Disclosure of Invention
In order to solve the defects in the prior art that KRAS mutant TCR-T medicines are not developed aiming at HLA-A1101 typing, the application provides a T Cell Receptor (TCR), wherein the T cell receptor can specifically identify A1101 restriction-KRASG12D mutation, and T cells (TCR-T) which are transduced with the TCR can be combined with an antigen short peptide KRASG12D -HLA-A1101 complex, specifically kill tumor cells aiming at tumor antigens, and can be used for treating malignant tumors carrying KRASG12D mutation. Meanwhile, the application also provides application of the T cell receptor.
In particular, the application relates to the following aspects:
1. A T Cell Receptor (TCR), wherein the TCR comprises an alpha chain comprising a variable region and/or a beta chain comprising a variable region,
The variable region of the alpha chain comprises complementarity determining region 1 (CDR 1) having the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and/or
The amino acid sequence is complementarity determining region 2 (CDR 2) as shown in SEQ ID NO.3 or SEQ ID NO. 4, and/or
The amino acid sequence is complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 5 or SEQ ID NO. 6.
2. The T Cell Receptor (TCR) of item 1, wherein,
The variable region of the beta strand comprises complementarity determining region 1 (CDR 1) having the amino acid sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8, and/or
The amino acid sequence is complementarity determining region 2 (CDR 2) as shown in SEQ ID NO 9 or SEQ ID NO 10, and/or
The amino acid sequence is complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 11 or SEQ ID NO. 12.
3. The T Cell Receptor (TCR) according to item 1 or 2, wherein,
The variable region of the alpha chain further comprises a first leader sequence, and/or
The variable region of the beta strand further comprises a second leader sequence,
Preferably, the amino acid sequence of the variable region of the alpha chain is as shown in SEQ ID NO. 13 or SEQ ID NO. 14, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 13 or SEQ ID NO. 14, and/or the amino acid sequence of the variable region of the beta chain is as shown in SEQ ID NO. 15 or SEQ ID NO. 16, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 15 or SEQ ID NO. 16,
Preferably, the alpha chain further comprises an alpha constant region and/or the beta chain further comprises a beta constant region, preferably the constant region is a mouse constant region or a human constant region.
4. The T Cell Receptor (TCR) of any one of claims 1-3, wherein the TCR is isolated or purified or recombinant;
Preferably, the TCR is human;
preferably, the TCR is monoclonal;
preferably, the TCR is single chain;
preferably, the TCR comprises two chains;
Preferably, the TCR is in a cell-bound form or in a soluble form, preferably in a soluble form;
preferably, the TCR binds to an antigen oligopeptide-HLA-A 1101 complex, preferably the amino acid sequence of the antigen oligopeptide is shown in SEQ ID NO. 1.
5. A nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the TCR of any one of claims 1-4, or an alpha or beta chain of the TCR.
6. The nucleic acid molecule according to item 5, wherein the nucleotide sequence encoding the alpha chain is the nucleotide sequence shown as SEQ ID NO. 17 or SEQ ID NO. 18, and/or
The nucleotide sequence of the coded beta chain is shown as SEQ ID NO. 19 or SEQ ID NO. 20.
7. A vector, wherein the vector comprises the nucleic acid molecule of item 5 or 6.
8. The vector according to item 7, wherein the vector is an expression vector;
preferably, the vector is a viral vector, preferably a retroviral vector;
Preferably, the viral vector is a lentiviral vector.
9. An engineered cell comprising the TCR of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-6, or the vector of any one of claims 7-8.
10. The engineered cell of claim 9, wherein the TCR is heterologous to the cell;
preferably, the engineered cell is a cell line;
preferably, the engineered cell is a primary cell obtained from a subject, preferably the subject is a mammalian subject, preferably a human;
preferably, the engineered cells are T cells or NK cells, preferably, the T cells are T cells isolated from peripheral blood;
Preferably, the T cells are cd8+ or cd4+.
11. A method of producing an engineered cell of any one of claims 9-10, comprising introducing the nucleic acid molecule of any one of claims 5-6 or the vector of any one of claims 7-8 into a cell in vitro or ex vivo.
12. The method of item 11, wherein the vector is a viral vector and the introducing is by transduction.
13. A pharmaceutical composition comprising the T Cell Receptor (TCR) of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-6, the vector of any one of claims 7-8, or the engineered cell of any one of claims 9-10;
Preferably, it further comprises a pharmaceutically acceptable carrier or adjuvant.
14. Use of the T Cell Receptor (TCR) of any one of claims 1-4, the nucleic acid molecule of any one of claims 5-6, the vector of any one of claims 7-8, the engineered cell of any one of claims 9-10, or the pharmaceutical composition of claim 13 in the manufacture of a medicament for treating a malignancy;
Preferably, the malignancy is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, prostate cancer.
Effects of the invention
The T Cell Receptor (TCR) can specifically identify the A1101 restriction-KRASG12D mutation, and the T cell (TCR-T) transduced with the TCR can be combined with an antigen short peptide KRASG12D -HLA-A1101 complex, specifically kills tumor cells aiming at tumor antigens, and can be used for treating malignant tumors carrying the KRASG12D mutation.
