CN118221830A - T cell antigen receptor and application thereof - Google Patents
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Abstract
The invention relates to the technical field of T cell antigen receptors, and discloses a T cell antigen receptor and application thereof. The T cell antigen receptor provided by the invention can specifically recognize and bind to HLa-a 11:01CMVpp65 501‑509 antigen complex. Experiments prove that the T cell antigen receptor provided by the invention can be specifically activated by CMV epitope peptide, has good activity of killing target cells in vitro, and can release cytokines such as IFN-gamma. In addition, through ALA-SCAN experiments and whole protein group comparison of raw message, the On-target-off-tumor off-target risk of the T cell antigen receptor provided by the invention is eliminated, so that the T cell antigen receptor has higher clinical application value and better safety.
Description
Technical Field
The invention relates to the technical field of T cell antigen receptors, in particular to a T cell antigen receptor and application thereof.
Background
Human Cytomegalovirus (CMV) is a widely occurring herpes virus with an infection rate of 50-100% in the adult population. In individuals with normal immune systems, infection with CMV typically does not show any symptoms. However, in individuals with suppressed immune systems, such as aids patients or patients who have received organ transplants, reactivation of CMV can lead to severe or even fatal disease. Traditional therapies for CMV include treatment with antiviral drugs, such as ganciclovir, sodium phosphonate, and cidofovir, but these drugs are often limited and often cause serious side effects.
Cell receptor therapy is an Adoptive cell therapy technique (Adoptive CELL TRANSFER THERAPY, ACT) which mainly uses the mode of collecting human autoimmune cells, introducing the human autoimmune cells into a cell receptor, culturing the human autoimmune cells in vitro to expand the number of the human autoimmune cells, and killing pathogens, cancer cells or mutated cells in blood and tissues in a mode of reinfusion into a patient. A T Cell antigen Receptor (TCR) is a surface protein capable of specifically recognizing an antigen on the surface of a T Cell, and a specific TCR sequence is introduced into the T Cell to obtain a T Cell that specifically recognizes the antigen and kills cells containing the antigen, thereby treating diseases caused by virus infection such as CMV by Cell Receptor therapy.
The major histocompatibility complex (Major Histocompatibility Complex, MHC) is a collective term for a group of genes encoding animal major histocompatibility antigens, and human MHC is known as human leukocyte antigen (Human Leukocyte antigen, HLA). HLA exhibits high polymorphism, and HLA typing varies among people in different countries and regions. HLA typing accounts for the largest proportion of HLA-a3 superfamily (including HLA-A11, HLA-A33, HLA-A68, HLA-A31, etc.) in the Chinese population, and accounts for about 52.7% of HLA typing in the Chinese population.
Although researchers have developed a variety of TCRs for CMV, there have been no reports on TCR sequences based on HLA-A3 superfamily restriction epitopes.
Disclosure of Invention
The invention aims to solve the problems of the prior art that TCR aiming at HLA main typing restriction epitopes of Chinese population and related high-efficiency CMV treatment methods are lacking, and provides a T cell antigen receptor and application thereof. The T cell antigen receptor provided by the invention can specifically recognize and combine with HLA-A 11:01CMV antigen complex, has good activity of killing target cells, can release cytokines such as IFN-gamma and the like, and eliminates the On-target/off-tumor off-target risk of the T cell antigen receptor through experiments.
In order to achieve the above object, the present invention provides, in one aspect, a T cell antigen receptor that specifically recognizes and binds to a human cytomegalovirus binding epitope comprising the amino acid sequence shown in SEQ ID No. 1.
In a second aspect the invention provides a nucleic acid molecule encoding the T cell antigen receptor of the first aspect.
In a third aspect the invention provides a nucleic acid expression vector comprising a coding gene and a vector sequence, wherein the coding gene is provided by a nucleic acid molecule according to the second aspect.
In a fourth aspect the invention provides a vector cell comprising the T cell antigen receptor of the first aspect and/or comprising the nucleic acid molecule of the second aspect and/or comprising the nucleic acid expression vector of the third aspect.
In a fifth aspect, the invention provides the use of a T cell antigen receptor according to the first aspect, and/or a nucleic acid molecule according to the second aspect, and/or a nucleic acid expression vector according to the third aspect, and/or a vector cell according to the fourth aspect, for the preparation of a medicament and/or vaccine for the prophylaxis and/or treatment of a disease caused by human cytomegalovirus infection.
In a sixth aspect, the present invention provides a pharmaceutical composition for preventing and/or treating a disease caused by human cytomegalovirus infection, comprising: the T cell antigen receptor of the first aspect, and/or the nucleic acid molecule of the second aspect, and/or the nucleic acid expression vector of the third aspect, and/or the vector cell of the fourth aspect.
Through the technical scheme, the invention at least has the following beneficial effects:
(1) The TCR molecule provided by the invention can recognize and combine with HLA-A 11:01CMV pp65 501-509 antigen complex, and is more suitable for people in China to use. After activated by the specific CMV epitope peptide, the TCR molecule has good activity of killing target cells in vitro, and can release cytokines such as IFN-gamma and the like, thereby having potential of being applied to CMV treatment/prevention through cell receptor therapy.
(2) The On-target/off-tumor off-target risk of the TCR molecule provided by the invention is eliminated through ALA-SCAN experiment and whole protein group comparison, so that the TCR molecule has clinical application value, and the TCR molecule is used with smaller off-target toxicity risk, so that the TCR molecule has better safety.
(3) The method for in vitro culture and amplification of the specific T cells containing the TCR molecules provided by the invention is simple and easy to operate, and the CMV pp65 epitope peptide is directly used for stimulating the PBMC to culture, so that the large-scale clinical application of the TCR molecules and the treatment/prevention method of the specific T cells containing the TCR molecules can be realized.
Drawings
FIG. 1 is a graph of CMV +CD8+ T cell duty cycle results of flow assays of T cells in example 1 after stimulation and stimulation with CMV epitope peptide.
FIG. 2 is a graph showing the results of ELISPot assay of T cell secretion of cytokine IFN-gamma in example 1.
FIG. 3 is a schematic diagram of the construction of an in vitro transcription plasmid vector in example 2.
FIG. 4 is a graph showing the results of gel electrophoresis of TCR mRNA obtained in example 2.
FIG. 5 is a map of HLA lentivirus expression plasmid employed in example 3.
FIG. 6 is a graph showing HLA expression level analysis of monoclonal cells for flow assay in example 3.
FIG. 7 is a flow chart of the flow assay of example 3 for electrotransport ZZ1 and ZZ2 NFAT-Luc Jurkat.
FIGS. 8A and 8B are graphs showing statistical results of NFAT-fluorescence values of ZZ1-NFAT-Luc Jurkat and ZZ2-NFAT-Luc Jurkat detected by a microplate reader in example 3.
FIG. 9 is a flow chart of the specific ratios of ZZ1 TCR-T and ZZ2 TCR-T for flow assays in example 4.
FIG. 10 is a graph showing the results of EuTDA detection of the killing efficiency of ZZ1 TCR-T and ZZ2 TCR-T on target cells in example 4.
FIGS. 11A and 11B are graphs showing the results of ELISA assays for ZZ1 TCR-T and ZZ2 TCR-T for cytokine IFN-gamma secretion in example 5.
FIG. 12 is a schematic representation of the position of an alanine mutation in an alanine scanning peptide library constructed in example 6.
FIG. 13 is a statistical plot of ELISPOT assay ZZ1 TCR-T secretion IFN-gamma spots in example 6.
FIG. 14 is a statistical plot of ELISPOT assay ZZ2 TCR-T secretion IFN-gamma spots in example 6.
FIG. 15 is a graph of positive duty cycle results of flow assay ZZ3 TCR-T CD8 +CMV+ in comparative example 1.
FIG. 16 is a chart showing the statistical result of NFAT-fluorescence values of ZZ3-NFAT-Luc Jurkat detected by a microplate reader in comparative example 1.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and the range or value is to be understood as encompassing the value near the range or value. For numerical ranges, one or more new numerical ranges may be obtained in combination with each other between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, and are to be considered as specifically disclosed herein.
The term "T cell antigen receptor (TCR)" as used herein refers to a protein molecule that T cells recognize a target cell surface specific antigen. The variable regions (V regions) of the α and β chains of TCRs comprise complementarity-DETERMINING REGIONS (CDRs) and antibody Framework Regions (FR). The amino acid sequence of the CDRs determines the specificity of the antibody for binding to antigen, and the main role of FR is to stabilize the spatial configuration of the CDRs to facilitate binding between the CDRs and the antigenic determinants.
In the present invention, "TCR" is an abbreviated form of "T cell antigen receptor" and both are synonymous; "TCR-T cells" are shorthand forms of "T cells containing TCR (molecules) and are synonymous; "HLA" is an abbreviated form of "human leukocyte antigen" and is synonymous; "CMV" is an abbreviated form of "human cytomegalovirus" and is synonymous; "MHC" is an abbreviated form of "major histocompatibility complex" and is synonymous; "fP2A" is an abbreviated form of "furin SGSG p2A" and has the same meaning, and the terms of the above groups having the same meaning are used interchangeably.
With the development of ACT technology, researchers in the field have developed a variety of different immune cell therapies for the treatment and/or prevention of cancers and viral infectious diseases that are difficult to effectively treat with existing drugs. Such as CAR-T therapy, TCR-T cell therapy, and the like. Among other advantages over CAR-T therapies, TCR-T cell therapies are that TCR-T cells can recognize not only cell membrane antigens, but also intracellular antigens presented by peptide-major histocompatibility complexes, free of target cell surface antigen expression, thereby allowing for more extensive recognition of target antigens, inducing a more durable immune synapse formation process, TCR can also be activated by a single target antigen molecule, and are more sensitive to low copy number antigens than CAR-T. However, TCRs only recognize peptide-HLA and kill target cells with matched HLA alleles, and HLA typing is highly polymorphic and exhibits a more pronounced regional profile. Thus, when designing and screening TCR molecules in TCR therapy, the selection of HLA-typed epitopes that they can recognize and bind has a great impact on the therapeutic effect of the TCR molecule on patients in a particular region.
