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WO2019173441A1 - Procédés de préparation de populations de cellules et de réactifs rétroviraux pour l'immunothérapie cellulaire adoptive - Google Patents

Procédés de préparation de populations de cellules et de réactifs rétroviraux pour l'immunothérapie cellulaire adoptive Download PDF

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WO2019173441A1
WO2019173441A1 PCT/US2019/020903 US2019020903W WO2019173441A1 WO 2019173441 A1 WO2019173441 A1 WO 2019173441A1 US 2019020903 W US2019020903 W US 2019020903W WO 2019173441 A1 WO2019173441 A1 WO 2019173441A1
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cells
tcr
amino acid
antigen
cancer
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Steven A. FELDMAN
Steven A. Rosenberg
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US Department of Health and Human Services
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/13—B-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4201—Neoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
    • A61K40/4205—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13051—Methods of production or purification of viral material

Definitions

  • Adoptive cell therapy using cells that have been genetically engineered to express an anti-cancer antigen T cell receptor (TCR) can produce positive clinical responses in some cancer patients. Nevertheless, obstacles to the successful use of TCR-engineered cells for the widespread treatment of cancer and other diseases remain. For example, the efficiency of the transfer of an exogenous TCR into host cells, e.g., peripheral blood lymphocytes (PBL) may be low. Accordingly, there is a need for improved methods of preparing populations of cells and reagents for adoptive cell therapies.
  • TCR anti-cancer antigen T cell receptor
  • An embodiment of the invention provides a method of preparing and testing replication incompetent viruses comprising a vector encoding a T cell receptor (TCR), or an antigen-binding portion thereof, having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in the nucleic acid of a cancer cell of a patient, each gene containing a cancer- specific mutation that encodes a mutated amino acid sequence; inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence; co culturing autologous T cells of the patient with the autologous APCs that present the mutated amino acid sequence; selecting the autologous T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a major histocompatability complex (MHC) molecule expressed
  • Another embodiment of the invention provides a method of preparing a population of cells which express a TCR, or an antigen-binding portion thereof, having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: preparing replication incompetent viruses according to any of the methods described herein; obtaining peripheral blood mononuclear cells (PBMC) from the patient; contacting the PBMC with the replication incompetent viruses under conditions sufficient to transduce the PBMC with the vector comprising the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof; and expressing the TCR, or the antigen-binding portion thereof, by the PBMC, thereby producing the population of cells which express the TCR, or the antigen-binding portion thereof, having antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.
  • PBMC peripheral blood mononuclear cells
  • Figure l is a schematic illustrating a method of preparing a population of cells which express a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation (neoantigen) according to an embodiment of the invention.
  • Figure 2A presents FACS plots showing the expression of neoantigen-reactive TCRs with a murine constant region (mTCRb) and CD8 by PBMC.
  • the neoantigen-reactive TCRs recognized mutated antigen H3F3B (Vbl6, TCR1) or mutated SKIV2L (Vb20, TCR2).
  • the numbers in the plots are the percentages of cells having each phenotype:
  • Untransduced (UT) PBMC served as a negative control.
  • Figure 2B is a graph showing the percentage of CD8+ cells transduced with neoantigen-specific TCR Vbl6 or Vb20 which upregulated 4-1BB expression following co culture with autologous APCs pulsed with either wildtype (wt) or mutated (mut) peptide for a given neoantigen (H3F3B or SKIV2L).
  • Figure 2C is a graph showing the percentage of cells transduced with neoantigen- specific TCR Vbl6 or Vb20 which upregulated IFN-g release (pg/mL) following co-culture with autologous APCs pulsed with either wildtype (wt) or mutated (mut) peptide for a given neoantigen (H3F3B or SKIV2L).
  • Figure 3 A is a graph showing the concentration (ng/mL) of residual TCR 1
  • TCR 2 squares vector DNA measured by qPCR after transfection of 293 GP cells and with or without treatment with BENZONASE endonuclease.
  • Figure 3B is a graph showing the concentration (ng/mL) of residual
  • BENZONASE endonuclease measured after BENZONASE endonuclease treatment, after transduction of PBMC with the TCR vector, and after a post-transduction wash during expansion of the numbers of transduced PBMC.
  • 293GP cells were treated with 50U/mL BENZONASE endonuclease at room temperature (RT) (squares) or 37° C (triangles). 293 GP cells treated with no BENZONASE endonuclease served as a negative control (circles).
  • Figure 3C is a graph showing the number of copies of RD114 DNA per 0.2 pg of total genomic DNA measured by qPCR at the indicated number of days post-transduction of PBMC.
  • PBMC were transduced with TCR1 without (open circles) or with 50 U/mL
  • FIGS. 4A-4C are graphs showing the amount of IFNy secreted by cells from Patient (Pt) D (Fig. 4A), Pt H (Fig. 4B), or Pt R (Fig.
  • Figures 4D-4F are graphs showing the amount of IFNy secreted by cells from Pt D (Fig. 4D), Pt H (Fig. 4E), or Pt R (Fig. 4F) which were untransduced (light grey bars) or transduced with nb5.2 a 26.1 TCR following co-culture with B cells pulsed with WT
  • ERBB2IP peptide or mutated ERBB2IP were included T cells cultured alone (no target) and co-cultures with (i) OKT3 antibody and (ii) B cells that have not been pulsed with mutated or wild type peptide. The supernatant used to transduce the cells was treated with 0 (black bars), 25 (checkered bars), or 50 (dark grey bars) U/mL BENZONASE endonuclease.
  • Figures 4G-4I are graphs showing the amount of IFNy secreted by cells from Pt D (Fig. 4G), Pt H (Fig. 4H), or Pt R (Fig. 41) which were untransduced (light grey bars) or transduced with nb5.2 a 30 TCR following co-culture with B cells pulsed with WT ERBB2IP peptide or mutated ERBB2IP.
  • Controls included T cells cultured alone (no target) and co- cultures with (i) OKT3 antibody and (ii) B cells that have not been pulsed with mutated or wild type peptide. The supernatant used to transduce the cells was treated with 0 (black bars), 25 (checkered bars), or 50 (dark grey bars) U/mL BENZONASE endonuclease.
  • Figures 5A-5B are graphs showing the amount of free DNA (ng/mL) measured in the supernatant at the indicated time points during transfection with the nb22 (circles) or nb5.2 a 30 (squares) TCR, including after treatment with 25 (Fig. 5A) or 50 U/mL (Fig. 5B) BENZONASE endonuclease.
  • Figures 6A-6B are graphs showing the amount of free DNA (ng/mL) measured in the supernatant at the indicated time points during transfection with the nb22 (circles) or nb5.2 a 30 (squares) TCR, including after treatment with 50 U/mL BENZONASE
  • FIG. 7 is a graph showing the concentration of BENZONASE endonuclease (ng/mL) in transduced cells at the indicated time points during transduction.
  • TCR-transduced cells were prepared using retroviral supernatant prepared with 0 (circles), 25 (squares), or 50 (triangles) U/mL BENZONASE endonuclease.