And the T cells transduced with the TCR of the application can be specifically activated by tumor cells expressing A11 and KRASG12D mutations, and have good specificity, and only recognize KRASG12D mutations, but not wild-type KRAS and KRASG12V mutations.
Detailed Description
The present application will now be described in detail with reference to the embodiments thereof as illustrated in the accompanying drawings, wherein like numerals refer to like features throughout. While specific embodiments of the application are shown in the drawings, it should be understood that the application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.
It should be noted that certain terms are used throughout the description and claims to refer to particular components. Those of skill in the art will understand that a person may refer to the same component by different names. The specification and claims do not identify differences in terms of components, but rather differences in terms of the functionality of the components. As referred to throughout the specification and claims, the terms "include" or "comprising" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description hereinafter sets forth a preferred embodiment for practicing the application, but is not intended to limit the scope of the application, as the description proceeds with reference to the general principles of the description. The scope of the application is defined by the appended claims.
The application provides a T Cell Receptor (TCR), wherein the TCR comprises an alpha chain comprising a variable region and/or a beta chain comprising a variable region, the variable region of the alpha chain comprising a complementarity determining region 1 (CDR 1) having an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2, and/or a complementarity determining region 2 (CDR 2) having an amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4, and/or a complementarity determining region 3 (CDR 3) having an amino acid sequence as shown in SEQ ID NO. 5 or SEQ ID NO. 6,
The amino acid sequence shown in SEQ ID NO.1 is TRDTTYY;
the amino acid sequence shown in SEQ ID NO. 2 is SSVSVY;
the amino acid sequence shown in SEQ ID NO. 3 is RNSFDEQN;
the amino acid sequence shown in SEQ ID NO. 4 is YLSGSTLV;
the amino acid sequence shown in SEQ ID NO. 5 is ALSEAAPGGSYIPT;
The amino acid sequence shown in SEQ ID NO. 6 is AVIGNDYKLS.
In one embodiment, the variable region of the β strand comprises complementarity determining region 1 (CDR 1) having an amino acid sequence as shown in SEQ ID NO. 7 or SEQ ID NO. 8, and/or complementarity determining region 2 (CDR 2) having an amino acid sequence as shown in SEQ ID NO. 9 or SEQ ID NO. 10, and/or complementarity determining region 3 (CDR 3) having an amino acid sequence as shown in SEQ ID NO. 11 or SEQ ID NO. 12,
The amino acid sequence shown in SEQ ID NO. 7 is MDHEN;
the amino acid sequence shown in SEQ ID NO. 8 is SNHLY;
the amino acid sequence shown in SEQ ID NO. 9 is SYDVKM;
the amino acid sequence shown in SEQ ID NO. 10 is FYNNEI;
The amino acid sequence shown in SEQ ID NO.11 is ASSLGPGQHNSPLH;
the amino acid sequence shown in SEQ ID NO. 12 is ASSGTGGIEAF.
In one embodiment, the variable region of the alpha chain further comprises a first leader sequence and/or the variable region of the beta chain further comprises a second leader sequence. The first leader sequence of the variable region of the alpha chain and the second leader sequence of the variable region of the beta chain are well known to those skilled in the art, for example, the first leader sequence of the variable region of the alpha chain may use the leader sequence shown as SEQ ID NO. 21 or SEQ ID NO. 22, the second leader sequence of the variable region of the beta chain may use the leader sequence shown as SEQ ID NO. 23 or SEQ ID NO. 24,
The amino acid sequence shown in SEQ ID NO. 21 is MLTASLLRAVIASICVVSSM;
the amino acid sequence shown in SEQ ID NO. 22 is MLLLLVPAFQVIFTLGGTR;
the amino acid sequence shown in SEQ ID NO. 23 is MGIRLLCRVAFCFLAVGLV;
the amino acid sequence shown in SEQ ID NO. 24 is MDTWLVCWAIFSLLKAGLT;
In one embodiment, the amino acid sequence of the alpha chain variable region is as shown in SEQ ID NO.13 or SEQ ID NO. 14, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO.13 or SEQ ID NO. 14, and/or the amino acid sequence of the beta chain variable region is as shown in SEQ ID NO. 15 or SEQ ID NO. 16, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO. 15 or SEQ ID NO. 16,
The amino acid sequence shown in SEQ ID NO. 13 is ：MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAAPGGSYIPTFGRGTSLIVHPY;
The amino acid sequence shown in SEQ ID NO. 14 is ：MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVESINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAVIGNDYKLSFGAGTTVTVRAN;
The amino acid sequence shown in SEQ ID NO. 15 is ：MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLGPGQHNSPLHFGNGTRLTVT;
The amino acid sequence shown in SEQ ID NO. 16 is ：MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSGTGGIEAFFGQGTRLTVV.
