JP2020180136A - Methods and compositions for treatment of melanoma - Google Patents

Abstract

To provide methods and compositions for the treatment of melanoma.SOLUTION: Provided herein are methods and compositions for the treatment of melanoma using anti-tumor immune cells treated with a PTD-MYC fusion protein (e.g., HIV TAT-MYC fusion protein).SELECTED DRAWING: Figure 1

Description

Background of the Invention Adoptive cell transfer (ACT) is a type of immunotherapy that involves the transfer of immune cells with antitumor activity into a patient. ACT typically involves isolating lymphocytes with antitumor activity from a patient, culturing the lymphocytes in vitro to grow the population, and then injecting the lymphocytes into a host that carries the cancer. Including. The lymphocytes used for adoption can come from either the stroma of the resected tumor (eg, tumor-infiltrating lymphocytes), lymph vessels or lymph nodes, or blood. In some cases, isolated lymphocytes are genetically modified to express antitumor T cell receptors (TCRs) or chimeric antigen receptors (CARs). Lymphocytes used for injection can be isolated from a donor (allogeneic ACT) or a cancer-carrying host (self-ACT).

In certain embodiments, methods for adoptive cell transfer for the treatment of melanoma are provided herein. In some embodiments, the step of administering to a subject a therapeutically effective amount of immune cells with antitumor activity, the immune cells are fused with a protein transduction domain (PTD) -MYC prior to administration to the subject. Methods for the treatment of melanoma in a subject, including the step of contacting with a polypeptide, are provided. In some embodiments, the immune cell comprises one or more lymphocytes. In some embodiments, the one or more lymphocytes comprises T cells and / or B cells. In some embodiments, the one or more lymphocytes comprises tumor infiltrating lymphocytes. In some embodiments, the melanoma is a metastatic melanoma. In some embodiments, the melanoma is a superficial extended melanoma, a nodular melanoma, a malignant mole-derived melanoma, or a terminal melanoma. In some embodiments, immune cells are obtained from donor subjects with melanoma. In some embodiments, the donor subject and the subject that receives the immune cell are the same (ie, self-ACT). In some embodiments, the donor subject and the subject that receives the immune cell are different (ie, allogeneic ACT).

In some embodiments, the PTD-MYC fusion polypeptide comprises (i) an HIV TAT protein transduction domain; and (ii) a MYC polypeptide sequence. In some embodiments, the PTD-MYC fusion polypeptide translocates to the nucleus of immune cells. In some embodiments, the PTD-MYC fusion polypeptide exhibits biological activity of MYC, such as activation of a MYC target gene. In some embodiments, the fusion peptide comprises SEQ ID NO: 1.

In certain embodiments, (a) (i) a protein transfectation domain; (ii) a MYC fusion peptide comprising a MYC polypeptide sequence; and (b) a melanoma-specific antigen isolated from a donor subject with a melanoma tumor. Compositions comprising one or more primary immune cells that are reactive against are described herein. In some embodiments, the MYC fusion peptide migrates to the nucleus of one or more primary immune cells. In some embodiments, the MYC fusion peptide exhibits the biological activity of MYC. In some embodiments, the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain with the MYC polypeptide. In some embodiments, the MYC fusion peptide has the following general structure:
Contains a MYC fusion peptide with a protein transduction domain-X-MYC sequence
Here, -X- is a molecule that links the protein transduction domain with the MYC sequence. In some embodiments, the protein transduced domain sequence is a TAT protein transduced domain sequence. In some embodiments, the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48]. In some embodiments, the MYC fusion peptide comprises SEQ ID NO: 1. In some embodiments, the MYC fusion peptide is acetylated. In some embodiments, one or more immune cells have antitumor activity against melanoma cells, in some embodiments, one or more immune cells have one or more lymphocytes. Including. In some embodiments, the one or more lymphocytes comprises T cells, B cells, NK cells, or a combination thereof. In some embodiments, T cells are naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, energy T cells, tolerant T cells, chimeric T cells, and antigen-specific. Selected from the group consisting of T cells. In some embodiments, B cells consist of a group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. Be selected. In some embodiments, the one or more lymphocytes are tumor infiltrating lymphocytes, T cell receptor modified lymphocytes, or chimeric antigen receptor modified lymphocytes. In some embodiments, the tumor-infiltrating lymphocyte has a CD8 + CD25 + signature. In some embodiments, the tumor infiltrating lymphocyte has a CD4 + CD25 + signature. In some embodiments, the one or more immune cells comprises a detectable moiety.

In certain embodiments, the step of administering one or more modified immune cells to a subject in need thereof, wherein the one or more modified immune cells comprises a MYC fusion peptide and is MYC fused. In a subject comprising a step, wherein the peptide is (i) a protein transfecting domain; (ii) contains a MYC polypeptide sequence and one or more modified immune cells are reactive to a tumor-specific antigen. Methods for treating melanoma are described herein. In some embodiments, the modified immune cell is derived from a primary immune cell isolated from a subject. In some embodiments, the modified immune cell is derived from a primary immune cell isolated from another donor subject having the same type of melanoma. In some embodiments, one or more modified immune cells are prepared by isolating the primary immune cells and then contacting them with the MYC fusion peptide in vitro. In some embodiments, the method further comprises increasing primary immune cells in vitro prior to contact with the MYC fusion peptide. In some embodiments, the method further comprises the step of increasing primary immune cells after contact with the MYC fusion peptide. In some embodiments, anti-CD3 antibodies are used to grow cells. In some embodiments, irradiated allogeneic feeder cells are used to grow cells. In some embodiments, cells grow in the presence of exogenous cytokines. In some embodiments, the cytokine is interleukin 2. In some embodiments, the MYC fusion peptide translocates to the nucleus of immune cells. In some embodiments, the MYC fusion peptide exhibits the biological activity of MYC. In some embodiments, the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain with the MYC polypeptide. In some embodiments, the MYC fusion peptide has the following general structure:
Contains a MYC fusion peptide with a protein transduction domain-X-MYC sequence
Here, -X- is a molecule that links the protein transduction domain with the MYC sequence. In some embodiments, the protein transduced domain sequence is a TAT protein transduced domain sequence. In some embodiments, the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48]. In some embodiments, the MYC fusion peptide comprises SEQ ID NO: 1. In some embodiments, the MYC fusion peptide is acetylated. In some embodiments, one or more modified immune cells have antitumor activity against melanoma cells in a subject. In some embodiments, one or more modified immune cells have antitumor activity against melanoma cells in a subject. In some embodiments, the modified immune cell comprises one or more anerogenic immune cells. In some embodiments, the one or more immune cells comprises one or more lymphocytes. In some embodiments, the one or more lymphocytes comprises T cells, B cells, NK cells, or a combination thereof. In some embodiments, T cells are naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, energy T cells, tolerant T cells, chimeric T cells, and antigen-specific. Selected from the group consisting of T cells. In some embodiments, B cells consist of a group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. Be selected. In some embodiments, the one or more lymphocytes are tumor infiltrating lymphocytes, T cell receptor modified lymphocytes, or chimeric antigen receptor modified lymphocytes. In some embodiments, the lymphocyte has a CD8 + CD28-CD152-signature. In some embodiments, the lymphocytes have a CD8 + CD25 + signature. In some embodiments, the lymphocytes have a CD4 + CD25 + signature. In some embodiments, the method further comprises the step of isolating primary immune cells from the donor subject. In some embodiments, the donor subject has melanoma. In some embodiments, the modified immune cell is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratumorally. In some embodiments, the method further comprises putting the subject in a lymph depleted state prior to administration of one or more modified immune cells. In some embodiments, the method further comprises the step of administering the cytokine to the subject. In some embodiments, the cytokine is administered before, during, or after administration of one or more modified immune cells. In some embodiments, the cytokines are interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFα, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14- 1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1 CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR4 Be selected. In some embodiments, melanoma is metastatic. In some embodiments, the subject is a human or animal. In some embodiments, the method further comprises the step of performing additional cancer treatment. In some embodiments, additional cancer treatments are selected from among chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anticancer nucleic acids or proteins, anticancer viruses or microorganisms, and combinations thereof. .. In some embodiments, the modified immune cell comprises a detectable moiety.

In certain embodiments, the step of contacting one or more immune cells with a MYC fusion polypeptide in vitro, wherein the immune cells are derived from a donor exposed to one or more tumor antigens. And a modified immune cell for the treatment of melanoma, comprising a step, wherein the MYC fusion peptide is (i) a protein transfectation domain; (ii) a MYC polypeptide sequence and is responsive to a tumor-specific antigen. Methods for preparing are also described herein. In some embodiments, the modified immune cell is derived from a primary immune cell isolated from a subject with melanoma. In some embodiments, the method further comprises increasing primary immune cells in vitro prior to contact with the MYC fusion peptide. In some embodiments, the method further comprises the step of increasing primary immune cells after contact with the MYC fusion peptide. In some embodiments, anti-CD3 antibodies are used to grow cells. In some embodiments, irradiated allogeneic feeder cells are used to grow cells. In some embodiments, cells grow in the presence of exogenous cytokines. In some embodiments, the cytokine is interleukin 2. In some embodiments, the MYC fusion peptide translocates to the nucleus of immune cells. In some embodiments, the MYC fusion peptide exhibits the biological activity of MYC. In some embodiments, the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain with the MYC polypeptide. In some embodiments, the MYC fusion peptide has the following general structure:
Contains a MYC fusion peptide with a protein transduction domain-X-MYC sequence
Here, -X- is a molecule that links the protein transduction domain with the MYC sequence. In some embodiments, the protein transduced domain sequence is a TAT protein transduced domain sequence. In some embodiments, the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48]. In some embodiments, the MYC fusion peptide comprises SEQ ID NO: 1. In some embodiments, the MYC fusion peptide is acetylated. In some embodiments, one or more modified immune cells have antitumor activity. In some embodiments, one or more modified immune cells have antitumor activity against melanoma cells in a subject. In some embodiments, the modified immune cell comprises one or more anerogenic immune cells. In some embodiments, the one or more immune cells comprises one or more lymphocytes. In some embodiments, the one or more lymphocytes comprises T cells, B cells, NK, or a combination thereof. In some embodiments, T cells are naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, anergic T cells, tolerant T cells, chimeric T cells, and antigen-specific. Selected from the group consisting of T cells. In some embodiments, B cells consist of a group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. Be selected. In some embodiments, the one or more lymphocytes are tumor infiltrating lymphocytes, T cell receptor modified lymphocytes, or chimeric antigen receptor modified lymphocytes. In some embodiments, the lymphocyte has a CD8 + CD28-CD152-signature. In some embodiments, the lymphocytes have a CD8 + CD25 + signature. In some embodiments, the lymphocytes have a CD4 + CD25 + signature.

In certain embodiments, (a) one or more isolated primary immune cells exposed to a melanoma cell line; and (b) (i) a protein transfecting domain; (ii) a MYC polypeptide sequence. Compositions comprising a MYC fusion peptide, wherein one or more primary immune cells are reactive to a melanoma-specific antigen, are also described herein.

In certain embodiments, any of the above compositions for use in the treatment of melanoma is also described herein. In certain embodiments, any of the above compositions for use in the manufacture of a medicament for use in the treatment of melanoma is also described herein.

Methods for increasing the effectiveness of adoptive or T cell therapy in a subject, including the step of administering any of the above compositions in certain embodiments, are also described herein.

In certain embodiments, modified tumor infiltrating lymphocytes comprising a MYC fusion peptide, wherein the MYC fusion peptide comprises (i) a protein transduction domain; (ii) a MYC polypeptide sequence are also described herein. .. In some embodiments, the tumor-infiltrating lymphocytes are derived from primary tumor-infiltrating lymphocytes isolated from a subject having cancer (eg, melanoma).

In certain embodiments, lymphocytes comprising a chimeric antigen receptor and a MYC fusion peptide, wherein the MYC fusion peptide comprises (i) a protein transduction domain; (ii) a MYC polypeptide sequence are also described herein. Will be done. In some embodiments, the lymphocytes are derived from primary lymphocytes isolated from a subject with cancer (eg, melanoma).

In certain embodiments, the step of contacting one or more primary immune cells with a MYC fusion polypeptide comprising (i) a protein transfection domain; (ii) a MYC polypeptide sequence, one or more. Composition for adoptive cell therapy, comprising steps, in which multiple primary immune cells are isolated from a patient with melanoma and one or more primary immune cells are responsive to a melanoma-specific antigen. Methods for preparing the article are also described herein.

Kits containing MYC fusion polypeptides and / or MYC fusion polypeptide-modified immune cells provided herein for use in the treatment of melanoma are also provided. In some embodiments, the kit comprises one or more reagents for the detection of administered MYC fusion polypeptide and / or MYC fusion polypeptide modified immune cells. In some embodiments, the kit comprises cells for treatment with the MYC fusion polypeptide provided herein, such as hematopoietic stem cells, donor leukocytes, T cells, or NK cells. In some embodiments, the kit comprises relevant instructions for using MYC fusion polypeptides and / or MYC fusion polypeptide-modified immune cells.

Illustrates the results for survival of melanoma tumor-bearing mice after injection of lymphocytes from tumor-carrying donor mice treated with TAT-MYC for 1 hour. Mice were treated with TAT-MYC lymphocytes, treated with control protein-treated lymphocytes, or left untreated. The date of death was recorded with the date of treatment as day 0. Illustrates the results for survival of melanoma tumor-bearing mice after injection of lymphocytes from tumor-carrying donor mice treated with TAT-MYC (repeated experiment shown in FIG. 1). Mice were treated with TAT-MYC lymphocytes, treated with control protein-treated lymphocytes, or left untreated. The date of death was recorded with the date of treatment as day 0. Illustrates the results for survival of melanoma tumor-bearing mice after injection of different amounts of lymphocytes from tumor-carrying donor mice treated with TAT-MYC. Mice were treated with TAT-MYC lymphocytes, treated with control protein-treated lymphocytes, or left untreated. The date of death was recorded with the date of treatment as day 0. Illustrates the results for survival of melanoma tumor-bearing mice after injection of different amounts of lymphocytes from tumor-carrying donor mice treated with TAT-MYC. Mice were treated with TAT-MYC lymphocytes, treated with control protein-treated lymphocytes, or left untreated. The date of death was recorded with the date of treatment as day 0.

