CCR7 affinity peptide, coding nucleic acid and application of CCR7 affinity peptide

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a CCR7 affinity peptide, a coding nucleic acid and application of the CCR7 affinity peptide. According to the invention, CCR7 is taken as a target molecule, and the CCR7 affinity peptide CRBP3 is obtained through a phage display dodecapeptide library high-throughput screening technology. In-vitro function experiments show that the CRBP3 can efficiently target CCR7 + DC, promote phagocytosis of CCR7 + DC on antigens, stimulate T cell proliferation and enhance T cell functions, and the CRBP3 does not block a CCR7-CCL19 / CCL21 pathway and does not inhibit migration of CCR7 + DC. In-vivo anti-tumor experiments show that the CCR7 affinity peptide coupled polypeptide vaccine can improve presentation of antigens, initiate powerful specific CD8 + T cellular immune response and remarkably inhibit growth of tumors, has the effects of inhibiting tumor recurrence for a long time and establishing immune memory, and can provide new effective treatment strategies and candidates for improving the anti-tumor effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a CCR7 affinity peptide, its encoded nucleic acid, and its applications. Background Technology

[0002] In recent years, tumor immunotherapy, represented by immune checkpoint inhibitors, tumor vaccines, and adoptive cell therapy, has achieved significant clinical efficacy. Numerous clinical trials have demonstrated that the clinical efficacy of tumor immunotherapy depends on the presence of tumor antigen-specific T lymphocytes in cancer patients, and tumor vaccines play a crucial role in activating and expanding antigen-specific T lymphocytes.

[0003] The biggest problem in the clinical application of tumor vaccines, especially tumor peptide vaccines, is the lack of specific antigen-presenting cell (APC) targeting, leading to the activation of CD8+ in vivo. + Poor efficacy of T-cell therapy and CD8 + The problem of poor persistence of T-cell immunity is a significant concern. Therefore, improving the APC targeting of tumor vaccines and enhancing their T-cell activation capacity and sustained anti-tumor activity could potentially solve the clinical efficacy issues of tumor vaccines. As a bridge connecting innate and adaptive immune responses, professional APCs (dendritic cells, DCs) are known to have the strongest antigen-presenting capacity for capturing, processing, and delivering antigens, and are the only professional antigen-presenting cells capable of activating naive T cells. They provide the first signal (antigen peptide / MHC complex and TCR-CD3 complex), the second signal (co-stimulatory signal), and the third signal (cytokine signal) for the full activation of T lymphocytes, thereby inducing a persistent antigen-specific T lymphocyte response, playing a crucial role in anti-tumor or anti-infective immunity. Therefore, immunotherapy targeting DCs is currently a widely studied and clinically effective immunotherapy strategy.

[0004] CCR7 belongs to the G protein-coupled receptor (GPCR) family and is highly expressed during dendritic cell (DC) maturation. It is also a key endocytic receptor driving DC lymph node homing and is mainly expressed on the surface of immune cells (such as dendritic cells, B lymphocytes, and T lymphocyte subsets), playing an important role in the immune system. Numerous studies have shown that CCR7... + DC (dendritic cell line) is significantly positively correlated with improved prognosis in patients with various cancers, such as colorectal cancer, breast cancer, cutaneous melanoma, and lung cancer. CCR7 + Patients with a higher DC ratio have better survival, indicating that CCR7 +Dendritic cells (DCs) play a crucial role in the antitumor immune response to various tumors. CCR7, as a key endocytic receptor and lymph node homing receptor, enables mature CCR7... + Dendritic cells (DCs) still possess strong phagocytic capacity and more pronounced lymph node homing ability, which are key to the sustained activation of specific T lymphocytes. Therefore, selectively targeting CCR7 on DCs with tumor vaccines may significantly enhance the in vivo activation of cytotoxic CD8+. + The ability of T cells. Furthermore, peptides have advantages such as low production cost, ease of synthesis and modification, and convenient conjugation with tumor vaccines and nanocarriers.

[0005] Currently, several studies have reported antibodies (ZL201880009163.2, ZL201680059274.5) and inhibitory peptides (CN202411309143.0, ZL202411022694.9) that can inhibit the CCR7-CCL21 / CCL19 signaling pathway. Among them, the CCR7 protein affinity peptide has a good affinity blocking effect on the CCR7-CC21 / CC19 protein interaction and can inhibit ERK1 / 2 phosphorylation and lymph node metastasis, but it has not been found to target CCR7. + Results of a study on DC-enhanced tumor immunotherapy.

[0006] Based on this, the present invention enhances the immune response to antigens by screening peptides that target and are compatible with CCR7, thereby improving the efficacy of immunoprophylaxis and treatment. Summary of the Invention

[0007] The main technical problem addressed by this invention is to provide a CCR7 affinity peptide, obtained through phage display library technology. This affinity peptide specifically targets CCR7 without affecting dendritic cell (DC) migration and possesses in vivo lymph node targeting capability. Upon conjugation with an antigen, it significantly enhances the phagocytic and cross-presentation capabilities of antigen-presenting cells, efficiently promoting the immune response to antigens. It can serve as a lead peptide for developing CCR7-targeting drugs and shows promising application prospects in anti-tumor and anti-infective immunoprophylaxis and therapy.

[0008] Secondly, the present invention provides a nucleic acid encoding a CCR7 affinity peptide.

[0009] Meanwhile, the present invention provides an immunogenic conjugate containing a CCR7 affinity peptide.

[0010] Furthermore, the present invention provides a biomaterial comprising a CCR7 affinity peptide or its encoded nucleic acid, or an immunogenic conjugate, and its application therein.

[0011] Finally, the present invention provides a drug for preventing and treating tumors, pathogenic microorganism infections or autoimmune diseases, and a reagent for detecting or diagnosing tumors, pathogenic microorganism infections or autoimmune diseases.

