WO2023010436A1 - Tcr expression construct, and preparation method therefor and use thereof - Google Patents

Abstract

Provided are a T cell receptor (TCR) expression construct for expressing a TCR, and a preparation method therefor and the use thereof. Also provided are a TCR and a TCR complex that are expressed by the expression construct, a T cell containing the TCR, a method for preparing the TCR, a method for treating tumors by using the TCR and the use thereof.

Description

TCR expression construct and its preparation method and use Technical field:

The present invention relates to a TCR expression construct for expressing a T cell receptor (TCR), a TCR and a TCR complex expressed by the expression construct, a T cell comprising the TCR, a method for preparing it, and an application thereof The TCR method for treating tumors and its use.

Background technique:

TCR is the only receptor that recognizes specific antigenic peptides on the histocompatibility complex (MHC). In the immune system, the combination of antigen-specific TCR and pMHC complexes triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of T cells and APCs interact. It causes a series of subsequent cell signal transmission and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells. Therefore, TCR is crucial to the cellular immune function of the immune system.

Like antibodies as antigen recognition molecules, TCRs can also be developed for diagnosis and therapy. TCR can be combined with other molecules (eg, anti-CD3 antibody) to redirect T cells so that they target cells presenting specific antigens for killing.

TCR-engineered T cells have limited proliferation and function in the immunosuppressive tumor microenvironment. Several investigators have attempted to improve the surface expression and effector functions of transduced TCRs by introducing multiple genetic modifications. For example, the introduction of murine sequences to replace human constant region sequences, or the addition of cysteine to the α and β chains to form additional disulfide bonds to stabilize the structure of the TCR

T cell receptor delta chain (CD3δ) is the main signal transduction element of T cell antigen receptor. It has been described that CD3δ expression is reduced in T cells from cancer patients. Reduced TCR-delta chain expression results in impaired T cell responsiveness. The function of CD3δ is clearly stated in the report, it participates in the construction of TCR-CD3 complex, can promote T cell function, and participate in T cell signal transduction. When constructing T cell receptors in the prior art, TCRαβ and CD3δ are usually separated into two vectors, or TCRαβ is put into two expression vectors, and then CD3δ is added separately, that is, VαCαδ and VβCβδ. This method cannot guarantee specificity TCRαβ was expressed simultaneously. At the same time, in the prior art, the human constant region sequence generally cannot avoid mismatching with the endogenous TCR.

Therefore, the prior art needs a new TCR construct, which can promote the expression and function of TCR-T, improve its anti-tumor efficiency, and overcome the problem of insufficient TCR expression caused by low transfection efficiency.

Contents of the invention

In one aspect the present invention relates to a novel TCR construct comprising in turn

(i) a nucleic acid encoding a TCR alpha chain;

(ii) a nucleic acid encoding a TCR beta chain; and

(iii) a nucleic acid encoding the CD3ζ chain;

Wherein there is a linker between the TCRα chain and the TCRβ chain, and there is a linker between the TCRβ chain and the CD3ζ chain, wherein both the TCRα chain and the TCRβ chain comprise a constant region, and both the constant regions are derived from a mouse; and the CD3ζ chain is derived from people.

One aspect of the invention relates to the following embodiments:

1. A new TCR expression construct comprising

(i) a nucleic acid encoding a TCR alpha chain;

(ii) a nucleic acid encoding a TCR beta chain; and

(iii) a nucleic acid encoding the CD3 delta chain;

Wherein the TCRα chain and the TCRβ chain both comprise constant regions, and both the constant regions are derived from mice; and the CD3δ chains are derived from humans; and

Optionally, a linker is present between a nucleic acid encoding a TCR alpha chain and a nucleic acid encoding a TCR beta chain, and/or a linker is present between a nucleic acid encoding a TCR beta chain and a nucleic acid encoding a CD3 delta chain.

2. The TCR expression construct of embodiment 1, wherein

The constant region of the TCR alpha chain comprises the amino acid sequence of SEQ ID NO:5, or comprises amino acids with 1, 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO:5 sequence, or comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 5, or consisting of said sequence composition; or

The nucleic acid encoding the TCR α chain comprises the nucleic acid encoding the TCR α chain constant region, the nucleic acid encoding the TCR α chain constant region comprises the nucleotide sequence of SEQ ID NO: 7, or comprises the nucleic acid sequence having 1, 2 compared with the nucleotide sequence of SEQ ID NO: 7 , 3, 4 or 5 variations (such as substitutions, insertions or deletions) of the nucleic acid sequence, or comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleic acid sequence of SEQ ID NO: 7 , 96%, 97%, 98% or 99% of, or consist of, a nucleic acid sequence.

3. The TCR expression construct of embodiment 1 or 2, wherein

The constant region of the TCR beta chain comprises the amino acid sequence of SEQ ID NO: 12, or comprises amino acids with 1, 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 12 sequence, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 12, or consisting of said sequence composition; or

The nucleic acid encoding the TCR β chain comprises the nucleic acid encoding the TCR β chain constant region, and the nucleic acid encoding the TCR β chain constant region comprises the nucleotide sequence of SEQ ID NO: 16, or comprises 1, 2 compared with the nucleotide sequence of SEQ ID NO: 16 , 3, 4 or 5 variations (such as substitutions, insertions or deletions) of the nucleic acid sequence, or comprising at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleic acid sequence of SEQ ID NO: 16 , 96%, 97%, 98% or 99% of, or consist of, a nucleic acid sequence.

4. The expression construct of any one of embodiments 1-3, wherein,

The CD3 delta chain comprises the amino acid sequence of SEQ ID NO: 21, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations (e.g. substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 21, or Comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 21, or consisting of said sequence composition;

or

The nucleic acid encoding the CD3 delta chain comprises the nucleic acid sequence of SEQ ID NO: 22, or comprises a nucleic acid having 1, 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions) compared to the nucleic acid sequence of SEQ ID NO: 22 sequence, or comprising a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence of SEQ ID NO: 22, or consisting of The nucleic acid sequence composition.

5. The TCR expression construct according to any one of embodiments 1-4, wherein the TCRα chain comprises a variable region and/or the TCRβ chain comprises a variable region, preferably, the variable region of the TCRα chain and/or the variable region of the TCRβ chain The variable regions are of human origin.

6. The TCR expression construct according to any one of embodiments 1-5, wherein the linker between the TCRα chain and the TCRβ chain, or the linker between the TCRβ chain and the CD3δ chain is a self-cleaving peptide, such as T2A or GSGP2A, preferably Preferably, the linker between the TCR α chain and the TCR β chain is GSGP2A and/or the linker between the TCR β chain and the CD3 δ chain is T2A, for example, T2A comprises or consists of the amino acid sequence of SEQ ID NO: 17 or encodes T2A Nucleic acid comprises the nucleotide sequence of SEQ ID NO:18 or is made up of said nucleotide sequence; GSGP2A comprises the aminoacid sequence of SEQ ID NO:19 or is made up of said sequence or the nucleic acid of encoding GSGP2A comprises the nucleotide sequence of SEQ ID NO:20 or is made up of The nucleic acid sequence composition.

7. The TCR expression construct of any one of embodiments 1-6, wherein the TCR is a TCR directed against MART-1.

8. The TCR expression construct of any one of embodiments 1-7, wherein

TCR alpha chain comprises TCR alpha chain variable region, and it comprises following CDR1, CDR2 and CDR3, and wherein CDR1 comprises the aminoacid sequence of SEQ ID NO:2 or is made up of it, and CDR2 comprises the aminoacid sequence of SEQ ID NO:3 or is made up of it, CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:4;

Or the TCR α chain comprises a TCR α chain variable region comprising the amino acid sequence of SEQ ID NO: 1, or comprising 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 1 (e.g. substitutions, insertions or deletions) of the amino acid sequence (in preferred embodiments, the variation does not occur in the CDRs), or comprising at least 90%, 91%, 92% of the amino acid sequence of SEQ ID NO:1 , 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence, or consists of said sequence; or

The TCRα chain comprises the amino acid sequence of SEQ ID NO: 13, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 13 (in In a preferred embodiment, the variation does not occur in the CDR), or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence, or consist of said sequence; or the nucleic acid encoding the TCR α chain comprises the nucleic acid sequence of SEQ ID NO:6, or comprises the nucleic acid sequence having 1 compared with the nucleic acid sequence of SEQ ID NO:6 , 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions) of the nucleic acid sequence, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of, or consisting of, a nucleic acid sequence.

