US20220315892A1 - Engineered Artificial Antigen Presenting Cells for Tumor Infiltrating Lymphocyte Expansion - Google Patents

Engineered Artificial Antigen Presenting Cells for Tumor Infiltrating Lymphocyte Expansion Download PDF

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US20220315892A1
US20220315892A1 US17/229,113 US202117229113A US2022315892A1 US 20220315892 A1 US20220315892 A1 US 20220315892A1 US 202117229113 A US202117229113 A US 202117229113A US 2022315892 A1 US2022315892 A1 US 2022315892A1
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protein
population
tils
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Anand Veerapathran
Aishwarya Gokuldass
Brian Rabinovich
Michael T. Lotze
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Iovance Biotherapeutics Inc
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Assigned to IOVANCE BIOTHERAPEUTICS, INC. reassignment IOVANCE BIOTHERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RABINOVICH, BRIAN, LOTZE, MICHAEL T., GOKULDASS, Aishwarya, Veerapathran, Anand
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Definitions

  • aAPCs artificial antigen presenting cells
  • TILs tumor infiltrating lymphocytes
  • REP can result in a 1,000-fold expansion of TILs over a 14-day period, it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT-3) and high doses of IL-2.
  • PBMCs peripheral blood mononuclear cells
  • OKT-3 anti-CD3 antibody
  • PBMCs have multiple drawbacks, including the large numbers of allogeneic PBMCs required, the need to obtain PBMCs by leukapheresis from multiple healthy donors, the resulting interdonor variability in PBMC viability after cryopreservation and variable TIL expansion results, the risk of undetected viral pathogens causing downstream patient infections, and the extensive and costly laboratory testing of each individual donor cell product to confirm sterility and quality (including viral contaminant testing) and to test expansion properties.
  • aAPCs developed for use in the expansion of TILs have suffered from poor performance when compared to PBMCs, including alterations of the phenotypic properties of the input TILs, as well as poor expansion performance and/or high variability in expansion results. Because of the large number of potential cells that might be adapted for use as aAPCs and the unpredictability of identifying suitable candidates, the focus of aAPC development for polyclonal TILs to date has been solely on the well-established K562 cell line. Butler and Hirano, Immunol. Rev. 2014, 257, 191-209.
  • K562 cells modified to express 4-1BBL were tested in pre-REP culture (but not in REP culture) to determine enhancement of TIL expansion from tumor digest, but PBMCs were still required to be used in conjunction with K562 cells to obtain TIL expansion.
  • PBMCs were still required to be used in conjunction with K562 cells to obtain TIL expansion.
  • Other engineered K562 cells modified to express CD64, CD86, and 4-1BBL were tested and achieved TIL expansion that was at best comparable to PBMCs, and most likely less than PBMCs, and also suffered from skewing of the polyclonal TIL phenotype to a less favorable CD8 + /CD4 + T cell ratio.
  • K562 modified aAPCs have not been shown to provide for consistent expansion of TILs with acceptable variability while also performing better than PBMCs in other measures including overall expansion cell counts.
  • Alternative aAPCs besides K562 cells have been successful in other cell expansion methods, but have not achieved the same performance as PBMCs with the unique polyclonal subset of cells that make up TILs. Maus, et al., Nat. Biotechnol. 2002, 20, 143-148; Suhoski, et al., Mot Ther. 2007, 15, 981-988.
  • MOLM-14 human leukemia cell line was established from the peripheral blood of a patient with relapsed acute monocytic leukemia, and initial phenotypic characterization indicated the presence of at least the following markers: CD4, CD9, CD11a, CD13, CD14, CD15, CD32, CD33, CD64, CD65, CD87, CD92, CD93, CD116, CD118, and CD155. Matsuo, et al., Leukemia 1997, 11, 1469-77. Additional phenotypic characterization of MOLM-14 found higher levels of HLA-A/B/C, CD64, CD80, ICOS-L, CD58, and lower levels of CD86. MOLM-14 cells and the closely-related MOLM-13 cells have not been previously reported as useful aAPCs for the expansion of cells for tumor immunotherapy applications.
  • the EM-3 human cell line was established from the bone marrow of a patient with Philadelphia chromosome-positive CIVIL. Konopka, et al., Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. EM-3 cells and the closely-related EM-2 cell line have not been previously reported as useful aAPCs for the expansion of cells for tumor immunotherapy applications. Phenotypic characterization for EM-3 cells indicates the presence of at least the following markers: CD13, CD15, and CD33.
  • the present invention provides the unexpected finding that engineered myeloid lineage cells, including MOLM-13, MOLM-14, EM-3, and EM-2 cells, transduced with additional costimulatory molecules, including CD86 (B7-2), 4-1BBL (CD137L), and OX40L (CD134L), provide for superior and highly efficient expansions of TILs in large numbers with minimal variability, reduced cost, and no reliance on human blood samples as a source of PBMCs, with the benefit of using an aAPC which can be produced efficiently from a master cell bank.
  • CD86 and 4-1BBL are costimulatory molecules that provide costimulatory signals for T cell activation.
  • the MOLM-14, MOLM-13, EM-3, and/or EM-2 cells transduced with additional costimulatory molecules are useful, for example, in the expansion of TILs for use in cancer immunotherapy and other therapies.
  • the invention provides an artificial antigen presenting cell (aAPC) comprising a myeloid cell transduced with one or more vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein.
  • aAPC artificial antigen presenting cell
  • each of the CD86 protein and the 4-1BBL protein are human proteins.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC can stimulate and expand a tumor infiltrating lymphocyte (TIL) contacted with the aAPC.
  • TIL tumor infiltrating lymphocyte
  • the nucleic acid molecule encoding CD86 may be comprised in a different viral vector to the nucleic acid molecule encoding 4-1BBL or the same viral vector.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 IU/mL and OKT-3 antibody at a concentration of about 30 ng/mL.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC can stimulate and expand a T cell contacted with the aAPC.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell endogenously expresses HLA-AB/C, ICOS-L, and CD58.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is essentially devoid of membrane-bound IL-15.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a MOLM-14 cell.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a MOLM-13 cell.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a EM-3 cell.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a EM-2 cell.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the CD86 protein comprises an amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence comprising one or more conservative amino acid substitutions thereof, and the 4-1BBL protein comprises SEQ ID NO:9, or an amino acid sequence comprising one or more conservative amino acid substitutions thereof.
  • the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the nucleic acid molecule encoding CD86 comprises a nucleic acid sequence as set forth in SEQ ID NO:16 and the nucleic acid molecule encoding 4-1BBL comprises a nucleic acid sequence as set forth in SEQ ID NO:19.
  • the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising the step of contacting a population of TILs with an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and wherein the population of TILs is expanded.
  • the method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the foregoing method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the foregoing method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the foregoing method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the foregoing method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the foregoing method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the foregoing method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the foregoing method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the foregoing method is an in vitro or an ex vivo method.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising a population of TILs with a myeloid artificial antigen presenting cell (aAPC), wherein the myeloid aAPC comprises at least two co-stimulatory ligands that specifically bind with at least two co-stimulatory molecules on the TILs, wherein binding of the co-stimulatory molecules with the co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs, and wherein the at least two co-stimulatory ligands comprise CD86 and 4-1BBL.
  • the foregoing method is an in vitro or ex vivo method.
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating cancer, wherein the TILs are a second population of TILs and are obtainable from a method comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating cells (TILs) for use in treating a cancer, wherein the population of TILs is a second population of TILs and is obtainable by a process comprising:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a second population and is obtainable by a method comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating cells (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a method comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and obtainable by a method comprising the steps of:
  • the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • the myeloid cells comprise MOLM-13 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cells express a CD86 protein and a 4-1BBL protein.
  • the myeloid cells comprise EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein.
  • the myeloid cells comprise EM-2 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-2 cells express a CD86 protein and a 4-1BBL protein.
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs are a third population of TILs and obtainable by a method comprising the steps of:
  • the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • the myeloid aAPCs comprise MOLM-13 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cells express a CD86 protein and a 4-1BBL protein.
  • the myeloid aAPCs comprise EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein.
  • the population of TILs is for use in the treating of a cancer selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma.
  • a cancer selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma.
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:
  • the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is about 1 to 300.
  • the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • the invention provides a kit for specifically inducing proliferation of a tumor infiltrating lymphocyte expressing a known co-stimulatory molecule, the kit comprising an effective amount of an aAPC, wherein said aAPC comprises a MOLM-14 cell or a EM-3 cell transduced using a lentiviral vector (LV), wherein the LV comprises a nucleic acid encoding at least one co-stimulatory ligand that specifically binds said known co-stimulatory molecule, wherein binding of the known co-stimulatory molecule with said co-stimulatory ligand stimulates and expands said T cell, the kit further comprising an applicator and an instructional material for the use of said kit.
  • aAPC comprises a MOLM-14 cell or a EM-3 cell transduced using a lentiviral vector (LV), wherein the LV comprises a nucleic acid encoding at least one co-stimulatory ligand that specifically binds said known co-stimulatory molecule, wherein
  • the invention provides a method for assessing the potency of tumor infiltrating lymphocytes (TILs) comprising the steps of:
  • FIG. 1 illustrates the results of rapid expansion of TILs using irradiated allogeneic PBMC feeder cells.
  • Each TIL line (M1015T and M1016T) (1.3 ⁇ 10 5 cells) was co-cultured with 46 different irradiated feeders (1.3 ⁇ 10 7 cells), IL-2 (3000 IU/mL) and OKT-3 (30 ng/mL) in a T25 flask for 7 days.
  • the fold expansion value for TILs was calculated on Day 7.
  • the figure shows the number of fold expansions for two TIL lines in separate stimulation experiments, with 46 different feeder lots tested, and highlights the variability of expansion results using PBMC feeder cells.
  • FIG. 2 illustrates a vector diagram of the pLV430G human 4-1BBL vector.
  • FIG. 3 illustrates a diagram of the 4-1BBL PCRP (polymerase chain reaction product) portion of the pLV430G human 4-1BBL vector.
  • FIG. 4 illustrates a vector diagram of the pLV430G human CD86 vector.
  • FIG. 5 illustrates a diagram of the CD86 PCRP portion of the pLV430G human CD86 vector.
  • FIG. 6 illustrates a vector diagram of the pDONR221 human CD86 donor vector.
  • FIG. 7 illustrates a vector diagram of the pDONR221 human 4-1BBL donor vector.
  • FIG. 8 illustrates a vector diagram of the pLV430G empty vector.
  • FIG. 9 illustrates a vector diagram of the pDONR221 empty vector.
  • FIG. 10 illustrates a vector diagram of the psPAX2 helper plasmid for lentivirus production.
  • FIG. 11 illustrates a vector diagram of the pCIGO-VSV.G helper plasmid for lentivirus production.
  • FIG. 12 illustrates the results of flow cytometry experiments on MOLM-14 cells before lentiviral transfection (“Untransfected”) and after transfection (“Transfected”), confirming the expression of CD137 and CD86 on engineered MOLM-14 cells.
  • FIG. 13 illustrates the results of rapid expansion of TILs using irradiated parental unmodified MOLM-14 cells (“Parent MOLM14”), engineered MOLM-14 cells (CD86/4-1BBL, “Engineered MOLM14”), or PBMC feeders (“Feeders”) for TIL lot M1032-T2.
  • TIL were co-cultured with PBMC feeders or parental or engineered MOLM14 cells at 1:100 ratios with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted and split on Day 6 and 11. Each dot represents cell numbers determined on Day 0, 6, 11 and 14 respectively. A logarithmic scale is used.
  • FIG. 14 illustrates results as shown in FIG. 13 , depicted using a linear scale.
  • FIG. 15 illustrates results for TIL lot M1033-T6 with other parameters as given in FIG. 13 , using a logarithmic scale.
  • FIG. 16 illustrates results as shown in FIG. 14 , depicted using a linear scale.
  • FIG. 17 illustrates the results of rapid expansions of TILs using engineered MOLM-14 cells expressing CD86 and 4-1BBL (“TIL+Engineered MOLM14 (CD86/41BB)+OKT3”) or irradiated PBMC feeders (“TIL+Feeders+OKT3”).
  • TIL were co-cultured with PBMC feeders or engineered MOLM-14 cells (aMOLM14) at 1:100 ratios plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted and split on Day 6 and 11. Each point represents cell numbers determined on Day 14.
  • FIG. 18 illustrates the results of experiments in which TILs (2 ⁇ 10 4 ) were cultured with different ratios (1:10, 1:30, and 1:100, denoted “10”, “30”, and “100”, respectively) of parental MOLM-14 (“MOLM14”) cells, MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), or PBMC feeders (“PBMC+”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in wells of a 24-well G-Rex plate.
  • a control was performed using only OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) (“PBMC-”). Each condition was cultured in triplicate.
  • FIG. 19 illustrates the results of TILs cultured with different ratios (1:30, 1:100, and 1:300, denoted “30”, “100”, and “300”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Viable cells were counted on day 11 and plotted. Other conditions are as in FIG. 18 .
  • FIG. 20 illustrates the results of TILs cultured with different ratios (1:50, 1:100, and 1:200, denoted “50”, “100”, and “200”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Cells were counted on day 14. Other conditions are as in FIG. 18 .
  • FIG. 21 illustrates the results of TILs cultured with different ratios (1:100, 1:200, 1:400, and 1:800, denoted “100”, “200”, “400”, and “800”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Cells were counted on day 14. Other conditions are as in FIG. 18 .
  • FIG. 22 illustrates a sunburst visualization showing fine distribution of Live, T cell receptor (TCR) ⁇ / ⁇ , CD4, CD8, CD27, CD28, and CD57 TILs expanded with PBMC feeders.
  • TCR Live, T cell receptor
  • FIG. 23 illustrates a sunburst visualization showing fine distribution of Live, TCR ⁇ / ⁇ , CD4, CD8, CD27, CD28, and CD57 TILs expanded with aMOLM14 aAPCs.
  • FIG. 24 depicts a flow cytometry contour plot showing memory subset (CD45RA+/ ⁇ , CCR7+/ ⁇ ) gated on Live, TCR ⁇ / ⁇ +, CD4 + , or CD8 + TILs.
  • FIG. 25 illustrates phenotypic characterization of the T cell subset, CD4 + and CD8 + post-REP TILs (expanded with aMOLM14 aAPCs) gated on CD3 + cells using a SPADE tree.
  • the color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, PD1, and CD137.
  • FIG. 26 illustrates phenotypic characterization of the T cell subset, CD4 + and CD8 + post-REP TILs (expanded with aMOLM14 aAPCs) gated on CD3 + cells using a SPADE tree.
  • the color gradient is proportional to the MFI CD69, CD154, KLRG1, and TIGIT
  • FIG. 27 illustrates oxygen consumption rate (OCR) of TIL after expansion with Feeders or aMOLM14 measured during a mitochondrial stress test. Each data point represents mean ⁇ standard error of the mean (SEM) measured in triplicate.
  • FIG. 28 illustrates extracellular acidification rate (ECAR) of TIL after expansion with Feeders or aMOLM14 measured during a mitochondrial stress test. Each data point represents mean ⁇ SEM measured in triplicate.
  • FIG. 29 illustrates a vector diagram of the destination vector pLV4301G.
  • FIG. 30 illustrates a vector diagram of donor vector 1, pMK 7c12 anti mFC scFv CoOp ECORV SacII L1R5.
  • FIG. 31 illustrates a vector diagram of donor vector 2, pMK hCD8a scaffold TN L5 L2.
  • FIG. 32 illustrates a vector diagram of final vector used for lentiviral production, pLV4301G 7C12 scFv mIgG hCD8 flag.
  • FIG. 33 illustrates a vector diagram of the destination vector pLV4301G.
  • FIG. 34 illustrates a vector diagram of donor vector 1, pMK 8B3 anti mFC scFv CoOp ECORV SacII L1R5.
  • FIG. 35 illustrates a vector diagram of donor vector 2, pMK hCD8a scaffold TN L5 L2.
  • FIG. 36 illustrates a vector diagram of final vector used for lentiviral production, pLV4301G 8B3 scFv mIgG hCD8 flag.
  • FIG. 37 illustrates the results of flow cytometry experiments on EM-3 cells before lentiviral transfection (“Untransfected”) and after transfection (“Transfected”), confirming the expression of CD137 and CD86 on engineered EM-3 cells.
  • FIG. 38 illustrates the results of experiments wherein TILs were co-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14.
  • FIG. 39 illustrates the results of experiments wherein TILs were co-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14.
  • FIG. 40 illustrates the results of experiments wherein TILs were co-cultured with aEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3000 IU/mL), with or without OKT-3 (30 ng/mL).
  • the bar graph shows cell numbers determined on Day 11.
  • FIG. 41 illustrates the results of TIL expansions with EM-3 aAPCs at different TIL:aAPC ratios.
  • FIG. 42 illustrates the results of TIL expansions with EM-3 aAPCs.
  • TILs (2 ⁇ 10 4 ) were co-cultured with five different PBMC feeder lots or aEM3 (in triplicate) at a 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. Viable cells were counted on Day 14. The graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14.
  • FIG. 43 illustrates the results of TIL expansions with EM-3 aAPCs and MOLM-14 aAPCs.
  • TILs (2 ⁇ 10 4 ) were co-cultured with five different PBMC feeder lots or aMOLM14 (in triplicate) or aEM3 (also in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate.
  • the graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14.
  • FIG. 44 illustrates a sunburst visualization to show fine distribution of Live, TCR ⁇ / ⁇ , CD4 + , and CD8 + TILs expanded with aEM3 aAPCs or PBMC feeders (TIL batch M1054).
  • FIG. 45 illustrates the sunburst visualization to show fine distribution of Live, TCR ⁇ / ⁇ , CD4 + , and CD8 + TILs expanded with aEM3 aAPCs or PBMC feeders (TIL batch M1055).
  • FIG. 46 illustrates the CD4 + and CD8 + SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3 + cells.
  • the color gradient is proportional to the MFI of LAG-3, TIM-3, PD-1, and CD137.
  • FIG. 47 illustrates the CD4 + and CD8 + SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3 + cells.
  • the color gradient is proportional to the MFI of CD69, CD154, KLRG1, and TIGIT.
  • FIG. 48 illustrates a summary of spare respiratory capacity measured by the Seahorse XF Mito stress test.
  • FIG. 49 illustrates a summary of glycolytic reserve measured by the Seahorse XF Mito stress test.
  • FIG. 50 illustrates a mitochondrial stain of live TILs expanded against PBMC or aEM3 using MitoTracker dye, which stains mitochondria in live cells and for which accumulation is dependent upon membrane potential.
  • TILs expanded against PBMC or aEM3 were stained L/D Aqua followed by MitoTracker red dye. Data shown are MitoTracker positive (MFI) cells gated on live population.
  • MFI MitoTracker positive
  • FIG. 51 illustrates results of a P815 BRLA for cytotoxic potency and functional activity, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs.
  • FIG. 52 illustrates results of a P815 BRLA for cytotoxic potency and functional activity, comparing TILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs.
  • FIG. 53 illustrates IFN- ⁇ release for two batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs.
  • S overnight stimulation
  • US unstimulated
  • FIG. 55 illustrates Granzyme B release for two batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs.
  • S overnight stimulation
  • US unstimulated
  • FIG. 57 illustrates a TIL expansion and treatment process.
  • aAPCs of the present invention may be used in both the pre-REP stage (top half of figure) or REP stage (bottom half of figure) and may be added when IL-2 is added to each cell culture.
  • Step 1 refers to the addition of 4 tumor fragments into 10 G-Rex 10 flasks.
  • step 2 approximately 40 ⁇ 10 6 TILs or greater are obtained.
  • a split occurs into 36 G-Rex 100 flasks for REP.
  • TILs are harvested by centrifugation at step 4.
  • Fresh TIL product is obtained at step 5 after a total process time of approximate 43 days, at which point TILs may be infused into a patient.
  • FIG. 58 illustrates a treatment protocol for use with TILs expanded with aAPCs.
  • Surgery occurs at the start, and lymphodepletion chemo refers to non-myeloablative lymphodepletion with chemotherapy as described elsewhere herein.
  • FIG. 59 illustrates Bioluminescent Redirected Lysis Assay (BRLA) results, showing percentage cytotoxicity of TIL batch M1033T-1 when co-cultured with P815 Clone G6 (with and without anti-CD3) at individual effector:target ratios.
  • BRLA Bioluminescent Redirected Lysis Assay
  • FIG. 60 illustrates enzyme-linked immunosorbent assay (ELISA) data showing amount of IFN- ⁇ released against different ratios of effector to target cells.
  • ELISA enzyme-linked immunosorbent assay
  • FIG. 61 illustrates LAMP1(%) expressed by TIL batch M1033T-1 when co-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of 1:1 effector to target cells for 4 hr and 24 hr co-culture.
  • FIG. 62 illustrates BRLA results for TIL batch M1030. Cytotoxicity (measured as LU 50 /1 ⁇ 10 6 TIL) by BRLA is 26 ⁇ 16.
  • FIG. 63 illustrates standard chromium release assay for TIL batch M1030. Cytotoxicity (measured as LU 50 /1 ⁇ 10 6 TIL) by the chromium release assay is 22.
  • FIG. 64 illustrates BRLA results for TIL batch M1053, showing the lytic units of the TILs by BRLA as 70 ⁇ 17.
  • FIG. 65 illustrates standard chromium release assay results for TIL batch M1053, also showing lytic unit of the TILs by chromium assay as 14 ⁇ 5. Comparison of this result with FIG. 64 shows the comparable performance of the BRLA and chromium release assay.
  • FIG. 66 illustrates the linear relationship between IFN- ⁇ release and cytotoxic potential of TILs.
  • FIG. 67 illustrates ELISpot results for IFN- ⁇ .
  • FIG. 68 illustrates enzymatic IFN- ⁇ release for TIL batch M1053.
  • FIG. 69 illustrates enzymatic IFN- ⁇ release for TIL batch M1030.
  • FIG. 70 illustrates ELISpot data showing Granzyme B release by M1053T and M1030T. This data confirms the potency of the TILs shown by the BRLA.
  • FIG. 71 illustrates enzymatic Granzyme B release for TIL batch M1053.
  • FIG. 72 illustrates enzymatic Granzyme B release for TIL batch M1030.
  • FIG. 73 illustrates ELISpot data showing TNF- ⁇ release by M1053T and M1030T. This data confirms the potency of the TILs shown by the BRLA.
  • FIG. 74 illustrates enzymatic TNF- ⁇ release for TIL batch M1053.
  • FIG. 75 illustrates enzymatic TNF- ⁇ release for TIL batch M1030.
  • FIG. 76 illustrates changes in cell populations of aEM3 cells (C712 (A) and 8B5 (B)) when weaning such cell populations off of FBS to hAB serum media.
  • FIG. 77 illustrates changes in cell populations of during freeze-thaw-recovery cycles with aEM3 cell populations suspended in various freezing media.
  • FIG. 78 illustrates the growth of aEM3 cells in gas permeable cell culture flasks over an eight-day time course.
  • FIG. 79 illustrates a flow panel analysis to determine the purity of aEM3 cells.
  • FIG. 80 illustrates the results of a flow panel analysis used to determine the purity of aEM3 cells.
  • FIG. 81 illustrates the differences in cytokine expression between aEM3 feeder cells and PBMC feeders stimulated by OKT3.
  • FIG. 82 illustrates that TIL may advantageously expanded (pre-REP) with serum free media (i.e., CTS Optmizer) to provide increased cell numbers as compared to CM1.
  • pre-REP serum free media
  • CTS Optmizer serum free media
  • FIG. 83 and FIG. 84 illustrate that TIL may advantageously expanded with serum free media (i.e., CTS Optmizer) to provide increased cell numbers as compared to CM1 at Day 11 (PreREP) ( FIG. 83 ) and Day 22 (Pre- and Post-REP) ( FIG. 84 ).
  • serum free media i.e., CTS Optmizer
  • FIG. 85 illustrates that aAPC cells (i.e., aEM3 cells) can be grown and using serum free media.
  • aAPC cells i.e., aEM3 cells
  • CTS OpTimizer and Prime-TCDM were found to be effective in growing aEM3 as compared to cDMEM (10% hSerum). Data shown were mean ⁇ SD of five separate experiments. The p value was calculated by the student t-test. *P ⁇ 0.05.
  • FIG. 86 and FIG. 87 illustrate the results of two experiments that demonstrate the rapid recovery of aEM3 cells from the TIL-R3 cell line on day 3 following cryopreservation.
  • FIG. 86 illustrates the total cell counts for experiment one and
  • FIG. 87 illustrates the total cell counts for experiment two.
  • FIG. 88 illustrates the growth of aEM3 cells from the TIL-R3 cell line following cryopreservation where the cells were plated and grown for 9 days. Cell counts were measured every three days post thaw.
  • FIG. 89 illustrates the growth of aEM3 cells from the TIL-R3 cell line following cryopreservation where the cells were plated in GREX 10 flasks and grown for 8 days. Cell counts were measured every four days post thaw.
  • FIG. 90 illustrates a vector diagram of the pLenti-C-Myc-DDK human OX40L vector.
  • FIG. 91 illustrates the results of flow cytometry analysis of TILs expanded in a REP with the aEM3 cell line and PBMC feeders, showing that TILs cultured with aEM3 promotes CD8 + TIL skewness.
  • FIG. 92 illustrates the numbers of viable cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.
  • FIG. 93 illustrates the numbers of CD3 + cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.
  • FIG. 94 illustrates the numbers of CD3 ⁇ cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.
  • FIG. 95 illustrates the results of telomere length analysis using a qPCR method.
  • FIG. 96 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.
  • FIG. 97 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.
  • FIG. 98 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.
  • SEQ ID NO:1 is an amino acid sequence for the heavy chain of muromonab.
  • SEQ ID NO:2 is an amino acid sequence for the light chain of muromonab.
  • SEQ ID NO:3 is an amino acid sequence for recombinant human IL-2.
  • SEQ ID NO:4 is an amino acid sequence for aldesleukin.
  • SEQ ID NO:5 is an amino acid sequence for recombinant human IL-7.
  • SEQ ID NO:6 is an amino acid sequence for recombinant human IL-15.
  • SEQ ID NO:7 is an amino acid sequence for recombinant IL-21.
  • SEQ ID NO:8 is the amino acid sequence of human CD86.
  • SEQ ID NO:9 is the amino acid sequence of human 4-1BBL (CD137L).
  • SEQ ID NO:10 is the amino acid sequence of human OX40L (CD134L).
  • SEQ ID NO:11 is the amino acid sequence of human CD28.
  • SEQ ID NO:12 is the amino acid sequence of human CTLA-4.
  • SEQ ID NO:13 is the amino acid sequence of human 4-1BB (CD137).
  • SEQ ID NO:14 is the amino acid sequence of human OX40 (CD134).
  • SEQ ID NO:15 is a nucleotide sequence for the pLV430G 4-1BBL empty vector.
  • SEQ ID NO:16 is a nucleotide sequence for the 4-1BBL CoOP portion of the pLV430G human 4-1BBL vector.
  • SEQ ID NO:17 is a nucleotide sequence for the 4-1BBL PCRP.
  • SEQ ID NO:18 is a nucleotide sequence for the pLV430G hCD86 empty vector.
  • SEQ ID NO:19 is a nucleotide sequence for the hCD86 CoOP portion of the pLV430G human hCD86 vector.
  • SEQ ID NO:20 is a nucleotide sequence for the hCD86 CoOP B1 B2 PCRP portion of the pLV430G human hCD86 vector.
  • SEQ ID NO:21 is a nucleotide sequence for the pDONR221 hCD86 vector.
  • SEQ ID NO:22 is a nucleotide sequence for the pDONR221 4-1BBL vector.
  • SEQ ID NO:23 is a nucleotide sequence for the pLV430G vector.
  • SEQ ID NO:24 is a nucleotide sequence for the pDONR221 vector.
  • SEQ ID NO:25 is a nucleotide sequence for the psPAX2 helper plasmid for lentiviral production.
  • SEQ ID NO:26 is a nucleotide sequence for the pCIGO-VSV.G helper plasmid for lentiviral production.
  • SEQ ID NO:27 is the amino acid sequence of the mFc-7C12 scFv clone.
  • SEQ ID NO:28 is the amino acid sequence of the mFc-8B3 scFv clone.
  • SEQ ID NO:29 is a nucleotide sequence for the mFC-7C12 scFv.
  • SEQ ID NO:30 is a nucleotide sequence for the mFC-8B3 scFv.
  • SEQ ID NO:31 is a nucleotide sequence for the destination vector pLV4301G.
  • SEQ ID NO:32 is a nucleotide sequence for the donor vector 1, pMK 7c12 anti mFC scFv CoOp ECORV SacII L1R5.
  • SEQ ID NO:33 is a nucleotide sequence for the donor vector 2, pMK hCD8a scaffold TN L5 L2.
  • SEQ ID NO:34 is a nucleotide sequence for the final vector used for lentiviral production, pLV4301G 7C12 scFv mIgG hCD8 flag.
  • SEQ ID NO:35 is a nucleotide sequence for the destination vector, pLV4301G.
  • SEQ ID NO:36 is a nucleotide sequence for the donor vector 1, pMK 8B3 anti mFC scFv CoOp ECORV SacII L1R5.
  • SEQ ID NO:37 is a nucleotide sequence for the donor vector 2, pMK hCD8a scaffold TN L5 L2.
  • SEQ ID NO:38 is a nucleotide sequence for the final vector used for lentiviral production, pLV4301G 8B3 scFv mIgG hCD8 flag.
  • SEQ ID NO:39 is a nucleotide sequence for pLenti-C-Myc-DDK OX40L vector for lentiviral production.
  • SEQ ID NO:40 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.
  • SEQ ID NO:41 is a nucleotide sequence for Tel-2b primer used for quantitative polymerase chain reaction measurements of telomere length.
  • SEQ ID NO:42 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.
  • SEQ ID NO:43 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.
  • co-administration encompass administration of two or more active pharmaceutical ingredients to a human subject so that both active pharmaceutical ingredients and/or their metabolites are present in the human subject at the same time.
  • Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present is also encompassed in the methods of the invention.
  • in vivo refers to an event that takes place in a subject's body.
  • in vitro refers to an event that takes places outside of a subject's body.
  • in vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.
  • ex vivo refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject's body. Aptly, the cell, tissue and/or organ may be returned to the subject's body in a method of surgery or treatment.
  • an antigen refers to a substance that induces an immune response.
  • an antigen is a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by major histocompatibility complex (MEW) molecules.
  • TCR T cell receptor
  • MEW major histocompatibility complex
  • the term “antigen”, as used herein, also encompasses T cell epitopes.
  • An antigen is additionally capable of being recognized by the immune system.
  • an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope.
  • An antigen can also have one or more epitopes (e.g., B- and T-epitopes).
  • an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.
  • an effective amount refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment.
  • a therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the human subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc. which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and/or cell migration).
  • the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.
  • a prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients.
  • the use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.
  • rapid expansion means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week.
  • rapid expansion protocols are described herein.
  • TILs tumor infiltrating lymphocytes
  • TILs include, but are not limited to, CD8 + cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + T cells, natural killer cells, dendritic cells and M1 macrophages.
  • TILs include both primary and secondary TILs.
  • Primary TILs are those that are obtained from patient tissue samples as outlined herein (sometimes referred to herein as “freshly harvested” or “a first population of TILs”)
  • secondary TILs are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”, or “second population of TILs” or “third population of TILs” where appropriate).
  • TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment.
  • TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR ⁇ , CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.
  • cryopreserved TILs herein is meant that TILs are treated and stored in the range of about ⁇ 150° C. to ⁇ 60° C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.
  • cryopreserved TILs herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.
  • population of cells is meant a number of cells that share common traits.
  • central memory T cell refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7 hi ) and CD62L (CD62 hi ).
  • the surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1.
  • Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering.
  • Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.
  • effector memory T cell refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7 lo ) and are heterogeneous or low for CD62L expression (CD62L lo ).
  • the surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R.
  • Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon- ⁇ , IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin.
  • sequence identity in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences
  • ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, available from DNASTAR are additional publicly available software programs that can be used to align sequences.
  • One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.
  • conservative amino acid substitutions means amino acid sequence modifications which do not abrogate the binding of an antibody to an antigen or a protein to its ligand.
  • Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix.
  • Class I Cys
  • Class II Ser, Thr, Pro, Ala, Gly
  • Class III Asn, Asp, Gln, Glu
  • Class IV His, Arg, Lys
  • Class V Class Ile, Leu, Val, Met
  • Class VI Phe, Tyr, Trp
  • substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution.
  • a predicted nonessential amino acid residue in a 4-1BBL or CD86 protein is preferably replaced with another amino acid residue from the same class.
  • retrovirus refers to RNA viruses that utilize reverse transcriptase during their replication cycle, wherein retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase.
  • the double-stranded DNA form is integrated into the chromosome of the infected cell (a “provirus”).
  • the provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.
  • LTRs long terminal repeats
  • the LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome.
  • Retroviridae Several genera included within the family Retroviridae, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, Gammaretrovirus, and Spumavirus. Some of the retroviruses are oncogenic (i.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in susceptible species. Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. Because they are integrated into the host DNA, they are capable of transmitting sequences of host DNA from cell to cell.
  • Example gammaretroviral vectors include those derived from the amphotropic Moloney murine leukemia virus (MLV-A), which use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes.
  • MLV-A amphotropic Moloney murine leukemia virus
  • the amphotropic MLV vector system has been well established and is a popular tool for gene delivery (See, e.g., Gordon and Anderson, Curr. Op. Biotechnol., 1994, 5, 611-616 and Miller, et al., Meth. Enzymol., 1993, 217, 581-599, the disclosures of which are incorporated herein by reference.
  • lentivirus refers to a genus that includes HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (Hy), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates.
  • HIV human immunodeficiency virus
  • visna-maedi which causes encephalitis (visna) or pneumonia (maedi) in sheep
  • viruses Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells).
  • anti-CD3 antibody refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells.
  • Anti-CD3 antibodies include OKT-3, also known as muromonab.
  • Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3 ⁇ .
  • Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.
  • OKT-3 refers to a monoclonal antibody or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof.
  • the amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).
  • a hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001.
  • a hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706.
  • IL-2 refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof.
  • IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated by reference herein.
  • the amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3).
  • IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors.
  • aldesleukin PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials
  • CELLGRO GMP CellGenix, Inc.
  • ProSpec-Tany TechnoGene Ltd. East Brunswick, N.J., USA
  • Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa.
  • the amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4).
  • the term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and pegylated IL-2 suitable for use in the invention is described in U.S. Patent Application Publication No.
  • IL-7 refers to a glycosylated tissue-derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery.
  • Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-7 recombinant protein, Cat. No. Gibco PHC0071).
  • the amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:5).
  • IL-15 refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. IL-15 shares ⁇ and ⁇ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa.
  • Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82).
  • the amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:6).
  • IL-21 refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4 + T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa.
  • Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein, Cat. No. 14-8219-80).
  • the amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:7).
  • myeloid cell refers to cells of the myeloid lineage or derived therefrom.
  • the myeloid lineage includes a number of morphologically, phenotypically, and functionally distinct cell types including different subsets of granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, erythrocytes, megakaryocytes, and mast cells.
  • the cell is a cell derived from a cell line of myeloid lineage.
  • MOLM-14 refers to a human leukemia cell line which was established from the peripheral blood of a patient with relapsed acute monocytic leukemia, and initial phenotypic characterization indicated the presence of at least the following markers: CD4, CD9, CD11a, CD13, CD14, CD15, CD32, CD33, CD64, CD65, CD87, CD92, CD93, CD116, CD118, and CD155. Matsuo, et al., Leukemia 1997, 11, 1469-77. Additional phenotypic characterization of MOLM-14 found higher levels of HLA-AB/C, CD64, CD80, ICOS-L, CD58, and lower levels of CD86.
  • MOLM-14 cell line is deposited at DSMZ under Accession No. ACC777.
  • the closely related MOLM-13 cell line is deposited at DSMZ under Accession No. ACC554.
  • MOLM-14 cell refers to a MOLM-14 cell and/or a cell derived from the deposited MOLM-14 parental cell line.
  • MOLM-13 cell refers to a MOLM-13 cell and/or a cell derived from the deposited MOLM-13 parental cell line.
  • EM-3 refers to a human cell line was established from the bone marrow of a patient with Philadelphia chromosome-positive CML. Konopka, et al., Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. Phenotypic characterization for EM-3 cells indicates the presence of at least the following markers: CD13, CD15, and CD33.
  • the EM-3 cell line is deposited at DSMZ under Accession No. ACC134 whilst the closely related EM-2 cell line is deposited at DSMZ under Accession No. ACC135.
  • EM-3 cell refers to a EM-3 cell and/or a cell derived from the deposited EM-3 parental cell line.
  • a CD86 protein may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:8 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:8, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • 4-1BBL or “CD137L” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:9 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:9, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • OX40L or “CD137L” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:10 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:10, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • biosimilar means a biological product, including a monoclonal antibody or fusion protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product.
  • a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency.
  • biosimilar is also used synonymously by other national and regional regulatory agencies.
  • Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast.
  • IL-2 proteins can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies.
  • aldesleukin PROLEUKIN
  • a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin.
  • EMA European Medicines Agency
  • a biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and/or efficacy.
  • the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product.
  • a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product.
  • a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product.
  • a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product.
  • a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product.
  • a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA.
  • the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies.
  • the term “biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator.
  • Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins.
  • a protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and/or substitutions of amino acids) which do not significantly affect the function of the polypeptide.
  • the biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%.
  • the biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and/or truncation which is/are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and/or efficacy of the medicinal product.
  • the biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and/or presentation, providing safety and efficacy of the medicinal product is not compromised.
  • the biosimilar may comprise differences in for example pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization.
  • PK pharmacokinetic
  • PD pharmacodynamic
  • biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product.
  • Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product.
  • biosimilar is also used synonymously by other national and regional regulatory agencies.
  • variant encompasses but is not limited to proteins, antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference protein or antibody by way of one or more substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the reference protein or antibody.
  • the variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference protein or antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.
  • the variant retains the ability to specifically bind to the antigen of the reference protein or antibody.
  • variant also includes pegylated antibodies or proteins.
  • Pegylation refers to a modified antibody, or a fragment thereof, or protein that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody, antibody fragment, or protein.
  • PEG polyethylene glycol
  • Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody or protein.
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C 1 -C 10 ) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody or protein to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies and proteins described herein, as described for example in European Patent Nos. EP 0154316 and EP 0401384.
  • the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art.
  • a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.
  • transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s).
  • the term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material.
  • compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”
  • the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses HLA-AB/C, CD64, CD80, ICOS-L, and CD58, and is modified to express one or more costimulatory molecules.
  • aAPC isolated artificial antigen presenting cell
  • the invention includes an aAPC comprising a MOLM-14 cell that is modified to express one or more costimulatory molecules.
  • the invention includes an aAPC comprising a MOLM-13 cell that is modified to express one or more costimulatory molecules.
  • the invention includes an aAPC comprising a MOLM-14 cell that endogenously expresses HLA-AB/C, CD64, CD80, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cell expresses CD86 and 4-1BBL.
  • the invention includes an aAPC comprising a MOLM-13 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cell expresses CD86 and 4-1BBL.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-14 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the MOLM-14 cell expresses CD86 and OX40L.
  • the invention includes an aAPC comprising a MOLM-13 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the MOLM-13 cell expresses CD86 and OX40L.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, and conservative amino acid substitutions thereof, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-13 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • an aAPC comprising a MOLM-14 or MOLM-13 cell may be modified to express both OX40L and 4-1BBL.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses HLA-AB/C, ICOS-L, and CD58, and is modified to express one or more costimulatory molecules, wherein the aAPC is derived from an EM-3 parental cell line.
  • aAPC isolated artificial antigen presenting cell
  • the invention includes an aAPC comprising an EM-3 cell that is modified to express one or more costimulatory molecules.
  • the invention includes an aAPC comprising an EM-2 cell that is modified to express one or more costimulatory molecules.
  • the invention includes an aAPC comprising an EM-3 cell that expresses HLA-AB/C, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising an EM-3 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cell expresses CD86 and 4-1BBL.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • aAPC comprising an EM-3 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • scFv single chain fragment variable
  • the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • a single chain fragment variable (scFv) binding domain such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 96 . In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 97 . In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 98 .
  • the invention includes an aAPC comprising an EM-3 cell that expresses HLA-AB/C, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising an EM-3 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the EM-3 cell expresses CD86 and OX40L.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-3 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • aAPC comprising an EM-3 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • scFv single chain fragment variable
  • the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell.
  • the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an EM-2 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • a single chain fragment variable (scFv) binding domain such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 96 . In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 97 . In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 98 .
  • an aAPC comprising an EM-3 or EM-2 cell may be modified to express both OX40L and 4-1BBL.
  • the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses CD58, and is modified to express one or more costimulatory molecules, wherein the aAPC is derived from a K562-lineage parental cell line.
  • aAPC isolated artificial antigen presenting cell
  • the invention includes an aAPC comprising a K562-lineage cell that is modified to express one or more costimulatory molecules.
  • the K562 lineage parental cell line is deposited under accession no. ATCC CCL-243 and also at European Collection of Authenticated. Cell Cultures (ECACCECACC 89121407).
  • the invention includes an aAPC comprising a K562-lineage cell that expresses CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • the invention includes an aAPC comprising a K562-lineage cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the K562-lineage cell expresses CD86 and 4-1BBL.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:11, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13, and conservative amino acid substitutions thereof.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13.
  • the invention includes an aAPC comprising a K562-lineage modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13.
  • the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13.
  • the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • the invention includes an aAPC comprising an K562-lineage cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • a single chain fragment variable (scFv) binding domain such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • a method of preparing an aAPC includes the step of stable incorporation of genes for production of CD86 and 4-1BBL. In an embodiment, a method of preparing an aAPC includes the step of retroviral transduction. In an embodiment, a method of preparing an aAPC includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J.
  • a method of preparing an aAPC includes the step of gamma-retroviral transduction.
  • Gamma-retroviral transduction systems are known in the art and are described, e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated by reference herein.
  • a method of preparing an aAPC includes the step of transposon-mediated gene transfer.
  • Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, a transposase provided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail).
  • a transposase provided as an mRNA e.g., an mRNA comprising a cap and poly-A tail.
  • Suitable transposon-mediated gene transfer systems including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100 ⁇ , and engineered enzymes with increased enzymatic activity, are described in, e.g., Bushett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of which
  • viruses modified and applied to such techniques include adenoviruses, adeno-associated viruses, herpes simplex viruses, and retroviruses.
  • nucleic acid molecules of interest may be cloned into a viral genome.
  • the resultant viral particle is capable of delivering the nucleic acid of interest into a cell via the viral entry mechanism.
  • modified retroviruses to introduce genetic material into cells to treat genetic defects and other diseases.
  • the present invention provides highly efficient methods, and compositions related thereto, for the stable transduction of cells with viral vectors and viral particles.
  • stable transduction it is meant where an integrated form of the viral vector has been inserted into the chromosomal DNA of the transduced cell.
  • the methods comprise exposing the cells to be transduced to contact with at least one molecule that binds the cell surface. This contacting step may occur prior to, during, or after the cells are exposed to the viral vector or viral particle.
  • viral vector will be used to denote any form of a nucleic acid derived from a virus and used to transfer genetic material into a cell via transduction.
  • the term encompasses viral vector nucleic acids, such as DNA and RNA, encapsidated forms of these nucleic acids, and viral particles in which the viral vector nucleic acids have been packaged.
  • cell surface binding molecules include polypeptides, nucleic acids, carbohydrates, lipids, and ions, all optionally complexed with other substances.
  • the molecules bind factors found on the surfaces of blood cells, such as CD1a, CD1b, CD1c, CD1d, CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD6, CD7, CD8 ⁇ , CD8 ⁇ , CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a, CD16b, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47, CD
  • CD2 CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD6, CD7, CD8 ⁇ , CD8 ⁇ , CD9, CD11a, CD18, CD25, CD26, CD27, CD28, CD29, CD30, CD37, CD38, CD39, CD43, CD44, CD45R, CD46, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD53, CD54, CD56, CD57, CD58, CD59, CDw60, CD62L, CD68, CD69, CDw70, CD71, CD73, CDw75, CDw76, CD84, CD85, CD86, CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD100, CD101, CD103, CD107a, CD107b, CDw108, CDw109, CD118, CD119, CD120b,
  • the cell to be transduced is a eukaryotic cell. More preferably, the cell is a primary cell. Cell lines, however, may also be transduced with the methods of the invention and, in many cases, more easily transduced.
  • the cell to be transduced is a primary lymphocyte (such as a T lymphocyte) or a macrophage (such as a monocytic macrophage), or is a precursor to either of these cells, such as a hematopoietic stem cell.
  • cells of the hematopoietic system are cells of the hematopoietic system, or, more generally, cells formed by hematopoiesis as well as the stem cells from which they form and cells associated with blood cell function.
  • Such cells include granulocytes and lymphocytes formed by hematopoiesis as well as the progenitor pluripotent, lymphoid, and myeloid stem cells.
  • Cells associated with blood cell function include cells that aid in the functioning of immune system cells, such as antigen presenting cells like dendritic cells, endothelial cells, monocytes, and Langerhans cells.
  • the cells are T lymphocytes (or T cells), such as those expressing CD4 and CD8 markers.
  • the cell is a primary CD4+T lymphocyte or a primary CD34+ hematopoietic stem cell.
  • the viral vectors for use in the invention may be pseudotyped with Vesicular Stomatitis Virus envelope G protein (as discussed below), any cell can be transduced via the methods of the present invention.
  • the cell is of a eukaryotic, multicellular species (e.g., as opposed to a unicellular yeast cell), and, even more preferably, is of mammalian origin, e.g., a human cell.
  • Such a “larger collection of cells” can comprise, for instance, a cell culture (either mixed or pure).
  • the invention may be used to transduce hematopoietic stem cells in vivo in the bone marrow.
  • Any combination of antibodies or other cell surface binding molecules, such as FLT-3 ligand, TPO and Kit ligand, or functional analogs thereof, or stromal cells expressing the cell surface binding molecule, could be added with vector upon direct injection into the bone marrow for high efficiency bone marrow transduction.
  • Transduction of mainly a cell type of interest can be accomplished by the choice of cell surface moiety to be bound.
  • transduction of cells expressing CD3 Such as certain T cells, will be enhanced when CD3 specific anti bodies are used to interact with the cells. This will occur in preference over other cell types in the population, such as granulocytes and monocytes that do not express CD3.
  • the invention also encompasses the transduction of purified or isolated cell types if desired.
  • the use of a purified or isolated cell type provides additional advantages Such as higher efficiencies of transduction due to higher vector concentrations relative to the cell to be transduced.
  • the present invention includes viral vectors, and compositions comprising them, for use in the disclosed methods.
  • the vectors are preferably retroviral (family Retroviridae) vectors, and more preferably lentiviral vectors.
  • retroviral vectors such as oncoviral and murine retroviral vectors, may also be used.
  • Additional vectors may be derived from other DNA viruses or viruses that can convert their genomes into DNA during some point of their lifecycle.
  • viruses are from the families Adenoviridae, Parvoviridae Hepandaviridae (including the hepatitis delta virus and the hepatitis E virus which is not normally classified in the Hepandaviridae), Papoviridae (including the polyomavirinae and the papillomavirinae), Herpesviridae, and Poxviridae.
  • Retroviridae i.e., a retrovirus
  • retroviridae i.e., a retrovirus
  • Oncovirinae Spumavirinae
  • Spumavirus Spumavirus
  • Lentivirinae Lentivirus
  • RNA virus of the subfamily Oncovirinae is desirably a human T-lymphotropic virus type 1 or 2 (i.e., HTLV-1 or HTLV-2) or bovine leukemia virus (BLV), an avian leukosissarcoma virus (e.g., Rous Sarcoma virus (RSV), avian myeloblastosis virus (AMV), avian erythroblastosis virus (AEV), and Rous-associated virus (RAV; RAV-0 to RAV-50), a mammalian C-type virus (e.g., Moloney murine leukemia virus (Mul V), Harvey murine sarcoma virus (HaMSV), Abelson murine leukemia virus (A-MuLV), AKR-Mul V, feline leukemia virus (FeLV), simian sarcoma virus, reticuloendotheliosis virus (REV), Spleen necrosis virus (SNV
  • RNA virus of the subfamily Lentivirus is desirably a human immunodeficiency virus type 1 or 2 (i.e., HIV-1 or HIV-2, wherein HIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)), or another virus related to HIV-1 or HIV-2 that has been identified and associated with AIDS or AIDS-like disease.
  • HIV human immunodeficiency virus type 1 or 2
  • HIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)
  • HIV-1 or HIV-2 which HIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)
  • HIV-1 or HIV-2 formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)
  • HIV lymphadenopathy associated
  • RNA virus of the subfamily Lentivirus preferably is a Visna/maedi virus (e.g., such as infect sheep), a feline immunodeficiency virus (FIV), bovine lentivirus, simian immunodeficiency virus (SIV), an equine infectious anemia virus (EIAV), and a caprine arthritisencephalitis virus (CAEV).
  • Visna/maedi virus e.g., such as infect sheep
  • FIV feline immunodeficiency virus
  • SIV simian immunodeficiency virus
  • EIAV equine infectious anemia virus
  • CAEV caprine arthritisencephalitis virus
  • a particularly preferred lentiviral vector is one derived from HIV, most preferably HI-1, HIV-2, or chimeric combinations thereof.
  • different serotypes of retroviruses, especially HIV may be used singly or in any combination to prepare vectors for use in the present invention.
  • Preferred vectors of the invention contains cis acting elements that are present in the wild-type virus, but not present in a “basic” lentiviral vector.
  • a “basic” lentiviral vector contains minimally, LTRS and packaging sequences in the 5′ leader and gag encoding Sequences, but can also optionally contain the RRE element to facilitate nuclear export of vector RNA in a Rev dependent manner.
  • a preferred vector additionally contains nucleotide aequences that enhance the efficiency of transduction into cells.
  • pN2cGFP a vector that contains the complete sequences of gag and pol.
  • Another example is a vector that contain sequences from about position 4551 to position 5096 in pol (reference positions from the pNL4-3 sequence, Accession number M19921, HIVNL43 9709 bp, kindly provided by C. E. Buckler, NIAID, NIH, Bethesda, Md.).
  • any cis-acting sequence from the wt-HIV that can improve vector transduction efficiency may be used.
  • Other examples of vectors capable of efficient transduction via the present invention are cr2HIV constructs as described in U.S. Pat. No. 5,885,806.
  • Viral vector constructs that may be used in the present invention are found in U.S. Pat. No. 5,885,806, which is hereby incorporated by reference as if fully set forth.
  • the constructs in U.S. Pat. No. 5,885,806 are merely examples that do not limit the scope of vectors that efficiently transduce cells. Instead, the constructs provide additional guidance to the skilled artisan that a viral vector for use with the present invention may contain minimal sequences from the wild-type virus or contain sequences up to almost the entire genome of wild-type virus, yet exclude an essential nucleic acid sequence required for replication and/or production of disease. Methods for determining precisely the sequences required for efficient transduction of cells are routine and well known in the art. For example, a systematic incorporation of viral sequences back into a “basic” vector or deleting sequences from vectors that contain virtually the entire HIV genome, such as cr2HIVs, is routine and well known in the art.