[0012] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0013] A CCR7 affinity peptide, said affinity peptide comprising the sequence of any of the following polypeptides:

[0014] (1) The polypeptide as shown in SEQ ID NO: 1;

[0015] (2) Derivative peptides obtained by mutating 1-3 amino acids of the polypeptide shown in SEQ ID NO: 1;

[0016] (3) A fusion peptide obtained by attaching a tag to the N-terminus and / or C-terminus of the peptide shown in SEQ ID NO: 1.

[0017] In a preferred embodiment of the present invention, the amino acid sequence of the CCR7 affinity peptide is as follows:

[0018] HVTFKFQWDRES (as shown in SEQ ID NO: 1).

[0019] In a preferred embodiment of the present invention, the derived polypeptide is an alanine scanning peptide.

[0020] Specifically, the amino acid sequence of the alanine scanning peptide is as follows:

[0021] HVTFKFQWDAES (as shown in SEQ ID NO: 10); or,

[0022] HVTFKFQWDREA (as shown in SEQ ID NO: 12).

[0023] In a preferred embodiment of the present invention, the configuration of each amino acid in the amino acid sequence of the CCR7 affinity peptide is independently selected from either the D or L configuration. For example, all amino acids in the amino acid sequence of the CCR7 affinity peptide are either D or L configurations. When a certain amino acid is specified as having a D configuration without specifying the configurations of other amino acids, it is assumed that the other amino acids are all L configurations.

[0024] As a preferred embodiment of the present invention, the CCR7 affinity peptide includes, but is not limited to, one or more of the following modifications: N-terminal modification, C-terminal modification, backbone modification, side chain modification, amino acid modification, etc.

[0025] Specifically, the modifications include, but are not limited to, one or more of the following forms:

[0026] Specifically, the modifications include, but are not limited to, one or more of the following forms:

[0027] (1) Polypeptide-protein coupling: KLH, BSA, OVA, etc.;

[0028] (2) Peptide epitope conjugation: OVA 257-264 E7 49-57 Novel epitope peptides, etc.;

[0029] (3) Peptide nanocarrier coupling: PLGA, fish oil, etc.;

[0030] (4) Isotope labeling: 13 C, 15 N, 18 Marked with 'O', etc.;

[0031] (5) N-terminal or side chain amino acid modification: acetylation, formylation, biotin labeling, trifluoroacetylation, benzoylation, 2-aminobenzoylation, maleimideation, chloroacetylation, bromoacetylation, succinylation, palmitation, malication, fatty acidation, formaldehydeation, chelation reaction (such as Hynic, DTPA, DOTA, Nota), chlorination, fluorination, bromination, nitro or methoxy substitution, fluorescent labeling (such as Cy fluorescein series (Cy3, Cy5, Cy7), Texas series, Alexa series, Rhodamine series, Bodipy, Rox, FAM, FITC, MCA, TAMRA, Dnp) and other modifications;

[0032] (6) C-terminal modification: amidation, esterification, aldehydeation, alcoholation, succinylation, fluorescent labeling (such as Cy series, rhodamine series, AMC, AFC, PNA, CMK, FMK) and other modifications;

[0033] (7) Polyethylene glycol (PEG) modification: PEG2, PEG4, PEG8, PEG12, PEG24, PEG36, PEG2000, PEG3400, PEG5000, PEG20K, PEG40K and other modifications;

[0034] (8) Phosphorylation modification: phosphorylation modification of L-type or D-type amino acids (such as threonine T, serine S), phosphorylation modification of single or multiple amino acids, etc.

[0035] (9) Alkylation modification: N-methylation, side chain methylation, N-ethylation, N-phenylpropylation, N-allylation, etc.;

[0036] (10) Cyclic peptide synthesis: end-to-end cyclization, side chain cyclization (lactone, lactam, ether bond, etc.), multiple disulfide bonds, monothioether cyclization, etc.

[0037] (11) Other special modifications: glycopeptides, sulfonation, MAPS and other modifications.

[0038] A nucleic acid encoding a CCR7 affinity peptide.

[0039] Specifically, the CCR7 affinity peptide encodes either DNA or RNA. Without altering the encoded amino acid sequence, it can be modified based on codon preferences.

[0040] An immunogenic conjugate containing a CCR7 affinity peptide.

[0041] In a preferred embodiment of the present invention, the immunogenic conjugate is an antigen conjugate obtained by conjugating a target antigen with a CCR7 affinity peptide.

[0042] Specifically, the target antigen is derived from humans, mice, or pathogenic microorganisms (including viruses, bacteria, mycoplasma, chlamydia, fungi, etc.).

[0043] Specifically, the target antigen can be an endogenous or exogenous antigen.

[0044] Specifically, the target antigen is a tumor-associated (including tumor-specific) antigen protein or antigenic peptide. This includes, but is not limited to, polypeptide-protein conjugates formed by coupling with KLH, BSA, OVA, etc., or conjugates with OVA. 257-264 E7 49-57 Peptide epitope peptide conjugates formed by coupling with new epitope peptides, etc.

[0045] Specifically, the target antigen is located at the N-terminus and / or C-terminus of the CCR7 affinity peptide.

[0046] Specifically, the target antigen is coupled to the CCR7 affinity peptide via a linker, including but not limited to one or more of the following: (GGGGS)n, (GGGS)n, (GGG)n, n≥1, and n is an integer.

[0047] An immunogenic conjugate encoding a nucleic acid containing a CCR7 affinity peptide.

[0048] Specifically, the nucleic acid encoded by the immunogenic conjugate is DNA or RNA. It can be modified according to codon preferences without altering the encoded amino acid sequence.

[0049] Specifically, the CCR7 affinity peptide or immunogenic conjugate can be prepared by chemical synthesis, including but not limited to any one of solid-phase synthesis, liquid-phase synthesis, and solid-liquid-phase synthesis. Synthetic strategies include N-terminal synthesis, C-terminal synthesis, and / or segmented synthesis. If conventional modifications are performed at the N-terminus and / or C-terminus during synthesis, the resulting polypeptide will contain modified groups.

