T cell receptor for disease treatment
阅读说明：本技术 应用于疾病治疗的t细胞受体 (T cell receptor for disease treatment ) 是由 于力 王智鼎 于 2021-10-14 设计创作，主要内容包括：本发明公开了应用于疾病治疗的T细胞受体,所述T细胞受体包含α和/或β链,所述α或β链包含SEQ ID NO.1-16任一项所述的CDR氨基酸序列或可变区氨基酸序列。本发明还公开了编码T细胞受体的核酸分子、表达载体、宿主细胞和组合物及其应用。(The invention discloses a T cell receptor for use in the treatment of disease, said T cell receptor comprising an alpha and/or beta chain comprising a CDR amino acid sequence or a variable region amino acid sequence as set out in any one of SEQ ID No.1 to 16. The invention also discloses nucleic acid molecules encoding T cell receptors, expression vectors, host cells and compositions and uses thereof.)
1. A T cell receptor comprising the variable domain va of the alpha chain and/or the variable domain V β of the beta chain, said va comprising the amino acid sequence of SEQ ID NO: 5, and said V β comprises the amino acid sequence set forth in SEQ ID NO: 13;
preferably, the T cell receptor comprises:
(a) SEQ ID NO:1, optionally as set forth in SEQ ID NO: 2, further optionally as shown in SEQ ID NO: 3 is shown in the specification;
(b) SEQ ID NO: 4, CDR2 a amino acid sequence set forth in seq id no;
(c) SEQ ID NO: 5, the CDR3 a amino acid sequence shown in seq id no;
(d) SEQ ID NO: 9, optionally as set forth in SEQ ID NO: 10, further optionally as set forth in SEQ ID NO: 11 is shown in the figure;
(e) SEQ ID NO: 12, the CDR2 β amino acid sequence shown in seq id no; and
(f) SEQ ID NO: 13, CDR3 β amino acid sequence;
preferably, the T cell receptor comprises V.alpha.having at least 80%, preferably 90%, preferably 95% identity with the amino acid sequence as set forth in any one of SEQ ID Nos. 6 to 8 and/or V.beta.having at least 80%, preferably 90%, preferably 95% identity with the amino acid sequence as set forth in any one of SEQ ID Nos. 14 to 16;
preferably, the T cell receptor comprises V alpha shown by an amino acid sequence shown in any one of SEQ ID NO 6-8 and/or V beta shown by an amino acid sequence shown by any one of SEQ ID NO 14-16;
preferably, the T cell receptor comprises V alpha shown by an amino acid sequence shown as SEQ ID NO. 8 and/or V beta shown by an amino acid sequence shown as SEQ ID NO. 14;
preferably, the T cell receptor is a soluble T cell receptor lacking a transmembrane domain;
preferably, the T cell receptor binds to MHC I and/or MHC II peptide complexes;
preferably, the T cell receptor further comprises a detectable label;
preferably, the detectable label comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, biotin;
preferably, the alpha chain further comprises an alpha constant region ca and/or the beta chain further comprises a beta constant region cbp;
preferably, the ca region and/or the cp region comprises the introduction of one or more cysteines capable of forming one or more non-native disulfide bridges between the a chain and the β chain.
2. The T-cell receptor of claim 1, wherein the alpha and beta chains further comprise a signal peptide.
3. A recombinant T cell receptor comprising the T cell receptor of claim 1 or 2, and/or a costimulatory region;
preferably, the costimulatory region comprises a costimulatory molecule selected from the group consisting of a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, and any combination thereof;
preferably, the co-stimulatory region comprises a CD28 polypeptide.
4. A nucleic acid encoding the T cell receptor of claim 1 or 2; or encodes the recombinant T cell receptor of claim 3;
preferably, the nucleotide sequence of the nucleic acid is codon optimized for expression in a host cell;
preferably, the host cell is optionally an immune system cell;
preferably, the immune system cell is a T cell.
5. An expression vector comprising the nucleic acid of claim 4 operably linked to an expression control sequence;
preferably, the expression vector is capable of delivering the polynucleotide to a host cell;
preferably, the vector is a viral vector;
preferably, the viral vector is a retroviral vector or a lentiviral expression vector.
6. A host cell comprising the nucleic acid of claim 4 or the expression vector of claim 5;
preferably, the T cell receptor is heterologous to the host cell;
preferably, the host cell is selected from hematopoietic progenitor cells or cells of the immune system;
preferably, the immune system cell is selected from the group consisting of an immune system cell that is a CD4+ T cell, a CD8+ T cell, a CD4-CD 8-double negative T cell, a γ δ T cell, a natural killer T cell, a macrophage, a dendritic cell, or any combination thereof;
preferably, the immune system cell is a T cell;
preferably, the T cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
7. A chimeric molecule comprising a T cell receptor or a portion thereof according to claim 1 or 2 conjugated to a non-cellular substrate, toxin and/or antibody;
preferably, the acellular substrate is selected from the group consisting of: nanoparticles, exosomes and other non-cellular substrates.
8. A composition comprising the T cell receptor of claim 1 or 2, the recombinant T cell receptor of claim 3, the nucleic acid of claim 4, the expression vector of claim 5, the host cell of claim 6, the chimeric molecule of claim 7;
preferably, the composition further comprises a pharmaceutically acceptable carrier.
9. A method for engineering an immune cell, comprising contacting the immune cell with the expression vector of claim 5;
preferably, the immune cell is a T cell, a peripheral blood lymphocyte, an NK cell, a constant NK cell, or an NKT cell;
preferably, the contacting is transfection or transduction;
preferably, the method further comprises sorting the immune cells to isolate TCR-engineered T cells;
preferably, the method further comprises T cell cloning by serial dilution, and expanding T cell clones by a rapid expansion protocol.