  • Figure 8 presents FACS plots showing the expression of neoantigen-reactive TCRs from patient 4217 based on detection of the murine constant region (mTCRP) after transduction into healthy donor PBMC.
  • the neoantigen-reactive TCRs recognized mutated antigen MAP3K2 S153F (4217 MAP3K2 S153F TCR1), UELVD F191L
  • the numbers in the plots are the percentages of cells having each phenotype: mTCRp+/CD8+ (Q6), mTCRp+/CD8- (Q5), mTCRp-/CD8+ (Q7), and mTCRp-/CD8- (Q8).
  • Figure 9 presents FACS plots showing the expression of neoantigen-reactive TCRs from patient 4275 based on detection of the murine constant region (mTCRP) after transduction into healthy donor PBMC.
  • the neoantigen-reactive TCRs recognized mutated antigen GPATCH R954H (4275 GPATCH R954H TCR1), WLS R445G (4275_WLS_ R445G TCR2), or WLS R445G (4275 WLS R445G TCR3).
  • the numbers in the plots are the percentages of cells having each phenotype: mTCRp+/CD8+ (Q6), mTCRp+/CD8- (Q5), mTCRp-/CD8+ (Q7), and mTCRp-/CD8- (Q8).
  • Figure 10 presents FACS plots showing the expression of neoantigen-reactive TCRs from patient 4271 based on detection of the murine constant region (mTCRP) after transduction into healthy donor PBMC.
  • the neoantigen-reactive TCRs recognized mutated antigen UPS47 F1156L (4271 UPS47 F1156L TCR2), CHD2 K1351R
  • the numbers in the plots are the percentages of cells having each phenotype: mTCRp+/CD8+ (Q6), mTCRp+/CD8- (Q5), mTCRp-/CD8+ (Q7), and mTCRp-/CD8- (Q8).
  • Figure 11 presents FACS plots showing the expression of neoantigen-reactive TCRs from patient 4251 based on detection of the murine constant region (mTCRP) after transduction into healthy donor PBMC.
  • the neoantigen-reactive TCRs recognized mutated antigen RNF213 P4766H (425l_RNF2l3_P4766H_TCR2), FMOD S332N
  • the numbers in the plots are the percentages of cells having each phenotype: mTCRp+/CD8+ (Q6), mTCRp+/CD8- (Q5), mTCRp-/CD8+ (Q7), and mTCRp-/CD8- (Q8).
  • Figure 12 presents a FACS plot showing the expression of a neoantigen-reactive
  • TCR from patient 4285 based on detection of the murine constant region (mTCRP) after transduction into healthy donor PBMC.
  • the neoantigen-reactive TCR recognized mutated antigen TP53 R175H (4285 TP53 R175H TCR1).
  • the numbers in the plot are the percentages of cells having each phenotype: mTCRp+/CD8+ (Q6), mTCRp+/CD8- (Q5), mTCRp-/CD8+ (Q7), and mTCRp-/CD8- (Q8).
  • Figure 13 is a graph showing the concentration (pg/mL) of IFN-g produced following co-culture of healthy donor T cells expressing TCR (post-transduction) with autologous patient B cells pulsed with 10 pg of either mutant or wild type peptide.
  • An embodiment of the invention provides a method of preparing replication incompetent viruses comprising a vector encoding a TCR, or an antigen-binding portion thereof, having antigenic specificity for a mutated amino acid sequence encoded by a cancer- specific mutation.
  • the invention may provide any one or more of a variety of advantages.
  • the invention may provide methods of preparing reagents (e.g., viruses) useful for preparing cells which express a TCR, or antigen-binding portions thereof, having antigenic specificity for mutated amino acid sequences encoded by cancer-specific mutations that are unique to the patient (also referred to as“neoantigen(s)”).
  • the inventive methods may produce reagents (e.g., viruses) and populations of cells in a manner which complies with Good Manufacturing Practices (GMP).
  • GMP Good Manufacturing Practices
  • the reagents may be biosafety tested to industry standards including testing for sterility, residual DNA vector, replication competent retrovirus (RCR), mycoplasma, endotoxin, human viruses and other adventitious agents.
  • the 293GP master cell bank may be fully validated.
  • the inventive methods may include testing the reagents for sterility.
  • inventive methods which comprise viral transduction of a population of PBMC with a vector encoding the TCR, or antigen binding portion thereof, may generate a cell population with a high frequency (e.g., up to about 90%) of cells which express the TCR, or antigen binding portion thereof.
  • the frequency of cells which express the TCR, or antigen binding portion thereof, obtained using the inventive methods which comprise viral transduction may greatly exceed that which can be achieved using methods of selecting mutation-specific tumor infiltrating lymphocytes (TIL), non-viral techniques such as transposons, or gene editing techniques using zinc finger nucleases, TALENs or CRISPR/Cas genome editing technologies, all of which may suffer from low efficiency gene delivery.
  • TIL tumor infiltrating lymphocytes
  • non-viral techniques such as transposons
  • gene editing techniques using zinc finger nucleases TALENs or CRISPR/Cas genome editing technologies
  • the method may comprise identifying one or more genes in the nucleic acid of a cancer cell of a patient, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence.
  • the cancer cell may be obtained from any bodily sample derived from a patient which contains or is expected to contain tumor or cancer cells.
  • the bodily sample may be any tissue sample such as blood, a tissue sample obtained from the primary tumor or from tumor metastases, or any other sample containing tumor or cancer cells.
  • the nucleic acid of the cancer cell may be DNA or RNA.
  • the method may further comprise sequencing nucleic acid such as DNA or RNA of normal, noncancerous cells and comparing the sequence of the cancer cell with the sequence of the normal, noncancerous cell.
  • the normal, noncancerous cell may be obtained from the patient or a different individual.
  • the cancer-specific mutation may be any mutation in any gene which encodes a mutated amino acid sequence (also referred to as a“non-silent mutation”) and which is expressed in a cancer cell but not in a normal, noncancerous cell.
  • a“non-silent mutation” also referred to as a“non-silent mutation”
  • Non-limiting examples of cancer-specific mutations that may be identified in the inventive methods include missense, nonsense, insertion, deletion, duplication, frameshift, and repeat expansion mutations.
  • the method comprises identifying at least one gene containing a cancer-specific mutation which encodes a mutated amino acid sequence.
  • the number of genes containing such a cancer-specific mutation that may be identified using the inventive methods is not limited and may include more than one gene (for example, about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values).
  • the method comprises identifying at least one cancer-specific mutation which encodes a mutated amino acid sequence.
  • cancer-specific mutations that may be identified using the inventive methods is not limited and may include more than one cancer-specific mutation (for example, about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values).
  • the cancer-specific mutations may be located in the same gene or in different genes.