Detailed Description of the Invention The present disclosure is not limited by the specific embodiments described herein, and they shall be a single example of the individual aspects of the present disclosure. Not all of the various aspects of the disclosure are described herein. As will be apparent to those skilled in the art, many modifications and variants of the present disclosure can be made without departing from that spirit and scope. In addition to those listed herein, functionally equivalent methods and devices within the scope of this disclosure will be apparent to those skilled in the art from the above description. Such modifications and variants shall be included within the scope of the appended claims. The present disclosure should be limited only by the appended claims, along with the full scope of the equivalents to which such claims are entitled.

It should be understood that the present disclosure is not limited to specific uses, methods, reagents, compounds, compositions, or biological systems, which, of course, can vary. It should also be understood that the terminology used herein is only for describing specific embodiments and is not limiting.

Further, one of ordinary skill in the art will recognize that if the features or aspects of the present disclosure are described by the Markush group, the disclosure will also be described by individual members or subgroups of members of the Markush group.

As will be appreciated by those skilled in the art, in fact, particularly with respect to the provision of written description, all ranges disclosed herein also include all possible sub-ranges and combinations of sub-ranges. Include. The listed range fully describes the range and can be divided into at least half, one-third, one-quarter, one-fifth, one-tenth, etc. Can be easily recognized. As a non-limiting example, each range described herein can be easily decomposed into a lower third, a central third, an upper third, and so on. As will be understood by those skilled in the art, words such as "to", "at least", "greater than", "less than", etc. all include explicit numbers and are later decomposed into subranges as described above. Refers to the range that can be done. Finally, as will be appreciated by those skilled in the art, the scope will include each individual member. Thus, for example, a group with 1 to 3 cells refers to a group with 1, 2, or 3 cells. Similarly, a group with 1 to 5 cells refers to a group with 1, 2, 3, 4, or 5 cells, and so on.

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 disclosure belongs.

I. Definitions The terminology used herein is merely to describe specific aspects and is not intended to limit this disclosure. As used herein, the singular forms "one (a)", "one (an)", and "the" do not explicitly indicate anything else in the context. As long as it is, the plural form is also included.

As used herein, the term "about" is used even if the value fluctuates by +/- 20%, +/- 15%, +/- 10%, or +/- 5%. It is meant to be within the scope of this disclosure. For example, "concentration of about 200 IU / mL" includes concentrations of 160 IU / mL to 240 IU / mL.

As used herein, the term "administration" of an agent to a subject includes any route by which the agent is introduced or delivered to the subject to perform its intended function. .. Administration can be performed by any suitable route, including intravenous, intramuscular, intraperitoneal, or subcutaneous. Administration includes self-administration and administration by others.

The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. The naturally encoded amino acids are 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine. , Tryptophan, tyrosine, and valine), and isoleucine and selenocysteine. Amino acid analogs are associated with the same basic chemical structure as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, ie hydrogen, carboxyl, amino, and R groups. Refers to an agent having α-carbon. Such analogs have a modified R group (like norleucine), or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, the amino acids that form the polypeptide are of type D. In some embodiments, the amino acids that form the polypeptide are L-type. In some embodiments, the first plurality of amino acids forming the polypeptide is of type D and the second plurality is of type L.

Amino acids are referred to herein by either a commonly known three-letter symbol or a one-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are likewise referred to by the generally accepted one-letter code.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms also apply to naturally occurring amino acid polymers, as well as to amino acids in which one or more amino acid residues are not naturally occurring, such as amino acid polymers that are amino acid analogs. These terms include amino acid chains of any length, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds.

As used herein, a "control" is an alternative sample used in an experiment for comparative purposes. The control can be "positive" or "negative". For example, if the purpose of the experiment is to determine the correlation of therapeutic substance efficacy for treatment for a particular type of disease, a positive control (known to show the desired therapeutic effect) is typically known. Compositions) and negative controls (subjects or samples that do not receive treatment or receive placebo) are used.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of agent sufficient to achieve the desired therapeutic effect. For therapeutic application, the amount of therapeutic peptide administered to a subject may depend on the type and severity of infection and individual characteristics such as general health, age, sex, body weight, and resistance to the drug. .. It may also depend on the severity, severity and type of the disease. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors.

As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA, and / or the transcribed mRNA is then translated into a peptide, polypeptide, or protein. Refers to the process. If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from a sample can be directly compared to the expression level of that gene from a control or reference sample. In another aspect, the expression level of a gene from a sample may be compared directly with the expression level of that gene from the same sample after administration of the compositions disclosed herein. The term "expression" also refers to one or more of the following events: (1) production of RNA templates from DNA sequences (eg, by transcription) in cells; (2) splicing, etc. in cells. Processing of RNA transcripts (by editing, 5'cap formation, and / or 3'end formation); (3) translation of RNA sequences into polypeptides or proteins in cells; (4) after translation of polypeptides or proteins in cells Modifications; (5) Presentation of polypeptide or protein on the cell surface; and (6) Secretion, presentation or release of polypeptide or protein from cells.

The term "linker" refers to a synthetic sequence (eg, an amino acid sequence) that connects or links two sequences, eg, links two polypeptide domains. In some embodiments, the linker contains a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

Terms such as "lyophilized" and "lyophilized", as used herein, first freeze the material to be dried (eg nanoparticles), then ice or a frozen solvent. Refers to the process of removal by sublimation in a vacuum environment. Excipients may be included in the pre-lyophilized formulation to enhance the stability of the lyophilized product during storage. The lyophilized sample may further contain additional excipients.

As used herein, the term immune cell refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin and are lymphocytes such as B cells and T cells; natural killer cells; monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils. , And myeloid cells such as granulocytes.

The term "lymphocyte" refers to all immature, mature, undifferentiated, and differentiated white lymphocyte populations, including tissue-specific and specialized types. It includes, as non-limiting examples, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include pre-B cells, progenitor B cells, early pro B cells, late pro B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, trait B cells. , Memory B cells, B-1 cells, B-2 cells, and all B cell lineages including the anerogenic AN1 / T3 cell population.

As used herein, the term T cell refers to naive T cell, CD4 + T cell, CD8 + T cell, memory T cell, activated T cell, anergic T cell, tolerant T cell, Includes chimeric T cells and antigen-specific T cells.

The term "(s) B cells" is a non-limiting example of pre-B cells, precursor B cells, early pro B cells, late pro B cells, large pre B cells, small pre B cells. , Immature B cells, mature B cells, naive B cells, trait B cells, activated B cells, energy B cells, tolerant B cells, chimeric B cells, antigen-specific B cells, memory B cells, B-1 cells, Refers to B-2 cells and anogenic AN1 / T3 cell populations. In some embodiments, the term B cell includes a B cell that expresses an immunoglobulin heavy chain and / or a light chain on its cell surface. In some embodiments, the term B cell includes B cells that express and secrete immunoglobulin heavy and / or light chains. In some embodiments, the term B cell includes cells that bind to antigens on the cell surface. In some embodiments disclosed herein, B cells or AN1 / T3 cells are used in the process described. In certain embodiments, such cells are optionally, eg, hematopoietic stem cells, naive B cells, B cells, pre-B cells, precursor B cells, early pro B cells, late pro B cells, large pre B cells, Express antibodies, including small pre-B cells, immature B cells, mature B cells, trait B cells, memory B cells, B-1 cells, B-2 cells, energy B cells, or energy AN1 / T3 cells. Is replaced by any animal cell that is suitable for, capable of expressing the antibody (eg, inducible expression), or capable of differentiating into cells suitable for expressing the antibody.

As used herein, "adoptive cell therapeutic composition" refers to any composition that contains cells suitable for adoption. In an exemplary embodiment, the composition for adoptive cell therapy comprises tumor infiltrating lymphocytes (TIL), TCR (ie, heterologous T cell receptor) modified lymphocytes, and CAR (ie, chimeric antigen receptor) modified lymphocytes. Includes cell types selected from the group consisting of spheres. In another embodiment, the composition for adoptive cell therapy comprises a cell type selected from the group consisting of T cells, CD8 + cells, CD4 + cells, NK cells, δγT cells, regulatory T cells, and peripheral blood mononuclear cells. .. In another embodiment, TIL, T cells, CD8 + cells, CD4 + cells, NK cells, δγT cells, regulatory T cells, or peripheral blood mononuclear cells form a composition for adoptive cell therapy. In one embodiment, the composition for adoptive cell therapy comprises T cells.

As used herein, "tumor-infiltrating lymphocytes" or TILs refer to white blood cells that have exited the bloodstream and migrated to the tumor.

The terms "MYC" and "MYC gene" are synonymous. They refer to the nucleic acid sequences that encode the MYC polypeptide. The MYC gene is at least 60% to 100% identical or homologous to the sequence of NCBI accession number NM_002467, eg, at least 60, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%. , 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percentage of about 70% to about 100%, identical, nucleotide sequences of at least 120 nucleotides. including. In some embodiments, the MYC gene is a protooncogene. In certain cases, the MYC gene is found at 8q24.21 on chromosome 8. In some cases, the MYC gene starts at 128,816,862 bp from pter and ends at 128,822,856 bp from pter. In some cases, the MYC gene is about 6 kb. In certain cases, the MYC gene encodes at least 8 separate mRNA sequences, 5 selectively spliced variants and 3 non-spliced variants.

である。
NP_002458.2（UniProtKB/Swiss-Prot：P01106.2）の配列は、

である。 The terms "MYC protein,""MYCpolypeptide," and "MYC sequence" are synonyms and NCBI accession number UniProtKB / Swiss-Prot: P01106.1 (MYC isoform 1) or NP_002458.2 (UniProtKB). / Swiss-Prot: P01106.2; Refers to polymers of amino acid residues disclosed in MYC Isoform 2), and their functional homologues, analogs, or fragments. UniProtKB / Swiss-Prot: The sequence of P01106.1 is

Is.
The sequence of NP_002458.2 (UniProtKB / Swiss-Prot: P01106.2) is

Is.

を含む。 In some embodiments, the MYC polypeptide is a complete MYC polypeptide sequence. In some embodiments, the MYC polypeptide is a partial MYC polypeptide sequence. In some embodiments, the MYC polypeptide comprises at least 400 contiguous amino acids with SEQ ID NO: 2 or 11. In some embodiments, the MYC polypeptide comprises at least 400 contiguous amino acids with SEQ ID NO: 2 or 11 and retains at least one MYC activity. In some embodiments, the MYC polypeptide comprises at least 400, at least 410, at least 420, at least 430, or at least 450 contiguous amino acids of SEQ ID NO: 2 or 11. In some embodiments, the MYC polypeptide comprises at least 400, at least 410, at least 420, at least 430, or at least 450 contiguous amino acids of SEQ ID NO: 2 or 11, and at least one type. Retains MYC activity. In some embodiments, the MYC polypeptide is c-MYC. In some embodiments, the MYC polypeptide sequence is the sequence shown below:

including.

を含む。 In some embodiments, the MYC polypeptide sequence is the sequence shown below:

including.

の配列を含む。いくつかの態様において、MYCポリペプチドは、転写因子（例えば、転写因子64）である。いくつかの態様において、MYCポリペプチドは、EボックスDNA結合ドメインを含有している。いくつかの態様において、MYCポリペプチドは、CACGTGを含む配列に結合する。いくつかの態様において、MYCポリペプチドは、細胞の生存および/または増殖のうちの一つまたは複数を促進する。いくつかの態様において、MYCポリペプチドは、前記のもののうちの一つまたは複数を含み、1個または複数個の翻訳後修飾（例えば、アセチル化）を含む。いくつかの態様において、MYCポリペプチドは、ポリペプチドのN末端またはC末端に、1個または複数個の付加的なアミノ酸残基を含む。いくつかの態様において、MYCポリペプチドは、融合タンパク質である。いくつかの態様において、MYCポリペプチドは、ポリペプチドのN末端またはC末端において1個または複数個の付加的なペプチドと連結されている。 In some embodiments, the MYC polypeptide is at least 40% to 100% identical to the sequence of NCBI accession number NP002458.2 or UniProt KB / Swiss-Prot accession number P01106.1, eg, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91% , 92%, 94%, 95%, 96%, 97%, 98%, 99%, or other percent of about 40% to about 100%, including amino acid sequences that are identical. In some embodiments, the MYC polypeptide refers to a 439 amino acid polymer, a post-translationally unmodified MYC polypeptide. In some embodiments, the MYC polypeptide refers to a post-translationally modified 439 amino acid polymer. In some embodiments, the MYC polypeptide is 48,804 kDa. In some embodiments, the MYC polypeptide contains a basic helix-loop-helix-leucine zipper (bHLH / LZ) domain. In some embodiments, the bHLH / LZ domain is

Contains an array of. In some embodiments, the MYC polypeptide is a transcription factor (eg, transcription factor 64). In some embodiments, the MYC polypeptide contains an E-box DNA binding domain. In some embodiments, the MYC polypeptide binds to a sequence containing CACGTG. In some embodiments, the MYC polypeptide promotes one or more of cell survival and / or proliferation. In some embodiments, the MYC polypeptide comprises one or more of the above and comprises one or more post-translational modifications (eg, acetylation). In some embodiments, the MYC polypeptide comprises one or more additional amino acid residues at the N-terminus or C-terminus of the polypeptide. In some embodiments, the MYC polypeptide is a fusion protein. In some embodiments, the MYC polypeptide is linked to one or more additional peptides at the N-terminus or C-terminus of the polypeptide.

Proteins suitable for use in the methods described herein include 1 to 15 amino acid changes, eg, 1, as compared to the amino acid sequences of the proteins described herein. Substitutions or deletions of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, Also included are functional variants, including proteins with additions. In other embodiments, the modified amino acid sequence is at least 75% identical to the amino acid sequence of the protein inhibitor described herein, eg, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99%, or 100% identical. Such sequence variant proteins are described herein as long as the modified amino acid sequence retains sufficient biological activity to be functional in the compositions and methods described herein. Suitable for the method used. If an amino acid substitution is made, the substitution can be a conservative amino acid substitution. Among the common naturally occurring amino acids, for example, "conservative amino acid substitutions" are exemplified by amino acid substitutions in each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, ( 2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartic acid and glutamic acid, (5) glutamine and asparagine, and (6) lysine, arginine, and histidine. The BLOSUM62 table is an amino acid substitution matrix derived from approximately 2,000 local multiple alignments of protein sequence segments representing highly conserved regions of more than 500 groups of related proteins (Henikoff et al., (1992), Proc.Natl Acad.Sci.USA, 89: 10915-10919). Therefore, BLOSUM62 substitution frequencies are used to define conservative amino acid substitutions, which, in some embodiments, are introduced into the amino acid sequences described or disclosed herein. Although it is possible to design amino acid substitutions based solely on chemical properties (as described above), the term "conservative amino acid substitutions" preferably refers to substitutions represented by BLOSUM62 values greater than -1. .. For example, if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3, the amino acid substitution is conservative. According to this system, preferred conservative amino acid substitutions are characterized by at least 1 (eg, 1, 2, or 3) BLOSUM62 values, and more preferred conservative amino acid substitutions are at least 2 (eg, 2 or 3) BLOSUM62. Characterized by a value.

