KR20190076297A - MART-1 Specific T Cell Receptor and Use Thereof - Google Patents

Abstract

The present invention relates to a nucleic acid for expressing the alpha chain or beta chain of a T-cell receptor that binds specifically to a cancer-specific antigen, MART-1; a polypeptide encoded by the same; and a composition for treating MART-1 associated diseases, comprising a cell expressing the T-cell receptor, as an active ingredient. The present invention provides a nucleotide sequence included in the genes of the alpha chain and beta chain of the MART-1 specific T-cell receptor, and an amino acid sequence of the peptide encoded thereby, and thus enables the production of cytotoxic T cells (CTL), suppressor T cells or natural killer (NK) cells, expressing recombinant T cell receptors enabling immunotherapy specific to cancer cells and tumor cells expressing MART-1. Accordingly, the present invention may provide a treatment method which can overcome or minimize the limitations of chemical and surgical methods often used in a conventional cancer treatment.

Description

MART-1 Specific T Cell Receptors and Their Uses {MART-1 Specific T Cell Receptor and Use Thereof}

The present invention relates to a nucleic acid for expressing an alpha chain or a beta chain of a T cell receptor specifically binding to a cancer-specific antigen MART-1, a polypeptide encoded thereby, and a cell expressing the T cell receptor 1 < / RTI > related diseases.

Recently, the number of cancer patients is increasing due to changes in eating habits, environmental changes, and aging. However, due to inherited imperfections of cancer cells, it is difficult to completely cure cancer with conventional anticancer drugs alone. As an alternative to this, immunotherapy is emerging recently. Immunotherapy has high selectivity and specificity for cancer, and there are few side effects.

In the case of T cells, a monoclonal T cell receptor (TCR) is expressed on the cell membrane to recognize the major histocompatibility complex / peptide (pMHC) of the antigen presenting cell (APC) Cellular adaptive immunity is very sophisticated and should be able to effectively remove infectious agents in the body or cancer cells. At this time, if the antigen-specific adaptive immune system does not function properly, it will cause serious problems in response to infectious diseases and cancer cell removal function.

The T cell receptor (TCR) operates in the form of a [beta] TCR complex consisting of an alpha beta heterodimer that specifically recognizes the antigen and CD3 molecules (CD3 [epsilon], CD3 [delta], CD3ζζ) for signaling, , And the?,?, And? Subunits of CD3 each have an Ig domain in an extracellular configuration. αβ if whether heterodimer is the antigen specifically makin the signaling carried into the cells to CD3 molecules as a medium, such signaling is the intracellular tyrosine kinase (LCK, ZAP70, LAT, PLCγ) phosphorylation, cytosolic Ca ^{2 +} concentration change and Is known to induce T cell activation through changes in the intracellular transcription system.

On the other hand, the Mart-1 gene, one of the major antigens expressed in melanoma cells, has a size of 18 Kb and is composed of 5 exons and has a total of 118 amino acid residues. Among them, the ELAGIGILTV (26-35) peptide has recently been studied as an epitope of a major T cell receptor (TCR) recognized by T cells in association with HLA-A2, as a target of recent anti-cancer immunotherapy (US 9128080 B2 , US 2013-0116167 A1, US 8088379 B2).

Although DMF5 is known to be an excellent T cell receptor for the treatment of cancer with tumor-specific T cells, it is known that DMF5 has only 30% anticancer effect in the first clinical trial. Therefore, DMF5 has a better effect than DMF5 The development of T-cell receptors is required.

Accordingly, the present inventors have made intensive efforts to identify a novel T cell receptor targeting MART-1. As a result, a novel T cell receptor targeting Mart-1 was identified, and the alpha- The amino acid sequences and nucleic acid sequences of the beta-chain were confirmed. Recombinant T cells expressing the Mart-1 specific T cell receptor on cytotoxic T cells (CTL) were prepared and the cancer cell-specific cell death The present invention has been completed.

US 9128080 B2 US 2013-0116167 A1 US 8088379 B2

S Yang et al., (2008) Gene Therapy 15: 1411-1423, Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition. Nishant Singh et al., (2015) The FASEB Journal vol. 29 no. 1 Supplement 571.30, Biophysical Characterization of TCR Variants with Reengineered Specificity and Affinity.

It is an object of the present invention to provide novel polypeptides which constitute the variable domains of the alpha or beta chains of the MART-1 specific T cell receptor.

Another object of the present invention is to provide a novel MART-1 specific T cell receptor and a cell wherein the MART-1 specific T cell receptor is expressed on the surface.

Yet another object of the present invention is to provide a nucleic acid encoding a variable domain of an alpha or beta chain of a MART-1 specific T cell receptor, a recombinant vector comprising the nucleic acid, and a cell into which the recombinant vector is introduced.

It is still another object of the present invention to provide a composition for treating a MART-1-related disease comprising the cell as an active ingredient.

In order to achieve the above object,

A polypeptide constituting all or part of the variable domain of the alpha or beta chain of the MART-1 specific T cell receptor represented by the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6.

The present invention also provides a polypeptide comprising the polypeptide represented by the amino acid sequence of SEQ ID NO: 1 as all or part of the variable domain of the alpha chain and the polypeptide represented by the amino acid sequence of SEQ ID NO: 2 as all or part of the variable domain of the beta chain Lt; RTI ID = 0.0 > MART-1 < / RTI > specific T cell receptor.

The present invention also provides a polypeptide comprising the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 as all or a part of the variable domain of the alpha chain and the polypeptide represented by the amino acid sequence of SEQ ID NO: 4 as all or a part of the variable domain of the beta chain Lt; RTI ID = 0.0 > MART-1 < / RTI > specific T cell receptor.

The present invention also provides a polypeptide comprising the polypeptide represented by the amino acid sequence of SEQ ID NO: 5 as all or a part of the variable domain of the alpha chain and the polypeptide represented by the amino acid sequence of SEQ ID NO: 6 as all or a part of the variable domain of the beta chain Lt; RTI ID = 0.0 > MART-1 < / RTI > specific T cell receptor.

The present invention also provides cells wherein the MART-1 specific T cell receptor is expressed on the surface.

The present invention also provides a nucleic acid encoding all or part of the variable domain of the alpha chain of the MART-1 specific T cell receptor represented by the nucleic acid sequence of SEQ ID NO: 7, 9 or 11.

The present invention also provides a nucleic acid encoding all or part of the variable domain of the beta chain of the MART-1 specific T cell receptor, represented by the nucleic acid sequence of SEQ ID NO: 8, 10 or 12.

The present invention also provides a recombinant vector comprising said nucleic acid.

The present invention also provides a cell into which said recombinant vector has been introduced.

The present invention also provides a pharmaceutical composition for treating a MART-1 related disease comprising the cell as an active ingredient.

According to the present invention, the nucleotide sequence contained in the alpha and beta chain genes of the MART-1 -specific T cell receptor and the amino acid sequence of the peptide encoded thereby can be used to identify cancer cells and tumor cells expressing MART-1 (CTL), inhibitory T cell or natural killer cell (NK cell) expressing a recombinant T cell receptor capable of specific immunotherapy to the cell, T cell therapy, it is possible to provide an effective treatment method capable of overcoming or supplementing the limitations of the chemical and surgical application which have been mainly used in conventional cancer treatment, 1 < / RTI > related diseases.