The amino acid sequence of the alpha chain variable region has at least 90% sequence identity to SEQ ID NO. 13 or SEQ ID NO. 14 and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity to SEQ ID NO. 13 or SEQ ID NO. 14. The amino acid sequence of the variable region of the β chain has at least 90% sequence identity to SEQ ID NO. 15 or SEQ ID NO. 16, and may be an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity to SEQ ID NO. 15 or SEQ ID NO. 16.
In one embodiment, the alpha chain further comprises an alpha constant region and/or the beta chain further comprises a beta constant region, preferably the constant region is a mouse constant region or a human constant region. For example, the amino acid sequence of the mouse alpha constant region is shown as SEQ ID NO:25, and/or the amino acid sequence of the mouse beta constant region is shown as SEQ ID NO:26, i.e., the constant regions for the alpha chain of the TCR described above may all have the same constant region, and similarly, the constant regions for the beta chain of all TCRs may also all have the same constant region. The amino acid shown in SEQ ID NO. 25 is ：IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS;
The amino acid shown in SEQ ID NO. 26 is ：EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS.
The constant region of the TCR may contain a short linking sequence in which the cysteine residues form a disulfide bond, thereby linking the two chains of the TCR. The TCR may have additional cysteine residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant region.
In one embodiment, the alpha chain variable region of the TCR has an amino acid sequence as set forth in SEQ ID NO. 13 and the beta chain variable region has an amino acid sequence as set forth in SEQ ID NO. 15, or the alpha chain variable region has an amino acid sequence as set forth in SEQ ID NO.14 and the beta chain variable region has an amino acid sequence as set forth in SEQ ID NO. 16.
In one embodiment, an artificial disulfide bond is introduced between residues of the α and β chain constant regions of the TCR, and the location of the disulfide bond that can be introduced is well known to those skilled in the art.
In one embodiment, the TCR is isolated or purified or recombinant.
In one embodiment, the TCR is human.
In one embodiment, the TCR is monoclonal.
In one embodiment, the TCR is single chain.
In one embodiment, the TCR comprises two chains.
TCRs may be obtained from biological sources, such as from cells (e.g. from T cells (e.g. cytotoxic T cells)), T cell hybridomas or other publicly available sources, e.g. TCRs may be derived from one of a number of animal species, such as humans, mice, rats or other mammals, such as typically from humans.
In some embodiments, the TCR may be in a cell-bound form or in a soluble form, preferably in a soluble form. The TCR is in soluble form, meaning a TCR which has mutations in its hydrophobic core region, preferably a mutation which results in an improved stability of the soluble TCR of the application.
The application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the TCR or an alpha or beta chain of the TCR.
In one embodiment, the nucleotide sequence encoding the alpha chain is the nucleotide sequence shown as SEQ ID NO. 17 or SEQ ID NO. 18 and/or the nucleotide sequence encoding the beta chain is the nucleotide sequence shown as SEQ ID NO. 19 or SEQ ID NO. 20.
The nucleotide sequence shown in SEQ ID NO. 17 is ：ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTTGTATCCAGCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATACTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTTTTGATGAGCAAAATGAAATAAGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAACTTCACCATCACAGCCTCACAAGTCGTGGACTCAGCAGTATACTTCTGTGCTCTGAGTGAGGCAGCACCAGGAGGAAGCTACATACCTACATTTGGAAGAGGAACCAGCCTTATTGTTCATCCGTATATCCAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTGCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGGCTGTGGAGCTCC;SEQ ID NO:18 and the nucleotide sequence shown in SEQ ID NO. 17 is ：ATGCTCCTGCTGCTCGTCCCAGCGTTCCAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTATTTATCAGGATCCACCCTGGTTGAAAGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCACTTGAGGAAACCCTCAGTCCATATAAGCGACACGGCTGAGTACTTCTGTGCTGTGATAGGGAACGACTACAAGCTCAGCTTTGGAGCCGGAACCACAGTAACTGTAAGAGCAAATATCCAGAATCCAGAGCCCGCCGTGTATCAGCTGAAGGACCCAAGGAGCCAGGATTCCACCCTGTGCCTGTTCACAGACTTTGATAGCCAGATCAACGTGCCCAAGACCATGGAGTCCGGCACCTTCATCACAGACAAGTGCGTGCTGGATATGAAGGCCATGGACTCTAAGAGCAACGGCGCCATCGCCTGGAGCAATCAGACCTCCTTCACATGCCAGGATATCTTTAAGGAGACCAATGCCACATATCCTTCCTCTGACGTGCCATGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACCGACATGAACCTGAATTTTCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGGCTGTGGAGCTCC;
The nucleotide sequence shown in SEQ ID NO. 19 is:
ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGCCTCGGCCCGGGACAGCATAATTCACCCCTCCACTTTGGGAATGGGACCAGGCTCACTGTGACAGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC;
The nucleotide sequence shown in SEQ ID NO. 20 is:
ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGGACTCAGCCATGTACTTCTGTGCCAGCAGTGGGACAGGGGGCATTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGATCTGAGGAACGTGACACCCCCTAAGGTGTCTCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAATAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGGGGCTTCTTTCCTGATCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCACAGGCCTACAAGGAGTCCAATTACTCTTATTGTCTGAGCTCCCGGCTGAGAGTGTCCGCCACATTTTGGCACAACCCTAGAAATCACTTCAGGTGCCAGGTGCAGTTTCACGGCCTGAGCGAGGAGGATAAGTGGCCAGAGGGATCCCCAAAGCCTGTGACCCAGAACATCTCTGCCGAGGCATGGGGAAGGGCAGACTGTGGAATCACATCCGCCTCTTATCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACTCC.