10. Use according to any one of the following:
(1) use of the T cell receptor of claim 1 or 2, the recombinant T cell receptor of claim 3, the nucleic acid of claim 4, the expression vector of claim 5, the host cell of claim 6, the chimeric molecule of claim 7, the composition of claim 8 for the preparation of a product for adoptive cell transfer therapy;
preferably, the adoptive cell transfer is adoptive T cell transfer;
preferably, the adoptive T cell transfer is allogeneic adoptive T cell transfer, autologous adoptive T cell transfer, or universal non-alloreactive adoptive T cell transfer;
(2) use of the T cell receptor of claim 1 or 2, the recombinant T cell receptor of claim 3, the nucleic acid of claim 4, the expression vector of claim 5, the host cell of claim 6, the chimeric molecule of claim 7, the composition of claim 8 for the preparation of a product for the diagnostic evaluation of peptide/MHC in tumor cells;
(3) use of the T cell receptor of claim 1 or 2, the recombinant T cell receptor of claim 3, the nucleic acid of claim 4, the expression vector of claim 5, the host cell of claim 6, the chimeric molecule of claim 7, the composition of claim 8 for the preparation of a targeting product for directing a therapeutic molecule to a tumor site;
(4) use of the T cell receptor of claim 1 or 2, the recombinant T cell receptor of claim 3, the nucleic acid of claim 4, the expression vector of claim 5, the host cell of claim 6, the chimeric molecule of claim 7, the composition of claim 8 for the preparation of a product for enhancing immunity;
(5) use of the T cell receptor of claim 1 or 2, the recombinant T cell receptor of claim 3, the nucleic acid of claim 4, the expression vector of claim 5, the host cell of claim 6, the chimeric molecule of claim 7, the composition of claim 8 for the preparation of a medicament for the treatment and/or prevention of a disease;
preferably, the disease is selected from a hematological malignancy or a solid tumor;
preferably, the hematological malignancy is selected from the group consisting of: acute myeloid leukemia, chronic myeloid leukemia, lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-hodgkin's lymphoma, and hodgkin's lymphoma;
preferably, the hematological malignancy is selected from acute myeloid leukemia, lymphoma;
preferably, the solid tumor is selected from the group consisting of: lung cancer, breast cancer, esophageal cancer, stomach cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial cancer, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.
Technical Field
The invention belongs to the fields of cellular immunology and genetic engineering, and relates to a T cell receptor applied to disease treatment.
Background
The T Cell Receptor (TCR) is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC), and this foreign or endogenous peptide may be the only indication of cellular abnormalities. In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.
TCRs exist in α β and γ δ forms, which are structurally similar, but have very different structural positions and possible functions. The α and β chains of native heterodimeric α β TCRs are transmembrane proteins, each chain comprising two extracellular domains, a membrane proximal constant domain and a membrane distal variable domain. Each of the constant and variable regions comprises an intrachain disulfide bond. The variable region contains highly polymorphic loops, analogous to the Complementarity Determining Regions (CDRs) of antibodies. The variable region of each TCR chain comprises variable and connecting segments, and in the case of the β chain also diversity segments. Each variable region consists of three CDRs (complementarity determining regions) embedded in a framework sequence, one of which is a hypervariable region designated CDR 3. The various types of alpha chain variable regions (V α) and the various types of beta chain variable regions (V β) are distinguished by their framework, CDR1 and CDR2 sequences, and the partially defined CDR3 sequence. Specific TRAV or TRBV numbers are assigned by IMGT nomenclature to either va or ν β s. The specificity of T cell receptors for recognized epitopes is largely determined by the CDR3 region.
Adoptive TCR gene therapy allows the patient's own T cells to have the desired specificity and generate sufficient numbers of T cells in a short time, thus avoiding their exhaustion. The TCR may be transduced to all T cells or T cell subsets such as CD8, central memory T cells or T cells with stem cell characteristics, which may ensure better persistence and function upon transfer. TCR-engineered T cells can be infused into cancer patients with lymphopenia, e.g., by chemotherapy or radiotherapy, inducing homeostatic expansion, thereby greatly enhancing engraftment and long-term persistence of transplanted T cells with high cure rates. In the natural state, the affinity of the T cell receptor is low, and the tumor cells have escape mechanisms, such as reducing the expression level of self MHC class I molecules, so that optimizing the affinity of TCR and finding out TCR with killing efficacy becomes a hot point of research in TCR-T therapy.
Disclosure of Invention
As described herein, the present invention relates to a T cell receptor having a high affinity and an effect of killing tumor cells. The invention further provides the following technical scheme:
in a first aspect the invention provides a T cell receptor,
a variable domain V α comprising an α chain and/or a variable domain V β comprising a β chain, said V α comprising SEQ ID NO: 5, and said V β comprises the amino acid sequence set forth in SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof.
Further, in the present invention,
the T cell receptor comprises:
(a) SEQ ID NO:1, optionally as set forth in SEQ ID NO: 2, further optionally as shown in SEQ ID NO: 3 is shown in the specification;
(b) SEQ ID NO: 4, CDR2 a amino acid sequence set forth in seq id no;
(c) SEQ ID NO: 5, the CDR3 a amino acid sequence shown in seq id no;
(d) SEQ ID NO: 9, optionally as set forth in SEQ ID NO: 10, further optionally as set forth in SEQ ID NO: 11 is shown in the figure;
(e) SEQ ID NO: 12, the CDR2 β amino acid sequence shown in seq id no; and
(f) SEQ ID NO: 13, CDR3 β amino acid sequence.
Further, the T cell receptor comprises V.alpha.having at least 80%, preferably 90%, preferably 95% identity with the amino acid sequence as set forth in any of SEQ ID NO 6-8 and/or V.beta.having at least 80%, preferably 90%, preferably 95% identity with the amino acid sequence as set forth in any of SEQ ID NO 14-16.