の配列を含む。ある種の場合において、MYCによってコードされた転写因子のEボックス配列への結合は、RNAポリメラーゼがEボックス配列の下流の遺伝子を転写することを可能にする。 The terms "E-box sequence" and "enhancer box sequence" are interchangeably used herein to mean the nucleotide sequence CANNTG, where N is any nucleotide. In some cases, the E-box sequence contains CACGTG. In certain cases, the basic helix-loop-helix domain of the transcription factor encoded by MYC binds to the E-box sequence. In certain cases, the E-box sequence is located upstream of the gene (eg, p21, Bc1-2, or ornithine decarboxylase). In certain cases, the MYC polypeptide contains an E-box DNA binding domain. In some cases, the E-box DNA binding domain is

Contains an array of. In certain cases, binding of a transcription factor encoded by MYC to the E-box sequence allows RNA polymerase to transcribe genes downstream of the E-box sequence.

The term "MYC activity" or "MYC biological activity" or "MYC with biological activity" includes one or more of enhancement or induction of cell survival, cell proliferation, and / or antibody production. included. For example, but not limited to, MYC activity includes enhanced proliferation of T cells activated by anti-CD3 and anti-CD28, and / or increased proliferation of long-term autologous hematopoietic stem cells. MYC activity includes invasion of cells into the nucleus, binding to nucleic acid sequences (eg, binding to E-box sequences), and / or induction of expression of MYC target genes.

Terms such as "patient," "subject," and "individual" are interchangeably used herein to refer to animals, typically mammals. In one embodiment, the patient, subject, or individual is a mammal. In one embodiment, the patient, subject, or individual is a human. In some embodiments, the patient, subject, or individual is an animal, such as, but not limited to, a domestic animal such as a horse, cow, mouse, sheep, dog, and cat.

The term "protein transfection domain (PTD)" or "transporter peptide sequence" (also known as cell-permeable protein (CPP) or membrane transfer sequence (MTS)) is much more independent of classical endositosis. As used herein interchangeably to refer to small peptides capable of transporting large molecules to cells. In some embodiments, nuclear localization signals that mediate further transfer of the molecule to the cell nucleus can be found within the protein transduction domain.

The terms "treating" or "treatment", as used herein, include the treatment of a disease in a subject such as a human and (i) inhibition of the disease, i.e., inhibition of its development. (Ii) alleviation of the disease, i.e. induction of disease regression; (iii) slowing the progression of the disease; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease. Including. With respect to melanoma, "treating" or "treatment" also includes tumor regression, slowing of tumor growth, inhibition of metastasis of melanoma tumors, inhibition of recurrence or recurrent melanoma, and / or maintenance of remission.

The various modes of treatment or prevention of the medical diseases and conditions described are not only complete treatment or prevention, but also incomplete treatment or prevention in which some biologically or medically appropriate results are achieved. It should also be understood that it means "substantial" including. The treatment may be a continuous long-term treatment of a chronic disease, or may be a single or small dose for the treatment of an acute condition.

The term "therapeutic" means treatment and / or prophylaxis, as used herein. Therapeutic effects are obtained by suppressing, ameliorating, or radically curing the disease state.

II. Overview The present disclosure comprises, in part, one or more immune cells having antitumor activity (eg, immune cells that modulate the response to a tumor, such as tumor infiltrating lymphocytes (TILs)). For treatment of melanoma in a subject by administration of, one or more immune cells are contacted with the PTD-MYC fusion polypeptide in vitro prior to administration to the subject. In some embodiments, immune cells are obtained from donor subjects with melanoma tumors. In some embodiments, the cell is self to the subject receiving the treatment. In some embodiments, the melanoma is a superficial extended melanoma, a nodular melanoma, a malignant mole-derived melanoma, or a terminal melanoma.

The present disclosure treats lymphocytes isolated from donor subjects with melanoma tumors with MYC polypeptides and MYC fusion polypeptides containing protein transfection domains (PTDs) such as the HIV TAT protein transfection domain. It is based, at least in part, on the finding that administration of treated lymphocytes to subjects carrying melanoma tumors significantly increases the survival of tumor-carrying subjects. The examples provided herein are when immune cells extracted from the lymph nodes of a melanoma-carrying mouse are treated with the TAT-MYC fusion protein in vitro prior to administration to a second melanoma-carrying mouse. Prove that it had a significantly increased therapeutic efficacy. These data support that adoptive cell transfer using antitumor immune cells treated with PTD-MYC fusion polypeptides can be used in the treatment of tumors such as melanoma tumors.

In some embodiments, a method for treating melanoma in a subject comprises administering immune cells in vitro contacted with a PTD-MYC fusion polypeptide. In some embodiments, immune cells for use in the methods of the invention are pre-stimulated by melanoma tumor antigens in vivo. In some embodiments, the immune cell is derived from a donor with melanoma. In some embodiments, immune cells are derived from donors with solid tumors, such as melanoma tumors. In some embodiments, immune cells are contacted with a melanoma tumor antigen in vivo. In some embodiments, immune cells are derived from a donor exposed to one or more melanoma tumor antigens. In some embodiments, immune cells are derived from a donor exposed to an antitumor vaccine. In some embodiments, the immune cell is a B cell, a T cell, an NK cell, or a combination thereof. In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL). In some embodiments, the immune cell is a chimeric antigen receptor (CAR) -T cell.

In some embodiments, a method for the treatment of melanoma in a subject is the step of administering one or more modified immune cells to the subject in need thereof, the one or more modified forms. Immune cells contain a MYC fusion peptide, the MYC fusion peptide (i) contains a protein transfection domain; (ii) contains a MYC polypeptide sequence, and one or more modified immune cells respond to a melanoma tumor-specific antigen. Including processes that are sex.

In some embodiments, the method for the treatment of melanoma in a subject is
(A) In vitro contact of immune cells with a MYC fusion polypeptide, wherein the immune cells are derived from a donor exposed to one or more melanoma tumor antigens, and the MYC fusion peptide is (i). ) Protein transfection domain; (ii) a step comprising a MYC polypeptide sequence; and (b) a step of administering contacted immune cells to a melanoma tumor-carrying subject, thereby treating the melanoma.

In some embodiments, in vitro contact of immune cells with the PTD-MYC fusion polypeptide is performed by culturing the immune cells in the presence of the MYC fusion polypeptide. In some embodiments, the immune cell comprises one or more cytokines and / or growth factors (eg, interleukin 2 (IL-2), IL-4, IL-7, IL-9, and IL-15. ) Is cultured in the presence of. In some embodiments, it does not increase immune cells prior to administration. In some embodiments, immune cells are expanded prior to administration. In some embodiments, the donor and the subject to be treated are the same.

In some embodiments, the immune cell is a tumor infiltrating lymphocyte. In some embodiments, the tumor infiltrating lymphocyte is an autologous tumor infiltrating lymphocyte. Thus, in some embodiments, a method for the treatment of melanoma in a subject is the step of administering lymphocytes in contact with a PTD-MYC fusion polypeptide in vitro, wherein the immune cells are lymphocyte-derived. Includes the step in which the lymphocytes are autologous tumor infiltrating lymphocytes from the subject.

In some embodiments, the method for the treatment of melanoma in a subject is
(A) In vitro contact of lymphocytes with PTD-MYC fusion polypeptide, wherein the lymphocytes are autologous tumor infiltrating lymphocytes of subject origin, and (b) contacted autotumor infiltrating lymphocytes. Includes the step of administering to the subject, thereby treating the melanoma.

Methods of Obtaining and Preparing Immune Cells for Translocation Immune cells for use in the methods provided herein can be obtained using any suitable method known in the art. In some embodiments, the immune cell is a primary immune cell. In some embodiments, the immune cell is a lymphocyte such as a T cell and a B cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is a mixture of lymphocytes and NK cells. In some embodiments, the immune cell is a peripheral blood mononuclear cell (PBMC). In some embodiments, the immune cell is a tumor-infiltrating T cell (eg, a tumor-infiltrating lymphocyte). In some embodiments, T cells are removed during surgery for melanoma or metastatic tumors in a subject. For example, in some embodiments, T cells are isolated after biopsy removal of tumor tissue. In some embodiments, immune cells are modified after being isolated from the donor. In some embodiments, the immune cell is a chimeric antigen receptor (CAR) -T cell.

In some embodiments, T cells are isolated from a sample containing a population of cells such as blood, lymph, or tissue biopsy sample. T cells can be isolated from a population of cells by any means known in the art. In one embodiment, the method comprises obtaining a bulk population of T cells from a tumor sample by any suitable method known in the art. For example, a bulk population of T cells can be obtained from a tumor sample by dissociating the tumor sample into a cell suspension from which a particular cell population can be selected. A suitable method for obtaining a bulk population of T cells is one of mechanical dissection of the tumor (eg, grinding), enzymatic dissection of the tumor (eg, digestion), and aspiration (eg, by needle). Multiple, but not limited to, these.

A bulk population of T cells obtained from a tumor sample may contain the appropriate type of T cells. Preferably, the bulk population of T cells obtained from the tumor sample comprises tumor infiltrating lymphocytes (TIL).

Tumor samples can be obtained from any mammal. Unless otherwise stated, the term "mammal" as used herein refers to the order Carnivora, including felines (cats) and canines (canidae); Refers to any mammal of the order Carnivora, including the family (Bovidae) and Canidae (Pig); or, but not limited to, mammals of the order Carnivora, including Equine (Equine). Mammals may be non-human primates of the order Primates, Ceboids, or Simoids (monkeys), or Simians (humans and apes). In some embodiments, the mammal may be a rodent mammal such as a mouse and a hamster. Preferably, the mammal is a non-human primate or human. An exemplary mammal is a human. In some embodiments, the subject receiving the immune cell is also the donor of the tumor sample (ie, self-ACT).

T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from units of blood collected from a subject using a number of techniques known to those of skill in the art, such as Ficoll separation. In one embodiment, cells can be obtained from the circulating blood of an individual by apheresis or leukocyte feresis. Apheresis products typically contain T cells, monocytes, granulocytes, B cells, and other lymphocytes, including nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In another embodiment, the wash solution may be deficient in calcium, magnesium, or many, if not all, of the divalent cations. The initial activation step in the absence of calcium results in expanded activation. As will be readily appreciated by those skilled in the art, the cleaning process is carried out by methods known to those skilled in the art, such as the use of semi-automatic "flow-through" centrifugation according to the manufacturer's instructions (eg, Cobe 2991 cell processor). Can be achieved. After washing, cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, the unwanted components of the apheresis sample may be removed and the cells may be resuspended directly in the culture medium.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example by centrifugation by a PERCOLL ™ gradient. Specific subpopulations of T cells, such as CD28 + T cells, CD4 + T cells, CDC, CD45RA + T cells, and CD45RO + T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, with beads conjugated to anti-CD3 / anti-CD28 (ie, 3x28), such as DYNABEADS® M-450 CD3 / CD28 T or XCYTE DYNABEADS®, desired. T cells are isolated by incubating for a sufficient period of time for positive selection of T cells. In one embodiment, the period is about 30 minutes. In a further embodiment, the period ranges from 30 minutes to 36 hours or more and all integer values in between. In a further embodiment, the period is at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours. In yet another embodiment, the period is 10 to 24 hours. In one embodiment, the incubation period is 24 hours. For isolation of T cells from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times simply leave T cells in situations where there are few T cells compared to other cell types, such as when tumor infiltrating lymphocytes (TILs) are isolated from tumor tissue or immunocompromised individuals. Can be used to separate. In addition, the use of longer incubation times can increase the efficiency of CD8 + T cell capture.

Concentration of the T cell population by negative selection can be achieved by a combination of antibodies against surface markers that are unique to the negatively selected cells. In one embodiment, the method is cell selection and / or selection via negative magnetic immunoadhesion or flow cytometry, using a cocktail of monoclonal antibodies against cell surface markers present in negatively selected cells. For example, to concentrate CD4 + cells by negative selection, monoclonal antibody cocktails typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

In addition, beads or columns coated with anti-CD14, or monocyte populations from blood products (ie, CD14 + cells) by a variety of methodologies, including the utilization of the phagocytic activity of these cells to facilitate removal. Can be depleted. Thus, in one embodiment, the invention uses paramagnetic particles of sufficient size to be phagocytosed by phagocytic monocytes. In certain embodiments, the paramagnetic particles are commercially available beads, such as those made by Life Technologies under the trademark Dynabeads ™. In one embodiment, the other non-specific cells are removed by coating the paramagnetic particles with an "irrelevant" protein (eg, serum protein or antibody). Unrelated proteins and antibodies include proteins and antibodies or fragments thereof that do not specifically target T cells to be isolated. In certain embodiments, irrelevant beads include sheep anti-mouse antibody, goat anti-mouse antibody, and beads coated with human serum albumin.

Briefly, such depletion of paramagnets causes T cells isolated from whole blood, aferesis peripheral blood, or tumors to be isolated from tumors at 22-37 ° C for about 30 minutes to 2 hours. Pre-incubate with paramagnetic particles with one or more unrelated antibodies coupled or with uncoupled paramagnetic particles in an amount that allows removal (approximately 20: 1 beads: cell ratio). Then, it is carried out by magnetically removing the cells attached to the paramagnetic particles or the cells that have phagocytosed the paramagnetic particles. Such separation can be performed using standard methods available in the art. For example, magnetic separation methodologies can be used that include a variety of commercially available products (eg, DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)). A variety of methodologies known to those of skill in the art, including flow cytometric analysis of CD14-positive cells, can be used to monitor the required depletion assurance before and after depletion.