  • viral vectors of interest such as the cytomegalovirus (CMV)
  • CMV cytomegalovirus
  • various accessory proteins encoded by, and sequences present in, the viral genetic material may be left in the vector or helper genomes if these proteins or sequences increase transduction efficiency in certain cell types. Numerous routine screens are available to determine whether certain genetic material increases transduction efficiency by incorporating the sequence in either the vector or helper genomes.
  • a preferred embodiment of the invention is to not include accessory proteins in either the vector or helper genomes. But this preference does not exclude embodiments of the invention where accessory proteins and other sequences are left in either the vector or a helper genome to increase transduction efficiency.
  • the viral vector for use in the transduction methods of the invention can also comprise and express one or more nucleic acid sequences under the control of a promoter present in the virus or under the control of a heterologous promoter introduced into the vector.
  • the promoters may further contain insulatory elements, such as erythroid DNAse hyper-sensitive sites, so as to flank the operon for tightly controlled gene expression.
  • Preferred promoters include the HIV-LTR, CMV promoter, PGK, Ul, EBER transcriptional units from Epstein Barr Virus, tRNA, U6 and U7. While Pol II promoters are preferred, Pol III promoters may also be used. Tissue specific promoters are also preferred embodiments.
  • the beta globin Locus Control Region enhancer and the alpha & beta globin promoters can provide tissue specific expression in erythrocytes and erythroid cells.
  • Another further preferred embodiment is to use cis-acting sequences that are associated with the promoters.
  • the Ul gene may be used to enhance antisense gene expression where non-promoter sequences are used to target the antisense or ribozymes molecule to a target spliced RNA as set out in U.S. Pat. No. 5,814,500, which is hereby incorporated by reference.
  • sequences and gene products are preferably biologically active agents capable of producing a biological effect in a cell.
  • the agent is a cell surface molecule.
  • the cells to be transduced are exposed to contact with the at least one molecule that binds the cell surface before, after, or simultaneously with application of the viral vector.
  • the cells can be cultured in media with CD3 and CD28 antibodies (coated onto the surface of the culture dish or immobilized on beads present in the culture) before, after, or in the presence of the viral vector to be transduced.
  • the cells are exposed to immobilized CD3 and/or CD28 only after or only upon initial contact with the viral vector. Under these conditions, the cells are not exposed to cell surface binding molecule(s) prior to actual transduction with the viral vector.
  • contact with a cell surface binding molecule occurs after exposure of the cells to a viral vector (transduction)
  • the contact preferably occurs within three days of transduction, more preferably within one to two days after transduction.
  • Incubation of the cells with the viral vector may be for different lengths of time, depending on the conditions and materials used. Factors that influence the incubation time include the cell, vector and MOI (multiplicity of infection) used, the molecule(s) and amounts used to bind the cell surface, whether and how said molecule(s) are immobilized or solubilized, and the level of transduction efficiency desired.
  • the incubation is for about eight to about 72 hours, more preferably for about 12 to about 48 hours. In a particularly preferred embodiment, the incubation is for about 24 hours and is optionally repeated once.
  • a preferred method of the invention is to Simultaneously introduce a viral vector in combination with a cell surface binding molecule (e.g. CD3 and/or CD28 antibodies or a FLT-3 ligand, TPO or Kit ligand) and avoid changing the medium for between about one and about eight days after transduction. More preferably, the medium is not changed for three days post transduction. Transduction can proceed for as long as the conditions permit without the process being significantly detrimental to the cells or the organism containing them. Additional examples of cell surface binding proteins for such use include those described hereinabove.
  • the MOI used is from about 1 to about 400, preferably less than 500.
  • the preferred MOI is from about 2 to about 50. More preferably, the MOI is from about 10 to about 30, although ranges of from about 1 to about 10, about 20, about 30, or about 40 are also contemplated. Most preferred is an MOI of about 20.
  • the copy number of viral vector per cell should be at least one. However, many copies of the vector per cell may also be used with the above described methods. The preferred range of copies per cell is from about 1 to about 100. The more preferred copy number is the minimum copy number that provides a therapeutic, prophylactic or biological impact resulting from vector transduction or the most efficient transduction.
  • a more preferred copy number is the maximum copy number that is tolerated by the cell without being significantly detrimental to the cell or the organism containing it. Both the minimum and maximum copy number per cell will vary depending upon the cell to be transduced as well as other cells that may be present. The optimum copy number is readily determined by those skilled in the art using routine methods. For example, cells are transduced at increasing increments of concentration or multiplicities of infection. The cells are then analyzed for copy number, therapeutic or biological impact and for detrimental effects on the transduced cells or a host containing them (e.g. safety and toxicity).
  • the cells may be cultured in the presence of the cell surface binding molecule(s) for various times before the cells are analyzed for the efficiency of transduction or otherwise used.
  • the cells may be cultured under any conditions that result in cell growth and proliferation, Such as incubation with interleukin-2 (IL-2) or incubation with the cell surface binding molecule(s) followed by IL-2.
  • IL-2 interleukin-2
  • the efficiency of transduction observed with the present invention is from about 75-100%. Preferably, the efficiency is at least about 75 to 90%. More preferred embodiments of the invention are where transduction efficiency is at least about 90 to 100%. Most preferred embodiments have transduction efficiencies of at least 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
  • the transduced cells may be used in research or for treatment or prevention of disease conditions in living subjects.
  • transduced cells include the introduction of the cells into a living organism.
  • unstimulated primary T cells isolated from an individual infected with, or at risk of being infected with HIV may be first transduced by a vector, like that described in U.S. Pat. No. 5,885,806, using the present methods and followed by injection of the transduced cells back into the individual.
  • the present invention is directed to methods, and compositions related thereto, for the stable transduction of cells with viral vectors to efficiencies of greater than about 75%.
  • Stably transduced cells may be distinguished from transiently transduced, or pseudotransduced cells, after about seven to ten days, or optionally after about 14 days, post transduction.
  • the methods relate to the fact that contact of the cells to be transduced with at least one molecule that binds the cell surface increases the efficiency of stable transduction.
  • the methods of the invention comprise the step of transduction with a viral vector in combination with contact with a cell surface binding molecule.
  • the contact may occur before, after or at the same time as transduction with the vector.
  • the invention is broadly applicable to any cell, and the use of any cell surface binding molecule.
  • Cells for use with the present methods include unstimulated primary cells, which are freshly isolated from an in vivo source as well as cell lines, which may have been previously cultured for various times in the presence of factors which maintain them in a proliferating state.
  • primary cells they are first obtained from an in vivo source followed optionally by selection for particular cell types.
  • PB peripheral blood
  • CB cord blood
  • CD4+ and/or CD8+ T cells are to be used
  • standard magnetic beads positive selection, plastic adherence negative selection, and/or other art recognized standard techniques may be used to isolate CD4+ and/or CD8+ cells away from contaminating PB cells. Purity of the isolated cell types may be determined by immunophenotyping and flow cytometry using standard techniques.
  • the primary cells may be used in the present methods to be transduced with Viral vectors at efficiencies of greater than 75%.
  • the invention is most advantageously used with primary lymphocytes, Such as T cells, transduced with an HIV-1 based vector capable of expressing heterologous genetic material of interest.
  • primary lymphocytes Such as T cells
  • primary hematopoietic stem cells such as CD34 positive cells.
  • the transduced primary cell can be introduced back into an in vivo environment, such as a patient.
  • the invention contemplates the use of the transfected cells in gene therapy to treat, or prevent, a disease by combating a genetic defect or targeting a viral infection.
  • the above isolation/purification steps would not be used. Instead, the cell to be transduced would be targeted by selection of at least one appropriate cell surface molecule or moiety found on that cell type and the preparation of one or more molecules capable of binding said moiety.
  • the cell surface moiety may be a receptor, marker, or other recognizable epitope on the surface of the targeted cells. Once selected, molecules that interact with the moiety, such as specific antibodies, may be prepared for use in the present invention.
  • CD4+ and/or CD8+ cells can either be first purified and then transduced by the methods of the invention with the use of immobilized CD3 and CD28 antibodies or alternatively be transduced as part of a mixed population, like peripheral blood cells (PBCs) or peripheral blood mononuclear cells (PBMNCs), by use of the same antibodies.
  • PBCs peripheral blood cells
  • PBMNCs peripheral blood mononuclear cells
  • Hematopoietic stem cells in total white blood cell populations which may be difficult to purify or isolate, may be transduced in the mixed populations by use of immobilized CD34 antibodies.
  • the cell surface binding molecules of the invention may target and bind any moiety found on the surface of the cell to be transduced.
  • the moieties are found as part of receptors, markers, or other proteinaceous or nonproteinaceous factors on the cell Surface.
  • the moieties include epitopes recognized by the cell surface binding molecule. These epitopes include those comprising a polypeptide sequence, a carbohydrate, a lipid, a nucleic acid, an ion and combinations thereof.
  • cell surface binding molecules include an antibody or an antigen binding fragment thereof and a ligand or binding domain for a cell surface receptor.
  • the cell surface binding molecule may itself be a polypeptide, a nucleic acid, a carbohydrate, a lipid, or an ion.
  • the molecule is an antibody or a fragment thereof, such as a Fab, or F, fragment. More preferably, the molecule is not used in a soluble form but is rather immobilized on a solid medium, such a bead, with which the cells to be transduced may be cultured, or the surface of a tissue culture dish, bag or plate, upon which the cells to be transduced may be cultured.
  • monoclonal antibodies that recognize CD3 and/or CD28 may be used in a cell culture bag in the presence of a viral vector.
  • the present invention includes compositions comprising a cell surface binding molecule for use as part of the disclosed methods.
  • An exemplary composition comprises the molecule and a viral vector to be transduced, optionally in the presence of the cells to be transduced.
  • the viral vectors may be derived from any source, but are preferably retroviral vectors. More preferably, they are lentiviral vectors.
  • a particularly preferred lentiviral vector is one derived from a Human Immunodeficiency Virus (HIV), most preferably HIV-1, HIV-2, or chimeric combinations thereof.
  • HIV Human Immunodeficiency Virus
  • different viral vectors may be simultaneously transduced into the same cell by use of the present methods.
  • one vector can be a replication deficient or conditionally replicating retroviral vector while a second vector can be a packaging construct that permits the first vector to be replicated/packaged and propagated.
  • various viral accessory proteins are to be encoded by a viral vector, they may be present in any one of the vectors being transduced into the cell.
  • the viral accessory proteins may be present in the transduction process via their presence in the viral particles used for transduction. Such viral particles may have an effective amount of the accessory proteins co-packaged to result in an increase in transduction efficiency.
  • the viral vector does not encode one or more of the accessory proteins.
  • a viral vector for use in the transduction methods of the invention can also comprise and express one or more nucleic acid sequences under the control of a promoter.
  • a nucleic acid sequence encodes a gene product that, upon expression, would alleviate or correct a genetic deficiency in the cell to be transduced.
  • the nucleic acid sequence encodes or constitutes a genetic antiviral agent that can prevent or treat viral infection.
  • genetic antiviral agent it is meant any substance that is encoded or constituted by genetic material. Examples of such agents are provided in U.S. Pat. No. 5,885,806.
  • ribozymes and antisense constructs include agents that function by inhibiting viral proteins, such as reverse transcriptase or proteases, competing with viral factors for binding or target sites, or targeting viral targets directly for degradation, Such as in the case of ribozymes and antisense constructs.
  • agents that function by inhibiting viral proteins such as reverse transcriptase or proteases, competing with viral factors for binding or target sites, or targeting viral targets directly for degradation,
  • viral proteins such as reverse transcriptase or proteases
  • Other examples of genetic antiviral agents include antisense, RNA decoys, transdominant mutants, interferons, toxins, nucleic acids that modulate or modify RNA splicing, immunogens, and ribozymes, such as “hammerhead” and external guide sequence (EGS) mediated forms thereof.
  • EGS external guide sequence
  • the cells to be transduced may be exposed to contact with the viral vector either before, after or simultaneously with contact with the cell surface binding molecule.
  • the cells can be first exposed to the vector for a period of time followed by introduction of the cell surface binding molecule.
  • Such cells may be newly isolated or prepared primary cells that have not been intentionally stimulated to enter the cell cycle.
  • the cells can be first exposed to the cell surface binding molecule for a period of time followed by contact with the viral vector.
  • excess vector is preferably not removed and the cells cultured under conditions conducive to cell growth and/or proliferation. Such conditions may be in the presence of the cell surface binding molecule or other stimulatory/activating factors, such as cytokines and lymphokines in the case of T cells.
  • excess vector may be removed after contact with the cell and before further culturing.
  • Another embodiment of the invention is to culture the cells in the presence of both viral vector and cell surface binding molecule simultaneously. Such cells are preferably not previously stimulated. After a period of time, the cells are cultured under growth or proliferation inducing conditions such as the continued presence of the cell surface binding molecule or other stimulatory/activating factors. Alternatively, excess vector may be removed before further culturing.
  • Incubation of the cells to be transduced with the viral vector may be for different lengths of time, depending on the conditions and materials used. Factors that influence the incubation time include the cell, vector and MOI (multiplicity of infection) used, the molecule(s) and amounts used to bind the cell surface, whether and how said molecule(s) are immobilized, and the level of transduction efficiency desired.
  • the cells are T lymphocytes
  • the MOI is about 20
  • the cell Surface binding molecules are CD3 and CD28 antibodies immobilized on beads, and the resultant efficiency at least 93%.
  • some of the above factors are directly correlated while others are inversely correlated. For example, a decrease in the MOI will likely decrease the level of efficiency while efficiency can likely be maintained if an increased amount of cell surface binding molecules is used.
  • the length of incubation viral vector and the cells to be transformed is preferably for 24 hours and optionally repeated once for lymphocytes and up to four times for hematopoietic stem cells.
  • the incubation may be for about 12 hours to about 96 hours.
  • incubation with a cell surface binding molecule occurs simultaneously with contact of the cells with the viral vector. Under such circumstances, the cell surface binding molecules may be left in contact with the cells when the vector is introduced. Alternatively, excess cell surface binding molecules may be first removed from the culture before introduction of the vector to the cells.
  • the cells After contact with the vector, the cells are cultured under conditions conducive to their growth or proliferation. Preferably, the conditions are continued culturing in the presence of the cell surface binding molecules.
  • the cells are initially cultured with the cell surface binding molecule followed by substitution with media containing another factor conducive to cell growth, such as interleukin-2.
  • media containing another factor conducive to cell growth such as interleukin-2.
  • Yet another embodiment would be to remove both the excess cell surface binding molecule and the excess vector followed by culturing in the presence of a factor conducive to growth or proliferation as well as enhancing further vector transduction.
  • Such factors include mitogens such as phytohemaglutinin (PHA) and cytokines, growth factors, activators, cell surface receptors, cell surface molecules, soluble factors, or combinations thereof, as well as active fragments of such molecules, alone or in combination with another protein or factor, or combinations thereof.
  • mitogens such as phytohemaglutinin (PHA) and cytokines
  • growth factors activators, cell surface receptors, cell surface molecules, soluble factors, or combinations thereof
  • active fragments of such molecules alone or in combination with another protein or factor, or combinations thereof.
  • EGF epidermal growth factor
  • TGF-alpha transforming growth factor alpha
  • TGF-beta angiotensin
  • BMP bone morphogenic protein
  • FGF acidic and basic bone morphogenic protein
  • VEGF vascular endothelial growth factor
  • PIGF human growth hormone
  • HGH human growth hormone
  • BGH bovine growth hormone
  • ARIA Ach receptor inducing activity
  • RANTES regulated on activation, normal T expressed and secreted
  • angiogenins hepatocyte growth factor
  • tumor necrosis factor beta tumor necrosis factor beta
  • TNF-alpha tumor necrosis factor alpha
  • angiopoietins 1 or 2 insulin, insulin growth factors I or II (IGF-I or IGF-2), ephrins, leptins, interleukins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (IL-1, IL-2,
  • the choice of culture conditions will depend on knowledge in the art concerning the cells transduced as well as the subsequent intended use of the cells. For example, the combination of IL-3, IL-6 and stem cell factor would not be a choice for transduced cells that are to be used in human transplantation. Similarly, the choice of culture conditions would preferably not be to the detriment of cell viability or transduction efficiency.
  • the post transduction incubation is for a period of about four hours, or for about one to about seven to ten days. More preferably from about 16 to about 20 hours or for about four, about five or about six days. About fourteen days of post-transduction incubation is also contemplated.
  • the efficiency of transduction observed with the present invention is from about 75-100%. Preferably, the efficiency is at least about 75 to 90%. More preferred embodiments of the invention are where transduction efficiency is at least about 90 to 95%. The most preferred embodiments have transduction efficiencies of at least 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
  • the transduced cells may be used in research or for treatment of disease conditions in living subjects. Particularly preferred as part of the invention are therapeutic uses for the transduced cells to produce gene products of interest or for direct introduction into a living organism as part of gene therapy.
  • primary T cells can be isolated and transduced with a viral vector.
  • the T cells are transduced with genes or nucleic acids capable of conditionally killing the T cell upon introduction into a host organism. This has applications in allogenic bone marrow transplantation to prevent graft versus host disease by killing T cells with a pro-drug approach.
  • the primary cells can be deficient in a gene product, and the deficiency correctable by the transduced viral vector. Such cells would be reintroduced into the living subject after transduction with the vector.
  • the transduced cells are preferably in a biologically acceptable solution or pharmaceutically acceptable formulation.
  • a biologically acceptable solution or pharmaceutically acceptable formulation may be made intravenously, intraperitoneally or by other injection and non-injection methods known in the art.
  • the dosages to be administered will vary depending on a variety of factors, but may be readily determined by the skilled practitioner.
  • a method of preparing an aAPC includes the step of stable incorporation of genes for transient production of CD86 and 4-1BBL.
  • a method of preparing an aAPC includes the step of electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. 1 1991, 60, 297-306, and U.S. Patent Application Publication No. 2014/0227237 A1, the disclosures of each of which are incorporated by reference herein.
  • a method of preparing an aAPC includes the step of calcium phosphate transfection.
  • a method of preparing an aAPC includes the step of liposomal transfection.
  • Liposomal transfection methods such as methods that employ a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S. Pat. Nos.
  • DOTMA cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride
  • DOPE dioleoyl phophotidylethanolamine
  • a method of preparing an aAPC includes the step of transfection using methods described in U.S. Pat. Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.
  • the aAPC is transduced by first using the Gateway cloning method (commercially available from ThermoFisher, Inc.) to prepare vector for lentiviral transduction, followed by lentiviral transduction using the vector and one or more associated helper plasmids, as is also described elsewhere herein.
  • the Gateway cloning method a gene is selected (such as CD86) and is then provided with primers and amplified using PCR technology with the help of an attB tagged primer pair.
  • the PCR fragment is then combined with a donor vector (pDONR, such as pDONR221) that includes attP sites to provide an entry clone, using the BP reaction.
  • pDONR such as pDONR221
  • An integration reaction between the attB and the attP sites combines the PCR fragment with the donor vector.
  • the resulting entry clone contains the gene of interest flanked by attL sites.
  • the LR reaction is then used to combine the entry clone with a destination vector to produce an expression vector.
  • a recombination reaction is used to link the entry clone with the destination vector (such as pLV430G) using the attL and attR sites and a clonase enzyme.
  • the attL sites are already found in the entry clone, while the destination vector includes the attR sites.
  • the LR reaction is carried out to transfer the sequence of interest into one or more destination vectors in simultaneous reactions.
  • the aAPCs described herein may be grown and maintained under serum-based media and/or serum free media.
  • aAPCs may be cultured in 24 well plates at a cell density of about 1 ⁇ 10 6 cells per well for 3 to 5 days. The cells may then be isolated and/or washed by centrifugation and resuspended in media or cryopreserved in an appropriate cryopreservation media (e.g., CryoStor 10 (BioLife Solutions)) and stored in a ⁇ 80° C. freezer.
  • an appropriate cryopreservation media e.g., CryoStor 10 (BioLife Solutions)
  • the aAPCs described herein may be grown in the presence of serum-based media.
  • the aAPCs described herein by may be grown in the presence of serum-based media that includes human serum (hSerum) containing media (e.g., cDMEM with 10% hSerum).
  • the aAPCs grown in the presence of serum-based media may be selected from the group consisting of aMOLM-13 cells, aMOLM-14 cells, and aEM3 cells.
  • the aAPCs described herein may be grown in the presence of serum free media.
  • the serum free media may be selected from the group consisting of CTS Optmizer (ThermoFisher), Xvivo-20 (Lonza), Prime T Cell CDM (Irvine), XFSM (MesenCult), and the like.
  • the aAPCs grown in the presence of serum free media may be selected from the group consisting of aMOLM-13 cells, aMOLM-14 cells, and aEM3 cells.
  • the invention includes a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising at least one TIL with an aAPC described herein, wherein said aAPC comprises at least one co-stimulatory ligand that specifically binds with a co-stimulatory molecule expressed on the cellular surface of the TILs, wherein binding of said co-stimulatory molecule with said co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs.
  • TILs tumor infiltrating lymphocytes
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs) using any of the aAPCs of the present disclosure, the method comprising the steps as described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated by reference herein.
  • the tumor may be placed in enzyme media and mechanically dissociated for approximately 1 minute.
  • the mixture may then be incubated for 30 minutes at 37° C. in 5% CO 2 and then mechanically disrupted again for approximately 1 minute.
  • the tumor may be mechanically disrupted a third time for approximately 1 minute.
  • 1 or 2 additional mechanical dissociations may be applied to the sample, with or without 30 additional minutes of incubation at 37° C. in 5% CO 2 .
  • a density gradient separation using Ficoll may be performed to remove these cells.
  • TIL cultures were initiated in 24-well plates (Costar 24-well cell culture cluster, flat bottom; Corning Incorporated, Corning, N.Y.), each well may be seeded with 1 ⁇ 10 6 tumor digest cells or one tumor fragment approximately 1 to 8 mm 3 in size in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.).
  • CM complete medium
  • IL-2 6000 IU/mL
  • Chiron Corp., Emeryville, Calif. complete medium
  • CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin.
  • Cultures may be initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm 2 gas-permeable silicon bottom (G-Rex 10; Wilson Wolf Manufacturing, New Brighton, each flask may be loaded with 10-40 ⁇ 10 6 viable tumor digest cells or 5-30 tumor fragments in 10-40 mL of CM with IL-2.
  • G-Rex 10 and 24-well plates may be incubated in a humidified incubator at 37° C. in 5% CO 2 and 5 days after culture initiation, half the media may be removed and replaced with fresh CM and IL-2 and after day 5, half the media may be changed every 2-3 days.
  • Rapid expansion protocol (REP) of TILs may be performed using T-175 flasks and gas-permeable bags or gas-permeable G-Rex flasks, as described elsewhere herein, using the aAPCs of the present disclosure.
  • REP Rapid expansion protocol
  • 1 ⁇ 10 6 TILs may be suspended in 150 mL of media in each flask.
  • the TIL may be cultured with aAPCs of the present disclosure at a ratio described herein, in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3).
  • the T-175 flasks may be incubated at 37° C. in 5% CO 2 . Half the media may be changed on day 5 using 50/50 medium with 3000 IU/mL of IL-2. On day 7, cells from 2 T-175 flasks may be combined in a 3L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may be added to the 300 mL of TIL suspension. The number of cells in each bag may be counted every day or two days, and fresh media may be added to keep the cell count between 0.5 and 2.0 ⁇ 10 6 cells/mL.
  • TILs For REP in 500 mL capacity flasks with 100 cm 2 gas-permeable silicon bottoms (e.g., G-Rex 100, Wilson Wolf Manufacturing, as described elsewhere herein), 5 ⁇ 10 6 or 10 ⁇ 10 6 TILs may be cultured with aAPCs at a ratio described herein (e.g., 1 to 100) in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). The G-Rex100 flasks may be incubated at 37° C. in 5% CO 2 .
  • G-Rex100 flasks may be incubated at 37° C. in 5% CO 2 .
  • AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may then be added to each flask.
  • G-Rex100 flasks may then be incubated at 37° C. in 5% CO 2 , and after four days, 150 mL of AIM-V with 3000 IU/mL of IL-2 may be added to each G-Rex100 flask. After this, the REP may be completed by harvesting cells on day 14 of culture.
  • TILs may be expanded advantageously in the presence of serum free media.
  • the TIL expansion methods described herein may include the use of serum free media rather than serum-based media (e.g., complete media or CM1).
  • the TIL expansion methods described herein may use serum free media rather than serum-based media.
  • the serum free media may be selected from the group consisting of CTS Optmizer (ThermoFisher), Xvivo-20 (Lonza), Prime T Cell CDM (Irvine), and the like.
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
  • the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising a population of TILs with a myeloid artificial antigen presenting cell (aAPC), wherein the myeloid aAPC comprises at least two co-stimulatory ligands that specifically bind with at least two co-stimulatory molecule on the TILs, wherein binding of the co-stimulatory molecules with the co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs, and wherein the at least two co-stimulatory ligands comprise CD86 and 4-1BBL.
  • TILs tumor infiltrating lymphocytes
  • the aAPC may further comprise OX40L in addition to 4-1BBL, or may comprise OX40L instead of 4-1BBL.
  • a method of expanding or treating a cancer includes a step wherein TILs are obtained from a patient tumor sample.
  • a patient tumor sample may be obtained using methods known in the art.
  • TILs may be cultured from enzymatic tumor digests and tumor fragments (about 1 to about 8 mm 3 in size) from sharp dissection.
  • Such tumor digests may be produced by incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator).
  • RPMI Roswell Park Memorial Institute
  • Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37° C. in 5% CO 2 , followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present.
  • a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells.
  • Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.
  • REP can be performed in a gas permeable container using the aAPCs of the present disclosure by any suitable method.
  • TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15).
  • the non-specific T cell receptor stimulus can include, for example, about 30 ng/mL of an anti-CD3 antibody, e.g. OKT-3, a monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, N.J., USA or Miltenyi Biotech, Auburn, Calif., USA) or UHCT-1 (commercially available from BioLegend, San Diego, Calif., USA).
  • TILs can be rapidly expanded by further stimulation of the TILs in vitro with one or more antigens, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 ⁇ M MART-1:26-35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T cell growth factor, such as 300 IU/mL IL-2 or IL-15.
  • HLA-A2 human leukocyte antigen A2
  • TIL may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof.
  • TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.
  • the TILs can be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.
  • a method for expanding TILs may include using about 5000 mL to about 25000 mL of cell culture medium, about 5000 mL to about 10000 mL of cell culture medium, or about 5800 mL to about 8700 mL of cell culture medium.
  • a method for expanding TILs may include using about 1000 mL to about 2000 mL of cell medium, about 2000 mL to about 3000 mL of cell culture medium, about 3000 mL to about 4000 mL of cell culture medium, about 4000 mL to about 5000 mL of cell culture medium, about 5000 mL to about 6000 mL of cell culture medium, about 6000 mL to about 7000 mL of cell culture medium, about 7000 mL to about 8000 mL of cell culture medium, about 8000 mL to about 9000 mL of cell culture medium, about 9000 mL to about 10000 mL of cell culture medium, about 10000 mL to about 15000 mL of cell culture medium, about 15000 mL to about 20000 mL of cell culture medium, or about 20000 mL to about 25000 mL of cell culture medium.
  • expanding the number of TILs uses no more than one type of cell culture medium.
  • Any suitable cell culture medium may be used, e.g., AIM-V cell medium (L-glutamine, 50 ⁇ M streptomycin sulfate, and 10 ⁇ M gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad, Calif., USA).
  • the inventive methods advantageously reduce the amount of medium and the number of types of medium required to expand the number of TIL.
  • expanding the number of TIL may comprise feeding the cells no more frequently than every third or fourth day. Expanding the number of cells in a gas permeable container simplifies the procedures necessary to expand the number of cells by reducing the feeding frequency necessary to expand the cells.
  • the rapid expansion is performed using a gas permeable container.
  • a gas permeable container Such embodiments allow for cell populations to expand from about 5 ⁇ 10 5 cells/cm 2 to between 10 ⁇ 10 6 and 30 ⁇ 10 6 cells/cm 2 .
  • this expansion occurs without feeding.
  • this expansion occurs without feeding so long as medium resides at a height of about 10 cm in a gas-permeable flask.
  • this is without feeding but with the addition of one or more cytokines.
  • the cytokine can be added as a bolus without any need to mix the cytokine with the medium.
  • Such containers, devices, and methods are known in the art and have been used to expand TILs, and include those described in U.S. Patent Application Publication No.
  • the gas permeable container is a G-Rex 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 10 cm 2 gas permeable culture surface. In an embodiment, the gas permeable container includes a 40 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 100 to 300 million TILs after 2 medium exchanges.
  • the gas permeable container is a G-Rex 100 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA).
  • the gas permeable container includes a 100 cm 2 gas permeable culture surface.
  • the gas permeable container includes a 450 mL cell culture medium capacity.
  • the gas permeable container provides 1 to 3 billion TILs after 2 medium exchanges.
  • the gas permeable container is a G-Rex 100M flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 100 cm 2 gas permeable culture surface. In an embodiment, the gas permeable container includes a 1000 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 1 to 3 billion TILs without medium exchange.
  • the gas permeable container is a G-Rex 100 L flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 100 cm 2 gas permeable culture surface. In an embodiment, the gas permeable container includes a 2000 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 1 to 3 billion TILs without medium exchange.
  • the gas permeable container is a G-Rex 24 well plate (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA).
  • the gas permeable container includes a plate with wells, wherein each well includes a 2 cm 2 gas permeable culture surface.
  • the gas permeable container includes a plate with wells, wherein each well includes a 8 mL cell culture medium capacity.
  • the gas permeable container provides 20 to 60 million cells per well after 2 medium exchanges.
  • the gas permeable container is a G-Rex 6 well plate (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA).
  • the gas permeable container includes a plate with wells, wherein each well includes a 10 cm 2 gas permeable culture surface.
  • the gas permeable container includes a plate with wells, wherein each well includes a 40 mL cell culture medium capacity.
  • the gas permeable container provides 100 to 300 million cells per well after 2 medium exchanges.
  • the cell medium in the first and/or second gas permeable container is unfiltered.
  • the use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells.
  • the cell medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME).
  • the duration of the method comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium therein; obtaining TILs from the tumor tissue sample; expanding the number of TILs in a second gas permeable container containing cell medium therein using aAPCs for a duration of about 14 to about 42 days, e.g., about 28 days.
  • the rapid expansion uses about 1 ⁇ 10 9 to about 1 ⁇ 10 11 aAPCs. In an embodiment, the rapid expansion uses about 1 ⁇ 10 9 aAPCs. In an embodiment, the rapid expansion uses about 1 ⁇ 10 10 aAPCs. In an embodiment, the rapid expansion uses about 1 ⁇ 10 11 aAPCs.
  • the ratio of TILs to aAPCs is selected from the group consisting of 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:225, 1:250, 1:275, 1:300, 1:350, 1:400, 1:450, and 1:500.
  • the ratio of TILs to aAPCs is about 1:90. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:95. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:100. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:105. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:110.
  • the ratio of TILs to aAPCs in the rapid expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500.
  • the ratio of TILs to aAPCs in the rapid expansion is between 1 to 50 and 1 to 300.
  • the ratio of TILs to aAPCs in the rapid expansion is between 1 to 100 and 1 to 200.
  • the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2.
  • the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.
  • the cell culture medium comprises an OKT-3 antibody. In a preferred embodiment, the cell culture medium comprises about 30 ng/mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 ⁇ g/mL of OKT-3 antibody.
  • the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody.
  • a rapid expansion process for TILs may be performed using T-175 flasks and gas permeable bags as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA).
  • TIL rapid expansion in T-175 flasks 1 ⁇ 10 6 TILs suspended in 150 mL of media may be added to each T-175 flask.
  • the TILs may be cultured with aAPCs at a ratio of 1 TIL to 100 aAPCs and the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU (international units) per mL of IL-2 and 30 ng per ml of anti-CD3 antibody (e.g., OKT-3).
  • the T-175 flasks may be incubated at 37° C. in 5% CO 2 . Half the media may be exchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2.
  • cells from two T-175 flasks may be combined in a 3 liter bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of TIL suspension.
  • the number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and 2.0 ⁇ 10 6 cells/mL.
  • 5 ⁇ 10 6 or 10 ⁇ 10 6 TIL may be cultured with aAPCs at a ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3 (OKT-3).
  • the G-Rex 100 flasks may be incubated at 37° C. in 5% CO 2 .
  • AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask.
  • the G-Rex 100 flasks may be incubated at 37° C. in 5% CO 2 and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-Rex 100 flask.
  • the cells may be harvested on day 14 of culture.
  • TILs may be prepared as follows. 2 mm 3 tumor fragments are cultured in complete media (CM) comprised of AIM-V medium (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, Va.), 100 U/mL penicillin (Invitrogen Life Technologies), 100 ⁇ g/mL streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc. Winchester, Va.) and 600 IU/mL rhlL-2 (Chiron, Emeryville, Calif.).
  • CM complete media
  • TILs established from fragments may be grown for 3-4 weeks in CM and expanded fresh or cryopreserved in heat-inactivated HAB serum with 10% dimethylsulfoxide (DMSO) and stored at ⁇ 180° C. until the time of study.
  • Tumor associated lymphocytes (TAL) obtained from ascites collections were seeded at 3 ⁇ 10 6 cells/well of a 24 well plate in CM. TIL growth was inspected about every other day using a low-power inverted microscope.
  • TILs are expanded in gas-permeable containers.
  • Gas-permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. U.S. Patent Application Publication No. 2005/0106717 A1, the disclosures of which are incorporated herein by reference.
  • TILs are expanded in gas-permeable bags.
  • TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare).
  • TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare).
  • the cell expansion system includes a gas permeable cell bag with a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, and about 30 L.
  • the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 50 and 150 mL, between 150 and 250 mL, between 250 and 350 mL, between 350 and 450 mL, between 450 and 550 mL, between 550 and 650 mL, between 650 and 750 mL, between 750 and 850 mL, between 850 and 950 mL, and between 950 and 1050 mL.
  • the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 1 L and 2 L, between 2 L and 3 L, between 3 L and 4 L, between 4 L and 5 L, between 5 L and 6 L, between 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, between 9 L and 10 L, between 10 L and 11 L, between 11 L and 12 L, between 12 L and 13 L, between 13 L and 14 L, between 14 L and 15 L, between 15 L and 16 L, between 16 L and 17 L, between 17 L and 18 L, between 18 L and 19 L, and between 19 L and 20 L.
  • a gas permeable cell bag with a volume range selected from the group consisting of between 1 L and 2 L, between 2 L and 3 L, between 3 L and 4 L, between 4 L and 5 L, between 5 L and 6 L, between 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, between 9 L and 10 L, between 10 L and 11 L, between 11 L
  • the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 0.5 L and 5 L, between 5 L and 10 L, between 10 L and 15 L, between 15 L and 20 L, between 20 L and 25 L, and between 25 L and 30 L.
  • the cell expansion system utilizes a rocking time of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days.
  • the cell expansion system utilizes a rocking time of between 30 minutes and 1 hour, between 1 hour and 12 hours, between 12 hours and 1 day, between 1 day and 7 days, between 7 days and 14 days, between 14 days and 21 days, and between 21 days and 28 days.
  • the cell expansion system utilizes a rocking rate of about 2 rocks/minute, about 5 rocks/minute, about 10 rocks/minute, about 20 rocks/minute, about 30 rocks/minute, and about 40 rocks/minute.
  • the cell expansion system utilizes a rocking rate of between 2 rocks/minute and 5 rocks/minute, 5 rocks/minute and 10 rocks/minute, 10 rocks/minute and 20 rocks/minute, 20 rocks/minute and 30 rocks/minute, and 30 rocks/minute and 40 rocks/minute.
  • the cell expansion system utilizes a rocking angle of about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In an embodiment, the cell expansion system utilizes a rocking angle of between 2° and 3°, between 3° and 4°, between 4° and 5°, between 5° and 6°, between 6° and 7°, between 7° and 8°, between 8° and 9°, between 9° and 10°, between 10° and 11°, and between 11° and 12°.
  • a method of expanding TILs using aAPCs further comprises a step wherein TILs are selected for superior tumor reactivity.
  • Any selection method known in the art may be used.
  • the methods described in U.S. Patent Application Publication No. 2016/0010058 A1 the disclosures of which are incorporated herein by reference, may be used for selection of TILs for superior tumor reactivity.
  • the aAPCs of the present invention may be used to expand T cells. Any of the foregoing embodiments of the present invention described for the expansion of TILs may also be applied to the expansion of T cells.
  • the aAPCs of the present invention may be used to expand CD8 + T cells.
  • the aAPCs of the present invention may be used to expand CD4 + T cells.
  • the aAPCs of the present invention may be used to expand T cells transduced with a chimeric antigen receptor (CAR-T).
  • the aAPCs of the present invention may be used to expand T cells comprising a modified T cell receptor (TCR).
  • the CAR-T cells may be targeted against any suitable antigen, including CD19, as described in the art, e.g., in U.S. Pat. Nos. 7,070,995; 7,446,190; 8,399,645; 8,916,381; and 9,328,156; the disclosures of which are incorporated by reference herein.
  • the modified TCR cells may be targeted against any suitable antigen, including NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof, as described in the art, e.g., in U.S. Pat. Nos. 8,367,804 and 7,569,664, the disclosures of which are incorporated by reference herein.
  • compositions and methods described herein can be used in a method for treating diseases. In an embodiment, they are for use in treating hyperproliferative disorders. They may also be used in treating other disorders as described herein and in the following paragraphs.
  • the TILs, populations and compositions thereof described herein may be for use in the treatment of a disease. In an embodiment, the TILs, populations and compositions described herein are for use in the treatment of a hyperproliferative disorder.
  • the hyperproliferative disorder is cancer.
  • the hyperproliferative disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma, pancreatic cancer, and glioblastoma.
  • the hyperproliferative disorder is a hematological malignancy.
  • the hematological malignancy is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, and mantle cell lymphoma.
  • the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing a rapid expansion of the first population of TILs using a population of artificial antigen presenting cells (aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs; and (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer.
  • TILs tumor infiltrating lymphocytes
  • the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • the rapid expansion is performed over a period not greater than 14 days.
  • the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing an initial expansion of the first population of TILs using a first population of artificial antigen presenting cells (aAPCs) in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 10-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second population of TILs using a second population of aAPCs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the first population of TILs; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion
  • the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • the rapid expansion is performed over a period not greater than 14 days.
  • the initial expansion is performed using a gas permeable container.
  • the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 10-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second population of TILs using a population of artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the first population of TILs; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion of the third population of TILs to a patient
  • the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • the rapid expansion is performed over a period not greater than 14 days.
  • the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure.
  • the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m 2 /d for 5 days (days 27 to 23 prior to TIL infusion).
  • the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.
  • Efficacy of the compounds and combinations of compounds described herein in treating, preventing and/or managing the indicated diseases or disorders can be tested using various models known in the art, which provide guidance for treatment of human disease.
  • models for determining efficacy of treatments for ovarian cancer are described, e.g., in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, et al., J. Ovarian Res. 2009, 2, 12.
  • Models for determining efficacy of treatments for pancreatic cancer are described in Herreros-Villanueva, et al., World J. Gastroenterol. 2012, 18, 1286-1294.
  • Models for determining efficacy of treatments for breast cancer are described, e.g., in Fantozzi, Breast Cancer Res. 2006, 8, 212.
  • Models for determining efficacy of treatments for melanoma are described, e.g., in Damsky, et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859.
  • Models for determining efficacy of treatments for lung cancer are described, e.g., in Meu Giveaway, et al., Genes & Development, 2005, 19, 643-664.
  • Models for determining efficacy of treatments for lung cancer are described, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60; and Sano, Head Neck Oncol. 2009, 1, 32.
  • the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure.
  • the invention provides a population of TILs obtainable by a method described herein for use in treating a cancer, wherein the population of TILs is for treating a patient which is pre-treated with non-myeloablative chemotherapy.
  • the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m 2 /d for 5 days (days 27 to 23 prior to TIL infusion).
  • the patient receives an intravenous infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN) intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.
  • IL-2 aldesleukin, commercially available as PROLEUKIN
  • lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the aAPC-expanded TILs of the invention.
  • a lymphodepletion step sometimes also referred to as “immunosuppressive conditioning”
  • lymphodepletion is achieved using administration of fludarabine or cyclophosphamide (the active form being referred to as mafosfamide) and combinations thereof.
  • fludarabine or cyclophosphamide the active form being referred to as mafosfamide
  • mafosfamide the active form being referred to as mafosfamide
  • Such methods are described in Gassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski, et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated by reference herein in their entireties.
  • the fludarabine is administered at a concentration of 0.5 ⁇ g/mL-10 ⁇ g/mL fludarabine. In some embodiments, the fludarabine is administered at a concentration of 1 ⁇ g/mL fludarabine. In some embodiments, the fludarabine treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the fludarabine is administered at a dosage of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day.
  • the fludarabine treatment is administered for 2-7 days at 35 mg/kg/day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 35 mg/kg/day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 25 mg/kg/day.
  • the mafosfamide, the active form of cyclophosphamide is obtained at a concentration of 0.5 ⁇ g/ml-10 ⁇ g/ml by administration of cyclophosphamide. In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 1 ⁇ g/mL by administration of cyclophosphamide. In some embodiments, the cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more.
  • the cyclophosphamide is administered at a dosage of 100 mg/m 2 /day, 150 mg/m 2 /day, 175 mg/m 2 /day, 200 mg/m 2 /day, 225 mg/m 2 /day, 250 mg/m 2 /day, 275 mg/m 2 /day, or 300 mg/m 2 /day.
  • the cyclophosphamide is administered intravenously (i.v.)
  • the cyclophosphamide treatment is administered for 2-7 days at 35 mg/kg/day.
  • the cyclophosphamide treatment is administered for 4-5 days at 250 mg/m 2 /day i.v.
  • the cyclophosphamide treatment is administered for 4 days at 250 mg/m 2 /day i.v.
  • lymphodepletion is performed by administering the fludarabine and the cyclophosphamide are together to a patient.
  • fludarabine is administered at 25 mg/m 2 /day i.v. and cyclophosphamide is administered at 250 mg/m 2 /day i.v. over 4 days.
  • the lymphodepletion is performed by administration of cyclophosphamide at a dose of 60 mg/m 2 /day for two days followed by administration of fludarabine at a dose of 25 mg/m 2 /day for five days.
  • TILs expanded using aAPCs of the present disclosure are administered to a patient as a pharmaceutical composition.
  • the pharmaceutical composition is a suspension of TILs in a sterile buffer.
  • TILs expanded using aAPCs of the present disclosure may be administered by any suitable route as known in the art.
  • the TILs are administered as a single infusion, such as an intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes.
  • Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.
  • TILs can be administered.
  • about 1.2 ⁇ 10 10 to about 4.3 ⁇ 10 10 of TILs are administered.
  • the number of the TILs provided in the pharmaceutical compositions of the invention is about 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇
  • the number of the TILs provided in the pharmaceutical compositions of the invention is in the range of 1 ⁇ 10 6 to 5 ⁇ 10 6 , 5 ⁇ 10 6 to 1 ⁇ 10 7 , 1 ⁇ 10 7 to 5 ⁇ 10 7 , 5 ⁇ 10 7 to 1 ⁇ 10 8 , 1 ⁇ 10 8 to 5 ⁇ 10 8 , 5 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 9 to 5 ⁇ 10 9 , 5 ⁇ 10 9 to 1 ⁇ 10 10 , 1 ⁇ 10 10 to 5 ⁇ 10 10 , 5 ⁇ 10 10 to 1 ⁇ 10 11 , 5 ⁇ 10 11 to 1 ⁇ 10 12 , 1 ⁇ 10 12 to 5 ⁇ 10 12 , and 5 ⁇ 10 12 to 1 ⁇ 10 13 .
  • the concentration of the TILs provided in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceutical composition.
  • the concentration of the TILs provided in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.5
  • the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition.
  • the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v of the pharmaceutical composition.
  • the amount of the TILs provided in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.00
  • the amount of the TILs provided in the pharmaceutical compositions of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07
  • the TILs provided in the pharmaceutical compositions of embodiments of the invention are effective over a wide dosage range.
  • the exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.
  • the clinically-established dosages of the TILs may also be used if appropriate.
  • the amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of TILs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.
  • TILs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs may continue as long as necessary.
  • an effective dosage of TILs is about 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 1 ⁇ 10 10 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9
  • an effective dosage of TILs is in the range of 1 ⁇ 10 6 to 5 ⁇ 10 6 , 5 ⁇ 10 6 to 1 ⁇ 10 7 , 1 ⁇ 10 7 to 5 ⁇ 10 7 , 5 ⁇ 10 7 to 1 ⁇ 10 8 , 1 ⁇ 10 8 to 5 ⁇ 10 8 , 5 ⁇ 10 8 to 1 ⁇ 10 9 , 1 ⁇ 10 9 to 5 ⁇ 10 9 , 5 ⁇ 10 9 to 1 ⁇ 10 10 , 1 ⁇ 10 10 to 5 ⁇ 10 10 , 5 ⁇ 10 10 to 1 ⁇ 10 11 , 5 ⁇ 10 11 to 1 ⁇ 10 12 , 1 ⁇ 10 12 to 5 ⁇ 10 12 , and 5 ⁇ 10 12 to 1 ⁇ 10 13 .
  • an effective dosage of TILs is in the range of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg, about 0.
  • an effective dosage of TILs is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.
  • An effective amount of the TILs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation.
  • Example 1 Variability in Expansion of Tumor Infiltrating Lymphocytes Using PBMC Feeder Cells
  • FIG. 1 illustrates typical results of rapid expansion of TILs using irradiated allogeneic PBMC feeder cells (PBMC feeders).
  • TIL lines labeled M1015T and M1016T were co-cultured with 46 different irradiated feeder cell lots (1.3 ⁇ 10 7 ), IL-2 (3000 IU/mL, recombinant human IL-2 (e.g., aldesleukin or equivalent), CellGenix, Inc., Portsmouth, N.H., USA) and OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in a T25 flask for 7 days.
  • the fold expansion value for TILs was calculated on Day 7. The figure shows the number of fold expansions for the two TIL lines in separate stimulation experiments. For each TIL line, 46 different PBMC feeder lots were tested.
  • the results range over more than 100-fold for each TIL line, and highlight the variability of expansion results using PBMC feeder cells.
  • the aAPCs of the present invention offer reduced variability in expansion performance compared to PBMC feeders, as well as other advantages, as shown in the following examples.
  • Phenotypic characterization was performed on various myeloid-lineage cell lines to identify potential candidates for further modification into aAPCs for TIL expansion. The results are summarized in Table 5.
  • the MOLM-14 cell line exhibited endogenous expression of CD64, and was selected for further development.
  • the EM-3 cell line was selected based on the observation of endogenous expression of ICOS-L (which was not observed for the EM-2 cell line, despite being taken from the same patient).
  • CML chronic myeloid leukemia
  • AML acute myeloid leukemia
  • MOLM-14 cells were obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH. To develop MOLM-14 based aAPCs, MOLM-14 cells were engineered with the costimulatory molecules CD86 and 4-1BBL (CD137L). Human CD86 (hCD86) and human 4-1BBL (h4-1BBL) genes were cloned into commercially-available PLV430G and co-transfected with PDONR221 vectors (Invitrogen/Thermo Fisher Scientific, Carlsbad, Calif., USA) using a lentiviral transduction method. The gateway cloning method was used as described in Katzen, Expert Opin. Drug Disc.
  • the 293T cell line human embryonic kidney cells transformed with large T antigen
  • the transfected cells were sorted (S3e Cell Sorter, Bio-Rad, Hercules, Calif., USA) using APC-conjugated CD86 and PE-conjugated CD137L to isolate and enrich the cells.
  • the enriched cells were checked for purity by flow cytometry.
  • the vectors and portions thereof used for cloning are depicted in FIG. 2 to FIG. 11 , and the nucleotide sequences for each vector are given in Table 6.
  • the pLV430G human 4-1BBL vector is illustrated in FIG. 2 , with the polymerase chain reaction product (PCRP) portion shown in FIG. 3 .
  • the pLV430G human CD86 vector is illustrated in FIG. 4 , with the PCRP portion shown in FIG. 5 .
  • the pDONR221 human CD86 donor and human 4-1BBL donor vectors are shown in FIG. 6 and FIG. 7 , respectively.
  • Diagrams of the empty pLV430G destination vector and empty pDONR221 donor vector for the Gateway cloning method are shown in FIG. 8 and FIG. 9 , respectively.
  • FIG. 10 and FIG. 11 illustrate vector diagrams of the psPAX2 and pCIGO-VSV.G helper plasmids used for lentivirus production.
  • MOLM-14 aAPCs also referred to herein as aMOLM14 aAPCs
  • flow cytometry Canto II flow cytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA
  • FIG. 12 aMOLM-14 aAPCs were ⁇ -irradiated at 100 Gy and frozen.
  • Engineered MOLM-14 cells were gamma-irradiated at 100 Gy before co-culturing with TILs.
  • REPs were initiated by culturing TILs with irradiated, engineered MOLM-14 cells at 1:100 ratios in CM2 media containing OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) for 14 days.
  • CM2 media containing OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) for 14 days.
  • the TIL expansion rates, phenotype for activation and differentiation stage markers, metabolism rate, cytotoxicity and re-rapid expansion protocol (re-REP) assay were measured.
  • results are shown in FIG. 13 , FIG. 14 , FIG. 15 , and FIG. 16 , where two expansions for two sets of patient TILs are compared.
  • the results with the CD86/4-1BBL modified MOLM-14 cells (labeled “TIL+Engineered MOLM14+OKT3”) are comparable to the PBMC feeders (labeled “TIL+Feeders+OKT3”).
  • results at day 14 are compared in FIG. 17 , where results from two additional patient TILs are shown.
  • the results indicate that MOLM-14 cells that were engineered with CD86 and 4-1BBL showed similar TIL expansion in the rapid expansion protocol when compared with allogeneic feeder cells. However, TILs cultured with parental MOLM-14 did not expand.
  • TILs expanded against MOLM-14 maintained a TIL phenotype and showed potency to kill P815 cells as measured using BRLA, which is described in detail in Example 9.
  • luciferin-transduced P815 target cells and TILs of interest were co-cultured with and without anti-CD3 to determine whether tumor reactivity of TILs is through TCR activation (specific killing) or non-specific killing.
  • FIG. 18 the results of expansions performed with low ratios of TILs to MOLM-14 aAPCs are shown in comparison to the results of expansions with PBMC feeders.
  • TILs (2 ⁇ 10 4 ) were cultured at different TIL to aAPC or PBMC ratios (1:10, 1:30, and 1:100, denoted “10”, “30”, and “100”, respectively) with parental MOLM-14 (“MOLM14”) cells, MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), or PBMC feeders (“PBMC+”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in a 24-well G-Rex plate.
  • MOLM14 parental MOLM-14
  • aMOLM14 MOLM-14 cells transduced to express CD86 and 4-1BBL
  • PBMC+ PBMC feeders
  • FIG. 19 the results of expansions performed with higher ratios of TILs to MOLM-14 aAPCs, and otherwise performed as described above for FIG. 18 , are shown in comparison to the results of expansions with PBMC feeders.