The inventors have also found in research that more research is currently focused on TCR-T therapies for cancer, especially solid tumor therapy, and less for TCR-T therapies for viral infectious diseases. Although some researchers developed some TCR molecules for CMV and corresponding TCR-T therapies, such as TCR molecules for CMV pp65 epitope disclosed in CN113121676a and TCR molecules for HCMV disclosed in CN111875698a, these currently developed TCR molecules for CMV are all specifically recognizing and binding to HLA-A x 02 epitope, which belongs to the high frequency HLa subtype of the european population, and is relatively low in the national population, thus not suitable for CMV treatment/prevention of the national population. Through a great deal of research, the inventor of the present invention found that a specific TCR molecule capable of specifically recognizing an HLA-A 11:01 subtype epitope (the HLa subtype is a high-frequency subtype of the population in China) targeting CMV can be obtained through screening, so that a product and a method more suitable for preventing and/or treating CMV in China can be further obtained. In addition, the inventor also discovers that through special design, after the TCR molecules are led into T cells in vitro, the expansion of specific T cells can be realized by a simpler method, so that the operation of the cell expansion step in TCR-T therapy is greatly simplified, and the large-scale application and popularization of the method are facilitated.
Based on the above findings, the first aspect of the present invention provides a T cell antigen receptor which specifically recognizes and binds to a human cytomegalovirus binding epitope comprising the amino acid sequence shown in SEQ ID No. 1.
ATVQGQNLK(SEQ ID NO:1)
SEQ ID NO.1 is the amino acid sequence of the HLA-A 11:01CMV pp65 501-509 antigen complex (i.e., amino acid sequence 501-509 of the HLA-A 11:01 restrictive CMV pp65 epitope antigen complex). That is, the TCRs provided by the present invention can specifically recognize and bind to the CMV antigen peptide-MHC molecule complex, activate TCR-T cells, thereby producing high levels of cytokines and killing target cells. Generally, TCRs provided by the present invention bind specifically to antigenic peptides from CMV through presentation by MHC.
Preferably, the TCR is a soluble TCR.
The specific structure and sequence of the TCR provided by the invention are not particularly limited, and any TCR capable of specifically binding to an amino acid sequence comprising SEQ ID NO. 1 is within the scope of the invention. According to a preferred embodiment of the invention, the TCR comprises at least one alpha chain variable region and/or at least one beta chain variable region.
Preferably, the TCR is a heterodimer comprising at least one alpha chain variable region and at least one beta chain variable region.
According to some preferred embodiments of the invention, wherein the α -chain variable region comprises a complementarity determining region of an α -chain and the β -chain variable region comprises a complementarity determining region of a β -chain.
Complementarity determining regions refer to sequence segments of a TCR sequence that are capable of forming a precise complement with an HLA-antigen complex, such that the TCR molecule can specifically recognize the HLA-antigen complex. In the TCR sequence provided by the invention, the complementarity determining region in the alpha chain/beta chain can be a continuous amino acid sequence or a discontinuous amino acid sequence, so long as the TCR sequence can specifically recognize and combine with the amino acid sequence shown in SEQ ID NO. 1.
The inventors of the present invention have skillfully found in the study that when the complementarity determining region in the alpha chain/beta chain of the TCR molecule has a specific amino acid sequence, the ability to specifically recognize and bind to the target sequence (e.g., SEQ ID NO:1, etc.) is enhanced.
In order to provide a TCR with better target sequence specific recognition and binding properties, according to some preferred embodiments of the invention, the complementarity determining region of the alpha chain includes an amino acid sequence having at least 80% homology (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be a range formed by any two of the above values and any intermediate value) with at least one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO: 12.
VSGNPY(SEQ ID NO:2)
YITGDNLV(SEQ ID NO:3)
CAVRDMEGGNNARLMF(SEQ ID NO:4)
DRGSQS(SEQ ID NO:10)
IYSNGD(SEQ ID NO:11)
CAVKGGYNKLIF(SEQ ID NO:12)
Preferably, the complementarity determining region of the α -chain comprises the amino acid sequence set forth in at least one of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.
According to some preferred embodiments of the invention, wherein the complementarity determining region of the β -strand comprises an amino acid sequence having at least 80% homology (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may also be a range formed by any two of the above values and any intermediate value) to at least one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO: 15.
SNHLY(SEQ ID NO:5)
FYNNEI(SEQ ID NO:6)
CASSEAYGGYNEQFF(SEQ ID NO:7)
SGHRS(SEQ ID NO:13)
YFSETQ(SEQ ID NO:14)
CASSLGGEGDRPQHF(SEQ ID NO:15)
Preferably, the complementarity determining region of the β strand comprises an amino acid sequence set forth in at least one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15.
According to some preferred embodiments of the present invention, wherein the complementarity determining regions of the α -chain comprise three discrete amino acid sequences designated as CDR1 α, CDR2 α, CDR3 α, respectively, in sequence, wherein CDR1 α comprises an amino acid sequence having at least 80% homology (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be a range formed by any two of the above values and any intermediate value) with the amino acid sequences shown in SEQ ID No.2 and/or SEQ ID No. 10; CDR 2. Alpha. Includes amino acid sequences having at least 80% homology to the amino acid sequences shown in SEQ ID NO:3 and/or SEQ ID NO:11 (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be a range formed by any two of the above values and any intermediate value); CDR 3. Alpha. Includes amino acid sequences having at least 80% homology to the amino acid sequences shown in SEQ ID NO.4 and/or SEQ ID NO. 12 (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be a range formed by any two of the above values and any intermediate value).
Preferably, CDR 1a comprises the amino acid sequence shown as SEQ ID NO.2 and/or SEQ ID NO. 10; CDR2 alpha includes the amino acid sequence shown as SEQ ID NO 3 and/or SEQ ID NO 11; CDR3. Alpha. Includes the amino acid sequences shown in SEQ ID NO. 4 and/or SEQ ID NO. 12.
More preferably, the amino acid sequence of CDR1 alpha is as shown in SEQ ID NO. 2 and/or SEQ ID NO. 10; the amino acid sequence of CDR2 alpha is shown as SEQ ID NO 3 and/or SEQ ID NO 11; the amino acid sequence of CDR3 alpha is shown as SEQ ID NO. 4 and/or SEQ ID NO. 12.
According to some preferred embodiments of the present invention, wherein the complementarity determining region of the β -strand comprises three discrete amino acid sequences designated as CDR1β, CDR2β, CDR3β, respectively, wherein CDR1β comprises an amino acid sequence having at least 80% homology (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be a range formed by any two of the above values and any intermediate value) with the amino acid sequence shown in SEQ ID No.5 and/or SEQ ID No. 13; CDR2β includes amino acid sequences having at least 80% homology to the amino acid sequences shown in SEQ ID NO. 6 and/or SEQ ID NO. 14 (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be a range formed by any two of the above values and any intermediate value); CDR3β includes amino acid sequences having at least 80% homology with the amino acid sequences shown in SEQ ID NO:7 and/or SEQ ID NO:15 (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be a range formed by any two of the above values and any intermediate value).
Preferably, CDR 1. Beta. Comprises the amino acid sequence shown as SEQ ID NO.5 and/or SEQ ID NO. 13; CDR 2. Beta. Includes the amino acid sequences shown in SEQ ID NO. 6 and/or SEQ ID NO. 14; CDR3. Beta. Includes the amino acid sequences shown in SEQ ID NO. 7 and/or SEQ ID NO. 15.
More preferably, the amino acid sequence of CDR 1. Beta. Is shown in SEQ ID NO. 5 and/or SEQ ID NO. 13; the amino acid sequence of CDR2 beta is shown as SEQ ID NO. 6 and/or SEQ ID NO. 14; the amino acid sequence of CDR3 beta is shown as SEQ ID NO 7 and/or SEQ ID NO 15.
The α chain variable region/β chain variable region included in the TCR provided by the present invention preferably includes complementarity determining regions composed of three discontinuous amino acid sequences, respectively, that is, the complementarity determining regions of the α chain variable region include the above CDR1α, CDR2α and CDR3α, and the complementarity determining regions of the β chain variable region include the above CDR1β, CDR2β and CDR3β, wherein the CDR1α, CDR2α, CDR3α and CDR1β, CDR2β and CDR3β may be arbitrarily combined according to the above sequences, so long as the TCR can specifically recognize and bind to HLA-A 11:01cmv pp65 501-509 antigen complex.
According to a preferred embodiment of the present invention, wherein the complementarity determining regions of the α -chain variable region include the amino acid sequences shown in SEQ ID NOS: 2-4; the complementarity determining regions of the β chain variable region include the amino acid sequences shown in SEQ ID NOS.5-7.
According to a preferred embodiment of the present invention, wherein the complementarity determining regions of the α -chain variable region include the amino acid sequences shown in SEQ ID NOS 10-12; the complementarity determining regions of the β chain variable region include the amino acid sequences shown in SEQ ID NOS 13-15.
According to a particularly preferred embodiment of the invention, the alpha chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8 and/or SEQ ID NO. 16.
MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDMEGGNNARLMFGDGTQLVVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSRAKRSGSG(SEQ ID NO:8)
MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKGGYNKLIFGAGTRLAVHPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSRAKRSGSG(SEQ ID NO:16)
According to a particularly preferred embodiment of the invention, the β chain variable region comprises the amino acid sequence shown in SEQ ID NO. 9 and/or SEQ ID NO. 17.