Due to the isolation of the desired cell population by positive or negative selection, the concentrations of cells and surfaces (eg, beads-like particles) can vary. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (ie, increase the concentration of cells) in order to ensure maximum contact between the cells and the cells. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, over 100 million cells / ml are used. In a further embodiment, 10 million cells / ml, 15 million cells / ml, 20 million cells / ml, 25 million cells / ml, 30 million cells / ml, 35 million cells / ml, 40 million cells. A cell concentration of / ml, 45 million cells / ml, or 50 million cells / ml is used. In yet another embodiment, from 75 million cells / ml, 80 million cells / ml, 85 million cells / ml, 90 million cells / ml, 95 million cells / ml, or 100 million cells / ml. The concentration of cells is used. In a further embodiment, a concentration of 125 million cells / ml or 150 million cells / ml can be used. The use of high concentrations can result in increased cell yield, cell activation, and cell proliferation. In addition, the use of high cell concentrations is from cells that can weakly express the target antigen of interest, such as CD28-negative T cells, or from samples in which many tumor cells are present (ie, leukemic blood, tumor tissue, etc.). Allows for more efficient capture of cells. Populations of such cells can have therapeutic value and are desirable to obtain. For example, the use of high concentrations of cells usually allows for more efficient selection of CD8 + T cells with weaker CD28 expression.

In related embodiments, it may be desirable to use lower cell concentrations. Significant dilution of the mixture of T cells and surfaces (eg, beads-like particles) minimizes the interaction between the particles and the cells. This selects cells that express a large amount of the desired antigen that should bind to the particles. For example, CD4 + T cells express higher levels of CD28 and are more efficiently captured than CD8 + T cells at low concentrations. In one embodiment, the concentration of cells used is 5 × 10 ⁶ / ml. In other embodiments, the concentration used is approximately 1 × 10 ⁵ / ml to 1 × 10 ⁶ / ml and any integer value in between.

T cells can also be frozen. The steps of freezing and subsequent thawing can provide a more uniform product by removing granulocytes in the cell population and, to some extent, monocytes. After a wash step to remove plasma and platelets, the cells can be suspended in a frozen solution. Many frozen solutions and parameters are known in the art and are useful in this context, but one method is PBS containing 20% DMSO and 8% human serum albumin, or other suitable cells. Containing the use of frozen medium, the cells are then frozen to -80 ° C at a rate of 1 degree per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may be used, and uncontrolled immediate freezing methods in -20 ° C or liquid nitrogen may be used.

The T cells for use in the present invention may be antigen-specific T cells. For example, tumor-specific T cells can be used. In certain embodiments, antigen-specific T cells can be isolated from a patient of interest, such as a patient with a melanoma tumor, such as a patient with a melanoma tumor. In some embodiments, the patient has melanoma.

In one embodiment, neoepitope for the subject is determined and T cells specific for these antigens are isolated. Antigen-specific cells for use in augmentation are, for example, US Patent Application Publication No. US 20040224402 or US Pat. No. 2, entitled Generation And Isolation of Antigen-Specific T Cells. It may be produced in vitro using a number of methods known in the art, such as those described in 6,040,177. Antigen-specific cells for use in the present invention are described, for example, in Current Protocols in Immunology or Current Protocols in Cell Biology, all published by John Wiley & Sons, Inc., Boston, Mass. It may be produced using a number of methods known in the art.

In related embodiments, it may be desirable to screen antigen-specific cells or otherwise positively select them (eg, via magnetic selection) before or after one or two augmentations. Selection or positive selection of antigen-specific cells can be performed using peptide-MHC tetramers (Altman, et al., Science. 1996 Oct.4; 274 (5284): 94-6). In another embodiment, an adaptive tetramer technology approach is used (Andersen et al., 2012 Nat Protoc. 7: 891-902). Tetramers are limited by the need to use binding peptides predicted based on prior hypotheses and constraints on specific HLA. Peptide-MHC tetramers can be produced using techniques known in the art and can be made from the MHC molecule of interest and the antigen of interest described herein. Specific epitopes used in this regard can be identified using a number of assays known in the art. For example, the ability of a polypeptide to bind MHC class I indirectly by monitoring its ability to promote the uptake of ¹²⁵ I-labeled β2 microglobulin (β2m) into the MHC class I / β2m / peptide heterotrimmer complex. Can be evaluated (see Parker et al., J. Immunol. 152: 163, 1994).

In some embodiments, T cells are recombinantly modified to express a modified or chimeric receptor (eg, chimeric antigen receptor (CAR) modified T cells).

In one embodiment, cells are directly labeled with epitope-specific reagents for flow cytometric isolation, followed by phenotyping and TCR characterization. In one embodiment, T cells are isolated by contacting them with T cell specific antibodies. Selection of antigen-specific T cells, or cells of the invention in general, is performed by MoFlo Sorter (DakoCytomation, Fort Collins, Colo.), FACSAria ™, FACSArray ™, FACSVantage ™, BD ™ LSR. II, and can be performed using any of a variety of commercially available cell sorters, including, but not limited to, FACSCalibur ™ (BD Biosciences, San Jose, Calif.).

In one embodiment, the method comprises selecting cells that also express CD3. The method may include the step of specifically selecting cells in an appropriate manner. Preferably, the selection is performed using flow cytometry. Flow cytometry can be performed using suitable methods known in the art. Appropriate antibodies and dyes can be used for flow cytometry. Preferably, the antibody is selected to specifically recognize and bind to a particular biomarker of choice. For example, specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 can be anti-CD3 antibody, anti-CD8 antibody, anti-TIM-3 antibody, anti-LAG-3 antibody, respectively. , Anti-4-1BB antibody, or anti-PD-1 antibody can be used. The antibody can be conjugated to beads (eg, magnetic beads) or a fluorescent dye. Preferably, the flow cytometry is fluorescence labeling cell fractionation (FACS). The TCR expressed on T cells can be selected based on its reactivity to autologous tumors. In addition, tumor-reactive T cells can be selected based on markers using the methods described in Patent Publication Nos. WO2014133567 and WO2014133568, which are incorporated herein by reference in their entirety. In addition, activated T cells can be selected based on surface expression of CD107a.

In one embodiment, the method further comprises increasing the number of T cells in the enriched cell population. Such methods are described in US Pat. No. 8,637,307, which is incorporated herein by reference in its entirety. T cells can be expanded before or after treatment of cells with the PTD-MYC polypeptide. The number of T cells is at least about 3 times (or 4 times, 5 times, 6 times, 7 times, 8 times, or 9 times), more preferably at least about 10 times (or 20 times, 30 times, 40 times). , 50 times, 60 times, 70 times, 80 times, or 90 times), more preferably at least about 100 times, more preferably at least about 1,000 times, most preferably at least about 100,000 times. The number of T cells can be increased using suitable methods known in the art. Exemplary methods of increasing the number of cells are described in Japanese Patent Publication No. WO 2003057171, US Pat. No. 8,034,334, and US Patent Application Publication No. 2012/0244133, each of which is herein by reference. Incorporated in.

In one embodiment, Exvivo T cell growth can be accomplished by T cell isolation and subsequent stimulation or activation, followed by further growth. In one embodiment of the invention, T cells can be stimulated or activated by a single agent. In another embodiment, T cells are stimulated or activated by two agents, one that induces a primary signal and the other that is a co-stimulatory signal. Ligand useful for stimulating a single signal or stimulating a primary signal, and accessory molecules that stimulate a second signal can be used in soluble form. The ligand can be attached to the surface of the cell, the Engineered Multivalent Signaling Platform (EMSP), or immobilized on the surface. In one embodiment, both the first and second agents are co-immobilized on the surface, eg, beads or cells. In one embodiment, the molecule that provides the primary activation signal can be a CD3 ligand and the co-stimulatory molecule can be a CD28 ligand or a 4-1BB ligand. In some embodiments, cells are expanded by stimulation with one or more antigens, such as melanoma tumor antigens or antigens derived from a patient's tumor.

In some embodiments, isolated immune cells are treated immediately after isolation with a PTD-MYC fusion polypeptide. In another embodiment, isolated immune cells are stored and frozen in a suitable buffer prior to being treated with the PTD-MYC fusion polypeptide. In some embodiments, isolated immune cells are immediately treated with a PTD-MYC fusion polypeptide after isolation and adequate buffering until the treated cells are needed for administration to a patient. Stored in liquid and frozen.

In certain embodiments, PTD-MYC fusion of isolated immune cells (eg, mixed herd immunity cells, or isolated forms such as tumor-infiltrating lymphocytes) for a period sufficient to be taken up by the cells. Contact with a composition containing the polypeptide. In some embodiments, the immune cells are subjected to less than about 24 hours, less than about 23 hours, less than about 22 hours, less than about 21 hours, less than about 20 hours, less than about 19 hours, less than about 18 hours, less than about 17 hours, Less than about 16 hours, less than about 15 hours, less than about 14 hours, less than about 13 hours, less than about 12 hours, less than about 11 hours, less than about 10 hours, less than about 9 hours, less than about 8 hours, less than about 7 hours, Contact with a composition containing the PTD-MYC fusion polypeptide for less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour.

In certain embodiments, the immune cells are subjected to less than about 55 minutes, less than about 50 minutes, less than about 45 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 29 minutes, less than about 28 minutes, Less than about 27 minutes, less than about 26 minutes, less than about 25 minutes, less than about 24 minutes, less than about 23 minutes, less than about 22 minutes, less than about 21 minutes, less than about 20 minutes, less than about 19 minutes, less than about 18 minutes, Less than about 17 minutes, less than about 16 minutes, less than about 15 minutes, less than about 14 minutes, less than about 13 minutes, less than about 12 minutes, less than about 11 minutes, or less than about 10 minutes, containing PTD-MYC fusion polypeptide Contact with the composition. In certain embodiments, the immune cells are contacted with a composition containing the PTD-MYC fusion polypeptide for approximately 1 hour.

In certain embodiments, immune cells are contacted with a composition containing the PTD-MYC fusion polypeptide for 24 hours or longer. In certain embodiments, the immune cells are subjected to less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, Contact with a composition containing the PTD-MYC fusion polypeptide for less than about 4 days, less than about 2 days, or less than about 1 day.

In certain embodiments that may be combined with any of the above embodiments, the cells are subjected to 0.5 μg / ml to 500 μg / ml, 0.5 μg / ml, at least 0.6 μg / ml, at least 0.7 μg / ml, at least 0.8 μg /. ml, at least 0.9 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 3 μg / ml, at least 4 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 7 μg / ml, at least 8 μg / ml, at least 9 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 30 μg / ml, at least 35 μg / ml, at least 40 μg / ml, at least 45 μg / ml, at least 50 μg / ml, at least 55 μg / ml, at least 60 μg / ml, at least 65 μg / ml, at least 70 μg / ml, at least 75 μg / ml, at least 80 μg / ml, at least 85 μg / ml, at least 90 μg / ml, at least 95 μg / ml, or at least 100 μg / ml Then contact with the MYC fusion polypeptide.

MYC Fusion Protein In some embodiments, the PTD-MYC fusion polypeptide comprises a protein transfection domain (PTD), a MYC polypeptide that promotes one or more of cell survival or proliferation, and optionally a protein. Includes a tag domain, eg, one or more amino acid sequences that facilitate purification of the fusion protein. In some embodiments, cells contacted with the MYC polypeptide have increased survival time (eg, compared to cells of the same type or similar that have not been contacted with MYC) and / or (eg, MYC). Shows increased proliferation (compared to identical or similar cells of the same type that are not in contact with).

In some embodiments, the fusion protein comprises (a) a protein transduction domain; and (b) a MYC polypeptide sequence. In some embodiments, the fusion peptide is represented by formula (I) :.
Protein transduction domain-A peptide of the MYC polypeptide sequence.

In some embodiments, the fusion peptides disclosed herein link (a) a protein transduction domain; (b) a MYC polypeptide sequence; and (c) a protein transduction domain to a MYC polypeptide sequence. Contains one or more molecules. In some embodiments, the fusion peptide is represented by formula (II) :.
Protein transduction domain-a peptide of the X-MYC polypeptide sequence,
Here, -X- is a molecule that links the protein transduction domain with the MYC polypeptide sequence. In some embodiments, -X- is at least one amino acid.

In some embodiments, the fusion peptides disclosed herein include (a) a protein transducing domain; (b) a MYC polypeptide sequence; (c) at least two protein tags; optionally, ( d) Includes linker. In some embodiments, the fusion peptide is formulated (III-VI):
Protein transfection domain-X-MYC polypeptide sequence-X-protein tag 1-X-protein tag 2 (formula (III)), or protein transfection domain-MYC polypeptide sequence-X-protein tag 1-X-protein Tag 2 (formula (IV)), or protein transfection domain-MYC polypeptide sequence-protein tag 1-X-protein tag 2 (formula (V)), or protein transfection domain-MYC polypeptide sequence-protein tag 1 -Protein tag 2 (Equation (VI))
Is a peptide of
Where -X- is a linker. In some embodiments, -X- is one or more amino acids.

In some embodiments, the fusion peptides disclosed herein comprise (a) a protein transducing domain; (b) a MYC polypeptide sequence; (c) a 6-histidine tag; (d) a V5 epitope tag. , Optionally include (e) linker. In some embodiments, the fusion peptide is of formula (VII-XIV):
Protein Transfection Domain-X-MYC Polypeptide Sequence-X-6-Histidine Tag-X-V5 Epitope Tag (Formula (VII)), or Protein Transfection Domain-MYC Polypeptide Sequence-X-6-Histidine Tag-X -V5 epitope tag (formula (VIII)), or protein transfection domain-MYC polypeptide sequence-6-histidine tag-X-V5 epitope tag (formula (IX)), or protein transfection domain-MYC polypeptide sequence- 6-Histidine tag-V5 epitope tag (formula (X)),
Protein transfection domain-X-MYC polypeptide sequence-X-V5 epitope tag-X-6-histidine tag (formula (XI)), or protein transfection domain-MYC polypeptide sequence-X-V5 epitope tag-X- 6-Histidine tag (formula (XII)), or protein transfection domain-MYC polypeptide sequence-V5 epitope tag-X-6-histidine tag (formula (XIII)), or protein transfection domain-MYC polypeptide sequence- V5 epitope tag-6-histidine tag (formula (XIV))
Is a peptide of
Where -X- is a linker. In some embodiments, -X- is one or more amino acids.

As mentioned above, in some embodiments, the MYC fusion protein comprises one or more linker sequences. The linker sequence can be used to link the protein transduction domain of the fusion protein, the MYC polypeptide sequence, the V5 epitope tag, and / or the 6-histidine tag. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the amino acid sequence of the linker comprises KGERNSKLE. In some embodiments, the linker comprises the amino acid sequence of RTG.

