NZ793899A - Engineered artificial antigen presenting cells for tumor infiltrating lymphocyte expansion - Google Patents

Engineered artificial antigen presenting cells for tumor infiltrating lymphocyte expansion

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Publication number
NZ793899A
NZ793899A NZ793899A NZ79389917A NZ793899A NZ 793899 A NZ793899 A NZ 793899A NZ 793899 A NZ793899 A NZ 793899A NZ 79389917 A NZ79389917 A NZ 79389917A NZ 793899 A NZ793899 A NZ 793899A
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New Zealand
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protein
tils
cell
population
seq
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NZ793899A
Inventor
Aishwarya Gokuldass
Michael T Lotze
Brian Rabinovich
Anand Veerapathran
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Iovance Biotherapeutics Inc
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Application filed by Iovance Biotherapeutics Inc filed Critical Iovance Biotherapeutics Inc
Publication of NZ793899A publication Critical patent/NZ793899A/en

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Abstract

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-lBBL and/or OX40L and transduce the myeloid cell to express CD86 and 4-lBBL 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. 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-lBBL and/or OX40L and transduce the myeloid cell to express CD86 and 4-lBBL 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

In some embodiments, compositions and methods relating to isolated artificial antigen presenting cells (aAPCs) are disclosed, including aAPCs comprising a d cell transduced with one or more viral vectors, such as a MOLM-14 or a EM-3 d 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-lBBL and/or OX40L and transduce the myeloid cell to express CD86 and 4-lBBL and/or OX40L proteins. In some embodiments, s of expanding tumor infiltrating lymphocytes (TILs) with aAPCs and s of treating cancers using TILs after expansion with aAPCs are also disclosed.
NZ 793899 ENGINEERED ARTIFICIAL ANTIGEN PRESENTING CELLS FOR TUMOR RATING LYMPHOCYTE EXPANSION CROSS-REFERENCE TO RELATED ATIONS This international application claims the benefit of priority to US. Provisional Application No. 62/481,831, filed Apr. 5, 2017, US. Provisional Application No. 62/475,053, filed Mar. 22, 2017, US. Provisional Application No. 62/438,600, filed Dec. 23, 2016, and US.
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 cytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. Gattinoni, el al., Nat. Rev. Immunol. 2006, 6, 3. A large number of TILs are ed 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. ILbased TIL expansion followed by a "rapid expansion process" (REP) has become a red method for TIL expansion because of its speed and efficiency. Dudley, el al., e 2002, 298, 850-54, , el al., J. Clin. Oncol. 2005, 23, 2346-57, Dudley, el al., J. Clin. Oncol. 2008, 26, 5233-39, Riddell, el al., Science 1992, 257, 23 8-41, Dudley, el al., J. Immunolher. 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 ), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT-3) and high doses of IL-2. , el al., J.
Immunolher. 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 le 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 ties.
Unfortunately, aAPCs developed for use in the expansion of TILs have suffered from poor mance 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- lBBL (CD137L) were tested in pre-REP e (but not in REP e) 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, el al., J. lher. 2011, 34, 65 1-661. Other engineered K562 cells modified to express CD64, CD86, and 4-lBBL 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 ble CD8+/CD4+ T cell ratio. Ye, el al., J. Translat. Med. 2011, 9, 131. ly, K562 cells modified to express CD86, 4-lBBL (CD137L), high affinity Fc receptor (CD64) and membrane- bound IL-l5 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-l5.
Forget, el al., J. Immunolher. 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-2l). Butler and Hirano, l. Rev. 2014, 25 7, 191-209.
Overall, K562 modified aAPCs have not been shown to e for consistent expansion of TILs with able ility 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 mance as PBMCs with the unique polyclonal subset of cells that make up TILs. Maus, el al., Nat. Biotechnol. 2002, 20, 143-148, i, el al., M0]. 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 terization ted 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, el 61]., Leukemia 1997, 11, 1469-77. Additional ypic characterization of MOLM-14 found higher levels ofHLA-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 ations.
The EM-3 human cell line was ished from the bone marrow of a t with Philadelphia chromosome-positive CML. Konopka, el 61]., Proc. Nat’lAcad. 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 therapy applications. Phenotypic characterization for EM-3 cells indicates the presence of at least the following markers: CD13, CD15, and CD33.
The t invention provides the unexpected g 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 OX4OL (CD134L), provide for superior and highly ent 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 ed 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.
SUMIVIARY 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 c 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-lBBL 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 le ng 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-lBBL 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-lBBL or the same viral vector.
In an ment, 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 c acid molecule encoding CD86 and a nucleic acid molecule encoding , and wherein the d 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 s, n the one or more viral s 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-lBBL 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 s comprise a nucleic acid molecule encoding CD86 and a nucleic acid le encoding 4-1BBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, wherein the d cell endogenously expresses HLA-A/B/C, ICOS-L, and CD58.
In an embodiment, the invention provides an aAPC sing 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, n the myeloid cell is essentially devoid of membrane-bound IL-15.
In an embodiment, the ion provides an aAPC comprising a myeloid cell transduced with one or more viral s, wherein the one or more viral s comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the d cell expresses a CD86 protein and a 4-1BBL protein, wherein the myeloid cell is a 4 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-l3 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 s se 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 es an aAPC comprising a d 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 s comprise a nucleic acid molecule ng CD86 and a nucleic acid molecule encoding 4-1BBL, and wherein the d 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 N08, or an amino acid sequence comprising one or more conservative amino acid substitutions thereof, and the 4-lBBL n comprises SEQ ID N09, 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-lBBL, 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 ng 4-lBBL comprises a nucleic acid sequence as set forth in SEQ ID NOIl9.
In an embodiment, the ion provides a method of expanding tumor infiltrating cytes (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 le encoding CD86 and a c acid molecule encoding 4-lBBL, wherein the myeloid cell expresses a CD86 protein and a 4-lBBL 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 ment, the invention provides a method of expanding a population of tumor inflltrating lymphocytes (TILs), the method comprising the steps of: (a) transducing a myeloid cell with one or more viral vectors to obtain a population of ial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a c acid le encoding CD86 and a nucleic acid le encoding 4-lBBL, and wherein the myeloid cell expresses a CD86 protein and a 4-lBBL protein, (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 .
In an embodiment, the invention provides a method of expanding a population of tumor inflltrating lymphocytes (TILs), the method sing 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 c acid molecule encoding 4-lBBL, and wherein the d cell expresses a CD86 protein and a 4-lBBL protein, (b) contacting the population of TlLs 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 (TlLs), the method comprising the steps of: (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen ting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-lBBL, and wherein the myeloid cell ses a CD86 protein and a 4-lBBL protein, (b) contacting the population of TlLs with the population of aAPCs in a cell e medium, wherein the population of APCs expands the population of TlLs by at least 50-fold over a period of 7 days in a cell culture medium.
In an ment, 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 (TlLs), the method comprising the steps of: (a) transducing a myeloid cell with one or more viral vectors to obtain a tion of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a nucleic acid le encoding 4-lBBL, and wherein the d cell expresses a CD86 n and a 4-lBBL protein, (b) contacting the population of TlLs with the population of aAPCs in a cell e , wherein the myeloid cell endogenously expresses HLA-A/B/C, ICOS-L, and CD58.
In an embodiment, the foregoing method is an in vitro or an ex vivo method.
In an embodiment, the ion es a method of expanding a population of tumor infiltrating lymphocytes (TlLs), 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 c acid molecule encoding CD86 and a nucleic acid le encoding 4-lBBL, and wherein the myeloid cell expresses a CD86 protein and a 4-lBBL protein, (b) contacting the population of TlLs with the population of aAPCs in a cell culture medium, wherein the myeloid cell is a MOLM-l4 cell.
In an embodiment, the foregoing method is an in vitro or an ex vivo method.
In an embodiment, the invention es a method of expanding a population of tumor infiltrating lymphocytes (TlLs), 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 s comprise a nucleic acid molecule encoding CD86 and a nucleic acid molecule encoding 4-lBBL, and wherein the myeloid cell ses a CD86 protein and a 4-lBBL protein, (b) contacting the population of TlLs with the tion of aAPCs in a cell culture medium, wherein the myeloid cell is a MOLM-l3 cell.
In an embodiment, the foregoing method is an in vitro or an ex vivo method.
In an ment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TlLs), the method comprising the steps of: (a) transducing a d cell with one or more viral s 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 le encoding 4-lBBL, and wherein the myeloid cell expresses a CD86 protein and a 4-lBBL protein, (b) contacting the population of TlLs 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 ment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes , 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-lBBL, and wherein the d cell expresses a CD86 protein and a 4-lBBL protein, (b) contacting the population of TlLs with the tion of aAPCs in a cell culture medium, wherein the d 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 (TlLs), 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 ), wherein the one or more viral vectors comprise a nucleic acid molecule encoding CD86 and a c acid molecule encoding 4-lBBL, and wherein the myeloid cell expresses a CD86 n and a 4-lBBL protein, (b) contacting the population of TlLs with the population of aAPCs in a cell e medium, wherein the CD86 protein comprises an amino acid sequence as set forth in SEQ ID N08, or comprises an amino acid sequence comprising one or more vative amino acid substitutions thereof, and the 4-lBBL protein comprises an amino acid sequence as set forth in SEQ ID N09, 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 cytes (TlLs), the method comprising the steps of: WO 81789 (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 ng 4-lBBL, and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and (b) ting 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-lBBL comprises a nucleic acid sequence as set forth in SEQ ID NO:l9.
In an embodiment, the ion provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) ucing a myeloid cell with one or more viral s 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-lBBL, and wherein the myeloid cell ses a CD86 protein and a 4-1BBL protein, and (b) contacting the tion of TILs with the population of aAPCs in a cell culture medium, wherein the ion is performed using a gas ble container.
In an embodiment, the invention provides a method of expanding a population of tumor infiltrating cytes (TILs), the method comprising the steps of: (a) transducing a d 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-lBBL, 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 l to 400.
In an embodiment, the ion 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 ting cells (aAPCs), wherein the one or more viral vectors se a c acid encoding CD86 and a c acid encoding , and wherein the myeloid cell expresses a CD86 protein and a 4-lBBL 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 ion provides a method of expanding tumor infiltrating cytes (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 mulatory 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-lBBL. 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 tion 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 tion 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 ive portion of the second population of TILs to a patient with the cancer, wherein the myeloid aAPCs nously expresses HLA-A/B/C, ICOS-L, and CD58, and wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-lBBL 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 TlLs using a tion of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, wherein the TlLs 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 tion of TILs after 7 days from the start of the rapid expansion; and wherein the myeloid aAPCs endogenously expresses HLA-A/B/C, , and CD5 8, and wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-lBBL protein.
In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TlLs) 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 TlLs, 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, n the myeloid aAPCs endogenously ses HLA-A/B/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 se MOLM-l4 cells transduced with one or more viral vectors, and wherein the one or more viral vectors se a nucleic acid encoding CD86 and a c acid encoding 4-lBBL, and n the MOLM-l4 cells express a CD86 protein and a 4-lBBL protein.
In an ment, the invention provides a population of tumor infiltrating cells (TlLs) for use in treating a cancer, wherein the tion of TlLs is a second population of TILs and is obtainable by a process comprising: (a) performing a rapid expansion of a first population of TlLs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second tion of TILs, wherein the first population of TILs are/have been obtained from a tumor resected from a patient, wherein the second population of TlLs 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 ses HLA-A/B/C, ICOS—L, and CD5 8, wherein the myeloid aAPCs are transduced to s a CD86 protein and a 4-lBBL protein, n the myeloid aAPCs comprise MOLM-l4 cells transduced with one or more viral vectors, and n the one or more viral vectors comprise a c acid encoding CD86 and a nucleic acid encoding 4-lBBL, and wherein the MOLM-l4 cells express a CD86 protein and a 4-lBBL protein.
In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TlLs) 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 ting cells (myeloid aAPCs) in a cell culture medium to obtain a second population of TlLs, 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 t with the cancer, wherein the myeloid aAPCs endogenously ses HLA-A/B/C, ICOS—L, and CD58, wherein the myeloid aAPCs are transduced to s a CD86 protein and a 4-lBBL 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-lBBL, and wherein the EM-3 cells express a CD86 protein and a 4- lBBL protein.
In an embodiment, the invention provides a population of tumor ating lymphocytes (TILs) for use in ng a cancer, the population of TILs being a second population of TILs and obtainable by a s comprising: (a) performing a rapid ion of a first population of TlLs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell e medium to obtain the second population of TILs, wherein the first population of TILs are/have been obtained from a tumor resected from a t, and wherein the second population of TlLs 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-A/B/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 se a nucleic acid ng CD86 and a nucleic acid encoding 4-lBBL, and wherein the EM-3 cells s a CD86 protein and a 4- lBBL protein.
In an embodiment, the invention provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TlLs) 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 tion of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a cell e medium to obtain a second population of TlLs, 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-A/B/C, ICOS—L, and CD5 8, 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) ming a rapid expansion of the first population of TILs using a population of myeloid ial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TILs, n 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-A/B/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 es 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 ion of the first population of TILs using a tion 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 r in number than the first population of TILs after 7 days from the start of the rapid ion, and (c) administering a therapeutically effective n of the second population of TILs to a patient with the cancer, wherein the myeloid aAPCs endogenously expresses HLA-A/B/C, ICOS—L, and CD5 8, 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 l 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 able by a process comprising: (a) performing a rapid ion of a first population of TlLs using a tion 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 TlLs is at least 50-fold greater in number than the first tion of TILs after 7 days from the start of the rapid expansion; and wherein the myeloid aAPCs endogenously express HLA-A/B/C, ICOS-L, and CD5 8, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein, and n the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an l concentration of about 30 ng/mL.
In an embodiment, the ion provides a method of treating a cancer with a population of tumor infiltrating lymphocytes (TlLs) 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 TlLs, n 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 eutically effective portion of the second population of TILs to a patient with the cancer, wherein the myeloid aAPCs endogenously expresses HLA-A/B/C, ICOS—L, and CD58, wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL n, and wherein the ion is performed using a gas permeable container.
In an embodiment, the invention es 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 TlLs 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 ed from a patient, and wherein the second tion of TlLs 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-A/B/C, ICOS-L, and CD5 8, 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 ating lymphocytes (TlLs) comprising the steps of: (a) ing a first population of TILs from a tumor resected from a patient, (b) ming 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 TlLs, wherein the second tion 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 ive portion of the second population of TILs to a patient with the cancer, n the myeloid aAPCs endogenously expresses HLA-A/B/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 n 1 to 200 and l to 400.
In an embodiment, the invention provides a tion of tumor infiltrating cells (TlLs) for use in treating a , the population of TILs being a second population of TlLs and obtainable by a process comprising the steps of: (a) performing a rapid expansion of a first population of TlLs 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 TlLs 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-A/B/C, ICOS—L, and CD5 8, 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 tion of aAPCs is between 1 to 200 and l to 400. In n ments, 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 d 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 eutically effective portion of the second population of TILs to a patient with the cancer, wherein the myeloid aAPCs endogenously expresses HLA-A/B/C, ICOS—L, and CD58, wherein the d aAPCs are transduced to express a CD86 protein and a 4-1BBL n, and wherein the ratio of the second population of TILs to the tion 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 ial n 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 ion; and (c) administering a therapeutically effective portion of the second tion of TILs to a patient with the ; wherein the myeloid aAPCs endogenously expresses HLA-A/B/C, ICOS—L, and CD5 8, wherein the d 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 oma.
In an embodiment, the invention provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, the tion 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 TlLs using a tion of myeloid ial antigen presenting cells (myeloid aAPCs) in a cell culture medium to obtain the second population of TlLs, wherein the first population of TILs is/has been obtained from a tumor resected from a t, 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 d aAPCs endogenously expresses HLA-A/B/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, all-cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck , 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 (TlLs) 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 tion 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 1L-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 ion 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 l expansion of the first tion 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 ting 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 1L-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-l4 cells transduced with one or more viral s; wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a c acid encoding 4-1BBL; and n the MOLM-l4 cells express a CD86 protein and a 4-lBBL protein.
WO 81789 In an ment, the ion 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 tion of TILs in a first cell culture medium to obtain a second tion 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 tion 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 d 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 1L-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-lBBL, and wherein the EM-3 cells express a CD86 protein and a 4-lBBL 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 ion 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 e 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 ment, the myeloid aAPCs comprise MOLM-l4 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-l4 cells s a CD86 protein and a 4-lBBL protein. In an embodiment, the myeloid cells comprise MOLM-l3 cells uced with one or more viral vectors, n the one or more viral s comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-lBBL, and wherein the MOLM-l3 cells express a CD86 protein and a 4-lBBL protein. In certain embodiments, the d 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 ng 4-lBBL, and wherein the EM-3 cells s a CD86 protein and a 4-lBBL 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- lBBL, and wherein the EM-2 cells express a CD86 n and a 4-lBBL 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 e medium comprises IL-2, (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen ting 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 r 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, n the non- myeloablative lymphodepletion regimen comprises the steps of stration of cyclophosphamide at a dose of 60 mg/mZ/day for two days ed by administration of fiudarabine at a dose of 25 mg/mZ/day for five days, (e) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer; and (f) ng the t with a high-dose IL-2 regimen, wherein the high-dose 1L-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin stered as a 15-minute bolus intravenous infusion every eight hours until tolerance; n the myeloid aAPCs comprise MOLM-l4 cells transduced with one or more viral vectors, wherein the one or more viral vectors se a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-l4 cells express a CD86 protein and a 4-lBBL protein.
In an ment, the invention provides a method of treating a cancer with a population of tumor ating 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 n the second cell culture medium comprises 1L-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/mZ/day for two days followed by administration of bine at a dose of 25 mg/mZ/day for five days, (e) stering a therapeutically effective n 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 1L-2 regimen comprises 600,000 or 720,000 IU/kg of eukin administered as a 15-minute bolus intravenous infusion every eight hours until tolerance; wherein the myeloid aAPCs comprise EM-3 cells uced 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-lBBL, and wherein the EM-3 cells express a CD86 protein and a 4-lBBL protein.
In an embodiment, the ion provides a population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TlLs 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 tion of TILs, wherein the first population of TILs is/ has been obtained from a tumor resected from a t, and wherein the second population of TlLs is at least 5-fold greater in number than the first population of TILs, and wherein the first cell culture medium comprises 1L-2, and (b) performing a rapid expansion of the second population of TlLs 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 ion, 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/mZ/day for two days followed by fiudarabine which is for administration at a dose of 25 mg/mZ/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 on every eight hours until tolerance. In certain embodiments, the tion 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-l4 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-lBBL, and wherein the MOLM-l4 cells express a CD86 protein and a 4-1BBL protein. the myeloid aAPCs comprise MOLM-l3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a c acid encoding , and wherein the MOLM-l3 cells express a CD86 protein and a 4-1BBL protein. In certain embodiments, the myeloid aAPCs se EM- 3 cells transduced with one or more viral s, wherein the one or more viral vectors comprise a c acid encoding CD86 and a nucleic acid encoding 4-lBBL, 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, r cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer, renal cancer, and renal cell carcinoma.
In an embodiment, the invention es a method of ng 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 ion of the first population of TILs in a first cell culture medium to obtain a second population of TILs, n 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 tion of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell e 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 1L-2 and OKT-3, (d) stering a therapeutically effective portion of the third tion of TILs to a patient with the cancer, wherein 1L-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 tion 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 r 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 d ial 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 n the second cell e medium comprises IL-2 and OKT-3, wherein 1L-2 is present at an initial concentration of about 3000 IU/mL and OKT-3 dy 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 tion of TILs, wherein the second population of TILs is at least 5-fold greater in number than the first tion of TlLs, and wherein the first cell culture medium ses IL-2, (c) performing a rapid expansion of the second population of TlLs using a tion of myeloid artificial antigen presenting cells (aAPCs) in a second cell culture medium to obtain a third population of TlLs, wherein the third tion of TlLs is at least 50-fold greater in number than the second population of TlLs after 7 days from the start of the rapid ion; and wherein the second cell culture medium comprises 1L-2 and OKT-3, (d) stering a therapeutically effective portion of the third population of TILs to a t with the cancer, wherein the rapid expansion is performed over a period not greater than 14 days.
In an embodiment, the invention provides a tion of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of TlLs 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 TlLs 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 TlLs 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) ming a rapid expansion of the second tion of TlLs 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 (TlLs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient, (b) performing an l expansion of the first population of TILs in a first cell e 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 ion of the second tion of TILs using a population of myeloid artificial antigen presenting cells (aAPCs) in a second cell e 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 1L-2 and OKT-3; (d) administering a therapeutically effective portion of the third population of TILs to a patient with the , wherein the initial expansion is performed using a gas permeable container.
In an embodiment, the invention provides a method of ng 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 t; (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 r 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 tion 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 1L-2 and OKT-3; (d) administering a therapeutically effective portion of the third population of TILs to a patient with the cancer; wherein the rapid expansion is med using a gas permeable container.