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEAYGGYNEQFFGPGTRLTVLEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:9)
MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSLGGEGDRPQHFGDGTRLSILEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS(SEQ ID NO:17)
Preferably, in the TCR, the amino acid sequence of the alpha chain variable region is shown in SEQ ID NO. 8, and the amino acid sequence of the beta chain variable region is shown in SEQ ID NO. 9.
Preferably, in the TCR, the amino acid sequence of the alpha chain variable region is shown as SEQ ID NO. 16 and the amino acid sequence of the beta chain variable region is shown as SEQ ID NO. 17.
In the TCRs provided by the invention, the α chain variable region and the β chain variable region may be joined by any means common to the art of joining α chain variable regions and β chain variable regions of TCR molecules to form a complete heterodimeric TCR molecule.
According to some preferred embodiments of the invention, wherein the TCR the α chain variable region is directly linked or indirectly linked to the β chain variable region. Direct linkage refers to the manner in which the amino acid sequences of the α chain variable region and the β chain variable region are directly linked (e.g., linked by disulfide bonds, etc.); indirect linkage refers to linkage between the α -chain variable region and the β -chain variable region by other amino acid sequences (which may be generally referred to as "linkage sequences"), for example, linkage of the α -chain variable region and the β -chain variable region to different fragments in the linkage sequence by disulfide bonds or the like, linkage of the α -chain variable region and the β -chain variable region to the same fragment in the linkage sequence by disulfide bonds or the like, or the like.
Preferably, in the TCR, the α chain variable region is indirectly linked to the β chain variable region.
According to some preferred embodiments of the present invention, wherein the alpha chain variable region may be linked to the beta chain variable region by fp2A sequence (amino acid sequence shown in SEQ ID NO: 55).
SGSGATNFSLLKQAGDVEENPGP(SEQ ID NO:55)
The present invention provides TCR molecules in which the alpha and beta chain constant region sequences are not particularly limited, so long as they are capable of binding to the alpha and beta chain variable regions having the characteristics described above to form a stable TCR molecule having the desired functions of the present invention.
To increase exogenous TCR expression efficiency, according to a preferred embodiment of the invention, wherein the TCR comprises an alpha chain constant region comprising an amino acid sequence having at least 80% homology to SEQ ID NO:26 (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be any two of the values defined above and any intermediate value); the β chain constant region includes an amino acid sequence having at least 80% homology to SEQ ID No. 27 (e.g., homology may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, 100%, or may be a range formed by any two of the above values and any intermediate value).
IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:26)
EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS(SEQ ID NO:27)
According to a particularly preferred embodiment of the invention, the TCR comprises an alpha chain constant region, an alpha chain variable region and a beta chain constant region, a beta chain variable region, the amino acid sequence of the alpha chain variable region being shown in SEQ ID NO. 8 and the amino acid sequence of the alpha chain constant region being shown in SEQ ID NO. 26; the amino acid sequence of the beta-chain variable region is shown as SEQ ID NO. 9, and the amino acid sequence of the beta-chain constant region is shown as SEQ ID NO. 27.
Preferably, the amino acid sequence of the TCR is shown in SEQ ID NO. 52.
According to a particularly preferred embodiment of the invention, the TCR comprises an alpha chain constant region, an alpha chain variable region and a beta chain constant region, a beta chain variable region, the amino acid sequence of the alpha chain variable region being shown in SEQ ID NO. 16 and the amino acid sequence of the alpha chain constant region being shown in SEQ ID NO. 26; the amino acid sequence of the beta-chain variable region is shown as SEQ ID NO. 17, and the amino acid sequence of the beta-chain constant region is shown as SEQ ID NO. 27.
Preferably, the amino acid sequence of the TCR is shown in SEQ ID NO. 53.
In a second aspect the invention provides a nucleic acid molecule encoding the T cell antigen receptor of the first aspect.
Any nucleic acid molecule (including DNA and RNA) capable of encoding a TCR provided by the invention is within the scope of the invention. Based on the degeneracy of the codons, one skilled in the art can design and synthesize different nucleic acid molecules capable of encoding TCRs provided by the present invention by themselves, based on the characteristics of the aforementioned TCR sequences.
According to a preferred embodiment of the invention, wherein the nucleic acid molecule comprises a nucleotide sequence as shown in at least one of SEQ ID NO:28 (encoding CDR 1. Alpha. As shown in SEQ ID NO: 2), SEQ ID NO:29 (encoding CDR 2. Alpha. As shown in SEQ ID NO: 3), SEQ ID NO:30 (encoding CDR 3. Alpha. As shown in SEQ ID NO: 4), SEQ ID NO:36 (encoding CDR 1. Alpha. As shown in SEQ ID NO: 10), SEQ ID NO:37 (encoding CDR 2. Alpha. As shown in SEQ ID NO: 11), SEQ ID NO:38 (encoding CDR 3. Alpha. As shown in SEQ ID NO: 12).
GTCTCCGGAAACCCCTAT(SEQ ID NO:28)
TACATCACTGGTGATAACCTGGTG(SEQ ID NO:29)
TGTGCGGTGCGTGACATGGAAGGGGGTAATAACGCTCGGCTGATGTTT(SEQ ID NO:30)
GACCGTGGCAGTCAGAGC(SEQ ID NO:36)
ATCTACAGCAATGGCGAT(SEQ ID NO:37)
TGCGCCGTTAAAGGGGGCTACAACAAGCTGATCTTC(SEQ ID NO:38)
According to a preferred embodiment of the present invention, wherein the nucleic acid molecule further comprises at least one of the nucleotide sequences shown in SEQ ID NO:31 (encoding CDR1β shown in SEQ ID NO: 5), SEQ ID NO:32 (encoding CDR2β shown in SEQ ID NO: 6), SEQ ID NO:33 (encoding CDR3β shown in SEQ ID NO: 7), SEQ ID NO:39 (encoding CDR1β shown in SEQ ID NO: 13), SEQ ID NO:40 (encoding CDR2β shown in SEQ ID NO: 14), SEQ ID NO:41 (encoding CDR3β shown in SEQ ID NO: 15).
AGCAACCACCTCTAC(SEQ ID NO:31)
TTTTACAACAACGAGATC(SEQ ID NO:32)
TGCGCCTCTTCCGAAGCCTACGGTGGCTACAACGAGCAGTTTTTC(SEQ ID NO:33)
TCTGGCCACCGCTCT(SEQ ID NO:39)
TACTTCTCCGAGACCCAG(SEQ ID NO:40)
TGCGCCTCCTCCTTGGGCGGGGAGGGCGACAGGCCTCAGCACTTT(SEQ ID NO:41)
According to a preferred embodiment of the present invention, wherein the nucleic acid molecule comprises a nucleotide sequence as shown in at least one of SEQ ID NO. 34 (encoding the alpha chain variable region of SEQ ID NO. 8), SEQ ID NO. 35 (encoding the beta chain variable region of SEQ ID NO. 9), SEQ ID NO. 42 (encoding the alpha chain variable region of SEQ ID NO. 16) and SEQ ID NO. 43 (encoding the beta chain variable region of SEQ ID NO. 17).