のアミノ酸配列を含む。 Protein transduction domain (PTD)
In some embodiments, the MYC fusion protein comprises a protein transduction domain. Peptide transport provides an alternative method for the delivery of small molecules, proteins, or nucleic acids to the intracellular compartment of a cell via the cell membrane. One non-limiting example of a well-characterized protein transduction domain (PTD) is a TAT-derived peptide. Frankel et al. (See, for example, US Pat. No. 5,804,604, US Pat. No. 5,747,641, US Pat. No. 5,674,980, US Pat. No. 5,670,617, and US Pat. No. 5,652,122) contain amino acids 48-57 of TAT. We have demonstrated the transport of cargo proteins (β-galactosidase or western wasabi peroxidase) to cells by conjugating the resulting peptides with cargo proteins. In some embodiments, the TAT

Contains the amino acid sequence of.

Another non-limiting example of PTD is penetratin. Penetratin is capable of transporting hydrophilic macromolecules through the cell membrane (Derossi et al., Trends Cell Biol., 8: 84-87 (1998), which is incorporated herein by reference in its entirety). Penetratin is a Drosophila transcription factor that translocates into cultured cells, a 16-amino acid peptide that corresponds to amino acids 43-58 in the homeodomain of Antennapedia.

In addition, another non-limiting example of PTD is VP22. The herpes simplex virus type 1 (HSV-1) -derived coat protein, VP22, has the ability to transport proteins and nucleic acids through the cell membrane (Elliot et al., All incorporated herein by reference). Cell 88: 223-233, 1997). Residues 267-300 of VP22 are required, but cannot be sufficient for transport. Since the region responsible for the transport function has not been identified, the entire VP22 protein is generally used to transport cargo proteins and nucleic acids through the cell membrane (Schwarze et al., Trends Pharmacol Sci, 21:45). -48,2000).

In some embodiments, the PTD-MYC fusion polypeptide comprises a protein transduction domain. For example, in some embodiments, but not limited to, the protein transduction domain is one or more of TAT, penetratin, VP22, vpr, EPTD, R9, R15, VP16, and Antennapedia. including. In some embodiments, the protein transduction domain comprises one or more protein transduction domains of TAT, penetratin, VP22, vpr, and EPTD. In some embodiments, the protein transduction domain comprises at least one protein transduction domain of TAT, penetratin, VP22, vpr, EPTD, R9, R15, VP16, and Antennapedia. In some embodiments, the protein transduction domain comprises a synthetic protein transduction domain (eg, polyarginine or PTD-5). In a specific embodiment, the protein transduction domain comprises a TAT protein transduction domain. In some embodiments, the protein transduction domain is covalently linked to the MYC polypeptide. In some embodiments, the protein transduction domain is linked to the MYC polypeptide via a peptide bond. In some embodiments, the protein transduction domain is linked to the MYC polypeptide via a linker sequence. In some embodiments, the linker comprises a short amino acid sequence. For example, in some embodiments, but not limited to, the linker sequence is 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, 5 amino acid length, 6 amino acid length, 7 amino acid length, 8 amino acid length, 9 amino acids. Amino acid length, or 10 amino acid length.

The MYC fusion proteins of the technology of the invention may be arranged in the desired order. For example, in some embodiments, the MYC fusion protein is (a) a protein transduction domain linked in-frame to a MYC polypeptide, (b) a MYC polypeptide linked in-frame to a V5 domain, and (c. ) 6-The order of the V5 domain linked with the histidine epitope tag in frame may be used. In some embodiments, the MYC fusion protein is (a) a MYC polypeptide linked in-frame with a protein transduction domain, (b) a protein transduction domain linked in-frame with a V5 domain, and (c). It has a component sequence of V5 domain linked in frame with the 6-histidine epitope tag. In some embodiments, additional amino acid sequences may be included between each sequence. In some embodiments, the additional amino acid may be included at the beginning and / or end of the polypeptide sequence.

In some embodiments, the protein transduction domain is a TAT protein transduction domain. In some embodiments, the protein transduction domain is TAT _[48-57] . In some embodiments, the protein transduction domain is TAT _[57-48] .

Protein Tag Domain In some embodiments, the MYC fusion protein comprises a protein tag domain comprising one or more amino acid sequences that facilitate purification of the fusion protein. In some embodiments, the protein tag domain comprises one or more of a polyhistidine tag and an epitope tag. For example, but not limited to, exemplary tags include V5, histidine tags (eg, 6-histidine tags), HA (hemoggregating elements) tags, FLAG tags, CBP (carmodulin binding peptides), CYDs (covalent binding). However, it contains one or more of dissociable NorpD peptides), StrepII, or HPCs (heavy chains of protein C). In some embodiments, the protein tag domain comprises about 10-20 amino acids in length. In some embodiments, the protein tag domain comprises 2-40 amino acids in length, eg, 6-20 amino acids in length. In some embodiments, two of the tags listed above (eg, V5 and HIS tags) are used together to form a protein tag domain.

のアミノ酸配列を含む。いくつかの態様において、V5タグは、

のアミノ酸配列を含む。 In some embodiments, the histidine tag is a 6-histidine tag. In some embodiments, the histidine tag comprises the sequence HHHHHH (SEQ ID NO: 8). In some embodiments, the fusion peptides disclosed herein include a V5 epitope tag. In some embodiments, the V5 tag

Contains the amino acid sequence of. In some embodiments, the V5 tag

Contains the amino acid sequence of.

Protein tags can be added to the fusion proteins disclosed herein by appropriate methods. For example, in some embodiments, but not limited to, the TAT-MYC polypeptide sequence is cloned into an expression vector encoding one or more protein tags, such as polyHis and / or V5 tags. In some embodiments, the polyhistidine tag and / or V5 tag is added by PCR (ie, the PCR primer comprises a polyhistidine sequence and / or a V5 sequence).

Construction of PTD-MYC Fusion Polypeptides The PTD-MYC fusion polypeptides disclosed herein (eg, TAT-MYC fusion polypeptides) can be constructed by methods well known in the art. For example, without limitation, the nucleotide sequence encoding the TAT-MYC fusion polypeptide can be generated by PCR. In some embodiments, the forward primer for the human MYC sequence comprises an in-frame N-terminal 9 amino acid sequence of the TAT protein transduction domain (eg, RKKRRQRRR). In some embodiments, reverse primers for human MYC sequences are designed to remove stop codons. In some embodiments, the PCR product is cloned into a suitable expression vector. In some embodiments, the expression vector comprises a polyhistidine tag and a V5 tag.

In some embodiments, the fusion peptides disclosed herein include (a) TAT and (b) c-MYC. In some embodiments, the fusion peptides disclosed herein include (a) TAT _[48-57] and (b) c-MYC. In some embodiments, the fusion peptides disclosed herein include (a) TAT _[57-48] and (b) c-MYC.

In some embodiments, the fusion peptides disclosed herein are (a) TAT, (b) c-MYC, (c) linker, (d) V5 tag, and (e) 6-histidine tag. Including. In some embodiments, the fusion peptides disclosed herein are (a) TAT _[48-57] , (b) c-MYC, (c) linker, (d) V5 tag, and (e). 6-Includes histidine tag. In some embodiments, the fusion peptides disclosed herein are (a) TAT _[57-48] , (b) c-MYC, (c) linker, (d) V5 tag, and (e). 6-Includes histidine tag.

In some embodiments, the PTD-MYC fusion polypeptide comprises SEQ ID NO: 1; in some embodiments, the PTD-MYC fusion polypeptide is SEQ ID NO: 1.

The fusion protein can be modified during or after synthesis to include one or more functional groups. For example, without limitation, proteins can be modified to contain one or more of acetyl, phosphoric acid, acetic acid, amide, alkyl, and / or methyl groups. This list is not exhaustive and is only exemplary. In some embodiments, the protein comprises at least one acetyl group.

PTD-MYC fusion polypeptides can be produced by suitable methods known in the art, such as recombinant protein expression in cells such as bacterial cells, insect cells, or mammalian cells. In some embodiments, the PTD-MYC fusion polypeptide is recombinantly made by microbial fermentation. In some embodiments, the microbial fermentation is carried out in a fermentation volume of about 1 to about 10,000 liters, eg, a fermentation volume of about 10 to about 1000 liters. For fermentation, suitable microbial host cells and culture medium can be used. In an exemplary embodiment, E. coli is used as a microbial host cell. In another embodiment, other microorganisms such as S. cerevisiae, P. pastoris, Lactobacillus, Bacilli, and Aspergilli may be used. In an exemplary embodiment, the microbial host cell is BL-21 Star ™ E. coli strain (Invitrogen). In an exemplary embodiment, the microbial host cell is a BLR DE3 E. coli strain.

In some embodiments, the host cell is modified to provide tRNA for the rare codon used to overcome the host microbial cell codon bias in order to improve translation of the expressed protein. In an exemplary embodiment, a host cell transformed with a plasmid such as pRARE (CamR) expressing tRNA for AGG codon, AGA codon, AUA codon, CUA codon, CCC codon, GGA codon (eg, E. coli). ). Additional suitable plasmids or constructs for providing tRNAs for a particular codon are known in the art and can be used in the methods provided.

Integrative or self-replicating vectors can be used to introduce the PTD-MYC fusion polypeptide expression cassette into selected host cells. In the expression cassette, the coding sequence for the PTD-MYC fusion polypeptide is functionally linked to a promoter such as an inducible promoter. An inducible promoter is a promoter that initiates increased levels of transcription from DNA under its control in response to any change in culture conditions, such as the presence or absence of nutrients or changes in temperature. In some embodiments, the nucleic acid encoding the PTD-MYC fusion polypeptide is a codon optimized for bacterial expression.

Illustrative promoters recognized by a variety of potential host cells are well known. These promoters, if present, are functionally linked to the DNA encoding the PTD-MYC fusion polypeptide by removing the promoter from the DNA of origin by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Can be done. Suitable promoters for use with microbial hosts include the β-lactamase and lactose promoter system (Chang et al., (1978) Nature, 275: 617-624; Goeddel et al., (1979) Nature, 281: 544). , Alkaline phosphatase, tryptophan (trp) promoter system (Goeddel (1980) Nucleic Acids Res. 8: 4057; EP 36,776), and tac promoter (deBoer et al., (1983) Proc. Natl. Acad. Sci. USA 80: Includes, but is not limited to, hybrid promoters such as 21-25). Promoters suitable for expression by selected host cells can be used. Appropriate nucleotides that allow one of ordinary skill in the art to functionally ligate the DNA encoding the PTD-MYC fusion polypeptide using a linker or adapter to provide the required restriction sites. The sequences are publicly available (see, eg, Siebenlist et al., (1980) Cell 20: 269). In an exemplary embodiment, the promoter for use in a bacterial system may contain a Shine-Dalgarno (S.D.) sequence operably linked to a coding sequence. In some embodiments, the inducible promoter is a lacZ promoter induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), which is well known in the art. Promoters and expression cassettes may be newly synthesized using well-known techniques for synthesizing DNA sequences of interest. In an exemplary embodiment, the expression vector for the expression of the PTD-MYC fusion polypeptide herein is pET101 / D-Topo (Invitrogen).

Due to the expression of the PTD-MYC fusion polypeptide, the microbial host containing the expression vector encoding the PTD-MYC fusion polypeptide typically grows densely in the fermentation reactor. In some embodiments, the reactor has a controlled feed of glucose. In some embodiments, the fermenter inoculum is first cultured in a medium supplemented with antibiotics (eg, overnight culture). The fermenter inoculum is then used to inoculate the fermenter culture for protein expression. Expression of recombinant protein is induced in at least about 15, generally at least about 20, at least 25, at least about 30, or more OD600 in fermenter cultures. In an exemplary embodiment, when the inducible promoter is the lacZ promoter, IPTG is added to the fermentation medium to induce expression of the PTD-MYC fusion polypeptide. Generally, IPTG is added to the fermenter culture at OD600, which represents the logarithmic growth phase.

In certain aspects of the provided method, induced protein expression can be maintained for about 2 to about 5 hours after induction and for about 2 to about 3 hours after induction. A longer induction period may be undesirable due to the degradation of recombinant proteins. The temperature of the reaction mixture during induction is preferably from about 28 ° C to about 37 ° C, generally from about 30 ° C to about 37 ° C. In certain embodiments, the induction is at about 37 ° C.

The PTD-MYC fusion polypeptide is typically expressed as a cytosol inclusion body in microbial cells. To collect inclusion bodies, after induction, cell pelletes are collected by centrifugation of the fermentation culture, frozen at -70 ° C or below, thawed and resuspended in disruption buffer. The cells are lysed by conventional methods such as sonication, homogenization and the like. The lysate is then placed in solubilization buffer, generally in the presence of urea at a concentration effective for solubilizing the protein, eg, about 5M, 6M, 7M, 8M, 9M, or higher. Resuspend. Resuspension may require mechanical breaking and stirring of pellets to achieve homogeneity. In some embodiments, the cell pellet is resuspended directly in urea buffer and mixed until homogeneous. In some embodiments, the resuspension / solubilization buffer is 8M urea, 50 mM phosphate pH 7.5 and the suspension is passed through a homogenizer.

In some embodiments, the homogenized suspension is sulfonylated. For example, in some embodiments, the homogenized suspension is adjusted to contain 200 mM sodium sulfite and 10 mM sodium tetrathionate. The solution is then mixed at room temperature until homogeneous. The mixed lysates are then mixed for an additional time (eg, 2-8 ° C. for 12 hours or longer) to complete the sulfonylation. The sulfonylated lysate was then centrifuged for 1 hour. The supernatant containing the sulfonylated PTD-MYC fusion polypeptide is then collected by centrifugation and the cell pellet is discarded. The supernatant is then passed through a filter, eg, a 0.22 μm membrane filter, to clarify the lysate.

The solubilized protein is then purified. Purification methods may include affinity chromatography, reverse phase chromatography, gel exclusion chromatography and the like. In some embodiments, affinity chromatography is used. For example, an epitope tag or a histidine 6 tag is provided on the protein for convenient purification. In the methods of the invention, the exemplary PTD-MYC fusion polypeptide comprises a histidine 6 tag for purification using Ni affinity chromatography with Ni resin.

In an exemplary embodiment, the Ni resin column is equilibrated with a buffer containing urea. In some embodiments, the equilibration buffer is a 6M urea, 50 mM phosphate, 500 mM NaCl, 10% glycerol solution. The sulfonylated and clarified supernatant containing the PTD-MYC fusion polypeptide is then loaded onto a Ni resin column. The column is then washed with wash buffer, such as 6M urea, 50 mM phosphate, 10% glycerol, 500 mM NaCl, pH 7.5. The column was then washed continuously with wash buffer with a decreasing salt concentration. For example, exemplary subsequent washes may include 6M urea, 50 mM phosphoric acid, 10% glycerol, and 2M NaCl, pH 7.5, followed by 6M urea, 50 mM phosphoric acid, 10% glycerol, 50 mM NaCl, And 30 mM imidazole, washed once more at pH 7.5.