Further, the T cell receptor comprises V alpha shown by an amino acid sequence shown in any one of SEQ ID NO 6-8 and/or V beta shown by an amino acid sequence shown by any one of SEQ ID NO 14-16.
Further, the T cell receptor comprises V alpha shown by an amino acid sequence shown by SEQ ID NO. 8 and/or V beta shown by an amino acid sequence shown by SEQ ID NO. 14.
Further, the T cell receptor is a soluble T cell receptor lacking a transmembrane domain.
Further, the T cell receptor binds to MHC I and/or MHC II peptide complexes.
Further, the T cell receptor further comprises a detectable label.
Further, the detectable label comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, biotin.
Further, the alpha chain further comprises an alpha constant region ca and/or the beta chain further comprises a beta constant region cbp.
Further, the ca region and/or the cp region comprises the introduction of one or more cysteines capable of forming one or more non-native disulfide bridges between the a chain and the β chain.
Further, the alpha and beta chains further comprise a signal peptide.
In a second aspect, the present invention provides a recombinant T cell receptor, wherein the recombinant T cell receptor comprises a T cell receptor according to the first aspect of the present invention, and/or a costimulatory region.
Further, the costimulatory region comprises a costimulatory molecule selected from the group consisting of a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, and any combination thereof.
Further, the co-stimulatory region comprises a CD28 polypeptide. In a third aspect, the present invention provides a nucleic acid encoding a T cell receptor according to the first aspect of the invention; or encodes a recombinant T cell receptor according to the second aspect of the invention.
Further, the nucleotide sequence of the nucleic acid is codon optimized for expression in a host cell.
Further, the host cell is optionally an immune system cell.
Further, the immune system cell is a T cell.
In a fourth aspect, the present invention provides an expression vector comprising a nucleic acid according to the third aspect of the invention.
Further, the expression vector is capable of delivering the polynucleotide to a host cell.
Further, the expression vector is a viral vector.
Further, the viral vector is a retroviral vector or a lentiviral expression vector.
In a fifth aspect, the invention provides a host cell comprising a nucleic acid according to the third aspect of the invention or an expression vector according to the fourth aspect of the invention.
Further, the T cell receptor is heterologous to the host cell.
Further, the host cell is selected from hematopoietic progenitor cells or immune system cells;
further, the immune system cell is selected from the group consisting of an immune system cell that is a CD4+ T cell, a CD8+ T cell, a CD4-CD 8-double negative T cell, a γ δ T cell, a natural killer T cell, a macrophage, a dendritic cell, or any combination thereof;
further, the immune system cell is a T cell;
further, the T cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.
In a sixth aspect, the invention provides a chimeric molecule comprising a T cell receptor according to the first aspect of the invention, or a portion thereof, conjugated to a non-cellular substrate, toxin and/or antibody.
Further, the non-cellular substrate is selected from the group consisting of: nanoparticles, exosomes and other non-cellular substrates.
In a seventh aspect, the present invention provides a composition comprising a T cell receptor according to the first aspect of the invention, a recombinant T cell receptor according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, an expression vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention, a chimeric molecule according to the sixth aspect of the invention.
Further, the composition further comprises a pharmaceutically acceptable carrier.
In an eighth aspect, the present invention provides a method for modifying an immune cell, which comprises contacting the immune cell with the expression vector of the fourth aspect of the present invention.
Further, the immune cell is a T cell, a peripheral blood lymphocyte, an NK cell, a constant NK cell, or an NKT cell.
Further, the contacting is transfection or transduction.
The method further comprises sorting the immune cells to isolate TCR-engineered T cells.
The method further comprises T cell cloning by serial dilution, and expanding T cell clones by a rapid expansion protocol. A ninth aspect of the invention provides the use of any one of:
(1) use of a T cell receptor according to the first aspect of the invention, a recombinant T cell receptor according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, an expression vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention, a chimeric molecule according to the sixth aspect of the invention, or a composition according to the seventh aspect of the invention, for the manufacture of a product for adoptive cell transfer therapy.
Further, the adoptive cell transfer is adoptive T cell transfer.
Further, the adoptive T cell transfer is allogeneic adoptive T cell transfer, autologous adoptive T cell transfer, or universal non-alloreactive adoptive T cell transfer.
(2) Use of a T cell receptor according to the first aspect of the invention, a recombinant T cell receptor according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, an expression vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention, a chimeric molecule according to the sixth aspect of the invention, or a composition according to the seventh aspect of the invention, for the preparation of a product for peptide/MHC diagnostic evaluation in a tumour cell.
(3) Use of a T cell receptor according to the first aspect of the invention, a recombinant T cell receptor according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, an expression vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention, a chimeric molecule according to the sixth aspect of the invention, or a composition according to the seventh aspect of the invention, for the preparation of a targeting product for directing a therapeutic molecule to a tumour site.
(4) Use of a T cell receptor according to the first aspect of the invention, a recombinant T cell receptor according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, an expression vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention, a chimeric molecule according to the sixth aspect of the invention, or a composition according to the seventh aspect of the invention, for the manufacture of a product for enhancing immunity.
(5) Use of a T cell receptor according to the first aspect of the invention, a recombinant T cell receptor according to the second aspect of the invention, a nucleic acid according to the third aspect of the invention, an expression vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention, a chimeric molecule according to the sixth aspect of the invention, or a composition according to the seventh aspect of the invention, for the manufacture of a medicament for the treatment and/or prevention of a disease.
Further, the disease is selected from a hematologic malignancy or a solid tumor;
further, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia, chronic myeloid leukemia, lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-hodgkin's lymphoma, and hodgkin's lymphoma.