  • the CD86/4-1BBL modified MOLM-14 aAPCs with OKT-3 and IL-2 significantly outperform PBMC feeders with OKT-3 and IL-2.
  • TIL to aMOLM14 ratios of 1:200 show enhanced TIL expansion compared to PBMC feeders under the same conditions.
  • TILs expanded with aMOLM14 or PBMC were compared by flow cytometry analysis to confirm that the TILs exhibited a similar phenotype and would be expected to perform similarly upon reinfusion into a patient.
  • TILs were first stained with L/D Aqua to determine viability.
  • cells were surface stained with TCR ⁇ / ⁇ PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28PE, CD27 APC-C7, and CD57-PerCP-Cy5.5.
  • Phenotype analysis was done by gating 10,000 to 100,000 cells according to forward light scattering (FSC)/side light scattering (SSC) using a Canto II flow cytometer (Becton, Dickinson, and Co., Franklin Lakes, N.J., USA). Data was analyzed by Cytobank software to create sunburst diagrams and SPADE (Spanning Tree Progression of Density Normalized Event) analyses. Gates were set based on fluorescence minus one (FMO) controls. TILs expanded against aMOLM14 increases CD8 + TILs when compared to PBMC feeders. Without being bound by theory, this enhanced CD8 + TIL percentage may be due to the presence of 4-1BBL engineered to MOLM14.
  • FIG. 24 depicts a flow cytometry contour plot showing a memory subset (CD45RA+/ ⁇ , CCR7+/ ⁇ ) gated on Live, TCR ⁇ / ⁇ +, CD4 + or CD8 + TILs, indicating that the memory subset obtained with PBMC feeders is replicated by the aMOLM14 aAPCs.
  • the CD4 and CD8 SPADE tree of TILs expanded with aMOLM14 aAPCs or PBMC feeders using CD3+ cells is shown in FIG. 25 and FIG. 26 .
  • the color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRG1 and TIGIT.
  • MFI mean fluorescence intensity
  • TILs expanded against aMOLM14 or PBMC were also analyzed for metabolic profiles.
  • Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TILs after expansion with irradiated PBMC feeders or aMOLM14 aAPCs were measured using a dual mitochondrial-glycolytic stress test. Briefly, cells were washed in assay medium (XF Assay Medium, Agilent Technologies, Santa Clara, Calif., USA), supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, at pH 7.4, and then 1 ⁇ 10 5 viable cells were plated onto an adhesive-coated (Cell-TakTM, Corning) XFp cell culture microplate.
  • assay medium XF Assay Medium, Agilent Technologies, Santa Clara, Calif., USA
  • Mitochondrial and glycolytic stress test experiments were performed using a Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, Calif., USA), sequentially injecting the following compounds at specified intervals for simultaneous analysis of mitochondrial and glycolytic respiration of the cells: 1 ⁇ M oligomycin; 0.5 ⁇ M FCCP; 50 mM 2-deoxyglucose; and 0.5 ⁇ M each of rotenone and antimycin A.
  • Results were analyzed using WAVE v2.3.0 software (Agilent Technologies, Santa Clara, Calif., USA) and GraphPad Prism v6.07 graphing software and are shown in FIG. 27 and FIG. 28 , where points represent mean ⁇ SEM measured in triplicate.
  • WAVE v2.3.0 software Align Technologies, Santa Clara, Calif., USA
  • GraphPad Prism v6.07 graphing software are shown in FIG. 27 and FIG. 28 , where points represent mean ⁇ SEM measured in triplicate.
  • Both TILs grown with aMOLM14 aAPCs and PBMC feeders show similar oxphos and glycolysis behavior. This data suggests that aMOLM14 does not alter the metabolic programming of TILs when compared with PBMC feeders.
  • EM-3 cells were obtained from Creative Bioarray, Inc. (Shirley, N.Y., USA). To develop an EM-3 based artificial APC, EM-3 cell lines were engineered with CD86, 4-1BBL, and antibody against IgG Fc region (Clone 7C12 or Clone 8B3). Human CD86 and human 4-1BBL/CD137 genes were cloned into commercially-available PLV430G and co-transfected with PDONR221 vectors (Invitrogen) using a lentiviral transduction method. The gateway cloning method was used as described in Katzen, Expert Opin. Drug Disc.
  • the 293T cell line was used for lentiviral production, and transduced to EM-3 cell lines.
  • the transfected cells were sorted (S3e Cell Sorter, BioRad, Hercules, Calif., USA) using APC-conjugated CD86 and PE-conjugated CD137L to isolate and enrich the cells.
  • the enriched cells were checked for purity by flow cytometry.
  • Single-chain Fv (scFv) antibody clones designated 7C12 and 8B3 were generated against Fc of mouse IgG1, IgG2a and IgG2b (Viva Biotech Ltd., Chicago, Ill., USA).
  • the amino acid sequences of these scFv clones are given in Table 7 (SEQ ID NO:27 and SEQ ID NO:28).
  • the generated scFv clones were screened for Fc binding efficiency against OKT-3, engineered towards pLV4301G containing eGFP as co-reporter to produce lentivirus.
  • the 293T cell line was used for packaging and lentiviral production.
  • Engineered EM-3 (CD86/CD137L) cells were transduced using the lentiviral system and sorted using eGFP.
  • EM37C12CD86CD137L and EM38B3CD86CD137L were regularly assessed for the consistent expression of each transduced molecule by flow cytometry.
  • DONR vector Molecular cloning of plasmids of interest may be performed as follows.
  • B site flanked PCR product or destination vector e.g., Gateway-adapted lentivector
  • DONR vector e.g., pDONR222
  • BR Clonase II Life Technologies
  • TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q.s. to bring volume to 5 ⁇ L).
  • TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q.s. to bring volume to 5 ⁇ L).
  • Incubate at room temperature for at least 1 hour. After incubation perform bacterial transformation either by heat shock method or electroporation.
  • the following cocktail may be used: recombined pDONR vector (e.g., pDON222-geneX) 50-100 ⁇ g, destination vector (e.g., Gateway adapted lentivector) 50-100 ⁇ g, LR Clonase II (Life Technologies) 1 ⁇ L, and TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q.s. to bring volume to 5 ⁇ L). Incubate at room temperature for at least 1 hour. After incubation, perform bacterial transformation either by chemical competent transformation/heat shock method.
  • pDONR vector e.g., pDON222-geneX
  • destination vector e.g., Gateway adapted lentivector
  • LR Clonase II Life Technologies
  • Transformation and selection of the cloned plasmid may be performed as follows.
  • the chemical competent transformation method may be performed as follows. Prepare nutrient agar plates (LB-Lennox or YT) with antibiotic for selection. Ensure that Recovery Medium (supplied by Lucigen, Middleton, Wis., USA) is readily available at room temperature.
  • sterile culture tubes may be chilled on ice (e.g., 17 mm ⁇ 100 mm tubes (14 mL tube)), one tube for each transformation reaction).
  • Colonies for Miniprep may be grown as follows. After colonies have formed from plating recovered transformation reaction of DNA manipulation (e.g. LR reaction), add 1 mL desired TB/antibiotics into desired number of 2 mL Eppendorf microtubes with punctured caps. Pick desired number of colonies using ART LTS 20 soft pipette tip (VWR 89031-352) or 10 ⁇ L Denville tip. Place tip in 2 mL Eppendorf microtube with punctured cap. Cut the tip so that it fits in tube, close cap, and place tubes on shaker (purple 15 mL tube holder with VWR brand 15 mL tubes).
  • ART LTS 20 soft pipette tip VWR 89031-352
  • Denville tip Place tip in 2 mL Eppendorf microtube with punctured cap. Cut the tip so that it fits in tube, close cap, and place tubes on shaker (purple 15 mL tube holder with VWR brand 15 mL tubes).
  • Lentiviral production may be performed as follows. The following media composition is prepared: 500 mL DMEM/F12 (Sigma); 25 mL FBS Heat Inactivated (HI) (Hyclone); 10 mM HEPES (Life Technologies); 1 ⁇ Primocin (Invivogen); 1 ⁇ Plasmocin (Invivogen); and 1 ⁇ 2-mermactoethanol (Life Technologies). Harvest T75 flasks (Thermo Fisher Scientific) containing 90% confluent 293T cells. Aspirate media. Add 10 ml PBS, rinse gently and aspirate off. Add 2 mL TrypLE Express (Life Technologies) and evenly distribute it over the cell layer, let sit for 3-5 minutes at 37° C. (cell culture incubator).
  • each T75 flask transfection requires 2 ⁇ g Baculo p35 plasmid (optional; only necessary if packaging a death gene), 2 ⁇ g VSV.G env plasmid (e.g., pMD2.G or PCIGO VSV-G); 4.7 ⁇ s Gag/polymerase plasmid (e.g., psPAX2 or pCMV-deltaR8.91), and 2.3 ⁇ g of the lentiviral vector described above.
  • Virus may be concentrated using the PEG-it method (System Biosciences, Inc., Palo Alto, Calif. 94303) for longer-term storage at ⁇ 80° C. Collect the supernatant from the transfection plates. Spin down the cell debris in the supernatant. The supernatant may also be filtered to completely remove any packaging cells. Add an amount of PEG-it solution equal to a quarter of the volume of supernatant to the supernatant. Incubate the suspension at 4° C. for overnight. Centrifuge at 3500 rpm (1500 g) at 4° C. for 30 minutes. Remove supernatant and centrifuge at 3500 rpm at 4° C. for 5 minutes. Remove remaining supernatant. Resuspend virus in desired amount of phosphate-buffered saline (PBS) and freeze aliquots at ⁇ 80° C.
  • PBS phosphate-buffered saline
  • Total volume of transduction per well should be approximately 500 ⁇ L with 3-10 ⁇ g/mL Polybrene (Hexadimethrine bromide, Sigma-Aldrich Co., St. Louis, Mo., USA).
  • the amount of concentrated virus added will depend on the MOI (multiplicity of infection) desired.
  • a typical MOI is 10:1 but this may vary depending on cell type.
  • the transfection well should contain 100 ⁇ L of standard media containing either 1 ⁇ 10 6 suspension cells or 50% confluent cells.
  • MOI of 10:1 e.g., virus activity is 1 ⁇ 10 8 IU/mL and the target is to infect 1 ⁇ 10 6 cells, then 1 ⁇ 10 7 virions or 100 ⁇ L of virus is needed). Add standard media to 500 ⁇ L.
  • Sorting of aAPCs may be performed as follows. Culture the cells in the media described above until the cell count reaches a minimum of 10-20 million. Take 1 ⁇ 10 6 cells for each condition and stain with the antibodies for the proteins transduced. Wash the cells and analyze by flow cytometry to test the stability of transduction. Once the expression of protein of interest has been analyzed and confirmed, prepare the rest of the cells for sorting. Sort the cells in an S3 sorter by gating on markers of interest. Culture the sorted cells using the media mentioned above. Before freezing the vial, test the stability of the protein expression of interest. Use Recovery cell culture Freezing media (Invitrogen), to make the cell bank of the same cells. Cells may be banked after each transduction and sorting procedure.
  • aEM3 aAPCs engineered EM-3 aAPCs (also referred to herein as aEM3 aAPCs) used for the experiments described herein, expression of CD86 and 4-1BBL was confirmed using flow cytometry (Canto II flow cytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA), with results shown in FIG. 37 .
  • flow cytometry Canto II flow cytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA
  • pLenti-C-Myc-DDK OX40L PS100064, Origene, SEQ ID NO:39, FIG. 90
  • VSV-G envelope plasmid pCIGO-VSV.G
  • Phoenix-GP ATCC CRL-3215
  • the supernatants were harvested 60 hours later and concentrated using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane.
  • aEM-3 cells were then infected with concentrated lentivirus and further expanded for five days.
  • the cells were stained with PE-conjugated anti-human OX40L, Brilliant Violet 421-conjugated anti-human CD137L (if 4-1BBL is included in the prior aEM-3 cells), and PE/Cy7 conjugated anti-human CD86 and sorted based on the expression of GFP, OX40L, CD137L (when included), and CD86 using a S3e Cell Sorter (Bio-Rad, Inc., Hercules, Calif., USA).
  • the purity of sorted cells was further validated using flow cytometry.
  • the enriched cells were checked for purity by flow cytometry.
  • TIL EM-3 aAPCs
  • aEM3 EM-3 aAPCs
  • aEM3 7C12 or 8B3
  • OKT-3 30 mg/mL
  • IL-2 3000 IU/mL
  • Cells were counted on Day 11 and 14.
  • the results are plotted for two batches of TILs in FIG. 38 and FIG. 39 .
  • TILs were co-cultured with aEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3000 IU/mL) with or without OKT-3 (30 mg/mL).
  • FIG. 40 where the bar graph shows cell numbers determined on Day 11.
  • FIG. 41 illustrates the results of TIL expansions with EM-3 aAPCs (aEM3) at different TIL:aAPC ratios.
  • the results show that aEM3 aAPCs perform comparably to and in some cases better than PBMCs, particularly at ratios of 1:200 at longer culture times (14 days).
  • FIG. 42 illustrates the low variability in cell counts from TIL expansions with EM-3 aAPCs (aEM3) in comparison to PBMC feeders.
  • TILs (2 ⁇ 10 4 ) were co-cultured with five different PBMC feeder lots or aEM3 (in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate.
  • the graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14.
  • TILs expanded against aEM3 or PBMC feeders were used for flow cytometry analysis using 4 different panels (differentiation panels 1 and 2, T cell activation panels 1 and 2). Briefly, TILs were first stained with L/D Aqua to determine viability. Next, cells were surface stained with TCR ⁇ / ⁇ PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28 PE, CD27 APC-Cy7, and CD57-PerCP-Cy5.5 for differentiation panel 1; CD45RA PE-Cy7, CD8a PerCP/Cy5, CCR7 PE, CD4 FITC, CD3 APC-Cy7, CD38 APC, and HLA-DR PB, for differentiation panel 2; CD137 PE-Cy7, CD8a PerCP-Cy5.5, Lag3 PE, CD4 FITC, CD3 APC-Cy7, PD1 APC, and Tim-3 BV421 for T cell activation panel 1; or CD69 PE-Cy7, CD8a Per
  • Phenotype analysis was done by gating 10,000 to 100,000 cells according to FSC/SSC using the Canto II flow cytometer. Data was analyzed using Cytobank software (Cytobank, Inc., Santa Clara, Calif., USA) to create sunburst diagrams and SPADE (Spanning-tree Progression Analysis of Density-normalized Events) plots. Gates were set based on fluorescence minus one (FMO) controls. SPADE plots were generated with the group of cells, characterized in a form of related nodes based on the expression level of surface markers. CD4 + and CD8 + TIL subsets were determined based on CD3 + gating, and trees were generated. Sunburst visualizations are shown in FIG. 44 and FIG. 45 . FIG.
  • FIG. 44 shows that TILs expanded against aEM3 aAPCs maintained the CD8 + phenotype when compared to the same TILs expanded against PBMC feeders.
  • FIG. 45 shows the results of a second batch of TILs from a different patient expanded against aEM3 aAPCs, where a clear increase of CD8 + cells (65.6%) is seen in comparison to the results from expansion using PBMC feeders (25%).
  • the CD4 and CD8 SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3 + cells is shown in FIG. 46 and FIG. 47 .
  • the color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRG1 and TIGIT.
  • MFI mean fluorescence intensity
  • the Seahorse XF Cell Mito Stress Test measures mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells, using modulators of respiration that target components of the electron transport chain in the mitochondria.
  • OCR oxygen consumption rate
  • the test compounds oligomycin, FCCP, and a mix of rotenone and antimycin A, described below
  • ATP production maximal respiration
  • non-mitochondrial respiration respectively.
  • Proton leak and spare respiratory capacity are then calculated using these parameters and basal respiration.
  • Each modulator targets a specific component of the electron transport chain.
  • Oligomycin inhibits ATP synthase (complex V) and the decrease in OCR following injection of oligomycin correlates to the mitochondrial respiration associated with cellular ATP production.
  • Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron flow through the electron transport chain is uninhibited and oxygen is maximally consumed by complex IV.
  • FCCP-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal respiration and basal respiration.
  • Spare respiratory capacity (SRC) is a measure of the ability of the cell to respond to increased energy demand.
  • the third injection is a mix of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination shuts down mitochondrial respiration and enables the calculation of nonmitochondrial respiration driven by processes outside the mitochondria.
  • FIG. 50 illustrates a mitochondrial stain of Live TILs expanded against PBMC feeders or aEM3 aAPCs.
  • MitoTracker dye stains mitochondria in live cells and its accumulation is dependent upon membrane potential.
  • TILs expanded against PBMC feeders or aEM3 were stained L/D Aqua followed by MitoTracker red dye. The data show MitoTracker positive (MFI) cells gated on live population,
  • the P815 BRLA is described in detail in Example 9. The results are shown in FIG. 51 and FIG. 52 , and show that TILs expanded with aAPCs have similar functional properties (and expected clinical efficacy) to those expanded with PBMC feeders.
  • IFN- ⁇ release and Granzyme B release from TILs expanded with PBMC feeders and aMOLM14 and aEM3 aAPCs as described above was also assessed following overnight stimulation with microbeads coated with anti-CD3/CD28/4-1BB.
  • the IFN- ⁇ release results are shown in FIG. 53 and FIG. 54
  • the Granzyme B release results are shown in FIG. 55 and FIG. 56 .
  • Significant and surprising increases in IFN- ⁇ release and Granzyme B release were observed for TILs expanded with aEM3 aAPCs relative to those expanded with PBMC feeders, but not for TILs expanded by aMOLM14 aAPCs. Without being bound by theory, this suggests that TILs cultured with aEM3 aAPCs may be more active in vivo as a cancer therapy. Most other differences observed were not statistically significant.
  • aEM3 and aMOLM14 aAPCs may be grown in the following media compositions to produce master cell banks, which may be further grown in this media for supply of aAPCs: 500 mL of Dulbecco's Modified Eagle Medium DMEM/F12 (Sigma-Aldrich, St. Louis, Mo., USA), 50 mL fetal bovine serum (FBS) Heat Inactivated (HI) (Hyclone); 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) (Life Technologies); 1 ⁇ Primocin (Invivogen); 1 ⁇ Plasmocin (Invivogen), and 1 ⁇ 2-mercaptoethanol (Life Technologies).
  • aAPCs described herein may also be grown from a master cell bank using any suitable method known in the art for the growth of cells.
  • aAPCs are thawed and are then expanded in a medium of 80-90% RPMI 1640+10-20% h.i. FBS (fetal bovine serum) by splitting saturated culture 1:2 to 1:3 every 2-3 days, seeding out at about 0.5-1 ⁇ 10 6 cells/mL in 24-well plates, and maintaining at about 0.5-1.5 ⁇ 10 6 cells/mL, with incubation at 37° C. and 5% CO 2 .
  • FBS fetal bovine serum
  • aAPCs of certain embodiments of the present invention in the production of human therapies are known in the art and include cell line characterization (HLA high resolution typing); cytokine release testing; testing of human serum to replace FBS to grow aAPC; testing freezing media to freeze aAPCs; master cell banking (including raw material testing and stability testing); standardization of irradiation (including irradiation dose (1000, 3000, 5000, 10000, 15000 rad), fresh versus frozen aAPCs, and with/without TILs); stability of aAPC; development of a panel to evaluate the contamination of aAPCs; development of molecular biology assays (qPCR, DNA sequencing); testing of TIL expansions from different tumor types, including melanoma, cervical, and head and neck cancer (using a G-Rex 5M); potency, purity, and identity testing; mycoplasma and sterility assays; microbiological testing (USP/EP sterility, bioburden and
  • TILs may be expanded using the aAPCs of certain embodiments of the present invention, such as aEM3 and aMOLM14 aAPCs, using any of the expansion methods described herein.
  • a method for expanding TILs is depicted in FIG. 57 .
  • the expansion of TILs using aAPCs may be further combined with any method of treating cancer in a patient described herein.
  • BRLA Bioluminescent Redirected Lysis Assay
  • Mouse mastocytoma P815 cells expressing the endogenous CD16 Fc receptor can bind anti-CD3c (OKT-3), providing a potent TCR activation signal as a target cell line.
  • the P815 Clone G6 was transduced with a lentiviral vector based on eGFP and firefly luciferase, sorted and cloned using the BD FACSAria II. Clone G6 was selected based on eGFP intensity analyzed using an Intellicyt iQue Screener. Target cells and TILs of interest were co-cultured+/ ⁇ OKT-3 to assess TCR activation (specific killing) or non-specific (lymphokine activated killing, LAK) respectively.
  • Interferon gamma release in the media supernatant of co-cultured TILs was analyzed by ELISA, and LAMP1 (CD107a, clone eBioH4A3) expression on TILs was analyzed on a flow cytometer to evaluate the cytotoxic potency of TILs.
  • FIG. 59 illustrates percent toxicity of TIL batch M1033T-1 co-cultured with P815 Clone G6 (with and without anti-CD3) at individual effector:target ratios by BRLA.
  • FIG. 60 illustrates enzyme-linked immunosorbent assay (ELISA) data showing the amount of IFN- ⁇ released against different ratios of effector to target cells.
  • FIG. 61 illustrates LAMP1(%) expressed by TIL batch M1033T-1 when co-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of 1:1 effector to target cells for 4 hours and 24 hours co-culture.
  • ELISA enzyme-linked immunosorbent assay
  • FIG. 62 illustrates BRLA for TIL batch M1030.
  • the cytotoxicity (measured as LU 50 /1 ⁇ 10 6 TIL) by BRLA is 26 ⁇ 16.
  • FIG. 63 illustrates the results of a standard chromium release assay for TIL batch M1030.
  • the cytotoxicity (measured as LU 50 /1 ⁇ 10 6 TIL) by chromium release assay is 22.
  • FIG. 64 illustrates BRLA results for TIL batch M1053, showing lytic units of the TILs by BRLA as 70 ⁇ 17.
  • FIG. 65 illustrates the results of a standard chromium release assay for TIL batch M1053, showing lytic unit of the TILs by chromium assay as 14 ⁇ 5. Comparison of two assay results shows the comparable performance of the BRLA result to the chromium release assay result.
  • FIG. 66 illustrates the linear relationship between IFN- ⁇ release and cytotoxic potential of TILs.
  • FIG. 67 illustrates ELISpot results for IFN- ⁇ .
  • FIG. 68 illustrates enzymatic IFN- ⁇ release for TIL batch M1053.
  • FIG. 69 illustrates enzymatic IFN- ⁇ release for TIL batch M1030.
  • FIG. 70 illustrates ELISpot data showing Granzyme B release by M1053T and M1030T.
  • FIG. 71 illustrates enzymatic Granzyme B release for TIL batch M1053.
  • FIG. 72 illustrates enzymatic Granzyme B release for TIL batch M1030.
  • FIG. 73 illustrates ELISpot data showing TNF- ⁇ release by M1053T and M1030T.
  • FIG. 74 illustrates enzymatic TNF- ⁇ release for TIL batch M1053.
  • FIG. 75 illustrates enzymatic TNF- ⁇ release for TIL batch M1030.
  • the data in FIG. 66 to FIG. 76 confirms the potency of these batches of TILs as also shown by the BRLA.
  • the BRLA requires no radionuclides and is as efficient and sensitive as traditional cytotoxicity assays.
  • Flow cytometric assessment of Lampl expression on TILs at individual time points demonstrates degranulation of cytotoxic T cells relative to the potency shown by BRLA.
  • the BRLA demonstrates similar to better potency than standard chromium release assay.
  • BRLA also enables evaluation of the potency of TIL lytic activity.
  • Comparison of BRLA with chromium release assay shows the efficiency and reliability of BRLA.
  • BRLA has a linear relationship with IFN ⁇ release by TILs. Release assay of IFN- ⁇ , TNF ⁇ and Granzyme B by ELISpot is consistent with the cytotoxic efficiency of the TILs evaluated by BRLA.
  • Example 11 Provides for Weaning EM3 Cells from FBS to hAB Serum
  • EM3 cells are weaned from FBS to hAB serum to avoid reactivity. As shown in FIG. 76 , aEM3 cells were successfully weaned off of FBS to hAB serum.
  • FIG. 77 demonstrates that the formulation of human AB serum (90%) and DMSO (10%) provided for unexpectedly increased EM3 cell numbers after 3 days of recovery.
  • aEM3 cells were cultured in gas permeable cell culture flasks (i.e., GREX flasks (Wilson Wolf Manufacturing)) and the effect on cell doubling time was observed over an 8 day time course. As shown in FIG. 78 , the GREX flasks provided for rapid growth of aEM3 cells.
  • gas permeable cell culture flasks i.e., GREX flasks (Wilson Wolf Manufacturing)
  • FIGS. 79 and 80 The results of such analysis are described in FIGS. 79 and 80 .
  • aEM3 cell populations were 53.5% and 43.2% eGFP+ for aEM3 7C12 and aEM3 8B5 cells, respectively.
  • Postsorting cell populations was improved to 96.8% and 96.3% eGFP+ for aEM3 7C12 and aEM3 8B5 cells, respectively ( FIG. 80 ).
  • Example 15 aEM3 Feeder Cells as an Alternative to PBMC Feeders
  • aEM3 cells may be used as an alternative for PBMC feeders, resulting in unexpectedly different properties for both TIL expansion process and the resulting TILs.
  • PBMCs and aEM3 cells were stimulated by treatment with OKT-3.
  • aEM3 cells displayed a comparatively different cytokine expression profile as compared to PBMCs.
  • the aEM3 cells of the present invention provide efficacious TILs (as shown herein) without reproducing the same cytokine secretion properties of TILs expanded with conventional PBMCs.
  • tissue fragments were cultured in a single well with CM1 or various serum free media with 300 IU/mL of IL-2. Cells were then counted on Day 11 before initiating REP.
  • the various serum free media used included Prime CDM (Irvine), CTS Optimizer (ThermoFisher), and Xvivo-20 (Lonza).
  • TIL expansion (PreREP) with CTS provided increased cell numbers as compared to CM1.
  • tissue fragments were cultured with CM1 or various serum free media with 6000 IU/mL of IL-2 until Day 11.
  • REP was then initiated on Day 11 using PBMC feeders, OKT-3, and IL-2, and culture was split on Day 16. Cultures were then terminated at the end of Day 22.
  • the various serum free media used included Prime CDM (Irvine), CTS Optimizer (ThermoFisher), and Xvivo-20 (Lonza).
  • Prime CDM Irvine
  • CTS Optimizer ThermoFisher
  • Xvivo-20 Livivo-20
  • Example 17 Growth of aAPCs in Serum Free Media as Compared to Serum-Based Media
  • aEM3 cells were cultured using various serum free media.
  • aEM3 cells were cultured in 24 well plates at 1 ⁇ 10 6 cells per well for 3 days using general cell culture protocols as described herein, with the exception that that one group of cells were provided with serum-based media (cDMEM (10% hSerum) and the other groups of cells were provided with serum free media.
  • serum-based media cDMEM (10% hSerum
  • CTS OpTmizer and Prime-TCDM serum free media provided cell growth that was comparable to serum-based media (i.e., cDMEM (10% hSerum). Therefore, serum free media is an effective alternative for growing and maintaining aAPCs as comapred to serum-based media.
  • Example 18 Provides, Maintenance, and Cryopreservation of aAPCs
  • aEM3 cells from a cell line designated TIL-Rs3 were propagated and cryopreserved.
  • Thawing and recovery of aEM3 cells may be accomplished using the following non-limiting procedure. Cyropreserved aEM3 cells are warmed slowly in pre-warmed media (37° C.) that is prepared from CTS OpTmizer Basal Media (Thermo Fisher), CTS OpTmizer Cell Supplement (Thermo Fisher), Gentamicin (Lonza), and Glutamax (Life Technologies). The suspended cells are then centrifuged at 1500 rpm for 5 minutes at 4° C. The resulting supernatant is discarded and the remaining aEM3 cells are resuspended in the foregoing media and plated (5 ⁇ 10 6 cells/10 mL per well of a 6 well plate).
  • Propagation of aEM3 cells may be accomplished using the following non-limiting procedure. Aliquots of the foregoing media are prepared in gas permeable cell culture flasks (i.e., GREX 10 flasks (Wilson Wolf Manufacturing)). The plated aEM3 cells are washed by centrifugation (i.e., 1500 rpm for 5 minutes at 4° C.), resuspended in media, and added to the GREX flasks at cell density of 1-2 ⁇ 10 6 cells/mL. The aEM3 cell suspension was diluted with 30 mL of media and the GREX flasks were then incubated for 3-4 days at 37° C. under CO 2 .
  • gas permeable cell culture flasks i.e., GREX 10 flasks (Wilson Wolf Manufacturing)
  • the plated aEM3 cells are washed by centrifugation (i.e., 1500 rpm for 5 minutes at 4° C.), resus
  • the GREX flasks were removed from the incubator and placed in a biological safety cabinet (BSC).
  • BSC biological safety cabinet
  • the cultured aEM3 cells are carefully extracted from the GREX flasks by pipette and the resulting extraction is centrifuged to provide the increased number of aEM3 cells, which may be resuspended at a cell density of 10-20 ⁇ 10 6 cells per GREX 10 flask.
  • An alternative cryopreservation of aEM3 cells may be accomplished using the following non-limiting procedure.
  • the foregoing GREX 10 flasks containing the aEM3 cells are removed from the incubator and placed in a BSC.
  • the cultured aEM3 cells are carefully extracted from the GREX flasks by pipette and the resulting extraction is centrifuged to provide the increased number of aEM3 cells, which is resuspended in a volume of CryStor 10 (Biolife Solutions) to provide a concentration of 10-100 ⁇ 10 6 cells/vial in cryovials.
  • the aEM3 cell suspensions may be placed in a freezing container and transferred to a ⁇ 80° C. freezer.
  • aEM3 cells from the TIL-R3 cell line (1-2 ⁇ 10 6 cells) were cryopreserved according to the procedure set forth in Example 18 using CS-10 cryopreservation media. Vials of such cells were then thawed and the cells were counted. Cell counts were taken pre-freeze, post-thaw, and 3 days after thaw (i.e., Post-Thaw Recovery). As shown in FIG. 86 and FIG. 87 , the total live cell counts recovered rapidly post thaw in two separate experiments.
  • TIL-R3 cells (1 ⁇ 10 6 cells) were thawed (Day 3 post-thaw) and plated at a density of 0.5 ⁇ 10 6 /cm 2 in each well of a 24 well plate. On day 0 and 3, viable cells were counted and recorded. On the first passage (Day 6), cells were split at the density of 2 ⁇ 10 6 cells/cm 2 or 0.5 ⁇ 10 6 cells/cm 2 . At the end of the first passage, a cell count was performed. The resulting cell counts are shown in FIG. 88 , which demonstrate both a recovery phase post-thaw and a growth phase.
  • TIL-R3 cells (20 ⁇ 10 6 cells) were cultured at a density of 2 ⁇ 10 6 /cm 2 in GREX 10 flasks according to the procedure described in Example 18. On days 4 and 8, live cells were counted and recorded. The resulting cell counts are shown in FIG. 89 , which demonstrates a growth phase for the cells following cryopreservation that reaches a plateau between days 4 and 8 when the cells reached a density of 13.9 ⁇ 10 6 cells/cm′.
  • PreREP TIL lines Fifteen different PreREP TIL lines (0.4 ⁇ 10 5 cells) were co-cultured with either aEM3 aAPCs (as described herein) or PBMC feeders (10 ⁇ 10 6 ), OKT3 (30 ng/mL) and IL-2 (3000 IU/mL) and cultures were split on Day 5 using 6 well Grex plates. Cultures were sampled at day 11 and analyzed by flow cytometry. The relative ratio of CD8 + cells was calculated by the formula (CD8% aEM3)/(CD8% PBMC). The results shown in FIG. 91 indicate that TILs cultured with aEM3 cells surprisingly promote CD8 + skewing and and an improved TIL product. Additional results of these experiments are shown in FIG. 92 , FIG. 93 , and FIG. 94 , where the results shown that TILs cultured with aEM3 aAPCs displayed comparable expansion and less non-CD3+ cell contamination in comparison to TILs cultured with PBMC feeders.
  • Genomic DNA was isolated from pre-REP or post-REP (magnetic bead sorted for CD3 + ) TILs for a qPCR (quantitative polymerase chain reaction) assay to measure telomere length.
  • the real time qPCR method is described in Cawthon, Nucleic Acids Res. 2002, 30(10), e47; and Yang, et al., Leukemia, 2013, 27, 897-906. Briefly, the telomere repeat copy number to single gene copy number (T/S) ratio was determined using an PCR thermal cycler (Bio-Rad Laboratories, Inc.) in a 96-well format. Ten ng of genomic DNA was used for either the telomere or hemoglobin (hgb) PCR reaction and the primers used were as follows:
  • Tel-1b primer (SEQ ID NO: 40) (CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT); Tel-2b primer (SEQ ID NO: 41) (GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT); hgb1 primer (SEQ ID NO: 42) (GCT TCT GAC ACA ACT GTG TTC ACT AGC); and hgb2 primer (SEQ ID NO: 43) (CAC CAA CTT CAT CCA CGT TCA CC).
  • each 96-well plate contained a five-point standard curve from 0.08 ng to 250 ng using genomic DNA isolated from the 1301 human T-cell leukemia cell line (available from Sigma and ATCC).
  • the T/S ratio ( ⁇ dCt) for each sample was calculated by subtracting the median hemoglobin threshold cycle (Ct) value from the median telomere Ct value.
  • the relative T/S ratio ( ⁇ ddCt) was determined by subtracting the T/S ratio of the 10.0 ng standard curve point from the T/S ratio of each unknown sample.
  • Results are shown in FIG. 95 . Each data point shown is the median measurement of relative T/S ratio. The results indicate that TILs cultured with aEM3 maintain their telomere length.

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Abstract

In some embodiments, compositions and methods relating to isolated artificial antigen presenting cells (aAPCs) are disclosed, including aAPCs comprising a myeloid cell transduced with one or more viral vectors, such as a MOLM-14 or a EM-3 myeloid cell, wherein the myeloid cell endogenously expresses HLA-A/B/C, ICOS-L, and CD58, and wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL and/or OX40L and transduce the myeloid cell to express CD86 and 4-1BBL and/or OX40L proteins. In some embodiments, methods of expanding tumor infiltrating lymphocytes (TILs) with aAPCs and methods of treating cancers using TILs after expansion with aAPCs are also disclosed.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of U.S. patent application Ser. No. 16/526,353, filed on Jul. 30, 2019, which is a continuation of U.S. patent application Ser. No. 15/800,967, filed Nov. 1, 2017, which is a continuation of International Application No. PCT/US17/59271, filed Oct. 31, 2017, which claims the benefit of priority to U.S. Provisional Application No. 62/481,831, filed Apr. 5, 2017, U.S. Provisional Application No. 62/475,053, filed Mar. 22, 2017, U.S. Provisional Application No. 62/438,600, filed Dec. 23, 2016, and U.S. Provisional Application No. 62/415,274, filed Oct. 31, 2016, the entireties of which are incorporated herein by reference.
    • FIELD OF THE INVENTION
  • Engineered artificial antigen presenting cells (aAPCs) for expansion of tumor infiltrating lymphocytes are disclosed.
    • BACKGROUND OF THE INVENTION
  • Treatment of bulky, refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. A large number of TILs are required for successful immunotherapy, and a robust and reliable process is needed for commercialization. This has been a challenge to achieve because of technical, logistical, and regulatory issues with cell expansion. IL-2-based TIL expansion followed by a “rapid expansion process” (REP) has become a preferred method for TIL expansion because of its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. However, although REP can result in a 1,000-fold expansion of TILs over a 14-day period, it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT-3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. Despite their high performance, PBMCs have multiple drawbacks, including the large numbers of allogeneic PBMCs required, the need to obtain PBMCs by leukapheresis from multiple healthy donors, the resulting interdonor variability in PBMC viability after cryopreservation and variable TIL expansion results, the risk of undetected viral pathogens causing downstream patient infections, and the extensive and costly laboratory testing of each individual donor cell product to confirm sterility and quality (including viral contaminant testing) and to test expansion properties.
  • Unfortunately, aAPCs developed for use in the expansion of TILs have suffered from poor performance when compared to PBMCs, including alterations of the phenotypic properties of the input TILs, as well as poor expansion performance and/or high variability in expansion results. Because of the large number of potential cells that might be adapted for use as aAPCs and the unpredictability of identifying suitable candidates, the focus of aAPC development for polyclonal TILs to date has been solely on the well-established K562 cell line. Butler and Hirano, Immunol. Rev. 2014, 257, 191-209. For example, K562 cells modified to express 4-1BBL (CD137L) were tested in pre-REP culture (but not in REP culture) to determine enhancement of TIL expansion from tumor digest, but PBMCs were still required to be used in conjunction with K562 cells to obtain TIL expansion. Friedman, et al., J. Immunother. 2011, 34, 651-661. Other engineered K562 cells modified to express CD64, CD86, and 4-1BBL were tested and achieved TIL expansion that was at best comparable to PBMCs, and most likely less than PBMCs, and also suffered from skewing of the polyclonal TIL phenotype to a less favorable CD8+/CD4+ T cell ratio. Ye, et al., J. Translat. Med. 2011, 9, 131. Recently, K562 cells modified to express CD86, 4-1BBL (CD137L), high affinity Fc receptor (CD64) and membrane-bound IL-15 have also been shown to propagate TIL (post-REP) at equivalent numbers compared to PBMC feeders, but with the additional complexity of membrane-bound IL-15. Forget, et al., J. Immunother. 2014, 37, 448-60. Other systems developed have lacked critical costimulatory molecules, have led to unfavorable T cell phenotypic skewing, or have required additional interleukins (such as IL-21). Butler and Hirano, Immunol. Rev. 2014, 257, 191-209. Overall, K562 modified aAPCs have not been shown to provide for consistent expansion of TILs with acceptable variability while also performing better than PBMCs in other measures including overall expansion cell counts. Alternative aAPCs besides K562 cells have been successful in other cell expansion methods, but have not achieved the same performance as PBMCs with the unique polyclonal subset of cells that make up TILs. Maus, et al., Nat. Biotechnol. 2002, 20, 143-148; Suhoski, et al., Mot Ther. 2007, 15, 981-988.
  • The MOLM-14 human leukemia cell line was established from the peripheral blood of a patient with relapsed acute monocytic leukemia, and initial phenotypic characterization indicated the presence of at least the following markers: CD4, CD9, CD11a, CD13, CD14, CD15, CD32, CD33, CD64, CD65, CD87, CD92, CD93, CD116, CD118, and CD155. Matsuo, et al., Leukemia 1997, 11, 1469-77. Additional phenotypic characterization of MOLM-14 found higher levels of HLA-A/B/C, CD64, CD80, ICOS-L, CD58, and lower levels of CD86. MOLM-14 cells and the closely-related MOLM-13 cells have not been previously reported as useful aAPCs for the expansion of cells for tumor immunotherapy applications.
  • The EM-3 human cell line was established from the bone marrow of a patient with Philadelphia chromosome-positive CIVIL. Konopka, et al., Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. EM-3 cells and the closely-related EM-2 cell line have not been previously reported as useful aAPCs for the expansion of cells for tumor immunotherapy applications. Phenotypic characterization for EM-3 cells indicates the presence of at least the following markers: CD13, CD15, and CD33.
  • The present invention provides the unexpected finding that engineered myeloid lineage cells, including MOLM-13, MOLM-14, EM-3, and EM-2 cells, transduced with additional costimulatory molecules, including CD86 (B7-2), 4-1BBL (CD137L), and OX40L (CD134L), provide for superior and highly efficient expansions of TILs in large numbers with minimal variability, reduced cost, and no reliance on human blood samples as a source of PBMCs, with the benefit of using an aAPC which can be produced efficiently from a master cell bank. CD86 and 4-1BBL are costimulatory molecules that provide costimulatory signals for T cell activation. The MOLM-14, MOLM-13, EM-3, and/or EM-2 cells transduced with additional costimulatory molecules are useful, for example, in the expansion of TILs for use in cancer immunotherapy and other therapies.
    • SUMMARY OF THE INVENTION
  • In an embodiment, the invention provides an artificial antigen presenting cell (aAPC) comprising a myeloid cell transduced with one or more vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein.
  • In an embodiment, each of the CD86 protein and the 4-1BBL protein are human proteins.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC can stimulate and expand a tumor infiltrating lymphocyte (TIL) contacted with the aAPC.
  • It will be apparent that in certain embodiments of the invention, the nucleic acid molecule encoding CD86 may be comprised in a different viral vector to the nucleic acid molecule encoding 4-1BBL or the same viral vector.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC expands a population of TILs by at least 50-fold over a period of 7 days in a cell culture medium comprising IL-2 at a concentration of about 3000 IU/mL and OKT-3 antibody at a concentration of about 30 ng/mL.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the aAPC can stimulate and expand a T cell contacted with the aAPC.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell endogenously expresses HLA-AB/C, ICOS-L, and CD58.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is essentially devoid of membrane-bound IL-15.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a MOLM-14 cell.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a MOLM-13 cell.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a EM-3 cell.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a EM-2 cell.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the CD86 protein comprises an amino acid sequence as set forth in SEQ ID NO:8, or an amino acid sequence comprising one or more conservative amino acid substitutions thereof, and the 4-1BBL protein comprises SEQ ID NO:9, or an amino acid sequence comprising one or more conservative amino acid substitutions thereof.
  • In an embodiment, the invention provides an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the nucleic acid molecule encoding CD86 comprises a nucleic acid sequence as set forth in SEQ ID NO:16 and the nucleic acid molecule encoding 4-1BBL comprises a nucleic acid sequence as set forth in SEQ ID NO:19.
  • In an embodiment, the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising the step of contacting a population of TILs with an aAPC comprising a myeloid cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and wherein the population of TILs is expanded. In an embodiment, the method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium.
  • In an embodiment, the foregoing method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL.
  • In an embodiment, the foregoing method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the population of APCs expands the population of TILs by at least 50-fold over a period of 7 days in a cell culture medium.
  • In an embodiment, the foregoing method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the myeloid cell endogenously expresses HLA-AB/C, ICOS-L, and CD58.
  • In an embodiment, the foregoing method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the myeloid cell is a MOLM-14 cell.
  • In an embodiment, the foregoing method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the myeloid cell is a MOLM-13 cell.
  • In an embodiment, the foregoing method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the myeloid cell is a EM-3 cell.
  • In an embodiment, the foregoing method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the myeloid cell is a EM-2 cell.
  • In an embodiment, the foregoing method is an in vitro or an ex vivo method.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the CD86 protein comprises an amino acid sequence as set forth in SEQ ID NO:8, or comprises an amino acid sequence comprising one or more conservative amino acid substitutions thereof, and the 4-1BBL protein comprises an amino acid sequence as set forth in SEQ ID NO:9, or comprises an amino acid sequence comprising one or conservative amino acid substitutions thereof.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the nucleic acid encoding CD86 comprises a nucleic acid sequence as set forth in SEQ ID NO:16 and the nucleic acid encoding 4-1BBL comprises a nucleic acid sequence as set forth in SEQ ID NO:19.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the expansion is performed using a gas permeable container.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the ratio of the population of TILs to the population of aAPCs is between 1 to 200 and 1 to 400.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the ratio of the population of TILs to the population of aAPCs is about 1 to 300.
  • In an embodiment, the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising a population of TILs with a myeloid artificial antigen presenting cell (aAPC), wherein the myeloid aAPC comprises at least two co-stimulatory ligands that specifically bind with at least two co-stimulatory molecules on the TILs, wherein binding of the co-stimulatory molecules with the co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs, and wherein the at least two co-stimulatory ligands comprise CD86 and 4-1BBL. In an embodiment, the foregoing method is an in vitro or ex vivo method.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, and wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating cancer, wherein the TILs are a second population of TILs and are obtainable from a method comprising the steps of:
      • (a) performing a rapid expansion of a first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the TILs are/have been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, and wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, wherein the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, and wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a population of tumor infiltrating cells (TILs) for use in treating a cancer, wherein the population of TILs is a second population of TILs and is obtainable by a process comprising:
      • (a) performing a rapid expansion of a first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the first population of TILs are/have been obtained from a tumor resected from a patient, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, wherein the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, and wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, wherein the myeloid aAPCs comprise EM-3 cells transduced with one or more viral vectors, and wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:
      • (a) performing a rapid expansion of a first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the first population of TILs are/have been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, wherein the myeloid aAPCs comprise EM-3 cells transduced with one or more viral vectors, and wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the rapid expansion is performed over a period not greater than 14 days.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a second population and is obtainable by a method comprising the steps of:
      • (a) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion, wherein the myeloid aAPCs endogenously express HLA-AB/C, ICOS-L and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the rapid expansion is performed over a period not greater than 14 days.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:
      • (a) performing a rapid expansion of a first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the first population of TILs are/have been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and wherein the myeloid aAPCs endogenously express HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the expansion is performed using a gas permeable container.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising:
      • (a) performing a rapid expansion of a first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the first population of TILs are/have been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and wherein the myeloid aAPCs endogenously express HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the expansion is performed using a gas permeable container.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the ratio of the second population of TILs to the population of aAPCs is between 1 to 200 and 1 to 400.
  • In an embodiment, the invention provides a population of tumor infiltrating cells (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a process comprising the steps of:
      • (a) performing a rapid expansion of a first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the first population of TILs is/has been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the ratio of the second population of TILs to the population of aAPCs is between 1 to 200 and 1 to 400. In certain embodiments, the ratio of the second population of TILs to the population of aAPCs is about 1 to 300.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and wherein the ratio of the second population of TILs to the population of aAPCs is about 1 to 300.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing a rapid expansion of the first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer;
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the population of TILs being a second population of TILs and obtainable by a method comprising the steps of:
      • (a) performing a rapid expansion of a first population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the first population of TILs is/has been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs after 7 days from the start of the rapid expansion; and
      • wherein the myeloid aAPCs endogenously expresses HLA-AB/C, ICOS-L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein the myeloid aAPCs comprise EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and obtainable by a method comprising the steps of:
      • (a) performing an initial expansion of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first population of TILs is/has been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (b) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain the third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3.
  • In an embodiment, the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. In an embodiment, the myeloid cells comprise MOLM-13 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cells express a CD86 protein and a 4-1BBL protein. In certain embodiments, the myeloid cells comprise EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein. In certain embodiments, the myeloid cells comprise EM-2 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-2 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
      • (d) treating the patient with a non-myeloablative lymphodepletion regimen, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administration of fludarabine at a dose of 25 mg/m2/day for five days;
      • (e) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer; and
      • (f) treating the patient with a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin administered as a 15-minute bolus intravenous infusion every eight hours until tolerance;
      • wherein the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
      • (d) treating the patient with a non-myeloablative lymphodepletion regimen, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administration of fludarabine at a dose of 25 mg/m2/day for five days;
      • (e) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer; and
      • (f) treating the patient with a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin administered as a 15-minute bolus intravenous infusion every eight hours until tolerance;
      • wherein the myeloid aAPCs comprise EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs are a third population of TILs and obtainable by a method comprising the steps of:
      • (a) an initial expansion of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first population of TILs is/has been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; and
      • (b) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain the third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
        and further wherein the population of TILs is for administration to a patient in combination with a non-myeloablative lymphodepletion regimen, wherein the non-myeloablative lymphodepletion regimen comprises cyclophosphamide which is for administration at a dose of 60 mg/m2/day for two days followed by fludarabine which is for administration at a dose of 25 mg/m2/day for five days and further wherein the population of TILs is for administration in combination with a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin for administration as a 15-minute bolus intravenous infusion every eight hours until tolerance. In certain embodiments, the population of TILs is for administration prior to the high-dose IL-2 regimen and subsequent to the non-myeloablative lymphodepletion regimen.
  • In certain embodiments, the myeloid aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. the myeloid aAPCs comprise MOLM-13 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cells express a CD86 protein and a 4-1BBL protein. In certain embodiments, the myeloid aAPCs comprise EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cells express a CD86 protein and a 4-1BBL protein.
  • In an embodiment, the population of TILs is for use in the treating of a cancer selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; and
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein IL-2 is present at an initial concentration of about 3000 IU/mL and OKT-3 antibody is present at an initial concentration of about 30 ng/mL in the second cell culture medium.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:
      • (a) performing an initial expansion of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first population of TILs is/has been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; and
      • (b) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain the third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; wherein IL-2 is present at an initial concentration of about 3000 IU/mL and OKT-3 antibody is present at an initial concentration of about 30 ng/mL in the second cell culture medium.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; and
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein the rapid expansion is performed over a period not greater than 14 days.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:
      • (a) performing an initial expansion of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first population of TILs is/has been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; and
      • (b) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain the third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; wherein the rapid expansion is performed over a period not greater than 14 days.
  • In embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; and
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein the initial expansion is performed using a gas permeable container.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; and
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein the rapid expansion is performed using a gas permeable container.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:
      • (a) performing an initial expansion of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first population of TILs is/has been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (b) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain the third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3; wherein the initial expansion and/or the rapid expansion is performed using a gas-permeable container.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is between 1 to 80 and 1 to 400.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
  • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is about 1 to 300.
  • In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TILs is a third population of TILs and is obtainable by a method comprising the steps:
      • (a) performing an initial expansion of a first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the first population of TILs is/has been obtained from a tumor resected from a patient, and wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (b) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain the third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3, and wherein the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is between 1 to 80 and 1 to 400.
  • In an embodiment, the the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is about 1 to 300.
  • In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) obtaining a first population of TILs from a tumor resected from a patient;
      • (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2;
      • (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 and OKT-3;
      • (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer,
      • wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma.
  • In an embodiment, the invention provides a kit for specifically inducing proliferation of a tumor infiltrating lymphocyte expressing a known co-stimulatory molecule, the kit comprising an effective amount of an aAPC, wherein said aAPC comprises a MOLM-14 cell or a EM-3 cell transduced using a lentiviral vector (LV), wherein the LV comprises a nucleic acid encoding at least one co-stimulatory ligand that specifically binds said known co-stimulatory molecule, wherein binding of the known co-stimulatory molecule with said co-stimulatory ligand stimulates and expands said T cell, the kit further comprising an applicator and an instructional material for the use of said kit.
  • In an embodiment, the invention provides a method for assessing the potency of tumor infiltrating lymphocytes (TILs) comprising the steps of:
      • (a) providing a plurality of mouse mastocytoma P815 cells expressing the endogenous CD16 Fc receptor, wherein the P815 cells are transduced with a lentiviral vector based on enhanced green fluorescent protein (GFP) and Firefly Luciferase;
      • (b) co-culturing the plurality of P815 cells TILs with and without OKT-3 to assess T cell receptor (TCR) activation (specific killing) or lymphokine activated killing (LAK, non-specific killing), respectively;
      • (c) incubating for four hours;
      • (d) adding Luciferin and incubating for 5 minutes;
      • (e) reading bioluminescence intensity using a luminometer; and
      • (f) and calculating percent cytotoxicity and survival.
    BRIEF DESCRIPTION OF THE DRAWINGS
  • The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.
  • FIG. 1 illustrates the results of rapid expansion of TILs using irradiated allogeneic PBMC feeder cells. Each TIL line (M1015T and M1016T) (1.3×105 cells) was co-cultured with 46 different irradiated feeders (1.3×107 cells), IL-2 (3000 IU/mL) and OKT-3 (30 ng/mL) in a T25 flask for 7 days. The fold expansion value for TILs was calculated on Day 7. The figure shows the number of fold expansions for two TIL lines in separate stimulation experiments, with 46 different feeder lots tested, and highlights the variability of expansion results using PBMC feeder cells.
