CN117797253A - Tumor vaccine capable of expanding T cell epitope and preparation method thereof - Google Patents
Tumor vaccine capable of expanding T cell epitope and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of tumor medicaments, in particular to a tumor vaccine capable of expanding T cell epitope and a preparation method thereof. The invention fuses specific antigen with XCL1, specifically selects GP100 as model antigen, constructsXCL1/GP100Fused plasmid. Then byXCL1/GP100Plasmid-immunized melanoma model mice, comparedGP100Fused withXCL1The post antigen can inhibit tumor growth more effectively, which verifies that XCL1 can enhance antigen presentation and enhance the immune effect of GP 100. In addition to that, useXCL1/ GP100Plasmid immunization of mice foundXCL1/GP100The plasmid can induce the organism to generate cellular immunity aiming at target antigen and also can generate cellular immunity aiming at non-immune target antigen OVA, which shows that the fusion of XCL1 can not only enhance antigen presentation, but also expand T cell antigen epitope.
Description
Technical Field
The invention relates to the field of tumor medicaments, in particular to a tumor vaccine capable of expanding T cell epitope and a preparation method thereof.
Background
The tumor vaccine can induce antigen-specific effector T cells, and can enhance the inhibition effect by being matched with an immunodetection point inhibitor, thereby enhancing the effect of tumor treatment. A large number of animal experiments and preclinical studies show that tumor vaccines can effectively inhibit tumor growth, but the effect of tumor vaccines is affected by factors such as antigen species, vaccine delivery systems, adjuvants and the like, and only 15% -20% of patients can benefit in clinical application.
Accurate selection of the targeted antigen is a critical factor in the effect of tumor vaccines. Currently, strategies for designing tumor vaccines (targeted antigens) are generally: 1) Selecting a protein which is highly expressed by tumor cells and lowly expressed by normal tissues or expressed only in the embryo development process of the body as an antigen 2) selecting a protein encoded by a newly appeared mutant gene in a tumor genome as an antigen, and adopting a plurality of tumor neoantigen polypeptides as targets.
Epitope spreading refers to the sequential generation of a response by the immune system of the body to a cryptic or other epitope of a particular antigen during a sustained response to that antigen. Khaled el-Shami et al found for the first time in EG.7OVA-engrafted tumor mice models, and produced in addition to responses to OVA antigen following immunization with OVA antigenResponses to other antigens are generated, i.e., T cell epitopes are expanded. Subsequent studies have shown that tumor immunotherapy may extend cryptic epitopes for this antigen, and possibly T cell recognition epitopes for other antigens, both in animal models and in clinical studies. Immune-derived CD8 + T cells can mobilize the body's own CD8 while clearing tumor cells expressing target antigens + T cells produce an immune response against other antigens that are newly released by tumor cells, thereby overcoming the immune escape phenomenon of tumor cells. Thus, T cell epitope spreading induced by immunotherapy may have an important role in the treatment of tumors. In addition, the antigen released by the damaged tumor cells may induce a broader, more pronounced immune response in the body during immune clearance.
DC Cells (DCs) are professional antigen presenting cells (Antigen presenting cells, APC) with the strongest organism functions, can efficiently ingest, process and present antigens, and immature DCs have stronger migration capacity, and mature DCs can effectively activate primitive T cells in the central link of starting, regulating and maintaining immune responses.
DC cells are divided into a number of sub-populations including traditional type I DC (conventional type I DC, cDC 1), type II DC (cDC 2), plasmacytoid DC (pDC), peripheral blood mononuclear cell-derived dendritic cells (Mo-DC), and the like, and the ability of the different sub-populations of DCs to initiate and activate T cells are also different. cDC1 is the most professional antigen cross-presenting cell that efficiently ingests tumor antigens and processes them into the form of MHC-I/antigen peptide complexes, which are then cross-presented to the original CD8 + T cells, thereby inducing CD8 + T cell differentiation produces antigen-specific effector T cells.
The development of cDC1 cells depends on the transcription factor Batf3, and cDC1 cells in mice mainly include primitive cd8α located in T cell regions within secondary lymphoid tissues + CD103-Clec9A + CD11c + Cells and CD8 alpha-CD 103 located in peripheral tissues and having migration ability + Clec9A + CD11c + And (3) cells. Whereas cDC1 cells in humansMainly CD141 (BDCA 3) + XCR1 + DCs cells. These DC cells express the receptor XCR1 on the cell surface and can construct fusion molecules of specific antigens and the chemokine XCR1, which can target XCR1 with specific antigens based on strong interactions between XCR1 and XCR1 + DC cells promote uptake of a specific antigen by cDC1 cells and induce the generation of antigen-specific CTL cells (a T cell that recognizes a specific antigen, kills cancer cells) and/or antibody responses, thereby enhancing antitumor effects.