In an embodiment, the invention provides a population of tumor ating 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 ed from a tumor resected from a patient, and wherein the second tion 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 e 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 ion, and n the second cell culture medium comprises IL-2 and OKT-3, wherein the initial expansion and/or the rapid ion 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 d 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 1L-2 and OKT-3, (d) administering a eutically 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 l to 400.
In an embodiment, the ion 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 l 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 tion of TILs using a population of myeloid artificial antigen presenting cells ) in a second cell culture medium to obtain a third population of TILs, n the third population of TILs is at least 50-fold greater in number than the second tion of TILs after 7 days from the start of the rapid expansion; and wherein the second cell culture medium comprises 1L-2 and OKT-3, (d) administering a therapeutically ive portion of the third population of TILs to a patient with the cancer, wherein the ratio of the second population of TILs to the tion 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 e medium comprises IL-2, (b) performing a rapid ion of the second tion 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 r 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 l 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 ating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a patient; (b) ming an initial expansion of the first population of TILs in a first cell culture medium to obtain a second population of TILs; n 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; n 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 n the second cell culture medium comprises 1L-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; r 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 ses a MOLM-14 cell or a EM-3 cell transduced using a lentiviral vector (LV), wherein the LV ses a nucleic acid encoding at least one co-stimulatory ligand that specifically binds said known co-stimulatory le, wherein g of the known co-stimulatory molecule with said co-stimulatory ligand stimulates and expands said T cell, the kit r comprising an ator and an ctional material for the use of said kit.
In an embodiment, the invention provides a method for ing 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 t OKT-3 to assess T cell receptor (TCR) activation (specific killing) or lymphokine activated killing (LAK, non- specific killing), tively, (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 xicity 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. illustrates the results of rapid expansion of TILs using irradiated allogeneic PBMC feeder cells. Each TIL line (M1015T and M1016T) (1.3 X 105 cells) was co-cultured with 46 different irradiated feeders (1.3 X 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. illustrates a vector diagram of the pLV43OG human 4-1BBL vector. illustrates a diagram of the 4-1BBL PCRP (polymerase chain reaction t) portion of the pLV43OG human 4-1BBL vector. illustrates a vector diagram of the pLV43OG human CD86 vector. rates a diagram of the CD86 PCRP portion of the pLV43OG human CD86 vector. rates a vector diagram of the pDONR221 human CD86 donor vector. illustrates a vector diagram of the pDONR221 human 4-1BBL donor vector. illustrates a vector diagram of the pLV43OG empty vector. illustrates a vector diagram of the pDONR221 empty . illustrates a vector diagram of the psPAX2 helper plasmid for lentivirus production. illustrates a vector diagram of the pCIGO-VSVG helper plasmid for lentivirus production. illustrates the results of flow cytometry experiments on 4 cells before lentiviral transfection ansfected") and after transfection ("Transfected"), confirming the expression of CD137 and CD86 on engineered 4 cells. illustrates the results of rapid expansion of TILs using irradiated parental unmodif1ed MOLM-l4 cells ("Parent MOLM14"), engineered MOLM-l4 cells (CD86/4-1BBL, "Engineered MOLM14"), or PBMC feeders ("Feeders") for TIL lot 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. illustrates s as shown in , depicted using a linear scale.
WO 81789 illustrates results for TIL lot M1033-T6 with other parameters as given in , using a logarithmic scale. illustrates results as shown in , depicted using a linear scale. illustrates the results of rapid expansions of TILs using engineered 4 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 4 cells (aMOLMl4) at 1:100 ratios plus OKT-3 (30 ng/mL) and IL-2 (3000 . Cells were counted and split on Day 6 and 11. Each point represents cell s determined on Day 14. illustrates the results of experiments in which TILs (2 X 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- lBBL ("aMOLM14"), or PBMC feeders ("PBMC+"), each with OKT-3 (30 ng/mL) and IL-2 (3 000 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 (3 000 IU/mL) ("PBMC-"). Each condition was cultured in cate.
Cultures were fed with fresh media and IL-2 on Day 4 and 7. Viable cells were d 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 t ‘t’ test. illustrates the results of TILs cultured with different ratios (1 :30, 1:100, and 1:300, denoted "30", "100", and "300", respectively) ofPBMC feeders ("PBMC"), al 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 (3 000 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 . illustrates the s of TILs cultured with different ratios (1 :50, 1:100, and 1:200, denoted "50", "100", and "200", respectively) ofPBMC 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 (3 000 IU/mL) in the single 24 well G- Rex culture plates. Cells were counted on day 14. Other conditions are as in . 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) ofPBMC s ("PBMC"), parental MOLM-14 cells ("MOLM14"), or MOLM-14 cells transduced to express CD86 and 4- 1BBL M14"), 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 . illustrates a sunburst visualization showing f1ne bution of Live, T cell receptor (TCR) OL/B, CD4, CD8, CD27, CD28, and CD57 TILs expanded with PBMC feeders. illustrates a sunburst visualization showing f1ne distribution of Live, TCR d/B, CD4, CD8, CD27, CD28, and CD57 TILs expanded with aMOLM14 aAPCs. depicts a flow cytometry contour plot showing memory subset (CD45RA+/—, CCR7+/—) gated on Live, TCR d/B +, CD4+, or CD8+ TlLs. illustrates phenotypic characterization of the T cell subset, CD4+ and CD8+ post-REP TlLs (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, THVI3, PDl, and CD137. illustrates phenotypic terization of the T cell subset, CD4+ and CD8+ post-REP TlLs (expanded with aMOLM14 aAPCs) gated on CD3+ cells using a SPADE tree.
The color gradient is proportional to the MFI CD69, CD154, KLRGl, and TIGIT rates oxygen consumption rate (OCR) of TIL after expansion with Feeders or aMOLM14 measured during a mitochondrial stress test. Each data point represents mean i standard error of the mean (SEM) measured in triplicate. illustrates ellular acidification rate (ECAR) of TIL after expansion with Feeders or aMOLM14 measured during a mitochondrial stress test. Each data point represents mean i SEM measured in cate. illustrates a vector diagram of the destination vector pLV4301G. rates a vector m of donor vector 1, prJK 7c12 anti mFC scFv CoOp ECORV SacII LlR5. illustrates a vector diagram of donor vector 2, prJK hCD8a scaffold TN L5 L2. illustrates a vector diagram of final vector used for lentiviral tion, pLV4301G 7C12 scFv mIgG hCD8 flag. illustrates a vector diagram of the destination vector pLV430lG. illustrates a vector m of donor vector 1, pMK 8B3 anti mFC scFv CoOp ECORV SacII L1R5. illustrates a vector diagram of donor vector 2, pMK hCD8a ld TN L5 L2. illustrates a vector diagram of final vector used for lentiviral production, pLV4301G 8B3 scFv mIgG hCD8 flag. ] illustrates the results of flow cytometry experiments on EM-3 cells before lentiviral transfection ("Untransfected") and after transfection ("Transfected"), confirming the sion of CD137 and CD86 on engineered EM-3 cells. 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. ] 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. illustrates the results of experiments wherein TILs were co-cultured with aEM3 or PBMC feeders at a 1:100 ratio with IL-2 (3 000 IU/mL), with or without OKT-3 (30 ng/mL).
The bar graph shows cell numbers determined on Day 11. illustrates the s of TIL expansions with EM-3 aAPCs at different TIL:aAPC ratios. illustrates the results of TIL expansions with EM-3 aAPCs. TILs (2 X 104) were co-cultured with five ent PBMC feeder lots or aEM3 (in triplicate) at a 1:100 ratio with IL-2 (3 000 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. illustrates the results of TIL expansions with EM-3 aAPCs and 4 aAPCs. TILs (2 X 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. illustrates a st visualization to show fine distribution of Live, TCR d/B, CD4+, and CD8+ TILs expanded with aEM3 aAPCs or PBMC feeders (TIL batch M1054). illustrates the st visualization to show fine distribution of Live, TCR d/B, CD4+, and CD8+ TILs expanded with aEM3 aAPCs or PBMC feeders (TIL batch M1055). ] illustrates the CD4+ and CD8+ SPADE tree of TILs expanded with aEM3 aAPCs or PBMC s using CD3+ cells. The color gradient is proportional to the MFI of LAG-3, TIM-3,PD-1, and CD137. 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, KLRGl, and TIGIT. illustrates a summary of spare respiratory capacity measured by the Seahorse XF Mito stress test. illustrates a summary of glycolytic reserve ed by the se XF Mito stress test. 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 ial. 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. rates results of a P815 BRLA for cytotoxic potency and functional activity, comparing TlLs expanded with PBMC feeders to TILs expanded using aMOLM14 aAPCs. illustrates results of a P815 BRLA for cytotoxic potency and onal activity, comparing TILs expanded with PBMC feeders to TILs expanded using aEM3 aAPCs. ] illustrates IFN-y release for two batches of TILs following overnight ation ("S") with microbeads coated with anti-CD3/CD28/4-1BB in comparison to unstimulated ("US") TILs, ing TILs expanded with PBMC feeders to TILs expanded using aMOLMl4 aAPCs. * p<0.05, ** p<0.005, *** p<0.00l, ns = not significant. illustrates IFN-y e 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 ed using aEM3 aAPCs. * p<0.05, ** p<0.005, *** p<0.00l, ns = not significant. ] illustrates Granzyme B release for two batches of TILs following overnight stimulation ("S") with microbeads coated with D3/CD28/4-1BB in comparison to unstimulated ("US") TILs, comparing TILs expanded with PBMC feeders to TILs expanded using aMOLMl4 aAPCs. * p<0.05, ** 5, *** l, ns = not significant. 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<0.00l, ns = not significant. 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 lO flasks. At step 2, approximately 40 X 106 TILs or greater are obtained. At step 3, a split occurs into 36 G-Rex 100 flasks for REP. TILs are ted by centrifugation at step 4. Fresh TIL product is obtained at step 5 after a total s time of approximate 43 days, at which point TILs may be infused into a patient. 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. illustrates Bioluminescent Redirected Lysis Assay (BRLA) s, showing percentage cytotoxicity of TIL batch MlO33T-l when co-cultured with P815 Clone G6 (with and without anti-CD3) at individual effector:target . illustrates enzyme-linked sorbent assay (ELISA) data showing amount of IFN—y released against different ratios of effector to target cells. illustrates LAMPl (%) expressed by TIL batch MlO33T-l when co-cultured with P815 Clone G6 in the presence of anti-CD3 at a ratio of 1:1 effector to target cells for 4hr and 24hr co-culture. illustrates BRLA results for TIL batch M1030. Cytotoxicity (measured as LU50/1 x 106 TIL) by BRLA is 26 16. illustrates standard chromium release assay for TIL batch M1030. xicity (measured as LU50/l X 106 TIL) by the chromium release assay is 22. illustrates BRLA results for TIL batch M1053, showing the lytic units of the TILs by BRLA as 70 :: l7. rates standard chromium release assay results for TIL batch M1053, also showing lytic unit of the TILs by um assay as 14 :: 5. Comparison of this result with shows the comparable performance of the BRLA and chromium release assay. rates the linear relationship between IFN—y release and cytotoxic potential of TILs. illustrates ELISpot results for IFN—y. illustrates enzymatic IFN—y release for TIL batch M1053. rates enzymatic IFN—y release for TIL batch M1030. illustrates ELISpot data showing Granzyme B e by M1053T and M103OT. This data confirms the potency of the TILs shown by the BRLA. illustrates enzymatic Granzyme B e for TIL batch M1053. illustrates enzymatic Granzyme B release for TIL batch M1030. illustrates ELISpot data showing TNF-Ot e by M1053T and M103OT.
This data confirms the potency of the TILs shown by the BRLA. rates enzymatic TI\F-0t e for TIL batch M1053. illustrates enzymatic TI\F-0t release for TIL batch M1030. illustrates s in cell populations of aEM3 cells (C712 (A) and 8B5 (B)) when weaning such cell populations off of FBS to hAB serum media. illustrates s in cell populations of during freeze-thaw-recovery cycles with aEM3 cell populations suspended in s freezing media. illustrates the growth of aEM3 cells in gas permeable cell culture flasks over an eight-day time course. illustrates a flow panel analysis to determine the purity of aEM3 cells. ] illustrates the results of a flow panel analysis used to determine the purity of aEM3 cells. illustrates the differences in cytokine expression between aEM3 feeder cells and PBMC feeders stimulated by OKT3. ] illustrates that TIL may advantageously expanded (pre-REP) with serum free media (i.e., CTS Optmizer) to provide increased cell numbers as compared to CMl. and illustrate that TIL may advantageously expanded with serum free media (1'.e., CTS Optmizer) to provide increased cell numbers as compared to CMl at Day 11 (PreREP) () and Day 22 (Pre- and EP) (). illustrates that aAPC cells (1'.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. and rate the results of two experiments that demonstrate the rapid recovery of aEM3 cells from the TIL-R3 cell line on day 3 following cryopreservation. illustrates the total cell counts for experiment one and illustrates the total cell counts for experiment two. 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. illustrates the growth of aEM3 cells from the TIL-R3 cell line following cryopreservation where the cells were plated in GREX lO flasks and grown for 8 days. Cell counts were measured every four days post thaw. rates a vector diagram of the pLenti-C-Myc-DDK human OX4OL vector. 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. ] illustrates the numbers of viable cells obtained from experiments wherein TILs were ed in a REP with the aEM3 cell line and PBMC feeders. illustrates the numbers of CD3+ cells obtained from experiments wherein TILs were ed in a REP with the aEM3 cell line and PBMC feeders. illustrates the numbers of CD3' cells obtained from experiments n TILs were expanded in a REP with the aEM3 cell line and PBMC feeders. illustrates the results of telomere length analysis using a qPCR method. illustrates a schematic diagram of an embodiment of an aAPC of the present illustrates a schematic diagram of an embodiment of an aAPC of the t invention. illustrates a schematic diagram of an embodiment of an aAPC of the present invention.
BRIEF PTION OF THE SEQUENCE LISTING SEQ ID NO:1 is an amino acid sequence for the heavy chain of muromonab.
SEQ ID \O:2 is an amino acid sequence for the light chain of muromonab.
SEQ ID \O:3 is an amino acid sequence for recombinant human IL-2.
SEQ ID \O:4 is an amino acid sequence for aldesleukin.
SEQ ID \O:5 is an amino acid sequence for recombinant human IL-7.
SEQ ID \O:6 is an amino acid sequence for recombinant human IL-15.
SEQ ID \O:7 is an amino acid ce for recombinant IL-21.
] SEQ ID \O:8 is the amino acid sequence of human CD86.
] SEQ ID \O:9 is the amino acid sequence of human 4-1BBL L).
SEQ ID \0: 10 is the amino acid sequence of human OX4OL (CD134L).
] SEQ ID \O:11 is the amino acid sequence of human CD28.
SEQ ID \0: 12 is the amino acid sequence of human CTLA-4.
SEQ ID \0: 13 is the amino acid sequence of human 4-1BB (CD137).
SEQ ID \0: 14 is the amino acid sequence of human 0X40 (CD134).
SEQ ID \0: 15 is a nucleotide sequence for the pLV43OG 4-1BBL empty vector.
SEQ ID \0: 16 is a nucleotide sequence for the 4-1BBL CoOP n of the pLV43OG human 4-1BBL vector.
SEQ ID NO: 17 is a tide sequence for the 4-1BBL PCRP.
SEQ ID NO: 18 is a nucleotide sequence for the G hCD86 empty vector.
SEQ ID NO: 19 is a nucleotide sequence for the hCD86 CoOP portion of the pLV43OG human hCD86 vector.
SEQ ID NO:2O is a nucleotide sequence for the hCD86 CoOP B1 B2 PCRP portion of the pLV43OG human hCD86 vector.
SEQ ID NO:21 is a nucleotide sequence for the pDOI\R221 hCD86 vector.
SEQ ID NO:22 is a nucleotide sequence for the pDOI\R221 4-1BBL vector.
SEQ ID NO:23 is a nucleotide sequence for the pLV43OG vector.
SEQ ID NO:24 is a nucleotide sequence for the pDONR221 .
SEQ ID NO:25 is a nucleotide sequence for the psPAX2 helper plasmid for lentiviral production.
SEQ ID NO:26 is a nucleotide ce for the pCIGO-VSVG helper plasmid for lentiviral production.
SEQ ID \O:27 is the amino acid sequence of the mFC-7C12 scFv clone.
SEQ ID \Oz28 is the amino acid sequence of the mFC-8B3 scFv clone.
SEQ ID \O:29 is a nucleotide sequence for the mFC-7C12 scFv.
] SEQ ID \O:3O is a nucleotide sequence for the mFC-8B3 scFv.
SEQ ID \Oz3l is a tide ce for the destination vector pLV430lG.
SEQ ID \O:32 is a nucleotide sequence for the donor vector 1, pMK 7c12 anti mFC scFv CoOp ECORV SacII LlRS.
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, pLV430lG 7C12 scFv mIgG hCD8 flag.
SEQ ID NO:35 is a nucleotide sequence for the destination vector, pLV430lG.
SEQ ID NO:36 is a nucleotide sequence for the donor vector 1, pMK 8B3 anti mFC scFv CoOp ECORV SacII LlRS.
SEQ ID NO:37 is a tide ce for the donor vector 2, pMK hCD8a scaffold TN L5 L2.
SEQ ID N038 is a nucleotide sequence for the final vector used for lentiviral tion, pLV430lG 8B3 scFv mIgG hCD8 flag.
SEQ ID NO:39 is a nucleotide sequence for pLenti-C-Myc-DDK OX4OL vector for lentiviral production.
SEQ ID NO:4O is a nucleotide sequence for Tel-lb primer used for quantitative polymerase chain reaction measurements of re .
SEQ ID NO:4l is a nucleotide ce for Tel-2b primer used for quantitative polymerase chain reaction measurements of telomere length.
SEQ ID NO:42 is a nucleotide sequence for Tel-lb primer used for quantitative polymerase chain reaction measurements of telomere length.
SEQ ID NO:43 is a nucleotide sequence for Tel-lb primer used for quantitative polymerase chain reaction measurements of telomere .
DETAILED PTION 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.
The terms "co-administration," "co-administering,77 (L administered in combination with," "administering in combination with," "simultaneous," and "concurrent," as used , 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 ition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a ition in which both agents are present is also encompassed in the s of the invention.
The term "in viva" refers to an event that takes place in a subject’s body.
The term "in vilro" 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 ass 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 d 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 (MHC) molecules. The term "antigen", as used , also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system. In some ments, an antigen is capable of ng a humoral immune response or a cellular immune response leading to the tion 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- es). In some embodiments, an antigen will ably react, lly in a highly specific and selective manner, with its corresponding dy 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 ent. 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 se in target cells (e.g., the reduction of platelet adhesion and/or cell migration).
The specific dose will vary depending on the particular nds 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 t. A prophylactic effect includes delaying or eliminating the appearance of a disease or ion, delaying or eliminating the onset of symptoms of a e 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 ed to e any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, ic and tion ng agents, and inert ingredients. The use of such pharmaceutically able 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 ably 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 lOO-fold over a period of a week. A number of rapid expansion protocols are described herein.
By "tumor inflltrating 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 e, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Thl7 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 , including, but not limited to bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs", or "second population of TILs" or "third tion of TILs" where riate).
TILs can generally be defined either biochemically, using cell surface markers, or onally, 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 dB, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-l, and CD25. onally, 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 0C to -60 0C. 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 d cryopreserved TILs" herein is meant a population of TlLs that was previously cryopreserved and then treated to return to room ature or , including but not limited to cell culture temperatures or temperatures wherein TlLs may be administered to a ] 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 CD45RO+ 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- lSR. Transcription factors for central memory T cells include BCL-6, BCL-6B, lVfl3D2, and BMIl. Central memory T cells primarily secret 1L-2 and CD4OL as effector molecules after TCR ring. Central memory T cells are inant in the CD4 compartment in blood, and in the human are proportionally enriched in lymph nodes and s.
The term "effector memory T cell" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45RO+, but have lost the constitutive expression of CCR7 (CCR710) and are heterogeneous or low for CD62L expression (CD62L10). The e phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL- lSR. Transcription factors for central memory T cells e BLIMPl. Effector memory T cells rapidly secret high levels of inflammatory cytokines following antigenic stimulation, including interferon-y, 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 s of perforin.
The terms "sequence identity,77 (Lpercent identity," and "sequence percent identity" in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or uences 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 t identity can be ed using sequence comparison software or algorithms or by visual tion. Various thms 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 US. Government’ s National Center for hnology Information BLAST web site. isons between two ces 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, California) or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. One d in the art can determine appropriate ters for maximal alignment by particular ent software. In certain embodiments, the default parameters of the alignment software are used.