1 is a schematic diagram showing a process for identifying an antigen-specific T cell receptor.
Figure 2 shows the results of three types of MART-1 specific T cell clones identified by flow cytometry.
FIG. 3 shows the results of screening MART-1-specific T cell clones through the ELIspot method.
FIG. 4 shows the results of confirming amplified TRAV and TRBV bands by PCR of MART-1-specific T cell clones.
FIG. 5 shows a vector map of the expression vector for MART-1-specific T cell expression and shows the amino acid sequence of the corresponding site.
FIG. 6 shows the results of confirming T cell surface expression of the recombinant T cell receptor according to the present invention.
FIG. 7 shows MART-1-specific cell death according to the ratio of the surface-expressed cells to the target cells of the recombinant T cell receptor according to the present invention.
FIG. 8 shows the cytotoxicity of two types of cancer cell lines (MeWo-K, UACC-6) of cells expressing the surface of the recombinant T cell receptor according to the present invention to DMF5 as a positive control.
FIG. 9 shows the results of confirming the tumor-reducing effect of melanoma-induced mice according to the administration of the cells expressing the surface of the recombinant T cell receptor according to the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, a cytotoxic T cell clone specifically recognizing ELAGIGILTV (26-35), a restriction peptide of HLA-A2 (human leukocyte antigen-A2) derived from MART-1 (melanoma antigen recognized by T cells 1) And the nucleic acid sequence and amino acid sequence of the alpha and beta variable domain of the T cell receptor were analyzed. Based on the analysis, cytotoxic T cells expressing the recombinant T cell receptor containing the variable domain were prepared, The tumor cell line specific cytotoxicity of the cytotoxic T cells and the tumor reduction effect in the tumor-induced mouse model were confirmed.

Accordingly, the present invention relates to polypeptides that constitute all or part of the variable domains of the alpha or beta chain of the MART-1 specific T cell receptor, represented by the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 6 in one aspect . In the present invention, SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 is a sequence derived from the alpha chain mutation portion of the T cell receptor specifically recognizing MART-1, Or at least 80%, 90%, or 95% amino acid sequence homology. For example, even if one to several amino acids are substituted, added or deleted in the amino acid sequence represented by SEQ ID NO: 1, 3 or 5, the peptide does not induce a change in the three-dimensional structure of the T cell receptor, And a function equivalent to that of a peptide. In the same context, SEQ ID NO: 2, 4, or 6 in the present invention is a sequence derived from the beta chain mutation site of a T cell receptor that specifically recognizes MART-1 and has the same sequence as SEQ ID NO: 2, , As well as homologous amino acid sequence homology of at least 80%, 90% or 95% homology. For example, even if one to several amino acids are substituted, added or deleted in the amino acid sequence represented by SEQ ID NO: 2, 4, or 6, the peptide does not induce a change in the three-dimensional structure of the T cell receptor, The peptide having the function equivalent to the peptide of the present invention. Such amino acid sequence is also included in the scope of the present invention.

The alpha-chain and beta-chain variable domains of the T cell receptor are the most diverse regions and correspond to those most strongly associated with the specificity of antigen recognition, so that the peptide sequences of the present invention are specific for MART-1 specific cytotoxic T- And thus, if any T cell has the peptide sequence of the present invention, the T cell can be regarded as a T cell with MART-1 specificity. Therefore, the present invention may be utilized as a specific diagnostic marker for cancer or tumor in which MART-1 is expressed.

Homogeneity comparisons are performed with a visual comparison program or a sequence comparison program that is more readily available. This commercially available computer program can calculate% homology between two or more sequences. For example, examples of other software that can perform sequence comparisons include the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410 ) And the GENEWORKS suite of comparison tools. Also, deleted, inserted or substituted sequences of amino acid residues that cause silent changes in existing sequences can be considered functionally equivalent. Intentional amino acid substitutions are based on the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of a residue as long as the second binding activity of the substrate is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid; Positively charged amino acids include lysine and arginine; And amino acids with non-electroactive headgroups of similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine. The present invention also includes homologous substitutions that are substitutions between amino acids that are basic for basic, acid for acid, and polar for polar. Non-homologous substitutions also relate to the inclusion of unnatural amino acids such as ornithine, diaminobutyric acid ori, noririn ornithine, pyriylalanine, thienylalanine, naphthylalanine and phenylglycine, But from other classes of alternative residues.

In another aspect, the invention relates to a MART-1 specific T cell receptor comprising the polypeptide as a variable domain of alpha and / or beta chains.

In the present invention, the T cell receptor is characterized in that the polypeptide represented by the amino acid sequence of SEQ ID NO: 1, 3 or 5 forms all or part of the variable domain of the alpha chain of the T cell receptor, Preferably, the polypeptide represented by the amino acid sequence of SEQ ID NO: 6 forms all or part of the variable domain of the beta chain of the T cell receptor, but is not limited thereto. For example, the alpha chain and the beta chain may express a sequence derived from the alpha chain mutation portion in the beta chain within a range that exhibits a similar effect, or may express a sequence derived from the beta chain mutation portion in the alpha chain to produce T Cell receptors.

Specifically, the T cell receptor comprises the polypeptide represented by the amino acid sequence of SEQ ID NO: 1 as all or a part of the variable domain of the alpha chain, the polypeptide represented by the amino acid sequence of SEQ ID NO: 2 as the variable domain of the beta chain Or the polypeptide represented by the amino acid sequence of SEQ ID NO: 3 is included in all or part of the variable domain of the alpha chain, and the polypeptide represented by the amino acid sequence of SEQ ID NO: 4 is included in the entirety or part of the variable domain of the beta chain Or a polypeptide represented by the amino acid sequence of SEQ ID NO: 5 as all or a part of the variable domain of the alpha chain and the polypeptide represented by the amino acid sequence of SEQ ID NO: 6 as the whole or part of the variable domain of the beta chain .

In the present invention, the term "T-cell receptor or TCR" is a molecule found on the surface of a T cell that is responsible for recognizing an antigen that binds to an MHC molecule and includes a molecule capable of recognizing the peptide when presented by the MHC molecule It is used in a customary sense. The molecule is either a heterodimer of two chains of alpha and beta (or alternatively gamma and delta) or a signal chain TCR construct. The TCR of the present invention is a hybrid TCR comprising a sequence derived from another species. For example, the mouse TCR may be more effectively expressed in human T cells than human TCRs, so the TCR may comprise a human variable region and a murine constant region.

The T cell receptor of the present invention recognizes all or part of HLA-A2 (human leukocyte antigen-A2) and ELAGIGILTV which is the 26th to 35th amino acid sequence of MART-1. T cell receptors may recognize portions of the sequence with one or more (e.g., up to 5) upstream or downstream amino acids.

In another aspect, the present invention provides a nucleic acid encoding all or a part of the variable domain of the alpha chain of the MART-1 specific T cell receptor represented by the nucleic acid sequence of SEQ ID NO: 7, 9 or 11; Or a nucleic acid encoding all or part of the variable domain of the beta chain of the MART-1 specific T cell receptor, represented by the nucleic acid sequence of SEQ ID NO: 8, 10 or 12.

The nucleic acid may comprise a mutant sequence encoding a mutant having an amino acid sequence homology of 80%, 90% or 95% or more with respect to the amino acid sequence of SEQ ID NO: 1 to 6, respectively. This is because, for example, if the peptide encoded by the nucleic acid does not induce a change in the three-dimensional structure of the T cell receptor of the present invention and these peptides have functions equivalent to those of the original peptide, And are included in the scope of the invention.

The mutated sequence may be added, deleted, or substituted with one or more bases. If the mutations include additions or deletions, they allow for balance (i.e., additions to each deletion) three times or so that the mutation does not cause a frame-shift to translation of the remainder of the sequence. Some or all of the mutations are "silent " in a send that does not affect the encoded protein sequence due to the degeneracy of the protein code. Some or all of the mutations cause conservative amino acid substitutions as described above. Variations can be concentrated or distributed in one or more regions, such as regions that encode constant regions, linkers, or regions of the framework of the alpha or beta chain.

The mutated sequence should be capable of encoding all or part of the sequence that binds the ELAGIGILTV: MHC complex.