[0050] A biomaterial comprising any of the following polypeptides or their encoded nucleic acids:

[0051] (1) CCR7 affinity peptide;

[0052] (2) Immunogenic conjugates containing CCR7 affinity peptide.

[0053] As a preferred embodiment of the present invention, the biological material includes, but is not limited to, one or more of the following: recombinant expression vectors (such as plasmid vectors, viral vectors), gene expression cassettes, recombinant bacteria, and host cells (such as prokaryotic cells, eukaryotic cells).

[0054] Applications of a CCR7 affinity peptide or its encoded nucleic acid, immunogenic conjugate or its encoded nucleic acid, or biomaterials, including but not limited to one or more of the following:

[0055] (1) Application in the preparation of drugs for the prevention and treatment of tumors, pathogenic microorganism infections or autoimmune diseases;

[0056] (2) Application in the preparation of reagents for the detection or diagnosis of tumors, pathogenic microbial infections or autoimmune diseases.

[0057] A drug for the prevention and treatment of tumors, pathogenic microorganism infections, or autoimmune diseases, wherein the drug comprises any one or more of the following active ingredients:

[0058] (1) CCR7 affinity peptide;

[0059] (2) The nucleic acid encoded by the CCR7 affinity peptide;

[0060] (3) Immunogenic conjugates containing CCR7 affinity peptide;

[0061] (4) Nucleic acid encoding an immunogenic conjugate containing the CCR7 affinity peptide;

[0062] (5) Biomaterials.

[0063] In a preferred embodiment of the present invention, the drug is a preventive and / or therapeutic vaccine, cell preparation, etc. The drug targets the CCR7 protein in vitro and in vivo, delivering the antigen (and other combined drugs) to enhance the immune response induced by the antigen, and is used for anti-tumor or anti-pathogenic microbial infection.

[0064] Specifically, the vaccine in question is a nucleic acid vaccine, such as a DNA vaccine or an RNA vaccine.

[0065] Specifically, the vaccines are polypeptide vaccines, recombinant protein vaccines, synthetic long peptide vaccines, mixed peptide pool vaccines, nano-vaccines, etc.

[0066] Specifically, the cell preparations include, but are not limited to, DC cell preparations and antigen-specific T cell preparations.

[0067] In a preferred embodiment of the present invention, the drug further comprises other pharmacologically active ingredients. Combined treatment is achieved through use in combination with these other pharmacologically active ingredients.

[0068] As a preferred embodiment of the present invention, the medicament further comprises a pharmaceutically acceptable adjuvant or immunomodulator, including but not limited to one or more of poly-ICLC, 1018ISS, Amplivax, MF59, AS03, AS04, AS15, BCG, CP-870, CP-893, CpG7909, CyaA, cyclic dinucleotides (such as STING), dSLIM, GM-CSF, IL-2, IC30, IC31, Montanide ISA51, etc.

[0069] In a preferred embodiment of the present invention, the drug is administered via local administration at a pharmaceutically acceptable dose.

[0070] In a preferred embodiment of the present invention, the dosage form of the drug is pharmaceutically acceptable, including but not limited to tablets, granules, capsules, powders, pills, sprays, powders for injection, and injection solutions. To prepare a specific dosage form, the drug also contains pharmaceutically acceptable excipients, such as coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH adjusters, and flavoring agents, the types of which can be rationally selected as needed.

[0071] A reagent for detecting or diagnosing tumors, pathogenic microbial infections, or autoimmune diseases, said reagent comprising any one or more of the following components:

[0072] (1) CCR7 affinity peptide;

[0073] (2) The nucleic acid encoded by the CCR7 affinity peptide;

[0074] (3) Immunogenic conjugates containing CCR7 affinity peptide;

[0075] (4) Nucleic acid encoding an immunogenic conjugate containing the CCR7 affinity peptide;

[0076] (5) Biomaterials;

[0077] (6) Fluorescein conjugates designed based on the polypeptide sequences in (1), (3), and (5) above;

[0078] (7) Primers and / or probes designed based on the sequences encoding nucleic acids in (2), (4) and (5) above.

[0079] In a preferred embodiment of the present invention, the reagents include, but are not limited to, immunoassay and / or diagnostic reagents, molecular assay and / or diagnostic reagents, etc. The reagents can bind to the CCR7 protein and are used to detect the affinity of the analyte for the CCR7 protein, or to qualitatively, quantitatively, or locally detect the expression, level, or location of the CCR7 protein in biological samples.

[0080] As a preferred embodiment of the present invention, the immunoassay and / or diagnostic reagent further includes one or more of the following: sample diluent, ELISA plate, blocking solution, washing solution, substrate, stop solution, negative control, and positive control.

[0081] As a preferred embodiment of the present invention, the molecular detection and / or diagnostic reagent further comprises one or more of polymerases (RNA-dependent and / or DNA-dependent polymerases), amplification reaction buffers, positive controls, negative controls, etc.

[0082] In a preferred embodiment of the present invention, the tumor is a solid tumor, including but not limited to melanoma, lung cancer, colorectal cancer, esophageal cancer, ovarian cancer, endometrial cancer, hepatocellular carcinoma, pancreatic cancer, breast cancer, biliary tract cancer, cerebral aneurysm, head and neck squamous cell carcinoma, and skin squamous cell carcinoma.

[0083] In a preferred embodiment of the present invention, the CCR7 affinity peptide or immunogenic conjugate in the drug or reagent is present in a free form or in the form of a pharmaceutically acceptable salt.

[0084] Specifically, CCR7 refers to a receptor protein on mammalian dendritic cells, such as human CCR7 (hCCR7) and mouse CCR7 (mCCR7). It can be a wild-type protein or a mutant protein that retains its activity.