  • FIG. 2 illustrates a vector diagram of the pLV430G human 4-1BBL vector.
  • FIG. 3 illustrates a diagram of the 4-1BBL PCRP (polymerase chain reaction product) portion of the pLV430G human 4-1BBL vector.
  • FIG. 4 illustrates a vector diagram of the pLV430G human CD86 vector.
  • FIG. 5 illustrates a diagram of the CD86 PCRP portion of the pLV430G human CD86 vector.
  • FIG. 6 illustrates a vector diagram of the pDONR221 human CD86 donor vector.
  • FIG. 7 illustrates a vector diagram of the pDONR221 human 4-1BBL donor vector.
  • FIG. 8 illustrates a vector diagram of the pLV430G empty vector.
  • FIG. 9 illustrates a vector diagram of the pDONR221 empty vector.
  • FIG. 10 illustrates a vector diagram of the psPAX2 helper plasmid for lentivirus production.
  • FIG. 11 illustrates a vector diagram of the pCIGO-VSV.G helper plasmid for lentivirus production.
  • FIG. 12 illustrates the results of flow cytometry experiments on MOLM-14 cells before lentiviral transfection (“Untransfected”) and after transfection (“Transfected”), confirming the expression of CD137 and CD86 on engineered MOLM-14 cells.
  • FIG. 13 illustrates the results of rapid expansion of TILs using irradiated parental unmodified MOLM-14 cells (“Parent MOLM14”), engineered MOLM-14 cells (CD86/4-1BBL, “Engineered MOLM14”), or PBMC feeders (“Feeders”) for TIL lot M1032-T2. TIL were co-cultured with PBMC feeders or parental or engineered MOLM14 cells at 1:100 ratios with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted and split on Day 6 and 11. Each dot represents cell numbers determined on Day 0, 6, 11 and 14 respectively. A logarithmic scale is used.
  • FIG. 14 illustrates results as shown in FIG. 13, depicted using a linear scale.
  • FIG. 15 illustrates results for TIL lot M1033-T6 with other parameters as given in FIG. 13, using a logarithmic scale.
  • FIG. 16 illustrates results as shown in FIG. 14, depicted using a linear scale.
  • FIG. 17 illustrates the results of rapid expansions of TILs using engineered MOLM-14 cells expressing CD86 and 4-1BBL (“TIL+Engineered MOLM14 (CD86/41BB)+OKT3”) or irradiated PBMC feeders (“TIL+Feeders+OKT3”). TIL were co-cultured with PBMC feeders or engineered MOLM-14 cells (aMOLM14) at 1:100 ratios plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted and split on Day 6 and 11. Each point represents cell numbers determined on Day 14.
  • FIG. 18 illustrates the results of experiments in which TILs (2×104) were cultured with different ratios (1:10, 1:30, and 1:100, denoted “10”, “30”, and “100”, respectively) of parental MOLM-14 (“MOLM14”) cells, MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), or PBMC feeders (“PBMC+”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in wells of a 24-well G-Rex plate. A control was performed using only OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) (“PBMC-”). Each condition was cultured in triplicate. Cultures were fed with fresh media and IL-2 on Day 4 and 7. Viable cells were counted on Day 7. The bar graph represented here shows the mean plus standard deviation (SD) of viable cell numbers counted on Day 11. The p-value was calculated by the student ‘t’ test.
  • FIG. 19 illustrates the results of TILs cultured with different ratios (1:30, 1:100, and 1:300, denoted “30”, “100”, and “300”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Viable cells were counted on day 11 and plotted. Other conditions are as in FIG. 18.
  • FIG. 20 illustrates the results of TILs cultured with different ratios (1:50, 1:100, and 1:200, denoted “50”, “100”, and “200”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Cells were counted on day 14. Other conditions are as in FIG. 18.
  • FIG. 21 illustrates the results of TILs cultured with different ratios (1:100, 1:200, 1:400, and 1:800, denoted “100”, “200”, “400”, and “800”, respectively) of PBMC feeders (“PBMC”), parental MOLM-14 cells (“MOLM14”), or MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in the single 24 well G-Rex culture plates. Cells were counted on day 14. Other conditions are as in FIG. 18.
  • FIG. 22 illustrates a sunburst visualization showing fine distribution of Live, T cell receptor (TCR) α/β, CD4, CD8, CD27, CD28, and CD57 TILs expanded with PBMC feeders.
  • FIG. 23 illustrates a sunburst visualization showing fine distribution of Live, TCR α/β, CD4, CD8, CD27, CD28, and CD57 TILs expanded with aMOLM14 aAPCs.
  • FIG. 24 depicts a flow cytometry contour plot showing memory subset (CD45RA+/−, CCR7+/−) gated on Live, TCR α/β+, CD4+, or CD8+ TILs.
  • FIG. 25 illustrates phenotypic characterization of the T cell subset, CD4+ and CD8+ post-REP TILs (expanded with aMOLM14 aAPCs) gated on CD3+ cells using a SPADE tree. The color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, PD1, and CD137.
  • FIG. 26 illustrates phenotypic characterization of the T cell subset, CD4+ and CD8+ post-REP TILs (expanded with aMOLM14 aAPCs) gated on CD3+ cells using a SPADE tree. The color gradient is proportional to the MFI CD69, CD154, KLRG1, and TIGIT
  • FIG. 27 illustrates oxygen consumption rate (OCR) of TIL after expansion with Feeders or aMOLM14 measured during a mitochondrial stress test. Each data point represents mean±standard error of the mean (SEM) measured in triplicate.
  • FIG. 28 illustrates extracellular acidification rate (ECAR) of TIL after expansion with Feeders or aMOLM14 measured during a mitochondrial stress test. Each data point represents mean±SEM measured in triplicate.
  • FIG. 29 illustrates a vector diagram of the destination vector pLV4301G.
  • FIG. 30 illustrates a vector diagram of donor vector 1, pMK 7c12 anti mFC scFv CoOp ECORV SacII L1R5.
  • FIG. 31 illustrates a vector diagram of donor vector 2, pMK hCD8a scaffold TN L5 L2.
  • FIG. 32 illustrates a vector diagram of final vector used for lentiviral production, pLV4301G 7C12 scFv mIgG hCD8 flag.
  • FIG. 33 illustrates a vector diagram of the destination vector pLV4301G.
  • FIG. 34 illustrates a vector diagram of donor vector 1, pMK 8B3 anti mFC scFv CoOp ECORV SacII L1R5.
  • FIG. 35 illustrates a vector diagram of donor vector 2, pMK hCD8a scaffold TN L5 L2.
  • FIG. 36 illustrates a vector diagram of final vector used for lentiviral production, pLV4301G 8B3 scFv mIgG hCD8 flag.
  • FIG. 37 illustrates the results of flow cytometry experiments on EM-3 cells before lentiviral transfection (“Untransfected”) and after transfection (“Transfected”), confirming the expression of CD137 and CD86 on engineered EM-3 cells.
  • FIG. 38 illustrates the results of experiments wherein TILs were co-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14.
  • FIG. 39 illustrates the results of experiments wherein TILs were co-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 plus OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14.
  • FIG. 40 illustrates the results of experiments wherein TILs were co-cultured with aEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3000 IU/mL), with or without OKT-3 (30 ng/mL). The bar graph shows cell numbers determined on Day 11.
  • FIG. 41 illustrates the results of TIL expansions with EM-3 aAPCs at different TIL:aAPC ratios.
  • FIG. 42 illustrates the results of TIL expansions with EM-3 aAPCs. TILs (2×104) were co-cultured with five different PBMC feeder lots or aEM3 (in triplicate) at a 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. Viable cells were counted on Day 14. The graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14.
  • FIG. 43 illustrates the results of TIL expansions with EM-3 aAPCs and MOLM-14 aAPCs. TILs (2×104) were co-cultured with five different PBMC feeder lots or aMOLM14 (in triplicate) or aEM3 (also in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. The graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14.
  • FIG. 44 illustrates a sunburst visualization to show fine distribution of Live, TCR α/β, CD4+, and CD8+ TILs expanded with aEM3 aAPCs or PBMC feeders (TIL batch M1054).
  • FIG. 45 illustrates the sunburst visualization to show fine distribution of Live, TCR α/β, CD4+, and CD8+ TILs expanded with aEM3 aAPCs or PBMC feeders (TIL batch M1055).
  • FIG. 46 illustrates the CD4+ and CD8+ SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3+ cells. The color gradient is proportional to the MFI of LAG-3, TIM-3, PD-1, and CD137.
  • FIG. 47 illustrates the CD4+ and CD8+ SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3+ cells. The color gradient is proportional to the MFI of CD69, CD154, KLRG1, and TIGIT.
  • FIG. 48 illustrates a summary of spare respiratory capacity measured by the Seahorse XF Mito stress test.
  • FIG. 49 illustrates a summary of glycolytic reserve measured by the Seahorse XF Mito stress test.
  • FIG. 50 illustrates a mitochondrial stain of live TILs expanded against PBMC or aEM3 using MitoTracker dye, which stains mitochondria in live cells and for which accumulation is dependent upon membrane potential. TILs expanded against PBMC or aEM3 were stained L/D Aqua followed by MitoTracker red dye. Data shown are MitoTracker positive (MFI) cells gated on live population.
  • FIG. 51 illustrates results of a P815 BRLA for cytotoxic potency and functional activity, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs.
  • FIG. 52 illustrates results of a P815 BRLA for cytotoxic potency and functional activity, comparing TILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs.
  • FIG. 53 illustrates IFN-γ release for two batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs. * p<0.05, ** p<0.005, *** p<0.001, ns=not significant.
  • FIG. 54 illustrates IFN-γ release for three batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs. * p<0.05, ** p<0.005, *** p<0.001, ns=not significant.
  • FIG. 55 illustrates Granzyme B release for two batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs. * p<0.05, ** p<0.005, *** p<0.001, ns=not significant.
  • FIG. 56 illustrates Granzyme B release for three batches of TILs following overnight stimulation (“S”) with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated (“US”) TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs. * p<0.05, ** p<0.005, *** p<0.001, ns=not significant.
  • FIG. 57 illustrates a TIL expansion and treatment process. aAPCs of the present invention may be used in both the pre-REP stage (top half of figure) or REP stage (bottom half of figure) and may be added when IL-2 is added to each cell culture. Step 1 refers to the addition of 4 tumor fragments into 10 G-Rex 10 flasks. At step 2, approximately 40×106 TILs or greater are obtained. At step 3, a split occurs into 36 G-Rex 100 flasks for REP. TILs are harvested by centrifugation at step 4. Fresh TIL product is obtained at step 5 after a total process time of approximate 43 days, at which point TILs may be infused into a patient.
  • FIG. 58 illustrates a treatment protocol for use with TILs expanded with aAPCs. Surgery (and tumor resection) occurs at the start, and lymphodepletion chemo refers to non-myeloablative lymphodepletion with chemotherapy as described elsewhere herein.
  • FIG. 59 illustrates Bioluminescent Redirected Lysis Assay (BRLA) results, showing percentage cytotoxicity of TIL batch M1033T-1 when co-cultured with P815 Clone G6 (with and without anti-CD3) at individual effector:target ratios.
  • FIG. 60 illustrates enzyme-linked immunosorbent assay (ELISA) data showing amount of IFN-γ released against different ratios of effector to target cells.
  • FIG. 61 illustrates LAMP1(%) expressed by TIL batch M1033T-1 when co-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of 1:1 effector to target cells for 4 hr and 24 hr co-culture.
  • FIG. 62 illustrates BRLA results for TIL batch M1030. Cytotoxicity (measured as LU50/1×106 TIL) by BRLA is 26±16.
  • FIG. 63 illustrates standard chromium release assay for TIL batch M1030. Cytotoxicity (measured as LU50/1×106 TIL) by the chromium release assay is 22.
  • FIG. 64 illustrates BRLA results for TIL batch M1053, showing the lytic units of the TILs by BRLA as 70±17.
  • FIG. 65 illustrates standard chromium release assay results for TIL batch M1053, also showing lytic unit of the TILs by chromium assay as 14±5. Comparison of this result with FIG. 64 shows the comparable performance of the BRLA and chromium release assay.
  • FIG. 66 illustrates the linear relationship between IFN-γ release and cytotoxic potential of TILs.
  • FIG. 67 illustrates ELISpot results for IFN-γ.
  • FIG. 68 illustrates enzymatic IFN-γ release for TIL batch M1053.
  • FIG. 69 illustrates enzymatic IFN-γ release for TIL batch M1030.
  • FIG. 70 illustrates ELISpot data showing Granzyme B release by M1053T and M1030T. This data confirms the potency of the TILs shown by the BRLA.
  • FIG. 71 illustrates enzymatic Granzyme B release for TIL batch M1053.
  • FIG. 72 illustrates enzymatic Granzyme B release for TIL batch M1030.
  • FIG. 73 illustrates ELISpot data showing TNF-α release by M1053T and M1030T. This data confirms the potency of the TILs shown by the BRLA.
  • FIG. 74 illustrates enzymatic TNF-α release for TIL batch M1053.
  • FIG. 75 illustrates enzymatic TNF-α release for TIL batch M1030.
  • FIG. 76 illustrates changes in cell populations of aEM3 cells (C712 (A) and 8B5 (B)) when weaning such cell populations off of FBS to hAB serum media.
  • FIG. 77 illustrates changes in cell populations of during freeze-thaw-recovery cycles with aEM3 cell populations suspended in various freezing media.
  • FIG. 78 illustrates the growth of aEM3 cells in gas permeable cell culture flasks over an eight-day time course.
  • FIG. 79 illustrates a flow panel analysis to determine the purity of aEM3 cells.
  • FIG. 80 illustrates the results of a flow panel analysis used to determine the purity of aEM3 cells.
  • FIG. 81 illustrates the differences in cytokine expression between aEM3 feeder cells and PBMC feeders stimulated by OKT3.
  • FIG. 82 illustrates that TIL may advantageously expanded (pre-REP) with serum free media (i.e., CTS Optmizer) to provide increased cell numbers as compared to CM1.
  • FIG. 83 and FIG. 84 illustrate that TIL may advantageously expanded with serum free media (i.e., CTS Optmizer) to provide increased cell numbers as compared to CM1 at Day 11 (PreREP) (FIG. 83) and Day 22 (Pre- and Post-REP) (FIG. 84).
  • FIG. 85 illustrates that aAPC cells (i.e., aEM3 cells) can be grown and using serum free media. Specifically, CTS OpTimizer and Prime-TCDM were found to be effective in growing aEM3 as compared to cDMEM (10% hSerum). Data shown were mean±SD of five separate experiments. The p value was calculated by the student t-test. *P<0.05.
  • FIG. 86 and FIG. 87 illustrate the results of two experiments that demonstrate the rapid recovery of aEM3 cells from the TIL-R3 cell line on day 3 following cryopreservation. FIG. 86 illustrates the total cell counts for experiment one and FIG. 87 illustrates the total cell counts for experiment two.
  • FIG. 88 illustrates the growth of aEM3 cells from the TIL-R3 cell line following cryopreservation where the cells were plated and grown for 9 days. Cell counts were measured every three days post thaw.
  • FIG. 89 illustrates the growth of aEM3 cells from the TIL-R3 cell line following cryopreservation where the cells were plated in GREX 10 flasks and grown for 8 days. Cell counts were measured every four days post thaw.
  • FIG. 90 illustrates a vector diagram of the pLenti-C-Myc-DDK human OX40L vector.
  • FIG. 91 illustrates the results of flow cytometry analysis of TILs expanded in a REP with the aEM3 cell line and PBMC feeders, showing that TILs cultured with aEM3 promotes CD8+ TIL skewness.
  • FIG. 92 illustrates the numbers of viable cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.
  • FIG. 93 illustrates the numbers of CD3+ cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.
  • FIG. 94 illustrates the numbers of CD3− cells obtained from experiments wherein TILs were expanded in a REP with the aEM3 cell line and PBMC feeders.
  • FIG. 95 illustrates the results of telomere length analysis using a qPCR method.
  • FIG. 96 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.
  • FIG. 97 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.
  • FIG. 98 illustrates a schematic diagram of an embodiment of an aAPC of the present invention.
    •  BRIEF DESCRIPTION OF THE SEQUENCE LISTING
  • SEQ ID NO:1 is an amino acid sequence for the heavy chain of muromonab.
  • SEQ ID NO:2 is an amino acid sequence for the light chain of muromonab.
  • SEQ ID NO:3 is an amino acid sequence for recombinant human IL-2.
  • SEQ ID NO:4 is an amino acid sequence for aldesleukin.
  • SEQ ID NO:5 is an amino acid sequence for recombinant human IL-7.
  • SEQ ID NO:6 is an amino acid sequence for recombinant human IL-15.
  • SEQ ID NO:7 is an amino acid sequence for recombinant IL-21.
  • SEQ ID NO:8 is the amino acid sequence of human CD86.
  • SEQ ID NO:9 is the amino acid sequence of human 4-1BBL (CD137L).
  • SEQ ID NO:10 is the amino acid sequence of human OX40L (CD134L).
  • SEQ ID NO:11 is the amino acid sequence of human CD28.
  • SEQ ID NO:12 is the amino acid sequence of human CTLA-4.
  • SEQ ID NO:13 is the amino acid sequence of human 4-1BB (CD137).
  • SEQ ID NO:14 is the amino acid sequence of human OX40 (CD134).
  • SEQ ID NO:15 is a nucleotide sequence for the pLV430G 4-1BBL empty vector.
  • SEQ ID NO:16 is a nucleotide sequence for the 4-1BBL CoOP portion of the pLV430G human 4-1BBL vector.
  • SEQ ID NO:17 is a nucleotide sequence for the 4-1BBL PCRP.
  • SEQ ID NO:18 is a nucleotide sequence for the pLV430G hCD86 empty vector.
  • SEQ ID NO:19 is a nucleotide sequence for the hCD86 CoOP portion of the pLV430G human hCD86 vector.
  • SEQ ID NO:20 is a nucleotide sequence for the hCD86 CoOP B1 B2 PCRP portion of the pLV430G human hCD86 vector.
  • SEQ ID NO:21 is a nucleotide sequence for the pDONR221 hCD86 vector.
  • SEQ ID NO:22 is a nucleotide sequence for the pDONR221 4-1BBL vector.
  • SEQ ID NO:23 is a nucleotide sequence for the pLV430G vector.
  • SEQ ID NO:24 is a nucleotide sequence for the pDONR221 vector.
  • SEQ ID NO:25 is a nucleotide sequence for the psPAX2 helper plasmid for lentiviral production.
  • SEQ ID NO:26 is a nucleotide sequence for the pCIGO-VSV.G helper plasmid for lentiviral production.
  • SEQ ID NO:27 is the amino acid sequence of the mFc-7C12 scFv clone.
  • SEQ ID NO:28 is the amino acid sequence of the mFc-8B3 scFv clone.
  • SEQ ID NO:29 is a nucleotide sequence for the mFC-7C12 scFv.
  • SEQ ID NO:30 is a nucleotide sequence for the mFC-8B3 scFv.
  • SEQ ID NO:31 is a nucleotide sequence for the destination vector pLV4301G.
  • SEQ ID NO:32 is a nucleotide sequence for the donor vector 1, pMK 7c12 anti mFC scFv CoOp ECORV SacII L1R5.
  • SEQ ID NO:33 is a nucleotide sequence for the donor vector 2, pMK hCD8a scaffold TN L5 L2.
  • SEQ ID NO:34 is a nucleotide sequence for the final vector used for lentiviral production, pLV4301G 7C12 scFv mIgG hCD8 flag.
  • SEQ ID NO:35 is a nucleotide sequence for the destination vector, pLV4301G.
  • SEQ ID NO:36 is a nucleotide sequence for the donor vector 1, pMK 8B3 anti mFC scFv CoOp ECORV SacII L1R5.
  • SEQ ID NO:37 is a nucleotide sequence for the donor vector 2, pMK hCD8a scaffold TN L5 L2.
  • SEQ ID NO:38 is a nucleotide sequence for the final vector used for lentiviral production, pLV4301G 8B3 scFv mIgG hCD8 flag.
  • SEQ ID NO:39 is a nucleotide sequence for pLenti-C-Myc-DDK OX40L vector for lentiviral production.
  • SEQ ID NO:40 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.
  • SEQ ID NO:41 is a nucleotide sequence for Tel-2b primer used for quantitative polymerase chain reaction measurements of telomere length.
  • SEQ ID NO:42 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.
  • SEQ ID NO:43 is a nucleotide sequence for Tel-1b primer used for quantitative polymerase chain reaction measurements of telomere length.
    • DETAILED DESCRIPTION OF THE INVENTION
  • Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties.
    • Definitions
  • The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients to a human subject so that both active pharmaceutical ingredients and/or their metabolites are present in the human subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present is also encompassed in the methods of the invention.
  • The term “in vivo” refers to an event that takes place in a subject's body.
  • The term “in vitro” refers to an event that takes places outside of a subject's body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.
  • The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject's body. Aptly, the cell, tissue and/or organ may be returned to the subject's body in a method of surgery or treatment.
  • The term “antigen” refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by major histocompatibility complex (MEW) molecules. The term “antigen”, as used herein, also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some embodiments, an antigen is capable of inducing a humoral immune response or a cellular immune response leading to the activation of B lymphocytes and/or T lymphocytes. In some cases, this may require that the antigen contains or is linked to a Th cell epitope. An antigen can also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen will preferably react, typically in a highly specific and selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be induced by other antigens.
  • The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the human subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc. which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells (e.g., the reduction of platelet adhesion and/or cell migration). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.
  • A “therapeutic effect” as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit in a human subject. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.
  • The term “rapid expansion” means an increase in the number of antigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold) over a period of a week, more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a period of a week, or most preferably at least about 100-fold over a period of a week. A number of rapid expansion protocols are described herein.
  • By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as white blood cells that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells and M1 macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to herein as “freshly harvested” or “a first population of TILs”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein, including, but not limited to bulk TILs and expanded TILs (“REP TILs” or “post-REP TILs”, or “second population of TILs” or “third population of TILs” where appropriate).
  • TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and effect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient.
  • By “cryopreserved TILs” herein is meant that TILs are treated and stored in the range of about −150° C. to −60° C. General methods for cryopreservation are also described elsewhere herein, including in the Examples. For clarity, “cryopreserved TILs” are distinguishable from frozen tissue samples which may be used as a source of primary TILs.
  • By “thawed cryopreserved TILs” herein is meant a population of TILs that was previously cryopreserved and then treated to return to room temperature or higher, including but not limited to cell culture temperatures or temperatures wherein TILs may be administered to a patient.
  • By “population of cells” (including TILs) herein is meant a number of cells that share common traits.
  • The term “central memory T cell” refers to a subset of T cells that in the human are CD45R0+ and constitutively express CCR7 (CCR7hi) and CD62L (CD62hi). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secret IL-2 and CD40L as effector molecules after TCR triggering. Central memory T cells are predominant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and tonsils.
  • The term “effector memory T cell” refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but have lost the constitutive expression of CCR7 (CCR7lo) and are heterogeneous or low for CD62L expression (CD62Llo). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-γ, IL-4, and IL-5. Effector memory T cells are predominant in the CD8 compartment in blood, and in the human are proportionally enriched in the lung, liver, and gut. CD8+ effector memory T cells carry large amounts of perforin.
  • The terms “sequence identity,” “percent identity,” and “sequence percent identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Suitable programs to determine percent sequence identity include for example the BLAST suite of programs available from the U.S. Government's National Center for Biotechnology Information BLAST web site. Comparisons between two sequences can be carried using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.
  • The term “conservative amino acid substitutions” means amino acid sequence modifications which do not abrogate the binding of an antibody to an antigen or a protein to its ligand. Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution. Thus, a predicted nonessential amino acid residue in a 4-1BBL or CD86 protein is preferably replaced with another amino acid residue from the same class. Methods of identifying amino acid conservative substitutions which do not eliminate antigen or ligand binding are well-known in the art (see, e.g., Brummell, et al., Biochemistry 1993, 32, 1180-1187; Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999); and Burks, et al., Proc. Natl. Acad. Sci. USA 1997, 94, 412-417).
  • The term “retrovirus” refers to RNA viruses that utilize reverse transcriptase during their replication cycle, wherein retroviral genomic RNA is converted into double-stranded DNA by reverse transcriptase. The double-stranded DNA form is integrated into the chromosome of the infected cell (a “provirus”). The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles. At each end of the provirus are structures called “long terminal repeats” or “LTRs.” The LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. Several genera included within the family Retroviridae, including Cisternavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lentivirus, Gammaretrovirus, and Spumavirus. Some of the retroviruses are oncogenic (i.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in susceptible species. Retroviruses infect a wide variety of species, and may be transmitted both horizontally and vertically. Because they are integrated into the host DNA, they are capable of transmitting sequences of host DNA from cell to cell. Example gammaretroviral vectors include those derived from the amphotropic Moloney murine leukemia virus (MLV-A), which use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes. The amphotropic MLV vector system has been well established and is a popular tool for gene delivery (See, e.g., Gordon and Anderson, Curr. Op. Biotechnol., 1994, 5, 611-616 and Miller, et al., Meth. Enzymol., 1993, 217, 581-599, the disclosures of which are incorporated herein by reference.
  • The term “lentivirus” refers to a genus that includes HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritis-encephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (Hy), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates. Diseases caused by these viruses are characterized by a long incubation period and protracted course. Usually, the viruses latently infect monocytes and macrophages, from which they spread to other cells. HIV, FIV, and SIV also readily infect T lymphocytes (i.e., T cells).
  • The term “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.
  • The term “OKT-3” (also referred to herein as “OKT3”) refers to a monoclonal antibody or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2). A hybridoma capable of producing OKT-3 is deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. A hybridoma capable of producing OKT-3 is also deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706.
  • TABLE 1
    Amino acid sequences of muromonab.
    Identifier
    (Description) Sequence (One-Letter Amino Acid Symbols)
    SEQ ID NO: 1 QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY 60
    (Muromonab heavy NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120
    chain) KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL 180
    YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG 240
    PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300
    STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360
    LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420
    QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450
    SEQ ID NO: 2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH 60
    (Muromonab light FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120
    chain) SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180
    TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 213
  • The term “IL-2” (also referred to herein as “IL2”) refers to the T cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated by reference herein. The amino acid sequence of recombinant human IL-2 suitable for use in the invention is given in Table 2 (SEQ ID NO:3). For example, the term IL-2 encompasses human, recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, available commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant IL-2 commercially supplied by CellGenix, Inc., Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a nonglycosylated human recombinant form of IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the invention is given in Table 2 (SEQ ID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated IL2 prodrug NKTR-214, available from Nektar Therapeutics, South San Francisco, Calif., USA. NKTR-214 and pegylated IL-2 suitable for use in the invention is described in U.S. Patent Application Publication No. US 2014/0328791 A1 and International Patent Application Publication No. WO 2012/065086 A1, the disclosures of which are incorporated by reference herein. Alternative forms of conjugated IL-2 suitable for use in the invention are described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated by reference herein. Formulations of IL-2 suitable for use in the invention are described in U.S. Pat. No. 6,706,289, the disclosure of which is incorporated by reference herein.
  • The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate the development of T cells. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and common gamma chain receptor, which in a series of signals important for T cell development within the thymus and survival within the periphery. Recombinant human IL-7 suitable for use in the invention is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-7 recombinant protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID NO:5).
  • The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated by reference herein. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the invention is given in Table 2 (SEQ ID NO:6).
  • The term “IL-21” (also referred to herein as “IL21”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated by reference herein. IL-21 is primarily produced by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular mass of 15.4 kDa. Recombinant human IL-21 is commercially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein, Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the invention is given in Table 2 (SEQ ID NO:7).
  • TABLE 2
    Amino acid sequences of interleukins.
    Identifier
    (Description) Sequence (One-Letter Amino Acid Symbols)
    SEQ ID NO: 3 MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL 60
    (recombinant EEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120
    human IL-2 RWITFCQSII STLT 134
    (rhIL-2))
    SEQ ID NO: 4 PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE 60
    (aldesleukin) ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120
    ITFSQSIIST LT 132
    SEQ ID NO: 5 MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA 60
    (recombinant ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL 120
    human IL-7 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153
    (rhIL-7))
    SEQ ID NO: 6 MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI 60
    (recombinant HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS 115
    human IL-15
    (rhIL-15))
    SEQ ID NO: 7 MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG 60
    (recombinant NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120
    human IL-21 HLSSRTHGSE DS 132
    (rhIL-21))
  • The term “myeloid cell” as used herein refers to cells of the myeloid lineage or derived therefrom. The myeloid lineage includes a number of morphologically, phenotypically, and functionally distinct cell types including different subsets of granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, erythrocytes, megakaryocytes, and mast cells. In certain embodiments, the cell is a cell derived from a cell line of myeloid lineage.
  • “MOLM-14” refers to a human leukemia cell line which was established from the peripheral blood of a patient with relapsed acute monocytic leukemia, and initial phenotypic characterization indicated the presence of at least the following markers: CD4, CD9, CD11a, CD13, CD14, CD15, CD32, CD33, CD64, CD65, CD87, CD92, CD93, CD116, CD118, and CD155. Matsuo, et al., Leukemia 1997, 11, 1469-77. Additional phenotypic characterization of MOLM-14 found higher levels of HLA-AB/C, CD64, CD80, ICOS-L, CD58, and lower levels of CD86. The MOLM-14 cell line is deposited at DSMZ under Accession No. ACC777. The closely related MOLM-13 cell line is deposited at DSMZ under Accession No. ACC554. As used herein the term “MOLM-14 cell” refers to a MOLM-14 cell and/or a cell derived from the deposited MOLM-14 parental cell line. As used herein the term “MOLM-13 cell” refers to a MOLM-13 cell and/or a cell derived from the deposited MOLM-13 parental cell line.
  • “EM-3” refers to a human cell line was established from the bone marrow of a patient with Philadelphia chromosome-positive CML. Konopka, et al., Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. Phenotypic characterization for EM-3 cells indicates the presence of at least the following markers: CD13, CD15, and CD33. The EM-3 cell line is deposited at DSMZ under Accession No. ACC134 whilst the closely related EM-2 cell line is deposited at DSMZ under Accession No. ACC135. As used herein the term “EM-3 cell” refers to a EM-3 cell and/or a cell derived from the deposited EM-3 parental cell line.
  • As used herein, the term “a CD86 protein” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:8 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:8, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • As used herein, the term “4-1BBL” or “CD137L” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:9 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:9, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • As used herein, the term “OX40L” or “CD137L” may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NO:10 or a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO:10, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • The term “biosimilar” means a biological product, including a monoclonal antibody or fusion protein, that is highly similar to a U.S. licensed reference biological product notwithstanding minor differences in clinically inactive components, and for which there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. Furthermore, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological source, such as a bacterium or yeast. They can consist of relatively small molecules such as human insulin or erythropoietin, or complex molecules such as monoclonal antibodies. For example, if the reference IL-2 protein is aldesleukin (PROLEUKIN), a protein approved by drug regulatory authorities with reference to aldesleukin is a “biosimilar to” aldesleukin or is a “biosimilar thereof” of aldesleukin. In Europe, a similar biological or “biosimilar” medicine is a biological medicine that is similar to another biological medicine that has already been authorized for use by the European Medicines Agency (EMA). The relevant legal basis for similar biological applications in Europe is Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC, as amended and therefore in Europe, the biosimilar may be authorized, approved for authorization or subject of an application for authorization under Article 6 of Regulation (EC) No 726/2004 and Article 10(4) of Directive 2001/83/EC. The already authorized original biological medicinal product may be referred to as a “reference medicinal product” in Europe. Some of the requirements for a product to be considered a biosimilar are outlined in the CHIMP Guideline on Similar Biological Medicinal Products. In addition, product specific guidelines, including guidelines relating to monoclonal antibody biosimilars, are provided on a product-by-product basis by the EMA and published on its website. A biosimilar as described herein may be similar to the reference medicinal product by way of quality characteristics, biological activity, mechanism of action, safety profiles and/or efficacy. In addition, the biosimilar may be used or be intended for use to treat the same conditions as the reference medicinal product. Thus, a biosimilar as described herein may be deemed to have similar or highly similar quality characteristics to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar biological activity to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have a similar or highly similar safety profile to a reference medicinal product. Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly similar efficacy to a reference medicinal product. As described herein, a biosimilar in Europe is compared to a reference medicinal product which has been authorized by the EMA. However, in some instances, the biosimilar may be compared to a biological medicinal product which has been authorized outside the European Economic Area (a non-EEA authorized “comparator”) in certain studies. Such studies include for example certain clinical and in vivo non-clinical studies. As used herein, the term “biosimilar” also relates to a biological medicinal product which has been or may be compared to a non-EEA authorized comparator. Certain biosimilars are proteins such as antibodies, antibody fragments (for example, antigen binding portions) and fusion proteins. A protein biosimilar may have an amino acid sequence that has minor modifications in the amino acid structure (including for example deletions, additions, and/or substitutions of amino acids) which do not significantly affect the function of the polypeptide. The biosimilar may comprise an amino acid sequence having a sequence identity of 97% or greater to the amino acid sequence of its reference medicinal product, e.g., 97%, 98%, 99% or 100%. The biosimilar may comprise one or more post-translational modifications, for example, although not limited to, glycosylation, oxidation, deamidation, and/or truncation which is/are different to the post-translational modifications of the reference medicinal product, provided that the differences do not result in a change in safety and/or efficacy of the medicinal product. The biosimilar may have an identical or different glycosylation pattern to the reference medicinal product. Particularly, although not exclusively, the biosimilar may have a different glycosylation pattern if the differences address or are intended to address safety concerns associated with the reference medicinal product. Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, pharmaceutical form, formulation, excipients and/or presentation, providing safety and efficacy of the medicinal product is not compromised. The biosimilar may comprise differences in for example pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles as compared to the reference medicinal product but is still deemed sufficiently similar to the reference medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the reference medicinal product, wherein the different binding characteristics are considered by a Regulatory Authority such as the EMA not to be a barrier for authorization as a similar biological product. The term “biosimilar” is also used synonymously by other national and regional regulatory agencies.
  • As used herein, the term “variant” encompasses but is not limited to proteins, antibodies or fusion proteins which comprise an amino acid sequence which differs from the amino acid sequence of a reference protein or antibody by way of one or more substitutions, deletions and/or additions at certain positions within or adjacent to the amino acid sequence of the reference protein or antibody. The variant may comprise one or more conservative substitutions in its amino acid sequence as compared to the amino acid sequence of a reference protein or antibody. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference protein or antibody. The term “variant” also includes pegylated antibodies or proteins.
  • “Pegylation” refers to a modified antibody, or a fragment thereof, or protein that typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody, antibody fragment, or protein. Pegylation may, for example, increase the biological (e.g., serum) half life of the antibody or protein. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. The antibody or protein to be pegylated may be an aglycosylated antibody. Methods for pegylation are known in the art and can be applied to the antibodies and proteins described herein, as described for example in European Patent Nos. EP 0154316 and EP 0401384.
  • The terms “about” and “approximately” mean within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, more preferably still within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the terms “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. Moreover, as used herein, the terms “about” and “approximately” mean that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.
  • The transitional terms “comprising,” “consisting essentially of,” and “consisting of,” when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of” excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of” limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. All compositions, methods, and kits described herein that embody the present invention can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”
    • Artificial Antigen Presenting Cells
  • In an embodiment, the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses HLA-AB/C, CD64, CD80, ICOS-L, and CD58, and is modified to express one or more costimulatory molecules. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell that is modified to express one or more costimulatory molecules. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell that is modified to express one or more costimulatory molecules.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell that endogenously expresses HLA-AB/C, CD64, CD80, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cell expresses CD86 and 4-1BBL. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-13 cell expresses CD86 and 4-1BBL. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the MOLM-14 cell expresses CD86 and OX40L. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the MOLM-13 cell expresses CD86 and OX40L. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, and conservative amino acid substitutions thereof, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-13 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the MOLM-14 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In any of the foregoing embodiments, it will be understood that an aAPC comprising a MOLM-14 or MOLM-13 cell may be modified to express both OX40L and 4-1BBL.
  • The sequences for human CD86, human 4-1BBL (CD137L), and human OX40L (CD134L) are given in Table 3.
  • TABLE 3
    Amino acid sequences for human CD86, human 4-1BBL, and human OX40L.
    Identifier
    (Description) Sequence (One-Letter Amino Acid Symbols)
    SEQ ID NO: 8 MGLSNILFVM AFLLSGAAPL KIQAYFNETA DLPCQFANSQ NQSLSELVVF WQDQENLVLN 60
    (human CD86) EVYLGKEKFD SVHSKYMGRT SFDSDSWTLR LHNLQIKDKG LYQCIIHHKK PTGMIRIHQM 120
    NSELSVLANF SQPEIVPISN ITENVYINLT CSSIHGYPEP KKMSVLLRTK NSTIEYDGIM 180
    QKSQDNVTEL YDVSISLSVS FPDVTSNMTI FCILETDKTR LLSSPFSIEL EDPQPPPDHI 240
    PWITAVLPTV IICVMVFCLI LWKWKKKKRP RNSYKCGTNT MEREESEQTK KREKIHIPER 300
    SDEAQRVFKS SKTSSCDKSD TCF 323
    SEQ ID NO: 9 MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA 60
    (human 4-1BBL, SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL 120
    CD137) TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA 180
    LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV 240
    TPEIPAGLPS PRSE 254
    SEQ ID NO: 10 MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSAL QVSHRYPRIQ 60
    (human OX40L, SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS QEVNISLHYQ 120
    CD134L) KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL ILIHQNPGEF 180
    CVL 183
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising a MOLM-13 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • The sequences for the ligands to which human CD86 binds (CD28 and CTLA-4), the ligand to which human 4-1BBL binds (4-1BB), and the ligand to which human OX40L binds (OX40) are given in Table 4.
  • TABLE 4
    Amino acid sequences for human CD28, human CTLA-4, human 4-1BB, and human OX40.
    Identifier
    (Description) Sequence (One-Letter Amino Acid Symbols)
    SEQ ID NO: 11 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD 60
    (human CD28) SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 120
    PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 160
    SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS 220
    SEQ ID NO: 12 MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQPAVVLASS RGIASFVCEY 60
    (human CTLA-4) ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR 120
    AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL 180
    LTAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN 223
    SEQ ID NO: 13 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 60
    (human 4-1BB) TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 120
    CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 180
    PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 240
    CSCRFPEEEE GGCEL 255
    SEQ ID NO: 14 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 60
    (human OX40) NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 120
    PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 180
    GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 240
    RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI 277
  • In an embodiment, the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses HLA-AB/C, ICOS-L, and CD58, and is modified to express one or more costimulatory molecules, wherein the aAPC is derived from an EM-3 parental cell line. In an embodiment, the invention includes an aAPC comprising an EM-3 cell that is modified to express one or more costimulatory molecules. In an embodiment, the invention includes an aAPC comprising an EM-2 cell that is modified to express one or more costimulatory molecules.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell that expresses HLA-AB/C, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the EM-3 cell expresses CD86 and 4-1BBL. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:13, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:13 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 96. In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 97. In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 98.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell that expresses HLA-AB/C, ICOS-L, and CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding OX40L, and wherein the EM-3 cell expresses CD86 and OX40L. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC comprising a EM-2 cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a OX40L protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:10, wherein the CD86 protein and the OX40L protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:14, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:14 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO:12. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an EM-2 cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
  • In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 96. In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 97. In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG. 98.
  • In any of the foregoing embodiments, it is understood that an aAPC comprising an EM-3 or EM-2 cell may be modified to express both OX40L and 4-1BBL.
  • In an embodiment, the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses CD58, and is modified to express one or more costimulatory molecules, wherein the aAPC is derived from a K562-lineage parental cell line. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell that is modified to express one or more costimulatory molecules. In an embodiment, the K562 lineage parental cell line is deposited under accession no. ATCC CCL-243 and also at European Collection of Authenticated. Cell Cultures (ECACCECACC 89121407).
  • In an embodiment, the invention includes an aAPC comprising a K562-lineage cell that expresses CD58, wherein the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell.
  • In an embodiment, the invention includes an aAPC comprising a K562-lineage cell transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the K562-lineage cell expresses CD86 and 4-1BBL. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-1BBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO:11, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth protein comprising an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13, and conservative amino acid substitutions thereof. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage modified to express a first protein that binds to a second protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes an aAPC comprising a K562-lineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:11 and a third protein that binds to a fourth protein comprising a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:12 or SEQ ID NO:13. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
  • In an embodiment, the invention includes an aAPC comprising an K562-lineage cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described herein, to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
    • Methods of Preparing Artificial Antigen Presenting Cells
  • In an embodiment, a method of preparing an aAPC includes the step of stable incorporation of genes for production of CD86 and 4-1BBL. In an embodiment, a method of preparing an aAPC includes the step of retroviral transduction. In an embodiment, a method of preparing an aAPC includes the step of lentiviral transduction. Lentiviral transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71, and U.S. Pat. No. 6,627,442, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of gamma-retroviral transduction. Gamma-retroviral transduction systems are known in the art and are described, e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, a transposase provided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail). Suitable transposon-mediated gene transfer systems, including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100×, and engineered enzymes with increased enzymatic activity, are described in, e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Pat. No. 6,489,458, the disclosures of each of which are incorporated by reference herein.
  • Examples of viruses modified and applied to such techniques include adenoviruses, adeno-associated viruses, herpes simplex viruses, and retroviruses. Generally, nucleic acid molecules of interest may be cloned into a viral genome. Upon replication and packaging of the viral genome, the resultant viral particle is capable of delivering the nucleic acid of interest into a cell via the viral entry mechanism.
  • Of particular interest is the use of modified retroviruses to introduce genetic material into cells to treat genetic defects and other diseases.
  • The present invention provides highly efficient methods, and compositions related thereto, for the stable transduction of cells with viral vectors and viral particles. By “stable transduction,” it is meant where an integrated form of the viral vector has been inserted into the chromosomal DNA of the transduced cell. The methods comprise exposing the cells to be transduced to contact with at least one molecule that binds the cell surface. This contacting step may occur prior to, during, or after the cells are exposed to the viral vector or viral particle. Hereinafter, the term “viral vector” will be used to denote any form of a nucleic acid derived from a virus and used to transfer genetic material into a cell via transduction. The term encompasses viral vector nucleic acids, such as DNA and RNA, encapsidated forms of these nucleic acids, and viral particles in which the viral vector nucleic acids have been packaged.
  • Additional examples of cell surface binding molecules include polypeptides, nucleic acids, carbohydrates, lipids, and ions, all optionally complexed with other substances. Preferably, the molecules bind factors found on the surfaces of blood cells, such as CD1a, CD1b, CD1c, CD1d, CD2, CD3γ, CD3δ, CD3∈, CD4, CD5, CD6, CD7, CD8α, CD8β, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a, CD16b, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CDw70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CD79α, CD79β, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CDw109, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CDw124, CD125, CD126, CDw127, CDw128a, CDw128b, CDw130, CDw131, CD 132, CD133, CD134, CD135, CD 136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166, and TCRξ. Small letters (e.g. “a” or “b”) indicate complex CD molecules composed of multiple gene products or belonging to families of structurally related proteins. The notation “w” refers to putative CD molecules that have not yet been fully confirmed. A more complete listing of CD molecules is found in Kishimoto, T. (ed). Current information on CD molecules is also found in Shaw, S. (ed)., Protein Reviews on the Web: An International WWW Resource/Journal at http://www.bsi.vt.edu/immunology.
  • More preferred are molecules that bind factors found on the Surfaces of lymphocytes, T cells and leukocytes, Such as CD2, CD3γ, CD3δ, CD3∈, CD5, CD6, CD7, CD8α, CD8β, CD9, CD11a, CD18, CD25, CD26, CD27, CD28, CD29, CD30, CD37, CD38, CD39, CD43, CD44, CD45R, CD46, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD53, CD54, CD56, CD57, CD58, CD59, CDw60, CD62L, CD68, CD69, CDw70, CD71, CD73, CDw75, CDw76, CD84, CD85, CD86, CD87, CD89, CD90, CD94, CD96, CD97, CD98, CD99, CD100, CD101, CD103, CD107a, CD107b, CDw108, CDw109, CD118, CD119, CD120b, CD121a, CD122, CDw124, CDw127, CDw128a, CDw130, CD132, CD134, CDw137, CD140a, CD140b, CD143, CD146, CD148, CD152, CD153, CD154, CD155, CD161, CD162, CD165, CD166, and TCRξ.
  • Of course any cell can be used in the practice of the invention. Preferably, the cell to be transduced is a eukaryotic cell. More preferably, the cell is a primary cell. Cell lines, however, may also be transduced with the methods of the invention and, in many cases, more easily transduced. In one preferred embodiment, the cell to be transduced is a primary lymphocyte (such as a T lymphocyte) or a macrophage (such as a monocytic macrophage), or is a precursor to either of these cells, such as a hematopoietic stem cell. Other preferred cells for transduction in general are cells of the hematopoietic system, or, more generally, cells formed by hematopoiesis as well as the stem cells from which they form and cells associated with blood cell function. Such cells include granulocytes and lymphocytes formed by hematopoiesis as well as the progenitor pluripotent, lymphoid, and myeloid stem cells. Cells associated with blood cell function include cells that aid in the functioning of immune system cells, such as antigen presenting cells like dendritic cells, endothelial cells, monocytes, and Langerhans cells. In a preferred embodiment, the cells are T lymphocytes (or T cells), such as those expressing CD4 and CD8 markers.
  • In particularly preferred embodiments, the cell is a primary CD4+T lymphocyte or a primary CD34+ hematopoietic stem cell. However, and given that the viral vectors for use in the invention may be pseudotyped with Vesicular Stomatitis Virus envelope G protein (as discussed below), any cell can be transduced via the methods of the present invention.
  • Preferably, the cell is of a eukaryotic, multicellular species (e.g., as opposed to a unicellular yeast cell), and, even more preferably, is of mammalian origin, e.g., a human cell.
  • Such a “larger collection of cells” can comprise, for instance, a cell culture (either mixed or pure).
  • Additional applications of the invention in cancer therapy are numerous, and one skilled in the art would be able to use the invention set out herein for the treatment of many types of cancers without undue experimentation.
  • Furthermore, in Vivo uses are not restricted to disease states and can be used to transduce normal cells. For example, the invention may be used to transduce hematopoietic stem cells in vivo in the bone marrow. Any combination of antibodies or other cell surface binding molecules, such as FLT-3 ligand, TPO and Kit ligand, or functional analogs thereof, or stromal cells expressing the cell surface binding molecule, could be added with vector upon direct injection into the bone marrow for high efficiency bone marrow transduction.
  • Transduction of mainly a cell type of interest can be accomplished by the choice of cell surface moiety to be bound. Thus in a mixed population of blood cells, for example, transduction of cells expressing CD3, Such as certain T cells, will be enhanced when CD3 specific anti bodies are used to interact with the cells. This will occur in preference over other cell types in the population, such as granulocytes and monocytes that do not express CD3.
  • The invention also encompasses the transduction of purified or isolated cell types if desired. The use of a purified or isolated cell type provides additional advantages Such as higher efficiencies of transduction due to higher vector concentrations relative to the cell to be transduced.
  • The present invention includes viral vectors, and compositions comprising them, for use in the disclosed methods. The vectors are preferably retroviral (family Retroviridae) vectors, and more preferably lentiviral vectors. Other retro viral vectors, such as oncoviral and murine retroviral vectors, may also be used. Additional vectors may be derived from other DNA viruses or viruses that can convert their genomes into DNA during some point of their lifecycle. Preferably the viruses are from the families Adenoviridae, Parvoviridae Hepandaviridae (including the hepatitis delta virus and the hepatitis E virus which is not normally classified in the Hepandaviridae), Papoviridae (including the polyomavirinae and the papillomavirinae), Herpesviridae, and Poxviridae.
  • Additional viruses of the family Retroviridae (i.e., a retrovirus), are of the genus or subfamily Oncovirinae, Spumavirinae, Spumavirus, Lentivirinae, and Lentivirus. An RNA virus of the subfamily Oncovirinae is desirably a human T-lymphotropic virus type 1 or 2 (i.e., HTLV-1 or HTLV-2) or bovine leukemia virus (BLV), an avian leukosissarcoma virus (e.g., Rous Sarcoma virus (RSV), avian myeloblastosis virus (AMV), avian erythroblastosis virus (AEV), and Rous-associated virus (RAV; RAV-0 to RAV-50), a mammalian C-type virus (e.g., Moloney murine leukemia virus (Mul V), Harvey murine sarcoma virus (HaMSV), Abelson murine leukemia virus (A-MuLV), AKR-Mul V, feline leukemia virus (FeLV), simian sarcoma virus, reticuloendotheliosis virus (REV), Spleen necrosis virus (SNV)), a B-type virus (e.g., mouse mammary tumor virus (MMTV)), and a D-type virus (e.g., Mason-Pfizer monkey virus (MPMV) and “SAIDS” viruses).
  • An RNA virus of the subfamily Lentivirus is desirably a human immunodeficiency virus type 1 or 2 (i.e., HIV-1 or HIV-2, wherein HIV-1 was formerly called lymphadenopathy associated virus 3 (HTLV-III) and acquired immune deficiency syndrome (AIDS)-related virus (ARV)), or another virus related to HIV-1 or HIV-2 that has been identified and associated with AIDS or AIDS-like disease. The acronym “HIV” or terms “AIDS virus” or “human Immunodeficiency virus” are used herein to refer to these HIV viruses, and HIV-related and -associated viruses, generically. Moreover, a RNA virus of the subfamily Lentivirus preferably is a Visna/maedi virus (e.g., such as infect sheep), a feline immunodeficiency virus (FIV), bovine lentivirus, simian immunodeficiency virus (SIV), an equine infectious anemia virus (EIAV), and a caprine arthritisencephalitis virus (CAEV).
  • A particularly preferred lentiviral vector is one derived from HIV, most preferably HI-1, HIV-2, or chimeric combinations thereof. Of course different serotypes of retroviruses, especially HIV, may be used singly or in any combination to prepare vectors for use in the present invention. Preferred vectors of the invention contains cis acting elements that are present in the wild-type virus, but not present in a “basic” lentiviral vector. A “basic” lentiviral vector contains minimally, LTRS and packaging sequences in the 5′ leader and gag encoding Sequences, but can also optionally contain the RRE element to facilitate nuclear export of vector RNA in a Rev dependent manner. A preferred vector additionally contains nucleotide aequences that enhance the efficiency of transduction into cells.
  • An example of such a vector is pN2cGFP, a vector that contains the complete sequences of gag and pol. Another example is a vector that contain sequences from about position 4551 to position 5096 in pol (reference positions from the pNL4-3 sequence, Accession number M19921, HIVNL43 9709 bp, kindly provided by C. E. Buckler, NIAID, NIH, Bethesda, Md.). However any cis-acting sequence from the wt-HIV that can improve vector transduction efficiency may be used. Other examples of vectors capable of efficient transduction via the present invention are cr2HIV constructs as described in U.S. Pat. No. 5,885,806.