However, the development of tumors is a dynamic process of the interaction of the immune system with tumor cells, and Robert Schreiber et al in 2002 proposed the theory of immune editing of "immune clearance, immune balance, immune escape". The theory states that: while the immune system plays a role in eliminating tumor cells, the tumor cells continuously generate new mutation, and the expression of tumor markers MHC-I and other molecules and the expression of immunosuppressive molecules are continuously regulated down so as to escape from the immune system for elimination. Antigens designed for specific tumor targets can induce organisms to produce specific CD8 + T cells thereby eliminate the corresponding tumor cells, but CD8 due to the rapid tumor cell division and mutation rate + T cells have difficulty in efficiently clearing tumor cells that have undergone new mutations. Scientists have made a great deal of effort to solve this problem, however, to date this has remained a bottleneck in the application of vaccines in clinical oncologic therapy.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a tumor vaccine capable of expanding T cell epitope and a preparation method thereof.
The invention provides application of XCL1 in preparing a medicine for expanding T cell epitope.
Furthermore, the invention discovers that the XCL1 fusion on the antigen can expand T cell epitope.
XCL1 is a lymphocyte chemotactic factor, and the full-length amino acid sequence of the XCL is shown as SEQ ID NO. 7. According to the research result of the invention, the XCL1 fusion molecule can increase the content of the wetted chemotactic factor XCL1 in the body after immunization, which increases the content in the bodyXCR1 + Number of cDC1 cells. Cells expressing the target antigen break down and release large amounts of antigen when cleared by the immune system due to XCR1 + The number of cDC1 cells is increased, and the non-target antigens are more easily ingested and carried by immune cells to secondary lymphoid tissues, so that the organism is induced to generate specific CD8 aiming at the non-target antigens + T cells. Therefore, the fusion application of XCL1 and specific antigen can play a role in expanding T cell epitope. Therefore, the invention proves that the XCL1 fusion can expand T cell epitope on antigen.
Vaccines against specific target antigens induce specific CD8 production in the body + T cells thereby clear the foci where the target antigen is located. However, when the facing disease is a tumor, vaccine-induced specific CD8 against immune target antigens is susceptible to immune escape due to tumor cell division and rapid mutation rate + T cells have difficulty in efficiently clearing tumor cells that have undergone new mutations. Therefore, the XCL1 fusion molecule provided by the invention induces organisms to generate specific cellular immunity aiming at other antigens (non-immune target antigens) expressed by tumor cells by expanding T cell antigen recognition epitopes, thereby overcoming the problem of vaccine invalidity caused by the problems of tumor heterogeneity, antigen modulation and the like.
In the use of the invention, the antigen comprises a tumour antigen.
In some embodiments, the antigen of the present invention comprises melanoma antigen GP100; the expanded T cell epitope comprises: on the basis of the epitope corresponding to the GP100 antigen, the epitope corresponding to the OVA antigen is expanded.
The invention also provides a medicine for expanding T cell epitopes, which comprises at least one of the following a-d:
a. a fusion protein of an antigen and XCL 1;
b. nucleic acids encoding the fusion proteins;
c. a vector comprising the nucleic acid;
d. a host transformed or transfected with the vector;
the invention also provides a preparation method of the drug for expanding the T cell epitope, which comprises at least one of the following steps of:
i. synthesizing nucleic acid for encoding antigen and XCL1 fusion protein;
ii. Constructing a vector containing the fusion nucleic acid sequence;
iii, constructing a host expressing the antigen and XCL1 fusion protein with said nucleic acid or vector;
iv expressing and purifying the fusion protein of antigen and XCL1 using said host.
Specifically, the present invention also provides a fusion protein comprising: XCL1 peptide, linker peptide and GP100 peptide.
In the fusion protein, the XCL1 may be of human or animal origin, for example, murine, rabbit, porcine, bovine, equine or ovine origin; it may be a wild-type protein or an optimized or mutated protein; it may be a full-length fragment or a truncated form, and the present invention is not limited thereto. In the embodiment of the invention, the XCL1 is humanized XCL1 #hXCL1Gene ID 6375); in some embodiments, the XCL1 protein is the 1 st to 114 th positions of XCL1, and the amino acid sequence is shown in SEQ ID NO: shown at 7.
In the fusion protein, GP100 is taken as an example, and the expansion effect of XCL1 on an epitope is verified. The GP100 is a melanin surface antigen, and in some embodiments, the amino acid sequence of the GP100 is murine GP100 (Gene ID: 20431) or human GP100 (Gene ID: 6490). In some specific embodiments, the alpha chain amino acid sequence of GP100 protein is intercepted as an immune antigen, the amino acid sequence of the alpha chain is intercepted in the amino acid sequence of wild type GP100 (shown as SEQ ID NO: 3) for enhancing antigenicity, the 25-467 th amino acid of the alpha chain is intercepted, and T210M mutation occurs, and the sequence is shown as SEQ ID NO: 8.