In one embodiment, the nucleotide sequence encoding the alpha chain and/or the nucleotide sequence encoding the beta chain is codon optimized. In general, codon optimization involves balancing the percentage of selected codons with the abundance of published human transfer RNAs such that none is overloaded or limited. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and the codon usage varies from organism to organism. Codon usage differences between the transfected gene and the host cell may affect protein expression and immunogenicity of the nucleic acid construct. Typically, for codon optimization, codons are selected to select those codons that are balanced with human usage frequency. Typically, the redundancy of amino acid codons is such that the different codons encode one amino acid. In some embodiments, when selecting codons for substitution, it may be desirable that the resulting mutation be a silent mutation such that the codon changes do not affect the amino acid sequence. Typically, the last nucleotide of a codon can remain unchanged without affecting the amino acid sequence.
The present application provides a vector comprising the nucleic acid molecule described above.
For example, one or more nucleic acids encoding one or both strands of the TCR described above may be cloned into a suitable expression vector or vectors, which may be any suitable recombinant expression vector, and may be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.
The vector may contain regulatory sequences (such as transcription and translation initiation and termination codons) which are specific for the type of host (e.g., bacterial, fungal, plant or animal) into which the vector is to be introduced, and whether the vector is DNA-based or RNA-based as appropriate and considered. The vector may also contain a non-native promoter operably linked to the nucleotide sequence encoding the TCR. The promoter may be a non-viral or viral promoter such as the Cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter and promoters found in the long terminal repeat of murine stem cell viruses, as well as other promoters known to the skilled artisan are contemplated.
The vector is an expression vector, preferably a viral vector, preferably a retroviral vector, further preferably a lentiviral vector.
The application also provides a host cell comprising the nucleic acid molecule described above, wherein for recombinant production of a TCR, the nucleic acid encoding the TCR may be isolated and inserted into one or more vectors for further cloning and/or expression in the host cell. Such nucleic acids can be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the alpha and beta chains of a TCR). In some embodiments, methods of preparing a TCR are provided, wherein the methods comprise culturing a host cell comprising a nucleic acid encoding a TCR as provided above under conditions suitable for expression of the TCR molecule, and optionally recovering the TCR from the host cell (or host cell culture medium).
The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, including primary transformed cells and progeny derived therefrom, regardless of the number of passages. The offspring may not be identical in nucleic acid content to the parent cell, but may contain mutations.
The application also provides an engineered cell comprising the T Cell Receptor (TCR), the nucleic acid molecule described above, or the vector described above.
In one embodiment, the TCR is heterologous to the cell.
In one embodiment, the engineered cell is a cell line.
In one embodiment, the engineered cell is a primary cell obtained from a subject, preferably the subject is a mammalian subject, preferably a human.
In one embodiment, the engineered cells are T cells, preferably T cells isolated from peripheral blood.
In one embodiment, the T cell is cd8+ or cd4+.
The engineered cell may be, for example, a population of cells or genetically engineered cells expressing a TCR, typically eukaryotic cells, such as mammalian cells, and typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., bone marrow or lymphoid cells (including lymphocytes, typically T cells and/or NK cells). Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (ipscs). Cells are typically primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, cd4+ cells, cd8+ cells, and subpopulations thereof.
Subtypes and subsets of T cells and/or cd4+ and/or cd8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and subtypes thereof (e.g., stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells), tumor Infiltrating Lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated constant T (MAIT) cells, naturally occurring and accommodation T (Treg) cells, and the like.
In some embodiments, the engineered cell is a Natural Killer (NK) cell, preferably the cell is a monocyte or granulocyte, such as a bone marrow cell, macrophage, neutrophil, dendritic cell, mast cell, eosinophil, and/or basophil.
The present application provides a method for producing the engineered cell described above, comprising introducing the nucleic acid molecule described above or the vector described above into a cell in vitro or ex vivo.
The vector is a viral vector and the introducing is by transduction.
The present application provides a pharmaceutical composition comprising the above T Cell Receptor (TCR), the above nucleic acid molecule, the above vector or the above engineered cell.
In one embodiment, it further comprises a pharmaceutically acceptable carrier or adjuvant.
By pharmaceutically acceptable carrier or adjuvant is meant an ingredient of the pharmaceutical composition that is non-toxic to the subject in addition to the active ingredient. Pharmaceutically acceptable carriers or adjuvants include, but are not limited to, buffers, excipients, stabilizers or preservatives.