Further, the hematological malignancy is selected from acute myeloid leukemia and lymphoma.
Further, the solid tumor is selected from the group consisting of: lung cancer, breast cancer, esophageal cancer, stomach cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial cancer, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.
Drawings
FIG. 1 is a functional assay of TCR-T killer leukemia cells U937.
Detailed Description
Before setting forth the present disclosure in more detail, it may be helpful to provide definitions of certain terms used herein. Unless otherwise explicitly defined, technical terms used herein have their normal meaning as understood in the art. Additional definitions are set forth throughout this disclosure.
The use of alternatives (e.g., "or") in connection with the present invention should be understood to mean any one, all or any combination of alternatives. The terms "comprising," "having," and "including" are used synonymously in the present invention, and these terms and their variants are intended to be interpreted non-limitingly.
As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a natural amino acid, i.e., an alpha-carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a natural amino acid. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a natural amino acid.
In the present invention, nucleic acids encoding the T cell receptor or the recombinant T cell receptor of the present invention can be constructed using PCR, recombinant engineering, or the like, or such a T cell receptor can be synthesized. The T cell receptor may further comprise other components, such as a tag, linker or transduction marker. In certain embodiments, a protein or polypeptide expressed or produced by a host cell (e.g., a T cell) is located on the cell surface, wherein the protein or polypeptide is anchored to the cell membrane (e.g., by a transmembrane domain) and comprises an extracellular portion or an intracellular portion or component (e.g., comprising a binding domain, and in certain embodiments, a linker, a spacer, or both) and an intracellular portion or component.
"nucleic acid," "nucleic acid molecule," or "polynucleotide" are used interchangeably herein and refer to a polymeric compound comprising covalently linked nucleotides, which may be composed of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine and xanthine, and pyrimidine bases include uracil, thymine and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which may be single-stranded or double-stranded cDNA, genomic DNA, and synthetic DNA. If single stranded, the nucleic acid molecule may be the coding strand or the non-coding strand (antisense strand). Nucleic acid molecules encoding an amino acid sequence include all nucleotide sequences that encode the same amino acid sequence. Certain forms of the nucleotide sequence may also include introns, such that the introns will be removed by either co-transcriptional or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence due to redundancy or degeneracy of the genetic code or by splicing.
Variants of the nucleic acid molecules of the present disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the nucleic acid molecules of the polynucleotides defined or referenced herein, or conditions under which a polynucleotide hybridizes under stringent hybridization conditions: 0.015M sodium chloride, 0.0015M sodium citrate or 0.015M sodium chloride at about 65-68 ℃, 0.0015M sodium citrate and 50% formamide at about 42 ℃. Nucleic acid molecule variants retain the ability to encode T cell receptors with functionality described herein (e.g., specific binding to a target molecule).
"percent sequence identity" refers to the relationship between two or more sequences as determined by comparing the sequences. Preferred methods of determining sequence identity are designed to give the best match between the compared sequences. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). Furthermore, non-homologous sequences may be omitted for comparison purposes. Unless otherwise indicated, the percentage of sequence identity referred to herein is calculated over the length of the reference sequence. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using BLAST programs (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX).
As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substituents of the polypeptide to the sequence of the second polypeptide (e.g., using GENEWORKSTM, Align, Clustal)TMBLAST algorithm, etc.). In certain embodiments, the BLAST algorithm is preferred.
In some embodiments, the term "variant" relates not only to at least one fragment, but also to a polypeptide or fragment thereof that includes an amino acid sequence that is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a reference amino acid sequence or fragment thereof, wherein amino acids other than those essential for biological activity or folding or structure of the polypeptide are deleted or substituted, one or more such essential amino acids are substituted in a conservative manner, and/or amino acids are additions to such a polypeptide to maintain the biological activity of the polypeptide.
In certain embodiments, the variants may additionally include chemical modifications, such as isotopic labeling or covalent modifications, such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, and the like. Methods of modifying polypeptides are known and will generally be employed so as not to eliminate or substantially reduce the desired activity of the polypeptide.
In one embodiment, the term "variant" of a nucleic acid molecule includes nucleic acids whose complementary strand hybridizes, e.g., under stringent conditions, to a reference or wild-type nucleic acid. The stringency of the hybridization reaction can be readily determined by one of ordinary skill in the art, and is generally an empirical calculation depending on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require less temperatures. Hybridization generally depends on the ability of denatured DNA to anneal to complementary strands present in an environment below its melting temperature: the higher the degree of homology desired between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures are less stringent.
In one embodiment, the term variant of a nucleic acid sequence, as used herein, refers to any nucleic acid sequence that, based on the degeneracy of the genetic code, encodes the same amino acid sequence as a reference nucleic acid sequence, and variants thereof.
"functional variant" refers to a polypeptide or polynucleotide that is structurally similar or substantially similar to a parent or reference compound of the present disclosure, but in some cases is slightly different in composition (e.g., one base, atom, or functional group is different, added or removed; or one or more amino acids are mutated, inserted, or deleted) such that the polypeptide or encoded polypeptide is capable of performing at least one functional efficiency of the encoded parent polypeptide with at least 50% of its function, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the active level of the fragment of the parent polypeptide.
As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to the host cell or subject, or the native activity of any gene, protein, compound, nucleic acid molecule, or host cell or subject that has been altered. Heterologous, non-endogenous or exogenous includes genes, proteins, compounds or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity or both are native and altered. In certain embodiments, a heterologous, non-endogenous or exogenous gene, protein or nucleic acid molecule (e.g., receptor, ligand, etc.) may not be endogenous to a host cell or subject, but rather the nucleic acid, protein or nucleic acid molecule encoding such gene may have been added to the host cell by conjugation, transformation, transfection, electroporation, or the like, where the added nucleic acid molecule may be integrated into the host cell genome or may exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). It will be understood that in the case of a host cell comprising a heterologous polynucleotide, the polynucleotide is "heterologous" to the progeny of the host cell, whether or not the progeny are themselves manipulated (e.g., transduced) to comprise the polynucleotide.