The term rvative 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 BLOSUIVI 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 ential amino acid residue in a 4-lBBL or CD86 protein is preferably replaced with r 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., ll, el al., Biochemistry 1993, 32, 1180-1 187, Kobayashi, et al., Protein Eng. 1999, 12, 879-884 (1999), and Burks, el 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 ofRNA 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 us regulatory signals including transcriptional l elements, polyadenylation signals and sequences needed for replication and integration of the viral genome. Several genera included within the family Relroviridae, including Cislernavirus A, Oncovirus A, Oncovirus B, Oncovirus C, Oncovirus D, Lenlivirus, Gammarelrovirus, and Spumavirus. Some of the retroviruses are oncogenic (1'.e., tumorigenic), while others are not. The oncoviruses induce sarcomas, leukemias, lymphomas, and mammary carcinomas in tible species. Retroviruses infect a wide variety of s, and may be transmitted both horizontally and vertically. Because they are ated 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 ropic Moloney murine leukemia virus (MLV-A), which use cell surface phosphate transporter receptors for entry and then permanently integrate into proliferating cell chromosomes. The ropic 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, el al., Melh. l., 1993, 217, 9, the disclosures of which are incorporated herein by nce.
] The term "lentivirus" refers to a genus that includes HIV (human immunodeficiency virus, ing 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 , feline immunodeficiency virus (FIV), 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 ency and encephalopathy in sub-human es. 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 or of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 dies also include the UHCTl clone, also known as T3 and CD38. Other anti-CD3 antibodies e, for example, otelixizumab, teplizumab, and visilizumab.
The term "OKT-3" (also referred to herein as "OKT3") refers to a onal antibody or variant thereof, including human, humanized, chimeric, or murine antibodies, directed t 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 tutions, glycoforms, or biosimilars f. The amino acid sequences of the heavy and light chains of muromonab are given in Table l (SEQ ID N011 and SEQ ID N012). 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 nab.
Identifier Sequence (One-Letter Amino Acid Symbols) (Description) SEQ ID NO:1 QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY (Muromonab heavy NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA chain) KTTAPSVYPL APVCGGTTGS SVTLGCLVKG TLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ SRDE SLTC LVKGFYPSDI AVEWESNGQP TPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO:2 QIVLTQSPAI MSASPGEKVT MTCSASSSVS QKSG TSPKRWIYDT SKLASGVPAH (Muromonab light FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG RADT APTVSIFPPS chain) SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 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 ing human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, e.g., in Nelson, J. l. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated by nce herein. The amino acid sequence of recombinant human 1L-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 1L-2 such as aldesleukin (PROLEUKIN, ble commercially from multiple suppliers in 22 million IU per single use vials), as well as the form of recombinant 1L-2 commercially supplied by niX, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYTb) and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-l, 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 eukin suitable for use in the invention is given in Table 2 (SEQ ID N04).
The term IL-2 also encompasses pegylated forms of IL-2, as described herein, including the pegylated 1L2 prodrug 14, available from Nektar Therapeutics, South San Francisco, CA, USA. NKTR—214 and pegylated IL-2 suitable for use in the invention is bed in US.
Patent Application Publication No. US 328791 A1 and International Patent Application Publication No. herein. ative forms of conjugated IL-2 suitable for use in the invention are described in US. Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4902,502, the disclosures of which are orated by reference herein. Formulations of 1L-2 suitable for use in the invention are described in US. Patent 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 tissuederived 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 dimer 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, ing ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-7 recombinant protein, Cat. No.
Gibco PHCOO7l). The amino acid sequence of recombinant human IL-7 suitable for use in the invention is given in Table 2 (SEQ ID N015).
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-lS shares [3 and y 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 nine) with a molecular mass of 12.8 kDa. Recombinant human IL-15 is commercially ble from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East ick, NJ, USA (Cat. No. CYTb) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-lS recombinant protein, Cat. No. 3482). The amino acid sequence of recombinant human lL-lS suitable for use in the invention is given in Table 2 (SEQ ID N016).
The term "IL-21" (also ed to herein as "IL2l") refers to the pleiotropic cytokine protein known as eukin-2l, and includes all forms of 1L-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 ted human CD4+ T cells. inant 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 cially available from multiple suppliers, including ProSpec-Tany TechnoGene Ltd., East ick, NJ, USA (Cat. No. CYTb) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-2l recombinant protein, Cat. No. 1480). The amino acid sequence of recombinant human lL-21 suitable for use in the invention is given in Table 2 (SEQ ID N07).
WO 81789 TABLE 2. Amino acid ces of interleukins.
Identifier Sequence (One-Letter Amino Acid Symbols) (Description) SEQ ID NO:3 MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATEJKHLQCL (recombinant LEEV KNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN human IL’2 RWITFCQSII STLT (rhILe2)) SEQ ID NO:4 PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKlLQCLEE (aldesleukin) ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFSQSIIST Lr1 SEQ ID NO:5 MDCDIEGKDG KQYESVLMVS IDQLLDSMKE NNEF NFFKRHICDA NKEGMFLFRA (recombinant ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSJEENKSL human IL’7 NDLC QEIK TCWNKILMGT KEH (rhILe7)) SEQ ID NO:6 MNWVNVISDL K "MOLM-14" refers to a human leukemia cell line which was ished 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, el 61]., 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. The MOLM-14 cell line is deposited at DSMZ under ion 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 d 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 elphia chromosome-positive CIVIL. a, el 61]., 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. ACCl34 whilst the closely related EM-2 cell line is deposited at DSMZ under Accession No. ACCl35. 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 n comprising an amino acid sequence as set forth in SEQ ID N08 or a protein sing 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-lBBL" or "CDl37L" may refer to a protein comprising an amino acid sequence as set forth in SEQ ID N09 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 "OX4OL" or "CDl37L" may refer to a protein comprising an amino acid sequence as set forth in SEQ ID NOle 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 milar" means a ical t, including a monoclonal antibody or fusion protein, that is highly similar to a US. ed 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 y of the product. rmore, a r 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 al regulatory agencies. Biological products or biological medicines are medicines that are made by or derived from a biological , 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 eukin. In Europe, a similar biological or "biosimilar" medicine is a biological medicine that is similar to another biological medicine that has y been authorized for use by the European Medicines Agency (EMA). The nt 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 t may be referred to as a "reference medicinal product" in Europe. Some of the requirements for a product to be considered a ilar are outlined in the CHMP 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 hed on its e. 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 eff1cacy. In addition, the biosimilar may be used or be ed for use to treat the same conditions as the reference medicinal product. Thus, a ilar as bed herein may be deemed to have similar or highly similar quality characteristics to a nce medicinal product.
Alternatively, or in addition, a biosimilar as described herein may be deemed to have similar or highly r biological activity to a reference medicinal product. Alternatively, or in addition, a ilar 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 nal product. As described herein, a biosimilar in Europe is compared to a reference medicinal t which has been authorized by the EMA. However, in some instances, the biosimilar may be compared to a biological nal 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 inical s. As used , the term "biosimilar" also relates to a biological medicinal t 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 ce that has minor ations 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, gh 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 nal 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 nce nal product.
Additionally, the biosimilar may deviate from the reference medicinal product in for example its strength, ceutical form, formulation, excipients and/or presentation, providing safety and efficacy of the medicinal product is not mised. The biosimilar may comprise differences in for example pharmacokinetic (PK) and/or pharmacodynamic (PD) profiles as compared to the reference nal product but is still deemed suff1ciently similar to the nce medicinal product as to be authorized or considered suitable for authorization. In certain circumstances, the biosimilar exhibits different binding characteristics as compared to the nce medicinal t, 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 dy by way of one or more tutions, deletions and/or additions at certain ons within or adjacent to the amino acid sequence of the reference protein or dy. The variant may comprise one or more conservative substitutions in its amino acid ce 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 y to cally bind to the n of the reference protein or antibody. The term "variant" also es pegylated antibodies or proteins.
"Pegylation" refers to a modified dy, or a nt 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 n. 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 (Cl-Clo) alkoxy- or aryloxy-polyethylene glycol or hylene 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 6 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, ably 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 ion 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, sion factors, ng 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 ments of very different sizes, shapes and dimensions may employ the described arrangements.
The transitional terms "comprising,77 (L 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 al other than those specified in the claim and, in the latter instance, impurities ordinary ated with the specified material(s). The term "consisting ially 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 ate embodiments, be more specifically defined by any of the transitional terms "comprising, 77 (4 ting 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 ses HLA-A/B/C, CD64, CD80, ICOS-L, and CD58, and is modified to express one or more costimulatory molecules. In an ment, the invention includes an aAPC comprising a MOLM-l4 cell that is modified to express one or more costimulatory molecules. In an embodiment, the ion es an aAPC comprising a 3 cell that is modified to express one or more costimulatory molecules.
In an ment, the invention includes an aAPC comprising a MOLM-l4 cell that endogenously expresses HLA-A/B/C, CD64, CD80, ICOS-L, and CD58, n the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID N08, and conservative amino acid substitutions thereof, and a 4-lBBL protein comprising an amino acid sequence as set forth in SEQ ID N09, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-lBBL protein are sed on the surface of the MOLM-l4 cell.
In an embodiment, the invention es an aAPC comprising a MOLM-l4 cell transduced with one or more viral vectors, n the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-lBBL, and wherein the MOLM-l4 cell expresses CD86 and 4-lBBL. In an embodiment, the invention includes an aAPC WO 81789 comprising a MOLM-l3 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-l3 cell ses CD86 and 4-lBBL. In an embodiment, the invention es a method of preparing any of the foregoing embodiments of aAPCs.
] In an embodiment, the invention includes an aAPC sing a MOLM-l4 cell d to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID N08 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-l4 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID N08, and conservative amino acid substitutions thereof, and a 4-lBBL protein comprising an amino acid sequence as set forth in SEQ ID N09, and conservative amino acid substitutions thereof, wherein the CD86 n and the 4-1BBL protein are expressed on the surface of the MOLM-l3 cell. In an embodiment, the ion includes a method of preparing any of the foregoing embodiments of aAPCs.
In an embodiment, the invention includes an aAPC comprising a MOLM-l4 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 N08 and a 4-lBBL n 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-lBBL protein are expressed on the surface of the 4 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l4 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 N08 and a 4-lBBL n 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-lBBL protein are expressed on the surface of the MOLM-l4 cell. In an embodiment, the invention includes an aAPC sing a MOLM-l4 cell modified to express a CD86 protein comprising a sequence with r than 97% identity to an amino acid sequence as set forth in SEQ ID NO:8 and a 4-lBBL 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-lBBL protein are expressed on the surface of the MOLM-l4 cell. In an embodiment, the WO 81789 invention includes an aAPC comprising a MOLM-l4 cell modified to s a CD86 protein comprising a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID N08 and a 4-lBBL protein comprising a sequence with greater than 96% ty to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-lBBL protein are sed on the surface of the MOLM-l4 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l4 cell modified to s a CD86 protein comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID N08 and a 4-lBBL n 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-lBBL protein are expressed on the surface of the MOLM-l4 cell. In an embodiment, the invention includes an aAPC sing a MOLM-l4 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 N08 and a 4- lBBL 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-lBBL protein are expressed on the surface of the MOLM-l4 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 3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID N08 and a 4-lBBL protein comprising an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 n and the 4-lBBL protein are expressed on the surface of the MOLM-l3 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID N08, and conservative amino acid substitutions thereof, and a 4-lBBL protein sing an amino acid sequence as set forth in SEQ ID N09, and conservative amino acid substitutions f, wherein the CD86 n and the 4-lBBL protein are expressed on the surface of the MOLM-l3 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 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 N08 and a 4-lBBL 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-lBBL protein are expressed on the surface of the MOLM-l3 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l3 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 N08 and a 4-lBBL protein comprising a ce with greater than 98% identity to an amino acid ce as set forth in SEQ ID NO:9, wherein the CD86 n and the 4-lBBL protein are expressed on the surface of the MOLM-l3 cell. In an embodiment, the ion includes an aAPC comprising a MOLM-l3 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-lBBL protein comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID NO:9, n the CD86 protein and the 4-lBBL protein are expressed on the surface of the MOLM-l3 cell. In an ment, the invention includes an aAPC sing a MOLM-l3 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 N08 and a 4-lBBL n comprising a ce with r than 96% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-lBBL n are expressed on the surface of the MOLM-l3 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l3 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 N08 and a 4-lBBL protein comprising a ce with greater than 95% ty to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-lBBL protein are expressed on the surface of the MOLM-l3 cell. In an embodiment, the invention es an aAPC comprising a MOLM-l3 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 N08 and a 4- lBBL 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-lBBL protein are expressed on the surface of the MOLM-l3 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-l4 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 OX4OL, and wherein the 4 cell expresses CD86 and OX4OL. In an embodiment, the invention es an aAPC comprising a MOLM-l3 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 OX4OL, and wherein the MOLM-l3 cell expresses CD86 and OX4OL. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
In an ment, the invention includes an aAPC comprising a MOLM-l4 cell d to express a CD86 protein comprising an amino acid ce as set forth in SEQ ID N08 and a OX4OL n comprising an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL protein are expressed on the surface of the 4 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l3 cell modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID N08, and conservative amino acid substitutions f, and a OX4OL protein sing an amino acid ce as set forth in SEQ ID NO: 10, and conservative amino acid tutions thereof, wherein the CD86 protein and the OX4OL protein are expressed on the surface of the MOLM-l3 cell. In an embodiment, the ion includes a method of ing any of the foregoing embodiments of aAPCs.
In an embodiment, the invention includes an aAPC comprising a MOLM-l4 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 N08 and a OX4OL 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 OX4OL n are expressed on the surface of the MOLM-l4 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l4 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 N08 and a OX4OL 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 OX4OL protein are expressed on the surface of the MOLM-l4 cell. In an embodiment, the invention includes an aAPC comprising a MOLM-l4 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 N08 and a OX4OL protein comprising a ce with greater than 97% WO 81789 identity to an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL protein are expressed on the surface of the MOLM-l4 cell. In an embodiment, the invention includes an aAPC sing a MOLM-l4 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 N08 and a OX4OL 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 OX4OL protein are expressed on the surface of the MOLM-l4 cell. In an ment, the invention includes an aAPC comprising a MOLM-l4 cell modified to express a CD86 protein sing a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL protein comprising a sequence with greater than 95% ty to an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL protein are expressed on the surface of the MOLM-l4 cell. In an embodiment, the invention includes an aAPC sing a MOLM-l4 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 N08 and a OX4OL protein comprising a ce with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL protein are expressed on the surface of the MOLM-l4 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-l4 or MOLM-l3 cell may be modified to express both OX4OL and .
The sequences for human CD86, human 4-lBBL (CDl37L), and human OX4OL (CDl34L) are given in Table 3.
TABLE 3. Amino acid sequences for human CD86, human 4-lBBL, and human OX4OL.
Identifier Sequence (One-Letter Amino Acid Symbols) (Description) SEQ ID NO:8 MGLSNILFVM AAPL KIQAYFNETA DLPCQFANSQ NQSLSELVVF WQDQENLVLN (human CD86) EVYLGKEKFD SVHSKYMGRT SFDSDSWTLR LHNLQIKDKG LYQCIIHHKK PTGMIRIHQM NSELSVLANF SQPEIVPISN ITENVYINLT CSSIHGYPEP KKMSVLLRTK DGIM VTEL YDVSISLSVS FPDVTSNMTI FCILETDKTR LLSSPFSIEL EDPQPPPDHI LPTV IICVMVFCLI LWKWKKKKRP RNSYKCGTNT MEREESEQTK KREKIHIPER SDEAQRVFKS SKTSSCDKSD TCF SEQ ID NO:9 ASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA (human 4*1BBL, SPGSAASPRL REGPELSPDD PAGLLDLRQG AQNV LLIDGPLSWY SDPGLAGVSL CD137) TGGLSYKEDT KAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ SAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE SEQ ID NO:lO MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSAL QVSHRYPRIQ (human OX40L, SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS QEVNISLHYQ CD134L) KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL ILIHQNPGEF ] In an embodiment, the ion 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 n 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 3 cell modified to express a first n that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID N013, and conservative amino acid substitutions thereof, and a third protein that binds to a fourth n comprising an amino acid ce as set forth in SEQ ID NO: 11 or SEQ ID NO: 12, and conservative amino acid substitutions thereof. In an embodiment, the invention es 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 ce as set forth in SEQ ID NO:11 or SEQ ID NO: 12. In an embodiment, the invention es 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 ce 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 s 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 ce as set forth in SEQ ID NO: 11 or SEQ ID NO:12. In an embodiment, the ion includes an aAPC comprising a MOLM-14 cell modified to express a first protein that binds to a second protein comprising a sequence with WO 81789 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 n 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 es 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 es an aAPC comprising a 4 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 es 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 r 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 sing a 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 ce 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 n 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 s 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 n 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 r than 95% identity to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO: 12. In an ment, the invention includes an aAPC comprising a MOLM-13 cell d 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 4 cell modified to eXpress a first n that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO: 14, and conservative amino acid tutions thereof, and a third protein that binds to a fourth protein sing an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO: 12, and conservative amino acid tutions thereof. In an embodiment, the invention includes an aAPC sing a MOLM-13 cell modified to eXpress a first protein that binds to a second protein sing an amino acid sequence as set forth in SEQ ID N014, 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 N012, and vative 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 sing 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-l4 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 n 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-l4 cell modified to s 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 sing 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-l4 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 es an aAPC comprising a 4 cell modified to express a first protein that binds to a second protein comprising a ce with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO: 14 and a third n that binds to a fourth protein sing 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 ion es an aAPC comprising a MOLM-l4 cell modified to s a first n that binds to a second protein sing 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-l3 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 ion includes an aAPC comprising a MOLM-l3 cell modified to s 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 ion includes an aAPC comprising a MOLM-l3 cell modified to express a first protein that binds to a second protein sing a sequence with greater than 97% ty to an amino acid sequence as set forth in SEQ ID NO: 14 and a third protein that binds to a fourth protein sing 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 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 n 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-l3 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 n comprising a sequence with r 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-l3 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-lBBL binds (4-lBB), and the ligand to which human OX4OL binds (0X40) are given in Table 4.
TABLE 4. Amino acid sequences for human CD28, human CTLA-4, human 4-1BB, and human 0X40.
Identlfler Sequence (One-Letter Amino Acid Symbols) (Descrlptlon) SEQ ID NO:II MLRLLLALNL HGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD (human CD28) SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP PYLDNEKSNG GKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS SEQ ID NO:I2 MACLGFQRHK AQLNLAHRTW PCTLLFFLLF IPVFCKAMHV AQPAVVLASS RGIASFVCEY (human CTLAi4) ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL LTAVSLSKML KKRSPLHTGV YVKMPPTEPE PYFI PIN SEQ ID NO:I3 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR (human 4*IBB) TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC DQKR HNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF HALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL SEQ ID NO:I4 MCVGARRLGR GPCAALJLLG TGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ (human 0X40) NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL RRDQRLPPDA GSFR TPIQEEQADA HSTLAKI In an embodiment, the invention includes an isolated artificial antigen presenting cell (aAPC) comprising a cell that expresses HLA-A/B/C, , and CD58, and is d 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 ment, 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-A/B/C, ICOS-L, and CD58, n the cell is modified to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID N08, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID N09, and vative amino acid substitutions thereof, and n 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 s, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding , and n 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 N08 and a 4-1BBL protein comprising an amino acid ce as set forth in SEQ ID NO:9, wherein the CD86 n and the 4-1BBL protein are sed 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 N08 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 ment, the invention includes an aAPC comprising a EM-3 cell modified to express a CD86 n comprising a sequence with r 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 ce 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 n comprising a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID N08 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 s a CD86 protein comprising a sequence with greater than 96% identity to an amino acid ce as set forth in SEQ ID N08 and a 4- lBBL 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 s a CD86 protein comprising a ce with greater than 95% identity to an amino acid sequence as set forth in SEQ ID N08 and a 4-1BBL protein comprising a ce with greater than 95% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 n and the 4-1BBL protein are expressed on the surface of the EM-3 cell. In an embodiment, the invention es 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 ce as set forth in SEQ ID N08 and a 4-1BBL protein comprising a sequence with greater than 90% identity to an amino acid ce 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 ment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
In an embodiment, the invention es 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 N013, 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 n 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% ty 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 n that binds to a second protein comprising a sequence with greater than 97% ty 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 ce 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 d to express a first protein that binds to a second protein sing 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 r 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 ce 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 sing an EM-3 cell d to express a first protein that binds to a second protein comprising a sequence with greater than 90% ty to an amino acid ce as set forth in SEQ ID NO: 13 and a third protein that binds to a fourth protein comprising a ce with greater than 90% ty 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 d 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 N08 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 n are sed 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 N08 and a 4-lBBL 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 n 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 N08 and a 4-lBBL n comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 n and the 4-1BBL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention es an aAPC sing a EM-2 cell modified to express a CD86 n sing a sequence with greater than 97% identity to an amino acid sequence as set forth in SEQ ID N08 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 n 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 N08 and a 4- lBBL protein comprising a sequence with greater than 96% identity to an amino acid ce as set forth in SEQ ID NO:9, wherein the CD86 protein and the 4-lBBL 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 N08 and a 4-1BBL protein sing 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-lBBL protein are expressed on the e 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 N08 and a 4-lBBL protein comprising a ce with greater than 90% identity to an amino acid sequence as set forth in SEQ ID NO:9, wherein the CD86 n and the 4-lBBL n 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 N013, 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 ment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
In an embodiment, the invention es an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second n 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 ce 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 ion includes an aAPC comprising an EM-2 cell modified to express a first protein that binds to a second protein sing a ce 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 ment, 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 n comprising a ce 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 sing 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 ce 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 s 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) g domain, such as clones 7C12 and 8B3 described , 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 . In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in . In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as ed in FIG.