  • identifying one or more genes in the nucleic acid of a cancer cell comprises sequencing the whole exome, the whole genome, or the whole transcriptome of the cancer cell. Sequencing may be carried out in any suitable manner known in the art. Examples of sequencing techniques that may be useful in the inventive methods include Next Generation Sequencing (NGS) (also referred to as“massively parallel sequencing
  • NGS refers to non-Sanger-based high- throughput DNA sequencing technologies. With NGS, millions or billions of DNA strands may be sequenced in parallel, yielding substantially more throughput and minimizing the need for the fragment-cloning methods that are often used in Sanger sequencing of genomes. In NGS, nucleic acid templates may be randomly read in parallel along the entire genome by breaking the entire genome into small pieces. NGS may, advantageously, provide nucleic acid sequence information of a whole genome, exome, or transcriptome in very short time periods, e.g., within about 1 to about 2 weeks, preferably within about 1 to about 7 days, or most preferably, within less than about 24 hours.
  • Non-limiting examples of NGS technologies and platforms include sequencing- by-synthesis (also known as“pyrosequencing”) (as implemented, e.g., using the GS-FLX 454 Genome Sequencer, 454 Life Sciences (Branford, CT), ILLUMINA SOLEXA Genome Analyzer (Illumina Inc., San Diego, CA), or the ILLEIMINA HISEQ 2000 Genome Analyzer (Illumina), or as described in, e.g., Ronaghi et ah, Science , 281(5375): 363-365 (1998)), sequencing-by-ligation (as implemented, e.g., using the SOLID platform (Life Technologies Corporation, Carlsbad, CA) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)), single-molecule sequencing (as implemented, e.g., using the PACBIO RS system (Pacific Biosciences (Menlo Park, CA) or the HELISCOPE platform (He
  • the method may comprise inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence.
  • APCs may include any cells which present peptide fragments of proteins in association with major histocompatibility complex (MHC) molecules on their cell surface.
  • MHC major histocompatibility complex
  • the APCs may include, for example, any one or more of macrophages, DCs, langerhans cells, B-lymphocytes, and T-cells.
  • the APCs are DCs.
  • the inventive methods may, advantageously, identify TCRs, and antigen-binding portions thereof, that have antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation that is presented in the context of an MHC molecule expressed by the patient.
  • the MHC molecule can be any MHC molecule expressed by the patient including, but not limited to, MHC Class I, MHC Class II, HLA-A, HLA-B, HLA-C, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR molecules.
  • inventive methods may, advantageously, identify mutated amino acid sequences presented in the context of any MHC molecule expressed by the patient without using, for example, epitope prediction algorithms to identify MHC molecules or mutated amino acid sequences, which may be useful only for a select few MHC class I alleles and may be constrained by the limited availability of reagents to select mutation-reactive T cells (e.g., an incomplete set of MHC tetramers).
  • the inventive methods advantageously identify mutated amino acid sequences presented in the context of any MHC molecule expressed by the patient and are not limited to any particular MHC molecule.
  • the autologous APCs are antigen-negative autologous APCs.
  • inducing autologous APCs of the patient to present the mutated amino acid sequence may be carried out using any suitable method known in the art.
  • inducing autologous APCs of the patient to present the mutated amino acid sequence comprises pulsing the autologous APCs with peptides comprising the mutated amino acid sequence or a pool of peptides, each peptide in the pool comprising a different mutated amino acid sequence.
  • Each of the mutated amino acid sequences in the pool may be encoded by a gene containing a cancer specific mutation.
  • the autologous APCs may be cultured with a peptide or a pool of peptides comprising the mutated amino acid sequence in a manner such that the APCs internalize the peptide(s) and display the mutated amino acid sequence(s), bound to an MHC molecule, on the cell membrane.
  • the method may comprise pulsing the autologous APCs with a pool of peptides, each peptide in the pool comprising a different mutated amino acid sequence.
  • the peptide(s) used to pulse the APCs may include the mutated amino acid(s) encoded by the cancer-specific mutation.
  • the peptide(s) may further comprise any suitable number of contiguous amino acids from the endogenous protein encoded by the identified gene on each of the carboxyl side and the amino side of the mutated amino acid(s).
  • the number of contiguous amino acids from the endogenous protein flanking each side of the mutation is not limited and may be, for example, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or a range defined by any two of the foregoing values.
  • the peptide(s) comprise(s) about 12 contiguous amino acids from the endogenous protein on each side of the mutated amino acid(s).
  • inducing autologous APCs of the patient to present the mutated amino acid sequence comprises introducing a nucleotide sequence encoding the mutated amino acid sequence into the APCs.
  • the nucleotide sequence is introduced into the APCs so that the APCs express and display the mutated amino acid sequence, bound to an MHC molecule, on the cell membrane.
  • the nucleotide sequence encoding the mutated amino acid may be RNA or DNA.
  • Introducing a nucleotide sequence into APCs may be carried out in any of a variety of different ways known in the art as described in, e.g., Solheim et al. supra.
  • Non-limiting examples of techniques that are useful for introducing a nucleotide sequence into APCs include transformation, transduction, transfection, and electroporation.
  • the method may comprise preparing more than one nucleotide sequence, each encoding a mutated amino acid sequence encoded by a different gene, and introducing each nucleotide sequence into a different population of autologous APCs.
  • multiple populations of autologous APCs, each population expressing and displaying a different mutated amino acid sequence may be obtained.
  • the method may comprise introducing a nucleotide sequence encoding the more than one gene.
  • the nucleotide sequence introduced into the autologous APCs is a tandem minigene (TMG) construct, each minigene comprising a different gene, each gene including a cancer-specific mutation that encodes a mutated amino acid sequence.
  • TMG tandem minigene
  • Each minigene may encode one mutation identified by the inventive methods flanked on each side of the mutation by any suitable number of contiguous amino acids from the endogenous protein encoded by the identified gene, as described herein with respect to other aspects of the invention.
  • the number of minigenes in the construct is not limited and may include for example, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, or more, or a range defined by any two of the foregoing values.
  • the APCs express the mutated amino acid sequences encoded by the TMG construct and display the mutated amino acid sequences, bound to an MHC molecule, on the cell membranes.
  • the method may comprise preparing more than one TMG construct, each construct encoding a different set of mutated amino acid sequences encoded by different genes, and introducing each TMG construct into a different population of autologous APCs.
  • multiple populations of autologous APCs, each population expressing and displaying mutated amino acid sequences encoded by different TMG constructs may be obtained.
  • the method may comprise co-culturing autologous T cells of the patient with the autologous APCs that present the mutated amino acid sequence.
  • the T cells can be obtained from numerous sources in the patient, including but not limited to tumor, blood, bone marrow, lymph node, the thymus, or other tissues or fluids.
  • the T cells can include any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Thl and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells (e.g., tumor infiltrating lymphocytes (TIL)), peripheral blood T cells, memory T cells, naive T cells, and the like.
  • the T cells may be CD8+ T cells, CD4+ T cells, or both CD4+ and CD8+ T cells.
  • the method may comprise co-culturing the autologous T cells and autologous APCs so that the T cells encounter the mutated amino acid sequence presented by the APCs in such a manner that the autologous T cells specifically bind to and immunologically recognize a mutated amino acid sequence presented by the APCs.
  • the autologous T cells are co- cultured in direct contact with the autologous APCs.