As used herein, the term "expression" refers to the process of producing a polypeptide based on the coding sequence of a nucleic acid molecule, e.g., a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. The expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). By "unrelated" is meant that the related genetic elements are not closely related to each other and that the function of one does not affect the other.
The term "introduced" in the context of inserting a nucleic acid molecule into a cell refers to "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic organism. Cells, wherein the nucleic acid molecule can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). In the present invention, the terms "engineered", "recombinant" or "non-natural" or "modified" are meant to include at least one genetically altered or modified organism, microorganism, cell, nucleic acid molecule or vector. Introducing an exogenous nucleic acid molecule, wherein the alteration or modification is introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introduced into expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or additions, deletions, substitutions or other functional disruptions of the genetic material of the cell by other nucleic acid molecules. Additional modifications include, for example, non-coding regulatory regions, wherein the modification alters expression of a polynucleotide, gene, or operon; for example, the expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, wherein such alterations or modifications can be introduced by genetic engineering. Genetic alteration may include, for example, modification by introduction of a nucleic acid molecule encoding one or more proteins or enzymes (which may include an expression control element, such as a promoter), or addition, deletion, substitution, or other functional disruption or supplementation of the genetic material of the cell. Exemplary modifications include modifications in the coding region of a heterologous or homologous polypeptide from a reference or parent molecule or a functional fragment thereof.
The term "variable region" or "variable domain" refers to the domain of the α -chain or β -chain of the TCR (or γ -chain and δ -chain for γ δ TCRs) that is involved in binding to an antigen. The variable domains of the alpha and beta chains of native TCRs (va and ν β, respectively) generally have similar structures, each domain comprising four generally conserved Framework Regions (FRs) and three CDRs. In a TCR, the framework regions separate the CDRs, and the CDRs are located between the framework regions (i.e., in the primary structure).
The variable domains of the alpha and beta chains of native TCRs (va and ν β, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FRs) and three CDRs. The V.alpha.domain is encoded by two separate DNA segments, a variable gene segment and a linker gene segment (V-J). The V β domain is encoded by three 5 independent DNA segments (variable gene segment, diversity gene segment and linker gene segment (V-D-J)). Human TCR V, D and J alleles, including their nucleotide and encoded amino acid sequences, are known in the art. A single va or V β domain may be sufficient to confer antigen binding specificity. In addition, V α or V β domains can be used to isolate TCRs that bind a particular antigen from TCRs that bind the antigen to screen a library of complementary V α or V β domains, respectively.
The terms "complementarity determining regions" and "CDRs" are synonymous with "hypervariable regions" or "HVRs" and are known in the art to refer to amino acid sequences in the TCR variable regions, generally conferring antigen specificity and/or binding affinity, and are separated from each other in primary structure by framework sequences. In some cases, the framework amino acids may also facilitate binding, e.g., may also contact the antigen or antigen-containing molecule. Typically, there are three CDRs in each variable region. For the TCR, CDR3 is considered to be the primary CDR responsible for recognition of the processing antigen. The CDR1 and CDR2 interact primarily or in some cases only with MHC. The variable domain sequences can be aligned to numbering schemes (e.g., Kabat, European Union, International Immunogenetics Information System (IMGT) and Aho (Kabat, EU, International Immunogenetics Information System (IMGT) and Aho)) and the equivalent residue positions can be annotated and different molecules compared using the antigen receptor numbering and receptor Classification (ANARCI) software tool (2016, Bioinformatics 15: 298-.
As used herein, "immune system cell" refers to any cell in the immune system derived from bone marrow hematopoietic stem cells that gives rise to two major lineages, namely myeloid progenitor cells (which give rise to myeloid cells (e.g., monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells, e.g., T cells, B cells, and Natural Killer (NK) cells.) exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD 8-double negative T cells, γ δ T cells, regulatory T cells, stem cell memory T cells, natural killer cells (e.g., NK cells or NK-T cells), B cells, and dendritic cells macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs" that can stimulate Major Histocompatibility Complex (MHC) receptors on the surface of APCs complexed with peptides when they interact with TCRs on the surface of T cells Specialized cells of live T cells.
A "T cell" or "T lymphocyte" is a cell of the immune system that matures in the thymus and produces TCR. T cells may be naive (no exposure to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA, but decreased expression of CD45RO compared to TCM), memory T cells (TM) (cells that have undergone antigen and long-lived) and effector cells (cells that have undergone antigen cytotoxicity). TM can be further divided into central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD127, CD45RO and CD95, and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 compared to naive T cells or TCM).
"hematopoietic progenitor cells" are cells derived from Hematopoietic Stem Cells (HSCs) or fetal tissue that are capable of further differentiation into mature cell types (e.g., cells of the T cell lineage). In certain embodiments, CD24loLin-CD117+Hematopoietic progenitor cells are useful. As defined herein, hematopoietic progenitor cells may include embryonic stem cells that are capable of further differentiation into cells of the T cell lineage. The hematopoietic progenitor cells can be from a variety of animals, including humans, mice, rats or other mammals. A "thymocyte progenitor cell" or "thymocyte" is a hematopoietic progenitor cell present in the thymus.
"hematopoietic stem cells" or "HSCs" refer to undifferentiated hematopoietic cells that are capable of self-renewal in vivo, proliferation in vitro essentially without limitation, and differentiation into other cell types, including cells of the T cell lineage. HSCs can be isolated, for example, but not limited to, from fetal liver, bone marrow and umbilical cord blood.