In an embodiment, the invention includes an aAPC comprising an EM-3 cell that expresses HLA-A/B/C, ICOS-L, and CD58, n the cell is modified to express a CD86 protein sing an amino acid sequence as set forth in SEQ ID N08, and conservative amino acid substitutions thereof, and a OX4OL protein sing an amino acid sequence as set forth in SEQ ID NO: 10, and vative amino acid substitutions thereof, and wherein the CD86 protein and the OX4OL 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 OX4OL, and wherein the EM-3 cell expresses CD86 and OX4OL. In an embodiment, the invention includes a method of preparing any of the ing ments of aAPCs.
In an embodiment, the ion includes an aAPC comprising an EM-3 cell modified to s a CD86 n comprising an amino acid sequence as set forth in SEQ ID N08 and a OX4OL protein comprising an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL 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 ce with greater than 99% identity to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL protein comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO: 10, n the CD86 protein and the OX4OL 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 N08 and a OX4OL 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 OX4OL 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 s a CD86 protein comprising a sequence with r than 97% identity to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL protein comprising a sequence with greater than 97% ty to an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL 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 N08 and a OX4OL 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 n and the OX4OL protein are expressed on the surface of the EM-3 cell. In an ment, the invention includes an aAPC sing a EM-3 cell modified to express a CD86 protein sing a sequence with r than 95% identity to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL protein comprising a ce with r than 95% identity to an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL 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 sing a sequence with greater than 90% identity to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL protein comprising a sequence with greater than 90% identity to an amino acid ce as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL 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 s 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 n 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 ion includes a method of preparing any of the foregoing embodiments of aAPCs.
In an ment, the ion includes an aAPC comprising an EM-3 cell modified to express a first protein that binds to a second protein comprising a sequence with r 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% ty to an amino acid sequence as set forth in SEQ ID NO:11 or SEQ ID NO: 12. In an embodiment, the ion includes an aAPC comprising an EM-3 cell d to express a first protein that binds to a second protein comprising a sequence with greater than 98% identity to an amino acid ce 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 ce 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 n comprising a sequence with greater than 97% ty 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 sing 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 ments of aAPCs.
In an embodiment, the invention includes an aAPC comprising an EM-3 cell modified to s a single chain fragment le (scFv) binding , 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 .
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 N08 and a OX4OL protein comprising an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL 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 ce as set forth in SEQ ID N08 and a OX4OL protein comprising a sequence with greater than 99% ty to an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC sing a EM-2 cell ed to express a CD86 protein comprising a sequence with greater than 98% identity to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL protein comprising a sequence with greater than 98% ty to an amino acid sequence as set forth in SEQ ID NO: 10, wherein the CD86 protein and the OX4OL 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 sing a sequence with greater than 97% ty to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL 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 OX4OL 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 r than 96% identity to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL 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 OX4OL protein are expressed on the surface of the EM-2 cell. In an embodiment, the invention includes an aAPC sing 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 N08 and a OX4OL protein sing 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 OX4OL 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 n comprising a sequence with greater than 90% ty to an amino acid sequence as set forth in SEQ ID N08 and a OX4OL 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 OX4OL protein are expressed on the surface of the EM-2 cell. In an embodiment, the ion 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 f, and a third protein that binds to a fourth protein comprising an amino acid ce as set forth in SEQ ID NO:11 or SEQ ID NO: 12, and conservative amino acid substitutions f. In an embodiment, the invention includes a method of preparing any of the foregoing embodiments of aAPCs.
In an ment, the invention es 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 sing a sequence with greater than 98% ty to an amino acid ce 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 n 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 ion 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 ce with greater than 95% ty to an amino acid ce 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% ty to an amino acid sequence as set forth in SEQ ID NO: 14 and a third protein that binds to a fourth protein sing 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 es 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 ion includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in . In an embodiment, the invention includes an aAPC sing an EM-3 or an EM-2 cell d as depicted in . In an embodiment, the invention includes an aAPC comprising an EM-3 or an EM-2 cell modified as depicted in FIG.
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 OX4OL and 4-1BBL.
In an embodiment, the invention includes an isolated artificial n presenting cell (aAPC) sing a cell that expresses CD58, and is modified to express one or more costimulatory molecules, wherein the aAPC is derived from a ineage 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 e parental cell line is deposited under accession no. ATCC 3 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 d to express a CD86 protein comprising an amino acid sequence as set forth in SEQ ID N08, and conservative amino acid substitutions thereof, and a 4-1BBL protein comprising an amino acid sequence as set forth in SEQ ID N09, and conservative amino acid substitutions thereof, and wherein the CD86 protein and the 4-1BBL n 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, n the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid ng 4-lBBL, and wherein the K5 62-lineage cell expresses CD86 and 4-lBBL. In an embodiment, the invention includes a method of preparing any of the foregoing ments 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 N08 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- e 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 n comprising a sequence with greater than 99% identity to an amino acid sequence as set forth in SEQ ID N08 and a 4-lBBL protein comprising a ce with greater than 99% identity to an amino acid sequence as set forth in SEQ ID NO:9, n the CD86 protein and the 4-lBBL protein are expressed on the surface of the K562-lineage cell.
In an embodiment, the invention es 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 N08 and a 4-lBBL n 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-lBBL 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 N08 and a 4-lBBL 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-lBBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention es an aAPC comprising a ineage 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 N08 and a 4-lBBL 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-lBBL protein are sed 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 n comprising a sequence with greater than 95% identity to an amino acid sequence as set forth in SEQ ID N08 and a 4-lBBL 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 n and the 4-lBBL protein are expressed on the surface of the K562-lineage cell. In an embodiment, the invention es an aAPC comprising a K562-lineage cell modified to express a CD86 protein comprising a sequence with greater than 90% ty to an amino acid ce as set forth in SEQ ID N08 and a 4-lBBL n 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-lBBL protein are expressed on the e 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 s a first protein that binds to a second protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 l, 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 ion includes a method of preparing any of the foregoing embodiments of aAPCs.
] In an embodiment, the invention includes an aAPC comprising a ineage cell modified to express a first protein that binds to a second protein comprising a sequence with greater than 99% ty to an amino acid sequence as set forth in SEQ ID N011 and a third protein that binds to a fourth n 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 N011 and a third protein that binds to a fourth protein comprising a sequence with greater than 98% identity to an amino acid ce 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 r than 97% ty to an amino acid sequence as set forth in SEQ ID N011 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 sing a K562-lineage cell modified to express a first protein that binds to a second protein sing a sequence with greater than 96% identity to an amino acid sequence as set forth in SEQ ID N011 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% ty to an amino acid sequence as set forth in SEQ ID N011 and a third protein that binds to a fourth protein comprising a sequence with greater than 95% ty 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 N011 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 ing any of the foregoing embodiments of aAPCs.
In an embodiment, the invention includes an aAPC sing an K562-lineage cell modified to express a single chain fragment variable (scFv) binding domain, such as clones 7C12 and 8B3 described , to bind the Fc domain of a monoclonal antibody, such as OKT-3, providing an additional proliferative signal.
Methods of Preparing ial Antigen Presenting Cells In an embodiment, a method of preparing an aAPC includes the step of stable oration of genes for production of CD86 and 4-1BBL. In an ment, 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, ei al., Proc. Nat ’l Acad. Sci. 2006, 103, 17372-77, Zufferey, ei al., Nat. Biotechnol. 1997, 15, 871-75, Dull, ei al., J. gy 1998, 72, 8463-71, and US. Patent No. 6,627,442, the disclosures of each of which are incorporated by reference herein. In an ment, a method of preparing an aAPC includes the step of gamma-retroviral uction. Gamma-retroviral transduction systems are known in the art and are described, e.g., Cepko and Pear, Cur. Prof. 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 oson-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is ed 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 id-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB 10, SB 1 1, and SB 100x, and engineered enzymes with increased enzymatic activity, are described in, e.g., Hackett, ei al., Mol. Therapy 2010, 18, 674-83 and US.
Patent No. 6,489,458, the disclosures of each of which are incorporated by reference herein.
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 oporation. Electroporation methods are known in the art and are described, e.g., in Tsong, Biophys. J. 1991, 60, 297-306, and US. Patent Application Publication No. 2014/0227237 Al, the disclosures of each of which are incorporated by reference herein. In an embodiment, a method of preparing an aAPC es 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, 7, Wigler, el al, Proc. Natl. Acad. Sci. 1979, 76, 376, and Chen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752, and in US.
Patent 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—[l-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are known in the art and are bed in Rose, el al, Biolechm'ques 1991, 10, 520-525 and Felgner, el al, Proc. Natl. Acad.
Sci. USA, 1987,84, 417 and in US. Patent Nos. 5,279,833, 635, 6,056,938, 6,110,490, 484, and 7,687,070, the disclosures of each of which are orated by nce herein. In an embodiment, a method of preparing an aAPC includes the step of transfection using methods described in US. Patent 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 nce herein.
In an embodiment, the aAPC is transduced by first using the Gateway g 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 on. 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 e 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 pLV43OG) using the attL and attR sites and a clonase enzyme. The attL sites are already found in the entry clone, while the ation 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 X 106 cells per well for 3 to 5 days.
The cells may then be ed and/or washed by centrifugation and ended in media or cryopreserved in an appropriate cryopreservation media (e.g., CryoStor lO (BioLife Solutions)) and stored in a -80 0C freezer.
In some embodiments, the aAPCs bed herein may be grown in the presence of 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-l3 cells, aMOLM-l4 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 Cult), 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-l3 cells, aMOLM-l4 cells, and aEM3 cells.
Methods of Expanding Tumor Inflltrating Lymphocytes and T Cells In an embodiment, the invention includes a method of ing tumor inflltrating lymphocytes , the method comprising contacting a population of TILs comprising at least one TIL with an aAPC bed 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 TlLs, wherein binding of said co-stimulatory molecule with said co- stimulatory ligand induces proliferation of the TlLs, 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, el al., J. Immunolherapy 2012, 35, 283-292, the disclosure of which is orated by reference herein. For e, the tumor may be placed in enzyme media and mechanically dissociated for approximately 1 minute. The e may then be incubated for 30 minutes at 37 0C in 5% C02 and then mechanically disrupted again for approximately 1 minute. After incubation for 30 minutes at 37 0C in 5% C02, the tumor may be mechanically disrupted a third time for approximately 1 minute. If after the third ical 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 0C in 5% C02.
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 l cell culture cluster, flat bottom, Corning Incorporated, Corning, NY), each well may be seeded with IX 106 tumor digest cells or one tumor nt imately 1 to 8 mm3 in size in 2 mL of complete medium (CM) with IL-2 (6000 IU/mL, Chiron Corp., Emeryville, CA). CM consists of RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25mM Hepes, and 10 mg/mL gentamicin. es may be initiated in gas-permeable flasks with a 40 mL capacity and a 10 cm2 rmeable 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 0C in 5% C02 and 5 days after culture initiation, half the media may be removed and replaced with fresh CM and 1L-2 and after day 5, half the media may be changed every 2—3 days.
Rapid ion protocol (REP) of TlLs may be performed using T-l75 flasks and gas- permeable bags or gas-permeable G—Rex flasks, as described elsewhere herein, using the aAPCs of the t sure. For REP in T-l75 flasks, l><106 TlLs 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 l 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-l75 flasks may be incubated at 37 0C in 5% C02. 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-l75 flasks may be ed in a 3L bag and 300mL of AIM-V with 5% human AB serum and 3000 IU/mL of IL-2 may be added to the 300mL 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 n 0.5 and 2.0><106 cells/mL.
For REP in 500 mL ty flasks with 100 cm2 rmeable silicon bottoms (e.g., G-Rex 100, Wilson Wolf Manufacturing, as described elsewhere herein), 5><106 or 6 TILs may be cultured with aAPCs at a ratio described herein (e.g., l to 100) in 400 mL of 50/50 , 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 0C in 5% C02. On day five, 250 mL of supernatant may be d 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 sion may be divided into three 100 mL aliquots that may be used to seed 3 G—Rex100 flasks. About 150 mL of AHVI-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 0C in 5% C02, and after four days, 150 mL of AHVI-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 , 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 CMl). 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 s 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 , and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL n, 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), n the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding , 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 l 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 ing a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) ucing 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-lBBL, and n the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and (b) ting the population of TILs with the tion 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 ng 4-lBBL, and wherein the myeloid cell expresses a CD86 n and a 4-1BBL n, and (b) contacting the population of TILs with the population of aAPCs in a cell culture medium, wherein the myeloid cell endogenously expresses HLA-A/B/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-lBBL, and n 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-l4 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 ting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding , and wherein the myeloid cell expresses a CD86 protein and a 4-1BBL protein, and (b) contacting the tion of TILs with the tion of aAPCs in a cell culture medium, wherein the d cell is a MOLM-l3 cell.
In an embodiment, the invention provides a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method sing the steps of: (c) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial antigen presenting cells (aAPCs), n the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-lBBL, 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 ial 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 n comprises an amino acid ce as set forth in SEQ ID N08, or conservative amino acid substitutions thereof, and the 4-1BBL protein comprises an amino acid sequence as set forth in SEQ ID N09, 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 n presenting cells (aAPCs), wherein the one or more viral vectors se a c 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) ting the population of TILs with the population of aAPCs in a cell culture medium, wherein the nucleic acid ng CD86 comprises a nucleic acid sequence as set forth in SEQ ID N019 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 es a method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) ucing a d cell with one or more viral vectors to obtain a population of artificial antigen presenting cells ), 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 ses 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 ion is performed using a gas permeable container.
In an embodiment, the ion provides a method of expanding a population of tumor infiltrating lymphocytes , the method comprising the steps of: (a) transducing a myeloid cell with one or more viral vectors to obtain a population of artificial n presenting cells (aAPCs), n the one or more viral vectors comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-lBBL, 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 tion of TILs to the population of aAPCs is between 1 to 200 and l 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 se a nucleic acid encoding CD86 and a nucleic acid encoding 4-lBBL, and wherein the d 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 e 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 , wherein the d aAPC comprises at least two co-stimulatory ligands that specifically bind with at least two co- stimulatory le on the TILs, wherein binding of the co-stimulatory molecules with the co- stimulatory ligand s 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 OX4OL in addition to 4-lBBL, or may comprise OX4OL instead of 4-lBBL.
In an ment, a method of expanding or treating a cancer includes a step wherein TlLs are obtained from a patient tumor sample. A patient tumor sample may be ed using methods known in the art. For example, TlLs may be cultured from tic 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, lO mcg/mL gentamicine, 3O units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanical dissociation (e.g., using a tissue iator). 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 0C in 5% C02, 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 med to remove these cells.
Alternative methods known in the art may be used, such as those described in US. Patent Application Publication No. 2012/0244133 Al, 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 TlLs or methods treating a .
In an embodiment, REP can be performed in a gas permeable container using the aAPCs of the present sure by any suitable method. For example, TlLs can be rapidly ed using non-specific T cell or stimulation in the ce of interleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific T cell receptor stimulus can include, for example, about ng/mL of an anti-CD3 antibody, e.g. OKT-3, a monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ, USA or Miltenyi Biotech, Auburn, CA, USA) or UHCT-l (commercially available from end, San Diego, CA, USA). TlLs can be rapidly expanded by further stimulation of the TILs in vitro with one or more antigens, including antigenic portions f, 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 uM MART-l :26-35 (27 L) or gpl -217 (210M), optionally in the presence of a T cell growth , such as 300 IU/mL IL-2 or IL-15. Other suitable antigens may e, e.g., NY-ESO-l, TRP-l, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TIL may also be rapidly expanded by mulation 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., e, 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 e medium, about 5000 mL to about 6000 mL of cell culture medium, about 6000 mL to about 7000 mL of cell e , 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 e . In an embodiment, expanding the number of TILs uses no more than one type of cell culture medium.
Any le cell culture medium may be used, e.g., AIM-V cell medium (L-glutamine, 50 uM streptomycin sulfate, and 10 uM gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad, CA, 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 g 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 X 105 cells/cm2 to between X 106 and 30 X 106 cells/cmz. In an embodiment, this expansion occurs without feeding. In an ment, 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 ne 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 US.
Patent Application Publication No. US 2014/03 77739 A1, International Patent Application Publication No. WO 10036 A1, US. Patent Application ation No. US 2013/0115617 A1, International Publication No. WO 88427 A1, US. Patent Application Publication No. US 2011/0136228 A1, US. Patent No. 8,809,050, International Patent Application Publication No. 2016/0208216 A1, US. Patent Application Publication No. US 2012/0244133 A1, International Patent Application Publication No. WO 29201 A1, US. Patent Application Publication No. US 2013/0102075 A1, US. Patent No. 8,956,860, International Patent Application Publication No. WO 73 835 A1, and US. Patent Application Publication No. US 2015/0175966 Al, the disclosures of which are incorporated herein by reference. Such processes are also described in Jin, el al., J. Immunolherapy 2012, 35, 283-292, the disclosure of which is orated by reference herein.
In an embodiment, the gas permeable container is a G—ReX 10 flask (Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). In an ment, the gas ble container includes a 10 cm2 gas permeable culture surface. In an embodiment, the gas ble container includes a 40 mL cell culture medium capacity. In an embodiment, the gas ble 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 ble 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 on, MN, USA). In an ment, 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 t medium ge.
In an embodiment, the gas ble container is a G—Rex 100L 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 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, MN, 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 ty. In an ment, the gas permeable container provides 20 to 60 n cells per well after 2 medium exchanges.
In an embodiment, the gas permeable container is a G—Rex 6 well plate (Wilson Wolf cturing Corporation, New Brighton, MN, 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 n cells per well after 2 medium exchanges.
In an embodiment, the cell medium in the nd/or second gas permeable ner is unflltered. 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, ing the tumor tissue sample in a first gas permeable container containing cell medium therein, obtaining TILs from the tumor tissue , 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.
WO 81789 In an embodiment, the rapid expansion uses about 1 X 109 to about 1 X 1011 aAPCs. In an embodiment, the rapid expansion uses about 1 X 109 aAPCs. In an embodiment, the rapid eXpansion uses about 1 X 1010 aAPCs. In an ment, the rapid eXpansion uses about 1 X 1011a[XP(3s In an embodiment, the ratio of TILs to aAPCs (TIL:aAPC) is ed from the group consisting 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 red 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 ment, 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 ug/mL of OKT-3 antibody. In an ment, the cell e 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-l75 flasks and gas permeable bags as previously described (Tran, el al., J. Immunolher. 2008, 31, 742-51, Dudley, er al., J. Immunolher. 2003, 26, 332-42) or gas permeable cultureware (G—Rex flasks, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA). For TIL rapid expansion in T-l75 flasks, l X 106 TILs suspended in 150 mL of media may be added to each T-l75 flask. The TILs may be cultured with aAPCs at a ratio of l TIL to 100 aAPCs and the cells were cultured in a l to 1 mixture of CM and AHVI-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-l75 flasks may be incubated at 370 C in 5% C02. Half the media may be ged on day 5 using 50/50 medium with 3000 IU per mL of IL-2. On day 7 cells from two T-l75 flasks may be combined in a 3 liter bag and 300 mL of AHVI 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 n 0.5 and 2.0 x 106 mL.
In an embodiment, for TIL rapid expansions in 500 mL capacity gas ble flasks with 100 cm gas-permeable silicon bottoms (G—Rex 100, commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5 X 106 or 10 X 106 TIL may be cultured with aAPCs at a ratio of l 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% C02. On day 5, 250 mL of supernatant may be removed and placed into centrifuge bottles and fuged at 1500 rpm (revolutions per minute, 491 X 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 t 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 1L-2 may be added to each flask. The G—Rex 100 flasks may be incubated at 370 C in 5% C02 and after 4 days 150 mL of AHVI-V with 3000 IU per mL of IL-2 may be added to each G—Rex 100 flask. The cells may be ted on day 14 of culture.