The pharmaceutical compositions may utilize timed release, delayed release and sustained release delivery systems such that delivery of the composition occurs prior to and for a sufficient time to cause sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can be used to avoid repeated administration of the composition, thereby increasing the convenience of the subject and the physician.
The application provides the use of the T Cell Receptor (TCR), the nucleic acid molecule, the vector, the engineered cell or the pharmaceutical composition in the preparation of a medicament for treating malignant tumors.
In one embodiment, the malignancy is colorectal cancer, pancreatic cancer, lung cancer, endometrial cancer, multiple myeloma, esophageal cancer, gastric cancer, ovarian cancer, prostate cancer, and the like.
In one embodiment, the colorectal cancer is metastatic colorectal cancer.
The T Cell Receptor (TCR) can specifically identify the A1101 restriction-KRASG12D mutation, and the T cell (TCR-T) transduced with the TCR can be combined with an antigen short peptide KRASG12D -HLA-A1101 complex, specifically kills tumor cells aiming at tumor antigens, and can be used for treating malignant tumors carrying the KRASG12D mutation.
And the T cells transduced with the TCR of the application can be specifically activated by tumor cells expressing A11 and KRASG12D mutations, and have good specificity, and only recognize KRASG12D mutations, but not wild-type KRAS and KRASG12V mutations.
Examples
The materials used in the test and the test methods are described generally and/or specifically in the examples which follow,% represents wt%, i.e. weight percent, unless otherwise specified. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1 cloning of KRASG12D antigen short peptide specific T cells and TCR Gene
Peripheral blood lymphocytes from healthy volunteers of genotype HLA-A1101 were stimulated with the synthetic short peptide SEQ ID NO 27: VVGADGVGK (Jiangsu St Biotech Co.). The VVVGADGVGK short peptide was renatured with biotin-labeled HLA-A1101 to prepare pHLA (short peptide-human leukocyte antigen complex) monomer. These monomers were combined with PE-labeled streptavidin (BD company) to form PE (phycoerythrin ) -labeled tetramer (wherein, pHLA monomer and tetramer were prepared by the method disclosed by NIH TETRAMER Core Facility, see pages https:// tetramer. Eye. Edu/support/protocols #1), the tetramer and anti-CD 8-FITC (fluorescein isothiocyanate) biscationic cells were enriched, the resulting biscationic cells were flow-sorted to give single cells, antigen-specific T cells, the single cells obtained by sorting were amplified with TCR alpha and beta strands, respectively, using the one-step RT-PCR kit (QIAGEN Kajie, catalog No. 210212), and the PCR products were sequenced. The sequencing result is compared with the sequence in the published database of IMGT (International immune genetics information System), so that the nucleotide sequence of the alpha chain variable region sequence and the nucleotide sequence of the beta chain variable region of TCR (TCR 104 and TCR 106) and the information of CDR1, CDR2 and CDR3 can be obtained. The nucleotide sequence of the alpha chain variable region of TCR104 is ：SEQ ID NO:28：ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTTGTATCCAGCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATACTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTTTTGATGAGCAAAATGAAATAAGTGGTCGGTATTCTTGGAACTTCCAGAAATCCACCAGTTCCTTCAACTTCACCATCACAGCCTCACAAGTCGTGGACTCAGCAGTATACTTCTGTGCTCTGAGTGAGGCAGCACCAGGAGGAAGCTACATACCTACATTTGGAAGAGGAACCAGCCTTATTGTTCATCCGTAT;
The nucleotide sequence of the β chain variable region of TCR104 is ：SEQ ID NO:29：ATGGGAATCAGGCTCCTGTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGCCTCGGCCCGGGACAGCATAATTCACCCCTCCACTTTGGGAATGGGACCAGGCTCACTGTGACA;
The amino acid sequence of complementarity determining region 1 (CDR 1) of the alpha chain of TCR104 is SEQ ID NO:1:TRDTTYY;
The amino acid sequence of complementarity determining region 2 (CDR 2) of the alpha chain of TCR104 is SEQ ID NO:3 RNSFDEQN;
The amino acid sequence of complementarity determining region 3 (CDR 3) of the alpha chain of TCR104 is SEQ ID NO 5: ALSEAAPGGSYITIT;
the amino acid sequence of complementarity determining region 1 (CDR 1) of the β -chain of TCR104 is SEQ ID NO 7: MDHEN;
The amino acid sequence of complementarity determining region 2 (CDR 2) of the β -chain of TCR104 is SEQ ID NO 9:SYDVKM;
The amino acid sequence of complementarity determining region 3 (CDR 3) of the β -chain of TCR104 is SEQ ID NO. 11:ASSLGGQHNSPLH;
The nucleotide sequence of the alpha chain variable region of TCR106 is ：SEQ ID NO:30：ATGCTCCTGCTGCTCGTCCCAGCGTTCCAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTATTTATCAGGATCCACCCTGGTTGAAAGCATCAACGGTTTTGAGGCTGAATTTAACAAGAGTCAAACTTCCTTCCACTTGAGGAAACCCTCAGTCCATATAAGCGACACGGCTGAGTACTTCTGTGCTGTGATAGGGAACGACTACAAGCTCAGCTTTGGAGCCGGAACCACAGTAACTGTAAGAGCAAAT;
The nucleotide sequence of the β chain variable region of TCR106 is ：SEQ ID NO:31：ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGGACTCAGCCATGTACTTCTGTGCCAGCAGTGGGACAGGGGGCATTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTA;
The amino acid sequence of complementarity determining region 1 (CDR 1) of the alpha chain of TCR106 is SEQ ID NO 2 SSVSVY;
the amino acid sequence of complementarity determining region 2 (CDR 2) of the alpha chain of TCR106 is SEQ ID NO 4:YLSGSTLV;
the amino acid sequence of complementarity determining region 3 (CDR 3) of the alpha chain of TCR106 is SEQ ID NO:6 AVIGNDYKLS;
The amino acid sequence of complementarity determining region 1 (CDR 1) of the β -strand of TCR106 is SEQ ID NO 8:SNHLY;
the amino acid sequence of complementarity determining region 2 (CDR 2) of the β -strand of TCR106 is SEQ ID NO 10:FYNEI;
The amino acid sequence of complementarity determining region 3 (CDR 3) of the β -chain of TCR106 is SEQ ID NO: 12. ASSEGGTIAF.