To better maintain activity, linker was included between XCL1 and antigen. The linker fragment is not limited in the present invention, and in some embodiments, G is used between the antigen and XCL1 protein 5 SG 5 The peptide fragments are fused.
In some embodiments, the amino acid sequence of the fusion protein is set forth in SEQ ID NO. 1.
In some embodiments, to facilitate the observation of fusion protein localization information in cells and in vivo, the fusion protein is further fused with red fluorescent protein mCherry.
The invention also provides nucleic acids encoding the fusion proteins. In some embodiments, the nucleic acid encoding the fusion protein has the sequence set forth in SEQ ID NO: 2.
The invention provides a vector, which comprises a vector skeleton and the nucleic acid.
pcDNA3.1 (-) is often used as a protein expression vector in mammalian cells.
In the vector, the vector skeleton comprises pcDNA3.1 (-).
The invention also provides a host transformed or transfected with the vector.
The host includes mammalian cells. The mammalian cells include cells of human origin and cells of murine origin.
The invention also provides application of the fusion protein, the nucleic acid, the vector and/or the host in preparing anti-melanoma medicines.
The invention also provides an anti-melanoma medicine, which comprises at least one of the following I-IV:
I. the fusion protein disclosed by the invention;
II. Nucleic acids of the invention;
III, the carrier of the invention;
IV, a host according to the invention.
In some embodiments, the method of preparing the above medicament comprises: fusing XCL1 and GP100 at the nucleic acid level, and then connecting the fused nucleic acid with a vector to construct a protein expression vector; the protein expression vector is then transformed or transfected into the corresponding cell to obtain the corresponding host. The preparation process involves nucleic acid, vector, protein and host, and can be used for preparing vaccine or other medicines for treating melanoma.
The invention also provides a method for treating melanoma, which comprises the step of administering the medicament.
The invention constructs a fusion protein of a specific antigen and XCL1, specifically selects GP100 as a model antigen, constructsXCL1/GP100Fused plasmid. Then byXCL1/GP100Plasmid-immunized melanoma model mice, including Pmel transgenic mice and wild-type C57 mice, can more effectively inhibit tumor growth than GP100 by fusion with XCL1, which verifies that XCL1 enhances antigen presentation and enhances the immune effect of GP 100. In addition to that, useXCL1/GP100The plasmid immunized mice were stimulated with OVA, GP100 and CSC to stimulate T cells of the mice, foundXCL1/GP100The plasmid can induce the organism to generate cellular immunity aiming at target antigen and also can generate cellular immunity aiming at non-immune target antigen OVA, which shows that the fusion of XCL1 can not only enhance antigen presentation, but also expand T cell antigen epitope.
Drawings
FIG. 1 shows a schematic diagram of a fusion gene;
FIG. 2 shows the fusion protein to human CD141 + Chemotaxis of DC cells;
FIG. 3 shows cells that ingest antigen in lymph nodes after immunization with XCL1/mCherry and mCherry proteins;
FIG. 4 showsXCL1/GP100Immunization induces specific cellular immunity against non-immune target antigens;
FIG. 5 shows that in the B16-OVA tumor-bearing mouse (Pmel-transgenic mouse) model,XCL1/GP100the immunity can effectively inhibit the growth of tumor;
FIG. 6 shows that in the B16-OVA oncological mouse (wild type C57 mouse) model,XCL1/GP100the immunity can effectively inhibit tumor growth.
Detailed Description
The invention provides a tumor vaccine capable of expanding T cell epitope and a preparation method thereof, and a person skilled in the art can refer to the content of the tumor vaccine and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 construction of XCL1/GP100 fusion molecules and XCL1/mCherry fusion indicator molecules
Since the homology of the amino acid sequence of human GP100 (Gene ID: 6490) and mouse GP100 (Gene ID: 20431) reaches 76%, the GP100 in the present invention is all human. The human GP100 protein consists of signal peptide (1-24), alpha chain (25-467) and beta chain (470-661). The amino acid sequence of the alpha chain of the GP100 protein is intercepted as an immune antigen, and simultaneously, in order to enhance antigenicity, the 210 th amino acid of the GP100 is modified and mutated from threonine (Thr, T) to methionine (Met, M).
The invention is based on human XCL1hXCL1Gene ID 6375) and sequence information of human GP100, the carboxy-terminus of full-length hXCL1 was designed to be passed through Glycine 5 -Serine-Glycine 5 （G 5 SG 5 ) The peptide fragment was linked to the amino terminus of the alpha chain (25-467, T210M) of GP100, thereby constructingXCL1Sequence and sequenceGP100Fusion molecules of sequences, designatedXCL1/GP100A nucleic acid fragment. (A in FIG. 1). The alpha chain of the unfused GP100 protein (25-467) was designated as the experimental control group (without T210M mutation)GP100Nucleic acid fragment (A in FIG. 1).