In an embodiment, TILs may be prepared as s. 2 mm3 tumor fragments are cultured in complete media (CM) comprised of AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mM glutamine (Mediatech, Inc. Manassas, VA), 100 U/mL llin (Invitrogen Life Technologies), 100 ug/mL streptomycin (Invitrogen Life Technologies), 5% nactivated human AB serum (Valley Biomedical, Inc. ster, VA) and 600 IU/mL rhIL-2 (Chiron, Emeryville, CA). For enzymatic digestion of solid tumors, tumor specimens were diced into RPMI—1640, washed and centrifuged at 800 rpm for 5 s 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 X 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, ing those described in US. Patent Application Publication No.
US. Patent Application Publication No. 2005/0106717 Al, the disclosures of which are incorporated herein by reference. In an embodiment, TILs are ed in gas-permeable bags.
In an embodiment, TILs are expanded using a cell ion 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 ion system includes a gas permeable cell bag with a volume selected from the group ting 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, n 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 ion 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, n 6 L and 7 L, between 7 L and 8 L, between 8 L and 9 L, n 9 L and 10 L, between 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, n 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 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, n 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 rocks/minute, about 10 rocks/minute, about 20 minute, about 30 rocks/minute, and about 40 rocks/minute. In an ment, 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 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 ment, the cell expansion system es 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°, n 9° and 10°, between 10° and 11°, and between 11° and 12°.
In an ment, 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 US. Patent Application Publication No. 2016/0010058 Al, 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 t invention bed 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 ion may be used to expand T cells comprising a modified T cell receptor (TCR). The CAR-T cells may be targeted against any le antigen, including CD19, as described in the art, e.g., in US. Patent 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 n, including NY- ESO-l, TRP-l, TRP-2, tyrosinase cancer antigen, 3, SSX-2, and VEGFRZ, or antigenic portions thereof, as described in the art, e.g., in US. Patent Nos. 8,367,804 and 7,569,664, the disclosures of which are incorporated by reference herein. s of Treating s 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 ers 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 , 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 ma, 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 inflltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first tion of TILs from a tumor ed from a patient, (b) performing a rapid expansion of the first population of TILs using a tion of cial antigen presenting cells (aAPCs) in a cell e 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 tion of TILs, and (c) administering a therapeutically effective portion of the second population of TILs to a patient with the cancer. In an ment, the aAPCs comprise MOLM-l4 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 , and wherein the MOLM-l4 cells express a CD86 protein and a 4-1BBL n. 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 tion of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) obtaining a first population of TILs from a tumor resected from a t; (b) performing an initial expansion of the first population of TILs using a first tion of artificial antigen presenting cells (aAPCs) in a first cell culture medium to obtain a second population of TILs, n the second population of TILs is at least d 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, n 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 ses 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 ner.
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 tion of TILs from a tumor resected from a patient, (b) performing an initial expansion of the first tion of TILs in a first cell culture medium to obtain a second population of TILs, wherein the second population of TILs is at least d 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-l4 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid ng CD86 and a nucleic acid encoding 4-lBBL, and wherein the MOLM-l4 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 ion 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 ment, the non- myeloablative chemotherapy is cyclophosphamide 6O mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine 25 mg/mZ/d for 5 days (days 27 to 23 prior to TIL infusion). In an embodiment, after non-myeloablative chemotherapy and TIL on (at day 0) according to the t sure, the patient receives an intravenous infusion of IL-2 intravenously at 0 IU/kg every 8 hours to physiologic tolerance.
] Efficacy of the compounds and combinations of compounds bed herein in treating, preventing and/or managing the indicated es 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 os- 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 cy 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. nolaryngol. 2009, 2, 55-60, and Sano, Head Neck Oncol. 2009, I, 32.
Non-Myeloablative Lymphodepletion with Chemotherapy In an embodiment, the invention es 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 , wherein the population of TILs is for treating a patient which is pre-treated with non- myeloablative chemotherapy. In an ment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) and fludarabine mg/mZ/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 t 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 kine 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 l, 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, el al., Cancer Immunol. lher. 2011, 60, 75—85, Muranski, el al., Nat. Clin. Pract. ., 2006, 3, 668—681, Dudley, el al., J. Clin. Oncol. 2008, 26, 5233-5239, and Dudley, el al., J. Clin. Oncol. 2005, 23, 357, all ofwhich are incorporated by nce herein in their entireties.
In some embodiments, the bine is administered at a tration of 0.5 ug/mL - ug/mL fludarabine. In some embodiments, the fludarabine is administered at a concentration of l ug/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 ments, the fludarabine is administered at a dosage of 10 mg/kg/day, 15 day, 20 mg/kg/day, 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 WO 81789 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 ug/ml -10 ug/ml by administration of cyclophosphamide. In some embodiments, amide, the active form of hosphamide, is obtained at a concentration of l ug/mL by administration of cyclophosphamide. In some ments, 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/mZ/day, l50 day, l75 mg/mZ/day, 200 mg/mZ/day, 225 mg/mZ/day, 250 mg/mZ/day, 275 mg/mZ/day, or 300 mg/mZ/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 ent is administered for 4-5 days at 250 mg/mZ/day i.v. In some embodiments, the cyclophosphamide treatment is administered for 4 days at 250 mg/mZ/day i.v.
In some embodiments, lymphodepletion is performed by administering the fludarabine and the cyclophosphamide are er to a patient. In some embodiments, fludarabine is administered at 25 mg/mZ/day iv. and cyclophosphamide is stered at 250 mg/mZ/day i.v. over 4 days.
In an embodiment, the lymphodepletion is performed by administration of cyclophosphamide at a dose of 60 mg/mZ/day for two days followed by administration of fludarabine at a dose of 25 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 . 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 s. Other le routes of administration include intraperitoneal, intrathecal, and intralymphatic administration.
Any suitable dose of TILs can be stered. Preferably, from about 2.3>< 1010 to about 1010 TILs are administered, with an average of around 7.8><1010 TILs, particularly if the cancer is melanoma. In an embodiment, about l.2>< 1010 to about 4.3><1010 of TILs are administered.
In some ments, the number of the TILs provided in the pharmaceutical compositions ofthe invention is about 1x106, 2x106, 3x106, 4x106, 5x106, 6><106, 7x106, 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, 8x109,9x109,1x1010,2x1010,3x1010,4x1010,5x1010,6x1010,7x1010,8x1010,9x1010,1x10", 1,3><10H,4><10",5><1011,6><1011,7><1011,8><1011,9><1011,1><1012,2><1012,3><1012,4><1012, ><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 ofthe ion is in the range of 1x106 to 5x106, 5x106 to 1x107, 1x107 to 5x107, 5x107 to 1x108, 1x108 to 5x108, 5x108 to 1x109, 1x109 to 5x109, 5x109 to , 1x1010 to 5x10"), 5x1010 to 1x10", 5x1011 to 1x10", 1x1012 to 5x10", and 5x1012 to 1x10".
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%, %, 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 ceutical 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%, , 18.25% 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%, .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%, , 0.006%, 0.005%, 0.004%, , , 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 ceutical 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 ceutical compositions ofthe 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 ofthe 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, l 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 The TILs provided in the pharmaceutical itions of embodiments of the invention are effective over a wide dosage range. The exact dosage will depend upon the route of stration, 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 ence of the attending physician. The clinically-established dosages of the TILs may also be used if appropriate. The amounts of the pharmaceutical compositions stered 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 ian.
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. stration of TILs may continue as long as necessary.
In some embodiments, an effective dosage of TILs is about l><106, 2><106, 3><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><1010,5X10"), 6><1010,7><1010,8><1010,9><1010,1><1011,2><1011,3><1011,4><1011,5><10H,6><1011,7><1011,8X10", 9X10",1><1012,2><1012,3><1012,4><1012,5><1012,6><1012,7><1012,8><1012,9><1012,1><1013,2><1013, 3x1013,4x1013, 5><1013,6><1013,7><1013, 8><1013, and 9x10". In some embodiments, an effective dosage of TILs is in the range of 1x106 to 5x106, 5x106 to 1x107, 1x107 to 5x107, 5x107 to 1x10", 1x108 to 5x10", 5x108 to 1x109, 1x109 to 5x109, 5x109 to 1x10", 1x1010 to 5x10"), 5x1010 to 1x10", 5x1011 to 1x10", 1x1012 to 5x10", and 5x1012 to 1x10".
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 TlLs may be administered in either single or multiple doses by any of the accepted modes of administration of agents having r ies, including asal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, topically, by transplantation, or by inhalation.
EXAMPLES The embodiments assed herein are now described with reference to the following examples. These examples are provided for the e 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 t as a result of the teachings provided herein.
Example I — Variability in Expansion of Tumor Infiltrating Lymphocytes using PBMC Feeder Cells The variability in TIL ion obtained by use ofPBMC feeder cells may be demonstrated by comparing the results of le TIL expansions on the same line of TILs obtained from a patient. illustrates typical results of rapid expansion of TILs using irradiated allogeneic PBMC feeder cells (PBMC feeders). Two TIL lines labeled MlOlST and MlOl6T (1.3 X 105 cells) were co-cultured with 46 different irradiated feeder cell lots (1.3 X 107), IL-2 (3000 IU/mL, recombinant human IL-2 (e.g., aldesleukin or equivalent), CellGenix, Inc., outh, NH, USA) and OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotec GmbH, ch 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 ld 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. e 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-l4 cell line exhibited endogenous expression of CD64, and was selected for further pment. The EM-3 cell line was selected based on the observation of endogenous expression of ICOS-L (which was not ed for the EM-2 cell line, despite being taken from the same patient).
TABLE 5. Summary of costimulatory molecules sed endogenously on candidate cell lines for aAPCs. CML refers to chronic myeloid leukemia, and AML refers to acute myeloid ia. "Pop" refers to the population of cells ed to express the marker (1/2 pop—— 50%).
Can hm _— Km-246 Km-8031 K562 MOLM-14 Myeloid blast Myeloid blast cn'sis, CML cn'sis, CML HLA—WC____—- CD64 ------ ——————— ——————— ——————— ——————— ——————— Example 3 — Preparation of MOLM-l4 Artificial Antigen Presenting Cells gaMOLMl4 aAPCsz MOLM-l4 cells were obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. To develop 4 based aAPCs, MOLM-l4 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 PLV43OG and nsfected with PDONR221 vectors (Invitrogen/Thermo Fisher ific, Carlsbad, CA, USA) using a lentiviral transduction method. The gateway cloning method was used as described in Katzen, Expert Opin. Drug Disc. 2007, 4, 57 1—5 89, to clone hCD86 and hCDl37L genes onto the PLV43OG and PDONR221 vectors. The 293T cell line (human embryonic kidney cells transformed with large T antigen) was used for iral production, transduced to MOLM-l4 cells. The transfected cells were sorted (S3e Cell Sorter, Bio-Rad, Hercules, CA, USA) using APC-conjugated CD86 and PE-conjugated CDl37L to e 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 to , and the nucleotide sequences for each vector are given in Table 6. The pLV43OG human 4-1BBL vector is illustrated in with the rase chain reaction product (PCRP) portion shown in The pLV43OG human CD86 vector is illustrated in with the PCRP portion shown in The pDONR221 human CD86 donor and human 4-lBBL donor vectors are shown in and ctively. Diagrams of the empty pLV43OG destination vector and empty pDONR221 donor vector for the y cloning method are shown in and respectively. and illustrate vector diagrams of the psPAX2 and pCIGO- VSVG helper plasmids used for lentivirus production.
TABLE 6. Nucleotide sequences for preparation of lentivirus for transduction of aAPCs.