ATGGCGTCTGCTCCTATCAGCATGCTAGCTATGTTGTTCACCTTGAGCGGATTGAGGGCTCAGTCCGTAGCGCAGCCGGAGGATCAGGTCAATGTGGCGGAGGGCAATCCCCTGACCGTTAAGTGCACTTACAGCGTCTCCGGAAACCCCTATCTGTTTTGGTACGTGCAGTACCCCAACAGGGGTCTGCAGTTCCTGTTAAAGTACATCACTGGTGATAACCTGGTGAAAGGCAGTTACGGCTTCGAGGCCGAGTTCAACAAGTCCCAGACCTCCTTTCACCTGAAGAAGCCCTCGGCTCTGGTCTCCGACTCTGCGCTGTACTTCTGTGCGGTGCGTGACATGGAAGGGGGTAATAACGCTCGGCTGATGTTTGGCGACGGGACCCAGCTGGTAGTCAAGCCCAACATCCAGAATCCAGATCCAGCCGTGTACCAGCTGCGCGACTCCAAAAGCAGCGACAAATCGGTTTGTCTATTCACCGACTTCGACTCCCAAACTAATGTCAGTCAGTCCAAGGACTCTGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGTCCATGGATTTCAAGTCCAACTCCGCCGTAGCATGGTCTAACAAGAGCGACTTTGCCTGCGCCAACGCGTTCAACAACTCTATCATCCCCGAGGACACGTTCTTTCCCTCTCCGGAGTCCTCCTGCGACGTGAAGCTCGTGGAGAAATCTTTTGAGACCGACACAAATTTGAACTTCCAGAACCTGTCAGTCATCGGCTTCCGCATCCTTCTGTTGAAGGTCGCGGGATTCAACCTCCTGATGACCCTCCGCCTTTGGTCTTCCCGGGCCAAGCGCAGCGGTTCGGGC(SEQ ID NO:34)
ATGGATACCTGGCTGGTGTGCTGGGCTATTTTCTCCCTGCTCAAGGCCGGTCTCACCGAACCGGAGGTGACCCAGACTCCCTCTCACCAGGTCACCCAGATGGGCCAGGAGGTGATTTTGCGCTGCGTGCCCATCAGCAACCACCTCTACTTCTATTGGTACCGCCAAATCCTAGGCCAGAAAGTGGAGTTCCTGGTGTCCTTTTACAACAACGAGATCAGCGAGAAAAGCGAGATTTTCGATGACCAGTTCAGCGTGGAGCGCCCTGACGGCTCCAACTTTACGCTCAAGATCCGATCCACCAAGCTGGAGGACTCGGCAATGTATTTCTGCGCCTCTTCCGAAGCCTACGGTGGCTACAACGAGCAGTTTTTCGGCCCGGGGACCCGCCTGACTGTGCTGGAGGACCTTAAGAACGTGTTCCCACCGAAGGTGGCGGTTTTCGAGCCCAGCGAGGCCGAGATAAGCCACACTCAGAAGGCGACTCTCGTCTGTCTGGCGACAGGCTTCTACCCTGATCATGTGGAGCTGAGTTGGTGGGTCAACGGTAAGGAGGTGCATTCGGGCGTGTCTACCGATCCTCAGCCACTTAAAGAACAGCCTGCCCTGAACGACTCTCGTTATTGTCTGTCATCCCGCCTGCGCGTCTCTGCCACGTTCTGGCAGAACCCGCGCAACCACTTCCGTTGCCAGGTGCAATTCTACGGATTATCCGAGAATGACGAGTGGACCCAGGACCGCGCCAAGCCTGTGACCCAGATCGTCTCTGCTGAAGCATGGGGCCGAGCAGACTGCGGGTTCACGTCTGAGAGCTATCAGCAGGGAGTGCTGTCCGCTACCATCTTGTACGAGATTCTGCTGGGCAAGGCTACACTGTACGCAGTGCTGGTTTCCGCCCTTGTACTGATGGCCATGGTGAAGCGCAAGGACAGTAGAGGC(SEQ ID NO:35)
ATGAAATCTCTTCGCGTGCTGCTGGTGATTTTGTGGCTCCAGCTGTCTTGGGTCTGGTCTCAGCAGAAGGAGGTGGAACAGAACTCCGGACCTTTGTCGGTGCCGGAGGGCGCCATTGCTTCCCTCAACTGCACTTATTCTGACCGTGGCAGTCAGAGCTTCTTTTGGTACCGCCAGTACAGTGGCAAGTCGCCGGAGCTGATCATGTTTATCTACAGCAATGGCGATAAGGAGGATGGGCGCTTCACGGCGCAGCTTAACAAAGCTTCCCAGTACGTGAGCCTTCTCATTCGCGACAGCCAGCCGAGTGACTCCGCAACCTACCTGTGCGCCGTTAAAGGGGGCTACAACAAGCTGATCTTCGGTGCCGGCACCCGCCTGGCCGTGCACCCGTATATCCAGAACCCCGAGCCAGCGGTGTACCAGTTGAAGGACCCGCGCTCGCAGGACTCCACCCTGTGCCTGTTCACCGATTTCGACTCCCAAATTAACGTGCCAAAGACTATGGAGAGCGGTACGTTTATCACCGACAAAACCGTGCTGGACATGAAGGCCATGGATTCCAAGAGCAACGGCGCTATCGCTTGGTCCAACCAGACCTCCTTCACCTGCCAGGACATCTTCAAGGAGACCAATGCCACCTATCCTAGTTCCGACGTGCCCTGTGACGCAACACTAACCGAGAAGTCATTCGAGACCGACATGAACCTCAACTTCCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTCAAGGTTGCCGGCTTCAACCTGCTGATGACCCTCCGGCTTTGGTCTTCGCGTGCCAAGCGCAGCGGTTCCGGC(SEQ ID NO:42)
ATGGGGTCCCGCCTGCTGTGCTGGGTACTCCTGTGCCTGCTGGGAGCTGGACCAGTGAAGGCTGGAGTTACTCAGACTCCCCGTTACCTCATAAAAACGCGTGGTCAGCAGGTCACTCTGTCATGCAGCCCCATCTCTGGCCACCGCTCTGTATCGTGGTACCAGCAGACACCTGGGCAGGGTCTGCAGTTCCTGTTTGAGTACTTCTCCGAGACCCAGCGCAACAAGGGCAACTTCCCGGGTAGATTCTCTGGGCGACAGTTCAGCAATAGCCGGTCTGAGATGAACGTGTCCACCCTCGAGCTTGGTGATTCCGCTCTGTACCTGTGCGCCTCCTCCTTGGGCGGGGAGGGCGACAGGCCTCAGCACTTTGGTGACGGCACTCGACTTAGTATCCTGGAGGACCTGCGCAATGTGACCCCACCCAAGGTGTCGCTGTTCGAGCCCTCCAAGGCAGAGATCGCCAACAAGCAGAAGGCGACTCTGGTCTGTCTGGCGCGTGGCTTTTTCCCCGACCATGTCGAGCTGAGCTGGTGGGTCAACGGTAAAGAGGTCCACTCAGGCGTCTCGACCGACCCCCAGGCCTACAAGGAGAGCAACTATTCCTACTGCTTGTCTTCCAGATTACGGGTGAGCGCAACTTTTTGGCACAACCCCCGCAACCACTTTAGGTGTCAGGTGCAGTTCCATGGACTTTCTGAAGAAGATAAATGGCCTGAAGGGAGCCCTAAGCCCGTCACCCAGAACATCAGCGCCGAGGCCTGGGGCCGCGCGGACTGTGGCATCACCTCCGCTAGCTACCACCAAGGCGTGCTGTCTGCGACCATCCTGTACGAGATTCTGCTAGGCAAAGCTACACTGTACGCCGTGTTGGTGAGCGGCCTGGTCCTGATGGCCATGGTGAAGAAGAAGAACTCC(SEQ ID NO:43) Preferably, the nucleic acid molecule further comprises at least one of the nucleotide sequences shown as SEQ ID NO. 56 (encoding the alpha chain constant region shown as SEQ ID NO. 26) and SEQ ID NO. 57 (encoding the beta chain constant region shown as SEQ ID NO. 27).
ATCCAGAACCCCGAGCCCGCCGTGTACCAGCTGAAGGACCCCAGGAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAGAGCGGCACCTTCATCACCGACAAGACCGTGCTGGACATGAAGGCCATGGACAGCAAGAGCAACGGCGCCATCGCCTGGAGCAACCAGACCAGCTTCACCTGCCAGGACATCTTCAAGGAGACCAACGCCACCTACCCCAGCAGCGACGTGCCCTGCGACGCCACCCTGACCGAGAAGAGCTTCGAGACCGACATGAACCTGAACTTCCAGAACCTGAGCGTGATGGGCCTGAGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGGCTGTGGAGCAGC(SEQ ID NO:56)
GAGGACCTGAGGAACGTGACCCCCCCCAAGGTGAGCCTGTTCGAGCCCAGCAAGGCCGAGATCGCCAACAAGCAGAAGGCCACCCTGGTGTGCCTGGCCAGGGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGAGCACCGACCCCCAGGCCTACAAGGAGAGCAACTACAGCTACTGCCTGAGCAGCAGGCTGAGGGTGAGCGCCACCTTCTGGCACAACCCCAGGAACCACTTCAGGTGCCAGGTGCAGTTCCACGGCCTGAGCGAGGAGGACAAGTGGCCCGAGGGCAGCCCCAAGCCCGTGACCCAGAACATCAGCGCCGAGGCCTGGGGCAGGGCCGACTGCGGCATCACCAGCGCCAGCTACCACCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGAGCGGCCTGGTGCTGATGGCCATGGTGAAGAAGAAGAACAGC(SEQ ID NO:57)
According to a particularly preferred embodiment of the invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 58.
ATGGCGTCTGCTCCTATCAGCATGCTAGCTATGTTGTTCACCTTGAGCGGATTGAGGGCTCAGTCCGTAGCGCAGCCGGAGGATCAGGTCAATGTGGCGGAGGGCAATCCCCTGACCGTTAAGTGCACTTACAGCGTCTCCGGAAACCCCTATCTGTTTTGGTACGTGCAGTACCCCAACAGGGGTCTGCAGTTCCTGTTAAAGTACATCACTGGTGATAACCTGGTGAAAGGCAGTTACGGCTTCGAGGCCGAGTTCAACAAGTCCCAGACCTCCTTTCACCTGAAGAAGCCCTCGGCTCTGGTCTCCGACTCTGCGCTGTACTTCTGTGCGGTGCGTGACATGGAAGGGGGTAATAACGCTCGGCTGATGTTTGGCGACGGGACCCAGCTGGTAGTCAAGCCCAACATCCAGAATCCAGATCCAGCCGTGTACCAGCTGCGCGACTCCAAAAGCAGCGACAAATCGGTTTGTCTATTCACCGACTTCGACTCCCAAACTAATGTCAGTCAGTCCAAGGACTCTGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGTCCATGGATTTCAAGTCCAACTCCGCCGTAGCATGGTCTAACAAGAGCGACTTTGCCTGCGCCAACGCGTTCAACAACTCTATCATCCCCGAGGACACGTTCTTTCCCTCTCCGGAGTCCTCCTGCGACGTGAAGCTCGTGGAGAAATCTTTTGAGACCGACACAAATTTGAACTTCCAGAACCTGTCAGTCATCGGCTTCCGCATCCTTCTGTTGAAGGTCGCGGGATTCAACCTCCTGATGACCCTCCGCCTTTGGTCTTCCCGGGCCAAGCGCAGCGGTTCGGGCGCTACCAACTTTTCGTTGCTGAAGCAGGCCGGGGATGTGGAGGAGAACCCGGGCCCTATGGATACCTGGCTGGTGTGCTGGGCTATTTTCTCCCTGCTCAAGGCCGGTCTCACCGAACCGGAGGTGACCCAGACTCCCTCTCACCAGGTCACCCAGATGGGCCAGGAGGTGATTTTGCGCTGCGTGCCCATCAGCAACCACCTCTACTTCTATTGGTACCGCCAAATCCTAGGCCAGAAAGTGGAGTTCCTGGTGTCCTTTTACAACAACGAGATCAGCGAGAAAAGCGAGATTTTCGATGACCAGTTCAGCGTGGAGCGCCCTGACGGCTCCAACTTTACGCTCAAGATCCGATCCACCAAGCTGGAGGACTCGGCAATGTATTTCTGCGCCTCTTCCGAAGCCTACGGTGGCTACAACGAGCAGTTTTTCGGCCCGGGGACCCGCCTGACTGTGCTGGAGGACCTTAAGAACGTGTTCCCACCGAAGGTGGCGGTTTTCGAGCCCAGCGAGGCCGAGATAAGCCACACTCAGAAGGCGACTCTCGTCTGTCTGGCGACAGGCTTCTACCCTGATCATGTGGAGCTGAGTTGGTGGGTCAACGGTAAGGAGGTGCATTCGGGCGTGTCTACCGATCCTCAGCCACTTAAAGAACAGCCTGCCCTGAACGACTCTCGTTATTGTCTGTCATCCCGCCTGCGCGTCTCTGCCACGTTCTGGCAGAACCCGCGCAACCACTTCCGTTGCCAGGTGCAATTCTACGGATTATCCGAGAATGACGAGTGGACCCAGGACCGCGCCAAGCCTGTGACCCAGATCGTCTCTGCTGAAGCATGGGGCCGAGCAGACTGCGGGTTCACGTCTGAGAGCTATCAGCAGGGAGTGCTGTCCGCTACCATCTTGTACGAGATTCTGCTGGGCAAGGCTACACTGTACGCAGTGCTGGTTTCCGCCCTTGTACTGATGGCCATGGTGAAGCGCAAGGACAGTAGAGGC(SEQ ID NO:58)
According to another particularly preferred embodiment of the invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 59.