[0085] The beneficial effects of this invention are:

[0086] This invention targets CCR7 and obtains the CCR7 affinity peptide CRBP3 (as shown in SEQ ID NO: 1) using a phage display dodecapeptide library high-throughput screening technique. To obtain a short peptide with low antigenicity and low cost, this invention mutates the CRBP3 peptide (as shown in SEQ ID NO: 2-13). In vitro affinity experiments show that the affinity peptide CRBP3 exhibits the best and most specific affinity activity. In vitro cross-presentation experiments demonstrate that the affinity peptide CRBP3 can efficiently target CCR7. + DC, promotes CCR7 + DCs phagocytose antigens, stimulating T cell proliferation. In vivo antitumor experiments showed that the peptide-conjugated vaccine prepared using the affinity peptide CRBP3 can effectively present antigens in vivo, triggering potent and specific CD8+ expression.+ T-cell immune response, promoting CD3 + CD8 + T cells infiltrate tumor sites, inhibit tumor growth, and establish long-term immune memory. They have promising applications in immunoprophylaxis and treatment, and provide new materials and methods for the preparation of immunotargeted drugs and cell preparations.

[0087] This invention conjugates CCR7 affinity peptides with target antigens to prepare immunogenic conjugates, which are then used to prepare drugs for the prevention and treatment of tumors, pathogenic microorganism infections, or autoimmune diseases, or as detection and diagnostic reagents. These conjugates have high clinical application value in the field of immunoprophylaxis and treatment. Attached Figure Description

[0088] Figure 1 The figure shows the experimental results of the specific affinity of the affinity peptide CRBP3 (SEQ ID NO: 1) for B16-mCCR7.

[0089] Figure 2 The figure shows the experimental results of the specific affinity of alanine scanning peptide (SEQ ID NO: 2-13) for B16-mCCR7.

[0090] Figure 3 The figure shows the experimental results of specific affinity of the affinity peptide CRBP3 (SEQ ID NO: 1) for immature and mature BMDCs.

[0091] Figure 4 Figures show the results of flow cytometry and confocal microscopy experiments on the endocytotic affinity peptide CRBP3 (SEQ ID NO: 1) of immature BMDCs and mature BMDCs.

[0092] Figure 5 Figure showing the experimental results of T cell proliferation and activation stimulated by CRBP3-OVA-coupled peptide enhancing the cross-presentation function of immature and mature BMDCs.

[0093] Figure 6 The figure shows the experimental results showing that CRBP3 does not affect DC migration.

[0094] Figure 7 The figure shows the experimental results of CRBP3 specifically targeting the popliteal lymph nodes.

[0095] Figure 8 CRBP3-OVA, an immunogenic conjugate of CCR7 affinity peptide 257-264 Figure showing the results of the immune activation experiment in the C57BL / 6 mouse model.

[0096] Figure 9 CRBP3-OVA, an immunogenic conjugate of CCR7 affinity peptide 257-264Figure showing the results of anti-tumor experiments on the B16-OVA mouse model.

[0097] Figure 10 CRBP3-E7, an immunogenic conjugate of the CCR7 affinity peptide. 49-57 Figure showing the results of anti-tumor and re-challenge model experiments on the TC-1 mouse model.

[0098] In the above figures, the significance indicators are as follows: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0099] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings obtained in the experimental examples have been briefly described above. It should be understood that the above drawings only show some experimental examples of the present invention and should not be considered as any limitation on the scope of protection of the claims. For those skilled in the art, other related drawings can be obtained based on these drawings without any creative effort. Detailed Implementation

[0100] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and experimental examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, all other implementation schemes obtained by those skilled in the art without creative effort, such as implementation schemes obtained by modification, variation or simple substitution, should fall within the scope of protection of the present invention.

[0101] Unless otherwise specified, the experimental methods used in the following examples and experimental cases are conventional methods; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the field and commercially available to the public unless otherwise specified; the terms and abbreviations used have their conventional meanings in the field, such as PBS for phosphate buffer and NS (Normal saline) for physiological saline.

[0102] Experimental Example

[0103] I. Screening of CCR7 affinity peptides using phage display peptide libraries

[0104] Since screening of phage display peptide libraries requires the corresponding proteins as a basis, B16 cells (B16-mCCR7 and B16 cells) that stably overexpress mCCR7 and control plasmids were constructed and used as prerequisites for screening the phage display dodecapeptide library. The screening process was carried out according to the standard procedure of New England Biolabs (NEB). The specific screening steps are as follows:

[0105] (1) First, collect B16 cells and B16-mCCR7 cells in good growth condition, wash twice with serum-free medium, and prepare a solution with a concentration of 1×10⁻⁶. 6 Cell suspension with cells / mL.

[0106] (2) Add 400 μL of blocking solution (5 mg / mL BSA) to the B16 cell suspension and place it on a rotating shaker at 4°C for 1 h.

[0107] (3) The blocked B16 cells were resuspended in 200 μL of PBS buffer, and then 10 μL of phage dodecapeptide library was added. The cells were then incubated on a rotating shaker at 4°C for 2 h.

[0108] (4) Place the supernatant from the previous step and B16-CCR7 cells on a rotating shaker and incubate at 4°C for 2 hours.

[0109] (5) Wash 5 times with PBST containing 0.1% Tween-20 (pH=5.0) and 2 times with PBST containing 0.1% Tween-20 (pH=7.4). Then add 100 μL of lysis buffer, blow and lyse several times after adding, and place at room temperature for 5 min for lysis.

[0110] (6) Add 400 μL of elution buffer to the above system, elute at room temperature for 30 min, and then add 60 μL of neutralization buffer.

[0111] (7) The above system is used for the next round of experiments. The screening process (1)-(6) is repeated for 4 rounds to enrich phages with repetitive sequences.

[0112] (8) After five rounds of screening, phage monoclonals that bind to the extracellular domain of the target protein mCCR7 were enriched in each round.

[0113] (9) Select positive clones for sequencing to obtain the dodecapeptide sequence, namely the CCR7 affinity peptide sequence, and name it CRBP3. The amino acid sequence of CRBP3 is: HVTFKFQWDRES (SEQ ID NO: 1), and all amino acids in the sequence are L configuration.

[0114] In order to analyze the effect of specific amino acids on the overall peptide activity, the present invention designed the following alanine scanning peptide based on the above CRBP3 sequence, and its amino acid sequence information is shown in Table 1.