  • the method may comprise selecting the autologous T cells that (a) were co- cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a MHC molecule expressed by the patient.
  • the phrase“antigenic specificity,” as used herein, means that a TCR, or the antigen-binding portion thereof, expressed by the autologous T cells can specifically bind to and immunologically recognize the mutated amino acid sequence encoded by the cancer-specific mutation.
  • the selecting may comprise identifying the T cells that have antigenic specificity for the mutated amino acid sequence and separating them from T cells that do not have antigenic specificity for the mutated amino acid sequence.
  • the method comprises expanding the numbers of autologous T cells, e.g., by co-culturing with a T cell growth factor, such as interleukin (IL)-2 or IL-15, or as described herein with respect to other aspects of the invention, prior to selecting the autologous T cells.
  • a T cell growth factor such as interleukin (IL)-2 or IL-15
  • the method does not comprise expanding the numbers of autologous T cells with a T cell growth factor, such as IL-2 or IL-15 prior to selecting the autologous T cells.
  • T cells having antigenic specificity for the mutated amino acid sequence may express any one or more of a variety of T cell activation markers which may be used to identify those T cells having antigenic specificity for the mutated amino acid sequence.
  • T cell activation markers may include, but are not limited to, programmed cell death 1 (PD-l), lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1BB, 0X40, and CD 107a.
  • selecting the autologous T cells that have antigenic specificity for the mutated amino acid sequence comprises selecting the T cells that express any one or more of PD-l, LAG-3, TIM-3, 4-1BB, 0X40, and CDl07a.
  • Cells expressing one or more T cell activation markers may be sorted on the basis of expression of the marker using any of a variety of techniques known in the art such as, for example, fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) as described in, e.g., Turcotte et al, Clin. Cancer Res., 20(2): 331-43 (2013) and Gros et al., J Clin. Invest., 124(5): 2246-59 (2014).
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • selecting the autologous T cells that have antigenic specificity for the mutated amino acid sequence comprises selecting the T cells (i) that secrete a greater amount of one or more cytokines upon co-culture with APCs that present the mutated amino acid sequence as compared to the amount of the one or more cytokines secreted by a negative control or (ii) in which at least twice as many of the numbers of T cells secrete one or more cytokines upon co-culture with APCs that present the mutated amino acid sequence as compared to the numbers of negative control T cells that secrete the one or more cytokines.
  • the one or more cytokines may comprise any cytokine the secretion of which by a T cell is characteristic of T cell activation (e.g., a TCR expressed by the T cells specifically binding to and immunologically recognizing the mutated amino acid sequence).
  • cytokines the secretion of which is characteristic of T cell activation, include IFN-g, IL-2, and tumor necrosis factor alpha (TNF-a),
  • GM-CSF granulocyte/monocyte colony stimulating factor
  • IL-4 granulocyte/monocyte colony stimulating factor
  • IL-5 granulocyte/monocyte colony stimulating factor
  • IL-9 granulocyte/monocyte colony stimulating factor
  • IL-10 granulocyte/monocyte colony stimulating factor
  • IL-17 granulocyte/monocyte colony stimulating factor
  • IL-22 granulocyte/monocyte colony stimulating factor
  • a TCR, or an antigen-binding portion thereof, or a T cell expressing the TCR, or the antigen-binding portion thereof may be considered to have“antigenic specificity” for the mutated amino acid sequence if the T cells, or T cells expressing the TCR, or the antigen-binding portion thereof, secrete at least twice as much IFN-g upon co-culture with (a) antigen-negative APCs pulsed with a concentration of a peptide comprising the mutated amino acid sequence (e.g., about 0.05 ng/mL to about 10 pg/mL, e.g., 0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 100 ng/mL, 1 pg/mL, 5 pg/mL, or 10 pg/mL) or (b) APCs into which a nucleotide sequence encoding the
  • an irrelevant peptide e.g., the wild-type amino acid sequence, or some other peptide with a different sequence from the mutated amino acid sequence
  • APCs into which a nucleotide sequence encoding an irrelevant peptide sequence has been introduced or (ii) untransduced T cells (e.g., derived from PBMC, which do not express the TCR, or antigen binding portion thereof) co-cultured with (a) antigen-negative APCs pulsed with the same concentration of a peptide comprising the mutated amino acid sequence or (b) APCs into which a nucleotide sequence encoding the mutated amino acid sequence has been introduced.
  • a TCR, or an antigen-binding portion thereof, or a T cell expressing the TCR, or the antigen-binding portion thereof may also have“antigenic specificity” for the mutated amino acid sequence if T cells, or T cells expressing the TCR, or the antigen-binding portion thereof, secrete a greater amount of IFN-g upon co-culture with antigen-negative APCs pulsed with higher concentrations of a peptide comprising the mutated amino acid sequence as compared to a negative control, for example, any of the negative controls described above.
  • IFN-g secretion may be measured by methods known in the art such as, for example, enzyme- linked immunosorbent assay (ELISA).
  • a TCR, or an antigen-binding portion thereof, or a T cell expressing the TCR, or the antigen-binding portion thereof may be considered to have “antigenic specificity” for the mutated amino acid sequence if at least twice as many of the numbers of T cells, or T cells expressing the TCR, or the antigen-binding portion thereof, secrete IFN-g upon co-culture with (a) antigen-negative APCs pulsed with a concentration of a peptide comprising the mutated amino acid sequence or (b) APCs into which a nucleotide sequence encoding the mutated amino acid sequence has been introduced as compared to the numbers of negative control T cells that secrete IFN-g.
  • the concentration of peptide and the negative control may be as described herein with respect to other aspects of the invention.
  • the numbers of cells secreting IFN-g may be measured by methods known in the art such as, for example, ELISPOT.
  • T cells having antigenic specificity for the mutated amino acid sequence may both (1) express any one or more T cells activation markers described herein and (2) secrete a greater amount of one or more cytokines as described herein, in an embodiment of the invention, T cells having antigenic specificity for the mutated amino acid sequence may express any one or more T cell activation markers without secreting a greater amount of one or more cytokines or may secrete a greater amount of one or more cytokines without expressing any one or more T cell activation markers.
  • selecting the autologous T cells that have antigenic specificity for the mutated amino acid sequence comprises selectively growing the autologous T cells that have antigenic specificity for the mutated amino acid sequence.
  • the method may comprise co-culturing the autologous T cells with autologous APCs in such a manner as to favor the growth of the T cells that have antigenic specificity for the mutated amino acid sequence over the T cells that do not have antigenic specificity for the mutated amino acid sequence. Accordingly, a population of T cells may be produced that has a higher proportion of T cells that have antigenic specificity for the mutated amino acid sequence as compared to T cells that do not have antigenic specificity for the mutated amino acid sequence.
  • the method further comprises obtaining multiple fragments of a tumor from the patient, separately co-culturing autologous T cells from each of the multiple fragments with the autologous APCs that present the mutated amino acid sequence as described herein with respect to other aspects of the invention, and separately assessing the T cells from each of the multiple fragments for antigenic specificity for the mutated amino acid sequence, as described herein with respect to other aspects of the invention.