XCL1The amino acid sequence of (2) is shown as SEQ ID NO. 7:
MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG
the amino acid sequence of the alpha chain (25-467, T210M) of GP100 is shown in SEQ ID NO: 8:
KVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTIMDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLV
XCL1/GP100the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1,
MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTGGGGGGSGGGGGKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTIMDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLV*
XCL1/GP100the coding nucleic acid sequence of the fusion protein is shown as SEQ ID NO. 2,
ATGAGGCTGCTGATCCTGGCCCTGCTGGGCATCTGCTCCCTGACAGCCTACATCGTGGAGGGCGTGGGCTCTGAGGTGAGCGACAAGCGCACCTGCGTGAGCCTGACCACACAGCGGCTGCCCGTGAGCAGAATCAAGACATATACCATCACAGAGGGCTCTCTGAGGGCCGTGATCTTCATCACAAAGCGCGGCCTGAAGGTGTGCGCAGATCCTCAGGCAACCTGGGTGCGCGACGTGGTGCGGTCCATGGATAGAAAGTCTAACACCAGGAACAATATGATCCAGACAAAGCCAACCGGCACACAGCAGTCCACCAATACAGCCGTGACCCTGACAGGAGGAGGAGGAGGAGGATCTGGAGGAGGAGGCGGCAAAGTACCCAGAAACCAGGACTGGCTTGGTGTCTCAAGGCAACTCAGAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGTGGACAGAAGCCCAGAGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGATGGGCCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACTTCCCTGGAAGCCAAAAGGTATTGCCAGATGGGCAGGTTATCTGGGTCAACAATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGCCAGTGTATCCCCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCATCTGGCTCTTGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGGGGCCAATACTGGCAAGTTCTAGGGGGCCCAGTGTCTGGGCTGAGCATTGGGACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTGTCTACCATCGCCGGGGATCCCGGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCCTTCACCATTATGGACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCGGGCCTTGGATGGAGGGAACAAGCACTTCCTGAGAAATCAGCCTCTGACCTTTGCCCTCCAGCTCCATGACCCTAGTGGCTATCTGGCTGAAGCTGACCTCTCCTACACCTGGGACTTTGGAGACAGTAGTGGAACCCTGATCTCTCGGGCACTTGTGGTCACTCATACTTACCTGGAGCCTGGCCCAGTCACTGCCCAGGTGGTCCTGCAGGCTGCCATTCCTCTCACCTCCTGTGGCTCCTCCCCAGTTCCAGGCACCACAGATGGGCACAGGCCAACTGCAGAGGCCCCTAACACCACAGCTGGCCAAGTGCCTACTACAGAAGTTGTGGGTACTACACCTGGTCAGGCGCCAACTGCAGAGCCCTCTGGAACCACATCTGTGCAGGTGCCAACCACTGAAGTCATAAGCACTGCACCTGTGCAGATGCCAACTGCAGAGAGCACAGGTATGACACCTGAGAAGGTGCCAGTTTCAGAGGTCATGGGTACCACACTGGCAGAGATGTCAACTCCAGAGGCTACAGGTATGACACCTGCAGAGGTATCAATTGTGGTGCTTTCTGGAACCACAGCTGCACAGGTAACAACTACAGAGTGGGTGGAGACCACAGCTAGAGAGCTACCTATCCCTGAGCCTGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAGTATTACAGGTTCCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGGCTGGTG TAA
the amino acid sequence of GP100 is shown as SEQ ID NO. 3,
MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLV*
the coding nucleic acid sequence of GP100 is shown as SEQ ID NO. 4,
ATGGATCTGGTGCTAAAAAGATGCCTTCTTCATTTGGCTGTGATAGGTGCTTTGCTGGCTGTGGGGGCTACAAAAGTACCCAGAAACCAGGACTGGCTTGGTGTCTCAAGGCAACTCAGAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGTGGACAGAAGCCCAGAGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGATGGGCCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACTTCCCTGGAAGCCAAAAGGTATTGCCAGATGGGCAGGTTATCTGGGTCAACAATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGCCAGTGTATCCCCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCATCTGGCTCTTGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGGGGCCAATACTGGCAAGTTCTAGGGGGCCCAGTGTCTGGGCTGAGCATTGGGACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTGTCTACCATCGCCGGGGATCCCGGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCCTTCACCATTACTGACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCGGGCCTTGGATGGAGGGAACAAGCACTTCCTGAGAAATCAGCCTCTGACCTTTGCCCTCCAGCTCCATGACCCTAGTGGCTATCTGGCTGAAGCTGACCTCTCCTACACCTGGGACTTTGGAGACAGTAGTGGAACCCTGATCTCTCGGGCACTTGTGGTCACTCATACTTACCTGGAGCCTGGCCCAGTCACTGCCCAGGTGGTCCTGCAGGCTGCCATTCCTCTCACCTCCTGTGGCTCCTCCCCAGTTCCAGGCACCACAGATGGGCACAGGCCAACTGCAGAGGCCCCTAACACCACAGCTGGCCAAGTGCCTACTACAGAAGTTGTGGGTACTACACCTGGTCAGGCGCCAACTGCAGAGCCCTCTGGAACCACATCTGTGCAGGTGCCAACCACTGAAGTCATAAGCACTGCACCTGTGCAGATGCCAACTGCAGAGAGCACAGGTATGACACCTGAGAAGGTGCCAGTTTCAGAGGTCATGGGTACCACACTGGCAGAGATGTCAACTCCAGAGGCTACAGGTATGACACCTGCAGAGGTATCAATTGTGGTGCTTTCTGGAACCACAGCTGCACAGGTAACAACTACAGAGTGGGTGGAGACCACAGCTAGAGAGCTACCTATCCCTGAGCCTGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAGTATTACAGGTTCCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGGCTGGTG TAA