ATGAAATCTCTTCGCGTGCTGCTGGTGATTTTGTGGCTCCAGCTGTCTTGGGTCTGGTCTCAGCAGAAGGAGGTGGAACAGAACTCCGGACCTTTGTCGGTGCCGGAGGGCGCCATTGCTTCCCTCAACTGCACTTATTCTGACCGTGGCAGTCAGAGCTTCTTTTGGTACCGCCAGTACAGTGGCAAGTCGCCGGAGCTGATCATGTTTATCTACAGCAATGGCGATAAGGAGGATGGGCGCTTCACGGCGCAGCTTAACAAAGCTTCCCAGTACGTGAGCCTTCTCATTCGCGACAGCCAGCCGAGTGACTCCGCAACCTACCTGTGCGCCGTTAAAGGGGGCTACAACAAGCTGATCTTCGGTGCCGGCACCCGCCTGGCCGTGCACCCGTATATCCAGAACCCCGAGCCAGCGGTGTACCAGTTGAAGGACCCGCGCTCGCAGGACTCCACCCTGTGCCTGTTCACCGATTTCGACTCCCAAATTAACGTGCCAAAGACTATGGAGAGCGGTACGTTTATCACCGACAAAACCGTGCTGGACATGAAGGCCATGGATTCCAAGAGCAACGGCGCTATCGCTTGGTCCAACCAGACCTCCTTCACCTGCCAGGACATCTTCAAGGAGACCAATGCCACCTATCCTAGTTCCGACGTGCCCTGTGACGCAACACTAACCGAGAAGTCATTCGAGACCGACATGAACCTCAACTTCCAGAACCTGTCTGTGATGGGCCTGCGCATCCTGCTGCTCAAGGTTGCCGGCTTCAACCTGCTGATGACCCTCCGGCTTTGGTCTTCGCGTGCCAAGCGCAGCGGTTCCGGCGCGACGAATTTCTCCTTACTAAAGCAAGCGGGAGACGTGGAGGAGAACCCGGGGCCCATGGGGTCCCGCCTGCTGTGCTGGGTACTCCTGTGCCTGCTGGGAGCTGGACCAGTGAAGGCTGGAGTTACTCAGACTCCCCGTTACCTCATAAAAACGCGTGGTCAGCAGGTCACTCTGTCATGCAGCCCCATCTCTGGCCACCGCTCTGTATCGTGGTACCAGCAGACACCTGGGCAGGGTCTGCAGTTCCTGTTTGAGTACTTCTCCGAGACCCAGCGCAACAAGGGCAACTTCCCGGGTAGATTCTCTGGGCGACAGTTCAGCAATAGCCGGTCTGAGATGAACGTGTCCACCCTCGAGCTTGGTGATTCCGCTCTGTACCTGTGCGCCTCCTCCTTGGGCGGGGAGGGCGACAGGCCTCAGCACTTTGGTGACGGCACTCGACTTAGTATCCTGGAGGACCTGCGCAATGTGACCCCACCCAAGGTGTCGCTGTTCGAGCCCTCCAAGGCAGAGATCGCCAACAAGCAGAAGGCGACTCTGGTCTGTCTGGCGCGTGGCTTTTTCCCCGACCATGTCGAGCTGAGCTGGTGGGTCAACGGTAAAGAGGTCCACTCAGGCGTCTCGACCGACCCCCAGGCCTACAAGGAGAGCAACTATTCCTACTGCTTGTCTTCCAGATTACGGGTGAGCGCAACTTTTTGGCACAACCCCCGCAACCACTTTAGGTGTCAGGTGCAGTTCCATGGACTTTCTGAAGAAGATAAATGGCCTGAAGGGAGCCCTAAGCCCGTCACCCAGAACATCAGCGCCGAGGCCTGGGGCCGCGCGGACTGTGGCATCACCTCCGCTAGCTACCACCAAGGCGTGCTGTCTGCGACCATCCTGTACGAGATTCTGCTAGGCAAAGCTACACTGTACGCCGTGTTGGTGAGCGGCCTGGTCCTGATGGCCATGGTGAAGAAGAAGAACTCC(SEQ ID NO:59)
In a third aspect the invention provides a nucleic acid expression vector comprising a coding gene and a vector sequence, wherein the coding gene is provided by a nucleic acid molecule according to the second aspect.
The nucleic acid expression vector provided by the invention is used for expressing the TCR provided by the invention in vitro. That is, expression of the TCR provided by the present invention can be achieved by introducing the nucleic acid expression vector into cells in vitro.
In the nucleic acid expression vector provided by the invention, the coding gene is a nucleic acid sequence for coding and expressing the TCR molecule provided by the invention, and the characteristics are as described above and are not described in detail herein. The vector sequence is typically provided by the original vector (typically a plasmid vector) of the nucleic acid expression vector, which functions to load the coding gene and introduce it into the cell to cause expression of the TCR molecule in the cell. Any vector commonly used in the art to construct in vitro expression systems may be suitable for use in the present invention.
The invention further provides a construction method of the nucleic acid expression vector. Any method for constructing an in vitro expression vector common in the art can be suitable for constructing the nucleic acid expression vector provided by the invention, so long as the encoding gene can be introduced into a vector sequence and can be expressed in vitro cells, and detailed description is omitted.
In a fourth aspect the invention provides a vector cell comprising the T cell antigen receptor of the first aspect and/or comprising the nucleic acid molecule of the second aspect and/or comprising the nucleic acid expression vector of the third aspect.
The vector cell provided by the invention can be used for expressing the TCR molecule provided by the invention in vitro, and can also be used in a corresponding CMV prevention/treatment method or used for preparing related medicines. Any cell common in the art that is capable of the above purpose may be suitable for the present invention. For example, T cells (particularly T cells derived from the patient's own body) may be selected as carrier cells and introduced with the TCR, nucleic acid molecule or nucleic acid expression vector described above for use in the preparation of cell pharmaceutical compositions for CMV therapy/prophylaxis and the like.
For another example, cells commonly used in the art for in vitro expression of proteins, such as prokaryotic expression vectors, may be selected as vector cells into which the above nucleic acid molecules or nucleic acid expression vectors are introduced to produce TCR molecules for introduction into T cells.
The invention further provides a preparation method of the carrier cell. Any method commonly used in the art for introducing a protein (e.g., a TCR of the invention), nucleic acid molecule or nucleic acid expression vector into a cell may be suitable for use in the invention, e.g., electrotransformation, lentiviral transfection, etc.
In a fifth aspect, the invention provides the use of a T cell antigen receptor according to the first aspect, and/or a nucleic acid molecule according to the second aspect, and/or a nucleic acid expression vector according to the third aspect, and/or a vector cell according to the fourth aspect, for the preparation of a medicament and/or vaccine for the prophylaxis and/or treatment of a disease caused by human cytomegalovirus infection.
The specific pharmaceutical forms and components of the drug are not particularly limited as long as the drug contains at least one of the T cell antigen receptor, the nucleic acid molecule, the nucleic acid expression vector and the vector cell provided by the invention.
According to an exemplary preferred embodiment of the present invention, wherein the medicament/vaccine may comprise a fusion protein as (major) active ingredient, the fusion protein comprising the aforementioned TCR.
Preferably, the fusion protein may further comprise an antibody.
More preferably, the antibodies include single chain antibodies (SINGLE CHAIN FRAGMENT variable, scFv), monoclonal antibodies, chimeric antibodies, multispecific antibodies. Preferably a single chain antibody.
More preferably, the antibody is a humanized antibody.
Preferably, the fusion protein may also contain a CD3 agonist.
Preferably, the fusion protein may also contain antibodies specifically targeting CD 3.
In a sixth aspect, the present invention provides a pharmaceutical composition for preventing and/or treating a disease caused by human cytomegalovirus infection, comprising: the T cell antigen receptor of the first aspect, and/or the nucleic acid molecule of the second aspect, and/or the nucleic acid expression vector of the third aspect, and/or the vector cell of the fourth aspect.