Identifier Sequence (Description) SEQ ID NO:15 gataaccct aattcgatag catatgcttc ccgttgggta acatatgcta ttgaattag (pLV430G human ‘tctgg atagtatata cccg ggaagcata: cgtt tagggttca 4*1BBL vector) gatgcc ggccacgatg cgtccggcgt agaggatcta atgtgagtta gctcactca :aggcacccc aggctttaca ctttatgctt ccggc:cgta tgttgtgtgg aattgtgag ataacaat ttcacacagg aaacagctat gacca:gat: acgccaagcg cgcaattaa gggaacaaaa gctggagctg caagc:taa: gtagtcttat gcaatactc caacatggta acga:gagtt agcaacatgc cttacaagga gagaaaaag ccgattggtg gaag:aaggt ggtacgatcg tgccttatta ggaaggcaa gacatggatt ggacgaacca ctgaa:tgcc gcattgcaga gta agctcgatac ataaacgggt ctctc:ggt: agaccagatc tgagcctgg ctaactaggg aacccactgc cctca ataaagcttg ccttgagtg tgtgcccgt ctgt:gtgtg ‘gtaa atcc ccc tggaaaatc tctagcagtg cgaac agggacttga aagcgaaag agctctctc gacgcaggac :tgc gaagcgcgca cggcaagagLQLQHOLQHOOHOOHOLQ cgactggtga gtacgccaaa :gac ggct agaaggagag HUHLQLQHOOSDHSDLQSD gagagcgtca gtat:aagcg aa: agatcgcgat gggaaaaaa: ccagggggaa agaaaaaata :aaaa catatagtat gggcaagcag SDLQ gcag ctgg :agaa acatcagaag gctg:agaca cagctacaac catcccttca atca gaagaactta gatcat:ata mum gcaaccctct tgca gagataaaag acaccaagga aagatag SD l0 aagagcaaaa accaccgcac agcaagcggc ‘acct LQ rho ggaggagata ttggagaagt gaat:a:ata SD SDSDOFtOLQFtLQLQLQOSDFtSDLQ gaaccattag caccaaggca aagagaagag :gcagag l0 gcag:gggaa gttccttggg ttct:gggag g l gcagcgtcaa ggtacaggcc agacaa:tat 0 :ggta: OLQ IOQJIO cagaacaatt LQLQH tattgaggcg catc :gcaac: ggca:caagc LQ aagaatcc:g gctg:ggaaa acctaaa $1) ctcc:gggga ctctggaaaa ctca:t:gca :gctg: 51) gctagttgga tctggaacag atttggaatc gacagagaaa FlFlQJQJQJQJLQQJLQF’FO $1)$1)xr acacgacctg I cacaagct:a atacac:cct :aa_:gaaga cagcaagaaa SD agaattat:g gaat:agata tggt:taaca l -gggcaag I gctgtggtat ataaaa:tat :aatga: ttgg:aggtt tg:a ctttctatag aa:agag_ ta:tcaccat [l gacccacc:c ccaaccccga ga 0 LO 511 ggaa:agaag [l agagagagac agagacagat ::cgat: ‘a tc:cgaccgt H I aaagaaaagg ggggat:ggg g:acagtg ccaaag acac cagacataca aactaaagaa :tacaaaaac aaattacaaa aa:tcaaaat :ttatcga ttatttag:c tccagaaaaa ggggggaatg ‘acccca cc:g:aggtt :ggcaagc gcttaagtaa cgccat:ttg caaggcatgg aaaatacata ac:gagaata gagaagt: gatcaagg:t aggaacagag agacagcaga gcca atat ctgtggtaag cagttcctgc cccggc:cag ggccaagaac tccc caga:gcggt cccgccc:ca gcagtttc:a gagaaccatc agatgtttcc cccc aaggacctga aatgaccc:g at:t gaactaacca tcgc ‘cttc tg:tcgcgcg cttctgc:cc ccgagctcaa taaaagagcc ccct ‘gcgc cctc cgatagac:g cgtcgcccgg atat caacaagttt gtacaaaaaa gcaggcttcg tgga atacgcctct gatgccagcc tggaccccga agctccttgg cc:cctgccc ctagagccag agcctgtaga gtgctgcctt gggctctgg: cgctggcctt ctccttctg 0 tgctgctggc ctc‘cctgc gctgtgtttc tggcttgtcc ttgggccgtg gccW cag cttctcc cc ccca gactgagaga cggacctgag ctgagccccO atg atcctgc gac -g gatctgagac agggcatgt: cgcccagctg gtggcccag W acg tgctgct gatcg cccctgagct ggtacagcga tcctggactg gctggcgtg H cactgacagg CC gcc tacaaagagg acaccaaaga actgg:ggtg gccaaggccO ccc a C9 tgt cagctggaac thgé ag: ggtggccggc :ccQ cctctgtgtc tcth catctgcagc tgctgcaggc gctgctgca0 th ccctgac ac tgc cctccagcct cagaaactcc gcattcggg H ttcaaggcag actgc 0 :gtc:gccg gggag:gcat ctgcacacaO agg ccagagc cagacacgcc fi0 tacag:gc :g ggcctg’‘ F’r 0W cac c CC aaa :tcca 0ccggcc:gc aagcgagtag gacccagct H tct :gtacaa ac th :ga :t 0 agt:aatt agtgccat :t gttcag:gg H tcc :agggct cact O "‘0 ttggc:tt Qatgaigtgg tatZQQQQQ 0 caagtctgta cagca :ct :g Wm tccct:tt accaa:tt :C ttt:gtctt H 999 :atacat ttaaaccc :a W Qaaaacaaa tactc:ctaa att:ta:gg O tta :gtcatt 99 atg :ta :g tcc:tccc atcatacaaa aaa:caaag W atg :tttaga aaact :cc :a N’@ "‘0 aacaggcc aaagtatg :C aacgaa:tg H :cttttg 99 :tt :gc :g 0 0 cct:t:ac tatcc:gcgt tga:gcctt H :gcatgt at :caatc :a Wm caggc:tt ccaac:taca aggcct’IS H a tacctgaacc fi :taccccgt cggccagg :C aag H :gctgac gcaaccccca 0 :ggCZgQgg ggcca:cagc gtg O aaccttttcg gC ICC :ctgc 2 ac ctagccgc :t caggtct ggagcaaaca fi m‘atcggcac ctat tacatcgttt ccatggctgc fiWggc:g_gc atcctgcgcg :tacgtc ccgtcggcgc fi A0 aatcc:gc ggacg tc:cgggg :C :cgtccc ct :ctccg :c fi cg accgaccacg gggcgcacct actccccg tc :gtgcc :t 0 "m catctgcc ggaccgtgtg cacttcgc :t cgcatgg agaccaccgt ccac caaatat:gc ccaaggtc :t c tc :cagcaat O ’caacgacc gaccttgagg tcaa aaagac: ag O gggaggag attaggt:aa ttgt t9:aggcata 0 m‘o‘ caccagca ccatggcgca atcactagag ttaagaccaa O A0 cagctgta gatcttagcc ac:ttttaaa agaaaag ggactggaag 0 :cccaacga agacaagatc tgctttttgc ttgtactggg tc:ctctgg: 0 :gagcctgg gagctctctg gc:aactagg gaacccactg ct:aagcctc :tgagtg cttcaagtag tg:gtgcccg tctgt:gtgt gactctggta cc cagaccc ttttagtcag tg:ggaaaat ctctagcagt agttca: ta’:cagtat ttataacttg caaagaaa :g aatatcagag gagagga fi agcttat taca aa:aaagcaa tagca:caca a fi :tcactg cattctagtt gtggtttg :C caaac:catc gtatct: O tcccgcccct aactccgccc atcccgcccc cgcc 0 cccatggctg ac:aattt tttat:tatg cag aggccga 0 tttcatcctg gagcagac tgcag:ctgt ggactgcaac 0 aactcttggc tgaagctc acaccaatgc 199 gggaca: OO aagactacgg acac caacg:caat cag aggggcc 0O gaccctcaag agggcattag caatagtgtt :ataaggccc N'O 0 ‘ gaaagggcct cgtgatacgc ctatt:ttat agg :taatg: W O agacgtcagg tggcactt cggggaaatg :gcgcggaac fi ‘w‘\ W aaatacattc aaatatgta ccgctcatga gacaataacc 0 H H attgaaaaag gaagagta :g agtat :caac att :ccgtg: 0 0 0 cggcattttg ccttcctg tttgc :cacc cac aaacgc: WWW aaga :Cagtt gggtgcacga gtggg:taca -CC a ‘0 ttgagagttt :cgccccgaa gaacg :tttc caa :ga:gag to ‘\Q gtggcg cggt attatcccg: gttg acgccg ggcaagagca 0Q attc :Cagaa :gacttgg gagtactcac cac :cacaga tgacag :aag atgc agtg c :gcca :aacca:gag tact :C :gac aacgatcgga ggaccgaagg agc :aaccgc atca :9 :aac :cgccttga: cgttgggaac C99 agc:c agcg :9 acac cacgatgcc: gcagcaatgg caacaacg aactac :tac :ctagcttcc cggcaacaat -aa caggaccact :ctgcgctcg gcccttccgg ctg Cng :9 agcg :gggtctcgc ggtatcattg cag aagccctccc gtatcg :agt :atctacacg acggggagtc caga tcgc :9 agat aggtgcctca ctgattaagc ctttactcat atatac :tta gattgattta aaacttcatt gtgaagatcc tttt :9 ataa gacc aaaatccctt tgagcgtcag accccg :aga aaagatcaaa ggatcttctt gag gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag C99 tttgccggat caagagctac caac :C :ttt :ccgaaggta actggcttca gcagagcg gataccaaat actgtccttc tag tgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg :ctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 9 actcaag acgatagtta ccggataagg cgcagcgg:c gggctgaacg gggggttcgt gcc ggag cgaacgacct acaccgaact gagataccta cagcgtgagc :tgagaaag cgccacgctt cccgaacgga gaaaggcgga caggtatccg gtaagcggca thQ aggagagcgc acgagggagc ttccaggggg aaacgccth tatctttata gtttcgccac ctctgacttg agcg:cga:t tttgtgatgc tcgtcagggg gag atggaaaaac gccagcaacg cggccttt:t acggttcctg gccttttgct aagctgtccc tgatggtcgt catc:acc:g cctggacagc atggcctgca cccgatgccg aatggggaag gccatccagc SEQ ID NO:I6 cctctgatgc cttggcctcc tgcccctaga (4*IBBL COOP) gtagagtgct gccttctcct tctgctgctg cctgcgctgt ccgtgtcagg cgccagagct ccag tgag ccccgatgat tgctggatct 0 agctggtggc cgtg ccct 0 gactggcth cgtg:cactg tgagctacaa 0 tggtggccaa ggccggcgtg tctttcagct fi‘o Q ccggcgaagg atccggctct cactgcatct cagcccc Q caggcgctgc tgcactggcc g acctgcctcc 0 0fl 0flW ‘0 0 actccgcatt cggg:ttcaa c tgtc ‘ccggccaQ tgcatctgca cacagagg c ctgacacaQ tgctggg cct gttcag a fi cctgcccag agtag SEQ ID NO:I7 t fi aagcaggct N'O gaatacg cct (4*IBBL PRCP) c ggcctcctg 0 agagcctgta g ttctccttcfi g cct t Q‘o‘o gcg 0 cctggatctg a mwo agctgagcc0 gccggac :gc g gtggccc ctgatcg atg ‘c ‘0 gctggcgfi'W ggcggcc :ga W aa A0 ggccaagg 0 tacgtgt :ct W actgcggag a fi‘o gcgaaggat0 tctctgg cac W gcccctgag a gcgctgctg 0 actgg 0 I acagtgg acc ctctagcgag ccgcattcg Q ggc agactgc -gc 0 C99ccagag a 0‘0 atctgcaca aggccaga gccagacacg H gacacaggg c ‘0 tgt fi'O ‘agtgacc cccgaaa :tc H aggacccag 0 tttcttgtac aaagtgg -cc SEQ ID NO:IB ‘ataaccc catatgctt0 acatatg cta A (pLV430G human ‘tctg g ccc gctacccgtt CD86 vector) ‘atgcc cgtccggcgfi'@ atgtgag :ta :aggcacccc ctttatgctt tgttgtg :gg ataacaa: aaacagctat acgccaagcg gctggagctg gtagtct :at ‘:cttg acgatgagtt cttacaagga accgtgcatg gaagtaaggt tgcctta :ta gtc: ggacgaacca caga :taag:gcc: agctcgatac ataaacgggt agaccagatc gg ctaactaggg aacccactgc ataaagc :tg tag: tgtgcccgt ctgt:gtgtg ctagaga :cc :cag: tggaaaatc tctagcagtg t :ga gaaaccagag agctctctc gacgcaggac gaagcgcgca LQLQHOLQHOOHOOHOLQ A0 ggcgg cgactggtga gtacgccaaa agcggaggct Q fifiLQLQF’FOOWF‘FWLQW ggtgc cacagcgtca gtat:aagcg gat ‘gaaaaaa: QHWOOH‘O agatcgc :aagg ccagggggaa agaaaaaata catatag :at gcaagcag WkQ :agaa cgattcgcag ttaa:cctgg acatcagaag :ggga cagctacaac catcccttca gaagaac :ta W‘x‘xw cagta gcaaccctct attg:gtgca gagataaaag :agac gagg aagagcaaaa accaccgcac gatctt cagacctgga ggaggagata ttggagaagt :aaagt agtaaaaatt ttag caccaaggca gcagag agaaaaaaga gcag:gggaa gttccttggg 0aggaag cactatgggc gcagcgtcaa ggtacaggcc :ggtat agtgcagcag cagaacaatt tattgag9C9 gcaact cacagtctgg ggca:caagc aagaatcctg cctaaa acag ctcc:gggga ctctggaaaa :gctgt gccttggaat gctagttgga tctggaacag gacctg gatggagtgg gaaa cacaagctta :gaaga atcgcaaaac cagcaagaaa agaattattg aatgggcaag aaca taacaaat:g gctgtggtat ttat tcataatgat ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt tctcgacggt atcggttt:a aaagaaaagg ggggattggg gggtacagtg aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aattcaaaat ga:t ttatttagtc tccagaaaaa ggggggaatg ‘ cctgtaggtt gc:a gcttaagtaa cgccattttg caaggcatgg aata gagaagttca gatcaaggtt aggaacagag acagcaga aacaggatat ctgtggtaag cagttcctgc cccggctcag ccaagaac cagatgcggt cccgccctca gcagtttcta gagaaccatc OSDSDI‘ISDELQI‘ILQSDSDOOSD:gtttcc aaggacctga aatgaccc:g tgccttattt gaac:aacca SDgttcgc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc aacccct cctc cgatagac:g cgtcgcccgg g:accgatat SD caagttt gcaggcttcg ccaccatggg cctgagcaac :cc:gttcg OF’I mom ggcctt ‘ ggagccgccc ctctgaagat ccaggcctac :caacgaga ccgacct ttcgccaaca gccagaacca gagcctg 51) ac:ggtgg VI :ctggca ggaccag aacc:ggtcc tgaacgaggt gtacctgc aagaaaagt I‘I 0 acagcgt caag ggcc ggaccagctt cgacag Qctggaccc I‘I I0 gctgca caacc:gcag atcaaggaca a9 LO LO 0 :gta :ccaccaca SD In aacccac cggca:g :c agaa:ccacc agatg 51) 0 ag :gc:ggcca $1) 0 :cagcca g :c gtgcccatca gc $1) $1) 0 I ac $1) 0 a:caacc I‘I I0 0ctgcag cagca’I SD 0 ggctaccccg a9 0 0 0 SD I0 aa :gc:gcgga 0 0 $1) gaacag cacca:0 51) LO tacgacggcg t9 $1) aa $1) cg:gaccg 511 I0 :gtacga 0 II 0 agcg t9 $11511 LQF’I mom II on cc I0 caacatga 0 0 :cttttg g $1) $1) accgacaaga cc 0 I‘ILQ II In :ggaaga tcccca cctcccgacc ac DSDOI‘ILQSDSDSDLQI‘ISD :cagcatcg 0 0 $1) 0 O O cca atcat ctgcg:g II LO gtgt:ctgcc t9 $1) ("I 0 II I0 $1) $1) gaagaagc cctaggaa cagcta0 SD Q tgcggcacca ac5118 II In 51) 0 I cgagcaga caagCg ggagaag u caca:ccccg a9 0Q In I0 0 cggg:g:tca agcagcaa gaccag0OF’ILQQJQJLQOQJQJQJQJOLQQJLQOQJQJ m aaga 0 0 0fi@ ccagct:tct agt ggtga: F'I I In gt:aattaag 0LQSDLQI‘II‘IOSDSDSDOOOI‘IOSDSDSDO OOLQOSDOOSDLQSDSDLQF‘IOOSDF‘ISDF‘IF‘ISDOO I 51) LG VI cagtgg:tcg :agggctttc ccccact II tggc:ttcag 0 C SD I‘I :9 ccaa gtctgtacag catct’I Io ccct:tttac :acc :9 :ggg :atacattta I 0 aaacaaagag :tac gtta :gtcattgga tgtta I 0atc I cc:tgccaca aatg :tttagaaaa cttcc* IIm acagccctat I I I gaaa :ggg :cttttggg: tttgc:g 0 ct:t:acaca Q :tat :gta tat: caatc:aag aggc:ttcac :cgcca :gtg atac ctgaacct: accccgttgc ccggcaacgg :gt: :gctgacgca acccccac: gc:ggggctt ggtcatgggc gaac cttttcggc: cctctgccc tcca:actgc ggaactccta :cgcag caggtctgga gcaaacat tcgggactga tgt: gcaaatatac tcca tggctgcta gc:g:gctgc caac:ggatc ct:tg :tacgtcccg tcggcgctg atcc:gcgga cgacccttc: ‘ggac:ct :cgtcccct: ctccg:ctc cg:tccgacc gaccacgggg cgcacctctc :ttacgcgg ctccccgtc: gtgcc:tc II cgga ccgtgtgcac ttcgc:tcac ctctgcacg cgcatggaga ccaccgtga cgcccaccaa atat:gccca aggtc:taca :aagaggac ctc: tg: aacgaccgac gca: aaga ctgtttgtt aaagactggg aggag:tgg ggaggagatt aggt:aaagg tctttgtact ctg aggcataaa: tgg:c:gcc accagcacca tggcgcaatc actagagcgg gg:accttt agaccaatga cttacaagg agctgtagat cttagccac: ttttaaaaga aaagggggg ctggaagggc taa:tcac*I ccaacgaaga caagatctgc tttttgcttg tactgggtc ctctgg:tag accagatc:g agcctgggag ctctctggC’ aactagggaa aagcctcaa: aaagc:tgcc :tgagtgctt caagtagtg gtgcccgtct tctggtaac: agaga:ccc: cagacccttt tagtcagtg ggaaaatctc agttca:gtc atc:tatta: :cagtattta gcaa agaaatgaat tgt:tattgc agcttataat ggttacaaa: aaagcaatag aagca:tttII :tcactgcat tctagttgtg gtttgtccaa IIIIIIIIOIIIISDSDIIIIIIIISDOII atg:c:ggcII ctagctatcc cgcccctaac tccgcccatc ttccgcccaII :ctccgcccc atggctgac: aatttttttt :tatg cggatccctII gagtggcttt catcctggag cagactttgc ag:ctgtgga aca:tgcctII :atgtgtaac tcttggctga agctcttaca ccaatgctgg cctcccaggg gcccaggaag actacgggag gctacaccaa cg:caatcag gtagctaccg ataagcggac cctcaagagg gcattagcaa tagtgtttat tgt:aattc: :gaagacgaa agggcctcgt gatacgccta tt:ttatagg gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc cc tat:tgttta :ttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgct: caataatatt gaaaaaggaa gagtatgagt at:caacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tttt gctcacccag aaacg Ctggt gaaagtaaaa gaag ggg tgcacgagtg atcg 19 gatct caacagcggt aagatccttg gagttt :cg ccccgaagaa cgttttccaa tga:g agcac :tttaaagtt tgtg cg cggtatt atcccgtgtt gacgccgggc aagag Caact cggtcgccgc atacactatt HOHOHHOHLQLQHLQQJHOOLQQJQJ :C agaa :ga cttggttgag ccag tcacagaaaa gcatcttacg gatggcatga a9 :aagaga attatgcagt ataa cca:g agtga :aacactgcg gccaacttac :C :gacaac gatcggagga ccgaaggagc taaccgcttt :ttgcacaac atgggggatc :9 :aac :cg ccttgatcgt tgggaaccgg agc:g aatga acca aacgacgagc :9 acaccac gatgcctgca gcaa caacgLQOfiLQLQOFffiF’FSDOSDLQF’FfiF’F :gcg caaactatta actggcgaac ac :tac :ct agcttcccgg caacaattaa tagac LO LO 51) F? ggaggcgg aaagttgcag accact :ct gcgctcggcc cttccggctg gctgg :tat tctggagccg agcg :gg gtctcgcggt atcattgcag cac:c LO LO 0 0 ccctcccgta :agt :at ctacacgacg gggagtcagg :gga agacagatcg agatagg actg attaagcatt tata t tgatttaaaa cttcattttt cttt ‘ataa :ct catgaccaaa atcccttaac gcgtcagacc :agaaaa gatcaaagga tcttcttgag atctgctgct gcaaacaaa aaaaccaccg ctaccagcgg gagctaccaa :C :ttt :CC gaaggtaact ggcttcagca gtccttctag gtagccgta ccac aaga tacctcgc:c :gc :aatcct gttaccagtg gctgctgcca actcaagacg atagttaccg gataaggcgc cacagcccag cttg acgacctaca gagaaagcgc cacg gaagggagaa :cggaacagg agagcgcacg agggagcttc ctgtcgggtt tcgccacctc tgacttgagc ggagcctatg gaaaaacgcc agcaacgcgg ctttttgaag ctga tQQtCQ cgggcatccc gatgccgccg gcgtcg SEQ ID NO:I9 gccttcctgc gagc (CD86 COOP) gacctgccct gccagttcgc tggcaggacc aggaaaacc: gag agcgtgcaca gcaagtaca: agcttcgaca ctgcacaacc tgcagatcaa ctgtaccagt ccacaag cccaccggca tgatcagaa: aacagcgagc ‘OSDOSDgLQOSDOU ggccaac agccagcccg agatcgtgcc gaga H caacctg tgcagcagca tccacggcta ccccgagccc aagaaaatga H gcggacc aacagcacca tcgagtacg cggcgtgatg cagaaaagcc H gaccgag tacgacgtga gcatcag 0 0 gagcgtgag ttccccgacg catgacc ttttgcatcc ‘gaaaccg caagacccg ctgctgtcca Q catcgag gaagatcccc 0 0 0 0 0 F’F 0 ccaccacat ccctggatca \ I gcccacc atcatctgcg ctgcctgat ctgtggaagt aagaag SD gaagcgg 0 0 F’F aggaacagct cac atgcaacggg gc 51) cagaccaag aagC999 aga ccccgagcg agccacgagg cccagcgg to H ttcaagagc agcaagacca SDHLQUOSDHSD caagagcg SD0‘0000‘00 acctgcttc SEQ ID NO:20 ‘tacaaa aagcaggc F’F cgccaccatc acatcc:g (CD86 PCRP) ttcctgctg ccggagccg ccctctg aac atccagg acttcaac ctgccctg 0 agttcgcca g aac cagagcct gcgaac:g caggacca SDSDSDOLQSDLQOSDSDOSDLQOHSD SDSDLQLQOFfSDSDLQF‘FSDOF‘F aaaacc:gg cctgaacgag gtgtacct gcaaagaa gtgcacag 0 agtaca:gg ccggaccagc ttcgacag acagctgg cacaacctg agatcaagg caagggc :9 taccagtg tcatccac accggcatg tcagaa:c0 ccagatg agcgagct ccgtg 19 cagcccgag tcgtgccca cagcaacSDSDSDOOOF’FOSDSDSDO n 0SDSDHSD ‘0‘00Q accgagaa tgtac agcagcatc acggctac0 cgagccc aaaatgag QLQLQSDOSDSDLQOSDO :caaCg agcaccatc agtacgacg cgtgatg aaaagcca acaac ac gacgtgagc tcagcc:g $1) cgtgagc