in order to facilitate the observation of fusion protein localization information both in cells and in vivo, the present invention is based on miceXcl1(mXcl1Gene ID 16963) and red fluorescent protein mCherry (Biovison, cat: 4993-100), constructionmXcl1AndmCherryis named as fusion molecule of (C)mXcl1/mCherryNucleic acid fragment，Meanwhile, the single mCherry is used as an experimental control group and is named asmCherryNucleic acid fragments, experiments and analyses were performed. (B in FIG. 1), and expressed in a mammalian cell system, the above-mentioned nucleic acid sequence was constructed into a vector of pcDNA3.1 (-), and strep sequence (AGCGCCTGGAGCCACCCTCAGTTCGAGAAG) and stop codon (UGA) were added at the end of the gene sequence. And then transfecting the plasmid expression vector into HEK293T cells for amplified expression. Finally, obtaining the fusion protein by purifying by using strep affinity chromatography.
mXcl1/mCherryThe nucleic acid fragment is shown in SEQ ID NO. 5:
ATGAGACTTCTCCTCCTGACTTTCCTGGGAGTCTGCTGCCTCACCCCATGGGTTGTGGAAGGTGTGGGGACTGAAGTCCTAGAAGAGAGTAGCTGTGTGAACTTACAAACCCAGCGGCTGCCAGTTCAAAAAATCAAGACCTATATCATCTGGGAGGGGGCCATGAGAGCTGTAATTTTTGTCACCAAACGAGGACTAAAAATTTGTGCTGATCCAGAAGCCAAATGGGTGAAAGCAGCGATCAAGACTGTGGATGGCAGGGCCAGTACCAGAAAGAACATGGCTGAAACTGTTCCCACAGGAGCCCAGAGGTCCACCAGCACAGCGATAACCCTGACTGGGGGCGGAGGCGGTGGATCAGGAGGTGGCGGAGGCGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG
the mCherry nucleic acid fragment is shown as SEQ ID NO. 6:
GTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAG
designed as aboveXCL1/GP100、GP100、mXcl1/mCherryAndmCherrynucleic acid fragments (as shown in FIG. 1) are all linked to eukaryotic expressionOn the vector pcDNA3.1 (-).
EXAMPLE 2 XCL1/GP100 fusion protein potent chemotactic human XCR1 + CD141 + DC cell
Preparation of cell lysate: the respective uses containXCL1/GP100 and GP100Expression vector for nucleic acid fragment transfected 293T cells, after 68 hours, washed 2 times with PBS, cells were collected under aseptic conditions every 10 6 The cells were added to 100. Mu.l of PBS and resuspended, and then lysed by repeated rapid thawing 3 times at-80 ℃.12000 Centrifugation was performed at 4℃for 20 min at rpm, the supernatant of the lysate was collected, protein quantification was performed using Pierce BCA Protein Assay Kit, and the concentration of the cell lysate was adjusted to 420. Mu.g/ml with RPMI-1640 medium containing 10% FBS.
Chemotaxis experiments: human lymphocytes were derived from a small fraction of subcutaneous draining lymph node tissue isolated during papillary thyroid tumor surgery, and after digestion with collagenase D (Roche, cat: 1088858001) a single cell suspension was obtained, resuspended in RPMI-1640 medium containing 10% FBS, and the cell concentration was adjusted to: 10 7 And each ml. 100. Mu.l of the cell suspension (i.e., 10 in the presence of the cell suspension) was added to the upper chamber of a Transwell cell culture plate (Corning, cat: 3421) having a pore size of 5. Mu.m 6 Individual cells); 600. Mu.l of the cell lysate of different plasmid origin prepared above (i.e.containing 250. Mu.g of cell lysate) was added to the chemotactic lower chamber. Cell culture medium was used as negative control. After chemotaxis for 90min at 37deg.C, cells in the lower chamber were collected for flow staining and counting, and fusion protein was analyzed for human CD141 + Chemotaxis of DCs.