9. The TCR expression construct of any one of embodiments 1-8, wherein

TCR beta chain comprises TCR beta chain variable region, and it comprises following CDR1, CDR2 and CDR3, and wherein CDR1 comprises the aminoacid sequence of SEQ ID NO:9 or is made up of it, and CDR2 comprises the aminoacid sequence of SEQ ID NO:10 or is made up of it, CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11; or

The TCR β chain comprises a TCR β chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or comprising 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 8 ( such as substitutions, insertions or deletions) of the amino acid sequence (in a preferred embodiment, the variation does not occur in the CDRs), or an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence, or consists of said sequence; or

The TCR beta chain comprises the amino acid sequence of SEQ ID NO: 14, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 14 (in In a preferred embodiment, the variation does not occur in the CDR), or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence, or consist of said sequence; or the nucleic acid encoding the TCR β chain comprises the nucleotide sequence of SEQ ID NO: 15, or comprises 1 compared with the nucleotide sequence of SEQ ID NO: 15 , 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions) of the nucleic acid sequence, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of, or consisting of, a nucleic acid sequence.

10. A TCR encoded by and produced by the TCR expression construct of any one of embodiments 1-9.

11. A TCR complex produced in a T cell by the TCR expression construct of any one of embodiments 1-9.

12. A vector, such as an expression vector, comprising the TCR expression construct according to any one of embodiment 19, preferably, the expression vector is a retroviral vector, such as pMSGV1.

13. An engineered cell comprising the TCR expression construct of any one of embodiments 1-9, or the vector of embodiment 12, preferably, said cell is a T cell, such as a human T cell, such as a CD4+ cell or a CD8+ cells, or a mixed cell population of CD4+ T cells and CD8+ T cells.

14. A method of producing engineered cells comprising

(1) Transform or transduce host cells with the TCR expression construct of any one of embodiments 1-9 or the vector of embodiment 11;

(2) cultivating host cells in a suitable medium;

(3) Separating and purifying the engineered cells from the culture medium or cells; preferably, the expression construct or vector is transferred into the cells by electroporation or transferred into the cells by transposition.

15. A composition comprising the TCR expression construct of any one of embodiments 1-9, the TCR of embodiment 10, the TCR complex of embodiment 11, the vector of embodiment 12 or the engineered cell of embodiment 13, Preferably, the composition is a pharmaceutical composition, and optionally pharmaceutically acceptable excipients.

16. A method for treating or preventing a disease, comprising the TCR expression construct of any one of embodiments 1-9, the TCR of embodiment 10, the TCR complex of embodiment 11, the vector of embodiment 12 or the embodiment 13 The engineered cell of or the composition or formulation of embodiment 15 is administered to a subject, eg the disease is a tumor, eg cancer, eg melanoma.

17. The method of embodiment 16, wherein the TCR expression construct of any one of embodiments 1-9, the TCR of embodiment 10, the TCR complex of embodiment 11, the vector of embodiment 12, or the TCR of embodiment 13. The engineered cell or the composition or formulation of Embodiment 15 is combined with one or more other therapeutic agents, such as chemotherapeutic agents or antibodies.

Description of drawings:

Figure 1: Shows the percentage of TCR+ cells in PBL cells transfected with TCR and different chains.

Figure 2: Figure 2A shows the secretion of IFNγ in the co-culture of transduced PBL and tumor cells; Figure 2B shows the secretion of IL-2 in the co-culture of transduced PBL and tumor cells; Assay to detect the proliferative ability of cells; Figure 2D shows the results of ⁵¹ Cr release assay to detect cytotoxicity.

Figure 3: Structural diagrams showing two MSGV-based retroviral vectors for transfer and expression of MART-1 TCR-α, β or MART-1 TCR-α, β plus δ genes.

Figure 4: Figure 4A shows the flow chart of PBL transduction, stimulation and analysis; Figure 4B shows the results of FACS detection on day 6 for cells positive for both CD3δ and CD8 or CD4 in T cells transduced with DMF5 or DMF5δ ; FIG. 4C shows the percentage of cells positive for CD3δ expression on day 12 among CD8 ⁺ TCR ⁺ T cells transduced with DMF5 or DMF5δ.

Figure 5: Figures 5A and 5B show the TCR expression results in T cells transduced with DMF5 or DMF5δ on the 6th day and the 12th day;

Figure 6: Figures 6A and 6B show the release of IL-2 and IFN-γ in cells transduced with DMF5 or DMF5ζ; Figure 6C shows the results of 51Cr release assays from cells transduced with DMF5 or DMF5ζ to detect cytotoxicity . Figure 6D shows cell proliferation after co-culture of cells transduced with DMF5 or DMF5ζ and tumor cells.

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Detailed ways:

definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

1. T cell receptor

T cell receptors (TCRs) are molecules present on the surface of T cells that are responsible for recognizing peptide-MHC complexes. Specific binding of TCRs to peptide-MHC complexes triggers T cell activation through a series of biochemical events mediated by associated enzymes, co-receptors, and accessory molecules. In 95% of T cells, TCR heterodimers consist of α and β chains, whereas in 5% of T cells, TCR heterodimers consist of γ and δ chains. Each chain of the TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane region (i.e., transmembrane region) and a short cytoplasmic tail at the C-terminus.

In the variable domains of the TCR α chain and β chain, each variable domain has three hypervariable regions or complementarity determining regions (CDRs), wherein CDR3 in each variable domain is the main one responsible for recognizing processed antigens CDR. CDR2 is thought to recognize MHC molecules.

The constant domain of the TCR domain consists of a short linker sequence in which cysteine residues form disulfide bonds, creating a link between the TCR alpha and beta chains.

During T cell maturation, TCR and CD3 form a TCR/CD3 complex. The TCR/CD3 complex formation process usually proceeds in the following order; first, the three peptide chains of CD3γ, δ, and ε become a stable complex core by forming two heterodimers of γ-ε and δ-ε, and TCRαβ( or TCRγδ), and then the δ-δ or δ-ε dimer binds to the TCRαβ (or TCRγδ)/CD3γεδε complex, and finally transfers to the surface of T cells. The signal is transmitted from the TCR into the cell through the TCR/CD3 complex.

Signaling from the TCR/CD3 complex is enhanced by simultaneous MHC binding to specific co-receptors. In helper T cells, this co-receptor is the CD4 molecule, which is specific for class II MHC; in cytotoxic T cells, this co-receptor is CD8, which is specific for class I MHC is specific.

2. Other definitions

The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit 10% less than the stated numerical value and an upper limit 10% greater than the stated numerical value.

When the term "and/or" is used to link two or more optional items, it should be understood as meaning any one of the optional items or any two or more items of the optional items.

As used herein, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Herein, when the term "comprising" or "comprises" is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.

As used herein, a region "derived from human" or "derived from mouse" means that the region has the amino acid sequence or encoding nucleic acid sequence of the region of natural human or native mouse, or is derived from the amino acid sequence or encoding nucleic acid sequence Sequence composition. A region "derived from" a human or a mouse also encompasses a region that is substantially identical to, or at least 90%, 91% identical to, the amino acid sequence or encoding nucleic acid sequence of that region of a native human or native mouse , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, and still have the same or similar activity and/or function as the region. For example, when "the CD3ζ chain is derived from humans" is defined, it means that the CD3ζ chain has the amino acid sequence of the natural human CD3ζ chain, or consists of the amino acid sequence; or refers to the amino acid sequence of the CD3ζ chain and the natural human CD3ζ chain. The amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity and still has the activity of native human CD3ζ chain and/or functions.

As known in the art, "polynucleotide" or "nucleic acid" used interchangeably herein refers to a chain of nucleotides of any length and includes DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a strand by a DNA or RNA polymerase.

Calculation of sequence identity between sequences is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a first and second amino acid sequence or nucleic acid sequence may be placed between a first and a second amino acid sequence or nucleic acid sequence for optimal alignment). Gaps may be introduced in one or both or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com available), use the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the distance between two amino acid sequences. percent identity. In yet another preferred embodiment, using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and Length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

It is also possible to use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), utilize the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11- 17) Determining the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.

The term "antigen presenting cell" or "APC" refers to cells of the immune system, such as helper cells (e.g., B-cells, dendritic cells, etc.), that present on their surface foreign antigens complexed with the major histocompatibility complex (MHC). ). T cells can recognize these complexes using their T cell receptor (TCR). APCs process antigens and present them to T cells.

As used herein, "vector" means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably herein and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. The progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the cells screened or selected in the originally transformed cells.