  • Additional examples of Viral vector constructs that may be used in the present invention are found in U.S. Pat. No. 5,885,806, which is hereby incorporated by reference as if fully set forth. The constructs in U.S. Pat. No. 5,885,806 are merely examples that do not limit the scope of vectors that efficiently transduce cells. Instead, the constructs provide additional guidance to the skilled artisan that a viral vector for use with the present invention may contain minimal sequences from the wild-type virus or contain sequences up to almost the entire genome of wild-type virus, yet exclude an essential nucleic acid sequence required for replication and/or production of disease. Methods for determining precisely the sequences required for efficient transduction of cells are routine and well known in the art. For example, a systematic incorporation of viral sequences back into a “basic” vector or deleting sequences from vectors that contain virtually the entire HIV genome, such as cr2HIVs, is routine and well known in the art.
  • Furthermore, placing sequences from other viral back bones into viral vectors of interest, such as the cytomegalovirus (CMV), is also well known in the art. Regardless of the actual viral vector used, various accessory proteins encoded by, and sequences present in, the viral genetic material may be left in the vector or helper genomes if these proteins or sequences increase transduction efficiency in certain cell types. Numerous routine screens are available to determine whether certain genetic material increases transduction efficiency by incorporating the sequence in either the vector or helper genomes. A preferred embodiment of the invention is to not include accessory proteins in either the vector or helper genomes. But this preference does not exclude embodiments of the invention where accessory proteins and other sequences are left in either the vector or a helper genome to increase transduction efficiency.
  • The viral vector for use in the transduction methods of the invention can also comprise and express one or more nucleic acid sequences under the control of a promoter present in the virus or under the control of a heterologous promoter introduced into the vector. The promoters may further contain insulatory elements, such as erythroid DNAse hyper-sensitive sites, so as to flank the operon for tightly controlled gene expression. Preferred promoters include the HIV-LTR, CMV promoter, PGK, Ul, EBER transcriptional units from Epstein Barr Virus, tRNA, U6 and U7. While Pol II promoters are preferred, Pol III promoters may also be used. Tissue specific promoters are also preferred embodiments. For example, the beta globin Locus Control Region enhancer and the alpha & beta globin promoters can provide tissue specific expression in erythrocytes and erythroid cells. Another further preferred embodiment is to use cis-acting sequences that are associated with the promoters. For example, The Ul gene may be used to enhance antisense gene expression where non-promoter sequences are used to target the antisense or ribozymes molecule to a target spliced RNA as set out in U.S. Pat. No. 5,814,500, which is hereby incorporated by reference.
  • Such sequences and gene products are preferably biologically active agents capable of producing a biological effect in a cell.
  • In one preferred embodiment, the agent is a cell surface molecule.
  • In the methods of the invention, the cells to be transduced are exposed to contact with the at least one molecule that binds the cell surface before, after, or simultaneously with application of the viral vector. For example, the cells can be cultured in media with CD3 and CD28 antibodies (coated onto the surface of the culture dish or immobilized on beads present in the culture) before, after, or in the presence of the viral vector to be transduced. Preferably, the cells are exposed to immobilized CD3 and/or CD28 only after or only upon initial contact with the viral vector. Under these conditions, the cells are not exposed to cell surface binding molecule(s) prior to actual transduction with the viral vector. In embodiments where contact with a cell surface binding molecule occurs after exposure of the cells to a viral vector (transduction), the contact preferably occurs within three days of transduction, more preferably within one to two days after transduction.
  • Incubation of the cells with the viral vector may be for different lengths of time, depending on the conditions and materials used. Factors that influence the incubation time include the cell, vector and MOI (multiplicity of infection) used, the molecule(s) and amounts used to bind the cell surface, whether and how said molecule(s) are immobilized or solubilized, and the level of transduction efficiency desired. Preferably, the incubation is for about eight to about 72 hours, more preferably for about 12 to about 48 hours. In a particularly preferred embodiment, the incubation is for about 24 hours and is optionally repeated once.
  • Contact between the cells to be transduced and a viral vector occurs at least once, but it may occur more than once, depending upon the cell type. For example, high efficiency transduction of CD34 positive stem cells have been accomplished with multiple transductions with vector. A preferred method of the invention is to Simultaneously introduce a viral vector in combination with a cell surface binding molecule (e.g. CD3 and/or CD28 antibodies or a FLT-3 ligand, TPO or Kit ligand) and avoid changing the medium for between about one and about eight days after transduction. More preferably, the medium is not changed for three days post transduction. Transduction can proceed for as long as the conditions permit without the process being significantly detrimental to the cells or the organism containing them. Additional examples of cell surface binding proteins for such use include those described hereinabove.
  • Similarly, the MOI used is from about 1 to about 400, preferably less than 500. Generally, the preferred MOI is from about 2 to about 50. More preferably, the MOI is from about 10 to about 30, although ranges of from about 1 to about 10, about 20, about 30, or about 40 are also contemplated. Most preferred is an MOI of about 20. Furthermore, the copy number of viral vector per cell should be at least one. However, many copies of the vector per cell may also be used with the above described methods. The preferred range of copies per cell is from about 1 to about 100. The more preferred copy number is the minimum copy number that provides a therapeutic, prophylactic or biological impact resulting from vector transduction or the most efficient transduction.
  • For therapeutic or prophylactic applications, a more preferred copy number is the maximum copy number that is tolerated by the cell without being significantly detrimental to the cell or the organism containing it. Both the minimum and maximum copy number per cell will vary depending upon the cell to be transduced as well as other cells that may be present. The optimum copy number is readily determined by those skilled in the art using routine methods. For example, cells are transduced at increasing increments of concentration or multiplicities of infection. The cells are then analyzed for copy number, therapeutic or biological impact and for detrimental effects on the transduced cells or a host containing them (e.g. safety and toxicity).
  • After incubation with the viral vector in vitro, the cells may be cultured in the presence of the cell surface binding molecule(s) for various times before the cells are analyzed for the efficiency of transduction or otherwise used. Alternatively, the cells may be cultured under any conditions that result in cell growth and proliferation, Such as incubation with interleukin-2 (IL-2) or incubation with the cell surface binding molecule(s) followed by IL-2.
  • The efficiency of transduction observed with the present invention is from about 75-100%. Preferably, the efficiency is at least about 75 to 90%. More preferred embodiments of the invention are where transduction efficiency is at least about 90 to 100%. Most preferred embodiments have transduction efficiencies of at least 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%. In addition to the above, the transduced cells may be used in research or for treatment or prevention of disease conditions in living subjects.
  • Therapeutic uses for the transduced cells include the introduction of the cells into a living organism. For example, unstimulated primary T cells isolated from an individual infected with, or at risk of being infected with HIV, may be first transduced by a vector, like that described in U.S. Pat. No. 5,885,806, using the present methods and followed by injection of the transduced cells back into the individual.
  • The present invention is directed to methods, and compositions related thereto, for the stable transduction of cells with viral vectors to efficiencies of greater than about 75%. Stably transduced cells may be distinguished from transiently transduced, or pseudotransduced cells, after about seven to ten days, or optionally after about 14 days, post transduction. The methods relate to the fact that contact of the cells to be transduced with at least one molecule that binds the cell surface increases the efficiency of stable transduction.
  • The methods of the invention comprise the step of transduction with a viral vector in combination with contact with a cell surface binding molecule. As noted above, the contact may occur before, after or at the same time as transduction with the vector. The invention is broadly applicable to any cell, and the use of any cell surface binding molecule. Cells for use with the present methods include unstimulated primary cells, which are freshly isolated from an in vivo source as well as cell lines, which may have been previously cultured for various times in the presence of factors which maintain them in a proliferating state.
  • In the case of primary cells, they are first obtained from an in vivo source followed optionally by selection for particular cell types. For example, if primary CD4+ and/or CD8+ T cells are to be used, peripheral blood (PB) or cord blood (“CB” from an umbilical source) samples are first obtained followed by enrichment for CD4+ and/or CD8+ cell types. Standard magnetic beads positive selection, plastic adherence negative selection, and/or other art recognized standard techniques may be used to isolate CD4+ and/or CD8+ cells away from contaminating PB cells. Purity of the isolated cell types may be determined by immunophenotyping and flow cytometry using standard techniques.
  • After isolation, the primary cells may be used in the present methods to be transduced with Viral vectors at efficiencies of greater than 75%. The invention is most advantageously used with primary lymphocytes, Such as T cells, transduced with an HIV-1 based vector capable of expressing heterologous genetic material of interest. Another preferred use is with primary hematopoietic stem cells, such as CD34 positive cells. In cases where the heterologous genetic material is or encodes a therapeutic or prophylactic product for use in vivo to treat or prevent a disease, the transduced primary cell can be introduced back into an in vivo environment, such as a patient. As such, the invention contemplates the use of the transfected cells in gene therapy to treat, or prevent, a disease by combating a genetic defect or targeting a viral infection.
  • For the transduction of primary cells in a mixed population, the above isolation/purification steps would not be used. Instead, the cell to be transduced would be targeted by selection of at least one appropriate cell surface molecule or moiety found on that cell type and the preparation of one or more molecules capable of binding said moiety. The cell surface moiety may be a receptor, marker, or other recognizable epitope on the surface of the targeted cells. Once selected, molecules that interact with the moiety, such as specific antibodies, may be prepared for use in the present invention.
  • For example, CD4+ and/or CD8+ cells can either be first purified and then transduced by the methods of the invention with the use of immobilized CD3 and CD28 antibodies or alternatively be transduced as part of a mixed population, like peripheral blood cells (PBCs) or peripheral blood mononuclear cells (PBMNCs), by use of the same antibodies. Hematopoietic stem cells in total white blood cell populations, which may be difficult to purify or isolate, may be transduced in the mixed populations by use of immobilized CD34 antibodies.
  • The cell surface binding molecules of the invention may target and bind any moiety found on the surface of the cell to be transduced. Preferably, the moieties are found as part of receptors, markers, or other proteinaceous or nonproteinaceous factors on the cell Surface. The moieties include epitopes recognized by the cell surface binding molecule. These epitopes include those comprising a polypeptide sequence, a carbohydrate, a lipid, a nucleic acid, an ion and combinations thereof.
  • Examples of cell surface binding molecules include an antibody or an antigen binding fragment thereof and a ligand or binding domain for a cell surface receptor. The cell surface binding molecule may itself be a polypeptide, a nucleic acid, a carbohydrate, a lipid, or an ion. Preferably, the molecule is an antibody or a fragment thereof, such as a Fab, or F, fragment. More preferably, the molecule is not used in a soluble form but is rather immobilized on a solid medium, such a bead, with which the cells to be transduced may be cultured, or the surface of a tissue culture dish, bag or plate, upon which the cells to be transduced may be cultured. In a preferred embodiment for the transduction of CD4+ or CD8+ cells, monoclonal antibodies that recognize CD3 and/or CD28 may be used in a cell culture bag in the presence of a viral vector.
  • The present invention includes compositions comprising a cell surface binding molecule for use as part of the disclosed methods. An exemplary composition comprises the molecule and a viral vector to be transduced, optionally in the presence of the cells to be transduced. The viral vectors may be derived from any source, but are preferably retroviral vectors. More preferably, they are lentiviral vectors. A particularly preferred lentiviral vector is one derived from a Human Immunodeficiency Virus (HIV), most preferably HIV-1, HIV-2, or chimeric combinations thereof. Of course different viral vectors may be simultaneously transduced into the same cell by use of the present methods. For example, one vector can be a replication deficient or conditionally replicating retroviral vector while a second vector can be a packaging construct that permits the first vector to be replicated/packaged and propagated. When various viral accessory proteins are to be encoded by a viral vector, they may be present in any one of the vectors being transduced into the cell. Alternatively, the viral accessory proteins may be present in the transduction process via their presence in the viral particles used for transduction. Such viral particles may have an effective amount of the accessory proteins co-packaged to result in an increase in transduction efficiency. In a preferred embodiment, the viral vector does not encode one or more of the accessory proteins.
  • A viral vector for use in the transduction methods of the invention can also comprise and express one or more nucleic acid sequences under the control of a promoter. In one embodiment of the invention, a nucleic acid sequence encodes a gene product that, upon expression, would alleviate or correct a genetic deficiency in the cell to be transduced. In another embodiment, the nucleic acid sequence encodes or constitutes a genetic antiviral agent that can prevent or treat viral infection. By “genetic antiviral agent”, it is meant any substance that is encoded or constituted by genetic material. Examples of such agents are provided in U.S. Pat. No. 5,885,806. They include agents that function by inhibiting viral proteins, such as reverse transcriptase or proteases, competing with viral factors for binding or target sites, or targeting viral targets directly for degradation, Such as in the case of ribozymes and antisense constructs. Other examples of genetic antiviral agents include antisense, RNA decoys, transdominant mutants, interferons, toxins, nucleic acids that modulate or modify RNA splicing, immunogens, and ribozymes, such as “hammerhead” and external guide sequence (EGS) mediated forms thereof.
  • The cells to be transduced may be exposed to contact with the viral vector either before, after or simultaneously with contact with the cell surface binding molecule. Thus the cells can be first exposed to the vector for a period of time followed by introduction of the cell surface binding molecule. Such cells may be newly isolated or prepared primary cells that have not been intentionally stimulated to enter the cell cycle. Alternatively, the cells can be first exposed to the cell surface binding molecule for a period of time followed by contact with the viral vector. After contact with the vector, excess vector is preferably not removed and the cells cultured under conditions conducive to cell growth and/or proliferation. Such conditions may be in the presence of the cell surface binding molecule or other stimulatory/activating factors, such as cytokines and lymphokines in the case of T cells. Alternatively, excess vector may be removed after contact with the cell and before further culturing.
  • Another embodiment of the invention is to culture the cells in the presence of both viral vector and cell surface binding molecule simultaneously. Such cells are preferably not previously stimulated. After a period of time, the cells are cultured under growth or proliferation inducing conditions such as the continued presence of the cell surface binding molecule or other stimulatory/activating factors. Alternatively, excess vector may be removed before further culturing.
  • Incubation of the cells to be transduced with the viral vector may be for different lengths of time, depending on the conditions and materials used. Factors that influence the incubation time include the cell, vector and MOI (multiplicity of infection) used, the molecule(s) and amounts used to bind the cell surface, whether and how said molecule(s) are immobilized, and the level of transduction efficiency desired. In a preferred embodiment of the invention, the cells are T lymphocytes, the vector HIV based, the MOI is about 20, the cell Surface binding molecules are CD3 and CD28 antibodies immobilized on beads, and the resultant efficiency at least 93%. As would be evident to the skilled person in the art, some of the above factors are directly correlated while others are inversely correlated. For example, a decrease in the MOI will likely decrease the level of efficiency while efficiency can likely be maintained if an increased amount of cell surface binding molecules is used.
  • The length of incubation viral vector and the cells to be transformed is preferably for 24 hours and optionally repeated once for lymphocytes and up to four times for hematopoietic stem cells. Similarly, and in embodiments where the cells are incubated with the cell surface binding molecule before introduction of the viral vector, the incubation may be for about 12 hours to about 96 hours. Preferably, incubation with a cell surface binding molecule occurs simultaneously with contact of the cells with the viral vector. Under such circumstances, the cell surface binding molecules may be left in contact with the cells when the vector is introduced. Alternatively, excess cell surface binding molecules may be first removed from the culture before introduction of the vector to the cells.
  • After contact with the vector, the cells are cultured under conditions conducive to their growth or proliferation. Preferably, the conditions are continued culturing in the presence of the cell surface binding molecules. Alternatively, the cells are initially cultured with the cell surface binding molecule followed by substitution with media containing another factor conducive to cell growth, such as interleukin-2. Yet another embodiment would be to remove both the excess cell surface binding molecule and the excess vector followed by culturing in the presence of a factor conducive to growth or proliferation as well as enhancing further vector transduction. Such factors include mitogens such as phytohemaglutinin (PHA) and cytokines, growth factors, activators, cell surface receptors, cell surface molecules, soluble factors, or combinations thereof, as well as active fragments of such molecules, alone or in combination with another protein or factor, or combinations thereof.
  • Examples of additional factors include epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), angiotensin, transforming growth factor beta (TGF-beta), GDF, bone morphogenic protein (BMP), fibroblast growth factor (FGF acidic and basic), vascular endothelial growth factor (VEGF), PIGF, human growth hormone (HGH), bovine growth hormone (BGH), heregulins, amphiregulin, Ach receptor inducing activity (ARIA), RANTES (regulated on activation, normal T expressed and secreted), angiogenins, hepatocyte growth factor, tumor necrosis factor beta (TNF-beta), tumor necrosis factor alpha (TNF-alpha), angiopoietins 1 or 2, insulin, insulin growth factors I or II (IGF-I or IGF-2), ephrins, leptins, interleukins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (IL-1, IL-2, IL-3, IL-4, L-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, or IL-15), G-CSF (granulocyte colony stimulating factor), GM-CSF (granulocyte-macrophage colony stimulating factor), M-CSF (macrophage colony stimulating factor), LIF (leukemia inhibitory factor), angiostatin, oncostatin, erythropoietin (EPO), interferon alpha (including subtypes), interferons beta, gamma, and omega, chemokines, macrophage inflammatory protein-I alpha or beta (MIP-1 alpha or beta), monocyte chemotactic protein-1 or -2 (MCP-1 or 2), GRO beta, MWF (macrophage migration inhibitory factor), MGSA (melanoma growth stimulatory activity), alpha inhibin HGF, PD-ECGF, bFGF, lymphotoxin, Mullerian inhibiting substance, FAS ligand, osteogenic protein, pleiotrophin/midkine, ciliary neurotrophic factor, androgen induced growth factor, autocrine motility factor, hedgehog protein, estrogen, progesterone, androgen, glucocorticoid receptor, RAR/RXR, thyroid receptor, TRAP/CD40, EDF (erythroid differentiating factor), Fic (growth factor inducible chemokine), IL-1RA, SDF, NGR or RGD ligand, NGF, thymosine-alphal, OSM, chemokine receptors, Stem cell factor (SCF), or combinations thereof. As evident to one skilled in the art, the choice of culture conditions will depend on knowledge in the art concerning the cells transduced as well as the subsequent intended use of the cells. For example, the combination of IL-3, IL-6 and stem cell factor would not be a choice for transduced cells that are to be used in human transplantation. Similarly, the choice of culture conditions would preferably not be to the detriment of cell viability or transduction efficiency.
  • Preferably, the post transduction incubation is for a period of about four hours, or for about one to about seven to ten days. More preferably from about 16 to about 20 hours or for about four, about five or about six days. About fourteen days of post-transduction incubation is also contemplated.
  • The efficiency of transduction observed with the present invention is from about 75-100%. Preferably, the efficiency is at least about 75 to 90%. More preferred embodiments of the invention are where transduction efficiency is at least about 90 to 95%. The most preferred embodiments have transduction efficiencies of at least 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
  • In addition to the above, the transduced cells may be used in research or for treatment of disease conditions in living subjects. Particularly preferred as part of the invention are therapeutic uses for the transduced cells to produce gene products of interest or for direct introduction into a living organism as part of gene therapy. For example, and as exemplified below, primary T cells can be isolated and transduced with a viral vector.
  • In another embodiment, the T cells are transduced with genes or nucleic acids capable of conditionally killing the T cell upon introduction into a host organism. This has applications in allogenic bone marrow transplantation to prevent graft versus host disease by killing T cells with a pro-drug approach.
  • Alternatively, the primary cells can be deficient in a gene product, and the deficiency correctable by the transduced viral vector. Such cells would be reintroduced into the living subject after transduction with the vector.
  • Thus, both in vitro and ex vivo applications of the invention are contemplated. For transfers into a living subject, the transduced cells are preferably in a biologically acceptable solution or pharmaceutically acceptable formulation. Such a transfer may be made intravenously, intraperitoneally or by other injection and non-injection methods known in the art. The dosages to be administered will vary depending on a variety of factors, but may be readily determined by the skilled practitioner. There are numerous applications of the present invention, with known or well designed payloads in the viral vector, where the benefits conferred by the transduced genetic material will outweigh any risk of negative effects.
  • In an embodiment, a method of preparing an aAPC includes the step of stable incorporation of genes for transient production of CD86 and 4-1BBL. In an embodiment, a method of preparing an aAPC includes the step of electroporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. 1 1991, 60, 297-306, and U.S. Patent Application Publication No. 2014/0227237 A1, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating, and endocytosis) are known in the art and are described in Graham and van der Eb, Virology 1973, 52, 456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and in U.S. Pat. No. 5,593,875, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of liposomal transfection. Liposomal transfection methods, such as methods that employ a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are described in Rose, et al., Biotechniques 1991, 10, 520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and in U.S. Pat. Nos. 5,279,833; 5,908,635; 6,056,938; 6,110,490; 6,534,484; and 7,687,070, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC includes the step of transfection using methods described in U.S. Pat. Nos. 5,766,902; 6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of each of which are incorporated by reference herein.
  • In an embodiment, the aAPC is transduced by first using the Gateway cloning method (commercially available from ThermoFisher, Inc.) to prepare vector for lentiviral transduction, followed by lentiviral transduction using the vector and one or more associated helper plasmids, as is also described elsewhere herein. In the Gateway cloning method, a gene is selected (such as CD86) and is then provided with primers and amplified using PCR technology with the help of an attB tagged primer pair. The PCR fragment is then combined with a donor vector (pDONR, such as pDONR221) that includes attP sites to provide an entry clone, using the BP reaction. An integration reaction between the attB and the attP sites combines the PCR fragment with the donor vector. The resulting entry clone contains the gene of interest flanked by attL sites. The LR reaction is then used to combine the entry clone with a destination vector to produce an expression vector. In the LR reaction, a recombination reaction is used to link the entry clone with the destination vector (such as pLV430G) using the attL and attR sites and a clonase enzyme. The attL sites are already found in the entry clone, while the destination vector includes the attR sites. The LR reaction is carried out to transfer the sequence of interest into one or more destination vectors in simultaneous reactions.
  • In some embodiments, the aAPCs described herein may be grown and maintained under serum-based media and/or serum free media. According to an exemplary method, aAPCs may be cultured in 24 well plates at a cell density of about 1×106 cells per well for 3 to 5 days. The cells may then be isolated and/or washed by centrifugation and resuspended in media or cryopreserved in an appropriate cryopreservation media (e.g., CryoStor 10 (BioLife Solutions)) and stored in a −80° C. freezer.
  • In some embodiments, the aAPCs described herein may be grown in the presence of serum-based media. In some embodiments, the aAPCs described herein by may be grown in the presence of serum-based media that includes human serum (hSerum) containing media (e.g., cDMEM with 10% hSerum). In some embodiments, the aAPCs grown in the presence of serum-based media may be selected from the group consisting of aMOLM-13 cells, aMOLM-14 cells, and aEM3 cells.
  • In some embodiments, the aAPCs described herein may be grown in the presence of serum free media. In some embodiments, the serum free media may be selected from the group consisting of CTS Optmizer (ThermoFisher), Xvivo-20 (Lonza), Prime T Cell CDM (Irvine), XFSM (MesenCult), and the like. In some embodiments, the aAPCs grown in the presence of serum free media may be selected from the group consisting of aMOLM-13 cells, aMOLM-14 cells, and aEM3 cells.
    • Methods of Expanding Tumor Infiltrating Lymphocytes and T Cells
  • In an embodiment, the invention includes a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising at least one TIL with an aAPC described herein, wherein said aAPC comprises at least one co-stimulatory ligand that specifically binds with a co-stimulatory molecule expressed on the cellular surface of the TILs, wherein binding of said co-stimulatory molecule with said co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs) using any of the aAPCs of the present disclosure, the method comprising the steps as described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated by reference herein. For example, the tumor may be placed in enzyme media and mechanically dissociated for approximately 1 minute. The mixture may then be incubated for 30 minutes at 37° C. in 5% CO2 and then mechanically disrupted again for approximately 1 minute. After incubation for 30 minutes at 37° C. in 5% CO2, the tumor may be mechanically disrupted a third time for approximately 1 minute. If after the third mechanical disruption, large pieces of tissue are present, 1 or 2 additional mechanical dissociations may be applied to the sample, with or without 30 additional minutes of incubation at 37° C. in 5% CO2. At the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using Ficoll may be performed to remove these cells. TIL cultures were initiated in 24-well plates (Costar 24-well cell culture cluster, flat bottom; Corning Incorporated, Corning, N.Y.), each well may be seeded with 1×106tumor digest cells or one tumor fragment approximately 1 to 8 mm3 in size in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mM Hepes, and 10 mg/mL gentamicin. Cultures may be initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm2 gas-permeable silicon bottom (G-Rex 10; Wilson Wolf Manufacturing, New Brighton, each flask may be loaded with 10-40×106 viable tumor digest cells or 5-30 tumor fragments in 10-40 mL of CM with IL-2. G-Rex 10 and 24-well plates may be incubated in a humidified incubator at 37° C. in 5% CO2 and 5 days after culture initiation, half the media may be removed and replaced with fresh CM and IL-2 and after day 5, half the media may be changed every 2-3 days. Rapid expansion protocol (REP) of TILs may be performed using T-175 flasks and gas-permeable bags or gas-permeable G-Rex flasks, as described elsewhere herein, using the aAPCs of the present disclosure. For REP in T-175 flasks, 1×106 TILs may be suspended in 150 mL of media in each flask. The TIL may be cultured with aAPCs of the present disclosure at a ratio described herein, in a 1 to 1 mixture of CM and AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). The T-175 flasks may be incubated at 37° C. in 5% CO2. Half the media may be changed on day 5 using 50/50 medium with 3000 IU/mL of IL-2. On day 7, cells from 2 T-175 flasks may be combined in a 3L bag and 300 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may be added to the 300 mL of TIL suspension. The number of cells in each bag may be counted every day or two days, and fresh media may be added to keep the cell count between 0.5 and 2.0×106 cells/mL. For REP in 500 mL capacity flasks with 100 cm2 gas-permeable silicon bottoms (e.g., G-Rex 100, Wilson Wolf Manufacturing, as described elsewhere herein), 5×106 or 10×106 TILs may be cultured with aAPCs at a ratio described herein (e.g., 1 to 100) in 400 mL of 50/50 medium, supplemented with 3000 IU/mL of IL-2 and 30 ng/mL of anti-CD3 antibody (OKT-3). The G-Rex100 flasks may be incubated at 37° C. in 5% CO2. On day five, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (491 g) for 10 minutes. The obtained TIL pellets may be resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL of IL-2 and added back to the G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100 flasks, on day seven the TIL in each G-Rex100 are suspended in the 300 mL of media present in each flask and the cell suspension may be divided into three 100 mL aliquots that may be used to seed 3 G-Rex100 flasks. About 150 mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may then be added to each flask. G-Rex100 flasks may then be incubated at 37° C. in 5% CO2, and after four days, 150 mL of AIM-V with 3000 IU/mL of IL-2 may be added to each G-Rex100 flask. After this, the REP may be completed by harvesting cells on day 14 of culture.
  • As described herein, TILs may be expanded advantageously in the presence of serum free media. In some embodiments, the TIL expansion methods described herein may include the use of serum free media rather than serum-based media (e.g., complete media or CM1). In some embodiments, the TIL expansion methods described herein may use serum free media rather than serum-based media. In some embodiments, the serum free media may be selected from the group consisting of CTS Optmizer (ThermoFisher), Xvivo-20 (Lonza), Prime T Cell CDM (Irvine), and the like.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the population of APCs expands the population of TILs by at least 50-fold over a period of 7 days in a cell culture medium.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the myeloid cell endogenously expresses HLA-AB/C, ICOS-L, and CD58.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the myeloid cell is a MOLM-14 cell.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the myeloid cell is a MOLM-13 cell.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (c) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (d) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the myeloid cell is a EM-3 cell.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the CD86 protein comprises an amino acid sequence as set forth in SEQ ID NO:8, or conservative amino acid substitutions thereof, and the 4-1BBL protein comprises an amino acid sequence as set forth in SEQ ID NO:9, or conservative amino acid substitutions thereof.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the nucleic acid encoding CD86 comprises a nucleic acid sequence as set forth in SEQ ID NO:19 and the nucleic acid encoding 4-1BBL comprises a nucleic acid sequence as set forth in SEQ ID NO:16.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the expansion is performed using a gas permeable container.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the ratio of the population of TILs to the population of aAPCs is between 1 to 200 and 1 to 400.
  • In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of:
      • (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and
      • (b) contacting the population of TILs with the population of aAPCs in a cell culture medium,
      • wherein the ratio of the population of TILs to the population of aAPCs is about 1 to 300.
  • In an embodiment, the invention provides a method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising a population of TILs with a myeloid artificial antigen presenting cell (aAPC), wherein the myeloid aAPC comprises at least two co-stimulatory ligands that specifically bind with at least two co-stimulatory molecule on the TILs, wherein binding of the co-stimulatory molecules with the co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs, and wherein the at least two co-stimulatory ligands comprise CD86 and 4-1BBL.
  • In any of the foregoing embodiments, the aAPC may further comprise OX40L in addition to 4-1BBL, or may comprise OX40L instead of 4-1BBL.
  • In an embodiment, a method of expanding or treating a cancer includes a step wherein TILs are obtained from a patient tumor sample. A patient tumor sample may be obtained using methods known in the art. For example, TILs may be cultured from enzymatic tumor digests and tumor fragments (about 1 to about 8 mm3 in size) from sharp dissection. Such tumor digests may be produced by incubation in enzymatic media (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicine, 30 units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37° C. in 5% CO2, followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide may be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated by reference herein. Any of the foregoing methods may be used in any of the embodiments described herein for methods of expanding TILs or methods treating a cancer.
  • In an embodiment, REP can be performed in a gas permeable container using the aAPCs of the present disclosure by any suitable method. For example, TILs can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific T cell receptor stimulus can include, for example, about 30 ng/mL of an anti-CD3 antibody, e.g. OKT-3, a monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, N.J., USA or Miltenyi Biotech, Auburn, Calif., USA) or UHCT-1 (commercially available from BioLegend, San Diego, Calif., USA). TILs can be rapidly expanded by further stimulation of the TILs in vitro with one or more antigens, including antigenic portions thereof, such as epitope(s), of the cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217 (210M), optionally in the presence of a T cell growth factor, such as 300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TIL may also be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the TILs can be further re-stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.
  • In an embodiment, a method for expanding TILs may include using about 5000 mL to about 25000 mL of cell culture medium, about 5000 mL to about 10000 mL of cell culture medium, or about 5800 mL to about 8700 mL of cell culture medium. In an embodiment, a method for expanding TILs may include using about 1000 mL to about 2000 mL of cell medium, about 2000 mL to about 3000 mL of cell culture medium, about 3000 mL to about 4000 mL of cell culture medium, about 4000 mL to about 5000 mL of cell culture medium, about 5000 mL to about 6000 mL of cell culture medium, about 6000 mL to about 7000 mL of cell culture medium, about 7000 mL to about 8000 mL of cell culture medium, about 8000 mL to about 9000 mL of cell culture medium, about 9000 mL to about 10000 mL of cell culture medium, about 10000 mL to about 15000 mL of cell culture medium, about 15000 mL to about 20000 mL of cell culture medium, or about 20000 mL to about 25000 mL of cell culture medium. In an embodiment, expanding the number of TILs uses no more than one type of cell culture medium. Any suitable cell culture medium may be used, e.g., AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad, Calif., USA). In this regard, the inventive methods advantageously reduce the amount of medium and the number of types of medium required to expand the number of TIL. In an embodiment, expanding the number of TIL may comprise feeding the cells no more frequently than every third or fourth day. Expanding the number of cells in a gas permeable container simplifies the procedures necessary to expand the number of cells by reducing the feeding frequency necessary to expand the cells.
  • In an embodiment, the rapid expansion is performed using a gas permeable container. Such embodiments allow for cell populations to expand from about 5×105 cells/cm2 to between 10×106 and 30×106 cells/cm2. In an embodiment, this expansion occurs without feeding. In an embodiment, this expansion occurs without feeding so long as medium resides at a height of about 10 cm in a gas-permeable flask. In an embodiment this is without feeding but with the addition of one or more cytokines. In an embodiment, the cytokine can be added as a bolus without any need to mix the cytokine with the medium. Such containers, devices, and methods are known in the art and have been used to expand TILs, and include those described in U.S. Patent Application Publication No. US 2014/0377739 A1, International Patent Application Publication No. WO 2014/210036 A1, U.S. Patent Application Publication No. US 2013/0115617 A1, International Publication No. WO 2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228 A1, U.S. Pat. No. 8,809,050, International Patent Application Publication No. WO 2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216 A1, U.S. Patent Application Publication No. US 2012/0244133 A1, International Patent Application Publication No. WO 2012/129201 A1, U.S. Patent Application Publication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860, International Patent Application Publication No. WO 2013/173835 A1, and U.S. Patent Application Publication No. US 2015/0175966 A1, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin, et al., J. Immunotherapy 2012, 35, 283-292, the disclosure of which is incorporated by reference herein.
  • In an embodiment, the gas permeable container is a G-Rex 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 10 cm2 gas permeable culture surface. In an embodiment, the gas permeable container includes a 40 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 100 to 300 million TILs after 2 medium exchanges.
  • In an embodiment, the gas permeable container is a G-Rex 100 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In an embodiment, the gas permeable container includes a 100 cm2 gas permeable culture surface. In an embodiment, the gas permeable container includes a 450 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 1 to 3 billion TILs after 2 medium exchanges.
  • In an embodiment, the gas permeable container is a G-Rex 100M flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 100 cm2 gas permeable culture surface. In an embodiment, the gas permeable container includes a 1000 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 1 to 3 billion TILs without medium exchange.
  • In an embodiment, the gas permeable container is a G-Rex 100 L flask (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a 100 cm2 gas permeable culture surface. In an embodiment, the gas permeable container includes a 2000 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 1 to 3 billion TILs without medium exchange.
  • In an embodiment, the gas permeable container is a G-Rex 24 well plate (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a plate with wells, wherein each well includes a 2 cm2 gas permeable culture surface. In an embodiment, the gas permeable container includes a plate with wells, wherein each well includes a 8 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 20 to 60 million cells per well after 2 medium exchanges.
  • In an embodiment, the gas permeable container is a G-Rex 6 well plate (Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). In an embodiment, the gas permeable container includes a plate with wells, wherein each well includes a 10 cm2 gas permeable culture surface. In an embodiment, the gas permeable container includes a plate with wells, wherein each well includes a 40 mL cell culture medium capacity. In an embodiment, the gas permeable container provides 100 to 300 million cells per well after 2 medium exchanges.
  • In an embodiment, the cell medium in the first and/or second gas permeable container is unfiltered. The use of unfiltered cell medium may simplify the procedures necessary to expand the number of cells. In an embodiment, the cell medium in the first and/or second gas permeable container lacks beta-mercaptoethanol (BME).
  • In an embodiment, the duration of the method comprising obtaining a tumor tissue sample from the mammal; culturing the tumor tissue sample in a first gas permeable container containing cell medium therein; obtaining TILs from the tumor tissue sample; expanding the number of TILs in a second gas permeable container containing cell medium therein using aAPCs for a duration of about 14 to about 42 days, e.g., about 28 days.
  • In an embodiment, the rapid expansion uses about 1×109 to about 1×1011 aAPCs. In an embodiment, the rapid expansion uses about 1×109 aAPCs. In an embodiment, the rapid expansion uses about 1×1010 aAPCs. In an embodiment, the rapid expansion uses about 1×1011 aAPCs.
  • In an embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is selected from the group consisting of 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:225, 1:250, 1:275, 1:300, 1:350, 1:400, 1:450, and 1:500. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:90. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:95. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:100. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:105. In a preferred embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is about 1:110.
  • In an embodiment, the ratio of TILs to aAPCs in the rapid expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILs to aAPCs in the rapid expansion is between 1 to 50 and 1 to 300. In an embodiment, the ratio of TILs to aAPCs in the rapid expansion is between 1 to 100 and 1 to 200.
  • In an embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment, the cell culture medium comprises between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.
  • In an embodiment, the cell culture medium comprises an OKT-3 antibody. In a preferred embodiment, the cell culture medium comprises about 30 ng/mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL of OKT-3 antibody. In an embodiment, the cell culture medium comprises between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, and between 50 ng/mL and 100 ng/mL of OKT-3 antibody.
  • In an embodiment, a rapid expansion process for TILs may be performed using T-175 flasks and gas permeable bags as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas permeable cultureware (G-Rex flasks, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA). For TIL rapid expansion in T-175 flasks, 1×106 TILs suspended in 150 mL of media may be added to each T-175 flask. The TILs may be cultured with aAPCs at a ratio of 1 TIL to 100 aAPCs and the cells were cultured in a 1 to 1 mixture of CM and AIM-V medium, supplemented with 3000 IU (international units) per mL of IL-2 and 30 ng per ml of anti-CD3 antibody (e.g., OKT-3). The T-175 flasks may be incubated at 37° C. in 5% CO2. Half the media may be exchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2. On day 7 cells from two T-175 flasks may be combined in a 3 liter bag and 300 mL of AIM V with 5% human AB serum and 3000 IU per mL of IL-2 was added to the 300 ml of TIL suspension. The number of cells in each bag was counted every day or two and fresh media was added to keep the cell count between 0.5 and 2.0×106 cells/mL.
  • In an embodiment, for TIL rapid expansions in 500 mL capacity gas permeable flasks with 100 cm gas-permeable silicon bottoms (G-Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5×106 or 10×106 TIL may be cultured with aAPCs at a ratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ng per mL of anti-CD3 (OKT-3). The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and centrifuged at 1500 rpm (revolutions per minute; 491×g) for 10 minutes. The TIL pellets may be re-suspended with 150 mL of fresh medium with 5% human AB serum, 3000 IU per mL of IL-2, and added back to the original G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100 flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300 mL of media present in each flask and the cell suspension may be divided into 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may be added to each flask. The G-Rex 100 flasks may be incubated at 37° C. in 5% CO2 and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 may be added to each G-Rex 100 flask. The cells may be harvested on day 14 of culture.
  • In an embodiment, TILs may be prepared as follows. 2 mm3 tumor fragments are cultured in complete media (CM) comprised of AIM-V medium (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, Va.), 100 U/mL penicillin (Invitrogen Life Technologies), 100 μg/mL streptomycin (Invitrogen Life Technologies), 5% heat-inactivated human AB serum (Valley Biomedical, Inc. Winchester, Va.) and 600 IU/mL rhlL-2 (Chiron, Emeryville, Calif.). For enzymatic digestion of solid tumors, tumor specimens were diced into RPMI-1640, washed and centrifuged at 800 rpm for 5 minutes at 15-22° C., and resuspended in enzymatic digestion buffer (0.2 mg/mL Collagenase and 30 units/ml of DNase in RPMI-1640) followed by overnight rotation at room temperature. TILs established from fragments may be grown for 3-4 weeks in CM and expanded fresh or cryopreserved in heat-inactivated HAB serum with 10% dimethylsulfoxide (DMSO) and stored at −180° C. until the time of study. Tumor associated lymphocytes (TAL) obtained from ascites collections were seeded at 3×106 cells/well of a 24 well plate in CM. TIL growth was inspected about every other day using a low-power inverted microscope.
  • In an embodiment, TILs are expanded in gas-permeable containers. Gas-permeable containers have been used to expand TILs using PBMCs using methods, compositions, and devices known in the art, including those described in U.S. Patent Application Publication No. U.S. Patent Application Publication No. 2005/0106717 A1, the disclosures of which are incorporated herein by reference. In an embodiment, TILs are expanded in gas-permeable bags. In an embodiment, TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In an embodiment, TILs are expanded using a cell expansion system that expands TILs in gas permeable bags, such as the WAVE Bioreactor System, also known as the Xuri Cell Expansion System W5 (GE Healthcare). In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume selected from the group consisting of about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, about 10 L, about 11 L, about 12 L, about 13 L, about 14 L, about 15 L, about 16 L, about 17 L, about 18 L, about 19 L, about 20 L, about 25 L, and about 30 L. In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 50 and 150 mL, between 150 and 250 mL, between 250 and 350 mL, between 350 and 450 mL, between 450 and 550 mL, between 550 and 650 mL, between 650 and 750 mL, between 750 and 850 mL, between 850 and 950 mL, and between 950 and 1050 mL. In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 1 L and 2 L, between 2 L and 3 L, between 3 L and 4 L, between 4 L and 5 L, between 5 L and 6 L, between 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, between 9 L and 10 L, between 10 L and 11 L, between 11 L and 12 L, between 12 L and 13 L, between 13 L and 14 L, between 14 L and 15 L, between 15 L and 16 L, between 16 L and 17 L, between 17 L and 18 L, between 18 L and 19 L, and between 19 L and 20 L. In an embodiment, the cell expansion system includes a gas permeable cell bag with a volume range selected from the group consisting of between 0.5 L and 5 L, between 5 L and 10 L, between 10 L and 15 L, between 15 L and 20 L, between 20 L and 25 L, and between 25 L and 30 L. In an embodiment, the cell expansion system utilizes a rocking time of about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, and about 28 days. In an embodiment, the cell expansion system utilizes a rocking time of between 30 minutes and 1 hour, between 1 hour and 12 hours, between 12 hours and 1 day, between 1 day and 7 days, between 7 days and 14 days, between 14 days and 21 days, and between 21 days and 28 days. In an embodiment, the cell expansion system utilizes a rocking rate of about 2 rocks/minute, about 5 rocks/minute, about 10 rocks/minute, about 20 rocks/minute, about 30 rocks/minute, and about 40 rocks/minute. In an embodiment, the cell expansion system utilizes a rocking rate of between 2 rocks/minute and 5 rocks/minute, 5 rocks/minute and 10 rocks/minute, 10 rocks/minute and 20 rocks/minute, 20 rocks/minute and 30 rocks/minute, and 30 rocks/minute and 40 rocks/minute. In an embodiment, the cell expansion system utilizes a rocking angle of about 2°, about 3°, about 4°, about 5°, about 6°, about 7°, about 8°, about 9°, about 10°, about 11°, and about 12°. In an embodiment, the cell expansion system utilizes a rocking angle of between 2° and 3°, between 3° and 4°, between 4° and 5°, between 5° and 6°, between 6° and 7°, between 7° and 8°, between 8° and 9°, between 9° and 10°, between 10° and 11°, and between 11° and 12°.
  • In an embodiment, a method of expanding TILs using aAPCs further comprises a step wherein TILs are selected for superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in U.S. Patent Application Publication No. 2016/0010058 A1, the disclosures of which are incorporated herein by reference, may be used for selection of TILs for superior tumor reactivity.
  • In an embodiment, the aAPCs of the present invention may be used to expand T cells. Any of the foregoing embodiments of the present invention described for the expansion of TILs may also be applied to the expansion of T cells. In an embodiment, the aAPCs of the present invention may be used to expand CD8+ T cells. In an embodiment, the aAPCs of the present invention may be used to expand CD4+ T cells. In an embodiment, the aAPCs of the present invention may be used to expand T cells transduced with a chimeric antigen receptor (CAR-T). In an embodiment, the aAPCs of the present invention may be used to expand T cells comprising a modified T cell receptor (TCR). The CAR-T cells may be targeted against any suitable antigen, including CD19, as described in the art, e.g., in U.S. Pat. Nos. 7,070,995; 7,446,190; 8,399,645; 8,916,381; and 9,328,156; the disclosures of which are incorporated by reference herein. The modified TCR cells may be targeted against any suitable antigen, including NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof, as described in the art, e.g., in U.S. Pat. Nos. 8,367,804 and 7,569,664, the disclosures of which are incorporated by reference herein.
    • Methods of Treating Cancers and Other Diseases
  • The compositions and methods described herein can be used in a method for treating diseases. In an embodiment, they are for use in treating hyperproliferative disorders. They may also be used in treating other disorders as described herein and in the following paragraphs. The TILs, populations and compositions thereof described herein may be for use in the treatment of a disease. In an embodiment, the TILs, populations and compositions described herein are for use in the treatment of a hyperproliferative disorder.
  • In some embodiments, the hyperproliferative disorder is cancer. In some embodiments, the hyperproliferative disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma, pancreatic cancer, and glioblastoma. In some embodiments, the hyperproliferative disorder is a hematological malignancy. In some embodiments, the hematological malignancy is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, and mantle cell lymphoma.
  • In an embodiment, the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing a rapid expansion of the first population of TILs using a population of artificial antigen presenting cells (aAPCs) in a cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs; and (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer. In an embodiment, the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. In an embodiment, the rapid expansion is performed over a period not greater than 14 days.
  • In an embodiment, the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing an initial expansion of the first population of TILs using a first population of artificial antigen presenting cells (aAPCs) in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 10-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second population of TILs using a second population of aAPCs in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the first population of TILs; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer. In an embodiment, the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. In an embodiment, the rapid expansion is performed over a period not greater than 14 days. In an embodiment, the initial expansion is performed using a gas permeable container.
  • In an embodiment, the invention includes a method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) performing an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least 10-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises IL-2; (c) performing a rapid expansion of the second population of TILs using a population of artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TILs, wherein the third population of TILs is at least 50-fold greater in number than the first population of TILs; and wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer. In an embodiment, the aAPCs comprise MOLM-14 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-14 cells express a CD86 protein and a 4-1BBL protein. In an embodiment, the rapid expansion is performed over a period not greater than 14 days.
  • In an embodiment, the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m2/d for 5 days (days 27 to 23 prior to TIL infusion). In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.
  • Efficacy of the compounds and combinations of compounds described herein in treating, preventing and/or managing the indicated diseases or disorders can be tested using various models known in the art, which provide guidance for treatment of human disease. For example, models for determining efficacy of treatments for ovarian cancer are described, e.g., in Mullany, et al., Endocrinology 2012, 153, 1585-92; and Fong, et al., J. Ovarian Res. 2009, 2, 12. Models for determining efficacy of treatments for pancreatic cancer are described in Herreros-Villanueva, et al., World J. Gastroenterol. 2012, 18, 1286-1294. Models for determining efficacy of treatments for breast cancer are described, e.g., in Fantozzi, Breast Cancer Res. 2006, 8, 212. Models for determining efficacy of treatments for melanoma are described, e.g., in Damsky, et al., Pigment Cell & Melanoma Res. 2010, 23, 853-859. Models for determining efficacy of treatments for lung cancer are described, e.g., in Meuwissen, et al., Genes & Development, 2005, 19, 643-664. Models for determining efficacy of treatments for lung cancer are described, e.g., in Kim, Clin. Exp. Otorhinolaryngol. 2009, 2, 55-60; and Sano, Head Neck Oncol. 2009, 1, 32.
  • Non-Myeloablative Lymphodepletion with Chemotherapy
  • In an embodiment, the invention includes a method of treating a cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure. In an embodiment, the invention provides a population of TILs obtainable by a method described herein for use in treating a cancer, wherein the population of TILs is for treating a patient which is pre-treated with non-myeloablative chemotherapy. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/m2/d for 5 days (days 27 to 23 prior to TIL infusion). In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN) intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.
  • Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (“cytokine sinks”). Accordingly, some embodiments of the invention utilize a lymphodepletion step (sometimes also referred to as “immunosuppressive conditioning”) on the patient prior to the introduction of the aAPC-expanded TILs of the invention.
  • In general, lymphodepletion is achieved using administration of fludarabine or cyclophosphamide (the active form being referred to as mafosfamide) and combinations thereof. Such methods are described in Gassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski, et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated by reference herein in their entireties.
  • In some embodiments, the fludarabine is administered at a concentration of 0.5 μg/mL-10 μg/mL fludarabine. In some embodiments, the fludarabine is administered at a concentration of 1 μg/mL fludarabine. In some embodiments, the fludarabine treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the fludarabine is administered at a dosage of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, the fludarabine treatment is administered for 2-7 days at 35 mg/kg/day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 35 mg/kg/day. In some embodiments, the fludarabine treatment is administered for 4-5 days at 25 mg/kg/day.
  • In some embodiments, the mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 0.5 μg/ml-10 μg/ml by administration of cyclophosphamide. In some embodiments, mafosfamide, the active form of cyclophosphamide, is obtained at a concentration of 1 μg/mL by administration of cyclophosphamide. In some embodiments, the cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In some embodiments, the cyclophosphamide is administered at a dosage of 100 mg/m2/day, 150 mg/m2/day, 175 mg/m2/day, 200 mg/m2/day, 225 mg/m2/day, 250 mg/m2/day, 275 mg/m2/day, or 300 mg/m2/day. In some embodiments, the cyclophosphamide is administered intravenously (i.v.) In some embodiments, the cyclophosphamide treatment is administered for 2-7 days at 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment is administered for 4-5 days at 250 mg/m2/day i.v. In some embodiments, the cyclophosphamide treatment is administered for 4 days at 250 mg/m2/day i.v.
  • In some embodiments, lymphodepletion is performed by administering the fludarabine and the cyclophosphamide are together to a patient. In some embodiments, fludarabine is administered at 25 mg/m2/day i.v. and cyclophosphamide is administered at 250 mg/m2/day i.v. over 4 days.
  • In an embodiment, the lymphodepletion is performed by administration of cyclophosphamide at a dose of 60 mg/m2/day for two days followed by administration of fludarabine at a dose of 25 mg/m2/day for five days.
    • Pharmaceutical Compositions, Dosages, and Dosing Regimens
  • In an embodiment, TILs expanded using aAPCs of the present disclosure are administered to a patient as a pharmaceutical composition. In an embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using aAPCs of the present disclosure may be administered by any suitable route as known in the art. Preferably, the TILs are administered as a single infusion, such as an intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.
  • Any suitable dose of TILs can be administered. Preferably, from about 2.3×1010 to about 13.7×1010 TILs are administered, with an average of around 7.8×1010 TILs, particularly if the cancer is melanoma. In an embodiment, about 1.2×1010 to about 4.3×1010 of TILs are administered.
  • In some embodiments, the number of the TILs provided in the pharmaceutical compositions of the invention is about 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×1012, 2×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012, 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013, 6×1013, 7×1013, 8×1013, and 9×1013. In an embodiment, the number of the TILs provided in the pharmaceutical compositions of the invention is in the range of 1×106 to 5×106, 5×106 to 1×107, 1×107 to 5×107, 5×107 to 1×108, 1×108 to 5×108, 5×108 to 1×109, 1×109 to 5×109, 5×109 to 1×1010, 1×1010 to 5×1010, 5×1010 to 1×1011, 5×1011 to 1×1012, 1×1012 to 5×1012, and 5×1012 to 1×1013.
  • In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceutical composition.
  • In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition.
  • In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition.
  • In some embodiments, the concentration of the TILs provided in the pharmaceutical compositions of the invention is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v of the pharmaceutical composition.
  • In some embodiments, the amount of the TILs provided in the pharmaceutical compositions of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.
  • In some embodiments, the amount of the TILs provided in the pharmaceutical compositions of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.
  • The TILs provided in the pharmaceutical compositions of embodiments of the invention are effective over a wide dosage range. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. The clinically-established dosages of the TILs may also be used if appropriate. The amounts of the pharmaceutical compositions administered using the methods herein, such as the dosages of TILs, will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.
  • In some embodiments, TILs may be administered in a single dose. Such administration may be by injection, e.g., intravenous injection. In some embodiments, TILs may be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year. Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of TILs may continue as long as necessary.
