that more than 20% of CD3+ positive cells could be targeted and delivered (B).

[0237] This invention utilizes phage display technology to construct a highly diverse peptide library, ensuring coverage of a broad sequence space potentially binding to T-cell receptors (TCRs), thereby improving the effectiveness and specificity of screening. Precise screening methods identify peptides that specifically bind to T cells, avoiding non-specific binding, and repeatedly verify the affinity between peptides and TCRs to determine the stability and efficiency of binding. Further validation of the specificity and affinity of the screened peptides ensures their effective binding to target T cells, avoiding non-specific reactions or side effects. Animal models are used to validate the activity and effects of the screened peptides on T cells, while safety assessments are conducted to ensure that the effects of these peptides on the immune system in vivo are controllable and safe. Standardized peptide production and preparation procedures have been established to ensure the reproducibility and stability of peptide therapeutic strategies, laying the foundation for future clinical applications. Functional validation of the screened peptides assesses their ability to activate, proliferate, and regulate effectors of T cells, providing practical value and clinical prospects for the application of this technology in the field of immunotherapy.

[0238] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for screening peptides that specifically target T cells, comprising: Provide T cells, said T cells having a subset selected from CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD152 (CTLA-4), CD153, CD154 (CTLA-4), ... At least one of the following surface antigen markers: CD40L, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, and CCR7; The T cells are brought into contact with a polypeptide display library, and target polypeptides that specifically bind to the T cells are screened from it.

2. The method according to claim 1, characterized in that, The T cells were obtained by purifying spleen mononuclear cells.

3. The method according to claim 1 or 2, characterized in that, The polypeptide display library is selected from phage display libraries, yeast display libraries, or engineered cell surface display libraries, with phage display libraries being preferred.

4. The method according to any one of claims 1-3, characterized in that, The polypeptide display library contains 10 9 The above are polypeptide sequences.

5. The method according to any one of claims 1-4, characterized in that, The method further includes: obtaining a candidate peptide display library that binds to the T cells, extracting DNA from the candidate peptide display library, and... Optionally, the DNA is amplified using primer pairs as shown in SEQ ID NO:1 and SEQ ID NO:2, and the amplification products are sequenced to obtain the sequence of the target polypeptide.

6. A polypeptide that specifically targets T cells, characterized in that, The polypeptide is obtained by the screening method according to any one of claims 1-5.

7. A polypeptide that specifically targets T cells, characterized in that, The polypeptide comprises the amino acid sequence shown in formula (I), WX1LX2X3GY (Formula (I)). Where X1, X2, or X3 represents any one of the amino acids selected from glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), tryptophan (W), serine (S), tyrosine (Y), cysteine ​​(C), phenylalanine (F), asparagine (N), glutamine (Q), threonine (T), aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), and histidine (H).

8. The polypeptide according to claim 6 or 7, characterized in that, The percentage of positive binding of the polypeptide to T cells is greater than 15%.

9. The polypeptide according to any one of claims 6-8, characterized in that, The T cells are CD2+, CD3+, CD4+, CD5+, CD7+, CD8+, CD25+, CD28+, CD45+, CD69+, CD152+, CD154+ and / or PD-1+ T cells.

10. The polypeptide according to any one of claims 6-9, characterized in that, The polypeptide also includes no more than 9 amino acids at the C-terminus of the amino acid sequence shown in Formula (I), and / or no more than 9 amino acids at the N-terminus of the amino acid sequence shown in Formula (I).

11. The polypeptide according to any one of claims 6-10, characterized in that, The polypeptide comprises an amino acid sequence selected from the following: WSWSLSSGYADV(SEQ ID NO:3), VTYNWSLLAGYV(SEQ ID NO:4), YTWTLERGYSVN(SEQ ID NO:5), TSGTMQTNPLPV(SEQ ID NO:6), STWSLYAGYTHN(SEQ ID NO:7), NSVHVYHKSFLF(SEQ ID NO:8), TERTMESMTRFA(SEQ ID NO:9), FSVPSTPRTVVV (SEQ ID NO:10) and EWKVLEGHTTRD (SEQ ID NO:11).

12. The polypeptide according to any one of claims 6-11, characterized in that, The polypeptide comprises an amino acid sequence selected from the following: WWSLSSGYADV (SEQ ID NO:3), VTYNWSLLAGYV (SEQ ID NO:4), YTWTLERGYSVN (SEQ ID NO:5), and STWSLYAGYTHN (SEQ ID NO:7).

13. A delivery system specifically targeting T cells, comprising the polypeptide of any one of claims 6-12.

14. The delivery system according to claim 13, characterized in that, The delivery system is selected from liposomes, lipid nanoparticles, micelles, exosomes, or any combination thereof that exhibit the polypeptide on their surface.

15. The delivery system according to claim 14, characterized in that, The delivery system also includes at least one reagent encapsulated therein.

16. The delivery system according to claim 15, characterized in that, The delivery system further includes a connector for linking the polypeptide to the at least one reagent.

17. The delivery system according to claim 15 or 16, characterized in that, The at least one reagent is selected from drugs for preventing or treating diseases, imaging agents, diagnostic agents, markers, and detection agents. Preferably, the drug is selected from small molecule drugs, polypeptide drugs, nucleic acid drugs, or any combination thereof.

18. A fusion protein comprising one or more polypeptides according to any one of claims 6-12.

19. The fusion protein according to claim 18, characterized in that, The fusion protein also includes one or more other targeting moieties.

20. The fusion protein according to claim 18 or 19, characterized in that, The other targeting regions include tumor antigen-binding regions.

21. The fusion protein according to any one of claims 18-20, characterized in that, The fusion protein also includes one or more domains selected from the following: hinge region, transmembrane region, at least one co-stimulatory region, and intracellular signal transduction region.

22. An isolated nucleic acid molecule encoding a polypeptide according to any one of claims 6-12 or a fusion protein according to any one of claims 18-21.

23. A carrier comprising the nucleic acid molecule of claim 22.

24. An engineered cell comprising the nucleic acid molecule of claim 22 or the vector of claim 23.

25. The engineered cell according to claim 24, characterized in that, The engineered cells are prokaryotic cells, eukaryotic cells, insect cells, plant cells, and animal cells, and preferably, the engineered cells are immune cells.

26. A composition comprising the polypeptide of any one of claims 6-12, the delivery system of any one of claims 13-17, the fusion protein of any one of claims 18-21, the nucleic acid molecule of claim 22, the vector of claim 23, or the engineered cell of any one of claims 24-25.

27. The composition according to claim 26, characterized in that, The composition also includes a pharmaceutically acceptable carrier, diluent, and / or excipient.

28. The use of the polypeptide of any one of claims 6-12, the delivery system of any one of claims 13-17, the fusion protein of any one of claims 18-21, the nucleic acid molecule of claim 22, the vector of claim 23, the engineered cell of any one of claims 24-25, or the composition of any one of claims 26-27 in the preparation of a drug targeting T cells.

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