[0115] Table 1. Polypeptides and their amino acid sequences

[0116] Serial Number name Single letter sequence Three-letter sequence SEQ ID NO: 1 CRBP3 HVTFKFQWDRES His-Val-Thr-Phe-Lys-Phe-Gln-Trp-Asp-Arg-Glu-Ser SEQ ID NO: 2 CRBP3-1A AVTFKFQWDRES Ala-Val-Thr-Phe-Lys-Phe-Gln-Trp-Asp-Arg-Glu-Ser SEQ ID NO: 3 CRBP3-2A HATFKFQWDRES His-Ala-Thr-Phe-Lys-Phe-Gln-Trp-Asp-Arg-Glu-Ser SEQ ID NO: 4 CRBP3-3A HVAFKFQWDRES His-Val-Ala-Phe-Lys-Phe-Gln-Trp-Asp-Arg-Glu-Ser SEQ ID NO: 5 CRBP3-4A HVTAKFQWDRES His-Val-Thr-Ala-Lys-Phe-Gln-Trp-Asp-Arg-Glu-Ser SEQ ID NO: 6 CRBP3-5A HVTFAFQWDRES His-Val-Thr-Phe-Ala-Phe-Gln-Trp-Asp-Arg-Glu-Ser SEQ ID NO: 7 CRBP3-6A HVTFKAQWDRES His-Val-Thr-Phe-Lys-Ala-Gln-Trp-Asp-Arg-Glu-Ser SEQ ID NO: 8 CRBP3-7A HVTFKFAWDRES His-Val-Thr-Phe-Lys-Phe-Ala-Trp-Asp-Arg-Glu-Ser SEQ ID NO: 9 CRBP3-8A HVTFKFQADRES His-Val-Thr-Phe-Lys-Phe-Gln-Ala-Asp-Arg-Glu-Ser SEQ ID NO: 10 CRBP3-9A HVTFKFQWARES His-Val-Thr-Phe-Lys-Phe-Gln-Trp-Ala-Arg-Glu-Ser SEQ ID NO: 11 CRBP3-10A HVTFKFQWDAES His-Val-Thr-Phe-Lys-Phe-Gln-Trp-Asp-Ala-Glu-Ser SEQ ID NO: 12 CRBP3-11A HVTFKFQWDRAS His-Val-Thr-Phe-Lys-Phe-Gln-Trp-Asp-Arg-Ala-Ser SEQ ID NO: 13 CRBP3-12A HVTFKFQWDREA His-Val-Thr-Phe-Lys-Phe-Gln-Trp-Asp-Arg-Glu-Ala

[0117] Note: All amino acids with uppercase letters in the table are L-configuration amino acids.

[0118] The peptides and alanine scanning peptides obtained from the above screening were synthesized in an Fmoc solid phase, purified by a high performance liquid chromatography system, and used in subsequent experiments after the mass spectrometry identification results were correct (the molecular weight met the theoretical value).

[0119] II. Affinity Experiment

[0120] (1) Digest B16-mCCR7 and B16 cells that have grown to the logarithmic growth phase to prepare single-cell suspensions and adjust the cell density for later use.

[0121] (2) Induction of mouse BMDCs: C57BL / 6J mice (6-8 weeks old, female) were selected, and cells from their bone marrow cavity were taken. Red blood cell lysis buffer was added and lysed at 4°C for 5 min. After lysis, the cells were washed twice with sterile PBS (pH 7.2). The cells were resuspended in RPMI 1640 complete medium, and recombinant mouse GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) cytokines were added. The cells were cultured in a cell culture incubator and the day of induction was recorded as day 0. Half of the medium was changed every day from day 3. Immature BMDCs were obtained on day 5 and mature BMDCs were obtained on day 8. The cell density was adjusted for later use.

[0122] (3) Add biotinylated affinity peptides to the cell suspension prepared in advance in (1) or (2) on ice and co-incubate, and wash twice with pre-cooled PBS (pH 7.2) to wash away non-specifically bound peptides.

[0123] (4) BMDCs cells were blocked with 5% rat serum at 4℃ for 15 min, and then anti-mouse CD11c-APC and SA-PE antibodies were added and incubated at 4℃ for 30 min; B16-mCCR7 and B16 cells were directly incubated with SA-PE antibody at 4℃ for 30 min.

[0124] (5) After washing twice with pre-cooled PBS (pH 7.2), the affinity of cells for peptides was detected by flow cytometry. The results are as follows: Figure 1-3 As shown.

[0125] from Figure 1 It can be seen that the CRBP3 peptide has a specific affinity for B16-mCCR7 cells.

[0126] from Figure 2It can be seen that the affinity of alanine scanning peptides CRBP3-4A, CRBP3-5A, CRBP3-6A, and CRBP3-8A for B16-mCCR7 cells decreased significantly, all below 20%; the affinity of peptides CRBP3-10A and CRBP3-12A for B16-mCCR7 cells was comparable to that of peptide CRBP3; the affinity of peptides CRBP3-1A, CRBP3-2A, CRBP3-3A, CRBP3-7A, CRBP3-9A, and CRBP3-11A decreased by nearly half.

[0127] from Figure 3 It can be seen that CRBP3 has a high affinity for both immature and mature mouse BMDCs.

[0128] III. Engulfment Experiment

[0129] To assess the endocytosis of peptides by bone marrow-derived dendritic cells (BMDCs), a phagocytosis assay was performed, which included the following steps:

[0130] (1) Co-incubate the biotin-labeled peptide (50 μM) with immature or mature BMDCs (1 × 10⁻⁶ mcg per group). 5 (1 cell), reacted at 37°C for 2 hours.

[0131] (2) After incubation, the cells were washed with buffer and blocked in FACS buffer containing 5% rat serum for 15 min to reduce nonspecific binding.

[0132] (3) The surface was stained with anti-mouse CD11c-APC antibody (clone number N418) for 30 min. After washing twice, the cells were fixed at room temperature for 30 min, and then washed twice with permeation buffer.