Herein, "subject" and "individual" refer to animals, preferably mammals, more preferably humans, who need to alleviate, prevent and/or treat novel coronavirus infection. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. The term includes human subjects who have or are at risk of having a novel coronavirus infection.

specific description

I. TCR expression constructs of the invention

In one aspect the present invention relates to a novel TCR expression construct comprising in sequence

(i) a nucleic acid encoding a TCR alpha chain;

(ii) a nucleic acid encoding a TCR beta chain; and

(iii) a nucleic acid encoding the CD3ζ chain;

Wherein the TCRα chain and the TCRβ chain both comprise a constant region, and the constant region is derived from a mouse; and the CD3ζ chain is derived from a human; and

Optionally, a linker is present between a nucleic acid encoding a TCR alpha chain and a nucleic acid encoding a TCR beta chain, and/or a linker is present between a nucleic acid encoding a TCR beta chain and a nucleic acid encoding a CD3ζ chain.

In some embodiments, the TCR alpha chain comprises a variable region. In some embodiments, the TCR beta chain comprises a variable region. In some embodiments, the variable region of the TCR alpha chain and/or the variable region of the TCR beta chain is of human origin.

In one embodiment of the present invention, the constant region of the TCRα chain comprises the amino acid sequence of SEQ ID NO: 5, or comprises 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 5 ( such as substitutions, insertions or deletions), or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO:5 or 99% of the amino acid sequence, or consists of said sequence.

In one embodiment of the present invention, the nucleic acid of coding TCR α chain comprises the nucleic acid of coding TCR α chain constant region, and the nucleic acid of described coding TCR α chain constant region comprises the nucleotide sequence of SEQ ID NO:7, or comprises the nucleic acid sequence with SEQ ID NO:7 Compared with the nucleic acid sequence having 1, 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions), or comprising at least 90%, 91%, 92% of the nucleic acid sequence of SEQ ID NO:7 , 93%, 94%, 95%, 96%, 97%, 98% or 99% of, or consist of, a nucleic acid sequence.

In one embodiment of the present invention, the constant region of the TCR β chain comprises the amino acid sequence of SEQ ID NO: 12, or comprises 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 12 ( Such as substitutions, insertions or deletions), or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 12 or 99% of the amino acid sequence, or consists of said sequence.

In one embodiment of the present invention, the nucleic acid encoding the TCR β chain comprises the nucleic acid encoding the TCR β chain constant region, and the nucleic acid encoding the TCR β chain constant region comprises the nucleotide sequence of SEQ ID NO: 16, or comprises the same sequence as SEQ ID NO: 16 Compared with the nucleic acid sequence having 1, 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions), or comprising at least 90%, 91%, 92% of the nucleic acid sequence of SEQ ID NO: 16 , 93%, 94%, 95%, 96%, 97%, 98% or 99% of, or consist of, a nucleic acid sequence.

In one embodiment of the invention, the CD3ζ chain comprises the amino acid sequence of SEQ ID NO: 21, or comprises 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 21 (such as substitutions, insertion or deletion), or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 21 The amino acid sequence of, or consists of said sequence.

In one embodiment of the invention, the CD3ζ chain is derived from human CD3δ.

In one embodiment of the invention, the nucleic acid encoding CD3ζ chain comprises the nucleotide sequence of SEQ ID NO:22, or comprises 1, 2, 3, 4 or 5 variations compared with the nucleotide sequence of SEQ ID NO:22 ( Such as substitution, insertion or deletion) nucleic acid sequence, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleic acid sequence of SEQ ID NO:22 Or 99% of nucleic acid sequences, or consist of said nucleic acid sequences.

In one embodiment of the invention, the linker between the TCRα chain and the TCRβ chain, or the linker between the TCRβ chain and the CD3ζ chain is a self-cleaving peptide, such as T2A or GSGP2A. In one embodiment, the linker between the TCR alpha chain and the TCR beta chain is GSGP2A. In one embodiment, the linker between the TCR β chain and the CD3 ζ chain is T2A. In one embodiment, the linker between the TCRα chain and the TCRβ chain is GSGP2A, and the linker between the TCRβ chain and the CD3ζ chain is T2A. In one embodiment, T2A comprises or consists of the amino acid sequence of SEQ ID NO: 17. In one embodiment, the nucleic acid encoding T2A comprises the nucleotide sequence of SEQ ID NO: 18 or consists of said nucleotide sequence. In one embodiment, GSGP2A comprises or consists of the amino acid sequence of SEQ ID NO: 19. In one embodiment, the nucleic acid encoding GSGP2A comprises the nucleotide sequence of SEQ ID NO: 20 or consists of said nucleotide sequence.

In a specific embodiment of the present invention, the TCR of the present invention is a TCR against MART-1.

Thus, in one embodiment, the TCR alpha chain comprises a TCR alpha chain variable region. In some embodiments, the variable region comprises CDR1, CDR2 and CDR3 as follows, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:2, and CDR2 comprises or consists of the amino acid sequence of SEQ ID NO:3 , CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:4. In one embodiment, the TCRα chain comprises a TCRα chain variable region comprising the amino acid sequence of SEQ ID NO: 1, or comprising an amino acid sequence having 1, 2, 3, An amino acid sequence with 4 or 5 variations (such as substitutions, insertions or deletions) (in preferred embodiments, the variations do not occur in the CDRs), or an amino acid sequence comprising at least 90% of the amino acid sequence of SEQ ID NO: 1, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence, or consists of said sequence. In one embodiment, the TCRα chain comprises the amino acid sequence of SEQ ID NO: 13, or comprises 1, 2, 3, 4 or 5 variations (such as substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 13 ) (in a preferred embodiment, the variation does not occur in the CDRs), or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence, or consists of said sequence. In one embodiment, the nucleic acid encoding the TCRα chain comprises the nucleotide sequence of SEQ ID NO:6, or comprises 1, 2, 3, 4 or 5 variations (such as substitutions, substitutions, insertion or deletion), or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence of SEQ ID NO:6 The nucleic acid sequence of, or consists of said nucleic acid sequence.

Thus, in one embodiment, the TCR beta chain comprises a TCR beta chain variable region. In some embodiments, the variable region comprises CDR1, CDR2 and CDR3 as follows, wherein CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:9, and CDR2 comprises or consists of the amino acid sequence of SEQ ID NO:10 , CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11. In one embodiment, the TCR beta chain comprises a TCR beta chain variable region comprising the amino acid sequence of SEQ ID NO:8, or comprising an amino acid sequence having 1, 2, 3, An amino acid sequence with 4 or 5 variations (such as substitutions, insertions or deletions) (in preferred embodiments, the variations do not occur in the CDRs), or an amino acid sequence comprising at least 90% of the amino acid sequence of SEQ ID NO: 8, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence, or consists of said sequence. In one embodiment, the TCR beta chain comprises the amino acid sequence of SEQ ID NO: 14, or comprises 1, 2, 3, 4 or 5 variations (e.g. substitutions, insertions or deletions) compared to the amino acid sequence of SEQ ID NO: 14 ) (in a preferred embodiment, the variation does not occur in the CDRs), or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence, or consists of said sequence. In one embodiment, the nucleic acid encoding the TCR beta chain comprises the nucleotide sequence of SEQ ID NO: 15, or comprises 1, 2, 3, 4 or 5 variations (such as substitutions, substitutions, insertion or deletion), or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence of SEQ ID NO: 15 The nucleic acid sequence of, or consists of said nucleic acid sequence.

In some embodiments, the TCR expression construct may further comprise nucleic acid encoding a marker (e.g., EGFRt or other marker as described) separated from the TCR chain by a linker (e.g., a splicing peptide such as T2A or GSGP2A). open. In some embodiments, the constructs can be arranged in any order such that the encoding marker sequence is 3' to the alpha and/or beta chain sequence, 5' to the alpha and/or beta chain sequence and/or between the alpha and beta sequences wherein, in some cases, each separate component is separated by a cleavable linker sequence (eg, T2A or P2A) or an IRES.

The TCR expression construct of the present invention can be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, the nucleic acid sequence encoding the expression construct of the present invention (or its fragments, or its derivatives) can be obtained completely by chemical synthesis.

In another aspect of the present invention, the present invention relates to a T cell receptor (TCR) produced by expression in T cells of the TCR expression construct of the present invention.

The invention also relates to a TCR complex produced in a T cell by the TCR expression construct of the invention.

The invention also encompasses variants of the TCR expression constructs, TCR or TCR complexes of the invention having one or more variations, such as substitutions, deletions or additions (preferably no more than 5 substitutions, deletions or additions), provided that It still has the same or similar activity. Preferably, said variation is a substitution. Preferably, the substitutions are conservative substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and/or chemical properties is exchanged for another amino acid with the same chemical or physical properties. For example, the conservative amino acid substitution can be an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid replaces another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain replaces another one with a polar side chain Amino acids (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc., said conservative substitutions may be based, for example, on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. sex to proceed.