  • selecting the autologous T cells may further comprise separately assessing autologous T cells for antigenic specificity for each of the multiple mutated amino acid sequences.
  • the inventive method may further comprise separately inducing autologous APCs of the patient to present each mutated amino acid sequence encoded by the construct (or included in the pool), as described herein with respect to other aspects of the invention (for example, by providing separate APC populations, each presenting a different mutated amino acid sequence encoded by the construct (or included in the pool)).
  • the method may further comprise separately co- culturing autologous T cells of the patient with the different populations of autologous APCs that present each mutated amino acid sequence, as described herein with respect to other aspects of the invention.
  • the method may further comprise separately selecting the autologous T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a MHC molecule expressed by the patient, as described herein with respect to other aspects of the invention.
  • the method may comprise determining which mutated amino acid sequence encoded by a TMG construct that encodes multiple mutated amino acid sequences (or included in the pool) are immunologically recognized by the autologous T cells (e.g., by process of elimination).
  • the method may further comprise isolating a nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, from the selected autologous T cells, wherein the TCR, or the antigen-binding portion thereof, has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.
  • the numbers selected autologous T cells that have antigenic specificity for the mutated amino acid sequence may be expanded. Expansion of the numbers of T cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent
  • expansion of the numbers of T cells is carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC).
  • the numbers of selected autologous T cells that have antigenic specificity for the mutated amino acid sequence are not expanded prior to isolating the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof.
  • the numbers of cells are expanded in gas-permeable GREX flasks (Wilson Wolf
  • The“the antigen-binding portion” of the TCR refers to any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to the mutated amino acid sequence encoded by the gene identified as described herein with respect to other aspects of the invention.
  • the term “antigen-binding portion” refers to any part or fragment of the TCR of the invention, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR).
  • Antigen-binding portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to the mutated amino acid sequence, or detect, treat, or prevent cancer, to a similar extent, the same extent, or to a higher extent, as compared to the parent TCR.
  • the functional portion can comprise, for instance, about 10%, about 25%, about 30%, about 50%, about 65%, about 80%, about 90%, about 95%, or more, of the parent TCR.
  • the antigen-binding portion can comprise an antigen-binding portion of either or both of the a and b chains of the TCR of the invention, such as a portion comprising one or more of the complementarity determining region (CDR)l, CDR2, and CDR3 of the variable region(s) of the a chain and/or b chain of the TCR of the invention.
  • CDR complementarity determining region
  • the antigen-binding portion can comprise the amino acid sequence of the CDR1 of the a chain (CDRla), the CDR2 of the a chain (CDR2a), the CDR3 of the a chain (CDR3a), the CDR1 of the b chain (CDR ⁇ ), the CDR2 of the b chain (CDR2b), the CDR3 of the b chain (CDR3b), or any combination thereof.
  • the antigen-binding portion comprises the amino acid sequences of CDRla, CDR2a, and CDR3a; the amino acid sequences of CDR ⁇ , OOIT2b, and CDR3b; or the amino acid sequences of all of CDRla, CDR2a, CDR3a, CDR ⁇ , CDR2b, and CDR3b of the inventive TCR.
  • the antigen-binding portion can comprise, for instance, the variable region of the inventive TCR comprising a combination of the CDR regions set forth above.
  • the antigen-binding portion can comprise the amino acid sequence of the variable region of the a chain (Va), the amino acid sequence of the variable region of the b chain (nb), or the amino acid sequences of both of the Va and nb of the inventive TCR.
  • the antigen-binding portion may comprise a combination of a variable region and a constant region.
  • the antigen-binding portion can comprise the entire length of the a or b chain, or both of the a and b chains, of the inventive TCR.
  • Isolating the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, from the selected autologous T cells may be carried out in any suitable manner known in the art.
  • the method may comprise isolating RNA from the autologous T cells and sequencing the TCR, or the antigen-binding portion thereof, using established molecular cloning techniques and reagents such as, for example, 5’ Rapid Amplification of cDNA Ends (RACE) polymerase chain reaction (PCR) using TCR-a and -b chain constant primers.
  • RACE Rapid Amplification of cDNA Ends
  • PCR polymerase chain reaction
  • the method may comprise inserting the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, into a viral recombinant expression vector using established molecular cloning techniques as described in, e.g., Green et al. (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed. (2012).
  • the term "recombinant expression vector” means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell.
  • the vector is not naturally-occurring as a whole. However, parts of the vector can be naturally- occurring.
  • the recombinant expression vector can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double- stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides.
  • the recombinant expression vector can comprise naturally-occurring, non-naturally-occurring internucleotide linkages, or both types of linkages.
  • the non-naturally occurring or altered nucleotides or intemucleotide linkages does not hinder the transcription or replication of the vector.
  • the viral recombinant expression vector of the invention can be any suitable viral recombinant expression vector, and can be used to transform or transfect any suitable packaging cell line.
  • the viral recombinant expression vector is a retroviral vector.
  • retroviral vectors include alpharetroviral vectors, betaretroviral vectors, deltaretroviral vectors, epsilonretroviral vectors, gammaretroviral vectors, and lentiviral vectors.
  • the viral recombinant expression vector is a gammaretroviral vector.
  • retroviral vectors include, but are not limited to, pMSGVl vector, or a pUMVC vector.
  • the gammaretroviral vector is a pMSGVl vector.
  • the vector is a transient retroviral vector.
  • the vector may be a self-inactivating vector (SIN) or a non-SIN vector.
  • SIN vector is a viral vector that contains a non-functional or modified 3' Long Terminal Repeat (LTR) sequence. This sequence may be copied to the 5' end of the vector genome during integration, resulting in the inactivation of promoter activity by both LTRs.
  • LTR Long Terminal Repeat
  • the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, which is inserted into the recombinant expression vector may be the naturally occurring (i.e., wild-type) nucleotide sequence which encodes the TCR, or the antigen binding portion thereof, which was isolated from the patient.
  • the nucleotide sequence may be modified, for example, to increase expression of the TCR or antigen binding portion thereof.
  • the nucleotide sequence encoding the TCR which is inserted into the expression vector encodes the variable region of the TCR obtained from the patient, i.e., a human TCR, and the constant region of a murine TCR such that the TCR is“murinized.”
  • the method may further comprise transfecting packaging cell line 293 GP with the viral vector and a nucleotide sequence that encodes a viral envelope protein.
  • Transfecting cells with one or more vectors may be carried out in any suitable manner known in the art. See, for example, Green et al, supra.
  • the 293GP cell line is a human embryonic kidney (HEK) 293 -based retroviral packaging cell line stably expressing the Moloney Murine Leukemia Virus gap-pol and amphotropic envelope viral proteins (Ghani et al., Gene Ther ., 14(24): 1705-11 (2007)).
  • the viral envelope protein may be any envelope protein suitable for preparing replication incompetent viruses.
  • envelope proteins include, but are not limited to, an RD114 envelope protein (e.g., the feline RD114 envelope protein), a Gibbon ape leukemia virus (GALV) envelope protein, an amphotropic envelope protein (e.g., the amphotropic 4070 envelope protein), a xenotropic envelope protein, an ectotropic envelope protein, or a vesicular stomatitis virus envelope G protein (VSV-G).