The results are shown in FIG. 2, with A being migration CD141 + Flow chart of DC cell ratios, panel B shows CD141 migration for different conditioned media treated groups + Fold statistics of DC cells compared to spontaneous migration group. As can be seen, XCL1/GP100 is effective in chemotactic for human CD141 compared to GP100 alone + DC cells, which indicate that the fusion protein still retains the chemotactic activity of XCL1, demonstrating the ability of the fusion protein to target professional antigen cross-presenting cell CD141 expressing XCR1 + DC。
Example 3 XCL1 fusion proteins are effective in promoting secondaryPrimary CD8 alpha in grade lymphoid tissue + DC acquisition antigen
To analyze the effect of XCL1 fusion on the antigen presenting process of professional antigen presenting cells, we prepared the red fluorescent proteins mCherry and XCL1/mCherry fusion proteins described in example 1. Then, 5 μg of XCL1/mCherry fusion protein or mCherry protein was subcutaneously injected into C57 mice, respectively, locally draining lymph nodes were obtained 1h and 48h after immunization, respectively, and then cell types and numbers of uptake antigens in the locally draining lymph nodes were analyzed by flow cytometry.
The results are shown in FIG. 3, wherein A shows MHC-II + Proportion of antigen uptake by cells and MHCII cells uptake of antigen in lymph nodes (mcherry + MHCII + ) Is a number of (3). Wherein B shows DC cells (CD 11c + MHCII + ) Proportion of antigen uptake and DC cells that uptake antigen in lymph nodes (mcherry + CD11c + MHCII + ) Is a number of (3). Wherein C represents CD8 alpha + DC cells (CD 8 alpha) + CD11c + MHCII + ) Proportion of antigen uptake and CD8α antigen uptake in lymph node + DC cells (mcherry) + CD8α + CD11c + MHCII + ) Is a number of (3).
Analytical experiments show that compared with the mCherry protein immune group, the XCL1/mCherry fusion protein immune group has more MHC-II + The cells obtained antigen (A in FIG. 3). Further analysis found that MHC-II in the acquisition of antigen + In cells, DC (MHCII) + CD11c + ) Cells carry more antigen information (B in fig. 3). And antigen-carrying DC cells in immunized mice increased over time and were 3.3 times more numerous in the XCL1/mCherry fusion protein immunized group than in the mCherry immunized group. Professional antigen cross-presenting cells CD8 alpha + DC is located in secondary lymphoid tissue and selectively expresses receptor XCR1, and the invention further analyzes the XCL1 fusion protein to CD8 alpha + As a result of the fact that DC cells deliver antigen information, XCL1/mCherry fusion proteins deliver antigen to CD8 a faster than mCherry proteins + DC cells, and can promote over timeMore CD8 alpha + DC cells acquired antigen information (C in FIG. 3), which was 6.3 times the number of mCherry immunized groups.
This phenomenon suggests that XCL1 fusion proteins can deliver more and faster antigen information to professional antigen cross-presenting cells, which will facilitate antigen cross-presentation and induce specific CD8 + Immune response of T cells.
EXAMPLE 4,XCL1/GP100Inducing mice to generate specific immune cells aiming at non-target antigens, and expanding T cell antigen epitope.
The tumor vaccine can induce the organism to generate specific cellular immunity aiming at the immune target antigen, and remove tumor cells expressing the immune target antigen, however, at the same time, immune escape possibly occurs due to heterogeneity of the tumor cells, so that the vaccine loses protection.
The GP100 antigen and the OVA antigen can be expressed in B16-OVA cells, wherein GP200GP100 is a marker of melanoma. Therefore, the invention is based on a B16-OVA melanoma mouse plantation tumor model, uses GP100 as an immune target antigen, uses OVA as an indicative non-immune target antigen, and analyzes the fusion pair CD8 of XCL1 + Expansion of epitopes of T cells.
In the invention, the method for establishing the B16-OVA melanoma planting model in the mice comprises the following steps: subcutaneous inoculation of corresponding mice with 5X 10 4 After B16-OVA cells had developed with macroscopic tumors, mice were immunized using a gene gun. The immunization method comprises the following steps: injection with a low pressure gene gun (GDS-80, wealtec) as described in example 1XCL1/GP100Plasmid(s)、GP100Plasmid or vector plasmid, 40 μg of plasmid per mouse was injected 3 times at 8 day intervals at 40psi pressure. Peripheral blood or spleen cells from mice were isolated 7 days after the 3 rd immunization for analysis.