After continuous application of wash buffer, the PTD-MYC fusion polypeptide is added to the elution buffer, eg, 6M urea, 50 mM phosphate, 10% glycerol, and 50 mM NaCl, pH 7.5 with a gradient of 100-300 mM imidazole. Elute from the column by addition and collect fractions. The protein-containing fraction to be pooled is then filtered through a 0.22 μm membrane. Assessment of protein yield can be measured using a suitable method, eg, spectrophotometry at UV wavelength 280.

In some embodiments, one or more additional purification methods can be used to further purify the isolated PTD-MYC fusion polypeptide. In an exemplary embodiment, the fraction pooled from the Ni Sepharose chromatography step is further purified by anion exchange chromatography using a Q Sepharose resin. In some embodiments, the pool is subjected to a second wash buffer (eg, 6M urea, 50 mM phosphate, 10% glycerol, 2M NaCl, pH 7.5) from the Ni Sepharose chromatography step and the conductivity of the Q Sepharose buffer. Prepared for loading on the Q-sepharose column by diluting the sample to (17.52 +/- 1 mS / cm), then loading the diluted pool onto the Q-sepharose column and then chase. ) Perform two chase steps with buffer (eg 6M urea, 50 mM phosphate, 300 mM NaCl, and 10% glycerol) until the UV trace reaches the baseline and the protein is shown to have eluted from the column. , Chase buffer is further applied continuously.

Method of treatment
PTD-MYC fusion polypeptide-modified immune cells are administered for the treatment of melanoma in patients. In some embodiments, the patient has metastatic melanoma. In some embodiments, the patient has received one or more agents for the treatment of melanoma prior to administration of PTD-MYC fusion polypeptide-modified immune cells. In some embodiments, the melanoma is a recurrent melanoma or a refractory melanoma. In some embodiments, the melanoma is a metastatic melanoma. In some embodiments, the melanoma is a superficial extended melanoma, a nodular melanoma, a malignant mole-derived melanoma, or a terminal melanoma. In some embodiments, melanoma is resistant to one or more agents for the treatment of melanoma.

In some embodiments, administration of PTD-MYC fusion polypeptide-modified immune cells inhibits the growth of melanoma tumors or reduces the volume of melanoma tumors. In some embodiments, administration of PTD-MYC fusion polypeptide-modified immune cells to a subject with melanoma alleviates one or more symptoms of melanoma. In some embodiments, administration of PTD-MYC fusion polypeptide-modified immune cells to a subject with melanoma increases the overall survival of the subject. In some embodiments, administration of PTD-MYC fusion polypeptide-modified immune cells to a subject with melanoma increases melanoma regression.

Administration of PTD-MYC fusion polypeptide-modified immune cells (eg, tumor-infiltrating lymphocytes treated with PTD-MYC fusion polypeptide) by the methods provided herein can be injected, infused, implanted, or transplanted. Can be performed in a manner suitable for administration of cells to a subject, including, but not limited to,. In some embodiments, the PTD-MYC fusion polypeptide-modified immune cells are administered to the patient subcutaneously, intradermally, intratumorally, intralymph nodeally, or in the spinal cord. It is administered intramuscularly, intramuscularly, intrathecally, by intravenous or lymphatic injection, or intraperitoneally. In some embodiments, PTD-MYC fusion polypeptide immune cells are administered into a cavity formed by excision of tumor tissue (ie, intracavitary delivery) or directly into the tumor prior to excision (ie,). , Intratumoral delivery). In one embodiment, the MYC fusion polypeptide immune cells are administered by intravenous injection.

In addition to PTD-MYC fusion polypeptide-modified immune cells, compositions for administration include pharmaceutically acceptable carriers, buffers, excipients, supplements, additives, preservatives, bulking agents, stables. It may contain agents and / or thickeners, and / or other agents, such as components commonly found in the corresponding product. Selection of suitable components and suitable manufacturing methods for formulating compositions for a particular route of administration is generally known in the art.

Adoptive cell therapeutic compositions comprising PTD-MYC fusion polypeptide-modified immune cells can be in any form, such as solid, semi-solid, or liquid suitable for administration. Formulations can be selected from, but are not limited to, the group consisting of solutions, emulsions, suspensions, tablets, pellets, and capsules. The composition is not limited to certain formulations, and the composition can be formulated into any known pharmaceutically acceptable formulation. Pharmaceutical compositions can be made by conventional processes known in the art.

In some embodiments, administration of the MYC fusion polypeptide modified immune cells, 10 ⁴ to 10 ¹⁰ cells per body weight 1 kg, e.g., ^{105 to 106} cells per body weight 1 kg (cells within these ranges Includes administration of) (including all integer values of the number). In some embodiments, cells are administered with or without the process of lymphatic depletion, eg, with cyclophosphamide.

MYC fusion polypeptide-modified immune cells can be administered in one or more doses. In one embodiment, a therapeutically effective amount of PTD-MYC fusion polypeptide-modified immune cell is administered in a single dose. In some embodiments, a single dose of PTD-MYC fusion polypeptide-modified immune cells has a therapeutic effect. In another embodiment, an effective amount of MYC fusion polypeptide-modified immune cells is administered multiple times over a period of time. The timing of dosing is within the discretion of the managing physician and includes, but is not limited to, the characteristics of the melanoma, including the patient's age, gender, or clinical status, and the type, degree, or location of the melanoma. No, it depends on various factors. Determining the optimal range of effective amounts of MYC fusion polypeptide-modified immune cells for the treatment of a particular disease or condition is within the skill of one of ordinary skill in the art, although individual needs will vary.

PTD-MYC fusion polypeptide-modified immune cells can be administered, for example, in the first 2 weeks, 3 weeks, 4 weeks, monthly, or during the treatment period 1-10 times. In some embodiments, the PTD-MYC fusion polypeptide-modified immune cell is administered 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, PTD-MYC fusion polypeptide-modified immune cells are administered weekly, every two weeks, every three weeks, or every month.

A therapeutically effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the age, health, and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment, and the nature of the desired effect.

In some embodiments, the patient receiving PTD-MYC modified immune cells is first pretreated with one or more cytokines and / or other immunomodulators. In some embodiments, patients who receive PTD-MYC modified immune cells are depleted of lymph prior to administration of PTD-MYC modified immune cells. The purpose of lymph depletion is to make room for infused lymphocytes, in particular by eliminating regulatory T cells and other non-specific T cells that compete for homeostatic cytokines.

In some embodiments, PTD-MYC modified immune cells are administered with additional therapeutic material. In some embodiments, additional therapeutic material is administered prior to, at the same time, intermittently or thereafter, prior to treatment with PTD-MYC modified immune cells. In some embodiments, additional therapeutic agents are immunomodulators such as interleukins (eg, IL-2, IL-7, IL-12), cytokines, chemokines, or immunomodulators. In some embodiments, the cytokines are interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFα, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14- 1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5 (= RANTES), CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2 CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCL8, CXCL9, CXCL9, CXCR1 It is selected from the cytokines selected from the group consisting of. In some embodiments, the additional therapeutic substance is an anti-cancer agent such as chemotherapy or radiation therapy.

In some embodiments, the modified immune cells administered for the treatment of melanoma are T cells with a genetically modified antigen receptor, including chimeric antigen receptor (CAR) -T cells. .. By modifying the specificity of the T cell receptor (TCR), eg, by introducing new TCR α and β chains with selected peptide specificity (eg, US Pat. No. 8,697,854; PCT Patent Publication WO2003020763, WO2004033685, WO2004044004, WO2005114215, WO2006000830, WO2008038002, WO2008039818, WO2004074322, WO2005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173; see US Pat. No. 8,088,379), T cells genetically For example, various strategies can be used. Chimeric antigen receptors (CARs) can be used to generate immune-responsive cells such as T cells specific for selected targets such as malignant cells, and a variety of receptor chimeric constructs have been described. (For example, US Pat. No. 5,843,728; No. 5,851,828; No. 5,912,170; No. 6,004,811; No. 6,284,240; No. 6,392,013; No. 6,410,014; No. 6,753,162; No. 8,211,422; PCT Gazette WO9215322). Methods for the preparation of CAR T cells are known in the art and are described herein to generate modified CAR T cells containing the MYC fusion polypeptides (eg, PTDs) described herein. Can be used in combination with the methods provided in the book.

In general, CAR is composed of an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a predetermined target-specific antigen binding domain. The antigen-binding domain of CAR is often an antibody or antibody fragment (eg, single chain variable fragment, scFv), but the binding domain is not particularly limited as long as it provides specific recognition of the target. For example, in some embodiments, the antigen binding domain may include a receptor such that CAR can bind to the ligand of that receptor. Alternatively, the antigen binding domain may include the ligand so that CAR can bind to the endogenous receptor of the ligand.

In some embodiments, T cells expressing the desired CAR are selected through co-culture with γ-irradiated activating and propagating cells (AaPC) that co-express melanoma antigens and costimulatory molecules. To. In some embodiments, modified CAR T cells are expanded by co-culture with AaPC in the presence of soluble factors such as IL-2 and IL-21. This increase can be performed, for example, to provide memory CAR + T cells. Thus, CAR T cells can be provided that have specific cytotoxic activity against antigen-carrying tumors (optionally with the production of the desired chemokine such as interferon γ).

In some embodiments, CAR T cells are contacted in vitro with the PTD-MYC fusion polypeptide provided herein to generate modified CAR T cells for the treatment of melanoma. Modified CAR T cells can be administered according to appropriate methods, including the methods for administration of the PTD-MYC fusion polypeptide modified immune cells described above.

Kits Pharmaceutical compositions comprising MYC fusion polypeptides and / or MYC fusion polypeptide-modified immune cells provided herein can be assembled into kits or pharmaceutical systems for use in the treatment of melanoma. A kit according to this embodiment may include a delivery means such as a box, carton, tube in which one or more containers such as vials, tubes, ampoules, bottles, syringes, or bags are sealed. The kit may also include relevant instructions for using MYC fusion polypeptides and / or MYC fusion polypeptide-modified immune cells.

In some embodiments, the kit comprises an effective amount of adoptive cell therapy, such as MYC fusion polypeptide modified immune cells. In some embodiments, the kit comprises one or more reagents for the detection of administered MYC fusion polypeptide and / or MYC fusion polypeptide modified immune cells. In some embodiments, the kit comprises cells for treatment with the MYC fusion polypeptide provided herein, such as hematopoietic stem cells, donor leukocytes, T cells, or NK cells. In some embodiments, the kit further comprises an effective amount of a Therapeutic agent administered in combination with the MYC fusion polypeptide and / or MYC fusion polypeptide modified immune cells provided herein. In some embodiments, the therapeutic substance is an anti-cancer agent.

The kits provided herein may also include devices for administering to a subject the MYC fusion polypeptide and / or MYC fusion polypeptide modified immune cells provided herein. Any of a variety of devices known in the art for administering polypeptides and cells to a subject may be included in the kits provided herein. Exemplary devices include subcutaneous injection needles, intravenous injection needles, catheters, needleless injections, such as subcutaneous injection needles, intravenous needles, catheters, needleless injection devices, inhalers, and eye drops. Not limited to liquid dispensers. Typically, the device for administering the MYC fusion polypeptide and / or MYC fusion polypeptide modified immune cells of the kit is compatible with the desired method of administration of the composition. For example, the composition delivered intravenously may be included in the kit along with a subcutaneous needle and syringe.

Example 1. Immune cells treated with TAT-MYC to produce TAT-MYC-treated lymphocytes for immunotherapy of melanoma tumors In this example, the HIV-1 trans-activating protein ( The ability of PTD-MYC fusion polypeptides containing the protein transfection domain of TAT) and MYC to modulate the immune response to melanoma cells in vivo was investigated. Specifically, we studied the ability of lymphoid cells derived from melanoma-carrying mice and treated with TAT-MYC to treat mice carrying melanoma tumors. The purpose of these studies is to derive TAT-MYC lymphocytes from melanoma-carrying mice, so that immune cells treated with TAT-MYC are effective for melanoma tumors when transplanted into melanoma-carrying mice. It was to determine whether or not the treatment would be appropriate.

Materials and methods
C57BL / 6J is the most widely used inbred strain and the first strain whose genome has been sequenced. This lineage is refractory to many tumors, but is a background that allows maximum expression of most mutations. C57BL / 6J mice are resistant to primary hearing attacks, have relatively low bone mineral density, and develop age-related hearing loss. They are also susceptible to diet-induced obesity, type 2 diabetes, and atherosclerosis. Macrophages from this line are resistant to the effects of anthrax lethal toxin.

Treatment group Weighed approximately 25 g and produced 15 C57BL / 6 mice (Jackson Laboratory Stock # 000664) carrying melanoma tumors, 5 in 3 cohorts, 1 in 1 cohort as untreated control. , Derived from tumor-carrying mice and treated with lymphoid cells treated with control TAT fusion protein, and divided into one cohort treated with TAT-MYC lymphocytes.

Generation of tumor-carrying donor mice and preparation of donor cells B16-F10 melanoma cells (ATCC CRL 6475, mouse skin melanoma) for implantation in D10 medium (DMEM, 10% FBS, Pen / Strep (10,000 units / ml)) ( Gibco Cat # 15140); L-glutamine (200 mM) (Gibco Cat # 25030); MEM non-essential amino acids (Gibco Cat # 11140)).

C57BL / 6j mice (Jackson Laboratory # 003548) were implanted with 1 × 10 ⁴ B16-F10 melanoma cells in 250 μL PBS via tail intravenous injection. Prior to injection, each test mouse was placed under a 250 W heating lamp for 1-2 minutes, followed by intravenous injection of melanoma cells. On the 14th day after transplantation, lymph nodes were collected from the injected mice and disrupted with a 10 mL syringe plunger.

Lymph nodes were collected from 5 mice for the first study. For the second, lymph nodes were collected from 10 mice. Cells were washed with C10, collected and spun at 260 xg for 5 minutes. After discarding the supernatant, cells were resuspended in 10 mL sterile TAC and spun at 260 xg for 5 minutes. After discarding the supernatant, cells were resuspended in 2 mL of sterile filtered PBS containing 5% BSA.