  • the envelope protein is a RD114 envelope protein.
  • the transfection may take place in a closed system.
  • transfecting the packaging cell line 293 GP comprises transfecting the 293GP cells with about 80 to about 200 pg of the viral vector encoding the TCR, or antigen-binding portion thereof (per TCR).
  • Transfecting the packaging cell line 293GP comprises transfecting the 293GP cells with about 40 to about 100 pg of the nucleotide sequence that encodes a viral envelope protein (per TCR).
  • transfecting the packaging cell line 293GP comprises transfecting the 293GP cells with about 114 pg of the viral vector encoding the TCR, or antigen-binding portion thereof, and about 57 pg of the nucleotide sequence that encodes a viral envelope protein (per TCR).
  • Transfection may be carried out in a vessel (e.g., dish) of any size.
  • transfection may be carried out in a vessel having a size of from about 78.5 cm 2 to about 6320 cm 2 or greater.
  • the amount of vector used for the transfection may be calculated based on the size of the vessel.
  • transfecting the packaging cell line 293GP comprises transfecting the 293GP cells with about 0.050 pg to about 0.200 pg of the viral vector encoding the TCR, or antigen-binding portion thereof, per square cm of vessel and about 0.005 pg to about 0.200 pg of the nucleotide sequence that encodes a viral envelope protein per square cm of vessel.
  • transfecting the packaging cell line 293GP comprises transfecting the 293GP cells with about 0.114 pg of the viral vector encoding the TCR, or antigen-binding portion thereof, per square cm of vessel and about 0.057 pg of the nucleotide sequence that encodes a viral envelope protein per square cm of vessel.
  • Transfecting the packaging cell line 293 GP may comprise culturing the 293 GP cells in the presence of one or more of the viral vector, the nucleotide sequence that encodes a viral envelope protein, cell medium supplement, and transfection reagent for a period of time and under conditions sufficient to transfect the cells.
  • the transfection may be carried out using any suitable transfection reagent and any suitable technique.
  • suitable transfection reagents may include, but are not limited to, calcium phosphate precipitates, cationic lipids and liposomes, polyamines, polyethylenimine (PEI), histone proteins, and polylysine complexes.
  • transfecting the packaging cell line 293 GP may comprise culturing the 293 GP cells in the presence of the viral vector, the nucleotide sequence that encodes a viral envelope protein, fetal bovine serum (FBS) and LIPOFECTAMINE transfection reagent (Thermo Fisher Scientific, Waltham, MA) for a period of time and under conditions sufficient to transfect the cells.
  • the time period may be, for example, about 24 to about 75 hours, e.g., about 40 to about 50 hours, or, preferably, about 48 hours.
  • the cells are transfected in suspension.
  • the method may further comprise culturing the transfected cell line 293 GP under conditions sufficient to generate a retroviral supernatant comprising replication incompetent viruses, wherein the replication incompetent viruses comprise a vector comprising the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof.
  • the term“replication incompetent viruses” refer to viruses that contain a viral envelope and have the ability to infect cells but are not replication competent.
  • the replication incompetent viruses are retroviruses. Examples of retroviruses include alpharetroviruses, betaretroviruses, deltaretroviruses,
  • the replication incompetent viruses are gammaretroviruses.
  • the method comprises harvesting the supernatant comprising replication incompetent viruses.
  • the supernatant may be treated with an endonuclease which degrades free DNA and RNA which may be present in the
  • the supernatant may be treated with the endonuclease for any period of time and under such conditions so as to degrade free DNA and RNA.
  • the supernatant may be treated with the endonuclease for about 0.5 to about 3 hours at a temperature of about 37° C.
  • the supernatant is treated with the endonuclease for about 1 hour at a temperature of about 37° C.
  • the amount of free DNA and RNA may be reduced or eliminated.
  • the method comprises filtering the
  • the filtering may be carried out as a step-filtration.
  • the filtration may reduce or eliminate residual 293GP packaging cells and/or aggregates from the supernatant.
  • the method may, optionally, further comprise freezing the supernatant, e.g., in aliquots.
  • the method may further comprise testing one or both of (i) the replication incompetent viruses and (ii) the transfected cell line 293 GP for the presence of one or more adventitious agents.
  • adventitious agents may include any of a variety of microorganisms that may have been unintentionally introduced into the manufacturing process of a biological medicinal product. Examples of adventitious agents may include, but are not limited to, bacteria, fungi, mycoplasma, spiroplasma, mycobacteria, rickettsia, protozoa, parasites, transmissible spongiform encephalopathy (TSE) agents, and replication competent viruses (e.g., SV40 and adenovirus El A).
  • TSE transmissible spongiform encephalopathy
  • the method may comprise testing both of (i) the replication incompetent viruses and (ii) the transfected cell line 293 GP for the presence of one or more adventitious agents. In an embodiment of the invention, the method further comprises testing one or both of (i) the replication incompetent viruses and (ii) the transfected cell line 293 GP for the presence of one or more of sterility, residual DNA vector, endotoxin and replication competent retroviruses (RCR).
  • RCR replication competent retroviruses
  • a master cell bank (MCB) may also be produced and tested for RCR. By producing a MCB, testing on those cells may not need to performed more than once. Assays for RCR may include the use of antibodies for specific retroviruses and/or qPCR for DNA signatures of retroviruses.
  • the replication incompetent viruses prepared by the inventive methods may be useful for preparing cells for adoptive cell therapies.
  • an embodiment of the invention provides a method of preparing a population of cells that express a TCR, or an antigen-binding portion thereof, having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • the method may comprise preparing replication incompetent viruses according to any of the methods described herein with respect to other aspects of the invention.
  • the method may further comprise obtaining PBMC from the patient.
  • the PBMC include T cells.
  • the T cells may be any type of T cell, for example, any of those described herein with respect to other aspects of the invention.
  • the method may further comprise contacting the PBMC with the replication incompetent viruses under conditions sufficient to transduce the PBMC with the vector comprising the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof.
  • the PBMC may be physically contacted with the replication incompetent viruses such that the replication incompetent viruses introduce the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, into the genome of the PBMC.
  • the method comprises diluting the viruses about 2-fold prior to contacting the PBMC with the viruses.
  • PBMC may be contacted with the viruses on plates, e.g., plates coated with RETRONECTIN reagent.
  • Transduction of the PBMC may be carried out in any suitable vessel, e.g., a multiwell plate or bag.
  • the transduction of the PBMC is carried out in a closed system.
  • about lxlO 6 to about 3xl0 6 PBMC may be transduced in each well of the multi well plate.
  • about 5 to about 10 multi -well plates may be employed per TCR.
  • about 2xl0 6 PBMC may be transduced in each well of the multiwell plate (for a total of about 9.6xl0 7 cells).
  • about 8 multi-well plates may be employed per TCR.