The nucleic acid sequence is double or single strand, and is RNA or DNA. The nucleic acid sequence may be codon-optimized. The usage of certain codons differs depending on the cell. This codon bias corresponds to the bias of the relative frequency of the specific tRNAs in the cell type. By varying the codons of the sequences they are regulated in agreement with the relative frequency of the corresponding tRNAs, which makes it possible to increase expression.

Many viruses, including HIV and other lentiviruses, use a large number of rare codons and are used as packaging components in mammalian producer cells by altering their corresponding to the commonly used mammalian codons, Lt; / RTI > can be achieved. The codon usage tables are also known in the art for various other organisms as well as for mammalian cells. In addition, codon optimization is associated with the removal of mRNA instability motifs and complex splice sites.

In the case of producing the peptide or nucleic acid according to the present invention, genetic engineering techniques and / or chemical synthesis methods can be used. For example, the nucleic acid can be isolated from cells or chemically synthesized. The nucleic acid may be amplified by a known method such as PCR. For example, a nucleic acid (which may be amplified to an appropriate amount by a publicly known method) is assembled into a suitable cell and introduced into an appropriate cell, or introduced into a suitable cell by gene gun, electroporation, etc., The nucleic acid and the peptide of the present invention can be obtained by culturing and expressing the nucleic acid-introduced cell. Usable vectors and cells, gene introduction conditions, culture conditions, methods for isolating genes or peptides, etc. are well known to those skilled in the art and can be appropriately selected and used. Chemical synthesis may also be used to prepare the nucleic acids and peptides of the present invention. These chemical synthesis methods are well known, and examples of chemical synthesis methods of genes include solid phase synthesis of DNA by the amidite method and the 1-4 phosphonate method, and the chemical synthesis method of peptides includes the Fmoc method and the like.

In another aspect, the present invention relates to a recombinant vector comprising the nucleic acid sequence.

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying other nucleic acids linked thereto. One type of vector is a "plasmid ", which refers to a circular double-stranded DNA loop that can ligate additional DNA segments. Another type of vector is a viral vector that can ligate additional DNA segments into the viral genome. Some vectors may self-replicate in this host cell when introduced into the host cell (e. G., A bacterial vector having the bacterial origin of replication and an episomal mammalian vector). Other vectors (e. G., Non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell and replicated with the host genome. In addition, some vectors may direct expression of the genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "recombinant vectors", "expression vectors"). In general, "plasmid" and "vector" may be used interchangeably, because expression vectors useful in recombinant DNA techniques are most commonly the vector types in which plasmids are most commonly used in the form of plasmids. However, the present invention is not limited to the use of other such virus vectors (e.g., adenoviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, baculovirus vectors) Lt; / RTI > expression vector.

The retroviruses are RNA viruses having a life cycle different from that of fungal viruses. In this regard, retroviruses are infectious entities that replicate through DNA intermediates. When a retrovirus infects cells, the genome is converted to DNA by reverse transcriptase. DNA copies are used as templates for the generation of viral encoded proteins essential for assembly of new RNA genomes and infectious viral particles. There are many retroviruses, such as murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), murine breast tumor virus (MMTV), Rous sarcoma virus (RSV) (A-MLV), alveolar bone marrow (A-MLV), malignant hematopoietic progenitor virus (FBS), malignant rodent leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV) All other retroviruses, including cytomegalovirus-29 (MC29) and avian dengue virus (AEV) and lentivirus. A detailed list of retroviruses is described in Coffin et al. (&Quot; Retroviruses " 1997 Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).

In addition, lentiviruses belong to retroviruses, but they can infect both differentiated and non-differentiated cells (Lewis et al (1992) EMBO J. 3053-3058).

To efficiently infect human cells, the viral particles are encased in amphotropic envelopes or gibbon ape leukemia virus envelopes. Increased CD3 molecule supply increases TCR expression in transgenic cells. Thus, the vector may comprise a gene for CD3-gamma, CD3-delta, CD3-epsilon and / or CD3-zeta, and in one embodiment contains only the gene for CD3-zeta. Genes are linked by self-cleavage sequences such as 2A self-cleavage sequences. In addition, one or more separate vectors provide for encoding the CD3 gene for co-transfer with a TCR-encoding vector.

In another aspect, the present invention relates to a cell in which the MART-1 specific T cell receptor is expressed on a surface or a cell into which the recombinant vector is introduced. The cell may be a cytotoxic T cell (CTL), an inhibitory T cell or a natural killer cell (NK cell), but is not limited thereto.

In the present invention, in the production of a cell in which the MART-1 specific T cell receptor is expressed on the surface, the polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 2 is introduced into the alpha chain or the beta chain of the T cell receptor Or may be introduced together. In addition, in the production of a cell in which another MART-1 specific T cell receptor is expressed on the surface, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 is introduced into the alpha chain or the beta chain of the T cell receptor, And in the production of a cell in which another MART-1 specific T cell receptor is expressed on the surface, the polypeptide of SEQ ID NO: 5 or the polypeptide of SEQ ID NO: 6 binds to the alpha or beta chain of the T cell receptor Respectively, or may be introduced together. When the polypeptide is introduced into the T cell receptor, it may be possible to induce the improvement of the therapeutic effect by appropriately combining it with a polypeptide which forms a variable domain of another kind of alpha chain or beta chain. In order to introduce the polypeptide into the alpha or beta chain of the T cell receptor, the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 may be incorporated into a recombinant vector and introduced into appropriate cells. The nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 may be incorporated into a recombinant vector and introduced into a suitable cell. On the other hand, it can be introduced into appropriate cells by a genetic gun or electroporation, and other gene introduction conditions and cell culture conditions, etc., can be appropriately selected by those skilled in the art.

As a method for introducing the recombinant vector, there may be exemplified a method of "transformation" or "transfection". In addition, any method of introducing DNA into cells in the art may be utilized. The terms "transformation" and "transfection" refer to the external application of purified recombinant DNA from another cell of a different genotype, which leads to the uptake and integration of the cell's genomic subject cell into the genome Lt; RTI ID = 0.0 > genomic < / RTI > The terms "transformed" or "transfected" are used interchangeably herein. For example, a T cell may be transfected with a DNA sequence encoding the TCR of modified or high affinity described herein before the T cell treatment of the adoptive T cell.

The cells express the T-cell receptor of the present invention. The cell may be a T-cell. Preferably, the cell may be derived from a T-cell isolated from the subject. T-cells are part of a mixed population of cells isolated from subjects such as a population of peripheral blood lymphocytes (PBL). T cells in the PBL population are activated by methods known in the art, such as using anti-CD3 and CD28 antibodies.

The T-cells are CD4 + T helper cells or CD8 + cytotoxic T cells. Cells are present in a mixed population of CD4 + T helper cells / CD8 + cytotoxic T cells. For example, polyclonal activation using an anti-CD3 antibody that selectively binds anti-CD28 antibody results in the proliferation of CD4 + and CD8 + T cells but also the proliferation of CD4 + 25 + regulatory T-cells. It is not desirable that TCR gene transfer to regulatory T cells inhibit gene-modified cytotoxicity and anti-viral activity of T helper cells. Therefore, the CD4 + CD25 + population is greatly reduced prior to TCR gene transfer.

In another aspect, the present invention relates to a composition for treating a MART-1 related disease comprising the cell as an active ingredient, and specifically, the composition may be a cell therapy agent. The present invention also relates to a therapeutic agent for the prophylaxis or treatment of a MART-1-related disease comprising the recombinant vector as an active ingredient.

According to the present invention, the cells into which the recombinant vector has been introduced can be cultured in vitro to obtain a large amount of MART-1-specific cytotoxic T cells. And by introducing it into a patient of a MART-1-related disease such as cancer or a tumor, cancer or tumor cells expressing MART-1 can be killed, thereby treating cancer or tumor.