In the pharmaceutical composition provided by the invention, the TCR, the nucleic acid molecule, the nucleic acid expression vector and the vector cell can be used as (one of) the (main) active components of the pharmaceutical composition. According to some preferred embodiments of the present invention, pharmaceutically acceptable excipients (e.g., buffers, preservatives, stabilizers, etc.) may also be included in the pharmaceutical composition.
According to some preferred embodiments of the present invention, other active ingredients may be included in the pharmaceutical composition, for example, drugs or the like (e.g., antiviral drugs, antibodies, vaccine formulations, etc. useful for preventing/treating CMV infection) that have no or substantially no adverse effect on their effects and that do not have (severe) side effects on humans when used together with the TCRs, nucleic acid molecules, nucleic acid expression vectors, and vector cells provided herein.
The present invention will be described in detail by examples. It should be understood that the following examples are illustrative only and are not intended to limit the invention.
In the following examples, experiments involving human sample collection and detection have been informed by relevant persons. Unless specifically stated, reagents/materials used were commercially available from either regular chemical/biological reagents or material suppliers, and the purity of the reagents was analytically pure.
In the following examples, the operating temperatures were room temperature (25.+ -. 5 ℃ C.) unless otherwise specified.
Example 1
The present example is presented to demonstrate the preparation, expansion and killing of TCR-T cells provided by the present invention.
Preparation and expansion of TCR-T cells
1. Experimental materials
2. Experimental method
Chemically synthesized CMV epitope peptides were used to stimulate Peripheral Blood Mononuclear Cells (PBMCs) derived from HLA-A 11:01 healthy donors and patients with different cancer species. After 2 rounds of CMV epitope peptide stimulation, specific T cells were detected using flow assays and enzyme linked immunosorbent assay (Enzyme linked immunospot assay, ELISpot) to confirm that CMV specific T cells were obtained. The specific method comprises the following steps:
HLA-A 11:01 donor peripheral blood was collected and PBMCs were isolated from peripheral blood using density gradient centrifugation. The isolated PBMC were inoculated into an orifice plate at a constant cell density, and cultured with X-VIVO15 (LONZA) containing cytokines such as human serum, GM-CSF (Xiaomenbao bioengineering Co., ltd.) and IL-2 (Beijing Shuanglu pharmaceutical Co., ltd.).
Dissolving HLA-A 11:01CMV epitope peptide (gold srey; specific sequence is shown as SEQ ID NO: 1) by using X-VIVO15 to prepare CMV epitope peptide solution with concentration of 100 mug/mL, filtering and sterilizing for later use. Cells were cultured to day 2 and 4, each stimulated with one sterile CMV epitope peptide solution at a final concentration of 5 μg/mL.
The total cell culture cycle was about 14 days, cells were observed every 2-3 days during the culture, half-cell exchange was performed using the medium, and viable cell density, cell suspension volume and cell viability were recorded using a cell counter. On day 14, T cells were flow detected by staining with murine anti-human CD8 antibody (Biolegend) and HLA-A x 11:01cmv epitope tetramer (MBL). The experimental results are shown in FIG. 1. As can be seen by comparison with the control T cell group (without CMV epitope peptide stimulation), CMV-specific T cells were confirmed after stimulation with CMV polypeptide. The CD8 positive and tetramer positive T cell populations of interest were sorted out using a flow sorter.
And (3) performing ELISPot detection by taking IFN-gamma as a cytokine to be detected. A certain amount of cells were taken and washed with X-VIVO15 medium. After resuspension of cells with X-VIVO15 containing 10% FBS, cells were seeded in Elispot-IFN-gamma assay plates (Mabtech) at a cell number of 2X 10 4 per well. Antibody incubation and development were performed 18-24 hours after stimulation with 10 μg/mL CMV epitope peptide (MBL). The well plate was placed at room temperature in a shade and dark place, and after the well plate was naturally dried, the immunoblotches were imaged and read using c.t.ls6, and the results are detailed in fig. 2.
As can be seen from FIGS. 1 and 2, the specific T cell expansion method provided by the invention can obtain HLA-A 11:01CMV specific T cells, and compared with a control T cell group (A-C hole in FIG. 2) which is not stimulated by CMV epitope peptide, the stimulated CMV pp65 group (D-F hole in FIG. 2) can detect significantly more spots, which indicates that CD8 + T cells which are expanded by in vitro stimulation can specifically recognize CMV pp65 epitope peptide and secrete cytokines such as IFN-gamma.
Example 2
This example is presented to illustrate the construction of the in vitro transcribed plasmid vector and mRNA preparation of the TCR provided by the invention.
1. Experimental materials
Name of the name | Manufacturer' s | Goods number |
Non-ribozyme water (non-DEPC treatment) | Invitrogen | AM9932 |
QIAGEN OneStep RT-PCR Kit(100) | Qiagen | 210212 |
Single cell sequence specific amplification kit | Nanjinouzan Biotechnology Co., ltd | P621-01 |
HotStarTaq Master Mix Kit(250) | Qiagen | 203443 |
Qubit 1X dsDNA HS Assay Kit | Invitrogen | Q33230 |
DL500DNA Marker | TaKaRa | 3590A |
6×Loading Buffer | TaKaRa | 9156 |
2. Experimental method
Referring to the plasmid spectrogram of FIG. 3, a nucleic acid expression vector of TCR was constructed by inserting a coding gene of TCR (abbreviated as TCR. Alpha./. Beta. Coding gene) into pUC57 plasmid (Gonsrui) as a vector plasmid. Wherein the coding gene of TCR comprises the coding genes of alpha chain and beta chain, the coding gene of alpha chain (TRA ORF part in the figure) comprises the coding gene of alpha chain variable region and the coding gene of alpha chain constant region; the coding gene of the beta chain (TRB ORF part in the figure) includes a coding gene of the variable region of the beta chain and a coding gene of the constant region of the beta chain; the coding genes of the alpha chain and the beta chain are connected through a 2A sequence (p 2A sequence) of the internal self-splitting porcine teschovirus (Porcine teschovirus), and the specific sequences are shown in Table 1.
TABLE 1
Construction of in vitro transcription plasmid vector: the TCR alpha/beta coding gene sequence (containing BamHI, sacI cleavage sites) designated as synthesized by Nanjing Jinsrui was cloned between BamHI, sacI cleavage sites of pUC57 plasmid containing Kozak sequence (GCCACC) downstream of BamHI cleavage site and double stop codon sequence (TGATAA) upstream of SacI cleavage site by BamHI, sacI restriction enzyme cleavage.
MRNA preparation is carried out by using the constructed in vitro transcription plasmid vector and adopting the following method:
linearizing in vitro transcription plasmid vector by adopting the system of Table 2, placing the reaction system at 50 ℃ for reaction overnight (not more than 18 h) after the configuration of the reaction system is completed, and heating to 80 ℃ for 20min after the reaction is completed to inactivate enzymes in the system.
TABLE 2
Name of the name | Additive amount |
Plasmid(s) | 5μg |
NEBufferTM r3.1 | 4μL |
BspQI | 2μL |
Non-ribozyme water | Final volume of 40. Mu.L |
UsingDNA Clean Beads (DNA magnetic Beads) were used to purify linearized plasmids in the reaction products. After electrophoresis verification and concentration detection of the purified product, an in vitro transcription system was prepared according to Table 3, and incubated at 37℃for 2.5h after the preparation was completed. After the incubation was completed, 4. Mu.L of DNase I was added to the reaction system and incubated at 37℃for 15min to digest the transcribed DNA template.
TABLE 3 Table 3
Name of the name | Additive amount |
Template (purified linearization plasmid) | 1μg |
10×Transcrption Buffer | 8μL |
UTP solution | 8μL |
ATP solution | 8μL |
CTP solution | 8μL |
GTP solution | 8μL |
T7 RNA Polymerase Mix | 8μL |
RNase-free ddH 2 O | Final volume of 80. Mu.L |
UsingDNA Clean Beads (DNA magnetic Beads) purify the in vitro transcription products. After electrophoresis verification and concentration detection, 100 mug of purified product is added with a proper volume of non-ribozyme water to prepare 140 mug of RNA solution, and the RNA solution is heated for 5min at 65 ℃ and then placed on ice to be cooled for 5min, thus obtaining the heat-denatured RNA solution. The capping system was then formulated as in Table 4 and reacted at 37℃for 1.5h after completion of the formulation.
TABLE 4 Table 4
Component (A) | Dosage of | Final concentration |
Heat denatured RNA | 140μL | 0.5μg/μL |
10x Capping buffer | 20μL | 0.5U/μL |
Vaccinia capping enzyme (10U/. Mu.L) | 10μL | 2.5U/μL |
MRNA Cap 2' -O-methyltransferase (50U/. Mu.L) | 10μL | 0.5mM |
GTP(10mM) | 10μL | 0.2mM |
SAM(4mM) | 10μL | 500ng/μL |
UsingThe reaction product was purified by DNA Clean Beads (DNA magnetic Beads) to obtain TCR mRNA after capping. After BspQI cleavage, electrophoresis was performed to verify the results, and the results are shown in FIG. 4. In the figure, samples ZZ1, ZZ2 and ZZ3 are the electrophoresis results of the digested products of mRNA (designated as ZZ1 mRNA, ZZ2 mRNA and ZZ3 mRNA, respectively) obtained by the above-mentioned treatments of the nucleic acid expression vectors I, II and III, respectively.
Example 3
This example is used to illustrate the results of the activation verification of TCRs provided by the present invention.