  • the method may further comprise expressing the TCR, or the antigen-binding portion thereof, by the PBMC, thereby producing the population of cells which express the TCR, or the antigen-binding portion thereof, having antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.
  • the method may further comprise confirming that transduction of the PBMC was successful. Confirming that transduction of the PBMC may be carried out in a variety of different ways. For example, expression of the TCR (e.g., a murine TCR constant region) may be detected by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the method further comprises expanding the numbers of PBMC that express the TCR, or the antigen-binding portion thereof.
  • the numbers of PBMC may be expanded, for example, as described herein with respect to other aspects of the invention.
  • the numbers of cells expressing different TCRs may be expanded separately.
  • the inventive methods may, advantageously, generate a large number of T cells having antigenic specificity for the mutated amino acid sequence.
  • the inventive methods may, advantageously, transduce the PBMC with the nucleotide sequence that encodes the TCR, or the antigen binding portion thereof, with high efficiency.
  • the method produces a population of cells, wherein about 80% to about 100% of the cells in the population produced by the method express the TCR, or the antigen-binding portion thereof.
  • about 80% to about 99%, about 80% to about 98%, about 80% to about 97%, about 80% to about 96%, about 80% to about 95%, about 80% to about 94%, about 80% to about 93%, about 80% to about 92%, about 80% to about 91%, about 80% to about 90%, or about 85% to about 95%, or more of the cells in the population produced by the method express the TCR.
  • the method may further comprise testing the population of cells produced by the method for the presence of one or more adventitious agents.
  • the adventitious agents may be as described herein with respect to other aspects of the invention.
  • the method may further comprise testing the population of cells produced by the method for the presence of one or more of sterility, endotoxin, residual endonuclease, e.g., BENZONASE nuclease, and RCR.
  • Another embodiment of the invention provides an isolated or purified population of cells prepared according to any of the methods described herein with respect to other aspects of the invention.
  • the population of cells can be a heterogeneous population comprising the PBMC expressing the isolated TCR, or the antigen-binding portion thereof, in addition to at least one other cell, e.g., a host cell (e.g., a PBMC), which does not express the isolated TCR, or the antigen-binding portion thereof, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc.
  • a host cell e.g., a PBMC
  • a cell other than a T cell e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a
  • the population of cells can be a substantially homogeneous population, in which the population comprises mainly of PBMC (e.g., consisting essentially of) expressing the isolated TCR, or the antigen-binding portion thereof.
  • the population also can be a clonal population of cells, in which all cells of the population are clones of a single PBMC expressing the isolated TCR, or the antigen-binding portion thereof, such that all cells of the population express the isolated TCR, or the antigen binding portion thereof.
  • the population of cells is a clonal population comprising PBMC expressing the isolated TCR, or the antigen-binding portion thereof, as described herein.
  • the inventive methods may, advantageously, provide a population of cells that comprises a high proportion of PBMC cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence. In an embodiment of the invention, about 80% to about 100% of the cells in the population express the TCR, or the antigen-binding portion thereof.
  • populations of cells that comprise a high proportion of PBMC cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence have a lower proportion of irrelevant cells that may hinder the function of the PBMC, e.g., the ability of the PBMC to target the destruction of cancer cells and/or treat or prevent cancer.
  • the inventive populations of cells can be formulated into a composition, such as a pharmaceutical composition.
  • the invention provides a pharmaceutical composition comprising any of the inventive populations of cells described herein and a pharmaceutically acceptable carrier.
  • the inventive pharmaceutical composition can comprise a population of cells in combination with another pharmaceutically active agent(s) or drug(s), such as a chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel,
  • the carrier is a pharmaceutically acceptable carrier.
  • the carrier can be any of those conventionally used for the particular inventive population of cells under consideration.
  • Such pharmaceutically acceptable carriers are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.
  • the choice of carrier will be determined in part by the particular population of cells, as well as by the particular method used to administer the population of cells.
  • Suitable formulations may include any of those for oral, parenteral, subcutaneous, intravenous, intramuscular, intratumoral, intraarterial, intrathecal, or interperitoneal administration. More than one route can be used to administer the inventive population of cells, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • the inventive population of cells is administered by injection, e.g., intravenously.
  • the inventive population of cells is to be administered, the
  • pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate.
  • the pharmaceutically acceptable carrier is supplemented with human serum albumin.
  • the TCRs, or the antigen-binding portions thereof are believed to bind specifically to a mutated amino acid sequence encoded by a cancer-specific mutation, such that the TCR, or the antigen-binding portion thereof, when expressed by a cell, is able to mediate an immune response against a target cell expressing the mutated amino acid sequence.
  • an embodiment of the invention provides a method of treating or preventing cancer in a mammal, comprising preparing a population of cells which express a TCR, or an antigen-binding portion thereof, having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation by any of the methods described herein with respect to other aspects of the invention; and administering the population of cells produced by the method to the mammal in an amount sufficient to treat or prevent cancer in the mammal.
  • the population of cells to be administered to the mammal may be included in a pharmaceutical composition, as described herein with respect to other aspects of the invention.
  • cells expressing multiple different TCRs identified by the inventive methods may be pooled into a single pharmaceutical composition for administration.
  • the pharmaceutical composition may comprise greater than about lxlO 8 cells expressing each TCR, preferably greater than about lxlO 9 cells expressing each TCR.
  • inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
  • treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented.
  • treatment or prevention can include promoting the regression of a tumor.
  • prevention can encompass delaying the onset of the cancer, or a symptom or condition thereof.
  • the amount or dose of the inventive population of cells or pharmaceutical composition administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame.
  • the dose of the inventive population of cells or pharmaceutical composition should be sufficient to bind to a mutated amino acid sequence encoded by a cancer-specific mutation, or detect, treat or prevent cancer in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer.
  • the dose will be determined by the efficacy of the particular population of cells or pharmaceutical composition administered and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.
  • an assay which comprises comparing the extent to which target cells are lysed or IFN-g is secreted by T cells expressing the TCR, or the antigen-binding portion thereof, upon administration of a given dose of such T cells to a mammal among a set of mammals of which is each given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal.
  • the extent to which target cells are lysed or IFN-g is secreted upon administration of a certain dose can be assayed by methods known in the art.
  • the dose of the inventive population of cells or pharmaceutical composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive population of cells or pharmaceutical composition.
  • the attending physician will decide the dosage of the inventive population of cells or pharmaceutical composition with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive population of cells or pharmaceutical composition to be administered, route of administration, and the severity of the condition being treated.
  • the number of cells administered per infusion may vary, for example, in the range of one million to 100 billion cells; however, amounts below or above this exemplary range are within the scope of the invention.
  • the daily dose of inventive host cells can be about 1 million to about 150 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 60 billion cells, about 80 billion cells, about 100 billion cells, about 120 billion cells, about 130 billion cells, about 150 billion cells, or a range defined by any two of the foregoing values), preferably about 10 million to about 130 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about
  • Another embodiment of the invention provides any of the isolated populations of cells or pharmaceutical compositions described herein for use in treating or preventing cancer in a mammal.