In some embodiments, the TCR variants of the present invention may be (i) TCRs with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A TCR with substituent groups in multiple amino acid residues, or (iii) a polypeptide formed by fusing the TCR of the present invention with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional The polypeptide formed by fusing the amino acid sequence to the polypeptide sequence (the fusion protein formed by fusing with the leader sequence, secretory sequence or 6His and other tag sequences). Such variants are within the purview of those skilled in the art from the teachings herein.

II. Expression vectors and cells comprising the TCR expression construct, and preparation methods

Provided herein is a vector comprising a TCR expression construct provided herein. In certain embodiments, the vector is an expression vector. In particular embodiments, the vector is a viral vector, such as a retroviral vector, lentiviral vector, baculoviral vector, herpesviral vector, adenoviral vector, adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a retroviral vector. For a detailed list of retroviruses see Coffin et al, "Retroviruses" 1997 Cold Spring Harbor Laboratory Press eds: JM Coffin, SM Hughes, HE Varmus, p758763.

In a particular embodiment, the viral vector of the present invention is a retroviral vector pMSGV1, derived from the long terminal repeat of pMSGV murine stem cell virus, containing an extended gag region and a Kozak sequence (articles by Rosenberg SA and Zhong XS, Primary Human Lymphocytes Transduced with NY-ESO-1 Antigen-Specific TCR Genes Recognize and Kill Diverse Human Tumor Cell Lines, Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD8+T cell-mediated ad ication).

Provided herein is an engineered cell comprising any of the TCR expression constructs provided herein or a vector comprising said expression construct. Cells are typically eukaryotic cells, such as mammalian cells, and often human cells. In some embodiments, the cells are derived from blood, bone marrow, lymphoid or lymphoid organs and are cells of the immune system, such as innate or adaptive immune cells, for example myeloid or lymphoid cells, including lymphocytes, typically T cells and /or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). Cells are typically primary cells, such as cells isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells comprise one or more subsets of T cells or other cell types, such as the complete T cell population, CD4+ cells, CD8+ cells and subsets thereof, such as those defined according to: function, activation state, Maturity, differentiation potential, expansion, recycling, localization and/or persistence capacity, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile and/or degree of differentiation. In reference to the subject being treated, the cells can be allogeneic and/or autologous. These methods include off-the-shelf methods. In some aspects, such as in the art, the cells are pluripotent and/or multipotent cells, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods comprise isolating cells from a subject, preparing, processing, culturing and/or engineering cells as described herein, and reintroducing them into the same patient either before or after cryopreservation.

In some embodiments, the engineered cell is a cell line, such as a T cell line. In certain embodiments, engineered cells are primary cells obtained from a subject. In some embodiments, the subject is a mammalian subject. In certain embodiments, the subject is a human. In certain embodiments, the engineered cells are T cells, preferably human T cells. In some embodiments, the cells are human CD4+ T cells or CD8+ T cells, or a mixed cell population of CD4+ T cells and CD8+ T cells.

Through conventional recombinant DNA technology, the TCR expression construct of the present invention or the vector containing the expression construct can be used to express or produce TCR or engineered cells containing the TCR. Typically the methods include:

(1) transforming or transducing a suitable host cell with a vector encoding the TCR expression construct of the present invention or comprising the expression construct;

(2) cultivating host cells in a suitable medium;

(3) Separating and purifying the engineered cells or TCR of the present invention from the culture medium or cells.

In some embodiments, the recombinant nucleic acid is transferred into the cell via electroporation. In some embodiments, the recombinant nucleic acid is transferred into the cell via transposition.

III. Drug Conjugates

In some embodiments, a drug, such as a small molecule compound, is added to a TCR of the invention, particularly a soluble TCR, thereby obtaining a drug conjugate. Linkage can be achieved by covalent bonds or non-covalent interactions, such as by electrostatic forces. Various linkers known in the art can be used in order to form drug conjugates.

IV. Compositions, methods and uses

One aspect of the invention pertains to a composition (eg, a pharmaceutical composition) or formulation comprising a TCR molecule of the invention, or a TCR complex, or an engineered cell expressing said TCR expression construct. The medicament typically includes one or more optional pharmaceutically acceptable adjuvants or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

"Pharmaceutically acceptable excipients" refer to non-toxic ingredients in pharmaceutical preparations other than the active ingredient to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

In some embodiments, the additional therapeutic agent may be a chemotherapeutic agent or an antibody, such as an antibody directed against an immune checkpoint.

Cells or TCRs can be administered using standard administration techniques, formulations and/or devices. Formulations and devices for storing and administering the compositions, such as syringes and vials, are provided. Administration of cells can be autologous or allogeneic. For example, immunoreactive cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject. Immunoreactive cells derived from peripheral blood or progeny thereof (e.g., of in vivo, ex vivo or in vitro origin) may be administered via local injection (including catheter administration), systemic injection, local injection, intravenous injection or parenteral administration . When administering a therapeutic composition (eg, a pharmaceutical composition containing genetically modified immunoreactive cells), it is usually formulated in a unit dose injectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic and intraperitoneal administration. In some embodiments, the population of cells is administered to a subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery.

Another aspect of the present invention relates to a method for treating or preventing diseases, which includes applying the TCR expression construct, TCR, TCR complex of the present invention or engineered cells comprising the TCR of the present invention, or administering the above-mentioned drugs or preparations to the subject. In some embodiments, the disease is a tumor, eg, cancer, eg, melanoma.

In some embodiments, the TCR expression construct of the present invention, TCR, TCR complex or engineered cells comprising the TCR of the present invention, or the drugs or preparations comprising the above can be used in combination with other therapeutic agents for the treatment of diseases . In some embodiments, the additional therapeutic agent may be a chemotherapeutic agent or an antibody, such as an antibody directed against an immune checkpoint.

As used herein, "treating" refers to the complete or partial amelioration or alleviation of a disease or condition or disorder, or symptoms, side effects or consequences, or phenotypes associated therewith. Desired therapeutic effects include, but are not limited to, prevention of disease occurrence or recurrence, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of the disease, prevention of cancer metastasis, slowing of the rate of disease progression, amelioration or alleviation of disease conditions and remission or improved prognosis. These terms do not imply a complete cure of the disease or complete elimination of any symptoms or an effect on all symptoms or outcomes.

As used herein, "preventing" includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, provided molecules and compositions are used to delay disease development or slow disease progression.

A "therapeutically effective amount" of a composition or formulation or cell refers to an amount and period of time necessary to achieve a desired therapeutic result, such as for the treatment of a disease, condition or disorder, and/or the pharmacokinetics or The amount of pharmacodynamic effect. A therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and weight of the subject, and the cell population administered. In some embodiments, provided methods involve administering an effective amount (eg, a therapeutically effective amount) of a binding molecule, cell, and/or composition. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Usually, but not necessarily, a prophylactically effective amount will be less than a therapeutically effective amount because prophylactic doses are used in a subject prior to or early in the course of disease.

The TCRs of the invention are also useful as diagnostic reagents. Binding between the MHC-peptide and the TCR of the invention specific for the MHC-peptide is detected by labeling the TCR of the invention with a detectable label, such as a label suitable for diagnostic purposes. Fluorescently labeled TCR multimers are suitable for FACS analysis and can be used to detect antigen-presenting cells carrying TCR-specific peptides.

The present invention also relates to the use of the TCR expression construct, TCR, TCR complex or engineered cells containing the TCR of the present invention in the preparation of medicines or pharmaceutical compositions or preparations for treating diseases.

Example

Melanoma is a devastating tumor and is common among patients. The incidence of melanoma is increasing every year. Although melanoma treatment has progressed significantly in recent years with the advent and widespread use of immunotherapy, notably adoptive T cell therapy, there are still many unresolved issues in this field. MART-1/Melan-A is a reliable and commonly shared melanocyte marker found in >90% of melanomas. Rosenberg et al. designed a retroviral vector encoding high-affinity TCR-α, β chain, called F5MART-1TCR [Johnson, L.A., et al., Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. J Immunol, 2006.177(9): p.6548-59], repelling tumor regression when administered to patients with metastatic melanoma [Goff, S.L., et al. al.,Enhanced receptor expression and in vitro effector function of a murine-human hybrid MART-1-reactive T cell receptor following a rapid expansion.Cancer Immunol Immunother,2010.59(10):p.1551-60.SHughes, M. al.,Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.Hum Gene Ther,2005.16(4):p.457-72], Morgan et al reported MART-1 specific A positive TCR (DMF4) resulted in a 13% objective response rate [Morgan, R.A., et al., Cancer regression in patients after transfer of genetically engineered lymphocytes. Science, 2006.314(5796): p.126-9]. And the objective response rate is usually slight. Optimized immunization approaches in adoptive cell transfer using transgenic T cells are needed for better clinical output.

In this example, taking melanoma as an example, the effect of the T cell receptor construct of the present invention is verified.