"embryonic stem cell," "ES cell," or "ESC" refers to an undifferentiated embryonic stem cell that has the ability to integrate into and become part of the germline of a developing embryo. Embryonic stem cells are capable of differentiating into hematopoietic progenitor cells and any tissue or organ.
The term "T cell receptor" (TCR) refers to a member of The immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane domain and a short cytoplasmic tail; see, e.g., Janeway, et al, immunology: The Immune System in Health and Disease,3rd Ed., Current Biology Publications, p.4:33,1997) that is capable of specifically binding to an antigenic peptide that binds to an MHC receptor. TCRs can be found on the cell surface or in soluble form, and typically consist of heterodimers with alpha and beta chains (referred to as TCR alpha and TCR beta 0, respectively) or gamma and delta chains (also referred to as TCR gamma and TCR delta, respectively). Like immunoglobulins, the extracellular portion of a TCR chain (e.g., the α and β 1 chains) comprises two immunoglobulin domains, one variable domain (e.g., the α chain variable domain or the V α, β chain variable domain or V β); usually based on terminal Kabat numbering at the N-position (Kabat, et al, "Sequences of Proteins of Immunological Interest," US depth. Health and Human Services, Public Health Service National Institutes of Health,1991,5thed.) and a constant domain adjacent to the cell membrane (e.g., an alpha chain constant domain or Calpha, typically based on amino acids of Kabat 117 to 259, a beta chain constant domain or Cbeta, typically based on amino acids of Kabat 117 to 295). Also like immunoglobulins, the variable domains comprise Complementarity Determining Regions (CDRs) separated by Framework Regions (FRs). In certain embodiments, the TCR is found on the surface of a T cell (or T lymphocyte) and is associated with a CD3 complex. The source of TCRs as used in the present disclosure may be from various animal species, such as human, mouse, rat, cat, dog, goat, horse or other mammal.
As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some cases, integrate into the genome itself.
In certain embodiments, the viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a target-specific polypeptide. The viral vector may be a retroviral vector or a lentiviral vector. The viral vector may also include a nucleic acid sequence encoding a transduction marker.
Viral vectors suitable for use with the compositions of the present invention include those identified for human gene therapy applications. Suitable viral vectors include RNA virus-based vectors, such as retroviral-derived vectors, e.g., moloney Murine Leukemia Virus (MLV) -derived vectors, and more complex retroviral-derived vectors, such as lentiviral-derived vectors. HIV-1 derived vectors belong to this class.
Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive strand RNA viruses (e.g., picornaviruses and viruses A), and double stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2 and Epstein-Barr and cytomegalovirus) and poxviruses (e.g., vaccinia, fowlpox, and canarypox) Type D virus, HTLV-BLV group, lentivirus and foamy virus.
A "retrovirus" is a virus having an RNA genome that is reverse transcribed into DNA using a reverse transcriptase, and the reverse transcribed DNA is then incorporated into the host cell genome. "Gamma retrovirus" refers to a genus of the family Retroviridae. Examples of gamma retroviruses include mouse stem cell virus, murine leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.
As used herein, "lentiviral vector" refers to an HIV-based lentiviral vector for gene delivery, which may be integrated or non-integrated, has a relatively large packaging capacity, and can transduce a variety of different cell types. Lentiviral vectors are typically generated following transient transfection of three or more plasmids (packaging, envelope and transfer) into producer cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with cell surface receptors. Upon entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The reverse transcription product is double-stranded linear viral DNA, a substrate for viral integration into the DNA of infected cells. "lentivirus" refers to a genus of retroviruses capable of infecting both dividing and non-dividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV1 and HIV 2); equine infectious anemia virus; feline Immunodeficiency Virus (FIV); bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV and visna virus (lentivirus ovine).
The invention also provides compositions, including pharmaceutical compositions and formulations, comprising the TCR, or antigen-binding fragment thereof, and engineered cells, and methods of use and uses of these molecules and compositions, such as for preventing/treating diseases, and/or methods of detection, diagnosis, and prognosis.
By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical formulation other than an active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
In some aspects, the selection of a carrier is determined in part by the particular cell or binding molecule and/or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are typically present in amounts of about 0.0001% to about 2% by weight of the total composition. Vehicles are described, for example, by Remington's Pharmaceutical Sciences 16 th edition, Osol, A. eds (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexa hydroxy quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol alcohol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG).
In some embodiments, the composition contains an amount of binding molecule and/or cell effective to treat or prevent the disease or condition, such as a therapeutically effective amount or a prophylactically effective amount. In some embodiments, therapeutic or prophylactic efficacy is monitored by periodic assessment of the treated subject. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosing regimens may be suitable and may be determined. The desired dose may be delivered by administering the composition as a single bolus, by administering the composition as multiple boluses, or by administering the composition as a continuous infusion.
The terms "treatment" and "prevention" as used herein, and words derived from such terms, do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention and one of ordinary skill in the art recognizes that there is a potential benefit or therapeutic effect. In this regard, the methods of the invention can provide any amount of any therapeutic or prophylactic level for a condition in a mammal. In addition, treatment or prevention as set forth in the methods of the invention can include treatment or prevention of one or more of the conditions or symptoms of the condition (e.g., leukemia) to be treated or prevented. Furthermore, for purposes herein, "preventing" may include delaying the onset of the disease or a symptom or condition thereof.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
Example 1 high throughput sequencing screening of TCR sequences
1. RNA extraction and quality control
Peripheral blood samples were collected from patients with acute myeloid leukemia who had no hepatitis A, hepatitis B, hepatitis C, hepatitis E, AIDS, syphilis, gonorrhea and tuberculosis infection.