[0133] (4) Add streptavidin-PE (SA-PE) staining for 30 min to detect endocytosed biotinylated peptides. Finally, wash twice and analyze using flow cytometry. Results are as follows: Figure 4 As shown.

[0134] The phagocytosis of affinity peptides by BMDCs was detected by confocal microscopy. The experimental steps are as follows:

[0135] (1) The biotin-labeled peptide was pre-incubated with streptavidin-PE (SA-PE) at a molar ratio of 1:3 to obtain the fluorescently labeled peptide.

[0136] (2) 5×10 5 Bone marrow-derived dendritic cells (BMDCs) were incubated on ice for 30 min with fluorescently labeled GA peptide or CRBP3 peptide.

[0137] (3) After washing three times, the cells were transferred to 37°C and cultured for 2 hours.

[0138] (4) Surface staining was performed using anti-mouse CD11c PerCP-eFluor 450 antibody (clone number N418).

[0139] (5) After washing again, the cell nuclei were stained with Hoechst 33342 dye (Beyotime Biotechnology) for 1 hour. Finally, imaging analysis was performed using a confocal laser scanning microscope (CLSM). The results are as follows: Figure 4 As shown.

[0140] from Figure 4 It can be seen that, compared with the control group, CRBP3 can be phagocytosed by immature or mature BMDCs, showing a significant difference.

[0141] IV. In vitro cross-presentation experiment

[0142] (1) BMDC load OVA 257-264 GA-OVA 257-264 CRBP3-OVA 257-264 Immature BMDCs induced to day 5 and mature BMDCs induced to day 8 were obtained separately. After washing with serum-free RPMI 1640 medium, they were co-incubated with different drug groups for 2 hours. After incubation, the cells were centrifuged, washed, and the cell density was adjusted to 102. 5 cell / mL for later use.

[0143] (2) Sorting CD8 from OT-1 mice + T cells were labeled with CFSE and the cell density was adjusted to 10-1. 6 cell / mL for later use.

[0144] (3) BMDCs loaded with different drugs and CD8 + T cells were co-incubated at a ratio of 1:10 for 72 hours.

[0145] (4) After co-incubation, cells were collected, and T cell proliferation was detected by flow cytometry. The supernatant was collected, and IFN-γ secretion was detected by ELISA. The results are as follows: Figure 5 As shown.

[0146] from Figure 5 It can be seen that, compared with the control group, CRBP3-OVA 257-264 It can significantly increase the OVA on the surface of BMDCs 257-264 The peptide / MHC complex (the first signal for T cell activation) enhances the cross-presentation capacity of immature and mature dendritic cells (DCs) and significantly promotes CD8 activation. +The proliferation of T lymphocytes and their ability to secrete IFN-γ.

[0147] V. In vitro DC chemotaxis experiment

[0148] CCR7 is a chemokine receptor. Whether targeting CCR7 affects DC migration and thus DC function needs to be verified using in vitro DC chemotaxis experiments.

[0149] Transwell migration assays were performed using 24-well plates with polycarbonate membranes (5 μm pores). Specifically, bone marrow-derived dendritic cells (BMDCs) on day 8 post-induction were resuspended in 200 μL of complete RPMI 1640 medium and seeded into the upper chamber. Experimental groups may or may not have had 50 μM CRBP3 peptide added. The lower chamber contained 600 μL of complete RPMI 1640 medium containing 100 ng / mL CCL19 or 100 ng / mL CCL21. Cells were incubated at 37°C in a 5% CO2 humidified incubator for 48 h. After incubation, cells that migrated to the lower chamber were collected and counted. The results are shown below. Figure 6 As shown.

[0150] from Figure 6 It can be seen that CRBP3 does not inhibit the migration of DC cells.

[0151] VI. In vivo lymph node targeting experiment

[0152] C57BL / 6 mice (6-8 weeks old) were used for in vivo experiments. Each mouse received an injection of 20 μL of CpG ODN1826 (30 μg / mL) into its paw pad. Seven days later, each mouse received an injection of 20 μL of a fluorescently labeled peptide (Cy7-CRBP3 or Cy7-GA, 20 μg / mouse) into its paw pad. Twenty-four hours after injection, lymph nodes were collected from the popliteal fossa, groin, and axillary region, and imaging analysis was performed using a small animal in vivo imaging system. The results are as follows: Figure 7 As shown.

[0153] from Figure 7 It can be seen that, compared with the Cy7-GA group, Cy7-CRBP3 can specifically accumulate in the popliteal lymph node, indicating that CRBP3 can specifically target the lymph node in vivo.

[0154] VII. In vivo immune activation and anti-tumor experiments

[0155] 1. In vivo immune activation model

[0156] (1) CRBP3-OVA 257-264 / CpG was administered starting on day 0, once every 7 days, for a total of 4 doses. The dosing regimen for mice was as follows:

[0157] Normal saline (A) group: 200 μL of normal saline was injected subcutaneously into each animal;

[0158] OVA 257-264 (B) Group: Each animal was subcutaneously injected with 20 μg of OVA 257-264 Peptide + CpG ODN 182630μg;

[0159] GA-OVA 257-264 (C) Group: Each animal was subcutaneously injected with 20 μg of GA-OVA 257-264 Peptide + CpG ODN 182630μg;

[0160] CRBP3-OVA 257-264 Group (D): Each animal was subcutaneously injected with 20 μg of CRBP3-OVA 257-264 Peptide + CpG ODN182630μg.

[0161] (2) The mice were sacrificed on the 4th day after the last immunization, and their spleen and lymph nodes were removed.

[0162] (3) Single-cell suspensions were prepared and labeled with antibodies. The secretion of IFN-γ in the spleen and lymph nodes was detected by flow cytometry. The expression of granzyme B (Grz B) and perforin in spleen tissue was detected by qRT-PCR. The results are as follows: Figure 8 As shown.