Materials and methods

1. Cells and cultures

All peripheral blood lymphocytes (PBL) used in this example were obtained from healthy volunteers. The medium used was X-VIVO 15 medium (LONZA, 04-418Q), which was supplemented with 10% fetal bovine serum (FBS, HyClone, sv30160.03), 1% penicillin and streptomycin solution (CORNING, 30-002 -CI), 2mML-Glutamine (Thermo Fisher Scientific, 15630080) and 25mM HEPES (Thermo Fisher Scientific, 15630-080).

Melanoma cell lines 624.38mel (MART-1 ⁺ , HLA-A0201 ⁺ ), 526mel (MART-1 ⁺ , HLA-A0201 ⁺ ), A375mel (MART-1 + , HLA-A0201 + ), A375mel (MART-1 ⁺ , HLA- A0201 ^- ), and 888mel (MART-1 ⁺ , HLA-A0201 ^- ). Cell lines were maintained in RPMI1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin solution) at 37°, 5% _CO2 .

2. The acquisition of each chain used in the examples and the construction of TCR

The TCR (MSGV1 DMF5) composed of α, β chains and the TCR (MSGV1 DMF5ζ) composed of retroviral vectors containing α, β, δ chains were constructed as follows. The schematic diagrams of the two TCRs are shown in Figure 3, and the construction methods are as follows.

2-1. Acquisition of TCR α chain and β chain mRNA

According to literature reports (Gene Transfer of Tumor-Reactive TCR Confers Both High Avidity and Tumor Reactivity to Nonreactive Peripheral Blood Mononuclear Cells and Tumor-Infiltrating Lymphocytes, Laura A et al., J Immunol November 1, 2006, 15978-6) ; DOI: https://doi.org/10.4049/jimmunol.177.9.6548), TILs were isolated from resected tumor tissues of melanoma patients and cultured in vitro to screen for single clones. The most reactive strain obtained was the DMF5 clone. Its TCRα chain sequence is SEQ ID NO:6, and TCRβ chain sequence is SEQ ID NO:15. Therefore, according to the TCR sequence, the company synthesized the cDNA of the α and β chains of its TCR respectively. Using the cDNA synthesized by the company as a template, use the kit T7 mScript Standard mRNA Production System (Cellscript; Cat.No.C-MSC11610 to synthesize the mRNA of the TCR α chain and β chain, and introduce the T7 promoter at the 5′ end to promote RNA in vitro transcription , the primers used are shown in Table 1.

Table 1: Amplification primers for TCR-α and TCR-β chains

Primer name the DMF5-αfwd (forward primer) 5'-CAGAAGGAGGTGGAGCAGA-3' C-αrev (reverse primer) 5'-GTTCACGGCACAGAGGTAGG-3' DMF5-βfed (forward primer) 5'-ATTGCTGGGATCACCCAGGC-3' C-βrev (reverse primer) 5'-GTCACTGCTGGCACAGAAGT-3

2-2. Acquisition of TCR-ε, δ, γ, ζ chain mRNA

Total RNA isolation reagent: TRIzol (Thermo Fisher Scientific, 10296010) was used to extract RNA from the MART-1 reactive TIL clone DMF5, which was reverse transcribed into cDNA using a reverse transcription kit (Thermo Fisher Scientific, K1691). Use kit T7 mScript Standard mRNA Production System (Cellscript; Cat.No.C-MSC11610) to synthesize TCR to synthesize TCR-ε, δ, γ, ζ chain mRNA, and introduce T7 promoter at the 5′ end to promote in vitro transcription of RNA , the applied primers are shown in Table 2.

Table 2: Forward (F) and reverse (R) primers used for mRNA preparation of each strand of ε, δ, γ, ζ

δ-F ATGGAACATAGCACGTTTCT δ-R TCACTTGTTCCGAGCCCAGT ε-F ATGCAGTCGGGCACTCACTG ε-R TCAGATGCGTCTCTGATTCA γ_F ATGTGCACATCAAAGTTTGA γ-R TTACATACTTCTGTAATACA ζ_F ATGAAGGTTCTGTGGGCTGC ζ-R GCGAGGGGGCAGGGCCTGCA

2-3. Construction of MSGV1 DMF5 and MSGV1 DMF5ζ vectors

The retroviral vector pMSGV1 used in this study, derived from the pMSGV murine stem cell virus long terminal repeat, contains an extended gag region and a Kozak sequence. (Articles by Rosenberg SA and Zhong XS, Primary Human Lymphocytes Transduced with NY_ESO-1 Antigen-Specific TCR Genes Recognize and Kill Diverse Human Tumor Cell Lines, Chimeric antigen receptors combining 4-1BB and CD28 XT PI signaling/AKL signaling domains auking activation and CD8+T cell-mediated tumor eradication). The vector MSGV1 DMF5 was generated by introducing the TCR-α chain cDNA (SEQ ID NO: 6), followed by the introduction of the furin T2A cleavage sequence (SEQ ID NO: 18) and the TCR-β chain cDNA (SEQ ID NO: 15) (Fig. 3).

By introducing TCR-α chain cDNA (SEQ ID NO: 6), followed by the introduction of GSGP2A cleavage sequence (SEQ ID NO: 20), TCR-β chain cDNA (SEQ ID NO: 15), T2A cleavage sequence (SEQ ID NO: 18 ) and TCR-ζ chain cDNA (SEQ ID NO: 22), resulting in vector MSGV1 DMF5ζ (Fig. 3). Wherein, the TCR-ζ chain cDNA was obtained by PCR reaction, and the primers are shown in Table 2.

PCR reaction system:

Reagent Volume (μl) 10×KOD buffer 5 2mM dNTPs 5 25mM MgSO4 3 Primer 1(10uM) 0.75 Primer 2(10uM) 0.75 cDNA 1 KOD-plus-Neu 1 ddH2O 33.5

Response program

Gene linker sequences were from Thosea asigna virus (GSGP2A) and porcine teschovirus-1 (T2A).

The sequence is as follows:

2_4. Obtaining GFPmRNA

The cDNA of GFP (SinoBiological, RG81059-U) purchased by the company was used to synthesize mRNA using the kit T7 mScript Standard mRNA Production System (Cellscript; Cat.No.C-MSC11610), and the primers were as follows:

Primer sequence GFP-F ATGGTGAGCAAGGGCGAGGA GFP-R CTTGTACAGCTCGTCCATGC

GFP nucleic acid sequence:

GFP protein sequence:

2-5CD3ζ siRNA and Control siRNA

In the subsequent part of the examples, the knockdown of CD3ζ or GFP in cells is used, that is, CD3ζ siRNA and GFP siRNA, as follows:

Use reagents RNAiMAX Transfection Reagent (Thermo Fisher Scientifc, 13778), siCD3δ (Thermo Fisher Scientifc, s534912), siControl (Thermo Fisher Scientifc, 4390843) to knock down for 48h.

Example 1.CD3ζ increases the expression of TCR in PBL

T-cell receptors (TCRs) expressing tumor antigen-specific TCRs—T cells as heterodimers composed of two distinct subunits α and β chains—could mediate complete regression in patients with tumors. The TCR complex consists of 6 polypeptides, 2 heterodimers, CD3γε and CD3δε, and 1 homodimer CD3δ [Golubovskaya, V., et al., Major Highlights of the CAR-TCR Summit, Boston, 2016. Anticancer Agents Med Chem, 2017.17(10): p.1344-1350.]. In order to verify which component of the TCR complex has a major effect on the T cell receptor, the mRNAs of DMF5 TCRα, β chains, and CD3ζ, δ, γ, and ε were obtained as described above. Clone JFK6 (another reported TIL cell line isolated from patient tumor tissue, highly active to MART-1, reference: Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor- infiltrating lymphocytes) were included in this study as a positive control.

The PBL was stimulated with 50 ng/ml soluble anti-CD3 antibody (OKT3, Biolegen, 317302 ₎ in the presence of interleukin-2 (Double Eagle Pharmaceuticals, 200 IU/ml) for 48 hours in a humid air atmosphere containing 5% CO . The mRNAs of the α and β chains of the MART-1-specific TCR (DMF5) obtained as described above and combinations of different CD3 chains (i.e. DMF5 with different chains ζ, δ, γ, ε or ζ+δ+γ+ ε combination) mRNA electroporation.

For electroporation of IVT RNA, electroporated PBLs stimulated with OKT3 plus IL-2 for 2 days were washed twice with OPTI-MEM and resuspended in OPTI-MEM at a final concentration of 2.5 × ¹⁰ cells/ml. MEM (Thermo Fisher Scientific, 51985091). (Materials used: electric cup; electrotransfer instrument, ECM830; electrotransfer conditions: 200V, 0.8ms)

A total of 2.0 μg of α and β chains and ε, δ, γ, and δ mRNAs prepared above were electroporated per 1×10 ⁶ cells.