In the present invention, the MART-1 related disease may be solid cancer or a tumor, and the solid cancer may be skin cancer or melanoma.

The above-mentioned cancer or tumor treatment may be combined with other cancer treatment such as an anti-cancer agent or radiation therapy. The cancer or tumor treatment can be applied to a wide range of cancers or tumors, and they are exemplified above, but are not limited thereto.

In addition, depending on the type of patient or cancer, the amino acid sequence that is effective for treatment may be different, and the effective amount to be introduced according to the individual terms can be determined.

The composition for preventing or treating MART-1-related diseases of the present invention may further comprise a pharmaceutically acceptable carrier.

Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical field and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances Water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide But are not limited to, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool.

In addition, the composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.

The composition according to the present invention can be prepared as an aqueous solution for parenteral administration, and preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

The composition of the present invention may be administered systemically or locally, and may be formulated into a formulation suitable for such administration by known techniques. For example, upon oral administration, it may be admixed with an inert diluent or edible carrier, sealed in a hard or soft gelatin capsule, or pressed into tablets. For oral administration, the active compound may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

Various formulations for injection, parenteral administration and the like can be prepared according to techniques known in the art or commonly used techniques. Formulations may be administered by intravenous infusion, subcutaneous infusion, intramuscular injection, intraperitoneal injection, transdermal administration, and the like

A suitable dose of the composition of the present invention can be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient have. For example, the dose of the composition of the present invention may be 5 × 10 ⁴ to 5 × 10 ⁹ cells when administered in the form of T cells to an adult, more preferably, the TCR expression level and morbidity 5 x 10 < ⁷ > to 5 x 10 < ⁸ > cells may be included. The dose may be 5 × 10 ⁵ to 1.5 × 10 ⁶ cells / kg per day, preferably 1.5 × 10 ⁶ to 5 × 10 ⁷ cells / kg per day, once to several times per day.

The present invention also provides the use of said recombinant vector or said cell for the preparation of a therapeutic agent for use in the prevention or treatment of MART-1 related diseases.

In the present invention, the term " prevention " relates to averting, delaying, impeding or hindering the disease.

In the present invention, the term " treatment " relates to caring for a subject afflicted with a disease in order to ameliorate, cure, or reduce the symptoms of the disease or to reduce or stop the progression of the disease.

The present invention also provides a method for treating a MART-1 related disease comprising administering to said individual a pharmaceutically effective amount of said vector or a composition for preventing or treating a MART-1 related disease comprising said cell.

A vector comprising the TCR of the present invention or a cell transformed with said vector (such as a T cell) can be used for the treatment of MART-1 related diseases through adoptive transfer. To do this, T cells are isolated so that genetically modified cells from the subject are transferred to adoption. The genetically modified cells are produced by selectively activating the subject's immunotherapy with T-cells, TCR gene transduction and adoptive transfer of TCR-transduced cells separated from the subject. The T-cells isolated from the subject are separated from different subjects who are homozygous. The cells are separated from donor subjects. For example, if a subject is or has received a solid organ transplant, the cell is derived from the subject from which the solid organ is derived.

Since the pharmaceutical compositions and administration methods used in the MART-1 related disease treatment method have been described above, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.

On the other hand, individuals to which the composition for preventing or treating MART-1-related diseases can be administered include humans and all animals. For example, it may be an animal such as a dog, a cat, or a mouse.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1: MART-1 specific TCR  T cell excavation

1-1. Monocyte-derived Dendritic cell  Ready

Blood from healthy donors of HLA-A2 + was mixed with PBS (2% FBS) at a ratio of 1: 1. 16 ml of lymphoprep (stemcell ^TM , USA) was placed in a 50 ml tube and slowly diluted blood was placed on it. Centrifuged at 400 xg for 30 minutes with a centrifuge (Hanil 514R, Korea) and stopped without forced deceleration. The top layer (plasma) was removed and the peripheral blood mononuclear cell (PBMC) layer was transferred to a new tube.

PBS (2% FBS) was added to the peripheral blood mononuclear cells (PBMC) to adjust the final volume to 50 ml. After centrifugation at 400 xg for 10 minutes by centrifugation, the supernatant was removed and the cells were washed with 30 ml of PBS (2% FBS). After further centrifugation at 400 xg for 10 minutes by centrifugation, the supernatant was removed and resuspended in 2 ml of erythrocyte lysis buffer (Sigma) and incubated for 3 minutes. 30 ml of PBS (2% FBS) was added and centrifuged at 400 xg for 10 minutes. After the supernatant was removed, RPMI1640 (welgene, Korea) and 1% human AB serum (Corning, USA) were added to the culture medium to dilute the cells to 1.0 × 10 ⁷ / ml. Cells were added to the 6-well plate in an amount of 2 ml (2.0 x 10 ⁷ ) per well and cultured for 3 hours. Cells that were not attached to the plate were removed by pipetting and then used to prepare naive T cells. 3 ml of dendritic cell medium (cellgenix), 1% human AB serum (corning), IL-4 (10 ng / ml, peprotech) and GM-CSF (800 U / ml, peprotech) And incubated for 2 days at 37 ° C in a CO ₂ incubator. After 2 days, 1.5 ml of dendritic cell medium (IL-4 (10 ng / ml), GM-CSF (1600 U / ml)) was added and cultured in a CO ₂ incubator at 37 ° C for 24 hours. All of the cells differentiated into immature DCs were collected and counted. (10 μg / ml), GM-CSF (800 U / ml), LPS (10 ng / ml), IFN-γ (100 U / ml) and MART- 1 peptide (ELAGIGILTV, 2.5 μg / ml) to dilute the cells to 0.5 × 10 ⁶ / ml, and cultured in a CO ₂ incubator at 37 ° C. for 16 hours to prepare mature dendritic cells.

1-2. Naive CD8 + T cell preparation

(Miltenyi biotec Cat # 130-096-495, Germany) and naive T cell isolation beads (miltenyi biotec Cat # 130-046-001, 130-bp) using PBS / 1% human AB serum as buffer. Naive CD8 + T cells were prepared according to the standard method described in the manufacturer's manual, 092-073, Germany. Culture medium to the cells (dendritic medium, 5% human AB serum, IL-7 (5ng / ml)) into a 3.0 × 10 ⁶ / ml by dilution with Pipette 2ml per well in 6-well plates and 37 ℃, CO ₂ incubator Lt; / RTI >

1-3. Dendritic cells  T cell Symbiosis culture

The prepared mature dendritic cells were diluted to 5.0 × 10 ⁵ / ml with dendritic medium (5% human AB serum), and the prepared naive CD8 + T cells were cultured in dendritic medium (5% human AB serum, IL-7 (5 ng / ml), IL-21 (60 ng / ml)) was added thereto and diluted to 2.0 × 10 ⁶ / ml. Dendritic cells and T cells were mixed at the same volume (1: 1), and then 500 쨉 l per well was dispensed into a 48-well plate and cultured at 37 째 C for 72 hours in a CO ₂ incubator.

1-4. T cell proliferation

After 72 hours from the start of the symbiotic culture, 500 μl of culture medium (dendritic medium, 5% human AB serum, IL-15 (10 ng / ml), IL-7 , And cultured in a CO ₂ incubator. After 72 hours, the plate was changed to a 12-well plate and an additional 1 ml of the culture medium (dendritic medium, 5% human AB serum, IL-15 (10 ng / ml), IL- Gt; 37 C < / RTI > in a CO ₂ incubator. After 48 hours, the plate was changed to a 6-well plate and an additional 1 ml of the culture medium (dendritic medium, 5% human AB serum, IL-15 (10 ng / ml), IL- Gt; 37 C < / RTI > in a CO ₂ incubator.