[0163] from Figure 8 It can be seen that CRBP3-OVA 257-264 (D) CD8 concentrations in the spleen and lymph nodes (LN) of the treatment group + IFN-γ + The proportion of T cells was significantly higher in the treatment groups than in other treatment groups; the expression of granzyme B and perforin in spleen tissue was significantly higher in the treatment groups than in other treatment groups.

[0164] The above experimental results show that CRBP3-OVA 257-264 Peptide vaccines can effectively present antigens in vivo, triggering potent and specific CD8 responses. + T-cell immune response.

[0165] 2. B16-OVA tumor-bearing mouse model

[0166] Before the experiment, a B16-OVA tumor-bearing mouse model was established. The method included the following steps:

[0167] (1) Collect B16 cells in the logarithmic growth phase and adjust the cell density to 1×10⁻⁶. 6 cells / mL.

[0168] (2) Select 6-8 week old female C57BL / 6 mice and inject 200 μL of the above-mentioned concentration of B16-OVA cells into their right back, i.e., inject 2 × 10⁻⁶ cells per mouse. 5 Each cell.

[0169] (3) The day of injection is recorded as day 0, CRBP3-OVA 257-264 / CpG was administered starting on day 3, once every 5 days, for a total of 4 doses. The dosing regimen for mice was as follows:

[0170] Normal saline (A) group: 200 μL of normal saline was injected subcutaneously into each animal;

[0171] OVA 257-264 (B) Group: Each animal was subcutaneously injected with 20 μg of OVA 257-264 Peptide + CpG ODN 182630μg;

[0172] GA-OVA 257-264 (C) Group: Each animal was subcutaneously injected with 20 μg of GA-OVA 257-264 Peptide + CpG ODN 182630μg;

[0173] CRBP3-OVA 257-264 Group (D): Each animal was subcutaneously injected with 20 μg of CRBP3-OVA 257-264 Peptide + CpG ODN182630μg.

[0174] (4) Three days after the last administration, the mice were sacrificed and their tumors, spleens and lymph nodes were removed.

[0175] (5) Prepare single-cell suspensions, label them with antibodies, and detect CD8+ in tumor sites by flow cytometry. + T cell infiltration and intracellular factor staining assays were used to detect IFN-γ secretion in tumors, spleen, and lymph nodes. The results are as follows: Figure 9 As shown.

[0176] from Figure 9 It can be seen that CRBP3-OVA 257-264 (D) The treatment group significantly inhibited tumor growth. CD33 from mouse tumor sites... + CD8 + T-cell infiltration was observed to be significantly different from other treatment groups, with CRBP3-OVA showing better T-cell infiltration. 257-264 (D) The treatment group was able to promote CD3 more effectively. + CD8 + T cells infiltrated the tumor site. Intracellular factor staining results showed that CRBP3-OVA... 257-264(D) CD8 sites of tumors, spleen, and draining lymph nodes (dLN) in the treatment group + IFN-γ + The proportion of T cells was significantly higher in the treatment groups than in other treatment groups.

[0177] The above experimental results show that CRBP3-OVA 257-264 Peptide vaccines can effectively present antigens in vivo, triggering potent and specific CD8 responses. + T-cell immune responses inhibit the occurrence and growth of tumors.

[0178] 3. TC-1 tumor-bearing mouse model

[0179] Before the experiment, a TC-1 tumor-bearing mouse model was established. The method included the following steps:

[0180] (1) Collect TC-1 cells in the logarithmic growth phase and adjust the cell density to 7.5 × 10⁻⁶. 5 cells / mL.

[0181] (2) Select 6-8 week old female C57BL / 6 mice and inject 200 μL of the above-mentioned concentration of TC-1 cells into their right back, i.e., 1.5 × 10⁻⁶ cells per mouse. 5 Each cell.

[0182] (3) The day of injection is recorded as day 0, CRBP3-E7 49-57 / CpG was administered starting on day 1, once every 5 days, for a total of 4 doses. The mouse grouping and administration details are as follows:

[0183] Normal saline group: 200 μL of normal saline was injected subcutaneously into each animal;

[0184] E7 49-57 Group: Each animal was subcutaneously injected with 20 μg of E7. 49-57 Peptide + CpG ODN 182630μg;

[0185] CRBP3-E7 49-57 Group: Each animal was subcutaneously injected with 20 μg of CRBP3-E7 49-57 Peptide + CpG ODN 182630μg.

[0186] (4)CRBP3-E7 49-57 Twenty days after complete tumor regression, 200 μL of TC-1 cells (three times the initial injection volume) were subcutaneously injected again into the back of each mouse, i.e., 4.5 × 10⁻⁶ cells per mouse. 5 Cells. Control group mice were loaded with 4.5 × 10⁶ cells. 5C57BL / 6 mice with TC-1 cells were observed for 14 days, and the tumor volume of each group of mice was recorded. Their spleens and lymph nodes were harvested for ex vivo experiments.

[0187] (5) Prepare single-cell suspensions, label them with antibodies, and detect memory CD8 in the spleen by flow cytometry. + The proportion of T cells, the results are as follows Figure 10 As shown.

[0188] from Figure 10 It can be seen that CRBP3-E7 49-57 The group significantly inhibited tumor growth, and the tumor completely regressed by day 21. Even after reloading with three times the volume of tumor cells, it still significantly inhibited tumor development, and the detection results of immune memory T cells in spleen tissue showed CRBP3-E7. 49-57 Group T EM The significantly increased cell proportion indicates that CRBP3-E7 49-57 The group established a long-lasting immune memory, which can prevent the occurrence and recurrence of tumors.

[0189] Example 1

[0190] This embodiment provides a CCR7 affinity peptide and its encoded nucleic acid. The amino acid sequence of the CCR7 affinity peptide is as follows:

[0191] HVTFKFQWDRES (as shown in SEQ ID NO: 1).

[0192] This embodiment also provides a biological material, specifically a viral vector, containing nucleic acid encoding the CCR7 affinity peptide.

[0193] This embodiment also provides a biomaterial, specifically a recombinant cell, which can express and produce the CCR7 affinity peptide.