Immediately after electroporation, cells were transferred to fresh medium with 200 IU/ml IL-2 and incubated at 37° for further experiments.

24 hours after electroporation, the expression of MART-1 TCR was determined by FACS analysis. Cells were double stained with FITC-conjugated CD8 (CD8-FITC(BD)) and APC-conjugated MART-1/HLA-A0201 tetramer antibody (Beckman-Coulter Immunomics), and the percentage of positive cells was indicated, To test the expression of TCR tetramer.

After adding antibody CD8-FITC and tetramer antibody according to the antibody instructions, incubate in the dark for 15 min. Add 3ml PBS to each tube, centrifuge at 500g for 10min, discard the supernatant and repeat the washing once. After the supernatant was discarded, 500 μl of 1% paraformaldehyde fixative was added, and the cells were resuspended for detection on the machine. As a result, as shown in Fig. 1, it was possible to confirm that the CD3δ chain is the most important factor for increasing TCR tetramer.

Example 2 Tumor-reactive DMF5 electroporated into PBLs followed by zeta-strand RNA promotes higher reactivity to melanoma tumors

We next focused on the effect of the CD3δ chain on electroporated human PBLs.

As shown in the previous materials and methods, as described in Example 1, the PBL was electroporated to transduce the mRNA of TCRα chain, β chain and δ chain or the GFP RNA prepared above to construct PBL, and the obtained PBL or positive Control JFK6 or negative control (i.e. PBL transfected with only GFP plasmid) was co-cultured with melanoma tumor cell lines 624.38mel, 526mel, A375mel and 888mel at 37°C for 24 hours, and these cells were evaluated by IFN-γ and IL-2 functional affinity.

Specifically, in each well of a 96-well plate, 1×10 ⁵ responder cells (transduced PBL or JFK cells) and 5×10 ⁴ tumor cells were incubated in a culture volume of 0.2 ml per well for 37 °C for 24 hours, and the supernatant was taken as the sample to be tested. Add the standard (R&D Systems, DY008) and the sample to be tested into the transparent enzyme-coated plate of the pre-coated interferon-γ (INF-γ) ELISA kit (R&D Systems, DY008), and after incubation for a sufficient time, Wash to remove unbound components, then add enzyme-labeled working solution, and after incubation for a sufficient time, wash to remove unbound components. Substrates A and B are added in turn, and the substrate (TMB) is converted into a blue product under the catalysis of horseradish peroxidase (HRP), and turns yellow under the action of acid. The depth of the color is related to the interferon-γ in the sample. (INF-γ) concentration is positively correlated, the OD value is measured at a wavelength of 450nm, and the content of interferon-γ (INF-γ) in the sample is calculated according to the OD value of the standard and the sample. Experiments were performed in triplicate. The method for detecting IL2 by ELISA is the same (R&D Systems, D2050).

The results are shown in Figure 2A and B, for expressing HLA-A0201-restricted melanoma cell lines 624.38mel (MART-1 ⁺ , HLA-A0201 ⁺ ), and 526mel (MART-1 ⁺ , HLA-A0201 ⁺ ), The α+β+δ chain was able to produce the greatest amount of IFN-γ, while the α+β chain and JKF6 produced a moderate level of response, and the GFP control had only a negligible response. However, IFNγ production was negligible in co-cultures with tumor cells lacking HLA-A 0201 (A375mel, 888mel; Figure 2A).

Cells transduced by α+β chain electroporation with CD3ζ chain were able to produce higher amounts of IL-2 (Fig. 2B).

Thymidine assay was performed on the cells to detect the proliferative ability of the cells (Fig. 2C). A total of 4 groups were performed:

α+β: PBL transduced with TCR α+β chain by electroporation.

α+β+δ: PBL transduced with TCR α+β chain and ζ chain by electroporation.

α+β+CD3ζ siRNA: PBL cells were first administered with RNAiMAX (Transfection Reagent (Thermo Fisher Scientifc), siCD3δ (thermo, s534912)) to knock down CD3ζ for 48 hours, and then electroporated into PBL transduced with TCRα+β chain.

α+β+siControl: PBL cells were first knocked down by RNAiMAX (Transfection Reagent (Thermo Fisher Scientifc,), siControl (thermo, 4390843)) for 48 hours, and then electroporated to transduce the PBL of the TCRα+β chain.

The specific measurement steps are as follows:

For proliferation assays by thymidine, the above transduced PBLs (effector cells) were co-cultured with individual tumor cells (624.38mel, 526mel, A375mel, 888mel, target cells) to produce an effector:target (E:T) ratio of 10 :1.

1 μCi ^of3H thymidine was added to the medium of PBL and individual tumor cells 16 hours before harvest and counted per minute (cpm). Wells were then harvested using an automated cell harvester (Pharmacia), scintillation fluid (Pharmacia) was added to the counter, and radioactivity was measured. Experiments were performed in triplicate.

Figure 2C shows that [3H]-thymidine incorporation level (CPM) is α+β+δ>α+β≈α+β+siControl>α+β+siCD3δ (Figure 2C).

A 51Cr release assay was further performed to detect cytotoxicity. A total of 4 groups were performed:

A total of 4 groups were performed, and the effector cells (E) in the 4 groups were as follows:

α+β: PBL transduced with TCR α+β chain by electroporation.

α+β+δ: PBL transduced with TCR α+β chain and δ chain by electroporation.

α+β+siCD3δ: PBL cells were first administered with RNAiMAX (Transfection Reagent (Thermo Fisher Scientifc, 13778), siCD3δ (thermo, s534912)) to knock down CD3ζ for 48 hours, and then electroporated into PBL transduced with TCRα+β chain.

α+β+siControl: PBL cells were first knocked down by RNAiMAX (Transfection Reagent (Thermo Fisher Scientifc, 13778), siControl (Thermo Fisher Scientifc, 4390843)) for 48 hours, and then electroporated to transduce the PBL of the TCRα+β chain.

1×10 ⁶ tumor target cells (624.38mel, 526mel, A375mel, 888mel) were labeled with 200uCi of Na ₂ ⁵¹ CrO ₄ (Amersham Biosciences) for 1 hour at 37° to obtain target cells (T).

The transduced PBL (E) obtained in the above four groups was then washed with complete medium (complete medium containing RPMI1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin solution), and mixed with the above-mentioned obtained Transduced PBLs (5×10 ³ ) were incubated at the (E:T) ratio shown in Figure 2B. After 4 hours of incubation at 37°, the supernatant was collected and counted on a scintillation counter.

The percentage of cell lysis was calculated by the following formula: percentage of specific lysis=(sample ⁵¹ Cr release−spontaneous ⁵¹ Cr release)/(total ⁵¹ Cr release−spontaneous ⁵¹ Cr release)×100.

The ability of electroporated PBLs to lyse HLA-A0201 melanoma targets at different E:T was analyzed using a ⁵¹ Cr release assay. Figure 2D shows the results. PBLs transduced with α+β+ζ chains could effectively kill the MART-1/HLA-A0201 double-positive melanoma cell line (Fig. 2D), while PBLs transduced with only α+β chains had a lower percentage of cell lysis. Difference.

The effect of PBL with siControl using α+β chain transduction was consistent with the effect of transducing α+β chain.

Furthermore, PBLs transduced with α+β chains but knocked down CD3 were the least effective.

Example 3. Higher expression of CD3ζ chain in transduced DMF5ζ T cells

The construction of two retroviral vectors MSGV1 DMF5 and MSGV1 DMF5δ is described in detail in Materials and methods. Figure 3 shows the structural diagrams of two MSGV-based retroviral vectors for the transfer and expression of MART-1 TCR-α, β or MART-1 TCR-α, β plus δ genes. In these vectors, both α and β chains are mediated by LTRs but linked by different 2A peptides (T2A or GSGP2A) in the absence or presence of a GSG linker. In the vector MSGV1F5Mδ, expression is via δ chain coupling.

After PBL was stimulated with OKT3Ab plus IL-2 for 2 days, it was transfected with the obtained vector MSGV1 DMF5 and MSGV1 DMF5δ virus constructs for 3 days. The specific process is as follows:

The retrovirus vector was transfected into the PG13 cell line, and two days later, the supernatant was collected as the retrovirus supernatant. The retroviral supernatant was added to the plate, which had been coated with 15 g/ml recombinant fibronectin fragment (Takara, T202), for 2 hours at 37° and then incubated overnight at 4°.