Example  2: Identification of MART-1 specific T cell clones

1 μl of MART-1 tetramer-PE (MBL Cat # TS-0009-1C) per 1.0 × 10 ⁵ T cell clone and 1 μl of anti-CD8 antibody-A488 (eBioscience Cat # 53-0086-42) % FBS / 0.05% sodium azide) and reacted on ice for 30 minutes. The cells were washed twice with 200 완 of buffer and resuspended in final 200 완 buffer. Fluorescence was measured using a flow cytometer (FACSVerse). Three MART-1-specific T cell clones And the results are shown in Fig.

Example  3: Isolation of MART-1 specific T cell clone

Three ensured MART-1 specific T cell clones were cultured by dilution. First, cytotoxic T lymphocytes (CTL) with peptide specific spots were diluted to 1 cell / well or 2-3 cells / well and cultured in a 96-well plate. The cells were incubated with mitimycin C-treated feeder cells (allo PBMC, LCL), 1% phytohaemagglutinin, and 100 IU / ml IL-2. After 3 to 4 weeks, the wells in which T-cell proliferation was observed were identified and the presence of peptide-specific cells was confirmed by IFN-γ ELIspot using the cells of these wells. The ELISPOT proceeded as follows. Anti-human IFN-y antibody (Mabtech Cat # 3420-3-250) was diluted 1000-fold with PBS and added to a 96-well ELISpot plate (Millipore Cat # MSIPN4510) in an amount of 100 占 퐇 per well and coated overnight at 4 占 폚. PBS (5% FBS) was added to the coated plate in an amount of 200 μl per well, and washing was repeated four times. 50 [mu] l of cultured T cells were plated in an ELISpot plate coated with antibody with 50 [mu] l of T2 or unbound T2 cells bound to MART-1 peptide, respectively, and cultured for 24 hours. 200 [mu] l of PBS (0.5% FBS) per well was added to the plate, and washing was repeated 6 times. Biotin-conjugated anti-IFN- [gamma] antibody (Mabtech Cat # 3420-6-250) was diluted 1,000-fold with PBS and 100 [mu] l per well was added to the plate and reacted at room temperature for 90 minutes. 200 [mu] l of PBS (0.5% FBS) per well was added to the plate and washing was repeated four times. Streptavidin-alkaline phosphatase (Mabtech Cat # 3310-8) was diluted 2000-fold with PBS, and 100 쨉 l per well was added to the plate, followed by reaction at room temperature for 60 minutes. 200 [mu] l of PBS per well was added to the plate, and washing was repeated four times. NBT / BCIP (Thermo Cat # 34042) was added to each well in an amount of 100 占 퐇 and then allowed to react until color development. Identified T-cell clones were used for the experiment by expanding the cell number through an additional expansion process.

Example  4: Identification of MART-1 specific T cell clone and expression on T cell surface

Total RNA was purified from pooled T cell clones. The purified total RNA was used as a template and random hexamer (dN6) was inserted into the primer and cDNA was synthesized by reverse transcriptase reaction. TRAV was amplified by PCR with 25 forward primers and one reverse primer (TRACrev), which were able to amplify most of the known TRAV genes using the synthesized cDNA as a template. In the other tube, TRBV was amplified by PCR with 17 forward primers and 1 reverse primer (TRBCrev), which can amplify most of the known TRBV genes using cDNA as a template. The primers used in the reaction and the reaction conditions are shown in Tables 1 to 3, respectively, and detailed experimental methods were referred to the literature (I Boria et al., (2008) BMC Immunology 9:50). The PCR reaction was carried out at 95 ° C for 3 minutes, 30 cycles at 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 minutes, and 72 ° C for 3 minutes.

cDNA synthesis Stock Vol (ul) RNA 40 RTase 200u / ul 2 RNase Inhibitor 40 u / ul 0.8 5x RT buffer 16 dNTP 10 mM 4 dN6 100p / ul 3.2 DTT 0.1 uM 4 NFW 10 Total 80

PCR Stock Vol (ul) cDNA 10 Primer for Mix 4p / ul 5 Primer Crev 4p / ul 5 dNTP 10 mM 2 Bufffer 10X 10 Hypersense Taq 2.5 u / ul One NFW 67 Total 100

The amplified product was electrophoresed with 1.5% agarose gel and stained with EtBr. As a result, 360 bp and 400 bp bands were confirmed as shown in FIG. 4. The DNA bands were then extracted from the agarose gel to identify the sequences and used for cloning into TCR expression vectors. The amino acid sequences of the alpha and beta chains of the three T cell receptors identified in the present invention are as follows. An artificial nucleic acid sequence for recombinant TCR expression from each amino acid sequence is also shown below.

SEQ ID NO: 1. Alpha of T cell receptor (MTM 1) Chain  Amino acid sequence

≪

SEQ ID NO: 2. Beta of T cell receptor (MTM 1) Chain  Amino acid sequence

CTI

SEQ ID NO: 3. Alpha of T cell receptor (MTM 2) Chain  Amino acid sequence

≪ RTI ID =

SEQ ID NO: 4. Beta of T cell receptor (MTM 2) Chain  Amino acid sequence

MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRIQGLGSPLHFGNGTRLTVT

SEQ ID NO: 5. Beta of T cell receptor (MTM 3) Chain  Amino acid sequence

≪ RTI ID =

SEQ ID NO: 6. Beta of T cell receptor (MTM 3) Chain  Amino acid sequence

≪

SEQ ID NO: 7. Alpha of T cell receptor (MTM 1) Chain  Nucleic acid sequence

ATGGAGAAGATGCTTGAGTGCGCTTTCATCGTGCTGTGGCTTCAGCTCGGCTGGCTCTCTGGAGAAGACCAAGTGACCCAGTCTCCAGAGGCCCTGCGACTTCAGGAAGGTGAGAGCTCCTCACTGAATTGCAGTTATACCGTTAGCGGCCTTCGGGGACTCTTCTGGTACCGGCAGGATCCCGGAAAGGGTCCCGAGTTTCTGTTCACCCTCTATAGCGCCGGTGAGGAAAAAGAGAAGGAGAGACTGAAAGCAACTCTCACAAAGAAAGAAAGTTTTCTGCACATTACTGCTCCTAAACCTGAGGATTCCGCTACCTATCTGTGCGCGGTGCAGGCCAGAGCAGCCGGGAATAAGCTGACATTTGGAGGCGGAACAAGGGTTCTCGTCAAACCCAAT

SEQ ID NO: 8. Beta of T cell receptor (MTM 1) Chain  Nucleic acid sequence

ATGACCATCCGCCTGCTGTGTTACATGGGCTTCTATTTCCTCGGCGCCGGTCTGATGGAGGCCGATATCTATCAGACCCCAAGGTATCTGGTGATTGGGACAGGTAAAAAGATCACCCTGGAATGCTCCCAGACAATGGGCCACGATAAGATGTATTGGTATCAGCAGGATCCGGGCATGGAGCTCCATCTGATCCACTATTCCTACGGAGTCAATTCTACCGAGAAGGGCGACCTGTCTAGCGAATCAACGGTCAGCAGAATCAGGACCGAGCACTTCCCACTGACCCTCGAAAGTGCTAGGCCCAGCCACACTAGTCAGTATCTCTGTGCATCTTCCGATCATCTGCTGGCCTCCGGGATGGAAACTCAGTATTTTGGGCCGGGAACTAGATTGCTGGTGCTT