cccgacgt at tgcatcctg aaaccgaca gacccgg I 0 x ctgtccag gatccccag cccctcccg ccacatc 0 0 tggatcac atctgcgtg tggtgt:ctg cctgatc m tggaagtg aacagctac agtgcggcac caacacc gaacgggag cgggagaag tccaca:ccc cgagcgg gacgaggcc ‘agcagc aagaccagca acaa gagcgac tgcttctag tacaaa gtggtcccc SEQ ID NO:2I ctttcctgcg ttatcccctg attctg:gga taaccgta accgccttt (pDONR22l CD86 taccgctcgc cgaa cgaccgagcg cagcgag tca gtgagcgag‘ aagcg vector) gcgcccaata cgcaaaccgc ctctccccgc gcgttgg ccg attcattaat gcagctggca cgacaggttt tgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgc:ta tgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgc:tc acaacgttca aatccgctcc attt ctca cgtt caccgacaaa ataa aacgaaaggc ccagtcttcc gac:gagcct ttcg ccct actctcgcgt tagc gttt aacgacggcc agtcttaagc tcgggcccca aataatgatt ctgttcgttg caacacattg atgagcaatg ctt:tt:ata SBA—Art fifio LQSDO OF’FO aagcaggctt cgccaccatg ggcctgagca :g cgtgLQOWOWWfiWOLQfiLQWgfiWfi $1) 51) LG rt:ttat cctggtcac gacgttg:aa:tgac tgatagtgacttgtacaaaaA A gcc ttcctgc:g ccggagccgc ccctctgaag atccaggcct gacc ccgac ctgccctgc agttcgccaa cagccagaac cagagcct voAo mm ggtg :ctgg caggaccag aaaacc:ggt cctgaacgag gtgtacct gttc acagc gtgcacagc agtaca:ggg ccggaccagc ttcgacag cctg Q A0 ctg cacaacc:g agatcaagga caagggcctg taccagtg 0 on caag aaccc accggca:g :cagaa:cca ccagatgaac agcgagct caac cagcccgag :cgtgcccat 0A0 A0fi cagcaacatc accgagaa cctg agcagca’A ctaccc cgagcccaac aaaatgag 0 A0 gacc agcacca:c cgtgatgcac aaaagcca A0 cgag gacgtgagc cgtgagcttc cccgacgt m gacc tgcatcc:g gacccggctg ctgtccag 00 Q cgag gatccccag ccacatcccc tggatcac on cacc :ctgcg:g cctgatcctc tggaagtg gcgg 0 0 F’F $1) 0 51) LG 0 fl2 caacaccatg gaacggga 0A0 aaag gaccaag LO Q m aga SD 0 QJQJQJOLQSDLQOSDSDOQJLQOF’F cgagcggagc gacgaggc0 agCQQQ caagagcagc cca cagcgacacc tgcttcta A0 acccagcttt cttgtacaaa :tgcttatca atttg:tg 0 $1) acgaacaggt cac:atcagt ccatccacct gatatcccct atagtgagtc gta:tacatg AASDAASD OSDLQLQ SDSDAOSD OQJAALQ FASDA‘AOA LQSDSDSD gctctggccc caaa gatg :tacattgca SD SD m atgaacaata aaactgtctg cttacataaa cag:aataca LQ FALQ m -caacgg aa acgtcgaggc taaa :tccaacatg g ct cgcga:aatg tcgggcaatc agg:gcgaca AA 0 (‘f’AO gcccgat‘cg ccagagttg: ttctgaaaca :ggcaaaggt A0 agatgagatg gtcagactaa ggaatttatg SWFAOSDLQOSIJQJgSDOLQOLQSDOSDSDLQ 0 tgac 0 F’F 0 :tttatccgt actcc:gatc atgcatggtt actcaccact 0 FAQ 51) attccag ta ttagaagaa atcctgattc agg:gaaaat A cttcctg cggt:gcat ctgt :tg:aattgt fiAO atttcg:ctc ctcagc aatcacgaat gaa:aacggt :gatgacgag cgtaa ggcctgttga acaagtctgg WHO SIJLQF‘F A0 cccattctca ccgga :cgtcactca :gg:gatttc 0A0 -gacgagggg aaat gttgtattga :gt:ggacga A LQA‘ASIJA‘AA‘AA‘AA‘AF‘FF‘FSDOLQSDSSDSA LQSDSD ccagga:ctt ccca ggaactgcct cgg:gagttt ctttt:caa aaata :tgataatcc :ga:atgaat ctcga:gag tttt cagaattggt :aa:tggttg ctgac:tga cgggacg caagctcatg accaaaatcc ttccactga ccgtcag ccgtagaaaa gatcaaagga tctgcgcgta atctc :gcaaacaaa accg gccgga:caa ctctttttcc gaagg:aact accaaa:act :gtagccgta accgcc:aca :gctaatcct ctcgtg:ctt actcaagacg atag ctgaacgggg cacagcccag cttg acag gagaaagcgc cacg gaaggg QJQAO ggta :cggaacagg agag agggag F’F cg cctggtat ctgtcgggtt tcgc tgactt tgatgctcg ggagcctatg gaaa agcaac ttcctggcc :gctca catg SEQ ID NO:22 ttatcccctg agtgag (pDONR22l 4* cg cagccgaa aagcgg lBBL vector) cg caaaccgc gcagctg cccgactgga ccg gagtttgtag cttctgctta t9 gcagttta ccgttgcttc acaacg aatccgctcc caccgacaaa ataa aacgaaaggc ttgatgcctg gcagttccct actctcgcgt gtaa aacgacggcc a9 tcttaagc tgatagtgac ctgttcgttg caacacattg ttgtacaaaa gctt CQ tg gaatacgcct gaagctcctt ggcctcctgc ccctagagcc agagcctgta gtggctggcc ttctccttct gctgctgctg gccgctgcct ccttgggccg gcgc cag agcttct cctg gatctg ccgccagccc cagactgag cctg agctgagccc cgatgatcct gccg gactgc :ggatctgag acagggcat ttcgcccag tggtggccca gaacgtgctg ctga :cgatg gccccctgag ctggtacag tggctggcgt :cactgaca ggcg gcctga gctacaaaga ggacaccaa mm SD‘ 00 (*0 LQF’F LQLQ fiLQ mg; tggccaaggc 0 0 tacg :gttct :tcagctgga actgcggag LO LO F’F LO LO gcgaagga:c fiLQLQWWWWWLQLQfiOWLQWOOLQ Q A0 cgtgtac Q9 ctctgtg tctc :ggcac :gcatctgca cccctgag rho gcgctgctgc ctggccctg acag :ggacc :gcctccagc cga on 0 51) LO 51) 51) 511 0 ccgcat:cgg :ttcaaggc agac :gctgc acctgtc:gc gag atctgcacac SDLQ a ggccaga gccagacacg cctggcagct acacaggg m tgggcc:g:t g agtgacc cccg51) 511 m :c cagccggcct cccagccc 51; aagcgag \ x acccagc :tcttgtac aaag ‘ LO m 0 $1) gaaa ca:tgctt Ff $1) $1) Ff Ff Ff LQ ‘ acgaaca LQ t c agtc $1) $1) $1) ‘ 51) aaatcat:at tgccatcc atagtga :cgta:tac atgg H 0 51; ctgtttcctg cacctctg :c:g :9 :tacatt gcac 51) SD ‘0 aaataatatc atcatgaac ‘cttacat t acaa LQ F’FLQ ‘0 LQSDLQSDSDF‘FOSDLQSDSDSDOLQSD @fimfifififififimoflmr‘rmr‘rr‘r \SD :tatgagcca tat:caacg mmom ggccgcga:t :tccaac atgg a:gg cta:aaatgg atgtcgggca agg:gcg acaa gcttgta:gg caagcccgat mm tg:ttc:gaa :ggcaaa ggta ccaatga:gt tacagatgag SD OOOLQOF’FOOF’FSDSD taaactggct SD OSDOSDSD ggaattt atgc OLQHSD fiOO 0‘0rho cgaccatcaa cca:tttatc atgatgca:g :tactcacc actg cg SD ccggaaaaac agcattccag aa:atcctga :cagg:gaa aata :tg atgcgctggc cctg at:cga:tcc tgt :tg:aat tgtcctt acagcga:cg cgtatttcg: cgcaatcacg aatgaa:aac ggtt 199 atgcgag:ga ttt:gatgac gc:ggcctgt tgaacaagtc tggaaag aac: tttcccattc cac:cg:cac tca :gg:gat ttctcac ataacct:a: ttt:gacgag taggttgtat tga :gt:gga cgag :c9 $1) 51) tcgcagaccg ataccagga’ ta:ggaactg cctcgg:gag ttttctcc:t cattacagaa ttt gta:tgataa tcc :ga:atg aataaattgc agtttca:: gatgctcga aa:cagaatt ggt :aa:tgg cact ggcagagca tacgctgac gcgcaagctc atgaccaaaa tccc :taacg tgagttacg gtcgttccac accccgtaga aaagatcaaa ggatcttc:t gagatcc ttttctgcgc gc::gcaaac aaaaaaacca ccgc :accag cggtggt tttgccgga caactctttt g Q :a actggcttca gcagagcg gataccaaa :gtagcc gtagttaWLQLQQJQJQJQJQJF’FF'FQJOOOLQF'FOLQ gC caccacttca agaactc:I‘HLQLQOHLQLQQHH HHLQSDHOSDHSDLQHOHH gcc :gctaat cctgtta gc:g ccagtggc taagtcgtg g acgatag Cngataagg cgcagcgg gggctgaacg WLQLQfiWOOO‘OfiW cacagcc cagcttg cgaacgacct aa gagataccta gagaaag cgccacg cccgaaggga gaaaggcg caggtatccg :cggaac aggagag gagc ttccaggg aaacgcctgg ctgtcgg gtttcgc ctctgacttg agcgtcga tttgtgatgc ggagcc: atggaaa gccagcaacg cggccttt acggttcctg I‘LQWWOfifi" cttttgc tcacatg SEQ ID NO:23 :aaccc: aattcga catatgcttc acatatgcta 0G agtctgg t ctactacccg gctacccgtt vector) :gatgcc 99Ccacg cgtccggcgt atgtgag :ta gcacccc agg cttt ctttatgctt tgttgtg :gg :aacaa: ttcacac aaacagctat HHHSDH acgccaagcg cctcactaaa 999 aaca gctggagctg gtagtct :at $1) 0 F’F 0 :gtag:cttg caacatg :a acga :gagtt cttacaagga agaaaaag accgtgcatg ccg attg :g gaag :aaggt tgcctta :ta gaaggcaa c: gacatgg :t ggacgaacca gcattgcaga atat:gta :gcc- agctcgatac ataaacgggt agaccagatc tgagcctgg :ctgg ctaactaggg aacccactgc ataaagc :tg ccttgagtg t9 t ctgt :gtgtg ctagaga :cc ctcagaccc :cag- t9 gaaaa:c tctagcagtg cgaa agggact :ga aagcgaaag aaccagag a9 ctctc:c gacgcaggac :tgc gaagcgcgca cggcaagagLQLQHOLQHOOHOOHOLQ ggcgg cgactggtga gtacgccaaa -gacHHHOSDSDHOLQOHHSDSDHSD agcggaggct agaaggagag ggtgc gag agcgtca gtat :aagcg agatcgcgat gggaaaaaa: :aagg ggaa agaaaaaata g :at gggcaagcag :agaa cgattcgcag ttaa :cctgg acatcagaag gctgtagaca :ggga cag ctacaac catcccttca gaagaac :ta gatcattata atacagta gcaaccctct attg :gtgca gagataaaag acaccaagga ctt:agac aag atagagg aagagcaaaa accaccgcac agcaagcggc ‘ctgatctt cag acctgga ggaggagata ttggagaagt gaattatata aatataaagt aatt ttag caccaaggca aagagaagag :ggtgcagag agaaaaaaga gcagtgggaa gttccttggg ttcttgggag gaag cactatgggc gcagcgtcaa :gacgctgac ggtacaggcc agacaattat gtat agtgcagcag aatt :gctgagggc tattgaggcg caacagcatc :gttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag tttgggg: A Q ctctggaaaa ctcatt:gca ctgt gccttggaat gtaataaa A 0 tctggaacag atttggaatc acacgacctg gatggagtgg ttaacaa: cacaagct:a :cct taattgaaga atcgcaaaac aa 0A 511511 agaattat:g gaattagata aatgggcaag tttgtggaat taacaaa: gctgtggtat ataaaa:tat tcataatgat agtaggaggc taagaata ctttctatag tgaatagagt taggcaggga OLGA SDF’FLQ ttttgctg:a tatcgtt: gacccacc:c ccaaccccga ggggacccga caggcccgaa aagaagg: LO LO agagagagac agagacagat ccattcgatt agtgaacgga :cggtt: aaagaaaagg ggggat:ggg gggtacagtg caggggaaag :aatagc WA 51151) cag aactaaagaa aaac aaattacaaa :tatcgaA F‘r A F‘r F‘r SD tccagaaaaa ggggggaatg aaagacccca A ggcaagcA $1) LQ O F‘r cgccat:t:g caaggca:gg aaaatacata 51) LO 511 51) LG rt 0 51) Ft aggaacagag agacagcaga SDFASDOFAOLQLQSDFASDSDOF’FOF’FF‘FOF’FSDSDSD ‘gcca :gtggta 051) SDLQ cccggc:cag ggccaagaac tccc 0 0Q 0 0 0 LQOLQ 051151) A0 $1) gagaacca:c t:cc ‘cccc gaactaacca atcagttcgc ‘cttc 0A 0 OF’F 00 00 gctcaa taaaagag cacaacccct gcgc 0@ fiA gcccgg gtaccgat cacaagt:tg aag :aaa:a tcaatata :agatt :gcataaaa SDLQ $1151) SIJF‘F 0 $1) 0 $1) $1) 0 :ggcgg cgcattagg SD r’r A0 :tccgg gga:tt o 0 r’r SD A agttagga aaa:ca :ggatatac ‘ ‘ LO LO 0 cat:tc A0 tcagttgc tcaatgta FASDSDOSDSDOSDOLQF’FOF’FF’F ggaagc:cgtaa 0 0 0 ‘ :cagc: :tt:aa A0 accgtaaa gaaaaataag cccgcc A0 SDAAAAAASDAAOAASDAAAASDSDOOSDOOLQF’FF’FSDSD IASDAALQAASDAAAALQOLQAALQSDSDAALQSDAA HSDAAOAAAAAASDAAOLQAASDAALQAAAAAALQ atccggcct: atgaatgc tcatccggaa caatgaaaga :atggg AA agtgttca cccttgttac atgagcaaac cgc:cAA A0 agtgaata ccacgacgat aca: :ggcg AA AA tacggtga aaacctggcc aagggttta: :c AA agccaatcc ctgggtgagt AALQAALQAALQAAOAAAAO A ttgatttaaa AA cttcgcccc cgttttcacc attatacgca aggcgacaag cga tcaggttcat gct: cca:gtcg acagtactgc agggcggggc :aaacgc cagataacag tgcgcgctga :t:tgcg gtatacccga aaagaggta: 0 F’F $1) F’F LG 51) $1) gacagcgaca gctcaaggca atatgat gcacaaccat cccgtcgtc cga aggaagggat cccgg:tta :gaaatgaa acaggggctg gtttaaggt: acaccta:a :cg- SDLQSDLQFAOFASDOOOSDSDOO :gtttgtgg :gatat:at: acacgcccg AOOAOQJFT’AOQJ cccc :ggccagtg gtcaga:aaa :ctcccg:g gca :cggggatg 0 0 LO 511 ‘ catgatgacc :atgg :cggggaag :ctcagccac LO 0 LO 511 $1) $1) $1) :g :gatgttct aatgtcaggc A 0 0 0 F’F A $1) F’F ac gac gtg:tt:aca :attatg:a :aatttaat FALQFAOSDLQLQF‘FF‘FFAF‘FLQF’FLQ :ta:atcatt :acg:ttct LQ Ff LO 0‘tgatt aagctagcct agtgccat:t FASD :cccccact :atg gatga:gtgg ttg tccc tt:c :aaacccta aaaacaaa tactc:ctaa LQ SD r’r o‘ttatg tcc::ccc atcatacaaa aacttccta aacagccc aaagtatg:c LO :tttgctg 0cct:::ac tatcc:gcgt ::caatcta A0 tt ccaac:taca aacc :taccccgt cggccagg:c gcaaccccca :ggc:gggg cagc gc:cctctgc ctagccgc:t ggagcaaaca AAm atcca:ac ac gttgtcctat ccatggctgc SDggc:g:gc c atcctgcgcg tg ccgtcggcgc A0 aatcc:gc ggacgaccct gg:c ctc ct:ctccgtc accgaccacg gggcgcacct ggactccccg tc:gtgcctt AAm ccg:tccg catctgcc ggaccgtgtg cacttcgc:t cg:cgcatgg agaccaccgt aacgcccac caaatat:gc ccaaggtc:t :aagagg ggac tc:cagcaat ’caacgaccA gaccttgagg catacttcaa agactgtttg tt:aaagact agtt m gggaggag t:aa aggtctttgt actaggaggc tg:aggcata aa:tggtctg A0 caccagca ccatggcgca atcactagag cggggtacct ttaagaccaa tgacttacaa A0 cagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca :cccaacga agacaagatc ttgc ttgtactggg tc:ctctggt tagaccagat OOLQOLQLQLQOFAFAFAFAOOF’FSDOF’FLQSDSDLQOSD :gagcctgg gagctctctg gctaactagg actg gctt :tgagtg cttcaagtag tgtgtgcccg tctgt:gtgt actagagatc cagaccc ttttagtcag aaat ctctagcagt gtca:c:tat ’:cagtat ttataacttg caaagaaa:g aatatcagag acttgt:tat agcttat taca aataaagcaa tagca:caca ataaagcatt ‘OQJOOWQJF’FOLQOOOLQW $110 511LGIIIIIIIIII :tcactg cattctagtt gtggtttg:c caaac:catc atca:g:ctg SDO ctagcta tcccgcccct aactccgccc atcccgcccc cagt:ccgcc :ctccgc cccatggctg actaattt: tttat:tatg ggccggatcc tttcatcctg gagcagac: tgcag:ctgt acaaca:tgc aactcttggc tgaagctc: :atgtgt acaccaatgc gtacc:ccca LQIIII LQ gagtggc g aagactacgg gaggctacac caacg:caat tg:g :a ataagcg gaccctcaag agggcattag caatagtgtt cc:tg acgc ttat ca_ga tggcactt: gtttctt agacgtcagg cggg gaaatg IIIIIIO SDLQIIII :gaagac gcct :ttttct aaatacattc aaatatgta ccgctcatga ctga caataat attgaaaaag gaagagta:g agtat:caac 0 0 :tttttg cggcattttg tg:: tttg c :cacc -gaaag gatgctg agtt gggtgcacga gtggg:taca :cgccccgaa gaacg :tttc Io IILQ :aagatcc ttgagagttt :ctgctat gtggcgcggt attatcccg: gttg acgccg cgcatacact agaa :gacttgg:: gagtactcac acggatggca tgacag:aag agaattatgc agtg c :gcca ccgcccaact :gac aacgatcgga ggaccgaagg aacatggggg atca:g:aac :cgccttga: cgttgggaac ccaaacgacg acac cacgatgcc: gcagcaatgg ttaactggcg aac:ac:tac :ctagcttcc cggcaacaat gataaagttg caggaccact :ctgcgctcg cttccgg ctg aaatctggag ccgg:gagcg :gggtctcgc g cag LQ aagccctccc gta:cg:agt :atctacacg gggagtc agg :gaacg aatagacaga tcgc:gagat aggtgcctca ‘attaagc attggt SDIIII I0 :ttactcat ata:ac:tta gattgattta catt tttaat aaggatcta :gaagatcc ttt::gataa gacc aaaatccctt aacgtg II ttcgttcca fiIO gagcgtcag accccg:aga aaagatcaaa ggatcttctt gag II ttt:ctgcg :aatctgct gct:gcaaac aaaaaaacca ccgctaccag C99 tttgccgga QIO aagagctac caac:c:ttt :ccgaaggta actggcttca gcagagcg SDLQ gataccaaa mctgtccttc tag:gtagcc gtagttaggc caccacttca agaactct OSDII agcaccgcc mcatacctcg ctc:gc:aat cctgttacca gtggctgctg gc taagtcgtc Q I :taccgggt tggactcaag gtta ccggataagg cgcagcgg gggctg$1) $1) 0 cagcttggag cgaacgacct I acaccgaa I 0 0 F’F QIO ggggttcgt ccacacagcc agc aaag cgccacgctt cccgaaggga gaaaggcg SD ta 0 :aagcggca gg:cggaac aggagagcgc acgagggagc gg 0 OF’F F‘FO fi'fiwo at :ttata tcctg:cgg gtttcgccac ctctgacttg agcgtcga 0 m cagggg gcggagcc: atggaaaaac gccagcaacg cggccttt 051) OF’F mono Io 0 0 :ttgct gcctt:t:g aagctgtccc tgatggtcgt ccIIIIIIgLQOIILQLQOIIIIIISDOSDLQI‘II‘II‘I m gca: g ccggaagcga gaagaatca ctcgcg SEQ ID NO:24 tgcg ttatcccc accgcc agtgagctga (pDONR22l taccgctcgc cgcagccgaa QtQaQCQaQQ aaQCQQ vector) gcgcccaata cgcaaaccg attc gcagctggca cgacagg:tt cccgactgg rmoormosn Loomsnou IOIOI0 gcaa SD cgcgtaccgc tagccaggaa gagtttg:a tcag :tctgctta gtttgatgcc tggcagt’I 051) @ IIm accc t:ca aatccgc:c QQaQ $110 gttgcttcccgacaaa caacaga:aa aacgaaagg ttcgIISDIILQIISD :gatgcctg gcagttccct actctcgcg tccca SD cgttgtaa aacgacggcc agtcttaag ttat: LOatagtgac atgcc ctgttcg:tg caacaca’I gtacaaaaa agctgaacga :caatata ttaaaIISDIILQIILQOSDIIII gaaacgtaa taa aaaacagact acataata0 0 $1) $1) 0 ("I 51) 0 ("I ("I agatggtatt agtgacc:g agtcgaccga cagccttcca aatg::ct:c gct gccaact:ag tcgaccga0 gccttccaaa tctc aaacggaa’ :cgtatcca gcctactcgc tattgtcct aatgccgtat taaatcataa aaagaaataa aaaaagagg tgcgagcctc ttttttg:gI‘fOSlJI‘II‘ISDLQOI‘IOOSDLQSDO gtaaaacaca acatatccag tcac:a:gaa gacaaaataa aaacatctac ctat:catat SDLQLQLQI‘II‘II‘II‘ILQI‘IOOO cgctagtgt catagtcctg aaaatca:ct gcatcaagaa caatttcaca actc::atac F’F :ttctctta caagtcg:tc ggcttca:ct ggattttcag cc:ctatact tactaaacgt gataaagttt ctgtaat:tc tactgta:cg agac tggctgtgta taagggagcc tgacatttat attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg gatcaccatc cgtcgcccgg caat aatatcactc tgtacatcca caaacagacg ataacggctc tctcttttat aggtg:aaac cttaaactgc att:caccag cccctgt:ct cgtcagcaaa gttc atttcaa:aa accgggcgac ctcagcca:c ccttcctgat :ttccgcttt ccagcgttcg gcacgcagac gacgggcttc attctgca:g :ta ccagaccgga gatattgaca tcata:a:gc cttgagcaac :agctgt gctgtcaac :gtcactgta atacgctgct tcatagcata cctctt:ttg :acttcg gtatacata :cagtatata ttcttatacc gcaaaaa:ca gcgcgcaaa: acgcatac:g :atctggct :ttagtaagc cacg cggcg:t:ac gccccgccc: gccactca:c cagtac:g :gtaattcat taagcattct gccgaca:gg aagcca:cac agacggca:g AOQJAOFT’AOOAO :gaacc:g atcgccagcg gcatcagcac cttgtcgcct :gcgta:aa: at::gcccat gtgaaaa 0 Aosnrr ggggcgaaga agttgtccat attggccacg :taaa:caa aac:ggtgaa AOOfiAOQJAOQJ ctcaccc c:ca :aaaccct: tagggaaa:a gccagg