Peripheral blood was slowly added to 5mL of Ficoll, and centrifuged at 2000rpm for 15 min. Sucking the middle leucocyte layer, adding 0.9% physiological saline, counting the number of mononuclear cells, centrifuging at 1000rpm for 5min, and sorting T cells by magnetic beads. RNA extraction was performed on the treated cell samples by TRIzol method, and the RNA integrity was checked by 2100 bioanalyzer (Agilent) using the Qubit RNA HS Assay kit for the quality of the extracted RNA.
2. Reverse transcription and library preparation
The library was constructed using the repterore Analysis Kit (Human TCR alpha, beta). All reagents were thawed on ice and mixed and centrifuged before the mix was dispensed. Mix required for hybridization primers was placed on ice (Table 1). Mixing, centrifuging, heating the reverse transcription primer to hybridize mix, incubating at 65 deg.C for 5min, and incubating on ice for at least 2 min. Reverse transcription mix (table 2) was prepared on ice, mixed and centrifuged mix.
TABLE 1 reaction System
Components
Volume of
RT dNTP Mixture
1μl
RT Primer H TCRα/β
1μl
RNA sample (100pg-1000ng)
Up to 6.2. mu.l
Nuclease-free water
Make up to 8.2. mu.l
TABLE 2 reverse transcription System
Components
Volume of
RT buffer A
4μl
RT buffer B
1μl
RT buffer C
3.8μl
RT Universal primer
1μl
RNase inhibitor
1μl
Reverse Transcriptase
1μl
Total volume
11.8μl
Mu.l of hybridization buffer was added to the hybridized RNA from the previous reaction, mixed, centrifuged, and the following program was run on a PCR instrument (T100, Bio-RAD) (Table 3):
TABLE 3 reaction conditions
3. First step PCR
Preparing reagents required for the first PCR (Table 4), thawing the reagents on ice, mixing the reagents uniformly, performing instant centrifugation, preparing mix for the first PCR reaction on ice, mixing the prepared mix uniformly by using a pipette, and performing centrifugation.
TABLE 4 first step PCR reaction System
Add 41. mu.l of the first step PCR mix to each tube, mix with 9. mu.l of the first strand cDNA product, centrifuge, and run the following program on a PCR instrument (T100, Bio-RAD):
TABLE 5 first step PCR reaction conditions
4. Second step PCR
Preparing reagents required by the second PCR (Table 6), thawing on ice, mixing uniformly, centrifuging instantly, preparing mix for the second PCR reaction on ice, mixing the prepared mix uniformly by using a pipettor, and centrifuging.
TABLE 6 second step PCR reaction System
A new PCR tube was prepared, and 1. mu.l of the second PCR primer, hTCRalpha #1-18 (accession No.: A1-A18), or hTCRbeta #1-18 (accession No.: B1-B18) was added to each tube. Add 39. mu.l mix of the second step reaction and 10. mu.l of the first step PCR product, mix, centrifuge and run the following program on a PCR instrument (T100, Bio-RAD):
TABLE 7 second PCR reaction conditions
5. Third step PCR
Preparation of the third PCR step required reagents were thawed on ice (Table 9), mixed and centrifuged instantaneously. And preparing mix for the second PCR reaction on ice, uniformly mixing the prepared mix by using a pipette, and centrifuging.
TABLE 8 third step PCR reaction System
Components
Volume of
PCR buffer
25μl
PCR dNTP Mixture
10μl
3rd universal primer
2.5μl
Nuclease-free water
6.5μl
DNA polymerase
1μl
Total volume
45μl
A new PCR tube was prepared, and 45. mu.l of the third PCR mix was added to each tube, 5. mu.l of the second PCR product was added, finger-bellied, homogenized, and centrifuged, and the following procedure was run on a PCR instrument (T100, Bio-RAD):
TABLE 9 third PCR reaction conditions
6. Agarose gel electrophoresis
Prepare 1.5% agarose gel, use 1 TAE buffer and nucleic acid dye (Beijing Kibobo Ying), DNA marker sample volume is 2 u l, add 1 u l 6 loading buffer to PCR products, the sample. 100V electrophoresis for 30-45 min. The product size was judged using a gel-to-image system (1708195, Bio-RAD). The hCRA/beta fragment is about 650 bp.
7. Purification with AGENCURT AMPure XP (BEACKMAN) magnetic beads
A8-line nucleic acid sample (0.2 mL) was prepared, and the library was diluted as shown in Table 10.
TABLE 10 dilution System
Components
Volume of
Third step PCR product
30μl
Nuclease-free water
20μl
Total volume
50μl
DNase-free 70% ethanol (sigma) was configured. The beads were equilibrated at room temperature for 30min before use, 22.5. mu.l of beads were added to the diluted library and mixed by pipette at least 10 times. Incubate for 5min at room temperature, then briefly centrifuge. And placing the 8 connecting pipes on a 96-hole magnetic frame for magnetic absorption for 5min, and observing the separation of liquid and magnetic beads. All supernatants were transferred to new 8-tubes. The unused magnetic beads were again mixed, 17.5. mu.l of the beads were added to the supernatant, and the mixture was mixed at least 10 times by a pipette. Incubate for 5min at room temperature, then briefly centrifuge. And placing the 8 connecting pipes on a 96-hole magnetic frame for magnetic absorption for 5min, and observing the separation of liquid and magnetic beads. Discard 85. mu.l of the supernatant and keep 5. mu.l of the supernatant to avoid the adsorption to the beads. The magnetic beads are not separated from the magnetic rack. Rinse with 200. mu.l of 70% ethanol. And (5) at room temperature for 30s, removing the alcohol, and repeating the alcohol washing step. Discard all supernatants, add 30. mu.l of 10mM Tris-HCl to each PCR tube, and mix at least 10 times with a pipette. Incubate for 2min at room temperature and then briefly centrifuge. The centrifuge tube was placed on a magnetic stand at room temperature for 5min to separate the liquid and the magnetic beads. Transfer 20. mu.l of the supernatant to a new PCR tube.