  • In some embodiments, an effective dosage of TILs is about 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 8×1011, 9×1011, 1×1012, 2×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012, 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013, 6×1013, 7×1013, 8×1013, and 9×1013. In some embodiments, an effective dosage of TILs is in the range of 1×106 to 5×106, 5×106 to 1×107, 1×107 to 5×107, 5×107 to 1×108, 1×108 to 5×108, 5×108 to 1×109, 1×109 to 5×109, 5×109 to 1×1010, 1×1010 to 5×1010, 5×1010 to 1×1011, 5×1011 to 1×1012, 1×1012 to 5×1012, and 5×1012 to 1×1013.
  • In some embodiments, an effective dosage of TILs is in the range of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.
  • In some embodiments, an effective dosage of TILs is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg, about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg to about 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.
  • An effective amount of the TILs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation.
    • EXAMPLES
  • The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.
    • Example 1—Variability in Expansion of Tumor Infiltrating Lymphocytes Using PBMC Feeder Cells
  • The variability in TIL expansion obtained by use of PBMC feeder cells may be demonstrated by comparing the results of multiple TIL expansions on the same line of TILs obtained from a patient. FIG. 1 illustrates typical results of rapid expansion of TILs using irradiated allogeneic PBMC feeder cells (PBMC feeders). Two TIL lines labeled M1015T and M1016T (1.3×105 cells) were co-cultured with 46 different irradiated feeder cell lots (1.3×107), IL-2 (3000 IU/mL, recombinant human IL-2 (e.g., aldesleukin or equivalent), CellGenix, Inc., Portsmouth, N.H., USA) and OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in a T25 flask for 7 days. The fold expansion value for TILs was calculated on Day 7. The figure shows the number of fold expansions for the two TIL lines in separate stimulation experiments. For each TIL line, 46 different PBMC feeder lots were tested. The results range over more than 100-fold for each TIL line, and highlight the variability of expansion results using PBMC feeder cells. The aAPCs of the present invention offer reduced variability in expansion performance compared to PBMC feeders, as well as other advantages, as shown in the following examples.
    • Example 2—Selection of Myeloid Cells for aAPC Development
  • Phenotypic characterization was performed on various myeloid-lineage cell lines to identify potential candidates for further modification into aAPCs for TIL expansion. The results are summarized in Table 5. The MOLM-14 cell line exhibited endogenous expression of CD64, and was selected for further development. The EM-3 cell line was selected based on the observation of endogenous expression of ICOS-L (which was not observed for the EM-2 cell line, despite being taken from the same patient).
  • TABLE 5
    Summary of costimulatory molecules expressed endogenously on candidate cell lines for aAPCs.
    CML refers to chronic myeloid leukemia, and AML refers to acute myeloid leukemia. “Pop”
    refers to the population of cells observed to express the marker (½ pop = 50%).
    Cell line
    EM-2 EM-3 K562
    Myeloid blast Myeloid blast KG1-246 KG1-8031 myeloid erythro- MOLM-14
    Origin crisis, CML crisis, CML AML AML leukemia, CML AML
    HLA-A/B/C + + + + − +
    CD64 − − − − − +
    CD80 − − − − − +
    ICOS-L − + − − − +
    4-1BBL − − − − − −
    PD-L1 − − − − − −
    CD58 + + + + + +
    CD86 − − − − − + (½ pop)
    • Example 3—Preparation of MOLM-14 Artificial Antigen Presenting Cells (aMOLM14 aAPCs)
  • MOLM-14 cells were obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH. To develop MOLM-14 based aAPCs, MOLM-14 cells were engineered with the costimulatory molecules CD86 and 4-1BBL (CD137L). Human CD86 (hCD86) and human 4-1BBL (h4-1BBL) genes were cloned into commercially-available PLV430G and co-transfected with PDONR221 vectors (Invitrogen/Thermo Fisher Scientific, Carlsbad, Calif., USA) using a lentiviral transduction method. The gateway cloning method was used as described in Katzen, Expert Opin. Drug Disc. 2007, 4, 571-589, to clone hCD86 and hCD137L genes onto the PLV430G and PDONR221 vectors. The 293T cell line (human embryonic kidney cells transformed with large T antigen) was used for lentiviral production, transduced to MOLM-14 cells. The transfected cells were sorted (S3e Cell Sorter, Bio-Rad, Hercules, Calif., USA) using APC-conjugated CD86 and PE-conjugated CD137L to isolate and enrich the cells. The enriched cells were checked for purity by flow cytometry.
  • The vectors and portions thereof used for cloning are depicted in FIG. 2 to FIG. 11, and the nucleotide sequences for each vector are given in Table 6. The pLV430G human 4-1BBL vector is illustrated in FIG. 2, with the polymerase chain reaction product (PCRP) portion shown in FIG. 3. The pLV430G human CD86 vector is illustrated in FIG. 4, with the PCRP portion shown in FIG. 5. The pDONR221 human CD86 donor and human 4-1BBL donor vectors are shown in FIG. 6 and FIG. 7, respectively. Diagrams of the empty pLV430G destination vector and empty pDONR221 donor vector for the Gateway cloning method are shown in FIG. 8 and FIG. 9, respectively. FIG. 10 and FIG. 11 illustrate vector diagrams of the psPAX2 and pCIGO-VSV.G helper plasmids used for lentivirus production.
  • TABLE 6
    Nucleotide sequences for preparation of lentivirus for transduction of aAPCs.
    Identifier
    (Description) Sequence
    SEQ ID NO: 15 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60
    (pLV430G human gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120
    4-1BBL vector) cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180
    taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240
    ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300
    cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360
    tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420
    accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480
    agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540
    ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600
    agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660
    ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720
    tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780
    gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840
    cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900
    agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960
    tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020
    ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080
    aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140
    taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200
    agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260
    cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320
    aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380
    tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440
    cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500
    tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560
    tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620
    gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680
    ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740
    acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800
    taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860
    aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920
    tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980
    tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040
    ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100
    ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160
    gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220
    ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280
    ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340
    caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400
    agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460
    ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520
    agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580
    atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640
    cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700
    caacaagttt gtacaaaaaa gcaggcttcg ccaccatgga atacgcctct gatgccagcc 2760
    tggaccccga agctccttgg cctcctgccc ctagagccag agcctgtaga gtgctgcctt 2820
    gggctctggt ggctggcctt ctccttctgc tgctgctggc cgctgcctgc gctgtgtttc 2880
    tggcttgtcc ttgggccgtg tcaggcgcca gagcttctcc tggatctgcc gccagcccca 2940
    gactgagaga gggacctgag ctgagccccg atgatcctgc cggactgctg gatctgagac 3000
    agggcatgtt cgcccagctg gtggcccaga acgtgctgct gatcgatggc cccctgagct 3060
    ggtacagcga tcctggactg gctggcgtgt cactgacagg cggcctgagc tacaaagagg 3120
    acaccaaaga actggtggtg gccaaggccg gcgtgtacta cgtgttcttt cagctggaac 3180
    tgcggagagt ggtggccggc gaaggatccg gctctgtgtc tctggcactg catctgcagc 3240
    ccctgagatc tgctgcaggc gctgctgcac tggccctgac agtggacctg cctccagcct 3300
    ctagcgaggc cagaaactcc gcattcgggt ttcaaggcag actgctgcac ctgtctgccg 3360
    gccagagact gggagtgcat ctgcacacag aggccagagc cagacacgcc tggcagctga 3420
    cacagggcgc tacagtgctg ggcctgttca gagtgacccc cgaaattcca gccggcctgc 3480
    ccagccctag aagcgagtag gacccagctt tcttgtacaa agtggtgatt cgagttaatt 3540
    aagctagcct agtgccattt gttcagtggt tcgtagggct ttcccccact gtttggcttt 3600
    cagttatatg gatgatgtgg tattgggggc caagtctgta cagcatcttg agtccctttt 3660
    taccgctgtt accaattttc ttttgtcttt gggtatacat ttaaacccta acaaaacaaa 3720
    gagatggggt tactctctaa attttatggg ttatgtcatt ggatgttatg ggtccttgcc 3780
    acaagaacac atcatacaaa aaatcaaaga atgttttaga aaacttccta ttaacaggcc 3840
    tattgattgg aaagtatgtc aacgaattgt gggtcttttg ggttttgctg ccccttttac 3900
    acaatgtggt tatcctgcgt tgatgccttt gtatgcatgt attcaatcta agcaggcttt 3960
    cactttctcg ccaacttaca aggcctttct gtgtaaacaa tacctgaacc tttaccccgt 4020
    tgcccggcaa cggccaggtc tgtgccaagt gtttgctgac gcaaccccca ctggctgggg 4080
    cttggtcatg ggccatcagc gcatgcgtgg aaccttttcg gctcctctgc cgatccatac 4140
    tgcggaactc ctagccgctt gttttgctcg cagcaggtct ggagcaaaca ttatcgggac 4200
    tgataactct gttgtcctat cccgcaaata tacatcgttt ccatggctgc taggctgtgc 4260
    tgccaactgg atcctgcgcg ggacgtcctt tgtttacgtc ccgtcggcgc tgaatcctgc 4320
    ggacgaccct tctcggggtc gcttgggact ctctcgtccc cttctccgtc tgccgttccg 4380
    accgaccacg gggcgcacct ctctttacgc ggactccccg tctgtgcctt ctcatctgcc 4440
    ggaccgtgtg cacttcgctt cacctctgca cgtcgcatgg agaccaccgt gaacgcccac 4500
    caaatattgc ccaaggtctt acataagagg actcttggac tctcagcaat gtcaacgacc 4560
    gaccttgagg catacttcaa agactgtttg tttaaagact gggaggagtt gggggaggag 4620
    attaggttaa aggtctttgt actaggaggc tgtaggcata aattggtctg cgcaccagca 4680
    ccatggcgca atcactagag cggggtacct ttaagaccaa tgacttacaa ggcagctgta 4740
    gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga 4800
    agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg 4860
    gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg 4920
    cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc 4980
    ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat 5040
    ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat 5100
    aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 5160
    cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta 5220
    tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 5280
    cccatggctg actaattttt tttatttatg cagaggccga ggccggatcc cttgagtggc 5340
    tttcatcctg gagcagactt tgcagtctgt ggactgcaac acaacattgc ctttatgtgt 5400
    aactcttggc tgaagctctt acaccaatgc tgggggacat gtacctccca ggggcccagg 5460
    aagactacgg gaggctacac caacgtcaat cagaggggcc tgtgtagcta ccgataagcg 5520
    gaccctcaag agggcattag caatagtgtt tataaggccc ccttgttaat tcttgaagac 5580
    gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 5640
    agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 5700
    aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 5760
    attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 5820
    cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 5880
    aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 5940
    ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 6000
    gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact 6060
    attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 6120
    tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 6180
    tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 6240
    atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 6300
    agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg 6360
    aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 6420
    caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 6480
    ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 6540
    gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 6600
    tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 6660
    atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 6720
    tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 6780
    accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 6840
    gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 6900
    caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 6960
    tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 7020
    ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 7080
    tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 7140
    gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 7200
    attgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 7260
    gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 7320
    gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 7380
    ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 7440
    ggcctttttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca 7500
    acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc 7560
    ctcgcgtcg 7569
    SEQ ID NO: 16 atggaatacg cctctgatgc cagcctggac cccgaagctc cttggcctcc tgcccctaga 60
    (4-1BBL CoOP) gccagagcct gtagagtgct gccttgggct ctggtggctg gccttctcct tctgctgctg 120
    ctggccgctg cctgcgctgt gtttctggct tgtccttggg ccgtgtcagg cgccagagct 180
    tctcctggat ctgccgccag ccccagactg agagagggac ctgagctgag ccccgatgat 240
    cctgccggac tgctggatct gagacagggc atgttcgccc agctggtggc ccagaacgtg 300
    ctgctgatcg atggccccct gagctggtac agcgatcctg gactggctgg cgtgtcactg 360
    acaggcggcc tgagctacaa agaggacacc aaagaactgg tggtggccaa ggccggcgtg 420
    tactacgtgt tctttcagct ggaactgcgg agagtggtgg ccggcgaagg atccggctct 480
    gtgtctctgg cactgcatct gcagcccctg agatctgctg caggcgctgc tgcactggcc 540
    ctgacagtgg acctgcctcc agcctctagc gaggccagaa actccgcatt cgggtttcaa 600
    ggcagactgc tgcacctgtc tgccggccag agactgggag tgcatctgca cacagaggcc 660
    agagccagac acgcctggca gctgacacag ggcgctacag tgctgggcct gttcagagtg 720
    acccccgaaa ttccagccgg cctgcccagc cctagaagcg agtag 765
    SEQ ID NO: 17 ggggacaagt ttgtacaaaa aagcaggctt cgccaccatg gaatacgcct ctgatgccag 60
    (4-1BBL PRCP) cctggacccc gaagctcctt ggcctcctgc ccctagagcc agagcctgta gagtgctgcc 120
    ttgggctctg gtggctggcc ttctccttct gctgctgctg gccgctgcct gcgctgtgtt 180
    tctggcttgt ccttgggccg tgtcaggcgc cagagcttct cctggatctg ccgccagccc 240
    cagactgaga gagggacctg agctgagccc cgatgatcct gccggactgc tggatctgag 300
    acagggcatg ttcgcccagc tggtggccca gaacgtgctg ctgatcgatg gccccctgag 360
    ctggtacagc gatcctggac tggctggcgt gtcactgaca ggcggcctga gctacaaaga 420
    ggacaccaaa gaactggtgg tggccaaggc cggcgtgtac tacgtgttct ttcagctgga 480
    actgcggaga gtggtggccg gcgaaggatc cggctctgtg tctctggcac tgcatctgca 540
    gcccctgaga tctgctgcag gcgctgctgc actggccctg acagtggacc tgcctccagc 600
    ctctagcgag gccagaaact ccgcattcgg gtttcaaggc agactgctgc acctgtctgc 660
    cggccagaga ctgggagtgc atctgcacac agaggccaga gccagacacg cctggcagct 720
    gacacagggc gctacagtgc tgggcctgtt cagagtgacc cccgaaattc cagccggcct 780
    gcccagccct agaagcgagt aggacccagc tttcttgtac aaagtggtcc cc 832
    SEQ ID NO: 18 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60
    (pLV430G human gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120
    CD86 vector) cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180
    taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240
    ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300
    cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360
    tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420
    accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480
    agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540
    ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600
    agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660
    ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720
    tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780
    gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840
    cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900
    agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960
    tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020
    ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080
    aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140
    taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200
    agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260
    cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320
    aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380
    tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440
    cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500
    tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560
    tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620
    gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680
    ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740
    acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800
    taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860
    aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920
    tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980
    tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040
    ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100
    ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160
    gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220
    ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280
    ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340
    caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400
    agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460
    ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520
    agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580
    atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640
    cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700
    caacaagttt gtacaaaaaa gcaggcttcg ccaccatggg cctgagcaac atcctgttcg 2760
    tgatggcctt cctgctgtcc ggagccgccc ctctgaagat ccaggcctac ttcaacgaga 2820
    ccgccgacct gccctgccag ttcgccaaca gccagaacca gagcctgagc gaactggtgg 2880
    tgttctggca ggaccaggaa aacctggtcc tgaacgaggt gtacctgggc aaagaaaagt 2940
    tcgacagcgt gcacagcaag tacatgggcc ggaccagctt cgacagcgac agctggaccc 3000
    tgcggctgca caacctgcag atcaaggaca agggcctgta ccagtgcatc atccaccaca 3060
    agaaacccac cggcatgatc agaatccacc agatgaacag cgagctgtcc gtgctggcca 3120
    acttcagcca gcccgagatc gtgcccatca gcaacatcac cgagaacgtg tacatcaacc 3180
    tgacctgcag cagcatccac ggctaccccg agcccaagaa aatgagcgtg ctgctgcgga 3240
    ccaagaacag caccatcgag tacgacggcg tgatgcagaa aagccaggac aacgtgaccg 3300
    agctgtacga cgtgagcatc agcctgagcg tgagcttccc cgacgtgacc agcaacatga 3360
    ccatcttttg catcctggaa accgacaaga cccggctgct gtccagcccc ttcagcatcg 3420
    agctggaaga tccccagccc cctcccgacc acatcccctg gatcaccgcc gtgctgccca 3480
    ccgtgatcat ctgcgtgatg gtgttctgcc tgatcctgtg gaagtggaag aagaagaagc 3540
    ggcctaggaa cagctacaag tgcggcacca acaccatgga acgggaggaa agcgagcaga 3600
    ccaagaagcg ggagaagatc cacatccccg agcggagcga cgaggcccag cgggtgttca 3660
    agaggagcaa gaccagcagc tgcgacaaga gcgacacctg cttctaggac ccagctttct 3720
    tgtacaaagt ggtgattcga gttaattaag ctagcctagt gccatttgtt cagtggttcg 3780
    tagggctttc ccccactgtt tggctttcag ttatatggat gatgtggtat tgggggccaa 3840
    gtctgtacag catcttgagt ccctttttac cgctgttacc aattttcttt tgtctttggg 3900
    tatacattta aaccctaaca aaacaaagag atggggttac tctctaaatt ttatgggtta 3960
    tgtcattgga tgttatgggt ccttgccaca agaacacatc atacaaaaaa tcaaagaatg 4020
    ttttagaaaa cttcctatta acaggcctat tgattggaaa gtatgtcaac gaattgtggg 4080
    tcttttgggt tttgctgccc cttttacaca atgtggttat cctgcgttga tgcctttgta 4140
    tgcatgtatt caatctaagc aggctttcac tttctcgcca acttacaagg cctttctgtg 4200
    taaacaatac ctgaaccttt accccgttgc ccggcaacgg ccaggtctgt gccaagtgtt 4260
    tgctgacgca acccccactg gctggggctt ggtcatgggc catcagcgca tgcgtggaac 4320
    cttttcggct cctctgccga tccatactgc ggaactccta gccgcttgtt ttgctcgcag 4380
    caggtctgga gcaaacatta tcgggactga taactctgtt gtcctatccc gcaaatatac 4440
    atcgtttcca tggctgctag gctgtgctgc caactggatc ctgcgcggga cgtcctttgt 4500
    ttacgtcccg tcggcgctga atcctgcgga cgacccttct cggggtcgct tgggactctc 4560
    tcgtcccctt ctccgtctgc cgttccgacc gaccacgggg cgcacctctc tttacgcgga 4620
    ctccccgtct gtgccttctc atctgccgga ccgtgtgcac ttcgcttcac ctctgcacgt 4680
    cgcatggaga ccaccgtgaa cgcccaccaa atattgccca aggtcttaca taagaggact 4740
    cttggactct cagcaatgtc aacgaccgac cttgaggcat acttcaaaga ctgtttgttt 4800
    aaagactggg aggagttggg ggaggagatt aggttaaagg tctttgtact aggaggctgt 4860
    aggcataaat tggtctgcgc accagcacca tggcgcaatc actagagcgg ggtaccttta 4920
    agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 4980
    ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 5040
    ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 5100
    aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 5160
    tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt 5220
    agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt 5280
    gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 5340
    ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 5400
    gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc ccgcccctaa 5460
    ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 5520
    aggccgaggc cggatccctt gagtggcttt catcctggag cagactttgc agtctgtgga 5580
    ctgcaacaca acattgcctt tatgtgtaac tcttggctga agctcttaca ccaatgctgg 5640
    gggacatgta cctcccaggg gcccaggaag actacgggag gctacaccaa cgtcaatcag 5700
    aggggcctgt gtagctaccg ataagcggac cctcaagagg gcattagcaa tagtgtttat 5760
    aaggccccct tgttaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg 5820
    ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 5880
    gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 5940
    aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 6000
    tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 6060
    aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 6120
    aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 6180
    tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 6240
    aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 6300
    tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 6360
    ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 6420
    taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 6480
    agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa 6540
    caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 6600
    tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 6660
    gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 6720
    cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 6780
    caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 6840
    ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 6900
    aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 6960
    gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 7020
    atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 7080
    tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 7140
    gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 7200
    actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 7260
    gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 7320
    agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 7380
    ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa 7440
    aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 7500
    cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 7560
    gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 7620
    cctttttacg gttcctggcc ttttgctggc ctttttgaag ctgtccctga tggtcgtcat 7680
    ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa 7740
    gaatcataat ggggaaggcc atccagcctc gcgtcg 7776
    SEQ ID NO: 19 atgggcctga gcaacatcct gttcgtgatg gccttcctgc tgtccggagc cgcccctctg 60
    (CD86 CoOP) aagatccagg cctacttcaa cgagaccgcc gacctgccct gccagttcgc caacagccag 120
    aaccagagcc tgagcgaact ggtggtgttc tggcaggacc aggaaaacct ggtcctgaac 180
    gaggtgtacc tgggcaaaga aaagttcgac agcgtgcaca gcaagtacat gggccggacc 240
    agcttcgaca gcgacagctg gaccctgcgg ctgcacaacc tgcagatcaa ggacaagggc 300
    ctgtaccagt gcatcatcca ccacaagaaa cccaccggca tgatcagaat ccaccagatg 360
    aacagcgagc tgtccgtgct ggccaacttc agccagcccg agatcgtgcc catcagcaac 420
    atcaccgaga acgtgtacat caacctgacc tgcagcagca tccacggcta ccccgagccc 480
    aagaaaatga gcgtgctgct gcggaccaag aacagcacca tcgagtacga cggcgtgatg 540
    cagaaaagcc aggacaacgt gaccgagctg tacgacgtga gcatcagcct gagcgtgagc 600
    ttccccgacg tgaccagcaa catgaccatc ttttgcatcc tggaaaccga caagacccgg 660
    ctgctgtcca gccccttcag catcgagctg gaagatcccc agccccctcc cgaccacatc 720
    ccctggatca ccgccgtgct gcccaccgtg atcatctgcg tgatggtgtt ctgcctgatc 780
    ctgtggaagt ggaagaagaa gaagcggcct aggaacagct acaagtgcgg caccaacacc 840
    atggaacggg aggaaagcga gcagaccaag aagcgggaga agatccacat ccccgagcgg 900
    agcgacgagg cccagcgggt gttcaagagc agcaagacca gcagctgcga caagagcgac 960
    acctgcttc 969
    SEQ ID NO: 20 ggggacaagt ttgtacaaaa aagcaggctt cgccaccatg ggcctgagca acatcctgtt 60
    (CD86 PCRP) cgtgatggcc ttcctgctgt ccggagccgc ccctctgaag atccaggcct acttcaacga 120
    gaccgccgac ctgccctgcc agttcgccaa cagccagaac cagagcctga gcgaactggt 180
    ggtgttctgg caggaccagg aaaacctggt cctgaacgag gtgtacctgg gcaaagaaaa 240
    gttcgacagc gtgcacagca agtacatggg ccggaccagc ttcgacagcg acagctggac 300
    cctgcggctg cacaacctgc agatcaagga caagggcctg taccagtgca tcatccacca 360
    caagaaaccc accggcatga tcagaatcca ccagatgaac agcgagctgt ccgtgctggc 420
    caacttcagc cagcccgaga tcgtgcccat cagcaacatc accgagaacg tgtacatcaa 480
    cctgacctgc agcagcatcc acggctaccc cgagcccaag aaaatgagcg tgctgctgcg 540
    gaccaagaac agcaccatcg agtacgacgg cgtgatgcag aaaagccagg acaacgtgac 600
    cgagctgtac gacgtgagca tcagcctgag cgtgagcttc cccgacgtga ccagcaacat 660
    gaccatcttt tgcatcctgg aaaccgacaa gacccggctg ctgtccagcc ccttcagcat 720
    cgagctggaa gatccccagc cccctcccga ccacatcccc tggatcaccg ccgtgctgcc 780
    caccgtgatc atctgcgtga tggtgttctg cctgatcctg tggaagtgga agaagaagaa 840
    gcggcctagg aacagctaca agtgcggcac caacaccatg gaacgggagg aaagcgagca 900
    gaccaagaag cgggagaaga tccacatccc cgagcggagc gacgaggccc agcgggtgtt 960
    caagagcagc aagaccagca gctgcgacaa gagcgacacc tgcttctagg acccagcttt 1020
    cttgtacaaa gtggtcccc 1039
    SEQ ID NO: 21 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60
    (pDONR221 CD86 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120
    vector) gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180
    cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240
    tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300
    gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360
    acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420
    caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480
    gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540
    aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600
    ctgttcgttg caacacattg atgagcaatg cttttttata atgcacaagt ttgtacaaaa 660
    aagcaggctt cgccaccatg ggcctgagca acatcctgtt cgtgatggcc ttcctgctgt 720
    ccggagccgc ccctctgaag atccaggcct acttcaacga gaccgccgac ctgccctgcc 780
    agttcgccaa cagccagaac cagagcctga gcgaactggt ggtgttctgg caggaccagg 840
    aaaacctggt cctgaacgag gtgtacctgg gcaaagaaaa gttcgacagc gtgcacagca 900
    agtacatggg ccggaccagc ttcgacagcg acagctggac cctgcggctg cacaacctgc 960
    agatcaagga caagggcctg taccagtgca tcatccacca caagaaaccc accggcatga 1020
    tcagaatcca ccagatgaac agcgagctgt ccgtgctggc caacttcagc cagcccgaga 1080
    tcgtgcccat cagcaacatc accgagaacg tgtacatcaa cctgacctgc agcagcatcc 1140
    acggctaccc cgagcccaag aaaatgagcg tgctgctgcg gaccaagaac agcaccatcg 1200
    agtacgacgg cgtgatgcag aaaagccagg acaacgtgac cgagctgtac gacgtgagca 1260
    tcagcctgag cgtgagcttc cccgacgtga ccagcaacat gaccatcttt tgcatcctgg 1320
    aaaccgacaa gacccggctg ctgtccagcc ccttcagcat cgagctggaa gatccccagc 1380
    cccctcccga ccacatcccc tggatcaccg ccgtgctgcc caccgtgatc atctgcgtga 1440
    tggtgttctg cctgatcctg tggaagtgga agaagaagaa gcggcctagg aacagctaca 1500
    agtgcggcac caacaccatg gaacgggagg aaagcgagca gaccaagaag cgggagaaga 1560
    tccacatccc cgagcggagc gacgaggccc agcgggtgtt caagagcagc aagaccagca 1620
    gctgcgacaa gagcgacacc tgcttctagg acccagcttt cttgtacaaa gtggtcatta 1680
    taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa 1740
    tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg gtcatagctg 1800
    tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa 1860
    taatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta 1920
    tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt 1980
    tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct 2040
    tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca 2100
    atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga 2160
    ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg 2220
    gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg 2280
    cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca 2340
    gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg 2400
    cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc 2460
    ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata 2520
    accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg 2580
    cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat 2640
    tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt 2700
    ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacactggc 2760
    agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga 2820
    gttacgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 2880
    atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 2940
    tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 3000
    gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 3060
    actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 3120
    gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 3180
    agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 3240
    ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 3300
    aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 3360
    cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 3420
    gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 3480
    cctttttacg gttcctggcc ttttgctggc cttttgctca catgtt 3526
    SEQ ID NO: 22 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60
    (pDONR221 4- taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120
    1BBL vector) gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180
    cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240
    tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300
    gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360
    acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420
    caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480
    gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540
    aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600
    ctgttcgttg caacacattg atgagcaatg cttttttata atgcacaagt ttgtacaaaa 660
    aagcaggctt cgccaccatg gaatacgcct ctgatgccag cctggacccc gaagctcctt 720
    ggcctcctgc ccctagagcc agagcctgta gagtgctgcc ttgggctctg gtggctggcc 780
    ttctccttct gctgctgctg gccgctgcct gcgctgtgtt tctggcttgt ccttgggccg 840
    tgtcaggcgc cagagcttct cctggatctg ccgccagccc cagactgaga gagggacctg 900
    agctgagccc cgatgatcct gccggactgc tggatctgag acagggcatg ttcgcccagc 960
    tggtggccca gaacgtgctg ctgatcgatg gccccctgag ctggtacagc gatcctggac 1020
    tggctggcgt gtcactgaca ggcggcctga gctacaaaga ggacaccaaa gaactggtgg 1080
    tggccaaggc cggcgtgtac tacgtgttct ttcagctgga actgcggaga gtggtggccg 1140
    gcgaaggatc cggctctgtg tctctggcac tgcatctgca gcccctgaga tctgctgcag 1200
    gcgctgctgc actggccctg acagtggacc tgcctccagc ctctagcgag gccagaaact 1260
    ccgcattcgg gtttcaaggc agactgctgc acctgtctgc cggccagaga ctgggagtgc 1320
    atctgcacac agaggccaga gccagacacg cctggcagct gacacagggc gctacagtgc 1380
    tgggcctgtt cagagtgacc cccgaaattc cagccggcct gcccagccct agaagcgagt 1440
    aggacccagc tttcttgtac aaagtggtca ttataagaaa gcattgctta tcaatttgtt 1500
    gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgccatcca gctgatatcc 1560
    cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc 1620
    aaaatctctg atgttacatt gcacaagata aaataatatc atcatgaaca ataaaactgt 1680
    ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga 1740
    ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata 1800
    atgtcgggca atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt 1860
    tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac 1920
    taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg 1980
    atgatgcatg gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag 2040
    aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc 2100
    attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg 2160
    cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg 2220
    gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt 2280
    cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa 2340
    taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc 2400
    tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg 2460
    gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct 2520
    aatcagaatt ggttaattgg ttgtaacact ggcagagcat tacgctgact tgaggggagg 2580
    gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag 2640
    accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 2700
    gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 2760
    caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 2820
    tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 2880
    ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 2940
    tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 3000
    gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 3060
    tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 3120
    gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 3180
    gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 3240
    ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 3300
    ggccttttgc tcacatgtt 3319
    SEQ ID NO: 23 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60
    (pLV430G gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120
    vector) cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180
    taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240
    ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300
    cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360
    tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420
    accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480
    agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540
    ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600
    agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660
    ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720
    tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780
    gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840
    cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900
    agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960
    tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020
    ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080
    aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140
    taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200
    agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260
    cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320
    aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380
    tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440
    cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500
    tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560
    tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620
    gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680
    ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740
    acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800
    taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860
    aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920
    tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980
    tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040
    ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100
    ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160
    gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220
    ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280
    ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340
    caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400
    agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460
    ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520
    agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580
    atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640
    cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700
    cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 2760
    aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 2820
    actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 2880
    gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 2940
    aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 3000
    gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 3060
    gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 3120
    cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 3180
    gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 3240
    tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 3300
    gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 3360
    tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 3420
    atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 3480
    gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 3540
    agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt 3600
    ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt 3660
    ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc 3720
    agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt 3780
    caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga 3840
    acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa 3900
    cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata 3960
    aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg 4020
    ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg 4080
    aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg 4140
    ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg 4200
    acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac 4260
    acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta 4320
    gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct 4380
    cgttcagctt tcttgtacaa agtggtgatt cgagttaatt aagctagcct agtgccattt 4440
    gttcagtggt tcgtagggct ttcccccact gtttggcttt cagttatatg gatgatgtgg 4500
    tattgggggc caagtctgta cagcatcttg agtccctttt taccgctgtt accaattttc 4560
    ttttgtcttt gggtatacat ttaaacccta acaaaacaaa gagatggggt tactctctaa 4620
    attttatggg ttatgtcatt ggatgttatg ggtccttgcc acaagaacac atcatacaaa 4680
    aaatcaaaga atgttttaga aaacttccta ttaacaggcc tattgattgg aaagtatgtc 4740
    aacgaattgt gggtcttttg ggttttgctg ccccttttac acaatgtggt tatcctgcgt 4800
    tgatgccttt gtatgcatgt attcaatcta agcaggcttt cactttctcg ccaacttaca 4860
    aggcctttct gtgtaaacaa tacctgaacc tttaccccgt tgcccggcaa cggccaggtc 4920
    tgtgccaagt gtttgctgac gcaaccccca ctggctgggg cttggtcatg ggccatcagc 4980
    gcatgcgtgg aaccttttcg gctcctctgc cgatccatac tgcggaactc ctagccgctt 5040
    gttttgctcg cagcaggtct ggagcaaaca ttatcgggac tgataactct gttgtcctat 5100
    cccgcaaata tacatcgttt ccatggctgc taggctgtgc tgccaactgg atcctgcgcg 5160
    ggacgtcctt tgtttacgtc ccgtcggcgc tgaatcctgc ggacgaccct tctcggggtc 5220
    gcttgggact ctctcgtccc cttctccgtc tgccgttccg accgaccacg gggcgcacct 5280
    ctctttacgc ggactccccg tctgtgcctt ctcatctgcc ggaccgtgtg cacttcgctt 5340
    cacctctgca cgtcgcatgg agaccaccgt gaacgcccac caaatattgc ccaaggtctt 5400
    acataagagg actcttggac tctcagcaat gtcaacgacc gaccttgagg catacttcaa 5460
    agactgtttg tttaaagact gggaggagtt gggggaggag attaggttaa aggtctttgt 5520
    actaggaggc tgtaggcata aattggtctg cgcaccagca ccatggcgca atcactagag 5580
    cggggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa 5640
    agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc 5700
    ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg 5760
    gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg 5820
    tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat 5880
    ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg 5940
    aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa 6000
    tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 6060
    caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc 6120
    atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 6180
    tttatttatg cagaggccga ggccggatcc cttgagtggc tttcatcctg gagcagactt 6240
    tgcagtctgt ggactgcaac acaacattgc ctttatgtgt aactcttggc tgaagctctt 6300
    acaccaatgc tgggggacat gtacctccca ggggcccagg aagactacgg gaggctacac 6360
    caacgtcaat cagaggggcc tgtgtagcta ccgataagcg gaccctcaag agggcattag 6420
    caatagtgtt tataaggccc ccttgttaat tcttgaagac gaaagggcct cgtgatacgc 6480
    ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 6540
    cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 6600
    ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 6660
    agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 6720
    tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 6780
    gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 6840
    gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 6900
    gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 6960
    gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 7020
    agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 7080
    ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 7140
    cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 7200
    gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 7260
    cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 7320
    gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 7380
    ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 7440
    acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 7500
    ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 7560
    aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 7620
    aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 7680
    ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 7740
    ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 7800
    actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 7860
    caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 7920
    gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 7980
    ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 8040
    cgaacgacct acaccgaact gagataccta cagcgtgagc attgagaaag cgccacgctt 8100
    cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 8160
    acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 8220
    ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 8280
    gccagcaacg cggccttttt acggttcctg gccttttgct ggcctttttg aagctgtccc 8340
    tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg 8400
    ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcg 8449
    SEQ ID NO: 24 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60
    (pDONR221 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120
    vector) gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180
    cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240
    tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300
    gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360
    acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420
    caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480
    gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540
    aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600
    ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 660
    agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa 720
    aaaacagact acataatact gtaaaacaca acatatccag tcactatgaa tcaactactt 780
    agatggtatt agtgacctgt agtcgaccga cagccttcca aatgttcttc gggtgatgct 840
    gccaacttag tcgaccgaca gccttccaaa tgttcttctc aaacggaatc gtcgtatcca 900
    gcctactcgc tattgtcctc aatgccgtat taaatcataa aaagaaataa gaaaaagagg 960
    tgcgagcctc ttttttgtgt gacaaaataa aaacatctac ctattcatat acgctagtgt 1020
    catagtcctg aaaatcatct gcatcaagaa caatttcaca actcttatac ttttctctta 1080
    caagtcgttc ggcttcatct ggattttcag cctctatact tactaaacgt gataaagttt 1140
    ctgtaatttc tactgtatcg acctgcagac tggctgtgta taagggagcc tgacatttat 1200
    attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca 1260
    gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc 1320
    cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc 1380
    agacgtgcac tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc 1440
    tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc 1500
    atttcaccag cccctgttct cgtcagcaaa agagccgttc atttcaataa accgggcgac 1560
    ctcagccatc ccttcctgat tttccgcttt ccagcgttcg gcacgcagac gacgggcttc 1620
    attctgcatg gttgtgctta ccagaccgga gatattgaca tcatatatgc cttgagcaac 1680
    tgatagctgt cgctgtcaac tgtcactgta atacgctgct tcatagcata cctctttttg 1740
    acatacttcg ggtatacata tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat 1800
    acgcatactg ttatctggct tttagtaagc cggatccacg cggcgtttac gccccgccct 1860
    gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac 1920
    agacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat 1980
    atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa 2040
    aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt 2100
    tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa 2160
    actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat 2220
    ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg 2280
    ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat 2340
    aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg 2400
    tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc 2460
    attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag 2520
    ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt 2580
    gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg 2640
    gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca 2700
    caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttagtc gactacaggt 2760
    cactaatacc atctaagtag ttgattcata gtgactggat atgttgtgtt ttacagtatt 2820
    atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg 2880
    tttctcgttc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt 2940
    gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata 3000
    tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt 3060
    ctcaaaatct ctgatgttac attgcacaag ataaaataat atcatcatga acaataaaac 3120
    tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt 3180
    cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg 3240
    ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag 3300
    agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca 3360
    gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc 3420
    ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag 3480
    aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt 3540
    tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc 3600
    aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta 3660
    atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg 3720
    attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat 3780
    taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca 3840
    tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat 3900
    atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt 3960
    tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg 4020
    acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt 4080
    cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 4140
    gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 4200
    taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 4260
    ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 4320
    tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 4380
    ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 4440
    cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 4500
    agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 4560
    gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 4620
    atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 4680
    gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 4740
    gctggccttt tgctcacatg t 4761
    SEQ ID NO: 25 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 60
    (psPAX2 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 120
    plasmid) gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 180
    atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 240
    ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 300
    cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 360
    agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 420
    cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 480
    tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 540
    agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 600
    gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 660
    gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 720
    ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 780
    tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 840
    tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 900
    gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 960
    caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 1020
    atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggt 1080
    cgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 1140
    ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 1200
    aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 1260
    actttccatt gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat 1320
    caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 1380
    tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 1440
    ttagtcatcg ctattaccat gggtcgaggt gagccccacg ttctgcttca ctctccccat 1500
    ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc 1560
    gatgggggcg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 1620
    gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 1680
    cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 1740
    gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc cgcctcgcgc cgcccgcccc 1800
    ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 1860
    gctgtaatta gcgcttggtt taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc 1920
    ttaaagggct ccgggagggc cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg 1980
    tgtgtgtgcg tggggagcgc cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg 2040
    ggcgcggcgc ggggctttgt gcgctccgcg tgtgcgcgag gggagcgcgg ccgggggcgg 2100
    tgccccgcgg tgcggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg 2160
    ggggggtgag cagggggtgt gggcgcggcg gtcgggctgt aacccccccc tgcaccgccc 2220
    tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtgcgg ggcgtggcgc 2280
    ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc 2340
    gcctcgggcc ggggagggct cgggggaggg gcgcggcggc cccggagcgc cggcggctgt 2400
    cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga 2460
    cttcctttgt cccaaatctg gcggagccga aatctgggag gcgccgccgc accccctcta 2520
    gcgggcgcgg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg agggccttcg 2580
    tgcgtcgccg cgccgccgtc cccttctcca tctccagcct cggggctgcc gcagggggac 2640
    ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc 2700
    tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac 2760
    gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgggccggc cgcgttgacg 2820
    cgcacggcaa gaggcgaggg gcggcgactg gtgagagatg ggtgcgagag cgtcagtatt 2880
    aagcggggga gaattagatc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa 2940
    atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc 3000
    tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct 3060
    tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt 3120
    gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca 3180
    aaacaaaagt aagaaaaaag cacagcaagc agcagctgac acaggacaca gcaatcaggt 3240
    cagccaaaat taccctatag tgcagaacat ccaggggcaa atggtacatc aggccatatc 3300
    acctagaact ttaaatgcat gggtaaaagt agtagaagag aaggctttca gcccagaagt 3360
    gatacccatg ttttcagcat tatcagaagg agccacccca caagatttaa acaccatgct 3420
    aaacacagtg gggggacatc aagcagccat gcaaatgtta aaagagacca tcaatgagga 3480
    agctgcagaa tgggatagag tgcatccagt gcatgcaggg cctattgcac caggccagat 3540
    gagagaacca aggggaagtg acatagcagg aactactagt acccttcagg aacaaatagg 3600
    atggatgaca cataatccac ctatcccagt aggagaaatc tataaaagat ggataatcct 3660
    gggattaaat aaaatagtaa gaatgtatag ccctaccagc attctggaca taagacaagg 3720
    accaaaggaa ccctttagag actatgtaga ccgattctat aaaactctaa gagccgagca 3780
    agcttcacaa gaggtaaaaa attggatgac agaaaccttg ttggtccaaa atgcgaaccc 3840
    agattgtaag actattttaa aagcattggg accaggagcg acactagaag aaatgatgac 3900
    agcatgtcag ggagtggggg gacccggcca taaagcaaga gttttggctg aagcaatgag 3960
    ccaagtaaca aatccagcta ccataatgat acagaaaggc aattttagga accaaagaaa 4020
    gactgttaag tgtttcaatt gtggcaaaga agggcacata gccaaaaatt gcagggcccc 4080
    taggaaaaag ggctgttgga aatgtggaaa ggaaggacac caaatgaaag attgtactga 4140
    gagacaggct aattttttag ggaagatctg gccttcccac aagggaaggc cagggaattt 4200
    tcttcagagc agaccagagc caacagcccc accagaagag agcttcaggt ttggggaaga 4260
    gacaacaact ccctctcaga aggaggagcc gatagacaag gaactgtatc ctttagcttc 4320
    cctcagatca ctctttggca gcgacccctc gtcacaataa agataggggg gcaattaaag 4380
    gaagctctat tagatacagg agcagatgat acagtattag aagaaatgaa tttgccagga 4440
    agatggaaac caaaaatgat agggggaatt ggaggtttta tcaaagtagg acagtatgat 4500
    cagatactca tagaaatctg cggacataaa gctataggta cagtattagt aggacctaca 4560
    cctgtcaaca taattggaag aaatctgttg actcagattg gctgcacttt aaattttccc 4620
    attagtccta ttgagactgt accagtaaaa ttaaagccag gaatggatgg cccaaaagtt 4680
    aaacaatggc cattgacaga agaaaaaata aaagcattag tagaaatttg tacagaaatg 4740
    gaaaaggaag gaaaaatttc aaaaattggg cctgaaaatc catacaatac tccagtattt 4800
    gccataaaga aaaaagacag tactaaatgg agaaaattag tagatttcag agaacttaat 4860
    aagagaactc aagatttctg ggaagttcaa ttaggaatac cacatcctgc agggttaaaa 4920
    cagaaaaaat cagtaacagt actggatgtg ggcgatgcat atttttcagt tcccttagat 4980
    aaagacttca ggaagtatac tgcatttacc atacctagta taaacaatga gacaccaggg 5040
    attagatatc agtacaatgt gcttccacag ggatggaaag gatcaccagc aatattccag 5100
    tgtagcatga caaaaatctt agagcctttt agaaaacaaa atccagacat agtcatctat 5160
    caatacatgg atgatttgta tgtaggatct gacttagaaa tagggcagca tagaacaaaa 5220
    atagaggaac tgagacaaca tctgttgagg tggggattta ccacaccaga caaaaaacat 5280
    cagaaagaac ctccattcct ttggatgggt tatgaactcc atcctgataa atggacagta 5340
    cagcctatag tgctgccaga aaaggacagc tggactgtca atgacataca gaaattagtg 5400
    ggaaaattga attgggcaag tcagatttat gcagggatta aagtaaggca attatgtaaa 5460
    cttcttaggg gaaccaaagc actaacagaa gtagtaccac taacagaaga agcagagcta 5520
    gaactggcag aaaacaggga gattctaaaa gaaccggtac atggagtgta ttatgaccca 5580
    tcaaaagact taatagcaga aatacagaag caggggcaag gccaatggac atatcaaatt 5640
    tatcaagagc catttaaaaa tctgaaaaca ggaaaatatg caagaatgaa gggtgcccac 5700
    actaatgatg tgaaacaatt aacagaggca gtacaaaaaa tagccacaga aagcatagta 5760
    atatggggaa agactcctaa atttaaatta cccatacaaa aggaaacatg ggaagcatgg 5820
    tggacagagt attggcaagc cacctggatt cctgagtggg agtttgtcaa tacccctccc 5880
    ttagtgaagt tatggtacca gttagagaaa gaacccataa taggagcaga aactttctat 5940
    gtagatgggg cagccaatag ggaaactaaa ttaggaaaag caggatatgt aactgacaga 6000
    ggaagacaaa aagttgtccc cctaacggac acaacaaatc agaagactga gttacaagca 6060
    attcatctag ctttgcagga ttcgggatta gaagtaaaca tagtgacaga ctcacaatat 6120
    gcattgggaa tcattcaagc acaaccagat aagagtgaat cagagttagt cagtcaaata 6180
    atagagcagt taataaaaaa ggaaaaagtc tacctggcat gggtaccagc acacaaagga 6240
    attggaggaa atgaacaagt agatgggttg gtcagtgctg gaatcaggaa agtactattt 6300
    ttagatggaa tagataaggc ccaagaagaa catgagaaat atcacagtaa ttggagagca 6360
    atggctagtg attttaacct accacctgta gtagcaaaag aaatagtagc cagctgtgat 6420
    aaatgtcagc taaaagggga agccatgcat ggacaagtag actgtagccc aggaatatgg 6480
    cagctagatt gtacacattt agaaggaaaa gttatcttgg tagcagttca tgtagccagt 6540
    ggatatatag aagcagaagt aattccagca gagacagggc aagaaacagc atacttcctc 6600
    ttaaaattag caggaagatg gccagtaaaa acagtacata cagacaatgg cagcaatttc 6660
    accagtacta cagttaaggc cgcctgttgg tgggcgggga tcaagcagga atttggcatt 6720
    ccctacaatc cccaaagtca aggagtaata gaatctatga ataaagaatt aaagaaaatt 6780
    ataggacagg taagagatca ggctgaacat cttaagacag cagtacaaat ggcagtattc 6840
    atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 6900
    gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 6960
    aattttcggg tttattacag ggacaggaga gatccagttt ggaaaggacc agcaaagctc 7020
    ctctggaaag gtgaaggggc agtagtaata caagataata gtgacataaa agtagtgcca 7080
    agaagaaaag caaagatcat cagggattat ggaaaacaga tggcaggtga tgattgtgtg 7140
    gcaagtagac aggatgagga ttaacacatg gaattctgca acaactgctg tttatccatt 7200
    tcagaattgg gtgtcgacat agcagaatag gcgttactcg acagaggaga gcaagaaatg 7260
    gagccagtag atcctagact agagccctgg aagcatccag gaagtcagcc taaaactgct 7320
    tgtaccaatt gctattgtaa aaagtgttgc tttcattgcc aagtttgttt catgacaaaa 7380
    gccttaggca tctcctatgg caggaagaag cggagacagc gacgaagagc tcatcagaac 7440
    agtcagactc atcaagcttc tctatcaaag cagtaagtag tacatgtaat gcaacctata 7500
    atagtagcaa tagtagcatt agtagtagca ataataatag caatagttgt gtggtccata 7560
    gtaatcatag aatataggaa aatggccgct gatcttcaga cctggaggag gagatatgag 7620
    ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt 7680
    agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg 7740
    agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cctcaatgac 7800
    gctgacggta caggccagac aattattgtc tggtatagtg caggaggaga acaatttgct 7860
    gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct 7920
    ccaagcaaga atcctagctg tggaaagata cctaaaggat caacagctcc tagggatttg 7980
    gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa 8040
    taaatctctg gaacagatct ggaatcacac gacctggatg gagtgggaca gagaaattaa 8100
    caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa 8160
    tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac 8220
    aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag 8280
    aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc 8340
    gtttcagacc cacctcccaa tcccgagggg acccgacagg cccgaaggaa tagaagaaga 8400
    aggtggagag agagacagag acagatccat tcgattagtg aacggatcct tggcacttat 8460
    ctgggacgat ctgcggagcc tgtgcctctt cagctaccac cgcttgagag acttactctt 8520
    gattgtaacg aggattgtgg aacttctggg acgcaggggg tgggaagccc tcaaatattg 8580
    gtggaatctc ctacaatatt ggagtcagga gctaaagaat agtgctgtta gcttgctcaa 8640
    tgccacagcc atagcagtag ctgaggggac agatagggtt atagaagtag tacaaggagc 8700
    ttgtagagct attcgccaca tacctagaag aataagacag ggcttggaaa ggattttgct 8760
    ataagctcga aacaaccggt acctctagaa ctatagctag cagatctttt tccctctgcc 8820
    aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa 8880
    tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat 8940
    gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat 9000
    gccatatgct ggctgccatg aacaaaggtg gctataaaga ggtcatcagt atatgaaaca 9060
    gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt 9120
    tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt 9180
    actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt 9240
    atgaagatcc ctcgacctgc agcccaagct tggcgtaatc atggtcatag ctgtttcctg 9300
    tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 9360
    aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 9420
    ctttccagtc gggaaacctg tcgtgccagc ggatccgcat ctcaattagt cagcaaccat 9480
    agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 9540
    gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga 9600
    gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac 9660
    ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 9720
    aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 9780
    catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 9840
    ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 9900
    gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 9960
    caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 10020
    tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 10080
    gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 10140
    ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 10200
    cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg 10260
    tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 10320
    tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 10380
    cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 10440
    agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 10500
    agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 10560
    gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 10620
    aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 10680
    ggattttggt catgagatta tca 10703
    SEQ ID NO: 26 gtcgacggat cgggagatca attccggcac ctgtcctacg agttgcatga taaagaagac 60
    (pCIGO-VSV.G agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt 120
    plasmid) gaaggctctc aagggcatcg gtcgatgcag gaaaaggaca agcagcgaaa attcacgccc 180
    ccttgggagg tggcggcata tgcaaaggat agcactccca ctctactact gggtatcata 240
    tgctgactgt atatgcatga ggatagcata tgctacccgg atacagatta ggatagcata 300
    tactacccag atatagatta ggatagcata tgctacccag atatagatta ggatagccta 360
    tgctacccag atataaatta ggatagcata tactacccag atatagatta ggatagcata 420
    tgctacccag atatagatta ggatagccta tgctacccag atatagatta ggatagcata 480
    tgctacccag atatagatta ggatagcata tgctatccag atatttgggt agtatatgct 540
    acccagatat aaattaggat agcatatact accctaatct ctattaggat agcatatgct 600
    acccggatac agattaggat agcatatact acccagatat agattaggat agcatatgct 660
    acccagatat agattaggat agcctatgct acccagatat aaattaggat agcatatact 720
    acccagatat agattaggat agcatatgct acccagatat agattaggat agcctatgct 780
    acccagatat agattaggat agcatatgct atccagatat ttgggtagta tatgctaccc 840
    atggcaacat tagcccaccg tgctctcagc gacctcgtga atatgaggac caacaaccct 900
    gtgcttggcg ctcaggcgca agtgtgtgta atttgtcctc cagatcgcag caatcgcgcc 960
    cctatcttgg cccgcccacc tacttatgca ggtattcccc ggggtgccat tagtggtttt 1020
    gtgggcaagt ggtttgaccg cagtggttag cggggttaca atcagccaag ttattacacc 1080
    cttattttac agtccaaaac cgcagggcgg cgtgtggggg ctgacgcgtg cccccactcc 1140
    acaatttcaa aaaaaagagt ggccacttgt ctttgtttat gggccccatt ggcgtggagc 1200
    cccgtttaat tttcgggggt gttagagaca accagtggag tccgctgctg tcggcgtcca 1260
    ctctctttcc ccttgttaca aatagagtgt aacaacatgg ttcacctgtc ttggtccctg 1320
    cctgggacac atcttaataa ccccagtatc atattgcact aggattatgt gttgcccata 1380
    gccataaatt cgtgtgagat ggacatccag tctttacggc ttgtccccac cccatggatt 1440
    tctattgtta aagatattca gaatgtttca ttcctacact agtatttatt gcccaagggg 1500
    tttgtgaggg ttatattggt gtcatagcac aatgccacca ctgaaccccc cgtccaaatt 1560
    ttattctggg ggcgtcacct gaaaccttgt tttcgagcac ctcacataca ccttactgtt 1620
    cacaactcag cagttattct attagctaaa cgaaggagaa tgaagaagca ggcgaagatt 1680
    caggagagtt cactgcccgc tccttgatct tcagccactg cccttgtgac taaaatggtt 1740
    cactaccctc gtggaatcct gaccccatgt aaataaaacc gtgacagctc atggggtggg 1800
    agatatcgct gttccttagg acccttttac taaccctaat tcgatagcat atgcttcccg 1860
    ttgggtaaca tatgctattg aattagggtt agtctggata gtatatacta ctacccggga 1920
    agcatatgct acccgtttag ggttaacaag ggggccttat aaacactatt gctaatgccc 1980
    tcttgagggt ccgcttatcg gtagctacac aggcccctct gattgacgtt ggtgtagcct 2040
    cccgtagtct tcctgggccc ctgggaggta catgtccccc agcattggtg taagagcttc 2100
    agccaagagt tacacataaa ggcaatgttg tgttgcagtc cacagactgc aaagtctgct 2160
    ccaggatgaa agccactcaa gggatcttca atattggcca ttagccatat tattcattgg 2220
    ttatatagca taaatcaata ttggctattg gccattgcat acgttgtatc tatatcataa 2280
    tatgtacatt tatattggct catgtccaat atgaccgcca tgttggcatt gattattgac 2340
    tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 2400
    cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 2460
    gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 2520
    atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 2580
    aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 2640
    catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 2700
    catggtgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg 2760
    atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 2820
    ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt 2880
    acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatca ctagaagctt 2940
    tattgcggta gtttatcaca gttaaattgc taacgcagtc agtgcttctg acacaacagt 3000
    ctcgaactta agctgcagaa gttggtcgtg aggcactggg caggtaagta tcaaggttac 3060
    aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg 3120
    tttctgatag gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt 3180
    ccactcccag ttcaattaca gctcttaagg ctagagtact taatacgact cactataggc 3240
    tagcggtacc gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcaacagag 3300
    atcgatctgt ttccttgaca ctatgaagtg ccttttgtac ttagcctttt tattcattgg 3360
    ggtgaattgc aagttcacca tagtttttcc acacaaccaa aaaggaaact ggaaaaatgt 3420
    tccttctaat taccattatt gcccgtcaag ctcagattta aattggcata atgacttaat 3480
    aggcacagcc atacaagtca aaatgcccaa gagtcacaag gctattcaag cagacggttg 3540
    gatgtgtcat gcttccaaat gggtcactac ttgtgatttc cgctggtatg gaccgaagta 3600
    tataacacag tccatccgat ccttcactcc atctgtagaa caatgcaagg aaagcattga 3660
    acaaacgaaa caaggaactt ggctgaatcc aggcttccct cctcaaagtt gtggatatgc 3720
    aactgtgacg gatgccgaag cagtgattgt ccaggtgact cctcaccatg tgctggttga 3780
    tgaatacaca ggagaatggg ttgattcaca gttcatcaac ggaaaatgca gcaattacat 3840
    atgccccact gtccataact ctacaacctg gcattctgac tataaggtca aagggctatg 3900
    tgattctaac ctcatttcca tggacatcac cttcttctca gaggacggag agctatcatc 3960
    cctgggaaag gagggcacag ggttcagaag taactacttt gcttatgaaa ctggaggcaa 4020
    ggcctgcaaa atgcaatact gcaagcattg gggagtcaga ctcccatcag gtgtctggtt 4080
    cgagatggct gataaggatc tctttgctgc agccagattc cctgaatgcc cagaagggtc 4140
    aagtatctct gctccatctc agacctcagt ggatgtaagt ctaattcagg acgttgagag 4200
    gatcttggat tattccctct gccaagaaac ctggagcaaa atcagagcgg gtcttccaat 4260
    ctctccagtg gatctcagct atcttgctcc taaaaaccca ggaaccggtc ctgctttcac 4320
    cataatcaat ggtaccctaa aatactttga gaccagatac atcagagtcg atattgctgc 4380
    tccaatcctc tcaagaatgg tcggaatgat cagtggaact accacagaaa gggaactgtg 4440
    ggatgactgg gcaccatatg aagacgtgga aattggaccc aatggagttc tgaggaccag 4500
    ttcaggatat aagtttcctt tatacatgat tggacatggt atgttggact ccgatcttca 4560
    tcttagctca aaggctcagg tgttcgaaca tcctcacatt caagacgctg cttcgcaact 4620
    tcctgatgat gagagtttat tttttggtga tactgggcta tccaaaaatc caatcgagct 4680
    tgtagaaggt tggttcagta gttggaaaag ctctattgcc tcttttttct ttatcatagg 4740
    gttaatcatt ggactattct tggttctccg agttggtatc catctttgca ttaaattaaa 4800
    gcacaccaag aaaagacaga tttatacaga catagagatg aaccgacttg gaaagtaact 4860
    caaatcctgc acaacagatt cttcatgttt ggaccaaatc aacttgtgat accatgctca 4920
    aagaggcctc aattatattt gagtttttaa tttttatgga attctgcaga tatccatcac 4980
    actggcggcc gctcgagcat gcatctagag ggccctattc tatagtgtca cctaaatgct 5040
    agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 5100
    tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 5160
    gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 5220
    caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 5280
    tctatggctt ctgaggcgga aagaaccagc tgcattaatg aatcggccaa cgcgcgggga 5340
    gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 5400
    tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 5460
    aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 5520
    gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 5580
    aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 5640
    ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 5700
    tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 5760
    tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 5820
    ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 5880
    tatcgccact ggcaggagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 5940
    ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 6000
    tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 6060
    aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 6120
    aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 6180
    aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 6240
    ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 6300
    acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 6360
    ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 6420
    gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 6480
    taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 6540
    tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 6600
    gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 6660
    cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 6720
    aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 6780
    cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 6840
    tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 6900
    gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 6960
    tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 7020
    gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 7080
    ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 7140
    cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 7200
    agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 7260
    gggttccgcg cacatttccc cgaaaagtgc cacctgac 7298
  • Expression of CD86 and 4-1BBL on engineered MOLM-14 aAPCs (also referred to herein as aMOLM14 aAPCs) was confirmed using flow cytometry (Canto II flow cytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA), with results shown in FIG. 12. aMOLM-14 aAPCs were γ-irradiated at 100 Gy and frozen.