EXAMPLE 2 construction of VVGADGVGK antigen short peptide specific TCR lentiviral vector and lentiviral packaging
(1) TCR lentiviral vector construction
VVVGADGVGK TCR alpha and beta chain variable region sequences were cloned into pLKO-based expression plasmids (Addgene), alpha or beta variable domains were cloned into PLKO-based expression plasmids containing murine alpha or beta constant regions by standard methods using a multi-piece recombinant cloning kit (nuuzan biotechnology company, cat# C113), the ligated plasmids were transformed into competent e.coli strain Stbl3 cells (shanghai virucide biotechnology limited) and inoculated onto LB/agar plates containing 100 μg/ml ampicillin. After overnight incubation at 37 ℃, individual colonies were picked and grown overnight at 37 ℃ with shaking in 10ml of LB containing 100 μg/ml ampicillin. The cloned plasmid was purified using a small-medium-volume kit (Tiangen Biotechnology Co. (TIANGEN) catalog # DP 118-02) and the plasmids were sequenced to give the VVVGADGVGK TCR (i.e., TCR104, TCR 106) plasmid.
(2) Lentivirus package
Test Medium 10% FBS (Lonsera, catalog number S711-001), DMEM (ThermoFisher, catalog number C11995500 BT).
293T cells (accession number CRL-1573, american type culture Collection, ATCC) were prepared and cultured in 10cm dishes to initiate plasmid transfection at no more than 80% full, with a 1:1 ratio of virus packaging plasmid to VVVGAVGVGK TCR plasmid, total 10. Mu.g. The above plasmid was added to serum-free DMEM medium and mixed with PEI (polyethylenimine), and then the mixture of the added plasmid was added to 293T cells for 37 degrees culture. After 72h, the cell supernatants were concentrated using a 100kd ultrafiltration tube to collect the viral vectors.
Example 3 construction and functional identification of Jurkat cell lines expressing VVVGADGVGK antigen-short peptide-specific TCR
Activated T cell Nuclear Factor (NFAT) reporter gene expression method
The following experiments were performed to demonstrate the activation response of TCR-transduced T cells specific for target cells. NFAT expression levels were detected as read-out of T cell activation using flow cytometry.
(1) Reagent(s)
Test Medium 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (Siemens Fedder, thermoFisher, catalog number C11875500 BT).
(2) Method of
Target cell preparation
The target cells used in this experiment were T2-A11 cells (T2 cells were deposited with the ATCC under accession number CRL-1992, T2-A11 cells were constructed on a T2 cell basis with reference to Cancer Biology & Therapy,8:21, 2025-2032). Target cells were prepared in experimental medium at a target cell concentration of 1.6X106 cells/ml, 50 μl per well was taken to give 80000 cells/well.
Effector cell preparation
The effector cells of this experiment were Jurkat-CD8-NFAT (JK 8 NF) cells transduced with the TCR of the application, and JK8NF cells not transfected with the TCR of the application were used as control.
JK8NF cells (Jurkat cells deposited with ATCC under accession number TIB-152, JK8NF cells were referred to CANCER RES 2006;66 (23): 11455-61, front. Immunol.11:633. Lentivirus carrying the TCR gene of the application obtained in example 2 was added at MOI (multiplicity of infection) =10, and after 72 hours, the transfection positive rate was identified by flow cytometry to about 100% (the result is shown in FIG. 1), and the concentration of effector cells after expansion culture was adjusted to 1.6X106 cells/ml, and 50. Mu.L per well was taken to give 80000 cells/well.
Preparation of short peptide solutions
The concentration of the short peptide (VVVGADGVGK) with the original concentration of 5mg/ml is diluted to 400 mug/ml, and then diluted downwards to 40 mug/ml, 4 mug/ml, 0.4 mug/ml, 0.04 mug/ml, 0.004 mug/ml, 0.0004 mug/ml and 0.00004 mug/ml according to the proportion of 10 times.