8. Library concentration quantification and quality control
Determination of library concentration Using the Qubit dsDNA HS kit (England Weitiz), using the equipment Qubit 2.0(life), 2. mu.l of the sample was taken for the determination. Usually the library concentration is concentrated in the range of 5-40 ng/. mu.l. Miseq sequencing can be performed if the sample concentration is more than or equal to 1.72 ng/. mu.l.
9. Library target fragment quality control
The target fragments of the human TCR alpha/beta library are concentrated in 650bp, the library fragments are subjected to quality control by using 1.5% agarose gel electrophoresis, the loading amount of a DNA marker is 2 mu l by using 1 TAE buffer and a nucleic acid dye (Beijing Kibobo), and 1 mu l of 6 loading buffer is added into a PCR product for spotting. 100V electrophoresis for 30-45 min. The product size was judged using a gel-to-image system (1708195, Bio-RAD).
10. Library mixing and quality control
Library mixes were normalized to the lowest concentration library of the batch, multiplied by 10. mu.l for the reference nanograms, and other libraries were sampled according to the nanograms to ensure that the total amount of each library was consistent, and diluted to 8.58 ng/. mu.l (20nM, 650bp) after mixing, in terms of nanograms to moles.
11. High throughput sequencing
Library 2 × 300bp double-end sequencing using Miseq (Illumina), library quantification using the Qubit 2.0(life) device again for the mixed library, final dilution to 10pM, mixing with 10pM denated PhiX (PhiX ratio 30%), and on-machine sequencing 600 μ l after mixing.
12. Data analysis
A primary analysis using MiXCR allows highly personalized TCR and immunoglobulin sequences to be analyzed. The MiXCR may be parametrically adjusted for different data types and optimize the analysis results and output. Further advanced analysis of multiple diversity indices, such as Shannon index (Shannon), Simpson index (Simpson), Inverse-Simpson index, and Gini index (Gini), etc. VDJtools were used for secondary TCR profiling analysis and diversity assessment.
13. Phage display technology screens for high affinity TCR sequences.
14. Results
The high affinity TCR is screened out by a phage display technology, and the amino acid sequence of the alpha chain variable region of the TCR is shown as SEQ ID NO:6-8, wherein the sequence of CDR1 is set forth in SEQ ID NO: 1-3; the sequence of CDR2 is set forth in SEQ ID NO: 4 is shown in the specification; the CDR3 sequences are shown in SEQ ID NO: 5, respectively. The amino acid sequence of the beta chain variable region of the TCR is as set forth in SEQ ID NO:14-16, wherein the sequence of CDR1 is as set forth in SEQ ID NO: 9-11; the sequence of CDR2 is set forth in SEQ ID NO: 12 is shown in the specification; the sequence of CDR3 is set forth in SEQ ID NO: shown at 13.
EXAMPLE 2 construction of TCR-T
1. Using high throughput sequencing and data analysis results, the relevant sequences were synthesized in their entirety and ligated into the pCDH vector through XbaI/SalI restriction endonuclease cleavage sites.
2. TCR-T cell preparation
Collecting peripheral blood of healthy people, slowly adding the peripheral blood into Ficoll, centrifuging at 2000rpm for 15 min. The middle leucocyte layer was aspirated, 0.9% physiological saline was added, the number of mononuclear cells was counted, and centrifugation was performed at 1000rpm for 5 min. Magnetic beads sort T cells. And simultaneously adding CD3/CD28 antibody coupled magnetic beads for stimulation, adding viruses for 2 times after 24 hours and 48 hours, adding IL-2 during virus infection, and culturing for 3-20 days to obtain the TCR-T cells.
Example 3 TCR-T killing function assay
Functional assays were performed on any of the TCR-Ts constructed from the sequences described in this study.
Target cells (U937) and negative reference target cells (Daudi) were counted, labeled Celltrace far red and CFSE staining, respectively. TCR-T (TCR V alpha and V beta variable region amino acid sequences are respectively shown in SEQ ID NO.6 and SEQ ID NO: 14) cells were counted as effector cells. Resuspend effector and target cells to the corresponding concentrations according to the planned effector-target ratio (3: 1, 9: 1, 18: 1).
Each effective target ratio in the round-bottom 96-well plate is paved into group 1 effector cells, group 2 target cells and group 3 effector cells + target cells, and each group has 4 multiple wells. Culturing for 2-24h, collecting cell samples, respectively collecting tube A: group 1 effector cells + group 2 target cells, tube B: group 3 effector cells + target cells, fixed with paraformaldehyde solution, and flow-loaded onto a machine. A Kiling Rate and an E: T Ratio are plotted. Wherein, Kiling
The results are shown in FIG. 1, in which TCR-T has a higher killing effect on U937 cell line, indicating that TCR-T can be used for treatment of leukemia.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
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Ile Ala Gly Ile Thr Gln Ala Pro Thr Ser Gln Ile Leu Ala Ala Gly
1 5 10 15
Arg Arg Met Thr Leu Arg Cys Thr Gln Asp Met Arg Ala Asn Ala Met
20 25 30
Tyr Trp Tyr Arg Gln Asp Leu Gly Leu Gly Leu Arg Leu Ile His Tyr
35 40 45
Ser Asn Thr Ala Gly Thr Thr Gly Lys Gly Glu Val Pro Asp Gly Tyr
50 55 60
Ser Val Ser Arg Ala Asn Thr Asp Asp Phe Pro Leu Thr Leu Ala Ser
65 70 75 80
Ala Val Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Asp Ala
85 90 95
Ser Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu
100 105 110
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