The virus transfection process was as follows: 0.5ml Retronectin (15ug/ml) was added to a 12-well plate, and incubated at room temperature in the dark for 2 hours. Discard the supernatant, add 0.5% human AB serum (PBS configuration), incubate for 30 min, and discard the supernatant. Add 0.5ml of PBL cells (1.6×10 ⁶ /ml) and 0.5ml of virus supernatant, seal the well plate with a sealing film, centrifuge at 700g for 1 hour, and put it into a 37° incubator for cultivation.

The obtained PBLs were then washed and transduced into plates with retrovirus at 1 x ¹⁰⁶ cells/ml (5 ml/well) at 37° overnight, and the next day, the transfer of cells was repeated. After transduction, PBLs were washed and resuspended in fresh cell T cell medium in a 5% _CO2 incubator at 37°.

After the transduced PBLs obtained above were co-cultured with the tumor cell line 624.38mel (MART-1 ⁺ , HLA-A0201 ⁺ ) for 24 hours (first stimulation) (E:T=10:1), at 6 On the first day, functional assays were performed on the PBLs using FACS. Specifically, with FACS as described in Example 1, by FITC-conjugated CD4 (CD4-FITC(BD)), FITC-conjugated CD8 (CD8-FITC(BD)) and APC-conjugated CD3δ antibody (BD) was used to stain the cells to detect cells positive for both CD3δ and CD8 or CD4. The results are shown in Figure 4B. CD3δ of cells transduced with DMF5δ was highly expressed in CD3-positive, CD4-positive and CD8-positive T cells.

On day 11, the transduced PBL cells were again co-cultured with the tumor cell line 624.38mel (MART-1+, HLA-A0201 ⁺ ) for 24 hours (second stimulation) (E:T=10:1) and measured Levels of CD3δ in the PBL. See Figure 4A for a schematic diagram of the experimental process.

Afterwards, CD3δ in total T cells, CD4+ T cells and CD8+ T cells were gated and quantified by FACS, respectively. Specific steps are as follows:

FITC-conjugated CD3 antibody (CD3-FITC(BD)), FITC-conjugated CD8 (CD8-FITC(BD)), FITC-conjugated CD4 (CD4-FITC(BD)), PE-conjugated The PBL cells obtained above at day 12 were stained with conjugated TCRδ antibody, APC-conjugated MART-1/HLA-A0201 tetramer antibody (Beckman-Coulter Immunomics) to measure surface CD3δ. Flow cytometry was performed using BD FacsCanto II Plus (BD Biosciences) and analyzed with FlowJo (Tree Star).

The results are shown in Figure 4C. On day 12, the expression of CD3δ in DMF5δ-transduced CD8-positive T cells was still higher than that in DMF5-transduced CD8-positive T cells.

Therefore, we successfully generated two functional HLA-A0201-restricted and MART-1-specific TCR vectors DMF5 and DMF5δ for subsequent experiments.

Example 4. Overexpression of CD3ζ chain can maintain and enhance MART-1 TCR expression level after two rounds of tumor antigen stimulation

To determine whether overexpression of the CD3δ chain that affects TCR expression is persistent, serial analyzes were performed to look for differential expression of TCRs on the cell surface.

As shown in the schematic diagram of the experimental process in Figure 4A, the PBL obtained in Example 3 was cultured until the 11th day, and the above-mentioned PBL (10:1) was re-stimulated with fresh tumor cells (624.38mel), and the specific operation process was as in Example 3 mentioned. On day 12, after two rounds of tumor stimulation, TCR expression was monitored by MART-1 tetramer staining PBL using CD8 antibody and MART-1 tetramer (FACS assay as described in Example 1). See Figures 5A and 5B for results. On the sixth day (after the first round of stimulation), the TCR-positive expression rates in PBL cells transduced with DMF5 and DMF5δ were 31.85% and 39.19%, respectively. The positive expression rates of TCR in PBL cells were 32.95% and 45.75%, respectively.

In summary, the expression level of MART-1 TCR was significantly higher in CD8-positive T cells transduced with DMF5δ (42.75% vs. 32.95%).

Example 5. Activation and proliferation of DMF5TCR engineered CD8+ T cells can be significantly enhanced by possessing the CD3ζ chain.

The role of tumor antigen-specific CD8+ T cells in tumor rejection is well established. Therefore our experiments detect the functional changes of CD8+ T cells.

T cells (days 3 and 4) were retrovirally transfected with DMF5 and DMF5ζ constructs as described in Example 3 (see Figure 4A for the specific process). On day 6, 2 days after transduction, the transduced T cells were mixed with a MART-1/HLA-A0201 double positive melanoma tumor line (624.38mel, 526mel) and two HLA-A0201 negative but MART- 1 Positive tumor line (A375mel, 888mel) was co-cultured for 24 hours (first stimulation).

The secretion of IL-2 and IFN-γ in the cells was detected as shown in Example 2. As shown in Figures 6A and 6B, the cells transduced with MSGV1DMF5ζ can effectively produce the release of IL-2 and IFN-γ.

51Cr release assay was performed as described in Example 2 to detect cytotoxicity. 10 ⁶ tumor cells (624.38mel, 526mel, A375mel, 888mel) were co-cultured overnight with transduced T cells at multiple E:T. Cytotoxicity of the transduced CD8+ T cells was evaluated by percentage lysis and the results are shown in Figure 6C. CD8+ T cells transfected with DMF5δ virus could efficiently kill the MART-1/HLA-A0201 double-positive melanoma cell line, while CD8+ T cells transduced with DMF5 alone had a poor percentage of cell lysis.

CFSE labeling method was used to detect T cell proliferation: the transduced effector cells PBL were co-cultured with 1×10 ⁶ cells/well and 1×10 ⁵ cells/well of each melanoma cell line in a 24-well round bottom plate (10:1 ), label the cells with 2.5 μM preheated CFSE (Thermo Fisher Scientific, C34554), and after 5 days of co-cultivation, collect the cells, wash them with PBS, and add the prepared CD3 antibody (BD) staining solution (CD3 antibody + PBS , 1:1000), CD8 antibody (BD) staining solution (CD8 antibody+PBS, 1:1000) and MART-1TCR (APC-conjugated MART-1/HLA-A0201 tetramer antibody (Beckman-Coulter Immunomics)) For staining, incubate at room temperature in the dark for 15 min, wash with PBS, and detect CFSE signal by flow cytometry. As shown in Figure 6D, under the stimulation of 624.38mel antigen-positive tumor cells, the proliferation of PBL cells transduced with DMF5δ was stronger than that of CD8 T cells transduced with DMF5.

Claims (44)