First, a B16-OVA melanoma plantation model was established in Pmel transgenic mice, followed by the use ofXCL1/GP100Plasmid and method for producing the sameGP100The plasmids were immunized separately with the mice in which the melanoma model was grown. Then separate respectivelyXCL1/GP100AndGP100peripheral blood cells of immunized mice are treated with erythrocyte lysateAfter (ACK) lysis, cells of the 2 groups were stimulated with 2. Mu.M CFSE label, then with OVA antigen (Sigma-Aldrich), GP100 antigen (MedChemexpress) and cell culture medium (medium) for 72h, respectively, and finally analyzed for CD8 by flow cytometry + T cell proliferation. The results are shown in FIG. 4A, compared toGP100The plasmid is used for the preparation of the plasmid,XCL1/GP100more CD8 in plasmid immunized group + T cells proliferate upon stimulation with GP100 antigen (target antigen), indicatingXCL1/GP100Plasmid immunization effectively amplifies pre-existing antigen-specific CD8 in organism + T cells. More importantly, thanGP100The plasmid is used for the preparation of the plasmid,XCL1/GP100mouse peripheral blood cells after plasmid immunization have more CD8 after OVA stimulation + Proliferation of T cells occurs, indicatingXCL1/GP100Plasmid immunization can induce the body to produce CD8 against OVA antigen (non-target antigen) + T cells (a in fig. 4).
Next, analysis is performed in an in vivo environmentXCL1/GP100Immune expansion T cell antigen recognition epitope condition. The present invention establishes a B16-OVA melanoma plantation model in wild type C57 (CD 45.2) mice, each using the method described in example 1XCL1/GP100Plasmid and pcDNA3.1 (-) vector plasmid (Mock) mice were immunized and peripheral blood cells of the immunized mice were isolated. Then the obtained 2 groups of blood cells are lysed by erythrocyte lysate (ACK) and marked by using 3 mu M CFSE, then the obtained blood cells are respectively delivered into a receptor (CD 45.1) mouse body by tail vein, 100 mu g OVA protein is injected into the abdominal cavity of the receptor mouse, and after 48 hours, the donor CD45.2 in the receptor mouse body is analyzed by using flow cytometry + CD8 + T cell proliferation. The results are shown in FIG. 4B, and can be seen fromXCL1/GP100Plasmid-immunized donor mice had more CD8 after stimulation of immune cells with OVA antigen + T-cell proliferation was observed, which was not observed in the empty plasmid immunized group (B in FIG. 4), indicatingXCL1/GP100Immune energy induces the body to produce specific CD8 against OVA antigen (non-target antigen) + T cells.
Then, B16-OVA melanoma was modeled in wild-type C57 mice, each using example 1The said processXCL1/GP100The mice were immunized with plasmid or pcDNA3.1 (-) empty vector (Mock), three times each, and spleen cells from the immunized mice were isolated. Then, non-specific cell stimulators (Cell stimulator cocktail, CSC), GP100 antigen and OVA antigen were added to the 2 isolated spleen cells, respectively, for 60h of stimulation culture, and then IFN-gamma was analyzed by flow cytometry + CD8 + T cell ratio. The results are shown in fig. 4C, which, after CSC stimulation,XCL1/GP100more CD8 was found in spleen tissue of the immunized group + T cells secrete the effector cytokine IFN- γ; more importantly, after stimulation with GP100 antigen (target antigen) or OVA antigen (non-target antigen), both were observedXCL1/GP100Spleen tissue of immunized mice had more CD8 + T cells secrete the effector cytokine IFN-gamma, indicatingXCL1/GP100Immunization can induce specific cellular immunity against immune target antigen (GP 100) and can induce the organism to generate specific cellular immunity against non-immune target antigen (OVA), and expand T cell antigen recognition epitope (C in figure 4).
The possible reasons and mechanisms for this effect were analyzed as follows: the XCL1 fusion molecule can effectively promote cDC1 cells to take up antigen and deliver antigen information into local drainage lymph nodes, induce specific cellular immunity aiming at immune target antigen, and kill tumor cells. At the same time, the XCL1 fusion molecule increases the content of chemotactic factor XCL1 infiltrated in tumor after immunization, and can increase the infiltration of XCR1 in tumor + Number of cDC1 cells. When tumor cells are broken to release a large amount of target antigens, the tumor cells are more easily taken up by professional antigen cross-presenting cells and then carried to secondary lymphoid tissues, and the organism is induced to generate specific CD8 aiming at non-immune target antigens + T cells.
While tumor cells expressing the target antigen are cleared, the disrupted tumor cells release a large amount of tumor antigen, which, if capable of eliciting a cascade of immune responses, induces specific CD8 against the non-immune target antigen + T cells can possibly overcome the vaccine invalidity caused by the problems of tumor heterogeneity, antigen modulation and the like, and enhance the anti-tumor effect. Fusion of XCL1 with antigen is possible as a cascade of immunological reactionsOccurrence provides a condition.