1. Experimental materials
Preparation of experimental cells:
In order to meet the functional verification requirement of HLA-A 11:01 restricted TCR sequences, transformation is performed on the basis of human lymphoma blast cells (K562), and stable transgenic cell lines capable of expressing different HLA subtypes are constructed. Because of the lack of expression of HLA class I and class II molecules on the surface of K562 cells, single HLA-expressing cell lines can be constructed artificially and used as target cells to verify HLA-restricted killing. The specific construction method is as follows:
HLA over-expression lentiviral packaging:
(1) 293FT cells with good growth state are inoculated into T175 culture flasks, 20mL of Opti-MEM complete culture medium (Thermo FISHER SCIENTIFIC) is added into each flask, the inoculum size is about 1.2X10- 7 cells/flask, the cells are cultured overnight at 37 ℃ until the cell confluency reaches 50-60%, the reagents prepared according to the formula of Table 5 (the preparation amount of the T175 culture flask is the same as that of the T175 culture flask) are added into each flask for transfection, and the mixture is placed into a 37 ℃ incubator for continuous culture after uniform mixing.
TABLE 5
* The HLA lentivirus expression plasmid map is shown in FIG. 5.
(2) Culture supernatant was collected 48h after transfection and stored at 4 ℃. Then 20mL of fresh Opti-MEM complete medium (Thermo FISHER SCIENTIFIC) was added to each flask, and the culture was continued at 37 ℃.
(3) Culturing for 72h, collecting culture medium supernatant again, combining with the 48h supernatant collected in the step (2), centrifuging at 4500rpm for 5min at 4 ℃, removing precipitate, filtering the obtained supernatant with a 0.22 μm vacuum filter flask, adding PEG8000 concentration reagent with 1/5 of the volume of the filtrate, mixing well, and concentrating and settling at 4 ℃ overnight.
(4) After the concentration and sedimentation are finished, the mixture is centrifuged at 4500rpm for 50min at 4 ℃, the sediment is collected and resuspended in 200. Mu.L of precooled D-PBS, and after the mixture is mixed, 50. Mu.L/tube is split into 1.5mL centrifuge tubes for standby at-80 ℃.
Lentiviruses infect cells of interest and obtain positive monoclonal:
(1) The cultured K562 wild-type cells are prepared into a cell suspension of 3X 10 5 cells/mL, inoculated into a 12-well plate according to the dosage of 1mL per well, and simultaneously added with 5 mu L of the polyglutamine infection promoting reagent and 50 mu L of the slow virus, and the mixture is uniformly mixed, cultured at 37 ℃ and passaged.
(2) After two generations, cell flow is adopted to detect HLA expression condition, and wild K562 cells are used as negative control. HLA positive rate and expression quantity are detected through a flow, and cells are subjected to flow sorting monoclonal, and cells with 5% of the HLA expression intensity are selected and placed in at least one 96-well plate for amplification culture.
(3) The HLA expression level of the monoclonal cells is detected again by using the wild type K562 cells as a negative control in a flow mode, compared with the wild type, the HLA expression level is obviously increased, the monoclonal cells with the highest expression level are selected as target cells for functional verification of HLA-A11:01 restriction type TCR sequences for subsequent experiments (the flow detection result is shown in figure 6, the HLA positive rate of the K562-HLA-A11:01 OE cells is 98.68%).
2. Experimental method
The activation state of the TCR pathway was verified by detecting Luciferase fluorescence values using genetically engineered NFAT-Luc Jurkat cells as follows:
(1) Obtaining NFAT-Luc Jurkat cells expressing TCR:
NFAT-Luc Jurkat cells were cultured to a density of 2×10 7/mL or more, cells were collected, washed 1-fold with DPBS (cytova), resuspended in R-solution (Thermo FISHER SCIENTIFIC) to prepare a 2×10 7/mL cell suspension, which was added to a 1.5mL EP tube at 100 μl/tube, and 5 μg CD8 mRNA (gold srey), 5 μg ZZ1 TCR mRNA, and 5 μg ZZ2 mRNA, respectively, were added to EP tubes where CD8 mRNA or TCR mRNA was to be electrotransferred, respectively, and labeled. 3mL of electrotransfer E2 solution (Thermo FISHER SCIENTIFIC) was added to the electrotransfer cup, the electrotransfer cup was placed into the electrotransfer apparatus (Thermo FISHER SCIENTIFIC) cup tank, and the cell mixture was aspirated with a 100. Mu.L transfer tip, electrotransfer conditions: 1400V,20ms,2pulse; the added mRNA was introduced into NFAT-Luc Jurkat cells to give Jurkat cells expressing CD8 or TCR.
The CD8 or TCR expressing Jurkat cells described above were stained with HLA-A 11:01CMV tetramer (MBL) 24h after electrotransformation and the staining results are shown in FIG. 7. It can be seen from the figure that the electrotransformation successfully obtained cells capable of expressing the corresponding transferred mRNA, and that specific T cell populations expressing both TCR mrnas obtained in example 2 could be detected by HLA-A 11:01cmv tetramerization.
(2) TCR activation validation:
NFAT-Luc Jurkat cells expressing ZZ1 TCR and ZZ2 TCR were incubated with HLA-overexpressed K562 cells prepared as described previously at a 20:1 quantitative ratio for 4h at 37 ℃. After the incubation was completed, 100. Mu.L of Bio-Lite detection reagent (Norflu) equilibrated to room temperature was added to each well after one wash of DPBS (Cytiva), and after 2-5min of standing in the dark, detection was performed with an enzyme-labeled instrument, and the results are shown in FIGS. 8A and 8B. As can be seen from the figure, only HLA-A 11:01K562 cells loaded with CMV pp65 epitope peptide activated Jurkat Luc fluorescence.
Example 4
The present example is presented to demonstrate the killing effect verification of TCR-T cells provided by the present invention.
1. Experimental materials
2. Experimental method
Peripheral blood was obtained from healthy donors, PBMCs from the donors were isolated by Ficoll density gradient centrifugation and resuspended in XVIVO medium. T cells were isolated by magnetic bead purification and activated by CD3/CD28 magnetic beads and resuspended in XVIVO medium containing 2.5% human autologous serum and 30IU/mL IL-2 for 3 days. Cells were collected, DPBS was washed 1 time and resuspended in R-solution to prepare a 2X 10 7/mL cell suspension, which was added to a 1.5mL EP tube at 100. Mu.L/serving, and 5. Mu.g ZZ1 TCR mRNA and ZZ2 TCR mRNA, respectively. E2 solution 3mL is added into an electrorotor, the electrorotor is placed into a electrorotor cup groove, a 100 mu L transfer gun head is used for sucking cell mixed solution, and electrotransformation conditions are as follows: 1400V,10ms,3pulse; TCR mRNA was introduced into activated T cells to give T cells expressing TCR.
T cells expressing TCR as described above were stained with HLA-A 11:01CMV tetramer (MBL) 24h after electrotransformation. The results are shown in detail in FIG. 9.
Culturing K562 cells to a cell density of more than 1×10 7/mL, collecting the cells, washing the cells with 1640 culture medium for 1 time and re-suspending the cells to prepare a cell suspension of 2×10 6/mL, sucking 200 mu L/hole to 48-hole cell culture plates of the cell suspension, adding 60 mu L/hole of 200 mu M polypeptide (SEQ ID NO: 1) and then adding 1640 culture medium to fill up to 400 mu L/hole, and standing at 37 ℃ for overnight incubation.
K562 cells were incubated at 37℃for 20 min with 1. Mu.L EuTDA dye at 8X 10 5/mL, centrifuged, and the cells were washed 3 times with 1mL of 1 XPBS. After the last wash, the cells were resuspended in T cell medium.
TCR-expressing T cells (TCR-T cells containing the ZZ1 TCR and ZZ2 TCR obtained in example 2, respectively, and flow-validated to confirm successful transfer of TCR mRNA into activated T cells) were incubated with the above K562 cells at a number ratio of 20:1, 10:1, 5:1, 2.5:1, 1.25:1, respectively, for 4h at 37 ℃. After incubation, 500g was centrifuged at room temperature for 5 minutes. mu.L of the supernatant was pipetted into a flat bottom 96-well plate of EuTDA Cytotoxicity Reagents kit, 200. Mu.L of Eu solution was added to each well and incubated at room temperature with shaking at 250rpm for 10 minutes. The fluorescence values at the excitation light 330/80 and the emission light 620/10 wavelength were recorded by a microplate reader within 5 hours, and the window values and the killing efficiency were calculated from each set of fluorescence values, and the calculation formula was as follows, and the results are shown in FIG. 10 ((A) is ZZ1 TCR-T) and (B) is ZZ2 TCR-T).
Example 5
This example is used to demonstrate the results of T cell peptide sensitivity evaluation of TCRs provided by the present invention.
1. Experimental materials
Name of the name | Manufacturer' s | Goods number |
D-PBS | Cytiva | SH30028.02 |
Human IFN-γPrecoated ELISA Kit | Shenzhen Daida bioengineering Co., ltd | 1110003 |
RPMI-1640 | Thermo Fisher Scientific | R5886 |
Fetal bovine serum | Thermo Fisher Scientific | SV30087.02 |
Green streptomycin | Thermo Fisher Scientific | 15140-122 |
X-VIVO 15 | Lonza | 04-418Q |
2. Experimental method
T cells expressing ZZ1 TCR and ZZ2 TCR were obtained by the method of example 3. The two TCR-T cells obtained were then incubated with K562 loaded with 10 -5 M to 10 -12 M CMV epitope peptide in a 1:1 quantitative ratio for 18-24h, respectively. After the incubation, each group of cells and supernatant were collected. EC50 was estimated by plotting a fitted curve using the flow-test cell cd3+4-1bb+/cd3+ ratio (%). The concentration of IFN-gamma in the supernatants of each group of cells was measured by ELISA and a fitted curve was drawn to estimate the EC50.