Lymph node cells were treated with TAT-MYC or with a control TAT fusion protein to generate TAT-MYC lymphocytes. The cells were divided into two 15 mL conical tubes (1 mL each) and 1 mL of 25 ug / ml control protein (TAT-CRE for Experiment 1 and TAT-GFP for Experiment 2) or 1 mL of 25 ug / ml TAT. -Processed by MYC lot C18. After 1 hour incubation at room temperature, each tube was washed 3 times with sterile PBS, transferred to a 5 mL sterile tube and placed on ice.

For each cohort of 5 C57BL / 6j mice, test mice were prepared by injecting 1 × 10 ⁴ B16-F10 melanoma cells in 250 uL PBS into the tail vein. After the injection, the mice were observed once a day. Weight changes, feed consumption, activity, and mortality were monitored. Then, 7 days after transplantation, TAT-MYC lymphocytes or control lymphoid cells were transplanted into mice injected with melanoma cells.

Symptoms were monitored daily. Mice were euthanized and deaths were recorded if severe symptoms were presented. Mice were either found dead or euthanized if they were found to have severe symptoms such as rough breathing, circular dorsal position, and immobility. .. The date of death was recorded with the date of treatment as day 0.

The results from Experiments 1 and 2 are shown in Figures 1 and 2, respectively. As shown in the figure, treatment of melanoma-carrying mice with TAT-MYC lymphocytes (TBX-3400) generated by contacting mouse lymphoid cells derived from melanoma-carrying mice with TAT-MYC is a control TAT fusion. The overall survival of mice was significantly improved compared to transplantation of lymphoid cells treated with protein. These results suggest that TAT-MYC treatment of immune cells is useful in the treatment of melanoma using adoptive cell transfer.

Example 2. Dose-responsive effect of TAT-MYC-treated lymphocytes for immunotherapy of melanoma tumors In this example, different doses of TAT-MYC-treated lymphocytes for immunotherapy of melanoma tumors. The therapeutic effect of was investigated. This experiment was performed in a similar manner to Example 1 above, except that several different doses of TAT-MYC treated lymphocytes were injected and compared. Two experiments were carried out. In the first experiment, Experiment 3, TAT-MYC lymphocytes were administered to melanoma-carrying mice via tail intravenous injection according to the following dosing group: cells 3.0 × 10 ⁶ cells / kg, cells 6.0 × 10 ⁶ cells / kg, cells 14.0 x 10 ⁶ cells / kg, and cells 70.0 x 10 ⁶ cells / kg. For the control group, 70.0 × 10 ⁶ TAT-Cre-treated cells were administered to mice or no cells were administered (NT). In the second and fourth experiments, TAT-MYC lymphocytes were administered to melanoma-bearing mice via tail vein injection according to the following dosing group: cells 4.0 x 10 ³ cells / kg, cells 4.0 x 10 ⁴ cells / kg, cells 4.0 x 10 ⁵ cells / kg, cells 4.0 x 10 ⁶ cells / kg, and cells 4.0 x 10 ⁷ cells / kg. For the control group, 4.0 × 10 ⁶ TAT-Cre-treated cells were administered to mice or no cells were administered (NT). The results from Experiments 3 and 4 are shown in Figures 3 and 4, respectively. As shown in the figure, treatment of melanoma-carrying mice with increased amounts of TAT-MYC lymphocytes (TBX-3400) in both experiments resulted in significantly improved overall survival. These experiments demonstrate both reproducibility and efficacy of TAT-MYC lymphocytes for treating melanoma-carrying subjects.

Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variants, changes, and substitutions will be conceived by those skilled in the art without departing from this disclosure. It should be understood that various alternatives of the aspects of the present disclosure described herein can be used in the practice of the present disclosure. The appended claims define the scope of the present disclosure, thereby including the methods and structures within the scope of these claims and their equivalents.

All patents, patent applications, provisional patent applications, and publications referred to or cited herein are in their entirety, including all drawings and tables, as long as they do not conflict with the express teachings of this specification. Incorporated by. Other aspects are shown in the appended claims.

Kits containing MYC fusion polypeptides and / or MYC fusion polypeptide-modified immune cells provided herein for use in the treatment of melanoma are also provided. In some embodiments, the kit comprises one or more reagents for the detection of administered MYC fusion polypeptide and / or MYC fusion polypeptide modified immune cells. In some embodiments, the kit comprises cells for treatment with the MYC fusion polypeptide provided herein, such as hematopoietic stem cells, donor leukocytes, T cells, or NK cells. In some embodiments, the kit comprises relevant instructions for using MYC fusion polypeptides and / or MYC fusion polypeptide-modified immune cells.
[Invention 1001]
(A) (i) Protein transduction domain; (ii) MYC polypeptide sequence
MYC fusion peptide, including;
(B) One or more primary immune cells isolated from donor subjects with melanoma tumors and reactive to melanoma-specific antigens
Composition containing.
[Invention 1002]
The composition of 1001 of the present invention, wherein the MYC fusion peptide migrates to the nucleus of the one or more primary immune cells.
[Invention 1003]
The composition of the invention 1001 or 1002, wherein the MYC fusion peptide exhibits the biological activity of MYC.
[Invention 1004]
The composition of any of 1001 to 1003 of the present invention, wherein the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain to the MYC polypeptide.
[Invention 1005]
The MYC fusion peptide has the following general structure:
Protein transduction domain-X-MYC sequence
Contains the MYC fusion peptide with
Here, the composition according to any one of 1001 to 1004 of the present invention, wherein -X- is a molecule linking the protein transduction domain and the MYC sequence.
[Invention 1006]
The composition according to any one of the present inventions 1001 to 1005, wherein the protein transduction domain sequence is a TAT protein transduction domain sequence.
[Invention 1007]
The composition of the present invention 1006, wherein the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48].
[Invention 1008]
The composition of any of 1001 to 1007 of the present invention, wherein the MYC fusion peptide comprises SEQ ID NO: 1.
[Invention 1009]
The composition of any of 1001 to 1008 of the present invention, wherein the MYC fusion peptide is acetylated.
[Invention 1010]
The composition of any of 1001 to 1009 of the present invention, wherein the one or more immune cells have antitumor activity against melanoma cells.
[Invention 1011]
The composition of any of 1001 to 1010 of the present invention, wherein the one or more immune cells comprises one or more lymphocytes.
[Invention 1012]
The composition of the present invention 1011, wherein the one or more lymphocytes comprises T cells, B cells, NK cells, or a combination thereof.
[Invention 1013]
A group in which the T cells consist of naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, energy T cells, tolerant T cells, chimeric T cells, and antigen-specific T cells. More selected, the composition of the invention 1012.
[Invention 1014]
The B cells are selected from the group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. The composition of invention 1012.
[Invention 1015]
The composition of any of the present inventions 1011-1014, wherein the one or more lymphocytes are tumor infiltrating lymphocytes, T cell receptor modified lymphocytes, or chimeric antigen receptor modified lymphocytes.
[Invention 1016]
The composition of the invention 1015, wherein the tumor-infiltrating lymphocytes have a CD8 ⁺ CD28 ^- CD152 ^- signature, CD8 + CD25 + signature, or CD4 + CD25 + signature.
[Invention 1017]
The composition according to any one of 1001 to 1016 of the present invention, which comprises a portion in which the one or more immune cells can be detected.
[Invention 1018]
A step of administering one or more modified immune cells to a subject in need thereof, wherein the one or more modified immune cells contain a MYC fusion peptide and the MYC fusion peptide The step, wherein (i) a protein transfectation domain; (ii) contains a MYC polypeptide sequence and the one or more modified immune cells are reactive to a tumor-specific antigen.
Methods for treating melanoma in a subject, including.
[Invention 1019]
The method of the present invention 1018, wherein the one or more modified immune cells are derived from primary immune cells isolated from the subject.
[Invention 1020]
The method of the present invention 1018, wherein the one or more modified immune cells are derived from a primary immune cell isolated from another donor subject having the same type of melanoma.
[Invention 1021]
The method of the invention 1019 or 1020, wherein the one or more modified immune cells are prepared by isolating the primary immune cells and then contacting them with the MYC fusion peptide in vitro.
[Invention 1022]
The method of any of 1019-1021 of the present invention, further comprising the step of increasing the primary immune cells in vitro prior to contact with the MYC fusion peptide.
[Invention 1023]
The method of any of 1019-1021 of the present invention, further comprising the step of increasing the primary immune cells after contact with the MYC fusion peptide.
[1024 of the present invention]
The method of 1022 or 1023 of the present invention, which uses an anti-CD3 antibody to grow said cells.
[Invention 1025]
The method of any of 1022 to 1024 of the present invention, wherein the irradiated allogeneic feeder cells are used to grow the cells.
[Invention 1026]
The method of any of 1022-1025 of the present invention, which enlarges the cells in the presence of exogenous cytokines.
[Invention 1027]
The method of the present invention 1026, wherein the cytokine is interleukin 2.
[Invention 1028]
The method of any of 1018-1027 of the present invention, wherein the MYC fusion peptide is transferred to the nucleus of the immune cell.
[Invention 1029]
The method of any of 1018-1028 of the present invention, wherein the MYC fusion peptide exhibits the biological activity of MYC.
[Invention 1030]
The method of any of 1018-1029 of the present invention, wherein the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain to the MYC polypeptide.
[Invention 1031]
The MYC fusion peptide has the following general structure:
Protein transduction domain-X-MYC sequence
Contains the MYC fusion peptide with
Here, the method of any of 1018-1030 of the present invention, wherein -X- is a molecule that links the protein transduction domain with the MYC sequence.
[Invention 1032]
The method of any of 1018-1031 of the present invention, wherein the protein transduction domain sequence is a TAT protein transduction domain sequence.
[Invention 1033]
The method of the present invention 1032, wherein the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48].
[Invention 1034]
The method of any of 1018-1033 of the present invention, wherein the MYC fusion peptide comprises SEQ ID NO: 1.
[Invention 1035]
The method of any of 1018-1034 of the present invention, wherein the MYC fusion peptide is acetylated.
[Invention 1036]
The method of any of 1018-1035 of the present invention, wherein the one or more modified immune cells have antitumor activity against melanoma cells in the subject.
[Invention 1037]
The method of any of 1018-1036 of the present invention, wherein the one or more modified immune cells comprises one or more anerogenic immune cells.
[Invention 1038]
The method of any of 1018-1037 of the present invention, wherein the one or more immune cells comprises one or more lymphocytes.
[Invention 1039]
The method of the present invention 1038, wherein the one or more lymphocytes comprises T cells, B cells, NK cells, or a combination thereof.
[Invention 1040]
A group in which the T cells consist of naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, energy T cells, tolerant T cells, chimeric T cells, and antigen-specific T cells. More selected, the method of the present invention 1039.
[Invention 1041]
The B cells are selected from the group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. The method of invention 1039.
[Invention 1042]
The method of the invention 1038, wherein said one or more lymphocytes comprises tumor infiltrating lymphocytes, T cell receptor modified lymphocytes, or chimeric antigen receptor modified lymphocytes.
[Invention 1043]
The method of 1042 of the present invention, wherein said lymphocytes have a CD8 ⁺ CD28 ^- CD152 ^- signature, CD8 + CD25 + signature, or CD4 + CD25 + signature.
[Invention 1044]
The method of any of 1019-1043 of the present invention, further comprising the step of isolating the primary immune cells from the donor subject.
[Invention 1045]
The method of the present invention 1044, wherein the donor subject has melanoma.
[Invention 1046]
The method of any of 1018-1045 of the present invention, wherein the one or more modified immune cells are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratumorally.
[Invention 1047]
The method of any of 1018-1046 of the present invention, further comprising the step of putting the subject into a lymph depleted state prior to administration of the one or more modified immune cells.
[Invention 1048]
The method of any of 1018-1047 of the present invention, further comprising the step of administering the cytokine to the subject.
[Invention 1049]
The method of the present invention 1048, wherein the cytokine is administered before, during, or after administration of the one or more modified immune cells.
[Invention 1050]
The cytokines are interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFα, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2. , CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26 , CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CX3 , CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 The method of invention 1048 or 1049.
[Invention 1051]
The method of any of the present inventions 1018-1050, wherein the melanoma is metastatic.
[Invention 1052]
The method of any of the present inventions 1018-1051, wherein the subject is a human or animal.
[Invention 1053]
The method of any of 1018-1052 of the present invention, further comprising the step of performing additional cancer treatment.
[Invention 1054]
The additional cancer treatment is selected from among chemotherapy, radiotherapy, immunotherapy, monoclonal antibodies, anticancer nucleic acids or proteins, anticancer viruses or microorganisms, and combinations thereof, according to the invention 1053. Method.
[Invention 1055]
The method of any of 1018-1054 of the present invention comprising a detectable portion of the one or more modified immune cells.
[Invention 1056]
In vitro contact of one or more immune cells with a MYC fusion polypeptide, wherein the immune cells are derived from a donor exposed to one or more tumor antigens and the MYC fusion The step in which the peptide comprises (i) a protein transfectation domain; (ii) a MYC polypeptide sequence and is reactive to a tumor-specific antigen.
A method for preparing modified immune cells for the treatment of melanoma, including.
[Invention 1057]
The method of 1055 of the present invention, wherein the one or more modified immune cells are derived from primary immune cells isolated from a subject having melanoma.
[Invention 1058]
The method of the invention 1055 or 1056, further comprising the step of growing the primary immune cells in vitro prior to contact with the MYC fusion peptide.
[Invention 1059]
The method of any of 1055-1057 of the present invention, further comprising the step of increasing the primary immune cells after contact with the MYC fusion peptide.
[Invention 1060]
The method of any of 1055-1059 of the present invention, wherein the anti-CD3 antibody is used to grow the cells.
[Invention 1061]
The method of any of 1055-1060 of the present invention, wherein the irradiated allogeneic feeder cells are used to grow the cells.
[Invention 1062]
The method of any of 1055-1061 of the present invention, which enlarges the cells in the presence of exogenous cytokines.
[Invention 1063]
The method of 1062 of the present invention, wherein the cytokine is interleukin 2.
[Invention 1064]
The method of any of 1055-1063 of the present invention, wherein the MYC fusion peptide is transferred to the nucleus of the immune cell.
[Invention 1065]
The method of any of 1055-1064 of the present invention, wherein the MYC fusion peptide exhibits the biological activity of MYC.
[Invention 1066]
The method of any of 1055-1065 of the present invention, wherein the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain to the MYC polypeptide.
[Invention 1067]
The MYC fusion peptide has the following general structure:
Protein transduction domain-X-MYC sequence
Contains the MYC fusion peptide with
Here, the method of any of 1055-1066 of the present invention, wherein -X- is a molecule that links the protein transduction domain with the MYC sequence.
[Invention 1068]
The method of any of 1055-1067 of the present invention, wherein the protein transduced domain sequence is a TAT protein transduced domain sequence.
[Invention 1069]
The method of 1068 of the present invention, wherein the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48].
[Invention 1070]
The method of any of 1055-1069 of the present invention, wherein the MYC fusion peptide comprises SEQ ID NO: 1.
[Invention 1071]
The method of any of 1055-1070 of the present invention, wherein the MYC fusion peptide is acetylated.
[Invention 1072]
The method of any of 1055 to 1071 of the present invention, wherein the one or more modified immune cells have antitumor activity.
[Invention 1073]
The method of any of 1055-1072 of the present invention, wherein the one or more modified immune cells have antitumor activity against melanoma cells in the subject.
[Invention 1074]
The method of any of 1055 to 1073 of the present invention, wherein the one or more modified immune cells comprises one or more anerogenic immune cells.
[Invention 1075]
The method of any of 1055-1074 of the present invention, wherein the one or more immune cells comprises one or more lymphocytes.
[Invention 1076]
The method of 1075 of the present invention, wherein the one or more lymphocytes comprises T cells, B cells, NK cells, or a combination thereof.
[Invention 1077]
A group in which the T cells consist of naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, energy T cells, tolerant T cells, chimeric T cells, and antigen-specific T cells. More selected, the method of the invention 1076.
[Invention 1078]
The B cells are selected from the group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. The method of invention 1076.
[Invention 1079]
The method of 1075 of the present invention, wherein the one or more lymphocytes comprises tumor infiltrating lymphocytes, T cell receptor modified lymphocytes, or chimeric antigen receptor modified lymphocytes.
[Invention 1080]
The method of the invention 1075, wherein said lymphocytes have a CD8 ⁺ CD28 ^- CD152 ^- signature, CD8 + CD25 + signature, or CD4 + CD25 + signature.
[Invention 1081]
Step of administering any composition of the present invention 1001 to 1017
Methods for increasing the effectiveness of adoptive or T cell therapy in a subject, including.
[Invention 1082]
The composition of any of 1001-1017 of the present invention for use in the treatment of melanoma.
[Invention 1083]
Tumor-infiltrating lymphocytes comprising a MYC fusion peptide, wherein the MYC fusion peptide comprises (i) a protein transduction domain; (ii) a MYC polypeptide sequence.
[Invention 1084]
A lymphocyte comprising a chimeric antigen receptor and a MYC fusion peptide, wherein the MYC fusion peptide comprises (i) a protein transduction domain; (ii) a MYC polypeptide sequence.
[Invention 1085]
A step of contacting one or more primary immune cells with a MYC fusion peptide comprising (i) a protein transfection domain; (ii) a MYC polypeptide sequence, wherein the one or more primary immune cells The step in which one or more primary immune cells are isolated from a patient with melanoma and are responsive to a melanoma-specific antigen.
A method for preparing a composition for adoptive cell therapy, including.
[Invention 1086]
(A) One or more isolated primary immune cells exposed to a melanoma cell line; and
(B) (i) Protein transduction domain; (ii) MYC polypeptide sequence
MYC fusion peptide, including
Including
A composition in which the one or more primary immune cells are reactive with a melanoma-specific antigen.