Example 5 XCL1/GP100 was effective in inhibiting tumor growth in a melanoma mouse tumor-bearing model
Subcutaneous inoculation of mice with 5X 10 4 B16-OVA cells, a mouse melanoma subcutaneous implantation tumor model is established, and after macroscopic tumor growth is achieved, a gene gun is used for immunization of the mouse. Specifically, respectively using those described in example 1XCL1/ GP100Plasmid(s),GP100The plasmid and pcDNA3.1 (-) vector plasmid (Mock, as a control) were immunized against the tumor-bearing mice, 40 μg of plasmid DNA per mouse, 1 time per 8 days, and 3 total times. During this period, tumor length (L) and width (W) were measured every 3-4 days with vernier calipers, and tumor volumes of mice were calculated: v (mm) 3 )=L×W 2 2; subcutaneous tumor formation was observed at the end of the experiment for 40 days, when tumor volume reached 2000 mm 3 Mice were sacrificed at that time and survival of the mice was recorded. Tumor volume fold change (fold) =tumor volume per measurement/tumor initial volume was analyzed.
As a result, as shown in FIGS. 5 and 6, in the model of Pmel-transgenic mouse melanoma, mice were vaccinated and immunized as shown in FIG. 5A, and observedXCL1/GP100Plasmid(s),GP100After the plasmids and pcDNA3.1 (-) vector plasmid (Mock) are respectively immunized, the tumor growth and survival condition of the mice are realized; in fig. 5, B and C are the tumor volume changes of each group of mice; in fig. 5, D is a mouse survival curve. Each group of 6 mice. In the wild type C57 mouse melanoma model, the mice were tumor inoculated and immunized as shown in FIG. 6A, and observedXCL1/GP100Plasmid(s),GP100After the plasmid and pcDNA3.1 (-) vector plasmid are respectively immunized, the tumor growth and survival condition of the mice are realized. Fig. 6B and C show the tumor volume change in each group of mice; d in FIG. 6 is a mouse survival curve. 11 mice per group. It can be seen that, in both Pmel transgenic mice and wild-type C57 mice, compared to the pcDNA3.1 (-) vector plasmid,GP100plasmid and method for producing the sameXCL1/GP100Plasmid immunization can inhibit the volume increase of tumors to a certain extent. However, the process is not limited to the above-mentioned process,XCL1/GP100the antitumor effect of the plasmid is obviously better than that of the GP100 plasmid. At the same timeThis indicatesXCL1/GP100The immunity can induce the anti-tumor immune response generated from the head, amplify the pre-existing anti-tumor response in the body and play a role in inhibiting the growth of tumor.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (14)
- Application of XCL1 in preparing medicines for expanding T cell epitope.
- 2. The use of claim 1, wherein the antigen comprises a tumor antigen.
- 3. The use according to claim 1 or 2, wherein the antigen comprises the melanoma antigen GP100; the expanded T cell epitope comprises: on the basis of the epitope corresponding to the GP100 antigen, the epitope corresponding to the OVA antigen is expanded.
- 4. The medicine for expanding the T cell epitope is characterized by comprising at least one of the following components:a. a fusion protein of an antigen and XCL 1;b. nucleic acids encoding the fusion proteins;c. a vector comprising the nucleic acid;d. a host transformed or transfected with the vector.
- 5. The preparation method of the drug for expanding the T cell epitope is characterized by comprising at least one of the following steps of:i. synthesizing nucleic acid for encoding antigen and XCL1 fusion protein;ii. Constructing a vector containing the fusion protein nucleic acid sequence;iii, constructing a host expressing the antigen and XCL1 fusion protein with said nucleic acid or vector;iv expressing and purifying the fusion protein of antigen and XCL1 using said host.
- 6. A fusion protein, comprising: XCL1 peptide, linker peptide and GP100 peptide.
- 7. The fusion protein according to claim 6, wherein the amino acid sequence of XCL1 is as set forth in SEQ ID NO: shown in figure 7; the amino acid sequence of the GP100 is shown as SEQ ID NO:3 or is shown as SEQ ID NO. 8.
- 8. A nucleic acid encoding the fusion protein of any one of claims 6 to 7.
- 9. A vector comprising a vector backbone and the nucleic acid of claim 8.
- 10. The vector of claim 9, wherein the vector backbone comprises pcdna3.1 (-).
- 11. A host transformed or transfected with the vector of claim 9 or 10.
- 12. The host of claim 11, comprising mammalian cells.
- 13. Use of a protein according to any one of claims 6 to 7, a nucleic acid according to claim 8, a vector according to claim 9 or 10 and/or a host according to claim 11 or 12 for the preparation of an anti-melanoma medicament.
- 14. An anti-melanoma medicine is characterized by comprising at least one of the following 1-4:I. the protein of any one of claims 6 to 7;II. The nucleic acid of claim 8;the vector of claim III, 9 or 10;IV, the host of claim 11 or 12.
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