K562 cells were collected, counted using a cytometer, and viable cell density and cell viability were recorded. Cells were resuspended after 1 pass using RPMI 1640 (Thermo) medium. The 24-well plates were seeded at an inoculum size K562 per well of 5X 10 5 cells, for a total of 9 groups. Different concentrations of CMV epitope peptide were added to the different groups such that the final peptide concentration was 10-12M、10- 11M、10-10M、10-9M、10-8M、10-7M、10-6M、10-5M, and the loading time was 4 hours. The TCR-T cells after the transformation were collected, counted using a cytometer, and viable cell density and cell viability were recorded. The tetramer assay was performed using X-VIVO 15 with 10% FBS (Cytiva) to resuspend cells and 2X 10 5 cells.
Epitope peptide-loaded K562 was collected and cell density was adjusted to 1X 10 6 cells/mL using XVIVO containing 10% FBS. After adjustment, 100. Mu.L of the cell suspension was added to a 96U-well plate, 3 multiplex wells per group. The TCR-T cell density was adjusted to 1X 10 6 cells/mL using XVIVO containing 10% FBS, 100. Mu.L of the cell suspension was added to a 96U-type well plate which had been seeded with K562 target cell suspension, and mixed well so that the effector cell to target cell number ratio was 1:1. After incubation of TCR-T with K562 overnight (about 18-24 hours) at 37℃the 96-well plate samples were centrifuged and the supernatant was subjected to ELISA-IFN-gamma assay (Dakko).
The ELISA standard lyophilized powder was dissolved according to the label specification, allowed to stand for 5-10 minutes, and then diluted with 1 Xdiluent to 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL, respectively.
After 10 and 100-fold dilution of the collected cell supernatants with the dilution solution, diluted samples were added to a 96-well ELISA plate at 100. Mu.L per well. Adding diluted antibody into the standard substance and the sample hole according to 50 mu L/hole, uniformly mixing, and covering a sealing plate film. Incubate at 37℃for 2 hours. After the incubation, the liquid in the wells was discarded, 1 Xwashing buffer was added at 300. Mu.L/well, and after 1 minute of residence, the liquid in the wells was discarded, and this step was repeated 4 times. Diluted avidin was added to the wells of the samples at 100. Mu.L/well, the membrane was covered and incubated at 37℃for 30 minutes. After the incubation, the well plate was washed again, TMB was added at 100. Mu.L/well, and incubated at 37℃for 5-30 minutes in the absence of light. After the completion of the color development, 100. Mu.L/well of a stop solution was added. Within 10 minutes after termination, plates were read simultaneously with detection wavelength 450nm and correction wavelength 630 nm. IFN-y concentrations were calculated for each group of cell supernatants based on standard curves, the results were counted using GRAPHPAD PRISM and fitted curves were drawn, and the experimental results are detailed in FIGS. 11A-11B. As can be seen from the figure, both TCR-T cells expressing the TCR mRNA obtained in example 2 recognize HLA-A 11:01K652 cells loaded with different concentrations of CMV pp65 antigen peptide and release cytokines such as IFN-gamma, indicating that both TCR-T cells specifically recognize and kill cells expressing CMV pp 65. The EC 50 of ZZ1 TCR-T cells was estimated to be about 10 -5 M by fitting the curve. Although the ZZ2 TCR-T cells did not reach IFN-gamma secretion peak, EC 50 could not be calculated, but experimental results showed that they also had better ability to recognize and kill HLA-A 11:01K652 cells loaded with CMV pp65 antigen peptide.
Example 6
This example is presented to illustrate the results of motif evaluation of TCRs provided by the present invention.
1. Experimental materials
2. Experimental method
The ALA-SCAN method is adopted to identify specific amino acid sites closely related to functions, stability and conformation of the TCR provided by the invention, and the constructed alanine scanning peptide library is shown in figure 12 (the specific sequence of the epitope peptide is shown in table 6).
TABLE 6
Epitope peptide numbering | ID | Sequence(s) |
1 | SEQ ID NO:61 | AAVQGQNLK |
2 | SEQ ID NO:62 | ATAQGQNLK |
3 | SEQ ID NO:63 | ATVAGQNLK |
4 | SEQ ID NO:64 | ATVQAQNLK |
5 | SEQ ID NO:65 | ATVQGANLK |
6 | SEQ ID NO:66 | ATVQGQALK |
7 | SEQ ID NO:67 | ATVQGQNAK |
8 | SEQ ID NO:68 | ATVQGQNLA |
T cells expressing ZZ1 TCR and ZZ2 TCR were obtained by the method of example 4.
K562 cells were cultured until the cell density reached more than 1X 10 7/mL, the cells were collected, 1640 medium was washed 1 time and resuspended to obtain a cell suspension of 2X 10 6/mL, 200. Mu.L/well of this cell suspension was aspirated and added to a 48-well cell culture plate, while 60. Mu.L of epitope peptide (SEQ ID NO: 58-65) was added to each well, 1640 medium was made up to 400. Mu.L/well, and incubated overnight at 37 ℃.
Two TCR expressing T cells were incubated with the K562 cells described above at a 1:2 quantitative ratio at 37℃for 24h, respectively, and the results are shown in FIGS. 13-14. From the figure, it can be seen that the motif recognized by the electrotransport ZZ2 TCR is-TVQGQNLK, and the motif recognized by the ZZ1 TCR is-QGQ-K. The results indicate that the motif of ZZ2 recognition is more conserved.
Comparative example 1
Using the method of example 3, NFAT-Luc Jurkat was electrotransformed into ZZ3 TCR mRNA obtained in example 2 to obtain Jurkat cells expressing ZZ3 TCR, the flow test results of which are shown in FIG. 15. From the experimental results, the ZZ3 Jurkat cells were devoid of CMV-Sup>A 11:01 tetramer positive population.
The ZZ3 TCR sequence (SEQ ID NO: 54) comprises an alpha chain variable region with an amino acid sequence shown as SEQ ID NO:24, a beta chain variable region with an amino acid sequence shown as SEQ ID NO:25, and an alpha chain constant region and a beta chain constant region with amino acid sequences shown as SEQ ID NO:26-27, respectively. Wherein, the alpha chain variable region comprises CDR1 alpha, CDR2 alpha and CDR3 alpha with amino acid sequences shown as SEQ ID NO:18-20 respectively (the coding genes are shown as SEQ ID NO:44-46 respectively); the beta chain variable region comprises CDR1 beta, CDR2 beta and CDR3 beta which have the amino acid sequences shown in SEQ ID NO:21-23 respectively (the coding genes of the beta chain variable region are shown in SEQ ID NO:47-49 respectively).
The results of the NFAT-Luc reporting system experiments are shown in fig. 16, which shows that the control group has fluorescence values in the range of 12 to 14, and the ZZ3 Jurkat has fluorescence value of 18, which is close to the control group, indicating that the TCR cannot be activated by K562-a 11:01-pep.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. The technical solution of the invention can be modified in various ways within the scope of the technical idea of the invention, including that the technical features are combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, which is within the scope of the protection of the invention.
Claims (10)
1. A T cell antigen receptor, wherein the T cell antigen receptor specifically recognizes and binds to a human cytomegalovirus binding epitope comprising the amino acid sequence shown in SEQ ID No. 1.
2. The T cell antigen receptor of claim 1, wherein the T cell antigen receptor comprises at least one alpha chain variable region and/or at least one beta chain variable region.
3. The T cell antigen receptor of claim 2, wherein the alpha chain variable region comprises a complementarity determining region of an alpha chain and the beta chain variable region comprises a complementarity determining region of a beta chain;
Wherein the complementarity determining region of the α -chain comprises an amino acid sequence having at least 80% homology with at least one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12; and/or the number of the groups of groups,
The complementarity determining region of the β strand comprises an amino acid sequence having at least 80% homology with at least one of SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15.
4. A T cell antigen receptor according to claim 2 or 3, wherein the alpha chain variable region comprises the amino acid sequence shown in SEQ ID No. 8 and/or SEQ ID No. 16;
and/or, the beta chain variable region comprises the amino acid sequence shown in SEQ ID NO 9 and/or SEQ ID NO 17.
5. The T cell antigen receptor according to any one of claims 1 to 4, wherein in the T cell antigen receptor, the α chain variable region is directly linked or indirectly linked to the β chain variable region;
preferably, in the T cell antigen receptor, the α -chain variable region is indirectly linked to the β -chain variable region, preferably the α -chain variable region is indirectly linked to the β -chain variable region via furin SGSG p2A sequence.
6. A nucleic acid molecule encoding the T cell antigen receptor of any one of claims 1-5.
7. A nucleic acid expression vector comprising a coding gene and a vector sequence, wherein the coding gene is provided by the nucleic acid molecule of claim 6.
8. A vector cell comprising the T cell antigen receptor of any one of claims 1-5, and/or comprising the nucleic acid molecule of claim 6, and/or comprising the nucleic acid expression vector of claim 7.
9. Use of a T cell antigen receptor according to any one of claims 1 to 5, and/or a nucleic acid molecule according to claim 6, and/or a nucleic acid expression vector according to claim 7, and/or a vector cell according to claim 8 for the manufacture of a medicament for the prevention and/or treatment of a disease caused by human cytomegalovirus infection, in particular for the manufacture of a medicament for the prevention and/or treatment of a disease caused by human cytomegalovirus infection using TCR-T therapy.
10. A pharmaceutical composition for preventing and/or treating a disease caused by human cytomegalovirus infection, comprising: the T cell antigen receptor of any one of claims 1-6, and/or the nucleic acid molecule of claim 7, and/or the nucleic acid expression vector of claim 8, and/or the vector cell of claim 9.
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