50 Microliters of each well was taken to give a final concentration of the short peptide in the 96-well plate of 100. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml, 0.1. Mu.g/ml, 0.01. Mu.g/ml, 0.001. Mu.g/ml, 0.0001. Mu.g/ml, 0.00001. Mu.g/ml, respectively.
Finally, 50. Mu.l of target cells, 50. Mu.l of effector cells, 50. Mu.l of a corresponding concentration of short peptide diluent and 50ul of culture were added to each well and incubated for 12h in a 37℃cell incubator based on a 96-well flat bottom plate.
(3) Results
The expression of NFAT by TCR-transduced T cells of the application in response to target cells loaded with VVVGADGVGK antigen oligopeptide was examined by the method described above. The expression level of NFAT was plotted using GRAPHPAD PRISM and the results are shown in fig. 2.
As can be seen from FIG. 2, T cells transduced with TCRs of the application have a good activation response to target cells loaded with their specific short peptides.
Example 4 in vitro functional identification of Primary T cells expressing VVVGADGVGK antigen-short peptide-specific TCRs
IFN gamma ELISPOT method
(1) Reagent(s)
Test Medium ELISPOT kit (BD, catalog number 551849), 10% FBS (Siemeco-Beehive, thermoFisher, catalog number 10099-044), RPMI1640 (Siemeco-Beehive, thermoFisher, catalog number C11875500 BT).
(2) Method of
Effector T cell preparation
Effector cells (T cells) of this experiment were T cells transduced with the inventive TCR, and T cells not transfected with the inventive TCR from the same volunteer were used as control T cells (TCR negative control group).
Peripheral blood from volunteers was subjected to density gradient centrifugation to obtain peripheral blood mononuclear cells, which were placed in wells at 5.0X105/500 μl per well of 24-well plate, 1X106 cells were collected together, T cells were stimulated with anti-CD 3/CD28 magnetic beads and cultured in a 37℃5% CO2 incubator. After 24h, cell clumping was observed, and after TCR lentivirus transduction obtained in example 2 was added at MOI (multiplicity of infection) =2, TCR transfection efficiency was confirmed by flow cytometry after expansion of 1640 medium containing 10% fbs containing 200IU/ml IL-2 until 3-4 days post transduction (the results are shown in fig. 3). The concentration of effector cells after the expansion culture was adjusted to 5.0X104 positive cells/ml, and 100. Mu.l/well was taken to obtain 5000 positive cells/well.
In the T2-A11 target cell positive control group, the number of TCR-T cells was 2000/well for T2-A11+KRASG12D.
Target cell preparation
The target cells panc0813 (purchased from: ATCC, cat# CRL-2551) used in this experiment themselves express the G12D mutation and do not express A11, so that panc0813-A11 cells are constructed by overexpressing the A11 gene by lentiviral vectors, panc0813-A11 cells are a target cell group, panc0813 cells are a target cell negative control group, and the number of cells is 50000 cells/well. Both groups were simultaneously added with HLAI class neutralizing antibodies (anti-HLA I, clone number W6/32, purchased from Biolegend, catalog number 311402) as controls for both groups. T2-A11 cells used in this experiment were used as a positive control for target cells (T2 cells deposited with ATCC under accession number CRL-1992, T2-A11 cells were constructed on a T2 cell basis with reference to Cancer Biology & Therapy,8:21, 2025-2032) and the number of cells used was 20000 cells/well.
Preparation of short peptide solutions
The original concentration of 5mg/ml of the short peptide (VVVGADGVGK) was diluted to 4. Mu.g/ml, and 50. Mu.l was taken to give a final concentration of 1. Mu.g/ml of the short peptide in a 96-well plate.
ELISPOT detection
The well plate was prepared according to the instructions provided by the manufacturer (BD, cat. No. 551849) by diluting anti-human IFN-gamma capture antibody at 1:400 with 5ml of sterile PBS per plate and then adding 50 microliters of the diluted capture antibody aliquots to each well. The well plate was incubated overnight at 4 ℃. After incubation, the well plate is washed to remove excess capture antibody. 200 microliters of PBS containing 10% FBS was added and the well plate was incubated at temperature for 2 hours to block the well plate. The blocking solution was decanted, flicked and the ELISPOT plate tapped to remove any remaining blocking solution.
The corresponding target cells and effector cells and corresponding short peptides were then added, the well plate incubated overnight (37 ℃ C./5% CO 2) the next day, the medium was discarded, the well plate was washed 2 times with double distilled water, 3 times with wash buffer, and the plates were tapped to remove residual wash buffer. The detection antibody was then diluted 1:400 with PBS containing 10% FBS and each well was added at 100. Mu.L/well. The well plate was incubated at room temperature for 2 hours, washed 3 more times with wash buffer, and the well plate was tapped on paper towels to remove excess wash buffer.