A novel TCR expression construct that sequentially contains in tandem (i) a nucleic acid encoding a TCR alpha chain; (ii) a nucleic acid encoding a TCR beta chain; and (iii) a nucleic acid encoding the CD3ζ chain; Wherein both the TCRα chain and the TCRβ chain contain constant regions, and both the constant regions are derived from mice; and the CD3ζ chain is derived from humans. The TCR expression construct of claim 1, wherein a linker exists between the nucleic acid encoding the TCRα chain and the nucleic acid encoding the TCRβ chain, and/or a linker exists between the nucleic acid encoding the TCRβ chain and the nucleic acid encoding the CD3ζ chain. The TCR expression construct of claim 1, wherein The constant region of the TCRα chain comprises the amino acid sequence of SEQ ID NO: 5, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 5, or comprises the amino acid sequence of SEQ ID NO: 5 The amino acid sequence of: 5 has or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence; or The nucleic acid encoding the TCR α chain comprises the nucleic acid encoding the TCR α chain constant region, the nucleic acid encoding the TCR α chain constant region comprises the nucleotide sequence of SEQ ID NO: 7, or comprises the nucleic acid sequence having 1, 2 compared with the nucleotide sequence of SEQ ID NO: 7 , 3, 4 or 5 variant nucleic acid sequences, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleic acid sequence of SEQ ID NO:7 % or 99% of, or consist of, a nucleic acid sequence. The TCR expression construct of claim 3, wherein said variation is a substitution, insertion or deletion. The TCR expression construct of claim 1, wherein The constant region of the TCR β chain comprises the amino acid sequence of SEQ ID NO: 12, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 12, or comprises the amino acid sequence of SEQ ID NO: 12 The amino acid sequence of: 12 has or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence; or The nucleic acid encoding the TCR β chain comprises the nucleic acid encoding the TCR β chain constant region, and the nucleic acid encoding the TCR β chain constant region comprises the nucleotide sequence of SEQ ID NO: 16, or comprises 1, 2 compared with the nucleotide sequence of SEQ ID NO: 16 , 3, 4 or 5 variant nucleic acid sequences, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the nucleic acid sequence of SEQ ID NO: 16 % or 99% of, or consist of, a nucleic acid sequence. The TCR expression construct of claim 5, wherein said variation is a substitution, insertion or deletion. The TCR expression construct of any one of claims 1-6, wherein, The CD3ζ chain comprises the amino acid sequence of SEQ ID NO: 21, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 21, or comprises the amino acid sequence of SEQ ID NO: 21 The amino acid sequence has or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence; or The nucleic acid encoding CD3ζ chain comprises the nucleotide sequence of SEQ ID NO: 22, or comprises the nucleotide sequence with 1, 2, 3, 4 or 5 variations compared with the nucleotide sequence of SEQ ID NO: 22, or comprises the nucleotide sequence with SEQ ID NO The nucleic acid sequence of: 22 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence, or consists of said nucleic acid sequence. The TCR expression construct of claim 7, wherein said variation is a substitution, insertion or deletion. The TCR expression construct of claim 1 or 2, wherein the TCR alpha chain comprises a variable region and/or the TCR beta chain comprises a variable region. The TCR expression construct of claim 9, wherein the variable region of the TCR alpha chain and/or the variable region of the TCR beta chain is derived from human. The TCR expression construct according to claim 1 or 2, wherein the linker between TCRα chain and TCRβ chain, or the linker between TCRβ chain and CD3ζ chain is a self-cleaving peptide. The TCR expression construct of claim 11, wherein the self-cleaving peptide is T2A or GSGP2A. The TCR expression construct of claim 12, wherein T2A comprises the amino acid sequence of SEQ ID NO: 17 or is made up of said sequence or the nucleic acid encoding T2A comprises the nucleotide sequence of SEQ ID NO: 18 or is made up of said nucleotide sequence. The TCR expression construct of claim 12 or 13, wherein GSGP2A comprises the aminoacid sequence of SEQ ID NO:19 or is made up of said sequence or the nucleic acid encoding GSGP2A comprises the nucleotide sequence of SEQ ID NO:20 or is made up of said nucleotide sequence. The TCR expression construct according to claim 1 or 2, wherein the linker between TCRα chain and TCRβ chain is GSGP2A and/or the linker between TCRβ chain and CD3ζ chain is T2A. The TCR expression construct of claim 15, wherein T2A comprises the amino acid sequence of SEQ ID NO: 17 or is made up of said sequence or the nucleic acid encoding T2A comprises the nucleotide sequence of SEQ ID NO: 18 or is made up of said nucleotide sequence. The TCR expression construct of claim 15, wherein GSGP2A comprises the amino acid sequence of SEQ ID NO: 19 or is made up of said sequence or the nucleic acid encoding GSGP2A comprises the nucleotide sequence of SEQ ID NO: 20 or is made up of said nucleotide sequence. The TCR expression construct of claim 1 or 2, wherein the TCR is a TCR directed against MART-1. The TCR expression construct of claim 18, wherein TCR alpha chain comprises TCR alpha chain variable region, and it comprises following CDR1, CDR2 and CDR3, and wherein CDR1 comprises the aminoacid sequence of SEQ ID NO:2 or is made up of it, and CDR2 comprises the aminoacid sequence of SEQ ID NO:3 or is made up of it, CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:4; Or the TCR α chain comprises a TCR α chain variable region comprising the amino acid sequence of SEQ ID NO: 1, or comprising 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 1 or comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence of SEQ ID NO: 1, or consists of said sequence; or The TCR alpha chain comprises the amino acid sequence of SEQ ID NO: 13, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 13, or comprises the amino acid sequence of SEQ ID NO: 13 The amino acid sequence has or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence; or The nucleic acid encoding TCR α chain comprises the nucleotide sequence of SEQ ID NO: 6, or comprises the nucleotide sequence with 1, 2, 3, 4 or 5 variations compared with the nucleotide sequence of SEQ ID NO: 6, or comprises the nucleotide sequence with SEQ ID NO The nucleic acid sequence of: 6 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence, or consists of said nucleic acid sequence. The TCR expression construct of claim 19, wherein said variation is a substitution, insertion or deletion. The TCR expression construct of claim 18, wherein TCR beta chain comprises TCR beta chain variable region, and it comprises following CDR1, CDR2 and CDR3, and wherein CDR1 comprises the aminoacid sequence of SEQ ID NO:9 or is made up of it, and CDR2 comprises the aminoacid sequence of SEQ ID NO:10 or is made up of it, CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11; or The TCR beta chain comprises a TCR beta chain variable region comprising the amino acid sequence of SEQ ID NO:8, or comprising 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO:8 an amino acid sequence, or comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8, or consists of said sequence; or The TCR beta chain comprises the amino acid sequence of SEQ ID NO: 14, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 14, or comprises the amino acid sequence of SEQ ID NO: 14 The amino acid sequence has or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence; or The nucleic acid encoding TCR β chain comprises the nucleotide sequence of SEQ ID NO: 15, or comprises the nucleotide sequence with 1, 2, 3, 4 or 5 variations compared with the nucleotide sequence of SEQ ID NO: 15, or comprises the nucleotide sequence with SEQ ID NO: The nucleic acid sequence of: 15 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence, or consists of said nucleic acid sequence. The TCR expression construct of claim 19, wherein TCR beta chain comprises TCR beta chain variable region, and it comprises following CDR1, CDR2 and CDR3, and wherein CDR1 comprises the aminoacid sequence of SEQ ID NO:9 or is made up of it, and CDR2 comprises the aminoacid sequence of SEQ ID NO:10 or is made up of it, CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11; or The TCR beta chain comprises a TCR beta chain variable region comprising the amino acid sequence of SEQ ID NO:8, or comprising 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO:8 an amino acid sequence, or comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8, or consists of said sequence; or The TCR beta chain comprises the amino acid sequence of SEQ ID NO: 14, or comprises an amino acid sequence with 1, 2, 3, 4 or 5 variations compared to the amino acid sequence of SEQ ID NO: 14, or comprises the amino acid sequence of SEQ ID NO: 14 The amino acid sequence has or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence; or The nucleic acid encoding TCR β chain comprises the nucleotide sequence of SEQ ID NO: 15, or comprises the nucleotide sequence with 1, 2, 3, 4 or 5 variations compared with the nucleotide sequence of SEQ ID NO: 15, or comprises the nucleotide sequence with SEQ ID NO: The nucleic acid sequence of: 15 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence, or consists of said nucleic acid sequence. The TCR expression construct of claim 21 or 22, wherein said variation is a substitution, insertion or deletion. A TCR encoded and produced by the TCR expression construct of any one of claims 1-23. A TCR complex produced in a T cell by the TCR expression construct of any one of claims 1-23. A vector comprising the TCR expression construct of any one of claims 1-23. The vector of claim 26, which is an expression vector. The vector of claim 27, wherein said expression vector is a retroviral vector. The vector of claim 28, wherein said retroviral vector is pMSGV1. An engineered cell comprising the TCR expression construct of any one of claims 1-23, or the vector of any one of claims 26-29. The engineered cell of claim 30, wherein said cell is a T cell. The engineered cell of claim 31, wherein said T cells are human T cells. The engineered cell according to claim 32, wherein said human T cells are CD4+ cells or CD8+ cells, or a mixed cell population of CD4+ T cells and CD8+ T cells. A method of producing engineered cells comprising (1) Transform or transduce host cells with the TCR expression construct of any one of claims 1-23 or the vector of any one of claims 26-29; (2) cultivating host cells in a suitable medium; (3) Separating and purifying engineered cells from culture medium or cells. 34. The method of claim 34; wherein the expression construct or vector is transferred into the cell via electroporation or transferred into the cell via transposition. A composition comprising the TCR expression construct of any one of claims 1-23, the TCR of claim 24, the TCR complex of claim 25, the vector of any one of claims 26-29, or the vector of any one of claims 30- The engineered cell of any of 33. The composition of claim 36, wherein said composition is a pharmaceutical composition. The composition according to claim 36 or 37, which further comprises a pharmaceutically acceptable auxiliary material. A method for treating or preventing a disease, comprising applying the TCR expression construct of any one of claims 1-23, the TCR of claim 24, the TCR complex of claim 25, the vector of any one of claims 26-29 Or the engineered cell of any one of claims 30-33 or the composition of any one of claims 36-38 administered to a subject. 39. The method of claim 39, wherein said disease is a tumor. 40. The method of claim 40, wherein said tumor is cancer. 41. The method of claim 41, wherein said cancer is melanoma. The method of any one of claims 39-42, wherein the TCR expression construct of any one of claims 1-23, the TCR of claim 24, the TCR complex of claim 25, the TCR complex of claims 26-29 The vector of any one or the engineered cell of any one of claims 30-33 or the composition of any one of claims 36-38 in combination with one or more other therapeutic agents. 43. The method of claim 43, wherein said therapeutic agent is a chemotherapeutic agent or an antibody.

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