  • the cancer may, advantageously, be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, cholangiocarcinoma, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma,
  • the mammal referred to in the inventive methods can be any mammal.
  • the term "mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). Preferably, the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • a more preferred mammal is the human.
  • the mammal is the patient expressing the cancer-specific mutation.
  • a nucleotide sequence that encodes a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation was isolated as follows for the experiments described in Examples 1-9.
  • One or more genes were identified in the nucleic acid of a cancer cell of a patient. Each gene contained a cancer-specific mutation that encodes a mutated amino acid sequence.
  • Autologous APCs of the patient were induced to present the mutated amino acid sequence.
  • Autologous T cells of the patient were co-cultured with the autologous APCs that present the mutated amino acid sequence.
  • the autologous T cells that (a) were co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a MHC molecule expressed by the patient were selected.
  • a nucleotide sequence that encodes the TCR was isolated from the selected autologous T cells. The nucleotide sequence was inserted into a viral vector.
  • This example demonstrates a method of preparing a population of cells which express a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • FIG. 1 A schematic illustrating a method of preparing a population of cells which express a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer- specific mutation (neoantigen) according to an embodiment of the invention is presented in Figure 1.
  • the human constant regions of the TCR were replaced with murine constant regions.
  • packaging cell line 293GP was transfected with the viral vector and a nucleotide sequence that encodes a viral envelope protein.
  • the transfected cell line 293 GP was cultured under conditions sufficient to generate a retroviral supernatant comprising replication incompetent viruses, wherein the replication incompetent viruses comprise a vector comprising the nucleotide sequence that encodes the TCR.
  • the replication incompetent viruses (in the supernatant) were tested for safety.
  • the PBMC were contacted with the replication incompetent viruses under conditions sufficient to transduce the PBMC with the vector comprising the nucleotide sequence that encodes the TCR.
  • the population of cells produced by the method was tested for safety.
  • This example demonstrates the pre-clinical validation of good laboratory practice (GLP) preparations for transient gammaretovrial vector production.
  • Autologous PBMC were transduced with a transient gammaretoviral vector produced as described in Example 1 ( Figure 1) and encoding TCR1 or TCR2.
  • the transduced PBMC were then co-cultured with autologous APCs pulsed with either wildtype (wt) or mutated (mut) peptide for a given neoantigen.
  • the neoantigen-reactive TCR1 (Vbl6) recognized mutated antigen H3F3B.
  • the neoantigen-reactive TCR2 (Vb20) recognized mutated SKIV2L (Vb20, TCR2).
  • Gammaretroviral vector encoding the TCR1 or TCR2 of Example 2 was manufactured by transient transfection of 293GP cells as described in Example 1 ( Figure 1). Samples were taken after transfection with or without BENZONASE endonuclease treatment. Residual vector DNA was measured by quantitative polymerase chain reaction (qPCR).
  • PBMC transduced with either TCR1 or TCR2 were cultured for up to 1 month. Cell samples were taken and cryopreserved at the time points indicated in Figure 3C.
  • This example demonstrates a method of preparing a population of cells which express a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • a population of cells which express a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation was prepared as follows:
  • Day 1 the plated 293GP cells were transfected with a retroviral vector encoding the RD114 envelope protein and a viral vector encoding a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • Day 2 The cell medium was changed.
  • Day 3 The supernatant was harvested and treated with BENZONASE endonuclease. PBMC were transduced.
  • Day 9 Co-culture of T cells (expressing the TCR having antigenic specificity for a mutated amino acid sequence) with antigen-positive target cells.
  • Retroviral supernatant was prepared as described in Example 4. The supernatant was treated with 0, 25, or 50 U/mL BENZONASE endonuclease.
  • the supernatant was used to transfect PBMC from three patients (Patients D, H, and R) with one of three TCRs (nb22, nb5.2 a 26.1, or nb5.2 a 30) which recognize mutated ERBBIP2 as also described in Example 4.
  • ETntransduced or transduced cells were co-cultured with B cells pulsed overnight with wild type ERBBIP2 or mutated ERBBIP2. IFNy secretion was measured. Controls included T cells cultured alone (no target) and co-cultures with (i) OKT3 antibody and (ii) B cells that have not been pulsed with mutated or wild type peptide.
  • Retroviral supernatant was prepared as described in Example 4 with retroviral vectors encoding the Ub22 or Ub5.2 a 30 TCR. The supernatant was treated with 0, 25, or 50 U/mL BENZONASE endonuclease. Free DNA in the supernatant and in a 100 mL dose was measured by qPCR. The results are shown in Table 1A (Ub5.2 a 30) and Table 1B (Ub22).
  • Retroviral supernatant was prepared as described in Example 4 with retroviral vectors encoding the nb22 or nb5.2 a 30 TCR. The supernatant was treated with 0 or 50 U/mL BENZONASE endonuclease. Free DNA in the supernatant and in a 100 mL dose was measured by qPCR. The results are shown in Table 2A (Ub5.2 a 30) and Table 2B (Ub22). TABLE 2 A
  • TCR-transduced cells were prepared as described in Example 4 using retroviral supernatant prepared using 0, 25, or 50 U/mL BENZONASE endonuclease.
  • This example demonstrates a method of preparing a population of cells which expresses a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation.
  • TCRs having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation were identified and isolated from 5 patients (Patients 4217, 4275, 4271, 4251, and 4285).
  • the human constant region of the TCR was replaced with a murine constant region.
  • a viral vector (pMSGV) encoding the TCR with the murine constant region was prepared.
  • Packaging cell line 293GP was transfected with the viral vector and a nucleotide sequence that encodes a viral envelope protein (RD114) at a Contract Manufacturing
  • the transfected cell line 293 GP was cultured under conditions sufficient to generate a retroviral supernatant comprising replication incompetent viruses, wherein the replication incompetent viruses comprise a vector comprising the nucleotide sequence that encodes the TCR.
  • Healthy donor PBMC were contacted with the replication incompetent viruses under conditions sufficient to transduce the PBMC with the vector comprising the nucleotide sequence that encodes the TCR.
  • TCR-transduced healthy donor T cells of Example 9 were co-cultured overnight with autologous patient B cells pulsed with 10 pg of either mutant or wild type peptide. IFN-g release was measured by ELISA assay and reported in pg/mL. The results are shown in Figure 13. As shown in Figure 13, the TCR-transduced healthy donor T cells of Example 9 specifically recognized the target mutated antigen. [0134] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

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Abstract

L'invention concerne des procédés de préparation de virus incompétents pour la réplication comprenant un vecteur codant pour un récepteur de lymphocyte T (TCR), ou une partie de celui-ci se liant à l'antigène, ayant une spécificité antigénique pour une séquence d'acides aminés mutée codée par une mutation spécifique du cancer. Des procédés associés de préparation d'une population de cellules qui expriment le TCR, ou une partie de celui-ci se liant à l'antigène, ou de populations de cellules isolées ou purifiées préparées par lesdits procédés, des compositions pharmaceutiques associées, et des méthodes de traitement ou de prévention du cancer chez un mammifère sont en outre décrits.
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