SEQ ID NO: 9. Alpha of T cell receptor (MTM 2) Chain  Nucleic acid sequence

ATGAAGTCACTTCGAGTGCTTCTGGTGATTCTCTGGCTTCAACTTAGCTGGGTATGGTCACAGCAGAAGGAAGTTGAACAAAACAGCGGCCCCCTGAGCGTCCCGGAAGGTGCAATAGCCTCTCTCAACTGTACTTATAGCGACCGCGTTTCCCAGTCTTTTTTCTGGTACAGACAGTACTCCGGTAAGAGTCCCGAGCTGATTATGTCCATCTATAGTAATGGCGACAAAGAAGATGGCCGGTTTACAGCTCAACTTAATAAGGCGTCACAATATGTCTCTCTTTTGATCAGGGACTCCCAGCCATCTGATTCTGCTACATATTTGTGCGCCGTTACCAATACGAATGCAGGGAAGAGCACTTTTGGGGACGGGACCACTCTCACTGTTAAACCAAAT

SEQ ID NO: 10. Beta of T cell receptor (MTM 2) Chain  Nucleic acid sequence

ATGGGGATTCGATTGCTGTGTCGGGTTGCCTTTTGCTTCTTGGCGGTGGGTCTTGTAGACGTTAAGGTTACCCAGTCTAGCCGCTACCTGGTAAAACGGACAGGAGAAAAAGTTTTCTTGGAGTGTGTCCAGGATATGGATCACGAGAATATGTTCTGGTACCGACAAGATCCTGGGCTGGGCCTTAGATTGATCTACTTTAGTTACGACGTTAAGATGAAGGAAAAAGGGGACATTCCAGAAGGTTATTCTGTCTCAAGAGAGAAGAAAGAAAGGTTCTCCTTGATTTTGGAAAGTGCAAGTACTAACCAAACATCTATGTACCTGTGCGCTTCCAGAATACAGGGACTGGGGTCCCCACTGCACTTTGGAAACGGAACCAGATTGACAGTCACA

SEQ ID NO: 11. Alpha of T cell receptor (MTM 3) Chain  Nucleic acid sequence

ATGAAATCTCTTAGAGTGCTCTTGGTGATTTTGTGGCTTCAGCTCTCCTGGGTATGGAGTCAACAAAAAGAAGTCGAACAGGACCCTGGCCCACTCTCAGTTCCCGAAGGAGCAATAGTGTCATTGAACTGCACTTATAGTAATTCCGCGTTCCAATACTTTATGTGGTATCGCCAATACTCACGGAAGGGCCCAGAGTTGCTGATGTACACGTATTCCTCAGGAAATAAGGAAGACGGACGATTTACTGCACAGGTGGGCAAATCCTCTAAGTATATCTCTTTGTTCATTAGGGATTCCCAGCCGTCTGATAGTGCTACATATTTGTGCGCTCTGGGAAACAACGCCGGTAATATGTTGACCTTTGGGGGTGGCACCCGCCTTATGGTGAAGCCGCAT

SEQ ID NO: 12. Beta of T cell receptor (MTM 3) Chain  Nucleic acid sequence

ATGGGTTGTCGCTTGCTCTGTTGCGCGGTCCTCTGTCTTTTGGGGGCCGTCCCGATTGATACCGAGGTCACCCAGACTCCAAAACACCTGGTTATGGGGATGACCAATAAAAAATCCCTGAAATGCGAACAACACATGGGTCATCGGGCAATGTATTGGTATAAGCAGAAGGCAAAGAAGCCGCCGGAGTTGATGTTCGTGTACTCCTATGAGAAATTGTCCATAAATGAGTCTGTACCTTCACGATTCTCCCCTGAATGCCCGAACAGTTCCCTGCTGAACCTCCACCTTCACGCCCTGCAGCCTGAGGATTCTGCGCTGTATCTTTGTGCCAGTAGTCCCACAAGTATTGCGGGCGAACAGTACTTCGGGCCAGGAACTAGGCTTACTGTAACA

The pLVX vector (Thermo Scientific, USA) was used as an expression vector for expression of the T cell receptor. The TRAV and TRBV fragments were ligated to the respective constant domains and ligated into a single polypeptide using the 2A peptide system. 2A peptides are about 20 amino acid sequences derived from foot-and-mouth disease viruses. When proteins are expressed, they cut themselves and divide the TCR α / β chain into two, resulting in a TCR α chain and a TCR β chain Lt; / RTI > expression vector to produce the same amount. In addition, the Furin cleavage site (purine cleavage site) and the V5 peptide tag and linker were added to allow for better expression of TCR (see Yang et al., (2008) Gene Therapy 15: 1411-1423) .

Expression vector was introduced into T cells by mRNA electroporation. First, mRNA was prepared as follows using mMESSAGE mMACHINE T7 ultra kit (ambion, Cat # AM1345). The expression vector was linearized with restriction enzyme Eco52I (Thermo, Cat # ER0331) and used as a template. 1 μg of linearized DNA, 10 μl of T7 2X NTP / ARCA, 2 μl of 10 × T7 reaction buffer, 2 μl of T7 enzyme mix, and nuclease-free water to a final volume of 20 μl, mixed well and reacted at 37 ° C for 1 hour. 1 μl of TURBO Dnase was added, mixed well and reacted at 37 ° C for 15 min. After completion of the reaction, 36 μl of nuclease-free water, 20 μl of 5 × E-PAP buffer, 10 μl of 25 mM MnCl 2, 10 μl of ATP solution and 4 μl of E-PAP were added and reacted at 37 ° C for 45 minutes. After completion of the reaction, 100 μl of nuclease-free water was added, 200 μl of phenol / chloroform (bioneer, Cat # C-9017) was added, mixed well and centrifuged at 18000 × g for 5 minutes. After collecting the upper layer, 10 μl of ammonium acetate stop solution and 400 μl of ethanol were added, mixed well and reacted at -20 ° C. for 30 minutes. After centrifugation at 23000 x g for 20 minutes, the supernatant was discarded, 1 ml of 70% ethanol was added, mixed well, and centrifuged at 23,000 x g for 5 minutes. The supernatant was discarded and the mRNA was dissolved well in nuclease-free water. The mRNA was then introduced into T cells using the neon transfection system (thermo) as follows. 1.0 × 10 ⁶ T cells were added to 100 μl T buffer, and 5 μg mRNA was mixed and electroporated at 1600 V, 20 ms and 1 pulse. T cells were added to the culture medium (RPMI1640, 20% FBS, 300 U / ml IL-2) and cultured in a CO2 incubator at 37 ° C for 3 hours. As a result, as shown in FIG. 6, it was confirmed that it was expressed at a level (99%) similar to that of the positive control group DMF5.

Example  5: Assessment of cancer cell killing efficacy of MART-1 specific T cell clone in vitro

Two target cells derived from malignant melanoma expressing MART-1 / HLA-A2 surface were prepared as follows to evaluate cancer cell killing efficacy. The concentration of cancer cells (target cells, UACC62, MeWo) was adjusted to 1.0 × 10 ⁷ cells / mL, and then 10 μM calcein AM red-orange was added. After incubation for 30 min in a cell incubator (37 ° C, 5% CO ₂ ), the cells were stained with calcein AM dye and washed three times with the culture medium to remove remaining calcein AM dye (centrifugation conditions: 200xg , 3 minutes, room temperature). The concentration of the stained cancer cells was adjusted to 1.0 × 10 ⁵ cells / mL using a complete RPMI medium containing 2.5 mM probenecid, and 100 μl (10,000 cells) per 96-well plate was dispensed. For the preparation of effector cells, PBMC or human primary T cells in culture were suspended in a complete RPMI medium containing 2.5 mM probenecid. In this case, the ratio of the number of working cells to the number of target cells (E: T) was set to 80: 1, 40: 1, 20: 1, 10: 1, 5: 1, 2.5: 1, 1.25: 1, and then the number of required working cells compared to the target cells was calculated and the concentration was determined. The cell death was performed by mixing 100 μl of calcein AM dye labeled target cells and 100 μl of working cells in a total volume of 200 μl for 4 hours. The positive control (maximum release) was set on a 96-well plate. The target cells were treated with 1% Triton X-100 in a well-dispensed well and stored for 10 minutes in a cell incubator. Lt; / RTI >

After completion of the apoptosis, the calcein AM dye flowing from the target cells killed by the working cells was measured at 577/600 nm (Ex / Em) using a fluorescent plate reader. To minimize interference from non-apoptotic cells, 50-100 μl of supernatant was taken after centrifugation and transferred to a new plate and measured. In this case, centrifugation was carried out at 400 xg for 3 minutes at room temperature.