Claims (86)

(A) (i) Protein transfection domain; (ii) MYC fusion peptide containing MYC polypeptide sequence; and (b) Isolated from donor subjects with melanoma tumors and reactive to melanoma-specific antigens A composition comprising one or more primary immune cells. The composition according to claim 1, wherein the MYC fusion peptide is transferred to the nucleus of the one or more primary immune cells. The composition according to claim 1 or 2, wherein the MYC fusion peptide exhibits the biological activity of MYC. The composition according to any one of claims 1 to 3, wherein the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain and the MYC polypeptide. The MYC fusion peptide has the following general structure:
Contains a MYC fusion peptide with a protein transduction domain-X-MYC sequence
The composition according to any one of claims 1 to 4, wherein -X- is a molecule that links the protein transduction domain and the MYC sequence. The composition according to any one of claims 1 to 5, wherein the protein transduction domain sequence is a TAT protein transduction domain sequence. The composition according to claim 6, wherein the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48]. The composition according to any one of claims 1 to 7, wherein the MYC fusion peptide contains SEQ ID NO: 1. The composition according to any one of claims 1 to 8, wherein the MYC fusion peptide is acetylated. The composition according to any one of claims 1 to 9, wherein the one or more immune cells have antitumor activity against melanoma cells. The composition according to any one of claims 1 to 10, wherein the one or more immune cells contain one or more lymphocytes. The composition according to claim 11, wherein the one or more lymphocytes contain T cells, B cells, NK cells, or a combination thereof. A group in which the T cells consist of naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, energy T cells, tolerant T cells, chimeric T cells, and antigen-specific T cells. The composition according to claim 12, which is more selected. The B cells are selected from the group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. Item 12. The composition according to Item 12. The invention according to any one of claims 11 to 14, wherein the one or more lymphocytes are tumor infiltrating lymphocytes, T cell receptor-modified lymphocytes, or chimeric antigen receptor-modified lymphocytes. Composition. The composition of claim 15, wherein the tumor infiltrating lymphocytes have a CD8 ⁺ CD28 ^- CD152 ^- signature, CD8 + CD25 + signature, or CD4 + CD25 + signature. The composition according to any one of claims 1 to 16, which comprises a portion in which the one or more immune cells can be detected. In the step of administering one or more modified immune cells to a subject who needs it, the one or more modified immune cells contain a MYC fusion peptide, and the MYC fusion peptide is present. (I) Protein transfection domain; (ii) A melanoma in a subject comprising the step, comprising the MYC polypeptide sequence and the one or more modified immune cells being responsive to a tumor-specific antigen. How to treat. The method of claim 18, wherein the one or more modified immune cells are derived from a primary immune cell isolated from the subject. 18. The method of claim 18, wherein the one or more modified immune cells are derived from primary immune cells isolated from another donor subject having the same type of melanoma. 19. The method of claim 19 or 20, wherein the one or more modified immune cells are prepared by isolating the primary immune cells and then contacting them in vitro with the MYC fusion peptide. The method of any one of claims 19-21, further comprising the step of increasing the primary immune cells in vitro prior to contact with the MYC fusion peptide. The method according to any one of claims 19 to 21, further comprising the step of increasing the primary immune cells after contact with the MYC fusion peptide. The method of claim 22 or 23, wherein the anti-CD3 antibody is used to grow the cells. The method according to any one of claims 22 to 24, wherein the irradiated allogeneic feeder cells are used to grow the cells. The method according to any one of claims 22 to 25, wherein the cells are grown in the presence of exogenous cytokines. 26. The method of claim 26, wherein the cytokine is interleukin 2. The method according to any one of claims 18 to 27, wherein the MYC fusion peptide is transferred to the nucleus of the immune cell. The method according to any one of claims 18 to 28, wherein the MYC fusion peptide exhibits the biological activity of MYC. The method of any one of claims 18-29, wherein the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain to the MYC polypeptide. The MYC fusion peptide has the following general structure:
Contains a MYC fusion peptide with a protein transduction domain-X-MYC sequence
The method according to any one of claims 18 to 30, wherein -X- is a molecule that links the protein transduction domain with the MYC sequence. The method according to any one of claims 18 to 31, wherein the protein transduction domain sequence is a TAT protein transduction domain sequence. The method of claim 32, wherein the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48]. The method according to any one of claims 18 to 33, wherein the MYC fusion peptide comprises SEQ ID NO: 1. The method according to any one of claims 18 to 34, wherein the MYC fusion peptide is acetylated. The method according to any one of claims 18 to 35, wherein the one or more modified immune cells have antitumor activity against melanoma cells in the subject. The method according to any one of claims 18 to 36, wherein the one or more modified immune cells comprises one or more anergy immune cells. The method according to any one of claims 18 to 37, wherein the one or more immune cells contain one or more lymphocytes. 38. The method of claim 38, wherein the one or more lymphocytes comprises T cells, B cells, NK cells, or a combination thereof. A group of T cells consisting of naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, energy T cells, tolerant T cells, chimeric T cells, and antigen-specific T cells. The method of claim 39, which is more selected. Claimed that the B cells are selected from the group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. Item 39. 38. The method of claim 38, wherein the one or more lymphocytes comprises tumor infiltrating lymphocytes, T cell receptor modified lymphocytes, or chimeric antigen receptor modified lymphocytes. 42. The method of claim 42, wherein the lymphocyte has a CD8 ⁺ CD28 ^- CD152 ^- signature, CD8 + CD25 + signature, or CD4 + CD25 + signature. The method according to any one of claims 19 to 43, further comprising the step of isolating the primary immune cells from the donor subject. 44. The method of claim 44, wherein the donor subject has melanoma. The method according to any one of claims 18 to 45, wherein the one or more modified immune cells are administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratumorally. The method according to any one of claims 18 to 46, further comprising a step of putting the subject into a lymph depleted state prior to administration of the one or more modified immune cells. The method according to any one of claims 18 to 47, further comprising the step of administering the cytokine to the subject. 48. The method of claim 48, wherein the cytokine is administered before, during, or after administration of the one or more modified immune cells. The cytokines are interferon α, interferon β, interferon γ, complement C5a, IL-2, TNFα, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2. , CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26 , CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CX3 , CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 Item 8. The method according to any one of claims 18 to 50, wherein the melanoma is metastatic. The method according to any one of claims 18 to 51, wherein the subject is a human or an animal. The method of any one of claims 18-52, further comprising the step of performing additional cancer treatment. 35. The additional cancer treatment is selected from among chemotherapy, radiotherapy, immunotherapy, monoclonal antibodies, anticancer nucleic acids or proteins, anticancer viruses or microorganisms, and combinations thereof. The method described. The method according to any one of claims 18 to 54, which comprises a portion in which the one or more modified immune cells can be detected. In vitro contact of one or more immune cells with a MYC fusion polypeptide, wherein the immune cells are derived from a donor exposed to one or more tumor antigens and the MYC fusion To prepare modified immune cells for the treatment of melanoma, comprising the steps in which the peptide is (i) a protein transfectation domain; (ii) a MYC polypeptide sequence and is responsive to a tumor-specific antigen. the method of. 55. The method of claim 55, wherein the one or more modified immune cells are derived from primary immune cells isolated from a subject having melanoma. 55. The method of claim 55 or 56, further comprising the step of growing the primary immune cells in vitro prior to contact with the MYC fusion peptide. The method according to any one of claims 55 to 57, further comprising the step of increasing the primary immune cells after contact with the MYC fusion peptide. The method of any one of claims 55-59, wherein the anti-CD3 antibody is used to grow the cells. The method of any one of claims 55-60, wherein the irradiated allogeneic feeder cells are used to grow the cells. The method of any one of claims 55-61, which grows the cells in the presence of exogenous cytokines. 62. The method of claim 62, wherein the cytokine is interleukin 2. The method according to any one of claims 55 to 63, wherein the MYC fusion peptide is transferred to the nucleus of the immune cell. The method according to any one of claims 55 to 64, wherein the MYC fusion peptide exhibits the biological activity of MYC. The method of any one of claims 55-65, wherein the MYC fusion peptide further comprises one or more molecules linking the protein transduction domain to the MYC polypeptide. The MYC fusion peptide has the following general structure:
Contains a MYC fusion peptide with a protein transduction domain-X-MYC sequence
The method according to any one of claims 55 to 66, wherein -X- is a molecule that links the protein transduction domain with the MYC sequence. The method according to any one of claims 55 to 67, wherein the protein transduction domain sequence is a TAT protein transduction domain sequence. The method of claim 68, wherein the TAT protein transduction domain sequence is selected from the group consisting of TAT [48-57] and TAT [57-48]. The method according to any one of claims 55 to 69, wherein the MYC fusion peptide comprises SEQ ID NO: 1. The method according to any one of claims 55 to 70, wherein the MYC fusion peptide is acetylated. The method according to any one of claims 55 to 71, wherein the one or more modified immune cells have antitumor activity. The method according to any one of claims 55 to 72, wherein the one or more modified immune cells have antitumor activity against melanoma cells in the subject. The method according to any one of claims 55 to 73, wherein the one or more modified immune cells comprises one or more anergy immune cells. The method according to any one of claims 55 to 74, wherein the one or more immune cells contain one or more lymphocytes. The method of claim 75, wherein the one or more lymphocytes comprises T cells, B cells, NK cells, or a combination thereof. A group of T cells consisting of naive T cells, CD4 + T cells, CD8 + T cells, memory T cells, activated T cells, energy T cells, tolerant T cells, chimeric T cells, and antigen-specific T cells. The method of claim 76, which is more selected. Claimed that the B cells are selected from the group consisting of naive B cells, trait B cells, activated B cells, memory B cells, anergic B cells, tolerant B cells, chimeric B cells, and antigen-specific B cells. Item 7. The method of claim 75, wherein the one or more lymphocytes comprises tumor infiltrating lymphocytes, T cell receptor modified lymphocytes, or chimeric antigen receptor modified lymphocytes. The method of claim 75, wherein the lymphocyte has a CD8 ⁺ CD28 ^- CD152 ^- signature, CD8 + CD25 + signature, or CD4 + CD25 + signature. A method for increasing the effectiveness of adoptive cell therapy or T cell therapy in a subject, comprising the step of administering the composition according to any one of claims 1-17. The composition according to any one of claims 1 to 17, for use in the treatment of melanoma. Tumor-infiltrating lymphocytes comprising a MYC fusion peptide, wherein the MYC fusion peptide comprises (i) a protein transduction domain; (ii) a MYC polypeptide sequence. A lymphocyte comprising a chimeric antigen receptor and a MYC fusion peptide, wherein the MYC fusion peptide comprises (i) a protein transduction domain; (ii) a MYC polypeptide sequence. A step of contacting one or more primary immune cells with a MYC fusion peptide comprising (i) a protein transfection domain; (ii) a MYC polypeptide sequence, wherein the one or more primary immune cells A method for preparing a composition for adoptive cell therapy, comprising the step of being isolated from a patient with melanoma and having one or more primary immune cells reactive to a melanoma-specific antigen. MYC fusion peptides comprising (a) one or more isolated primary immune cells exposed to a melanoma cell line; and (b) (i) protein transduction domain; (ii) MYC polypeptide sequence. Including
A composition in which the one or more primary immune cells are reactive with a melanoma-specific antigen.

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