    • Example 4—Expansion of Tumor Infiltrating Lymphocytes Using MOLM-14 Artificial Antigen Presenting Cells
  • Engineered MOLM-14 cells were gamma-irradiated at 100 Gy before co-culturing with TILs. REPs were initiated by culturing TILs with irradiated, engineered MOLM-14 cells at 1:100 ratios in CM2 media containing OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) for 14 days. At REP harvest, the TIL expansion rates, phenotype for activation and differentiation stage markers, metabolism rate, cytotoxicity and re-rapid expansion protocol (re-REP) assay were measured.
  • The results are shown in FIG. 13, FIG. 14, FIG. 15, and FIG. 16, where two expansions for two sets of patient TILs are compared. The results with the CD86/4-1BBL modified MOLM-14 cells (labeled “TIL+Engineered MOLM14+OKT3”) are comparable to the PBMC feeders (labeled “TIL+Feeders+OKT3”).
  • The results at day 14 are compared in FIG. 17, where results from two additional patient TILs are shown. The results indicate that MOLM-14 cells that were engineered with CD86 and 4-1BBL showed similar TIL expansion in the rapid expansion protocol when compared with allogeneic feeder cells. However, TILs cultured with parental MOLM-14 did not expand.
  • In addition, TILs expanded against MOLM-14 maintained a TIL phenotype and showed potency to kill P815 cells as measured using BRLA, which is described in detail in Example 9. Briefly, luciferin-transduced P815 target cells and TILs of interest were co-cultured with and without anti-CD3 to determine whether tumor reactivity of TILs is through TCR activation (specific killing) or non-specific killing. Following 4 hours of incubation, luciferin was added to the wells and incubated for 5 minutes. After the incubation, bioluminescence intensity was read using a luminometer. The percentage cytotoxicity and percentage survival were calculated using the following formula: % Survival=(experimental survival-minimum)/(maximum signal-minimum signal)×100 or % Cytotoxicity=100−(% Survival).
  • In FIG. 18, the results of expansions performed with low ratios of TILs to MOLM-14 aAPCs are shown in comparison to the results of expansions with PBMC feeders. TILs (2×104) were cultured at different TIL to aAPC or PBMC ratios (1:10, 1:30, and 1:100, denoted “10”, “30”, and “100”, respectively) with parental MOLM-14 (“MOLM14”) cells, MOLM-14 cells transduced to express CD86 and 4-1BBL (“aMOLM14”), or PBMC feeders (“PBMC+”), each with OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) in a 24-well G-Rex plate. A control was performed using only OKT-3 (30 ng/mL) and IL-2 (3000 IU/mL) (“PBMC-”). Each condition was cultured in triplicate. Cultures were fed with fresh media and IL-2 on Day 4 and 7. Viable cells were counted on Day 7. FIG. 18 shows the mean plus standard deviation (SD) of viable cell numbers counted on Day 11, with ap-value calculated by the student t-test. Additional control experiments were performed using TILs alone, PBMCs alone, and aMOLM-14 cells alone, all of which resulted in undetectable cell numbers (data not shown). The results show that a ratio of 1:100 (TIL:aMOLM14) with OKT-3 and IL-2 yields a similar expansion when compared to PBMC feeders with OKT-3 and IL-2 (p=0.0598).
  • In FIG. 19, the results of expansions performed with higher ratios of TILs to MOLM-14 aAPCs, and otherwise performed as described above for FIG. 18, are shown in comparison to the results of expansions with PBMC feeders. At a ratio of 1:300, the CD86/4-1BBL modified MOLM-14 aAPCs with OKT-3 and IL-2 significantly outperform PBMC feeders with OKT-3 and IL-2. These results were verified using different TIL batches in repeat experiments shown in FIG. 20 and FIG. 21. In particular, as seen in FIG. 21, TIL to aMOLM14 ratios of 1:200 show enhanced TIL expansion compared to PBMC feeders under the same conditions. These results confirm that aMOLM14 aAPCs are unexpectedly superior in terms of expanding the TIL numbers than PBMCs particularly when using TIL:aMOLM14 ratios of 1:200 to 1:300.
  • In FIG. 22 and FIG. 23, TILs expanded with aMOLM14 or PBMC were compared by flow cytometry analysis to confirm that the TILs exhibited a similar phenotype and would be expected to perform similarly upon reinfusion into a patient. Briefly, TILs were first stained with L/D Aqua to determine viability. Next, cells were surface stained with TCR α/β PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28PE, CD27 APC-C7, and CD57-PerCP-Cy5.5. Phenotype analysis was done by gating 10,000 to 100,000 cells according to forward light scattering (FSC)/side light scattering (SSC) using a Canto II flow cytometer (Becton, Dickinson, and Co., Franklin Lakes, N.J., USA). Data was analyzed by Cytobank software to create sunburst diagrams and SPADE (Spanning Tree Progression of Density Normalized Event) analyses. Gates were set based on fluorescence minus one (FMO) controls. TILs expanded against aMOLM14 increases CD8+ TILs when compared to PBMC feeders. Without being bound by theory, this enhanced CD8+ TIL percentage may be due to the presence of 4-1BBL engineered to MOLM14. There is no difference in the expression of CD28, CD57, and CD27 differentiation markers. Additional flow cytometry data is shown in FIG. 24, and depicts a flow cytometry contour plot showing a memory subset (CD45RA+/−, CCR7+/−) gated on Live, TCR α/β+, CD4+ or CD8+ TILs, indicating that the memory subset obtained with PBMC feeders is replicated by the aMOLM14 aAPCs.
  • The CD4 and CD8 SPADE tree of TILs expanded with aMOLM14 aAPCs or PBMC feeders using CD3+ cells is shown in FIG. 25 and FIG. 26. The color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRG1 and TIGIT. Without being bound by theory, the results show that two batches of TILs expanded against aMOLM14 had undergone activation, but there was no difference in MFI between the aMOLM14 aAPCs and PBMC feeders, indicating that the aMOLM14 aAPCs effectively replicate the TIL phenotypic results obtained with PBMC feeders.
  • TILs expanded against aMOLM14 or PBMC were also analyzed for metabolic profiles. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TILs after expansion with irradiated PBMC feeders or aMOLM14 aAPCs were measured using a dual mitochondrial-glycolytic stress test. Briefly, cells were washed in assay medium (XF Assay Medium, Agilent Technologies, Santa Clara, Calif., USA), supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, at pH 7.4, and then 1×105 viable cells were plated onto an adhesive-coated (Cell-Tak™, Corning) XFp cell culture microplate. Plates were spun to adhere the cells to the plate, then equilibrated at 37° C. in a humidified, non-CO2 incubator prior to analysis of cellular metabolism. Mitochondrial and glycolytic stress test experiments were performed using a Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, Calif., USA), sequentially injecting the following compounds at specified intervals for simultaneous analysis of mitochondrial and glycolytic respiration of the cells: 1 μM oligomycin; 0.5 μM FCCP; 50 mM 2-deoxyglucose; and 0.5 μM each of rotenone and antimycin A. Results were analyzed using WAVE v2.3.0 software (Agilent Technologies, Santa Clara, Calif., USA) and GraphPad Prism v6.07 graphing software and are shown in FIG. 27 and FIG. 28, where points represent mean±SEM measured in triplicate. Both TILs grown with aMOLM14 aAPCs and PBMC feeders show similar oxphos and glycolysis behavior. This data suggests that aMOLM14 does not alter the metabolic programming of TILs when compared with PBMC feeders.
    • Example 5—Preparation of EM-3 Artificial Antigen Presenting Cells (aEM3 aAPCs)
  • EM-3 cells were obtained from Creative Bioarray, Inc. (Shirley, N.Y., USA). To develop an EM-3 based artificial APC, EM-3 cell lines were engineered with CD86, 4-1BBL, and antibody against IgG Fc region (Clone 7C12 or Clone 8B3). Human CD86 and human 4-1BBL/CD137 genes were cloned into commercially-available PLV430G and co-transfected with PDONR221 vectors (Invitrogen) using a lentiviral transduction method. The gateway cloning method was used as described in Katzen, Expert Opin. Drug Disc. 2007, 4, 571-589, to clone hCD86 and hCD137L genes onto the PLV430G and PDONR221 vectors. The 293T cell line was used for lentiviral production, and transduced to EM-3 cell lines. The transfected cells were sorted (S3e Cell Sorter, BioRad, Hercules, Calif., USA) using APC-conjugated CD86 and PE-conjugated CD137L to isolate and enrich the cells. The enriched cells were checked for purity by flow cytometry. Single-chain Fv (scFv) antibody clones designated 7C12 and 8B3 were generated against Fc of mouse IgG1, IgG2a and IgG2b (Viva Biotech Ltd., Chicago, Ill., USA). The amino acid sequences of these scFv clones are given in Table 7 (SEQ ID NO:27 and SEQ ID NO:28). The generated scFv clones were screened for Fc binding efficiency against OKT-3, engineered towards pLV4301G containing eGFP as co-reporter to produce lentivirus. The 293T cell line was used for packaging and lentiviral production. Engineered EM-3 (CD86/CD137L) cells were transduced using the lentiviral system and sorted using eGFP. EM37C12CD86CD137L and EM38B3CD86CD137L were regularly assessed for the consistent expression of each transduced molecule by flow cytometry.
  • TABLE 7
    Amino acid sequences of scFv clones 7C12 and 8B3.
    Identifier
    (Description) Sequence (One-Letter Amino Acid Symbols)
    SEQ ID NO: 27 QVQLVQSGGG LVKPGGSLRL SCAASGFNFN DQYMSWIRQA PGKGLEWVSF ISGSGGTTYY 60
    (mFC-7C12 TDSVKGRFTI SRDNTKDSLY LQMNSLTVED TAVYYCARGG NYYTSVGRGT LVTVSAGGGG 120
    scFv) SGAPDIQMTQ SPGTLSLSPG ERAILSCRAS QSVSGYLAWY QQKPGQAPRL LIYGASSRAT 180
    GIPDRFSGSG SGTDFTLTIS SLRPEDIGTY YCKQYINAPF TFGGGTKVEI K 231
    SEQ ID NO: 28 QVQLQQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGW ISPYNGNTDY 60
    (mFC-8B3 scFv) AQKVQGRVTL TTDTSTSTAY MELRSLRSDD TAVYYCATGG GTWYSDLWGR GTLVTVSAGG 120
    GGSGGGGSGG GGSGAPEIVL TQSPSTLSAS VGDRVSITCR ASQSIGGSLA WYQQKPGKAP 180
    KLLISEASTL ERGVPSRFSG SGSGTDFTLT ISSLQPEDVA TYYCQKYNSV PLTFGPGTKV 240
    EIK 243
  • A non-limiting protocol for preparation of aEM3 aAPCs, which may also be adapted for use with aMOLM14 aAPCs, is described in the following paragraphs.
  • Molecular cloning of plasmids of interest may be performed as follows. To generate DONR vector the following cocktail may be used: B site flanked PCR product or destination vector (e.g., Gateway-adapted lentivector) 50-100 μg; DONR vector (e.g., pDONR222) 50-100 μg; BR Clonase II (Life Technologies) 1 μL; and TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q.s. to bring volume to 5 μL). Incubate at room temperature for at least 1 hour. After incubation perform bacterial transformation either by heat shock method or electroporation. To generate destination vector, the following cocktail may be used: recombined pDONR vector (e.g., pDON222-geneX) 50-100 μg, destination vector (e.g., Gateway adapted lentivector) 50-100 μg, LR Clonase II (Life Technologies) 1 μL, and TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q.s. to bring volume to 5 μL). Incubate at room temperature for at least 1 hour. After incubation, perform bacterial transformation either by chemical competent transformation/heat shock method.
  • Transformation and selection of the cloned plasmid may be performed as follows. The chemical competent transformation method may be performed as follows. Prepare nutrient agar plates (LB-Lennox or YT) with antibiotic for selection. Ensure that Recovery Medium (supplied by Lucigen, Middleton, Wis., USA) is readily available at room temperature. Optionally, sterile culture tubes may be chilled on ice (e.g., 17 mm×100 mm tubes (14 mL tube)), one tube for each transformation reaction). Remove E. cloni cells (Lucigen) from an −80° C. freezer and thaw completely on wet ice (5-15 minutes). Optionally add 40 μL of E. cloni cells to the chilled culture tube. Add 1-4 μL of DNA sample to the 40 μL of cells. Flick with finger (do not pipet up and down to mix, which can introduce air bubbles and warm the cells). Incubate the cell/DNA mixture on ice for 30 minutes. Heat shock cells by placing the culture tubes in a 42° C. water bath for 45 seconds. Return the 1.7 mL tube or culture tubes to ice for 2 minutes. Add 350 μL room temperature Recovery Medium to the cells or 960 μL of room temperature Recovery Medium to the cells in the culture tube. Place the tubes in a shaking incubator at 250 rpm for 1 hour at 37° C. Plate up to 100% of the transformation mixture on LB-Lennox or YT agar plates containing the appropriate antibiotic. The plating volume may need to be optimized depending on DNA. Incubate the plates overnight at 37° C. Transformed clones can be further grown in any rich culture medium (e.g., LB or TB).
  • Colonies for Miniprep (Qiagen, Inc., Valencia, Calif., USA) may be grown as follows. After colonies have formed from plating recovered transformation reaction of DNA manipulation (e.g. LR reaction), add 1 mL desired TB/antibiotics into desired number of 2 mL Eppendorf microtubes with punctured caps. Pick desired number of colonies using ART LTS 20 soft pipette tip (VWR 89031-352) or 10 μL Denville tip. Place tip in 2 mL Eppendorf microtube with punctured cap. Cut the tip so that it fits in tube, close cap, and place tubes on shaker (purple 15 mL tube holder with VWR brand 15 mL tubes). Shake overnight (for no more than 16 hours) at 225 rpm/37° C. After overnight incubation, place each tip in a 1 mL tube in a ClavePak 96 plate from Denville with sterile water in it (to save the tip for making bacterial stock production after the plasmids are screened and selected). Perform Miniprep according to the Qiagen Mini prep kit protocol (Qiagen, Inc., Valencia, Calif., USA). Once the plasmids are eluted, restriction digestion is performed to select the right clones. After selecting the plasmids, use the tips saved from the same plasmids clone to grow the E. coli with the plasmid to make bacterial stock.
  • Lentiviral production may be performed as follows. The following media composition is prepared: 500 mL DMEM/F12 (Sigma); 25 mL FBS Heat Inactivated (HI) (Hyclone); 10 mM HEPES (Life Technologies); 1× Primocin (Invivogen); 1× Plasmocin (Invivogen); and 1×2-mermactoethanol (Life Technologies). Harvest T75 flasks (Thermo Fisher Scientific) containing 90% confluent 293T cells. Aspirate media. Add 10 ml PBS, rinse gently and aspirate off. Add 2 mL TrypLE Express (Life Technologies) and evenly distribute it over the cell layer, let sit for 3-5 minutes at 37° C. (cell culture incubator). Add 10 mL media and disperse cells by pipetting up and down. Combine if there are multiple flasks. Count cells. If using a hemacytometer to determine concentration, cells/mL=(# counted cells×dilution factor×104). To split back into T75 flasks, determine the time at which the cells will need to be fully confluent and dilute accordingly. (Cells double every 16-18 hours, so 3 days=1/27 dilution). Generally, a multiplication factor of 2.5 per day may be used where confluence is 2×105 cells/cm2. Bring volume up to 25 mL of media. To plate for titration of stocks, each well of the assay requires 5×104 cells in 0.4 mL of media. Adjust 293T cells to 2×104/mL in media. Plate 1 mL per well in a 24 well plate. For example, cells plated Monday may be infected on Tuesday and run on the flow cytometer on Friday, and cells plated Thursday are infected Friday and run on the flow cytometer on Monday. To plate for packaging transfections, seed T75 flasks with 6.8×106 cells one day before transfection or 1.7×106 cells on the morning of transfection. (Seeding on the day of transfection may reduce the variation in transfection efficiency). Bring volume in flask up to 25 mL with media. For example, flasks set up Monday are transfected Tuesday, and virus is collected on Thursday and Friday. In some cases (e.g., high titering constructs), the second collection can be omitted. To package lentiviral vectors, each T75 flask transfection requires 2 μg Baculo p35 plasmid (optional; only necessary if packaging a death gene), 2 μg VSV.G env plasmid (e.g., pMD2.G or PCIGO VSV-G); 4.7 μs Gag/polymerase plasmid (e.g., psPAX2 or pCMV-deltaR8.91), and 2.3 μg of the lentiviral vector described above. Determine the amount of VSV and R8.2/9.1 (+/−Baculo) plasmids needed for all samples (make a mixture of these DNAs if preparing many samples). Each T75 transfection requires 90 μL LipofectAmine 2000 (Thermo Fisher Scientific) in 2 mL Opti-MEM medium (Thermo Fisher Scientific). Make a mix containing enough Opti-Mem and LipofectAmine 2000 for all samples. Mix gently and let sit for 5 minutes at room temp, and label as tube A. For each transfection, add packaging DNA and specific lentiviral vector DNA to 500 μL room temperature Opti-MEM medium to a microtube and mix, and label as tube B. Add the 500 μL of DNA from tube B to the 2 mL of the LipofectAmine 2000 mix in tube A and mix gently, and incubate for 20-30 minutes at room temperature. Aspirate media from packaging flasks. Add the 2.5 mL of DNA/Lipofectamine complexes to 5 mL Opti-MEM medium and add to cells (do not pipet directly on cells since 293T cells are only semi adherent). Process plates in small groups to avoid drying. Incubate overnight and change media the next day in the morning. Collect the supernatant after 24 hours of media change. Supernatants can be harvested in a single collection, 48 hours after transfection or as 2 collections, 48 and 72 hours after transfection (in which case, harvests are pooled). If double collection is desired, collect supernatants by pipet on the first day, and replace with 20 mL of fresh media. To avoid flasks drying, work with only 5 flasks at a time. Keep collected supernatants at 4° C. until pooling the next day. Cool supernatants again on the following day and pool as appropriate. Spin the supernatants at 2000 rpm for 5 minutes to sediment any contaminating 293T cells. Filter harvested supernatants through a 0.45 μm or 0.8 μm filter unit containing a pre-filter disc. Use a large enough filtration unit so that the filtration speed is relatively fast. Store at 4° C. until ready to concentrate.
  • Virus may be concentrated using the PEG-it method (System Biosciences, Inc., Palo Alto, Calif. 94303) for longer-term storage at −80° C. Collect the supernatant from the transfection plates. Spin down the cell debris in the supernatant. The supernatant may also be filtered to completely remove any packaging cells. Add an amount of PEG-it solution equal to a quarter of the volume of supernatant to the supernatant. Incubate the suspension at 4° C. for overnight. Centrifuge at 3500 rpm (1500 g) at 4° C. for 30 minutes. Remove supernatant and centrifuge at 3500 rpm at 4° C. for 5 minutes. Remove remaining supernatant. Resuspend virus in desired amount of phosphate-buffered saline (PBS) and freeze aliquots at −80° C.
  • Transduction of cell line using lentivirus may be performed as follows. Adjust cells to be transduced to either: 1×106 suspension cells per well in 24 well plate (1 well per transduction) or 50% confluence for adherent cells (1 well per transduction) in 24 well plate. For suspended cells, adjust concentration of cells to 1×107/mL and plate 100 μL per well in 24 well plate (1 well per transduction). For adherent cells, plate to achieve 50% confluence on day of transduction based on cells/cm′ (e.g., for 293T cells, confluence=2×105/cm′). Total volume of transduction per well should be approximately 500 μL with 3-10 μg/mL Polybrene (Hexadimethrine bromide, Sigma-Aldrich Co., St. Louis, Mo., USA). The amount of concentrated virus added will depend on the MOI (multiplicity of infection) desired. A typical MOI is 10:1 but this may vary depending on cell type. The transfection well should contain 100 μL of standard media containing either 1×106 suspension cells or 50% confluent cells. For a MOI of 10:1 (e.g., virus activity is 1×108 IU/mL and the target is to infect 1×106 cells, then 1×107 virions or 100 μL of virus is needed). Add standard media to 500 μL. Add Polybrene to 3 μg/mL (primary cells) to 10 μg/mL (tumor cell lines). Spin plate(s) at 1800 rpm for 1.5 to 2 hours at 30° C. Incubate plate(s) at 37° C./5% CO2 using a Tissue Culture incubator for 5 hours to overnight. Change media. After 72 hours of transduction, if enough cells are available, perform flow cytometric analysis to test the transduction efficiency.
  • Sorting of aAPCs may be performed as follows. Culture the cells in the media described above until the cell count reaches a minimum of 10-20 million. Take 1×106 cells for each condition and stain with the antibodies for the proteins transduced. Wash the cells and analyze by flow cytometry to test the stability of transduction. Once the expression of protein of interest has been analyzed and confirmed, prepare the rest of the cells for sorting. Sort the cells in an S3 sorter by gating on markers of interest. Culture the sorted cells using the media mentioned above. Before freezing the vial, test the stability of the protein expression of interest. Use Recovery cell culture Freezing media (Invitrogen), to make the cell bank of the same cells. Cells may be banked after each transduction and sorting procedure.
  • Nucleotide sequence information for the 7C12 and 8B3 scFv clones (SEQ ID NO:29 and SEQ ID NO:30) and their lentiviral vectors are given in Table 8. Sequences used for generation of the pLV4301G 7C12 scFv mIgG hCD8 flag vector are provided as SED IQ NO:31 to SEQ ID NO:34 and are depicted in FIG. 29 to FIG. 32. Sequences used for generation of the pLV4301G 8B3 scFv mIgG hCD8 flag vector are provided as SEQ ID NO:35 to SEQ ID NO:38 and are depicted in FIG. 33 to FIG. 36.
  • TABLE 8
    Nucleotide sequences for preparation of lentivirus for transduction of aAPCs.
    Identifier
    (Description) Sequence
    SEQ ID NO: 29 caggtgcagc tggtgcagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60
    (mFC-7C12 tcctgtgcag cctctggatt caatttcaat gaccagtaca tgagttggat ccgccaggct 120
    scFv) ccagggaagg ggctggagtg ggtttcattc attagtggta gtggtggtac cacatactac 180
    acagactctg tgaagggccg gttcaccatc tccagggaca acaccaagga ctcattgtat 240
    ttgcaaatga acagcctgac agtcgaggac acggccgtgt actactgtgc gagaggaggg 300
    aattattata cttcggtggg ccggggcacc ctggtcaccg tctcggccgg tggcggcgga 360
    tctggcgcgc cagacatcca gatgacccag tctccaggca ccctgtcttt gtctccaggg 420
    gaaagagcca tcctctcctg cagggccagt cagagtgtta gcggctacct agcctggtat 480
    caacagaaac ctggccaggc tcccaggctc ctcatctatg gtgcatccag cagggccact 540
    ggcatcccag acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc 600
    agcctgcggc ctgaagatat tggaacatat tactgtaaac agtacattaa tgccccattc 660
    actttcggcg gcgggaccaa ggtggagatc aaa 693
    SEQ ID NO: 30 caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60
    (mFC-8B3 scFv) tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120
    cctggacaag ggcttgagtg gatgggatgg atcagccctt acaatggtaa cacagattat 180
    gcacagaagg tccagggcag agtcaccttg accacagaca catccacgag cacagcctac 240
    atggagctga ggagcctgag atccgacgac acggccgtgt attactgtgc gacaggtggc 300
    gggacctggt actccgatct ctggggccgt ggcaccctgg tcaccgtctc ggccggtggc 360
    ggtggcagcg gcggtggtgg gtccggtggc ggcggatctg gcgcgccaga aattgtgctg 420
    actcagtctc cctccaccct gtctgcatct gtaggagaca gagtcagcat cacttgccgg 480
    gccagtcaga gtattggtgg gtcgttggcc tggtatcaac aaaagccagg gaaagcccct 540
    aagctcctga tctctgaggc gtctacttta gagaggggcg tcccatcaag attcagcggc 600
    agtggatctg ggacagattt cactctcacc atcaggagcc tgcagcctga agatgttgca 660
    acttattact gtcaaaaata taacagtgtc ccgctcactt tcggccctgg gaccaaggtg 720
    gagatcaaa 729
    SEQ ID NO: 31 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60
    (destination gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120
    vector cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180
    pLV4301G) taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240
    ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300
    cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360
    tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420
    accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480
    agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540
    ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600
    agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660
    ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720
    tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780
    gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840
    cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900
    agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960
    tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020
    ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080
    aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140
    taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200
    agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260
    cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320
    aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380
    tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440
    cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500
    tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560
    tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620
    gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680
    ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740
    acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800
    taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860
    aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920
    tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980
    tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040
    ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100
    ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160
    gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220
    ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280
    ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340
    caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400
    agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460
    ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520
    agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580
    atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640
    cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700
    cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 2760
    aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 2820
    actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 2880
    gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 2940
    aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 3000
    gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 3060
    gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 3120
    cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 3180
    gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 3240
    tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 3300
    gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 3360
    tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 3420
    atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 3480
    gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 3540
    agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaatggat 3600
    ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa 3660
    gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg 3720
    tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat 3780
    atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc 3840
    tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc 3900
    tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag 3960
    agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg 4020
    acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact 4080
    ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag 4140
    tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat 4200
    caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag 4260
    ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct 4320
    gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt 4380
    cagctttctt gtacaaagtg gtgattcgag ttaattaagt taacgaattc cccccctctc 4440
    cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 4500
    tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 4560
    gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 4620
    gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 4680
    ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 4740
    aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 4800
    gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 4860
    aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct 4920
    ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 4980
    tttcctttga aaaacacgat gataatatgg ccacaaccat gggaggcgga agcggcggag 5040
    gctcccctcg aggcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 5100
    tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 5160
    agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 5220
    ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 5280
    accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 5340
    aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 5400
    tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 5460
    gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 5520
    ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 5580
    gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 5640
    tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 5700
    agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 5760
    acgagctgta caagtaacgc gtcccgggtc tagagctagc ggtaccatgc attacgtagt 5820
    cgacgactta attaagctag cctagtgcca tttgttcagt ggttcgtagg gctttccccc 5880
    actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 5940
    ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 6000
    ctaacaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 6060
    atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 6120
    ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 6180
    ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 6240
    ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 6300
    acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 6360
    ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 6420
    tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 6480
    acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 6540
    tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 6600
    cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 6660
    gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 6720
    cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 6780
    cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 6840
    aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 6900
    gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 6960
    ctgcgcacca gcaccatggc gcaatcacta gagcggggta cctttaagac caatgactta 7020
    caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat 7080
    tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca 7140
    gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 7200
    cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 7260
    atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct 7320
    tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt 7380
    tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 7440
    atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 7500
    ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 7560
    gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgga 7620
    tcccttgagt ggctttcatc ctggagcaga ctttgcagtc tgtggactgc aacacaacat 7680
    tgcctttatg tgtaactctt ggctgaagct cttacaccaa tgctggggga catgtacctc 7740
    ccaggggccc aggaagacta cgggaggcta caccaacgtc aatcagaggg gcctgtgtag 7800
    ctaccgataa gcggaccctc aagagggcat tagcaatagt gtttataagg cccccttgtt 7860
    aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 7920
    ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 7980
    tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 8040
    atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 8100
    attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 8160
    gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 8220
    agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 8280
    aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 8340
    cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 8400
    cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 8460
    actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 8520
    cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 8580
    ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 8640
    ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 8700
    gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 8760
    gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 8820
    ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 8880
    cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 8940
    caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 9000
    taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 9060
    cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 9120
    cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 9180
    gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 9240
    aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 9300
    cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 9360
    tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 9420
    acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 9480
    ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 9540
    ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 9600
    tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 9660
    tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 9720
    ctggcctttt gctggccttt ttgaagctgt ccctgatggt cgtcatctac ctgcctggac 9780
    agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg 9840
    aaggccatcc agcctcgcgt cg 9862
    SEQ ID NO: 32 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
    (donor vector attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
    1, pMK 7c12 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180
    anti mFC scFV gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240
    CoOp ECORV gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300
    SacII L1R5) acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 360
    aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg 420
    atgagcaatg cttttttata atgccaactt tgtacaaaaa agctgaacga tatcgccacc 480
    atgggcagca cagccattct ggccctgctg ctggcagtgc tgcagggcgt gtcagctcag 540
    gtgcagctgg tgcagtctgg cggcggactc gtgaaacctg gcggcagcct gagactgagc 600
    tgtgccgcca gcggcttcaa cttcaacgac cagtacatga gctggatccg gcaggcccct 660
    ggcaagggac tggaatgggt gtccttcatc agcggcagcg gcggcaccac ctactacacc 720
    gatagcgtga agggccggtt caccatcagc cgggacaaca ccaaggacag cctgtacctg 780
    cagatgaaca gcctgaccgt ggaagatacc gccgtgtact actgcgccag aggcggcaat 840
    tactacacca gcgtgggcag aggcaccctc gtgacagtgt ctgctggcgg aggcggatca 900
    ggcggcggag gatcaggggg aggcggaagc ggagcacccg atatccagat gacacagagc 960
    cccggcaccc tgtctctgag ccctggcgaa agagccatcc tgagctgcag agccagccag 1020
    agcgtgtccg gatacctggc ttggtatcag cagaagcccg gccaggcccc cagactgctg 1080
    atctatggcg ccaggaggag agccacaggc atccccgata gattcagcgg ctctggcagc 1140
    ggcaccgact tcaccctgac aatcagctcc ctgcggcccg aggacatcgg cacctactat 1200
    tgcaagcagt acatcaacgc ccccttcacc ttcggcggag gcaccaaggt ggaaatcaag 1260
    ccgcgggcca actttgtata caaaagtgga acgagaaacg taaaatgata taaatatcaa 1320
    tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat 1380
    ccagtcacta tgaatcaact acttagatgg tattagtgac ctgtactggg cctcatgggc 1440
    cttcctttca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaacatgg 1500
    tcatagctgt ttccttgcgt attgggcgct ctccgcttcc tcgctcactg actcgctgcg 1560
    ctcggtcgtt cgggtaaagc ctggggtgcc taatgagcaa aaggccagca aaaggccagg 1620
    aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgccgccc tgacgagcat 1680
    cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 1740
    gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 1800
    tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 1860
    tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accgcccgtt 1920
    cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 1980
    gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2040
    ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 2100
    ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 2160
    ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 2220
    agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 2280
    aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 2340
    atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 2400
    tctgacagtt attagaaaaa ttcatccagc agacgataaa acgcaatacg ctggctatcc 2460
    ggtgccgcaa tgccatacag caccagaaaa cgatccgccc attcgccgcc cagttcttcc 2520
    gcaatatcac gggtggccag cgcaatatcc tgataacgat ccgccacgcc cagacggccg 2580
    caatcaataa agccgctaaa acggccattt tccaccataa tgttcggcag gcacgcatca 2640
    ccatgggtca ccaccagatc ttcgccatcc ggcatgctcg ctttcagacg cgcaaacagc 2700
    tctgccggtg ccaggccctg atgttcttca tccagatcat cctgatccac caggcccgct 2760
    tccatacggg tacgcgcacg ttcaatacga tgtttcgcct gatgatcaaa cggacaggtc 2820
    gccgggtcca gggtatgcag acgacgcatg gcatccgcca taatgctcac tttttctgcc 2880
    ggcgccagat ggctagacag cagatcctga cccggcactt cgcccagcag cagccaatca 2940
    cggcccgctt cggtcaccac atccagcacc gccgcacacg gaacaccggt ggtggccagc 3000
    cagctcagac gcgccgcttc atcctgcagc tcgttcagcg caccgctcag atcggttttc 3060
    acaaacagca ccggacgacc ctgcgcgctc agacgaaaca ccgccgcatc agagcagcca 3120
    atggtctgct gcgcccaatc atagccaaac agacgttcca cccacgctgc cgggctaccc 3180
    gcatgcaggc catcctgttc aatcatactc ttcctttttc aatattattg aagcatttat 3240
    cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 3300
    ggggttccgc gcacatttcc ccgaaaagtg ccac 3334
    SEQ ID NO: 33 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
    (donor vector attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
    2, pMK hCD8a gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180
    scaffold TN L5 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240
    L2) gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300
    acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 360
    aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg 420
    atgagcaatg cttttttata atgcccaact ttgtatacaa aagtggcccg cggacaacaa 480
    cccctgcccc cagacctcct accccagccc ctacaattgc cagccagcct ctgagcctga 540
    ggcccgaggc ttgtagacct gctgctggcg gagccgtgca caccagagga ctggatttcg 600
    cctgcgacat ctacatctgg gcccctctgg ccggcacatg tggcgtgctg ctgctgagcc 660
    tcgtgatcac cctgtactgc ggctccacca gcggctccgg caagcccggc tctggcgagg 720
    gctccaccag cggcgactac aaggacgacg atgacaagta ataggatatc ggttcagctt 780
    tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg 840
    tcactatcag tcaaaataaa atcattattt ctgggcctca tgggccttcc tttcactgcc 900
    cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct 960
    tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt 1020
    aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 1080
    gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 1140
    tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 1200
    agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1260
    ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 1320
    taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1380
    gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1440
    gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1500
    ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 1560
    ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1620
    gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 1680
    caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 1740
    taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 1800
    aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttattag 1860
    aaaaattcat ccagcagacg ataaaacgca atacgctggc tatccggtgc cgcaatgcca 1920
    tacagcacca gaaaacgatc cgcccattcg ccgcccagtt cttccgcaat atcacgggtg 1980
    gccagcgcaa tatcctgata acgatccgcc acgcccagac ggccgcaatc aataaagccg 2040
    ctaaaacggc cattttccac cataatgttc ggcaggcacg catcaccatg ggtcaccacc 2100
    agatcttcgc catccggcat gctcgctttc agacgcgcaa acagctctgc cggtgccagg 2160
    ccctgatgtt cttcatccag atcatcctga tccaccaggc ccgcttccat acgggtacgc 2220
    gcacgttcaa tacgatgttt cgcctgatga tcaaacggac aggtcgccgg gtccagggta 2280
    tgcagacgac gcatggcatc cgccataatg ctcacttttt ctgccggcgc cagatggcta 2340
    gacagcagat cctgacccgg cacttcgccc agcagcagcc aatcacggcc cgcttcggtc 2400
    accacatcca gcaccgccgc acacggaaca ccggtggtgg ccagccagct cagacgcgcc 2460
    gcttcatcct gcagctcgtt cagcgcaccg ctcagatcgg ttttcacaaa cagcaccgga 2520
    cgaccctgcg cgctcagacg aaacaccgcc gcatcagagc agccaatggt ctgctgcgcc 2580
    caatcatagc caaacagacg ttccacccac gctgccgggc tacccgcatg caggccatcc 2640
    tgttcaatca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2700
    atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2760
    tttccccgaa aagtgccac 2779
    SEQ ID NO: 34 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60
    (Final vector gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120
    used for cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180
    lentiviral taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240
    production, ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300
    pLV4301G 7C12 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360
    scFV mIgG hCD8 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420
    flag) accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480
    agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540
    ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600
    agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660
    ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720
    tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780
    gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840
    cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900
    agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960
    tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020
    ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080
    aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140
    taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200
    agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260
    cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320
    aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380
    tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440
    cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500
    tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560
    tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620
    gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680
    ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740
    acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800
    taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860
    aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920
    tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980
    tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040
    ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100
    ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160
    gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220
    ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280
    ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340
    caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400
    agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460
    ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520
    agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580
    atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640
    cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700
    caccaacttt gtacaaaaaa gctgaacgat atcgccacca tgggcagcac agccattctg 2760
    gccctgctgc tggcagtgct gcagggcgtg tcagctcagg tgcagctggt gcagtctggc 2820
    ggcggactcg tgaaacctgg cggcagcctg agactgagct gtgccgccag cggcttcaac 2880
    ttcaacgacc agtacatgag ctggatccgg caggcccctg gcaagggact ggaatgggtg 2940
    tccttcatca gcggcagcgg cggcaccacc tactacaccg atagcgtgaa gggccggttc 3000
    accatcagcc gggacaacac caaggacagc ctgtacctgc agatgaacag cctgaccgtg 3060
    gaagataccg ccgtgtacta ctgcgccaga ggcggcaatt actacaccag cgtgggcaga 3120
    ggcaccctcg tgacagtgtc tgctggcgga ggcggatcag gcggcggagg atcaggggga 3180
    ggcggaagcg gagcacccga tatccagatg acacagagcc ccggcaccct gtctctgagc 3240
    cctggcgaaa gagccatcct gagctgcaga gccagccaga gcgtgtccgg atacctggct 3300
    tggtatcagc agaagcccgg ccaggccccc agactgctga tctatggcgc cagcagcaga 3360
    gccacaggca tccccgatag attcagcggc tctggcagcg gcaccgactt caccctgaca 3420
    atcagctccc tgcggcccga ggacatcggc acctactatt gcaagcagta catcaacgcc 3480
    cccttcacct tcggcggagg caccaaggtg gaaatcaagc cgcgggccaa ctttgtatac 3540
    aaaagtggcc cgcggacaac aacccctgcc cccagacctc ctaccccagc ccctacaatt 3600
    gccagccagc ctctgagcct gaggcccgag gcttgtagac ctgctgctgg cggagccgtg 3660
    cacaccagag gactggattt cgcctgcgac atctacatct gggcccctct ggccggcaca 3720
    tgtggcgtgc tgctgctgag cctcgtgatc accctgtact gcggctccac cagcggctcc 3780
    ggcaagcccg gctctggcga gggctccacc agcggcgact acaaggacga cgatgacaag 3840
    taataggata tcggttcagc tttcttgtac aaagttggga ttcgagttaa ttaagttaac 3900
    gaattccccc cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa 3960
    ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg 4020
    agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc 4080
    gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct 4140
    tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaaccgccc acctggcgac 4200
    aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc 4260
    cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta 4320
    ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg 4380
    cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaacgtcta ggccccccga 4440
    accacgggga cgtggttttc ctttgaaaaa cacgatgata atatggccac aaccatggga 4500
    ggcggaagcg gcggaggctc ccctcgaggc accatggtga gcaagggcga ggagctgttc 4560
    accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 4620
    gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 4680
    accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg 4740
    cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 4800
    cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 4860
    cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 4920
    gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 4980
    aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 5040
    cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 5100
    ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc 5160
    aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 5220
    atcactctcg gcatggacga gctgtacaag taacgcgtcc cgggtctaga gctagcggta 5280
    ccatgcatta cgtagtcgac gacttaatta agctagccta gtgccatttg ttcagtggtt 5340
    cgtagggctt tcccccactg tttggctttc agttatatgg atgatgtggt attgggggcc 5400
    aagtctgtac agcatcttga gtcccttttt accgctgtta ccaattttct tttgtctttg 5460
    ggtatacatt taaaccctaa caaaacaaag agatggggtt actctctaaa ttttatgggt 5520
    tatgtcattg gatgttatgg gtccttgcca caagaacaca tcatacaaaa aatcaaagaa 5580
    tgttttagaa aacttcctat taacaggcct attgattgga aagtatgtca acgaattgtg 5640
    ggtcttttgg gttttgctgc cccttttaca caatgtggtt atcctgcgtt gatgcctttg 5700
    tatgcatgta ttcaatctaa gcaggctttc actttctcgc caacttacaa ggcctttctg 5760
    tgtaaacaat acctgaacct ttaccccgtt gcccggcaac ggccaggtct gtgccaagtg 5820
    tttgctgacg caacccccac tggctggggc ttggtcatgg gccatcagcg catgcgtgga 5880
    accttttcgg ctcctctgcc gatccatact gcggaactcc tagccgcttg ttttgctcgc 5940
    agcaggtctg gagcaaacat tatcgggact gataactctg ttgtcctatc ccgcaaatat 6000
    acatcgtttc catggctgct aggctgtgct gccaactgga tcctgcgcgg gacgtccttt 6060
    gtttacgtcc cgtcggcgct gaatcctgcg gacgaccctt ctcggggtcg cttgggactc 6120
    tctcgtcccc ttctccgtct gccgttccga ccgaccacgg ggcgcacctc tctttacgcg 6180
    gactccccgt ctgtgccttc tcatctgccg gaccgtgtgc acttcgcttc acctctgcac 6240
    gtcgcatgga gaccaccgtg aacgcccacc aaatattgcc caaggtctta cataagagga 6300
    ctcttggact ctcagcaatg tcaacgaccg accttgaggc atacttcaaa gactgtttgt 6360
    ttaaagactg ggaggagttg ggggaggaga ttaggttaaa ggtctttgta ctaggaggct 6420
    gtaggcataa attggtctgc gcaccagcac catggcgcaa tcactagagc ggggtacctt 6480
    taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 6540
    gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt 6600
    ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 6660
    ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 6720
    actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta 6780
    gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga 6840
    gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 6900
    atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 6960
    atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct 7020
    aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 7080
    agaggccgag gccggatccc ttgagtggct ttcatcctgg agcagacttt gcagtctgtg 7140
    gactgcaaca caacattgcc tttatgtgta actcttggct gaagctctta caccaatgct 7200
    gggggacatg tacctcccag gggcccagga agactacggg aggctacacc aacgtcaatc 7260
    agaggggcct gtgtagctac cgataagcgg accctcaaga gggcattagc aatagtgttt 7320
    ataaggcccc cttgttaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata 7380
    ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 7440
    gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 7500
    acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 7560
    tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 7620
    agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 7680
    cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 7740
    aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 7800
    gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 7860
    agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 7920
    aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 7980
    gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 8040
    ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc 8100
    aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 8160
    aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 8220
    tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 8280
    agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 8340
    ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 8400
    ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 8460
    ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 8520
    acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 8580
    agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 8640
    ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 8700
    cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 8760
    gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 8820
    cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 8880
    gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 8940
    caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag 9000
    aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 9060
    tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 9120
    gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 9180
    ggccttttta cggttcctgg ccttttgctg gcctttttga agctgtccct gatggtcgtc 9240
    atctacctgc ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag 9300
    aagaatcata atggggaagg ccatccagcc tcgcgtcg 9338
    SEQ ID NO: 35 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60
    (destination gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120
    vector, cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180
    pLV4301G) taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240
    ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300
    cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360
    tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420
    accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480
    agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540
    ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600
    agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660
    ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720
    tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780
    gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840
    cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900
    agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960
    tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020
    ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080
    aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140
    taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200
    agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260
    cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320
    aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380
    tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440
    cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500
    tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560
    tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620
    gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680
    ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740
    acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800
    taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860
    aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920
    tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980
    tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040
    ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100
    ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160
    gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220
    ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280
    ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340
    caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400
    agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460
    ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520
    agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580
    atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640
    cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700
    cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt 2760
    aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc 2820
    actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat 2880
    gtgtggattt tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag 2940
    aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt 3000
    gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg 3060
    gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt 3120
    cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg 3180
    gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt 3240
    tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa 3300
    gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg 3360
    tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat 3420
    atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag 3480
    gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc 3540
    agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaatggat 3600
    ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa 3660
    gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg 3720
    tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat 3780
    atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc 3840
    tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc 3900
    tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag 3960
    agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg 4020
    acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact 4080
    ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag 4140
    tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat 4200
    caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag 4260
    ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct 4320
    gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt 4380
    cagctttctt gtacaaagtg gtgattcgag ttaattaagt taacgaattc cccccctctc 4440
    cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg 4500
    tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg 4560
    gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag 4620
    gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt 4680
    ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc 4740
    aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga 4800
    gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga 4860
    aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct 4920
    ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 4980
    tttcctttga aaaacacgat gataatatgg ccacaaccat gggaggcgga agcggcggag 5040
    gctcccctcg aggcaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 5100
    tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 5160
    agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 5220
    ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 5280
    accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 5340
    aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 5400
    tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 5460
    gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 5520
    ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 5580
    gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 5640
    tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 5700
    agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 5760
    acgagctgta caagtaacgc gtcccgggtc tagagctagc ggtaccatgc attacgtagt 5820
    cgacgactta attaagctag cctagtgcca tttgttcagt ggttcgtagg gctttccccc 5880
    actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 5940
    ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 6000
    ctaacaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt 6060
    atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 6120
    ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 6180
    ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat 6240
    ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 6300
    acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 6360
    ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc 6420
    tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 6480
    acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc 6540
    tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 6600
    cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 6660
    gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 6720
    cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 6780
    cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc 6840
    aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 6900
    gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt 6960
    ctgcgcacca gcaccatggc gcaatcacta gagcggggta cctttaagac caatgactta 7020
    caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat 7080
    tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca 7140
    gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 7200
    cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 7260
    atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct 7320
    tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt 7380
    tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 7440
    atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 7500
    ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc 7560
    gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgga 7620
    tcccttgagt ggctttcatc ctggagcaga ctttgcagtc tgtggactgc aacacaacat 7680
    tgcctttatg tgtaactctt ggctgaagct cttacaccaa tgctggggga catgtacctc 7740
    ccaggggccc aggaagacta cgggaggcta caccaacgtc aatcagaggg gcctgtgtag 7800
    ctaccgataa gcggaccctc aagagggcat tagcaatagt gtttataagg cccccttgtt 7860
    aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 7920
    ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 7980
    tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 8040
    atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 8100
    attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 8160
    gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 8220
    agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 8280
    aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 8340
    cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 8400
    cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 8460
    actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 8520
    cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 8580
    ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 8640
    ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 8700
    gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 8760
    gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 8820
    ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 8880
    cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 8940
    caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 9000
    taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 9060
    cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 9120
    cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 9180
    gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 9240
    aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 9300
    cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 9360
    tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 9420
    acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 9480
    ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 9540
    ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 9600
    tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 9660
    tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 9720
    ctggcctttt gctggccttt ttgaagctgt ccctgatggt cgtcatctac ctgcctggac 9780
    agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg 9840
    aaggccatcc agcctcgcgt cg 9862
    SEQ ID NO: 36 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
    (donor vector attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
    1, pMK 8B3 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180
    anti mFC scFV gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240
    CoOp ECORV gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300
    SacII L1R5) acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 360
    aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg 420
    atgagcaatg cttttttata atgccaactt tgtacaaaaa agctgaacga tatcgccacc 480
    atgggcagca cagccattct ggccctgctg ctggcagtgc tgcagggcgt gtcagctcag 540
    gtgcagctgc agcagtctgg cgccgaagtg aagaaacccg gcagcagcgt gaaggtgtcc 600
    tgcaaggcta gcggcggcac cttcaggagc tacgccattt cttgggtgcg ccaggcccct 660
    ggacagggcc tggaatggat gggctggatc agcccctaca acggcaacac cgactacgcc 720
    cagaaagtgc agggcagagt gaccctgacc accgacacca gcacctccac cgcctacatg 780
    gaactgcgga gcctgagaag cgacgacacc gccgtgtact actgtgccac aggcggcgga 840
    acctggtaca gcgatctgtg gggcagaggc accctcgtga cagtgtctgc tggcggcgga 900
    ggatctggcg gaggcggaag tggcggggga ggaagcggag cacctgagat cgtgctgacc 960
    cagagcccta gcacactgag cgccagcgtg ggcgacagag tgtccatcac ctgtagagcc 1020
    agccagagca tcggaggcag cctggcctgg tatcagcaga agcctggcaa ggcccccaag 1080
    ctgctgatct ctgaggccag caccctggaa agaggcgtgc ccagcagatt ttccggcagc 1140
    ggctctggca ccgacttcac cctgacaatc agcagcctgc agcccgagga cgtggccacc 1200
    tactactgcc agaagtacaa cagcgtgccc ctgaccttcg gccctggcac caaggtggaa 1260
    atcaagccgc gggccaactt tgtatacaaa agtggaacga gaaacgtaaa atgatataaa 1320
    tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca 1380
    acatatccag tcactatgaa tcaactactt agatggtatt agtgacctgt actgggcctc 1440
    atgggccttc ctttcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 1500
    acatggtcat agctgtttcc ttgcgtattg ggcgctctcc gcttcctcgc tcactgactc 1560
    gctgcgctcg gtcgttcggg taaagcctgg ggtgcctaat gagcaaaagg ccagcaaaag 1620
    gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg ccgccctgac 1680
    gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 1740
    taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 1800
    accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 1860
    tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 1920
    cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 1980
    agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 2040
    gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca 2100
    gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 2160
    tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 2220
    acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 2280
    cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 2340
    acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 2400
    acttggtctg acagttatta gaaaaattca tccagcagac gataaaacgc aatacgctgg 2460
    ctatccggtg ccgcaatgcc atacagcacc agaaaacgat ccgcccattc gccgcccagt 2520
    tcttccgcaa tatcacgggt ggccagcgca atatcctgat aacgatccgc cacgcccaga 2580
    cggccgcaat caataaagcc gctaaaacgg ccattttcca ccataatgtt cggcaggcac 2640
    gcatcaccat gggtcaccac cagatcttcg ccatccggca tgctcgcttt cagacgcgca 2700
    aacagctctg ccggtgccag gccctgatgt tcttcatcca gatcatcctg atccaccagg 2760
    cccgcttcca tacgggtacg cgcacgttca atacgatgtt tcgcctgatg atcaaacgga 2820
    caggtcgccg ggtccagggt atgcagacga cgcatggcat ccgccataat gctcactttt 2880
    tctgccggcg ccagatggct agacagcaga tcctgacccg gcacttcgcc cagcagcagc 2940
    caatcacggc ccgcttcggt caccacatcc agcaccgccg cacacggaac accggtggtg 3000
    gccagccagc tcagacgcgc cgcttcatcc tgcagctcgt tcagcgcacc gctcagatcg 3060
    gttttcacaa acagcaccgg acgaccctgc gcgctcagac gaaacaccgc cgcatcagag 3120
    cagccaatgg tctgctgcgc ccaatcatag ccaaacagac gttccaccca cgctgccggg 3180
    ctacccgcat gcaggccatc ctgttcaatc atactcttcc tttttcaata ttattgaagc 3240
    atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 3300
    caaatagggg ttccgcgcac atttccccga aaagtgccac 3340
    SEQ ID NO: 37 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
    (donor vector attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
    2, pMK hCD8a gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 180
    scaffold TN L5 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 240
    L2) gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 300
    acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca 360
    aggccgcata aataatgatt ttattttgac tgatagtgac ctgttcgttg caacaaattg 420
    atgagcaatg cttttttata atgcccaact ttgtatacaa aagtggcccg cggacaacaa 480
    cccctgcccc cagacctcct accccagccc ctacaattgc cagccagcct ctgagcctga 540
    ggcccgaggc ttgtagacct gctgctggcg gagccgtgca caccagagga ctggatttcg 600
    cctgcgacat ctacatctgg gcccctctgg ccggcacatg tggcgtgctg ctgctgagcc 660
    tcgtgatcac cctgtactgc ggctccacca gcggctccgg caagcccggc tctggcgagg 720
    gctccaccag cggcgactac aaggacgacg atgacaagta ataggatatc ggttcagctt 780
    tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc aacgaacagg 840
    tcactatcag tcaaaataaa atcattattt ctgggcctca tgggccttcc tttcactgcc 900
    cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata gctgtttcct 960
    tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt 1020
    aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 1080
    gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 1140
    tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 1200
    agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 1260
    ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 1320
    taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 1380
    gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 1440
    gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 1500
    ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 1560
    ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 1620
    gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 1680
    caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 1740
    taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 1800
    aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttattag 1860
    aaaaattcat ccagcagacg ataaaacgca atacgctggc tatccggtgc cgcaatgcca 1920
    tacagcacca gaaaacgatc cgcccattcg ccgcccagtt cttccgcaat atcacgggtg 1980
    gccagcgcaa tatcctgata acgatccgcc acgcccagac ggccgcaatc aataaagccg 2040
    ctaaaacggc cattttccac cataatgttc ggcaggcacg catcaccatg ggtcaccacc 2100
    agatcttcgc catccggcat gctcgctttc agacgcgcaa acagctctgc cggtgccagg 2160
    ccctgatgtt cttcatccag atcatcctga tccaccaggc ccgcttccat acgggtacgc 2220
    gcacgttcaa tacgatgttt cgcctgatga tcaaacggac aggtcgccgg gtccagggta 2280
    tgcagacgac gcatggcatc cgccataatg ctcacttttt ctgccggcgc cagatggcta 2340
    gacagcagat cctgacccgg cacttcgccc agcagcagcc aatcacggcc cgcttcggtc 2400
    accacatcca gcaccgccgc acacggaaca ccggtggtgg ccagccagct cagacgcgcc 2460
    gcttcatcct gcagctcgtt cagcgcaccg ctcagatcgg ttttcacaaa cagcaccgga 2520
    cgaccctgcg cgctcagacg aaacaccgcc gcatcagagc agccaatggt ctgctgcgcc 2580
    caatcatagc caaacagacg ttccacccac gctgccgggc tacccgcatg caggccatcc 2640
    tgttcaatca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2700
    atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2760
    tttccccgaa aagtgccac 2779
    SEQ ID NO: 38 cgataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattagg 60
    (Final vector gttagtctgg atagtatata ctactacccg ggaagcatat gctacccgtt tagggttcac 120
    used for cggtgatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactcat 180
    lentiviral taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 240
    production, ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac 300
    pLV4301G 8B3 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct 360
    scFV mIgG hCD8 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc 420
    flag) accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac 480
    agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat 540
    ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg 600
    agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc 660
    ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct 720
    tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg 780
    gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg 840
    cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag 900
    agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat 960
    tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag 1020
    ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca 1080
    aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata 1140
    taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga 1200
    agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc 1260
    cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata 1320
    aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag 1380
    tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag 1440
    cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat 1500
    tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc 1560
    tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa 1620
    gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca 1680
    ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc 1740
    acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct 1800
    taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata 1860
    aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat 1920
    tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag 1980
    tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga 2040
    ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat 2100
    ccattcgatt agtgaacgga tctcgacggt atcggtttta aaagaaaagg ggggattggg 2160
    gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa 2220
    ttacaaaaac aaattacaaa aattcaaaat tttatcgatt ttatttagtc tccagaaaaa 2280
    ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg 2340
    caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt aggaacagag 2400
    agacaggaga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 2460
    ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta gagaaccatc 2520
    agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 2580
    atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 2640
    cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg gtaccgatat 2700
    caccaacttt gtacaaaaaa gctgaacgat atcgccacca tgggcagcac agccattctg 2760
    gccctgctgc tggcagtgct gcagggcgtg tcagctcagg tgcagctgca gcagtctggc 2820
    gccgaagtga agaaacccgg cagcagcgtg aaggtgtcct gcaaggctag cggcggcacc 2880
    ttcaggagct acgccatttc ttgggtgcgc caggcccctg gacagggcct ggaatggatg 2940
    ggctggatca gcccctacaa cggcaacacc gactacgccc agaaagtgca gggcagagtg 3000
    accctgacca ccgacaccag cacctccacc gcctacatgg aactgcggag cctgagaagc 3060
    gacgacaccg ccgtgtacta ctgtgccaca ggcggcggaa cctggtacag cgatctgtgg 3120
    ggcagaggca ccctcgtgac agtgtctgct ggcggcggag gatctggcgg aggcggaagt 3180
    ggcgggggag gaagcggagc acctgagatc gtgctgaccc agagccctag cacactgagc 3240
    gccagcgtgg gcgacagagt gtccatcacc tgtagagcca gccagagcat cggaggcagc 3300
    ctggcctggt atcagcagaa gcctggcaag gcccccaagc tgctgatctc tgaggccagc 3360
    accctggaaa gaggcgtgcc cagcagattt tccggcagcg gctctggcac cgacttcacc 3420
    ctgacaatca gcagcctgca gcccgaggac gtggccacct actactgcca gaagtacaac 3480
    agcgtgcccc tgaccttcgg ccctggcacc aaggtggaaa tcaagccgcg ggccaacttt 3540
    gtatacaaaa gtggcccgcg gacaacaacc cctgccccca gacctcctac cccagcccct 3600
    acaattgcca gccagcctct gagcctgagg cccgaggctt gtagacctgc tgctggcgga 3660
    gccgtgcaca ccagaggact ggatttcgcc tgcgacatct acatctgggc ccctctggcc 3720
    ggcacatgtg gcgtgctgct gctgagcctc gtgatcaccc tgtactgcgg ctccaccagc 3780
    ggctccggca agcccggctc tggcgagggc tccaccagcg gcgactacaa ggacgacgat 3840
    gacaagtaat aggatatcgg ttcagctttc ttgtacaaag ttgggattcg agttaattaa 3900
    gttaacgaat tccccccctc tccctccccc ccccctaacg ttactggccg aagccgcttg 3960
    gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc 4020
    aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc 4080
    cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa 4140
    gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct 4200
    ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca 4260
    caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca 4320
    agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat 4380
    ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc 4440
    ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc 4500
    atgggaggcg gaagcggcgg aggctcccct cgaggcacca tggtgagcaa gggcgaggag 4560
    ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 4620
    ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 4680
    atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 4740
    ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 4800
    gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 4860
    aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 4920
    ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 4980
    agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 5040
    atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 5100
    cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 5160
    ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 5220
    gccgggatca ctctcggcat ggacgagctg tacaagtaac gcgtcccggg tctagagcta 5280
    gcggtaccat gcattacgta gtcgacgact taattaagct agcctagtgc catttgttca 5340
    gtggttcgta gggctttccc ccactgtttg gctttcagtt atatggatga tgtggtattg 5400
    ggggccaagt ctgtacagca tcttgagtcc ctttttaccg ctgttaccaa ttttcttttg 5460
    tctttgggta tacatttaaa ccctaacaaa acaaagagat ggggttactc tctaaatttt 5520
    atgggttatg tcattggatg ttatgggtcc ttgccacaag aacacatcat acaaaaaatc 5580
    aaagaatgtt ttagaaaact tcctattaac aggcctattg attggaaagt atgtcaacga 5640
    attgtgggtc ttttgggttt tgctgcccct tttacacaat gtggttatcc tgcgttgatg 5700
    cctttgtatg catgtattca atctaagcag gctttcactt tctcgccaac ttacaaggcc 5760
    tttctgtgta aacaatacct gaacctttac cccgttgccc ggcaacggcc aggtctgtgc 5820
    caagtgtttg ctgacgcaac ccccactggc tggggcttgg tcatgggcca tcagcgcatg 5880
    cgtggaacct tttcggctcc tctgccgatc catactgcgg aactcctagc cgcttgtttt 5940
    gctcgcagca ggtctggagc aaacattatc gggactgata actctgttgt cctatcccgc 6000
    aaatatacat cgtttccatg gctgctaggc tgtgctgcca actggatcct gcgcgggacg 6060
    tcctttgttt acgtcccgtc ggcgctgaat cctgcggacg acccttctcg gggtcgcttg 6120
    ggactctctc gtccccttct ccgtctgccg ttccgaccga ccacggggcg cacctctctt 6180
    tacgcggact ccccgtctgt gccttctcat ctgccggacc gtgtgcactt cgcttcacct 6240
    ctgcacgtcg catggagacc accgtgaacg cccaccaaat attgcccaag gtcttacata 6300
    agaggactct tggactctca gcaatgtcaa cgaccgacct tgaggcatac ttcaaagact 6360
    gtttgtttaa agactgggag gagttggggg aggagattag gttaaaggtc tttgtactag 6420
    gaggctgtag gcataaattg gtctgcgcac cagcaccatg gcgcaatcac tagagcgggg 6480
    tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa 6540
    aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta 6600
    ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc 6660
    cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 6720
    tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 6780
    gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat 6840
    cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 6900
    tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 6960
    tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc 7020
    gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat 7080
    ttatgcagag gccgaggccg gatcccttga gtggctttca tcctggagca gactttgcag 7140
    tctgtggact gcaacacaac attgccttta tgtgtaactc ttggctgaag ctcttacacc 7200
    aatgctgggg gacatgtacc tcccaggggc ccaggaagac tacgggaggc tacaccaacg 7260
    tcaatcagag gggcctgtgt agctaccgat aagcggaccc tcaagagggc attagcaata 7320
    gtgtttataa ggcccccttg ttaattcttg aagacgaaag ggcctcgtga tacgcctatt 7380
    tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 7440
    aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 7500
    catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 7560
    tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 7620
    tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 7680
    ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 7740
    ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 7800
    cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 7860
    ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 7920
    tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 7980
    gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 8040
    ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc 8100
    aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 8160
    acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 8220
    tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 8280
    cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 8340
    gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 8400
    taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 8460
    tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 8520
    cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 8580
    ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 8640
    accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 8700
    cttcaggaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 8760
    cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 8820
    tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 8880
    taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 8940
    gacctacacc gaactgagat acctacagcg tgagcattga gaaagcgcca cgcttcccga 9000
    agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 9060
    ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 9120
    acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 9180
    caacgcggcc tttttacggt tcctggcctt ttgctggcct ttttgaagct gtccctgatg 9240
    gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga 9300
    agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcg 9344
    SEQ ID NO: 39 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60
    (pLenti-C-Myc- atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120
    DDK OX40L) gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180
    tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat cgcgttgaca 240
    ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300
    tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360
    cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420
    ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480
    gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540
    ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600
    catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660
    tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 720
    ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 780
    cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc 840
    tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 900
    agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 960
    ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 1020
    cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 1080
    gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 1140
    tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 1200
    gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa 1260
    attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 1320
    gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 1380
    aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 1440
    aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 1500
    aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat 1560
    atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1620
    ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1680
    ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1740
    atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1800
    ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1860
    cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1920
    atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1980
    agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 2040
    attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 2100
    aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 2160
    ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2220
    ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2280
    ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2340
    gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2400
    cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2460
    gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2520
    taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2580
    cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg 2640
    ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2700
    cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2760
    caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2820
    gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2880
    tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 2940
    ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt 3000
    attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 3060
    gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3120
    ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca 3180
    aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 3240
    tcagaatttt gtaatacgac tcactatagg gcggccggga attcgtcgac tggatccggt 3300
    accgaggaga tctgccgccg cgatcgccat ggaaagggtc caacccctgg aagagaatgt 3360
    gggaaatgca gccaggccaa gattcgagag gaacaagcta ttgctggtgg cctctgtaat 3420
    tcagggactg gggctgctcc tgtgcttcac ctacatctgc ctgcacttct ctgctcttca 3480
    ggtatcacat cggtatcctc gaattcaaag tatcaaagta caatttaccg aatataagaa 3540
    ggagaaaggt ttcatcctca cttcccaaaa ggaggatgaa atcatgaagg tgcagaacaa 3600
    ctcagtcatc atcaactgtg atgggtttta tctcatctcc ctgaagggct acttctccca 3660
    ggaagtcaac attagccttc attaccagaa ggatgaggag cccctcttcc aactgaagaa 3720
    ggtcaggtct gtcaactcct tgatggtggc ctctctgact tacaaagaca aagtctactt 3780
    gaatgtgacc actgacaata cctccctgga tgacttccat gtgaatggcg gagaactgat 3840
    tcttatccat caaaatcctg gtgaattctg tgtccttacg cgtacgcggc cgctcgagca 3900
    gaaactcatc tcagaagagg atctggcagc aaatgatatc ctggattaca aggatgacga 3960
    cgataaggtt taaacggccg gccgcggtct gtacaagtag gattcgtcga gggacctaat 4020
    aacttcgtat agcatacatt atacgaagtt atacatgttt aagggttccg gttccactag 4080
    gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga 4140
    ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 4200
    cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 4260
    tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 4320
    actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 4380
    tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 4440
    gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 4500
    aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 4560
    tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 4620
    ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 4680
    tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa aacatggagc 4740
    aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga 4800
    ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa 4860
    ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 4920
    ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc 4980
    tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg 5040
    ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac 5100
    ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga 5160
    gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg 5220
    tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 5280
    cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 5340
    gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 5400
    tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 5460
    ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 5520
    gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 5580
    cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 5640
    gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 5700
    tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 5760
    cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 5820
    cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 5880
    gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 5940
    agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 6000
    cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 6060
    tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 6120
    tttggtcatg attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc 6180
    attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc 6240
    agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg 6300
    tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg 6360
    aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc 6420
    acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc 6480
    gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat 6540
    atcaccagct caccgtcttt cattgccata cggaactccg gatgagcatt catcaggcgg 6600
    gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa 6660
    aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat 6720
    gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt 6780
    tttttctcca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 6840
    atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt cccgcgcaca 6900
    tttccccgaa aagtgccacc tgac 6924
  • In the preparations of engineered EM-3 aAPCs (also referred to herein as aEM3 aAPCs) used for the experiments described herein, expression of CD86 and 4-1BBL was confirmed using flow cytometry (Canto II flow cytometer, Becton, Dickinson, and Co., Franklin Lakes, N.J., USA), with results shown in FIG. 37. aEM3 aAPCs were γ-irradiated at 100 Gy and frozen.
  • aEM-3 cells previously transduced to express CD86, antibody against IgG Fc region, and 4-1BBL (or optionally without 4-1BBL), as described above, were genetically engineered with a co-stimulatory human OX-40L using a similar lentiviral transduction approach. To generate lentivirus containing human OX-40L, pLenti-C-Myc-DDK OX40L (PS100064, Origene, SEQ ID NO:39, FIG. 90) vector together with the VSV-G envelope plasmid (pCIGO-VSV.G) were co-transfected into a Phoenix-GP (ATCC CRL-3215) cell line using PolyJet (Signagen Laboratories, Rockville, Md., USA). The supernatants were harvested 60 hours later and concentrated using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane. aEM-3 cells were then infected with concentrated lentivirus and further expanded for five days. The cells were stained with PE-conjugated anti-human OX40L, Brilliant Violet 421-conjugated anti-human CD137L (if 4-1BBL is included in the prior aEM-3 cells), and PE/Cy7 conjugated anti-human CD86 and sorted based on the expression of GFP, OX40L, CD137L (when included), and CD86 using a S3e Cell Sorter (Bio-Rad, Inc., Hercules, Calif., USA). The purity of sorted cells was further validated using flow cytometry. The enriched cells were checked for purity by flow cytometry.
    • Example 6—Expansion of Tumor Infiltrating Lymphocytes Using EM-3 Artificial Antigen Presenting Cells
  • Experiments were performed to test the ability of EM-3 aAPCs (aEM3) to expand TILs. TIL were co-cultured with aEM3 (7C12 or 8B3) at a ratio of 1:100 ratio plus OKT-3 (30 mg/mL) and IL-2 (3000 IU/mL). Cells were counted on Day 11 and 14. The results are plotted for two batches of TILs in FIG. 38 and FIG. 39. In addition, TILs were co-cultured with aEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3000 IU/mL) with or without OKT-3 (30 mg/mL). The results are plotted in FIG. 40, where the bar graph shows cell numbers determined on Day 11.
  • FIG. 41 illustrates the results of TIL expansions with EM-3 aAPCs (aEM3) at different TIL:aAPC ratios. The results show that aEM3 aAPCs perform comparably to and in some cases better than PBMCs, particularly at ratios of 1:200 at longer culture times (14 days).
  • FIG. 42 illustrates the low variability in cell counts from TIL expansions with EM-3 aAPCs (aEM3) in comparison to PBMC feeders. TILs (2×104) were co-cultured with five different PBMC feeder lots or aEM3 (in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. The graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14. FIG. 43 compares the results of TIL expansions with EM-3 aAPCs and MOLM-14 aAPCs, to illustrate variability in cell counts for both aEM3 and aMOLM14 in comparison to TILs (2×104) were co-cultured with five different PBMC feeder lots or aMOLM14 (in triplicate) or aEM3 (also in triplicate) at 1:100 ratio with IL-2 (3000 IU/mL) in a G-Rex 24 well plate. Viable cells were counted on day 14, and the graph shows viable cell numbers (mean) with 95% confidence interval. The aEM3 and aMOLM14 results indicate that much greater consistency can be obtained with both aAPCs compared to the PBMC feeder approach preferred in the prior art.
  • TILs expanded against aEM3 or PBMC feeders were used for flow cytometry analysis using 4 different panels ( differentiation panels 1 and 2, T cell activation panels 1 and 2). Briefly, TILs were first stained with L/D Aqua to determine viability. Next, cells were surface stained with TCR α/β PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28 PE, CD27 APC-Cy7, and CD57-PerCP-Cy5.5 for differentiation panel 1; CD45RA PE-Cy7, CD8a PerCP/Cy5, CCR7 PE, CD4 FITC, CD3 APC-Cy7, CD38 APC, and HLA-DR PB, for differentiation panel 2; CD137 PE-Cy7, CD8a PerCP-Cy5.5, Lag3 PE, CD4 FITC, CD3 APC-Cy7, PD1 APC, and Tim-3 BV421 for T cell activation panel 1; or CD69 PE-Cy7, CD8a PerCP/Cy5.5, TIGIT PE, CD4 FITC, CD3 APC-Cy7, KLRG1 ALEXA 647, and CD154 BV421 for T cell activation panel 2. Phenotype analysis was done by gating 10,000 to 100,000 cells according to FSC/SSC using the Canto II flow cytometer. Data was analyzed using Cytobank software (Cytobank, Inc., Santa Clara, Calif., USA) to create sunburst diagrams and SPADE (Spanning-tree Progression Analysis of Density-normalized Events) plots. Gates were set based on fluorescence minus one (FMO) controls. SPADE plots were generated with the group of cells, characterized in a form of related nodes based on the expression level of surface markers. CD4+ and CD8+ TIL subsets were determined based on CD3+ gating, and trees were generated. Sunburst visualizations are shown in FIG. 44 and FIG. 45. FIG. 44 shows that TILs expanded against aEM3 aAPCs maintained the CD8+ phenotype when compared to the same TILs expanded against PBMC feeders. FIG. 45 shows the results of a second batch of TILs from a different patient expanded against aEM3 aAPCs, where a clear increase of CD8+ cells (65.6%) is seen in comparison to the results from expansion using PBMC feeders (25%).
  • The CD4 and CD8 SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3+ cells is shown in FIG. 46 and FIG. 47. The color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRG1 and TIGIT. Without being bound by theory, the results show that TILs expanded with aEM3 aAPCs had undergone activation, but there was no difference in MFI between the aEM3 aAPCs and PBMC feeders, indicating that the aEM3 aAPCs effectively replicate the phenotypic results obtained with PBMC feeders.
  • Spare respiratory capacity (SRC) and glycolytic reserve were also evaluated for TILs expanded with aEM3 aAPCs in comparison to PBMC feeders, with results shown in FIG. 48 and FIG. 49. The Seahorse XF Cell Mito Stress Test measures mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells, using modulators of respiration that target components of the electron transport chain in the mitochondria. The test compounds (oligomycin, FCCP, and a mix of rotenone and antimycin A, described below) are serially injected to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. Proton leak and spare respiratory capacity are then calculated using these parameters and basal respiration. Each modulator targets a specific component of the electron transport chain. Oligomycin inhibits ATP synthase (complex V) and the decrease in OCR following injection of oligomycin correlates to the mitochondrial respiration associated with cellular ATP production. Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential. As a result, electron flow through the electron transport chain is uninhibited and oxygen is maximally consumed by complex IV. The FCCP-stimulated OCR can then be used to calculate spare respiratory capacity, defined as the difference between maximal respiration and basal respiration. Spare respiratory capacity (SRC) is a measure of the ability of the cell to respond to increased energy demand. The third injection is a mix of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination shuts down mitochondrial respiration and enables the calculation of nonmitochondrial respiration driven by processes outside the mitochondria.
  • FIG. 50 illustrates a mitochondrial stain of Live TILs expanded against PBMC feeders or aEM3 aAPCs. MitoTracker dye stains mitochondria in live cells and its accumulation is dependent upon membrane potential. TILs expanded against PBMC feeders or aEM3 were stained L/D Aqua followed by MitoTracker red dye. The data show MitoTracker positive (MFI) cells gated on live population,
    • Example 7—Comparison of Engineered MOLM-14 (aMOLM14) and EM-3 (aEM3) aAPCs
  • TILs expanded with PBMC feeders and aMOLM14 and aEM3 aAPCs, as described in the previous examples, were assessed for functional activity using the BRLA for cytotoxic potency. The P815 BRLA is described in detail in Example 9. The results are shown in FIG. 51 and FIG. 52, and show that TILs expanded with aAPCs have similar functional properties (and expected clinical efficacy) to those expanded with PBMC feeders.
  • IFN-γ release and Granzyme B release from TILs expanded with PBMC feeders and aMOLM14 and aEM3 aAPCs as described above was also assessed following overnight stimulation with microbeads coated with anti-CD3/CD28/4-1BB. The IFN-γ release results are shown in FIG. 53 and FIG. 54, and the Granzyme B release results are shown in FIG. 55 and FIG. 56. Significant and surprising increases in IFN-γ release and Granzyme B release were observed for TILs expanded with aEM3 aAPCs relative to those expanded with PBMC feeders, but not for TILs expanded by aMOLM14 aAPCs. Without being bound by theory, this suggests that TILs cultured with aEM3 aAPCs may be more active in vivo as a cancer therapy. Most other differences observed were not statistically significant.
  • The results of TIL expansions with aEM3 and aMOLM14 aAPCs are summarized in Table 9.
  • TABLE 9
    Summary of TIL expansion results with aAPCs.
    Fold Expansion Relative CD8 (%) CD4 (%) Relative Relative
    aAPC TIL# PBMC aAPC expansion PBMC aAPC PBMC aAPC CD8 CD4
    aMOLM14 M1032-T2 2112 1936 0.92 53 65 44 27 1.226 0.614
    M1033-T6 1761 1598 0.91 50 57 36 40 1.140 1.111
    M1021T-5 2053 2024 0.99 91 82 8 17 0.901 2.125
    M1030T-4  860  853 0.99 46 78 51 12 1.696 0.235
    M1045  858*  758* 0.88 — — — — — —
    M1021T-1 1866 1620 0.87 — — — — — —
    M1032T-1 2423 2049 0.85 — — — — — —
    M1042 1278 1704 1.33  8  8 88 89 0.919 1.015
    M1043 1601 1587 0.99 90 87 5  5 0.968 0.947
    aEM3 M1054 2058 1647 0.80 98 96 2  2 0.981 1.400
    M1055  729 1533 2.10 25 66 70 31 2.694 0.441
    M1021T-1 2985 2805 0.94 87 75 10 20 0.862 2.000
    M1045 1336 1047 0.78 — — — — — —
    • Example 8—Preparation of Master Cell Banks for aEM3 and aMOLM14 aAPCs
  • aEM3 and aMOLM14 aAPCs may be grown in the following media compositions to produce master cell banks, which may be further grown in this media for supply of aAPCs: 500 mL of Dulbecco's Modified Eagle Medium DMEM/F12 (Sigma-Aldrich, St. Louis, Mo., USA), 50 mL fetal bovine serum (FBS) Heat Inactivated (HI) (Hyclone); 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) (Life Technologies); 1× Primocin (Invivogen); 1× Plasmocin (Invivogen), and 1×2-mercaptoethanol (Life Technologies).
  • The aAPCs described herein, including aEM3 and aMOLM14 aAPCs, may also be grown from a master cell bank using any suitable method known in the art for the growth of cells. In an embodiment, aAPCs are thawed and are then expanded in a medium of 80-90% RPMI 1640+10-20% h.i. FBS (fetal bovine serum) by splitting saturated culture 1:2 to 1:3 every 2-3 days, seeding out at about 0.5-1×106 cells/mL in 24-well plates, and maintaining at about 0.5-1.5×106 cells/mL, with incubation at 37° C. and 5% CO2.
  • Further steps that may be employed to use the aAPCs of certain embodiments of the present invention in the production of human therapies are known in the art and include cell line characterization (HLA high resolution typing); cytokine release testing; testing of human serum to replace FBS to grow aAPC; testing freezing media to freeze aAPCs; master cell banking (including raw material testing and stability testing); standardization of irradiation (including irradiation dose (1000, 3000, 5000, 10000, 15000 rad), fresh versus frozen aAPCs, and with/without TILs); stability of aAPC; development of a panel to evaluate the contamination of aAPCs; development of molecular biology assays (qPCR, DNA sequencing); testing of TIL expansions from different tumor types, including melanoma, cervical, and head and neck cancer (using a G-Rex 5M); potency, purity, and identity testing; mycoplasma and sterility assays; microbiological testing (USP/EP sterility, bioburden and endotoxin assays); and adventitious viral agent testing.
    • Example 9—Methods of Expanding TILs and Treating Cancer with Expanded TILs
  • TILs may be expanded using the aAPCs of certain embodiments of the present invention, such as aEM3 and aMOLM14 aAPCs, using any of the expansion methods described herein. For example, a method for expanding TILs is depicted in FIG. 57. The expansion of TILs using aAPCs may be further combined with any method of treating cancer in a patient described herein. A method for expanding TILs and treating a patient with expanded TILs, wherein the expansion makes use of aAPCs (including aEM3 and aMOLM14 aAPCs), is shown in FIG. 58.
    • Example 10—P815 Bioluminescent Redirected Lysis Assay
  • In this example, the development of a surrogate target cell line to evaluate the lytic potential of TILs in a Bioluminescent Redirected Lysis Assay (BRLA) is described. The BRLA enables assessment of T cell mediated killing in the absence of autologous tumor cells. Cytolytic activity can be assessed with and without engaging the T cell receptor in one to four hours, assessing T cell killing engaging the T cell receptor and without so-called lymphokine activated killer activities (LAK).
  • Mouse mastocytoma P815 cells expressing the endogenous CD16 Fc receptor can bind anti-CD3c (OKT-3), providing a potent TCR activation signal as a target cell line. The P815 Clone G6 was transduced with a lentiviral vector based on eGFP and firefly luciferase, sorted and cloned using the BD FACSAria II. Clone G6 was selected based on eGFP intensity analyzed using an Intellicyt iQue Screener. Target cells and TILs of interest were co-cultured+/−OKT-3 to assess TCR activation (specific killing) or non-specific (lymphokine activated killing, LAK) respectively. Following 4 hours of incubation, firefly luciferin ((4S)-2-(6-hydroxy-1,3-benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid, commercially available from multiple sources) was added to the wells and incubated for 5 minutes. Bioluminescence intensity was read using a luminometer. Percent cytotoxicity and survival were calculated using the following formula: % Survival=(experimental survival−minimum)/(maximum signal−minimum signal)×100; % Cytotoxicity=100−(% Survival). Interferon gamma release in the media supernatant of co-cultured TILs was analyzed by ELISA, and LAMP1 (CD107a, clone eBioH4A3) expression on TILs was analyzed on a flow cytometer to evaluate the cytotoxic potency of TILs.
  • Results are shown in FIG. 59 to FIG. 75. FIG. 59 illustrates percent toxicity of TIL batch M1033T-1 co-cultured with P815 Clone G6 (with and without anti-CD3) at individual effector:target ratios by BRLA. FIG. 60 illustrates enzyme-linked immunosorbent assay (ELISA) data showing the amount of IFN-γ released against different ratios of effector to target cells. FIG. 61 illustrates LAMP1(%) expressed by TIL batch M1033T-1 when co-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of 1:1 effector to target cells for 4 hours and 24 hours co-culture.
  • The results were confirmed using a second TIL batch as shown in FIG. 62, which illustrates BRLA for TIL batch M1030. The cytotoxicity (measured as LU50/1×106 TIL) by BRLA is 26±16. FIG. 63 illustrates the results of a standard chromium release assay for TIL batch M1030. The cytotoxicity (measured as LU50/1×106 TIL) by chromium release assay is 22.
  • Results were further confirmed using a third TIL batch. FIG. 64 illustrates BRLA results for TIL batch M1053, showing lytic units of the TILs by BRLA as 70±17. FIG. 65 illustrates the results of a standard chromium release assay for TIL batch M1053, showing lytic unit of the TILs by chromium assay as 14±5. Comparison of two assay results shows the comparable performance of the BRLA result to the chromium release assay result.
  • FIG. 66 illustrates the linear relationship between IFN-γ release and cytotoxic potential of TILs. FIG. 67 illustrates ELISpot results for IFN-γ. FIG. 68 illustrates enzymatic IFN-γ release for TIL batch M1053. FIG. 69 illustrates enzymatic IFN-γ release for TIL batch M1030. FIG. 70 illustrates ELISpot data showing Granzyme B release by M1053T and M1030T. FIG. 71 illustrates enzymatic Granzyme B release for TIL batch M1053. FIG. 72 illustrates enzymatic Granzyme B release for TIL batch M1030. FIG. 73 illustrates ELISpot data showing TNF-α release by M1053T and M1030T. FIG. 74 illustrates enzymatic TNF-α release for TIL batch M1053. FIG. 75 illustrates enzymatic TNF-α release for TIL batch M1030. The data in FIG. 66 to FIG. 76 confirms the potency of these batches of TILs as also shown by the BRLA.
  • In conclusion, the BRLA requires no radionuclides and is as efficient and sensitive as traditional cytotoxicity assays. Flow cytometric assessment of Lampl expression on TILs at individual time points demonstrates degranulation of cytotoxic T cells relative to the potency shown by BRLA. The BRLA demonstrates similar to better potency than standard chromium release assay. BRLA also enables evaluation of the potency of TIL lytic activity. Comparison of BRLA with chromium release assay shows the efficiency and reliability of BRLA. BRLA has a linear relationship with IFNγ release by TILs. Release assay of IFN-γ, TNFα and Granzyme B by ELISpot is consistent with the cytotoxic efficiency of the TILs evaluated by BRLA.
    • Example 11—Process for Weaning EM3 Cells from FBS to hAB Serum
  • In order to avoid reactivity, some cell lines may need to be weaned from one medium to another. Here, EM3 cells are weaned from FBS to hAB serum to avoid reactivity. As shown in FIG. 76, aEM3 cells were successfully weaned off of FBS to hAB serum.
    • Example 12—Freezing Media Formulation Optimization
  • To cryobank EM3 cells cultured as described herein, methods were freezing media formulation were optimized. As shown in FIG. 77, three freezing media were used and their effect on cell numbers were counted. The cell media utilized included CryStor 10 (Biolife Solutions (CS10)) (A), hAB [90%] and DMSO [10%] (B), and hAB [20%] with DMSO [10%] and cDMEM2 [70%] (C). FIG. 77 demonstrates that the formulation of human AB serum (90%) and DMSO (10%) provided for unexpectedly increased EM3 cell numbers after 3 days of recovery.
    • Example 13—Growth of aEM3 Cells in GREX Flasks
  • aEM3 cells were cultured in gas permeable cell culture flasks (i.e., GREX flasks (Wilson Wolf Manufacturing)) and the effect on cell doubling time was observed over an 8 day time course. As shown in FIG. 78, the GREX flasks provided for rapid growth of aEM3 cells.
    • Example 14—Flow Panel Analysis to Determine aEM3 Cell Purity
  • To determine the purity of cells cultured according to the processes described herein, a flow panel analysis was used to determine the purity of aEM3 aAPCs. The results of such analysis are described in FIGS. 79 and 80. As shown in FIG. 80, before sorting aEM3 cell populations were 53.5% and 43.2% eGFP+ for aEM3 7C12 and aEM3 8B5 cells, respectively. Postsorting, cell populations was improved to 96.8% and 96.3% eGFP+ for aEM3 7C12 and aEM3 8B5 cells, respectively (FIG. 80).
    • Example 15—aEM3 Feeder Cells as an Alternative to PBMC Feeders
  • As described herein, aEM3 cells may be used as an alternative for PBMC feeders, resulting in unexpectedly different properties for both TIL expansion process and the resulting TILs. To compare differences in cytokine expression, PBMCs and aEM3 cells were stimulated by treatment with OKT-3. As shown in FIG. 81, aEM3 cells displayed a comparatively different cytokine expression profile as compared to PBMCs. Surprisingly, the aEM3 cells of the present invention provide efficacious TILs (as shown herein) without reproducing the same cytokine secretion properties of TILs expanded with conventional PBMCs.
    • Example 16—Comparison Between Complete Media and Serum Free Media TIL Expansion
  • In order to optimize the TIL expansion protocols, several TIL expansion expirements were peformed as described herein, but with serum free media rather than complete media (CM1).
  • In one experiment, tissue fragments were cultured in a single well with CM1 or various serum free media with 300 IU/mL of IL-2. Cells were then counted on Day 11 before initiating REP. The various serum free media used included Prime CDM (Irvine), CTS Optimizer (ThermoFisher), and Xvivo-20 (Lonza). As shown in FIG. 82, TIL expansion (PreREP) with CTS provided increased cell numbers as compared to CM1.
  • Additionally, tissue fragments were cultured with CM1 or various serum free media with 6000 IU/mL of IL-2 until Day 11. REP was then initiated on Day 11 using PBMC feeders, OKT-3, and IL-2, and culture was split on Day 16. Cultures were then terminated at the end of Day 22. The various serum free media used included Prime CDM (Irvine), CTS Optimizer (ThermoFisher), and Xvivo-20 (Lonza). As shown in FIG. 83 and FIG. 84, when counting cells at Days 11 and Day 22, respectively, TIL expansion (PreREP) with Prime CDM provided increased cell numbers as compared to CM1.
    • Example 17—Growth of aAPCs in Serum Free Media as Compared to Serum-Based Media
  • In order to optimize aAPC growth and maintenance protocols in the absence of serum, aEM3 cells were cultured using various serum free media.
  • aEM3 cells were cultured in 24 well plates at 1×106 cells per well for 3 days using general cell culture protocols as described herein, with the exception that that one group of cells were provided with serum-based media (cDMEM (10% hSerum) and the other groups of cells were provided with serum free media. The serum free media utilized for the study included CTS OpTmizer (ThermoFisher), Xvivo 20 (Lonza), Prime-TCDM (Irvine), and XFSM (MesenCult) media. Cells were then counted on Day 3.
  • As shown in FIG. 85, CTS OpTmizer and Prime-TCDM serum free media provided cell growth that was comparable to serum-based media (i.e., cDMEM (10% hSerum). Therefore, serum free media is an effective alternative for growing and maintaining aAPCs as comapred to serum-based media.
    • Example 18—Propagation, Maintenance, and Cryopreservation of aAPCs
  • In this example, procedures are provided for the preparation and preservation of aAPCs. Specifically, aEM3 cells from a cell line designated TIL-Rs3 were propagated and cryopreserved.
  • Thawing and recovery of aEM3 cells may be accomplished using the following non-limiting procedure. Cyropreserved aEM3 cells are warmed slowly in pre-warmed media (37° C.) that is prepared from CTS OpTmizer Basal Media (Thermo Fisher), CTS OpTmizer Cell Supplement (Thermo Fisher), Gentamicin (Lonza), and Glutamax (Life Technologies). The suspended cells are then centrifuged at 1500 rpm for 5 minutes at 4° C. The resulting supernatant is discarded and the remaining aEM3 cells are resuspended in the foregoing media and plated (5×106 cells/10 mL per well of a 6 well plate).
  • Propagation of aEM3 cells may be accomplished using the following non-limiting procedure. Aliquots of the foregoing media are prepared in gas permeable cell culture flasks (i.e., GREX 10 flasks (Wilson Wolf Manufacturing)). The plated aEM3 cells are washed by centrifugation (i.e., 1500 rpm for 5 minutes at 4° C.), resuspended in media, and added to the GREX flasks at cell density of 1-2×106 cells/mL. The aEM3 cell suspension was diluted with 30 mL of media and the GREX flasks were then incubated for 3-4 days at 37° C. under CO2. After 3-4 days, the GREX flasks were removed from the incubator and placed in a biological safety cabinet (BSC). The cultured aEM3 cells are carefully extracted from the GREX flasks by pipette and the resulting extraction is centrifuged to provide the increased number of aEM3 cells, which may be resuspended at a cell density of 10-20×106 cells per GREX 10 flask.
  • An alternative cryopreservation of aEM3 cells may be accomplished using the following non-limiting procedure. The foregoing GREX 10 flasks containing the aEM3 cells are removed from the incubator and placed in a BSC. The cultured aEM3 cells are carefully extracted from the GREX flasks by pipette and the resulting extraction is centrifuged to provide the increased number of aEM3 cells, which is resuspended in a volume of CryStor 10 (Biolife Solutions) to provide a concentration of 10-100×106 cells/vial in cryovials. The aEM3 cell suspensions may be placed in a freezing container and transferred to a −80° C. freezer.
    • Example 19—Demonstration of Rapid Recovery of aEM3 Cells Following Cryopreservation
  • aEM3 cells from the TIL-R3 cell line (1-2×106 cells) were cryopreserved according to the procedure set forth in Example 18 using CS-10 cryopreservation media. Vials of such cells were then thawed and the cells were counted. Cell counts were taken pre-freeze, post-thaw, and 3 days after thaw (i.e., Post-Thaw Recovery). As shown in FIG. 86 and FIG. 87, the total live cell counts recovered rapidly post thaw in two separate experiments.
  • TIL-R3 cells (1×106 cells) were thawed (Day 3 post-thaw) and plated at a density of 0.5×106/cm2 in each well of a 24 well plate. On day 0 and 3, viable cells were counted and recorded. On the first passage (Day 6), cells were split at the density of 2×106 cells/cm2 or 0.5×106 cells/cm2. At the end of the first passage, a cell count was performed. The resulting cell counts are shown in FIG. 88, which demonstrate both a recovery phase post-thaw and a growth phase.
  • Furthermore, TIL-R3 cells (20×106 cells) were cultured at a density of 2×106/cm2 in GREX 10 flasks according to the procedure described in Example 18. On days 4 and 8, live cells were counted and recorded. The resulting cell counts are shown in FIG. 89, which demonstrates a growth phase for the cells following cryopreservation that reaches a plateau between days 4 and 8 when the cells reached a density of 13.9×106 cells/cm′.
    • Example 20—CD8 Skewness, Expansion Performance, and CD3 Contamination of TILs Cultured with aEM3 aAPCs
  • Fifteen different PreREP TIL lines (0.4×105 cells) were co-cultured with either aEM3 aAPCs (as described herein) or PBMC feeders (10×106), OKT3 (30 ng/mL) and IL-2 (3000 IU/mL) and cultures were split on Day 5 using 6 well Grex plates. Cultures were sampled at day 11 and analyzed by flow cytometry. The relative ratio of CD8+ cells was calculated by the formula (CD8% aEM3)/(CD8% PBMC). The results shown in FIG. 91 indicate that TILs cultured with aEM3 cells surprisingly promote CD8+ skewing and and an improved TIL product. Additional results of these experiments are shown in FIG. 92, FIG. 93, and FIG. 94, where the results shown that TILs cultured with aEM3 aAPCs displayed comparable expansion and less non-CD3+ cell contamination in comparison to TILs cultured with PBMC feeders.
    • Example 21—Telomere Length Measurement
  • Genomic DNA was isolated from pre-REP or post-REP (magnetic bead sorted for CD3+) TILs for a qPCR (quantitative polymerase chain reaction) assay to measure telomere length. The real time qPCR method is described in Cawthon, Nucleic Acids Res. 2002, 30(10), e47; and Yang, et al., Leukemia, 2013, 27, 897-906. Briefly, the telomere repeat copy number to single gene copy number (T/S) ratio was determined using an PCR thermal cycler (Bio-Rad Laboratories, Inc.) in a 96-well format. Ten ng of genomic DNA was used for either the telomere or hemoglobin (hgb) PCR reaction and the primers used were as follows:
  • Tel-1b primer
    (SEQ ID NO: 40)
    (CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG
    GTT);
    Tel-2b primer
    (SEQ ID NO: 41)
    (GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC
    CCT);
    hgb1 primer
    (SEQ ID NO: 42)
    (GCT TCT GAC ACA ACT GTG TTC ACT AGC);
    and
    hgb2 primer
    (SEQ ID NO: 43)
    (CAC CAA CTT CAT CCA CGT TCA CC).
  • All samples were analyzed by both the telomere and hemoglobin reactions, and the analysis was performed in triplicate on the same plate. In addition to the test samples, each 96-well plate contained a five-point standard curve from 0.08 ng to 250 ng using genomic DNA isolated from the 1301 human T-cell leukemia cell line (available from Sigma and ATCC). The T/S ratio (−dCt) for each sample was calculated by subtracting the median hemoglobin threshold cycle (Ct) value from the median telomere Ct value. The relative T/S ratio (−ddCt) was determined by subtracting the T/S ratio of the 10.0 ng standard curve point from the T/S ratio of each unknown sample.
  • Results are shown in FIG. 95. Each data point shown is the median measurement of relative T/S ratio. The results indicate that TILs cultured with aEM3 maintain their telomere length.

Claims (21)

1.-99. (canceled)
100. A method of treating a subject having cancer with a population of lymphocytes, the method comprising:
(a) obtaining a first population of lymphocytes from a tumor resected from a patient;
(b) performing an initial expansion of the first population of lymphocytes in a first cell culture medium to obtain a second population of lymphocytes, wherein the second population of lymphocytes is at least 5-fold greater in number than the first population of lymphocytes, and wherein the first cell culture medium comprises IL-2;
(c) performing a rapid expansion of the second population of lymphocytes in a second cell culture medium to obtain a third population of lymphocytes, wherein the third population of lymphocytes is at least 50-fold greater in number than the second population of lymphocytes after about 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises IL-2 or OKT-3,
(d) transducing one of the first, the second, or the third population of lymphocytes with one or more viral vectors comprising a nucleic acid encoding a cell surface binding molecule, and/or one or more nucleic acids encoding one or more costimulatory molecules, wherein the first, the second, or the third population of lymphocytes expresses the cell surface binding molecule and the one or more costimulatory molecules; and
(e) administering a therapeutically effective portion of the third population of lymphocytes to a subject with the cancer.
101. The method of claim 100, wherein the lymphocytes comprise tumor-infiltrating lymphocytes (TILs).
102. The method of claim 100, wherein the one or more costimulatory molecules are independently selected from the group consisting of 4-1BB (CD137), OX40 (CD134), CD1a, CD1b, CD1c, CD1d, CD2, CD3γ, CD3δ, CD3∈, CD4, CD5, CD6, CD7, CD8α, CD8β, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a, CD16b, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD3δ, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CDw70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CD79α, CD79β, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CDw109, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CDw124, CD125, CD126, CDw127, CDw128a, CDw128b, CDw130, CDw131, CD132, CD133, CD135, CD136, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166, and TCRξ.
103. The method of claim 102, wherein the one or more costimulatory molecules are independently selected from the group consisting of CD28, 4-1BB (CD137), and OX40 (CD134).
104. The method of claim 100, wherein the cell culture medium comprises IL-2.
105. The method of claim 104, wherein the IL-2 is at an initial concentration of about 3000 IU/mL.
106. The method of claim 100, wherein the cell culture medium comprises OKT-3 antibody.
107. The method of claim 106, wherein the OKT-3 antibody is at an initial concentration of about 30 ng/mL.
108. The method of claim 100, wherein the rapid expansion is performed over a period not greater than 14 days.
109. The method of claim 100, wherein one or both of the initial expansion and the rapid expansion is performed using a gas permeable container.
110. The method of claim 100, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, renal cell carcinoma, pancreatic cancer, and glioblastoma.
111. The method of claim 100, wherein the one or more viral vectors comprise a lentiviral vector.
112. The method of claim 100, wherein the rapid expansion is performed using a population of antigen presenting cells (APCs).
113. The method of claim 112, wherein the population of APCs expands the population of lymphocytes by at least 50-fold over a period of about 7 days.
114. The method of claim 112, wherein the population of APCs endogenously express HLA-AB/C, ICOS-L, and CD58.
115. The method of claim 112, wherein the population of APCs are transduced to express an anti-OKT-3 antibody scFv binding domain.
116. The method of claim 112, wherein the ratio of the second population of lymphocytes to the population of APCs is between about 1 to 200 and about 1 to 400.
117. The method of claim 100, wherein the population of lymphocytes are cryopreserved.
118. The method of claim 101, wherein the TILs are cryopreserved.
119. The method of claim 100, wherein the cell surface binding molecule comprises a single chain fragment variable (scFv) binding domain.
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