Negative control (spontaneous release): The value measured in the supernatant of the control group cultured only in the target cell, positive control (maximum release): Target cells were incubated with 1% triton X-100 for 10 min to induce apoptosis of all cells. Then, the values measured in the supernatant were defined and specific lysis of target cells by the working cells was performed. Was calculated by the following equation.

((test sample) - (spontaneous release)) / ((maximum release) - (spontaneous release)) x 100

As a result of evaluating the killing efficacy of two target cells derived from malignant melanoma expressing MART-1 / HLA-A2 surface on cancer cells, as shown in Fig. 7, the number of working cells: the number of cells of target cells The ratio of MART-1-specific cells reached 30%, indicating that the expression of the recombinant T cell receptor according to the present invention resulted in significant cytotoxicity to cancer cells there was. In addition, as shown in FIG. 8, MART-1-specific cell death was observed in all two cancer cell lines (UACC62, MeWo) higher than that of DMF5, which is a positive control, as compared with DMF5, which is a positive control.

Example  6: Assessment of cancer cell killing activity of MART-1 specific T cell clone using animal model

Tumor cells (UACC62) were injected subcutaneously at a dose of 4 × 10 ⁵ cells / mice using a 1 cc syringe (26 gauge) at the site between the right clavicular and chest wall of NSG mice. On the 14th day after tumor transplantation, when the tumor size average was 50 to 60 mm ³ , the recombinant T cell receptor-expressing T cell treatment agent according to the present invention was administered at a concentration of 1 × 10 ⁷ cells / , IL-2 was also injected through the abdominal cavity at a concentration of 1 × 10 ⁵ IU / 100 μl on the day of treatment.

Evaluation of cancer cell killing effect of MART-1 specific T cell clone using animal model t-Test result for statistical significance evaluation Group
(n = 3) Δt = Tumor weight (mg) Day 0 2 4 7 9 11 15 Negative control group 0.0
± 0.0 72.5
± 3.2 191.1
± 8.1 402.4
± 20.1 605.1
± 34.3 902.3
± 44.5 2146.5
± 65.9 Positive control group DMF5 0.0
± 0.0 61.3
± 3.3
* 160.2
± 8.4
* 329.6
± 18.5
** 494.3
± 30.7
* 753.2
± 39.7
* 1941.2
± 51.0
* 15.5% 16.2% 18.1% 18.3% 16.5% 9.6% Experimental group MTM1 0.0
± 0.0 59.2
± 4.3
* 154.3
± 12.7
* 306.8
± 16.5
** 462.9
± 31.0
** 714.3
± 61.0
* 1864.5
± 100.0
* 18.3% 19.3% 23.8% 23.5% 20.8% 13.1%

* P <0.05, ** p < 0.01 (vs negative ^{control), † △ t = Vt -} Vo, Vt (Measurement of the tumor volume), Vo (Initial tumor volume), ‡ Inhibition Rate (%, vs negative control)