Streptavidin-alkaline phosphatase was diluted 1:200 with PBS containing 10% FBS, 100. Mu.L of diluted streptavidin-alkaline phosphatase was added to each well and the well plate was incubated for 1 hour at room temperature. The plate was then tapped on paper towels to remove excess wash buffer and PBS by washing 3 times with wash buffer 3 times with PBS. After washing, 100. Mu.l/well of BCIP/NBT solution provided by the kit was added for development. The plate was covered with tinfoil during development in the dark and left to stand for 5-15 minutes. During this period the spots of the developed well plate were routinely examined and the optimal time for termination of the reaction was determined. The BCIP/NBT solution was removed and the well plate was rinsed with double distilled water to stop the development reaction, spun dry, then the bottom of the well plate was removed, the well plate was dried at room temperature until each well was completely dried, and spots formed in the membrane in the well plate were counted using an immunospot plate counter. The function of TCR-transduced T cells of the invention was examined by ELISPOT experiments (as described above). The number of ELISPOT spots observed in each well was plotted using GRAPHPAD PRISM.
(3) Results
As can be seen from FIG. 4, T cells TCR104 and TCR106 expressing a VVVGADGVGK-A1101 antigen-specific TCR are strongly activated to the panc0813-A11 cell line, whereas control T cells are not. Recognition of the panc0813-a11 cell line by TCR104 and TCR106 can be inhibited following the addition of HLA class I neutralizing antibodies.
Example 5 Cross-reaction identification of VVGADGVGK antigen short peptide specific TCR activated T Nuclear Factor (NFAT) reporter expression methods
The following experiments were performed to demonstrate that VVVGADGVGK antigen-short peptide-specific TCRs do not cross-react to wild-type KRAS and KRASG12D mutations.
NFAT expression levels were detected as read-out of T cell activation using flow cytometry.
(1) Reagent(s)
Test Medium 10% FBS (Lonsera, catalog number S711-001), RPMI1640 (Siemens Fedder, thermoFisher, catalog number C11875500 BT).
(2) Method of
Target cell preparation
The target cells used in this experiment were T2-A11 cells (T2 cells were deposited with the ATCC under accession number CRL-1992, T2-A11 cells were constructed on a T2 cell basis with reference to Cancer Biology & Therapy,8:21, 2025-2032). Target cells were prepared in experimental medium at a target cell concentration of 1.6X106 cells/ml, 50 μl per well was taken to give 80000 cells/well.
Effector cell preparation
The effector cells of this experiment were Jurkat-CD8-NFAT (JK 8 NF) cells transduced with the TCR of the application, and JK8NF cells not transfected with the TCR of the application were used as control.
JK8NF cells (Jurkat cells deposited with ATCC under accession number TIB-152, JK8NF cells were referred to CANCER RES 2006;66 (23): 11455-61, front. Immunol.11:633. Lentivirus carrying the TCR gene of the application obtained in example 2 was added at MOI (multiplicity of infection) =10, and after 72 hours, the transfection positive rate was identified by flow cytometry to about 100% (the result is shown in FIG. 1), and the concentration of effector cells after expansion culture was adjusted to 1.6X106 cells/ml, and 50. Mu.L per well was taken to give 80000 cells/well.
Preparation of short peptide solutions
The original concentration of 5mg/ml of short peptide (VVVGADGVGK (G12D), VVVGAGGVGK (WT), VVVGAVGVGK (G12V)) was diluted to 400. Mu.g/ml, and then diluted down to 40. Mu.g/ml, 4. Mu.g/ml, 0.4. Mu.g/ml, 0.04. Mu.g/ml, 0.004. Mu.g/ml, 0.0004. Mu.g/ml, 0.00004. Mu.g/ml in a 10-fold ratio.
50 Microliters of each well was taken to give a final concentration of the short peptide in the 96-well plate of 100. Mu.g/ml, 10. Mu.g/ml, 1. Mu.g/ml, 0.1. Mu.g/ml, 0.01. Mu.g/ml, 0.001. Mu.g/ml, 0.0001. Mu.g/ml, 0.00001. Mu.g/ml, respectively.
Finally, 50. Mu.l of target cells, 50. Mu.l of effector cells, 50. Mu.l of a corresponding concentration of short peptide diluent and 50ul of culture were added to each well and incubated for 12h in a 37℃cell incubator based on a 96-well flat bottom plate.
(3) Results
The expression of NFAT by TCR-transduced T cells of the application in response to target cells bearing KRASG12D,KRASWT and KRASG12D antigen peptides was examined by the method described above. The expression level of NFAT was plotted using GRAPHPAD PRISM and the results are shown in fig. 5.
As can be seen from fig. 5, the TCRs of the present application have good specificity, recognizing only KRASG12D mutations, but not wild-type KRAS and KRASG12D mutations.
In conclusion, the T cells transduced with the TCR of the application are specifically activated by tumor cells expressing the complex of A11+KRASG12D mutation, and have good specificity, and only recognize KRASG12D mutation, but not wild-type KRAS and KRASG12V mutation.
The above description is only a preferred embodiment of the present application, and is not intended to limit the application in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present application still fall within the protection scope of the technical solution of the present application.
Sequence listing