[0194] This embodiment also provides the application of CCR7 affinity peptides or biomaterials in the preparation of drugs for the prevention and treatment of tumors, pathogenic microorganism infections or autoimmune diseases.

[0195] This embodiment also provides a drug for the prevention and treatment of tumors, comprising a pharmaceutically effective amount of CCR7 affinity peptide and appropriate amounts of adjuvants and immunomodulators.

[0196] In other embodiments of the present invention, the CCR7 affinity peptide may be industrially produced with one or more of the following modifications: N-terminal modification, C-terminal modification, backbone modification, side chain modification, amino acid modification, etc.

[0197] Example 2

[0198] This embodiment provides a CCR7 affinity peptide and its encoded nucleic acid. Specifically, the CCR7 affinity peptide is an alanine scanning peptide with the following amino acid sequence:

[0199] HVTFKFQWDAES (as shown in SEQ ID NO: 10); or,

[0200] HVTFKFQWDREA (as shown in SEQ ID NO: 12).

[0201] This embodiment also provides a biological material, specifically a plasmid vector containing nucleic acid encoding the CCR7 affinity peptide.

[0202] This embodiment also provides a biomaterial, specifically a recombinant cell, which can express and produce the CCR7 affinity peptide.

[0203] This embodiment also provides the application of CCR7 affinity peptide or its encoded nucleic acid, or biological material, in the preparation of reagents for detecting and / or diagnosing tumors, pathogenic microorganism infections, or autoimmune diseases.

[0204] This embodiment also provides a reagent for detecting and / or diagnosing tumors, comprising an appropriate amount of CCR7 affinity peptide, as well as sample diluent, ELISA plate, blocking solution, washing solution, substrate, stop solution, negative control, positive control, etc.

[0205] This embodiment provides a reagent for detecting and / or diagnosing tumors, comprising primers and / or probes designed and synthesized based on the nucleic acid encoding the CCR7 affinity peptide, as well as DNA polymerase, amplification reaction buffer, positive control, negative control, etc.

[0206] Example 3

[0207] This embodiment provides an immunogenic conjugate containing a CCR7 affinity peptide and its encoded nucleic acid. The immunogenic conjugate is derived from a target antigen (OVA). 257-264 It is obtained by coupling with CCR7 affinity peptide, as described in Example 1 or 2.

[0208] This embodiment also provides a drug for preventing and treating tumors, pathogenic microorganism infections, or autoimmune diseases, comprising a pharmacodynamic amount of an immunogenic conjugate and appropriate amounts of adjuvants and immunomodulators.

[0209] This embodiment also provides a drug for the prevention and treatment of tumors, specifically an mRNA vaccine, comprising a pharmaceutically effective amount of mRNA delivered by lipid nanoparticles (LNPs), and appropriate amounts of adjuvants and immunomodulators, wherein the mRNA is codon-optimized and can encode an immunogenic conjugate.

[0210] In other embodiments of the present invention, the target antigen is selected from E7. 49-57 Novel epitope peptides, etc.

[0211] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A CCR7 affinity peptide, characterized in that: The affinity peptide comprises the sequence of any of the following polypeptides: (1) The polypeptide as shown in SEQ ID NO: 1; (2) Derivative peptides obtained by mutating 1-3 amino acids of the polypeptide shown in SEQ ID NO: 1; (3) A fusion peptide obtained by attaching a tag to the N-terminus and / or C-terminus of the peptide shown in SEQ ID NO:

1.

2. The CCR7 affinity peptide according to claim 1, characterized in that: The amino acid sequence of the CCR7 affinity peptide is as follows: HVTFKFQWDRES; or HVTFKFQWDAES; or, HVTFKFQWDREA.

3. A nucleic acid encoding the CCR7 affinity peptide as described in any one of claims 1-2.

4. An immunogenic conjugate comprising the CCR7 affinity peptide as described in any one of claims 1-2.

5. The immunogenic conjugate according to claim 4, characterized in that: The immunogenic conjugate is an antigen conjugate obtained by conjugating the target antigen with the CCR7 affinity peptide.

6. A nucleic acid encoding an immunogenic conjugate as described in any one of claims 4-5.

7. A biomaterial, characterized in that: The biomaterial comprises any of the following polypeptides or their encoded nucleic acids: (1) The CCR7 affinity peptide as described in any one of claims 1-2; (2) The immunogenic conjugate as described in any one of claims 4-5.

8. The application of a CCR7 affinity peptide as described in any one of claims 1-2, a nucleic acid encoded as described in claim 3, an immunogenic conjugate as described in any one of claims 4-5, a nucleic acid encoded as described in claim 6, or a biomaterial as described in claim 7, characterized in that: The application includes, but is not limited to, one or more of the following aspects: (1) Application in the preparation of drugs for the prevention and treatment of tumors, pathogenic microorganism infections or autoimmune diseases; (2) Application in the preparation of reagents for the detection or diagnosis of tumors, pathogenic microbial infections or autoimmune diseases.

9. A drug for preventing and treating tumors, pathogenic microorganism infections, or autoimmune diseases, characterized in that: The drug contains any one or more of the following active ingredients: (1) The CCR7 affinity peptide as described in any one of claims 1-2; (2) The encoded nucleic acid as described in claim 3; (3) The immunogenic conjugate as described in any one of claims 4-5; (4) The encoded nucleic acid as described in claim 6; (5) The biomaterial as described in claim 7.

10. A reagent for detecting or diagnosing tumors, pathogenic microbial infections, or autoimmune diseases, characterized in that: The reagent contains any one or more of the following components: (1) The CCR7 affinity peptide as described in any one of claims 1-2; (2) The encoded nucleic acid as described in claim 3; (3) The immunogenic conjugate as described in any one of claims 4-5; (4) The encoded nucleic acid as described in claim 6; (5) The biomaterial as described in claim 7; (6) Fluorescein conjugates designed based on the polypeptide sequences in (1), (3), and (5) above; (7) Primers and / or probes designed based on the sequences encoding nucleic acids in (2), (4) and (5) above.

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