As a result, as shown in FIG. 9 and Table 4, it was confirmed that the tumor size was decreased by 24% compared with the negative control in the test group administered with the T cell treatment agent, and showed an excellent tumor size reduction compared to the positive control group, DMF5 .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Medytox Inc. <120> MART-1 Specific T Cell Receptor and Use Thereof <130> P16-B258 <160> 12 <170> KoPatentin 3.0 <210> 1 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Screened Amino Acid Sequence of Alpha Chain of T Cell          Receptor (MTM1) <400> 1 Met Glu Lys Met Leu Glu Cys Ala Phe Ile Val Leu Trp Leu Gln Leu   1 5 10 15 Gly Trp Leu Ser Gly Glu Asp Gln Val Thr Gln Ser Pro Glu Ala Leu              20 25 30 Arg Leu Gln Glu Gly Glu Ser Ser Ser Leu Asn Cys Ser Tyr Thr Val          35 40 45 Ser Gly Leu Arg Gly Leu Phe Trp Tyr Arg Gln Asp Pro Gly Lys Gly      50 55 60 Pro Glu Phe Leu Phe Thr Leu Tyr Ser Ala Gly Glu Glu Lys Glu Lys  65 70 75 80 Glu Arg Leu Lys Ala Thr Leu Thr Lys Lys Glu Ser Phe Leu His Ile                  85 90 95 Thr Ala Pro Lys Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln             100 105 110 Ala Arg Ala Ala Gly Asn Lys Leu Thr Phe Gly Gly Gly Thr Arg Val         115 120 125 Leu Val Lys Pro Asn     130 <210> 2 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> Screened Amino Acid Sequence of Beta Chain of T Cell          Receptor (MTM1) <400> 2 Met Thr Ile Arg Leu Leu Cys Tyr Met Gly Phe Tyr Phe Leu Gly Ala   1 5 10 15 Gly Leu Met Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile              20 25 30 Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His          35 40 45 Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu      50 55 60 Ile His Tyr Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser  65 70 75 80 Ser Glu Ser Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr                  85 90 95 Leu Glu Ser Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser             100 105 110 Ser Asp His Leu Leu Ala Ser Gly Met Glu Thr Gln Tyr Phe Gly Pro         115 120 125 Gly Thr Arg Leu Leu Val Leu     130 135 <210> 3 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Screened Amino Acid Sequence of Alpha Chain of T Cell          Receptor (MTM2) <400> 3 Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser   1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu              20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp          35 40 45 Arg Val Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser      50 55 60 Pro Glu Leu Ile Met Ser Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly  65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu                  85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val             100 105 110 Thr Asn Thr Asn Aly Gly Lys Ser Thr Phe Gly Asp Gly Thr Thr Leu         115 120 125 Thr Val Lys Pro Asn     130 <210> 4 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Screened Amino Acid Sequence of Beta Chain of T Cell          Receptor (MTM2) <400> 4 Met Gly Ile Arg Leu Leu Cys Arg Val Ala Phe Cys Phe Leu Ala Val   1 5 10 15 Gly Leu Val Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys              20 25 30 Arg Thr Gly Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His          35 40 45 Glu Asn Met Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu      50 55 60 Ile Tyr Phe Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro  65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile                  85 90 95 Leu Glu Ser Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser             100 105 110 Arg Ile Gln Gly Leu Gly Ser Pro Leu His Phe Gly Asn Gly Thr Arg         115 120 125 Leu Thr Val Thr     130 <210> 5 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Screened Amino Acid Sequence of Alpha Chain of T Cell          Receptor (MTM3) <400> 5 Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser   1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu              20 25 30 Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn          35 40 45 Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly      50 55 60 Pro Glu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly  65 70 75 80 Arg Phe Thr Ala Gln Val Gly Lys Ser Ser Lys Tyr Ile Ser Leu Phe                  85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Leu             100 105 110 Gly Asn Asn Aly Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu         115 120 125 Met Val Lys Pro His     130 <210> 6 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Screened Amino Acid Sequence of Beta Chain of T Cell          Receptor (MTM3) <400> 6 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala   1 5 10 15 Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met              20 25 30 Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His          35 40 45 Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu      50 55 60 Met Phe Val Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro  65 70 75 80 Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His                  85 90 95 Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser             100 105 110 Ser Pro Thr Ser Ile Ala Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg         115 120 125 Leu Thr Val Thr     130 <210> 7 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Screened Nucleic Acid Sequence of Alpha Chain of T Cell          Receptor (MTM1) <400> 7 atggagaaga tgcttgagtg cgctttcatc gtgctgtggc ttcagctcgg ctggctctct 60 ggagaagacc aagtgaccca gtctccagag gccctgcgac ttcaggaagg tgagagctcc 120 tcactgaatt gcagttatac cgttagcggc cttcggggac tcttctggta ccggcaggat 180 cccggaaagg gtcccgagtt tctgttcacc ctctatagcg ccggtgagga aaaagagaag 240 gagagactga aagcaactct cacaaagaaa gaaagttttc tgcacattac tgctcctaaa 300 cctgaggatt ccgctaccta tctgtgcgcg gtgcaggcca gagcagccgg gaataagctg 360 acatttggag gcggaacaag ggttctcgtc aaacccaat 399 <210> 8 <211> 405 <212> DNA <213> Artificial Sequence <220> <223> Screened Nucleic Acid Sequence of Beta Chain of T Cell          Receptor (MTM1) <400> 8 atgaccatcc gcctgctgtg ttacatgggc ttctatttcc tcggcgccgg tctgatggag 60 gccgatatct atcagacccc aaggtatctg gtgattggga caggtaaaaa gatcaccctg 120 gaatgctccc agacaatggg ccacgataag atgtattggt atcagcagga tccgggcatg 180 gagctccatc tgatccacta ttcctacgga gtcaattcta ccgagaaggg cgacctgtct 240 agcgaatcaa cggtcagcag aatcaggacc gagcacttcc cactgaccct cgaaagtgct 300 aggcccagcc acactagtca gtatctctgt gcatcttccg atcatctgct ggcctccggg 360 atggaaactc agtattttgg gccgggaact agattgctgg tgctt 405 <210> 9 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Screened Nucleic Acid Sequence of Alpha Chain of T Cell          Receptor (MTM2) <400> 9 atgaagtcac ttcgagtgct tctggtgatt ctctggcttc aacttagctg ggtatggtca 60 cagcagaagg aagttgaaca aaacagcggc cccctgagcg tcccggaagg tgcaatagcc 120 tctctcaact gtacttatag cgaccgcgtt tcccagtctt ttttctggta cagacagtac 180 tccggtaaga gtcccgagct gattatgtcc atctatagta atggcgacaa agaagatggc 240 cggtttacag ctcaacttaa taaggcgtca caatatgtct ctcttttgat cagggactcc 300 cagccatctg attctgctac atatttgtgc gccgttacca atacgaatgc agggaagagc 360 acttttgggg acgggaccac tctcactgtt aaaccaaat 399 <210> 10 <211> 396 <212> DNA <213> Artificial Sequence <220> <223> Screened Nucleic Acid Sequence of Beta Chain of T Cell          Receptor (MTM2) <400> 10 atggggattc gattgctgtg tcgggttgcc ttttgcttct tggcggtggg tcttgtagac 60 gttaaggtta cccagtctag ccgctacctg gtaaaacgga caggagaaaa agttttcttg 120 gagtgtgtcc aggatatgga tcacgagaat atgttctggt accgacaaga tcctgggctg 180 ggccttagat tgatctactt tagttacgac gttaagatga aggaaaaagg ggacattcca 240 gaaggttatt ctgtctcaag agagaagaaa gaaaggttct ccttgatttt ggaaagtgca 300 agtactaacc aaacatctat gtacctgtgc gcttccagaa tacagggact ggggtcccca 360 ctgcactttg gaaacggaac cagattgaca gtcaca 396 <210> 11 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> Screened Nucleic Acid Sequence of Alpha Chain of T Cell          Receptor (MTM3) <400> 11 atgaaatctc ttagagtgct cttggtgatt ttgtggcttc agctctcctg ggtatggagt 60 caacaaaaag aagtcgaaca ggaccctggc ccactctcag ttcccgaagg agcaatagtg 120 tcattgaact gcacttatag taattccgcg ttccaatact ttatgtggta tcgccaatac 180 tcacggaagg gcccagagtt gctgatgtac acgtattcct caggaaataa ggaagacgga 240 cgatttactg cacaggtggg caaatcctct aagtatatct ctttgttcat tagggattcc 300 cagccgtctg atagtgctac atatttgtgc gctctgggaa acaacgccgg taatatgttg 360 acctttgggg gtggcacccg ccttatggtg aagccgcat 399 <210> 12 <211> 396 <212> DNA <213> Artificial Sequence <220> <223> Screened Nucleic Acid Sequence of Beta Chain of T Cell          Receptor (MTM3) <400> 12 atgggttgtc gcttgctctg ttgcgcggtc ctctgtcttt tgggggccgt cccgattgat 60 accgaggtca cccagactcc aaaacacctg gttatgggga tgaccaataa aaaatccctg 120 aaatgcgaac aacacatggg tcatcgggca atgtattggt ataagcagaa ggcaaagaag 180 ccgccggagt tgatgttcgt gtactcctat gagaaattgt ccataaatga gtctgtacct 240 tcacgattct cccctgaatg cccgaacagt tccctgctga acctccacct tcacgccctg 300 cagcctgagg attctgcgct gtatctttgt gccagtagtc ccacaagtat tgcgggcgaa 360 cagtacttcg ggccaggaac taggcttact gtaaca 396

Claims (17)

A polypeptide comprising all or part of the variable domain of the alpha or beta chain of the MART-1 specific T cell receptor, represented by the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6. Comprising a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 in whole or in part of the variable domain of the alpha chain and a polypeptide represented by the amino acid sequence of SEQ ID NO: 2 as all or part of the variable domain of the beta chain, 1 specific T cell receptor. A polypeptide represented by the amino acid sequence of SEQ ID NO: 3 as all or part of the variable domain of the alpha chain and a polypeptide represented by the amino acid sequence of SEQ ID NO: 4 as all or part of the variable domain of the beta chain. 1 specific T cell receptor. A polypeptide represented by the amino acid sequence of SEQ ID NO: 5 as all or a part of the variable domain of the alpha chain and a polypeptide represented by the amino acid sequence of SEQ ID NO: 6 as all or part of the variable domain of the beta chain. 1 specific T cell receptor. A cell wherein the MART-1 specific T cell receptor of any one of claims 2 to 4 is expressed on the surface. 6. The cell according to claim 5, wherein the cell is a cytotoxic T cell (CTL), an inhibitory T cell or a natural killer cell (NK cell). A nucleic acid encoding all or a portion of the variable domain of the alpha chain of the MART-1 specific T cell receptor represented by the nucleic acid sequence of SEQ ID NO: 7, 9 or 11. A nucleic acid encoding all or part of the variable domain of the beta chain of the MART-1 specific T cell receptor, represented by the nucleic acid sequence of SEQ ID NO: 8, 10 or 12. 8. A recombinant vector comprising the nucleic acid of any of claims 7 and 8. A cell into which the recombinant vector of claim 9 has been introduced. 11. The cell according to claim 10, wherein the cell is a cytotoxic T cell (CTL), an inhibitory T cell or a natural killer cell (NK cell). A pharmaceutical composition for treating a MART-1 related disease comprising the cell of claim 5 as an active ingredient. 13. The composition of claim 12, wherein the MART-1 related disease is solid cancer. 14. The composition of claim 13, wherein the solid cancer is skin cancer or melanoma. A pharmaceutical composition for treating a MART-1 related disease comprising the cell of claim 10 as an active ingredient. 16. The composition of claim 15, wherein the MART-1 related disease is solid cancer. 17. The composition of claim 16, wherein the solid cancer is skin cancer or melanoma.

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