fig) AOfiAO ggattggctg agacgaaaaa :caccgtaac acgccacatc ttgcgaa:at :g:gtagaa ac:gccggaa :cgtcg A0 :attcactcc agagcgatga aaacg:::ca ca: ggaaaacggt g A0 A0 :gaacactat ccca:atcac cagctcaccg ct:tca:tg cca:acggaa :ccgga’A A gcattcatca cgggcaag aatgtgaata aggccg : aaaacttg:g :tattt AA :ttacggtct aaaaaggc a:cc cgg tc:ggtta:a gtacat:g gcaactgact aa:gcc:c aaaatg :ct :acga at:ggga:at :caacggt gtatatccag at:ttt:t ctcca: :ta ct:cc -ag ctcc:gaaaa ctcgataaAOAOOAOQJOQJ :caaaaaata cccggtag tga:c: ca:ta : gaaagttgga 0 0 F‘F 0 F‘F F‘F $1) 0 :gccgatcaa tc:cat:t tcgccaa :ggcccagg gc:tcccggt :caacagg :tat:c g_ga ct:ccg:ca :at’:a :cggcgca :gctgc caact:agtc gg: cc :ctaagta ctggat atg:tg:gtt :acagtat: a :g SDLQLQLQSDOOF’FSDSDFASDFASDSDF‘FSD :ttttatg "Hmong; :ata:t gatat::ata HHLQHSDSDHLQSDSDH ca::ttacg t :c gctttctt :ataag aaagca::gc :t: g ga caggtcac $1) 511 WV 0 511 WV :at:tgccat ccagctgata t 0 ag gagtcgta ctggcagctc :ggcccg:g: c ct :gatg:tac atcatca:ga aaac t :a ataaacag: cca:at:caa cgggaaacg: c :taaa:tcc aacatgg :aaa :gggctcgcg a caatcagg: gcgacaa :a :gggaagccc gatgcgccag a $1) $1) $1) 0 $1) :ggc aaaggtagcg ‘ccaatga :gt:acagat gaga:ggtca g ctgacggaa tttatgcctc :ccgaccat caagca:ttt atccgtactc c :ggttactc gcga ccccggaaa aacagcattc cagg:attag a tgattcagg: gaaaata:tg :gatgcgct ggcagtgttc ctgcgccggt tcctgt:tg: aattgtcctt :aacagcga :cgcgtattt cg:c:cgctc a gaa: aacggtt:gg gag :gattt:gat gacgagcgta a tgttgaacaa gtctggaaag SDatgcataa ac:tttgcca ttctcaccgg a cactca:gg[A gatttctcac Agataacct :a:ttt:gac gaggggaaat tattga:gt[A ggacgag:cg aga ccag gcca ctgcctcgg H gagttttctc :tcattaca gaaacggctt tt:caaaaat taatcc:ga H atgaataaat :t:gatgctc tttt attggt:aa H tggttgtaac ‘cagag ca:tacgctg ac:tgacggg ctcatgacca aaatccc:ta 0fifiOOWmfioflfimfifififififimfiflmfimflfiflflfi OOAQ LQF’FO (*ka 000‘tttca ‘agtta cgcgtcgttc cactgagcgt agaaaagatc aaaggatctt tcc :t:ttttctg cgcgtaatct aacaaaaaaa ccaccgc:ac agcggtggt ccg ga:caagagc ttttccgaag ggct cagcagagc c aa:actgttc gccgtagtta cact caagaactc :g cc:acatacc aatcctgtta ccagtggctg :gccagtgg cg tg:cttaccg aagacgatag ttaccggata aggcgcagcg gt acggggggt: cttg gagcgaacga cctacaccga ac ctacagcgtg aagcgccacg cttcccgaag ggagaaaggc 9g ccggtaagcg aacaggagag cgcacgaggg agcttccagg 9g tggtatc:t: tcgc cacctctgac ttgagcgtcg at tgctcgtcag cctatggaaa aacgccagca acgcggcctt tt ctggcct:t: tgctcacatg t SEQ ID NO:25 aaaagg ccttttaaat taaaaatgaa g tc aatctaaag: (psPAX2 atatatgag aaact:ggtc tgacagttac caatgcttaa tcagtgaggc acctatc:ca plasmid) gcgatctgt tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg gag gcttaccatc tggccccagt atga gaga cccacgc:ca ccggctcca attta:cagc aataaaccag ccagccggaa cggccgagcg cagaagtgg: cctgcaact tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaag: agttcgcca ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtg:ca tcg ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccc tgttg:gcaa aaaagcggtt agctccttcg gtcctccgat caga agtaagttg gtcatgccaHLQSDHLQHLQLQOHH tcacc:agat ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttac: ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattc:ga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaact:taaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgt gagatccagt tcga:gtaac ccactcgtgc ctga tcttcagcat cttttactt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ‘cgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtc:catga gcggatacat atttgaatgt atttagaaaa ataaacaaa: AgggttCCQ cgcacatttc cccgaaaagt tggt cgacattgat tattgactag ttattaatag taatcaatta cggggtca:t tagc ccatatatgg agttccgcg: tacataactt acgg:aaatg gcccgcct ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatac gccaataggg actttccatt gacgtcaatg ggtggactat ttacggtaaa acat caagtgtatc atatgccaag tacgccccct attgacgtca atggcccgcc tggcattatg cccagtaca: gaccttatg gact:tccta catctacgta ttagtcatcg ctattacca: gagccccacg 0 51) ccat ctcccccccc tccccacccc (—AA0 ttta:ttatt F’F tttgtgcag catgggcccg 999 FALQFASDOOOLQLQLQLQLQFAFAOLQLQLQ A0 A0 caQQCQQQQC m 0 gagggQCgQ ccgegeccag C9 A0 AA SD A0 AA@ ttt ccaa:cagag 0 tg A0 A0 A00A0 OAOOOAOOQJFT’AO ccta:aaaaa 0 0 CQQCQQQCQ A0 agtcgctgc A0 A0 cgctccgcgc A0 cgcccg 0 0 0 A0 gctctgact 0 A0 fir? 51) SD tgagcg Q0 0A 0 N'OAO A A r’r rho ctgtaatta 0 (—A A0 A A0 A0 tcgt A 0 r’r A0 taaag A0 A0 SD (—AA0A0 F’FA0 0 F’F A0 0 ggg (*0 r’r A0 tgtg A0 A0 $1) 0 0 OLQLQOO A0 0A0 ccg 000000000000 A0A0AASD (—AA0 0 (—AA0 A0 cgcg A0 A0 0 r‘r AA A0 tgtg 0 A0 A0 A0 cc 0 (—A 0 A0 A0 LQ A0 A0 (—AA0 aaca A0 A0 A0 A0 C Q A0 0 51) A0 A0 A0 Q (—A [AAO (—AA0 0A0 51) 0 gtcc 0 (—A 0 0 0 0 A0 0 (—A A0 51) LG A0 (—AA0A0A0 (—AoA00A0 0A0A0 999 A0 A0‘A‘AAO0A0[AAO 0 A0 A0 A0 A0 (—AA0 A0 FA A0 0 0Q A0 A0 A0 (—AA0 L0 A0 0 A0 A0 A0 0 LO 0 A0 A0 A0 $1) L0 A0 A0 AA A0 51) A0 A0 A0 0 A0 0 0 A0 0 0 (—AA0 00 A0 A0 0 0 (—A (—A F’r SD AA A0 51; AA@ A0 0 0 A0 51; A0 A0 0 A0 FALQO A0 A0 A0 SD A0 A—AA0 oA—AA0A0 0 0 0 0 F‘F F‘F 0 (*0 00 0 $1) 0 AA 0 (—AA0 A0 0 s1; 0 m 499 AggCQ ggg OOSDSDSDLQLQLQLQOSDOOF’F AA 0 A0 OOLQSDSDFAOOOLQLQLQLQOLQOLQOLQO :gttcatgcc ttc AA 0 51; 0 :catcatttt ggcaaag AA 0 0 AOAOFT’AO A0 thaQaQ ccggcgactg gatgggaaaa aat:cgg LQAQOA‘AA‘AOAOOAOQm :atgggcaag cagggag gtag acaaatactg tcagacagga :tagatcatt ata:aataca gcatcaaagg aag acaccaa aagcttta aaacaaaagt cacagcaag cagctgac A aaat :gcagaaca ccaggggcaa OHSD acctagaact gggtaaaag agtagaagag AA gatacccatg agccacccca OLQSDLQOLQF‘FLQO AA aaacacagtg gtta agctgcagaa gcatgcaggg AASD gagagaacca aactactagt LQ atggatgaca aggagaaatc gggattaaat ccctaccagc ggaa ccgattctat agcttcacaa cttg agattgtaag accaggagcg agcatgtcag A0 gacccg taaagcaaga ccaagtaaca AOrAAosnsnvosnrAsn ccataa A9; acagaaaggc accaaagaaa gactgttaag gtggcaaagWWWLQOQJLQF’FLQF’FF’FLQF’FF’FO agggcacata cccc taggaaaaag aatgtggaaa ggaaggacac attgtactga gagacaggct sn ggaaga:ctg gccttcccac cagggaattt tcttcagagc caacagcccc accagaagag ttggggaaga gacaacaact agcc gatagacaag ctttagcttc cctcagatca gcgacccctc gtcacaataa gcaattaaag gaagctctat agcaga:gat acagtattag SD tttgccagga agatggaaac agggggaatt ggaggtttta acagtatgat cagatactca cggaca:aaa gctataggta aggacctaca cctgtcaaca 51: 51:00 aaatctgttg attg FT’AOAO AOFA’FT’AOQ) aaattttccc attagtccta ttgagactg accagtaaaa ttaaagccag $1) cccaaaagtt aaacaatggc cattgacag agaaaaaata aaagcattag tacagaaatg gaaaaggaag gaaaaatttc aaaaat:ggg cctgaaaatc ata tccagtattt gccataaaga aaaaagacag tactaaatgg agaaaattag tca aagatttctg ttaggaatac ctg0A00A0 agaacttaat aagagaactc ggaagt:caa agggttaaaa cagaaaaaat cagtaacagt ac:ggatgtg ggcgatgcat atttttcag tcccttagat aaagacttca ggaagtatac tgcatttacc atacctagta atg gacaccaggg attagatatc agtacaatgt gc:tccacag ggatggaaag cag aatattccag tgtagcatg caaaaatctt agagcctttt agaaaacaaa atccagaca agtcatctat caatacatg atgatttgta tg:aggatct gacttagaaa agc tagaacaaaa atagagg$1) 51) :gagacaaca tc:gttgagg tggggattta ccacaccag caaaaaacat cagaaagfig) $1151) ctccattcct gggt tatgaactcc atcctgata atggacagta cagcc:a :gctgccag aaaggacagc tggactgtca atgacatac gaaattagtg t Ff A0 attgggcaa tcagatttat cagggatta aagtaaggc attatgtaaa cttct:a mmmoomm aag taacagaag agcagagcta ac:aacagaa tagtaccac gaactgg 00 mm aaaacaggg ga:tctaaaa aaccggtac atggagtgt ttatgaccca tcaaaag$1) snrmoo ‘orrrrguoormo :aatagcag aa:acagaag caggggcaag gccaatgga atatcaaatt SD catttaaaaOLQF’FFfSDF’FOF‘FSDOSDOLQSDOSDF‘FSDSDSDOLQSD tc:gaaaaca tatg caagaatga ccac SDLQSD :gaaacaat aacagaggca gtacaaaaaa tagccacag aagcatagta agactccta at:taaatta cccatacaaa aggaaacat a:gg ‘0 attggcaag cacctccatt cctgagtggg agtttgtca tacccctccc SD ‘0 :atggtacc gt:agacaaa gaacccataa taggagcag aactttc:a: cagccaata aa ttaggaaaag caggatatg caga O‘OSDSDF‘FSD‘OSD aagttgtcc :aacc gac acaacaaatc agaagactg gttacaagca ctttgcagg gaagtaaaca tagtgacag ctcacaa:a: :cattcaag aagagtgaat cagagttag cagtcaaata :aataaaaa tacctggcat gggtaccag agga aag gtcagtgctg gaatcagga agtac:a:t: :agataagg catgagaaat gta ttggagagca attttaacc tagcaaaag aaatagtag cagctgtga: :aaaagggg :ag actgtagcc aggaa:a:gg gtacacatt aaaa ttatct:gg tagcagtt0 tgtagccag: aagcagaag cagca agacagggc aagaaa atact:cc caggaag 511 WV ‘taaaa acagtacata cagaca 5110 fig) m cagcaat: cagttaagg 4ttgg tgggcgggga tcaagc atttggca cccaaagtca taata caatcta:ga ataaag$1151) aaagaaaa ‘atca aacat cttaagacag cagtac a::c aaa ggatt gggggtaca gtccagcfiLQQJQJLQ m‘omfimm aagaa:ag:a caacagaca: acaaactaaa aattacaaa aacaaat aaaaa::caa tttattacag acagcaga atccag:tt ggaaag m $1) 0 agcaaagc:c gtgaag‘ggc taata caaga:aata gtcacaF? $1) $1) agtag:gcca caaagatca’ attat 9 gaaaacaga tgccag K) F’F tgattg:g:g ‘caagtagac aggatg ttaacacatg 9 aattctgca m 0 rho ca:t tcagaattgg gtgtcg gaatag gcgttac:cg acagag ‘0 SD m gcaagaaa:g agccagtag ‘ccctgg aagca:ccag gaagtc mg; SDF’FLQ mrrofifiOfiOSDLQSD$1)0051)fifiQJLQ0$1)0051)SDOF’FSDSDF’FSDSDLQSDSDOSDSDSDSDSDSDSDF’FOSDF’F taaaac:gct ‘taccaatt :gttgc tttca:tgcc aagttt cct:aggca aagaag cggagacagc gacgaag tcagactc :a:caaag cagtaag:ag tacatg SD :agcaa :agtagca ataataa:ag :catag :ggccgc: gatct:caga acaattgg :a:a:aaata :aaag:ag:a cacccacc aaggcaaaga gaagag:gg_ gcagagagaa cttggg agcagc aggaagcact gacggta caggcc :gtc :ggta:ag:g LOgctatt gaggcg gcaac:caca 0 $1) gcaaga atccta cctaaaggat sum :gctg:gcct 51) :ctctg gaacag gacctgga:g SD I :acaca 51) LG 0 F’F F’F $1) :gaagaatcg caaaaccagc I :gctct ggaaaaLQSDSDOLQOSDF‘F SD caagaa ttattg 19 tggaattggt $1) ::ggctg tggtata :ag:a ggaggcttgg <1an rrmrrsn LO 511 OLQfiLQLQfifiWWWWLQWLQfiO ::tgttc agttttt gctgtac :tagg cagggatatt :cagacc cacctcccaa acccgacagg cccgaaggaa :ggagag agagacagag acagatcca ag:g aacggatcct ggacgat ctgcggagcc :ct: cagctaccac cgcttgagag :gtaacg aggattg:gg :ggg acgcaggggg tgggaagccc gaatctc ctacaatatt ggagtcagga gctaaagaat agtgctgtta ‘ cacagcc atagcag:ag ggac agataggg:t gtag gagc ttg:agagct attcgccaca :acctagaag aataagacag ggcttggaaa ggattt:gct ataagctcga aacaaccggt agaa ctatagctag cagatctttt :ccctc:gcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta gaaa ttca ttgcaatagt gtgttggaat tttttgtg:c tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat ttgg tttagagttt atat gccatatgct ggctgccatg aacaaaggtg gctataaaga ggtcatcagt atatgaaaca gccccctgct g:ccattcct :attccatag aaaagccttg acttgaggtt agat:tttt tatattttgt ::tgtgt:at :tttttcttt aacatcccta aaatt:tcct taca:gttt actagccaga ::tttcc:cc :ctcctgact actcccagtc atag :gtcc ctct:ctct a:gaagatcc c:cgacc:gc agcccaagct tggcgtaatc ctgt:tcctg at:g ::atccgctc acaattccac acaacatacg ataaagtgta aagcctgggg :gcctaa:ga gtgagctaac tcacattaat tcac:gcccg c:ttccag:c gggaaacctg :cgtgccagc ggatccgcat cagcaacca: agtcccgccc c:aactccgc ccatcccgcc cctaactccg ccca:tctcc gccccatggc :gactaa:tt :ttttattta tgcagaggcc cggcctctga gctattcc aagtag:gag gaggcttttt tggaggccta aaaagctaac t cagctta:aa :ggttacaaa taaagcaata tttcacaaa: a ::tcac:gca :tctagttgt tcca tgta:ctta: c :ccgctgcat :aatgaatcg gccaacgcgc ggtt:gcgta t ::ccgc::cc :cgctcactg actcgctgcg cggc:gcggc g agctcac:ca aaggcggtaa tacggttatc ggggataacg c catgtgagca aaaggccagc aaaaggccag aaggccgcg: t ::tcca:agg ctccgccccc ctgacgagca cgacgctcaa g gcgaaacccg acaggactat aaagatacca cctggaagc: c c:ctcc:g ccgaccctgc cgc:taccgg gcctttctcc c cgtggcgc tctcaatgct cacgctgtag tcggtgtagg caagctgg tgtgtgcacg aaccccccgt cgctgcgcc: c:atcg:c gagtccaacc cgg:aagaca gcag taacagga agcagagcga ggtatgtagg gagttcttga taactacc tacactagaa ggacagtatt gctctgctga c:tcgg agagttggta gctcttgatc ‘gcaaacaa accaccgctg t:tttt tgcaagcagc aga:tacgcg cagaaaaaaa ggatctcaag gatctt acggggtctg acgctcagtg gaacgaaaac tcacgttaag catgag tca SEQ ID NO:26 ‘ cgggag attccggcac ctg:cctacg taaagaagac *VSV.G ‘ gcggcg tgcc ccgcgcccac tgactgggtt plasmid) aagggcat gtcgatgcag gaaaaggaca ‘ attcacgccc tggcggca tgcaaaggat ccca gtatcata ‘ctgactgt ata:gcatga gcatagcata tgc:acccgg atagcata tactacccag at:a atagcata tgc:acccag atagccta tgctacccag ata:aaat:a atagcata tac:acccag atagcata tgctacccag ata:agat:a atagccta ccag ta tgctacccag ata:agat:a atagcata ccag m tatatgct acccaga:a: ggat cata:act accctaatct u m catatgct acccgga:ac aga:taggat ct acccagatat m 51) m ct a :a H aga:taggat ct acccagatat $1) $1) m catatact acccaga :a H ggat cata:gct acccagatat to $1) H ‘cctatgct acccaga:a H aga:taggat cata:gct :ccagatat m ‘0 SD tatgctaccc atggcaaca H tagcccaccg ctctcagc $1) 0 0 fl 0 LG fl LO 511 H m 0 caacaaccct gtgcttggcg ctcaggcgca ‘tgtg:gta 51) m caatcgcgcc cctatct:gg cccgcccacc tactta:gca fiWfifiQJQJgOOOLQSDLQSD 0 51) LG rt LO LO 51) LG:ttgtcctcgtattcccc tttt gtgggcaag: ggt:tgaccg ::ag ggggttaca "m m t:ac agtccaaaac cgcagggcgg gtgtggggg LQLQ ttattacacccccccactcc aaaaaagagt gccac::gt :ttgtttat x A0 A0 I ggcgtggagc gggt ttagagaca 0 Q tcggcgtcca cct:gt:aca aatagag:gt acaacatgg fififififiifiifiifiififiifiifiifii HQ m 0 ‘ ttggtccctg ccccag:atc act gttgcccata gacatccag ctttacggc "‘0 0x cccatggatt aatgt::ca :cctacact fi‘o gcccaagggg tcatagcac atgccacca "‘0 ‘0 cgtccaaatt :gt :tcgagcac H 0 ccttactgtt cacaactcag attagc: cgaaggagaa ‘0 51) ggcgaagatt caggagagtt tccttga:ct tcagccactg 0 0 taaaatggtt cac:accctc cacccca:gt aaataaaacc atggggtggg aga:atcgct accctt::ac taaccctaat cccg ttgggtaaca attg ggtt agtctggata LQWLQOWOOQJWQJWWLQOQJLQOWF’FQJQJQJOQJQJQJQJQJO n 0 ctacccg ga agcatatgct acccgt:tag ggttaacaag ggggccttat gctaatgccc tct:gagggt atcg gtagctacac aggcccctct ggtgtagcct cccgtagtct tcctgggccc ggta catgtccccc taagagcttc agccaagagt tacaca:aaa ggcaatgttg tgttgcagtc aaagtctg ccaggatgaa agccac:caa gggatcttca atattggcca tta:atagca taaatcaata ttggctattg gccattgcat tatgtacatt ggct catgtccaat atgaccgcca tgttggcatt gattattg tag:tattaa tagtaa:caa ttacggggtc attagttcat agcccatata tggagttccg cgt:acataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg :cccatagt aacgccaata gggactt:cc attgacg:ca SDHOHSDOAASDLQSDOOSDSD LG 51):gggtggag tatttacggt aactgccca cttggcagta g:gt atcatatgcc agtccgccc cctat:gacg caatgacgg taaatggccc gcctggcatt atgcccagta SD51) :gacctta cgggactttc :acttggca gtacatctac gtattag:ca tcgctat:ac :ggtgatg cggtt:tggc gtacaccaa tgggcg:gga tagcggt:tg ggg A :tccaagt ctccacccca :gacgtcaa tgggag:ttg ttttggcacc aac c:ttcca aaatg:cgta :aaccccgc cccgttgacg caaatgggcg gtaggcg:g ogg:gggag gtcta:ataa cagagc:cg tttagtgaac cgtcaga:ca ctagaagct SD :tgcggta gttta:caca rsnorrsnrr :taaat:gc taacgcagtc agtgcttctg acacaacag aactta agctgcagaa :tggtcgtg A IA 0 LG A0 agg caggtaagta ’ A 51) 51) LO 51) caggtt taaggagacc atagaaact agacagagaa gactc: Q 0 [A [A 0 :gatag gcacc:attg :c:tac:ga gcctttc:ct ccacag [A A0 0 $11 0 :cccag ttcaa:taca ctcttaagg taatacgact cacta: Q A0 51) Q 0ggtacc gagctcgga: 00AOA0§1J cactag:aa ‘ tg:gctggaa ttcaac LG 51) IA c atctgt ttcct:gacaLQ :a:gaagtg ttagcc::tt tattca cagaccHSDHSDLQAASDAASDSDLQMA [A A0 gg:gaattgc aagttcacca t:cc aaaggaaact ggaaaa AA A0 :cct:ctaat tacca:tat: ccccgtcaag aa:tggcata atgac: aggcacagcc atacaagtca aaa:gcccaa gc:attcaag "SD F‘FSD ga:g:gtcat aaa: ggg:cac:ac ‘tga cgctgg:atg gaccga LQ rt acag cga: cct:cac:cc agaa caatgcaagg aaagca [A A0 acaaacgaaa caaggaact: cgc:gaa:cc aggcttccc: cc:caaagtt ctgga: [A A0 aactgtgacg gatgccgaag cag:gat:gt ccaggtgac: cc:caccatg tgctgc AA A0 WOWQJLQWWLQLQOF’FLQOF’FF’FF’FLQLQ :gaa:acaca ggagaatggg ttgattcaca gttcatcaac ggaaaa:gca gcaat:acat atgccccac: tccataac: ctacaacctg gcattctgac ta:aaggtca aagggc :gat:ctaac ctcatttcca tggacatcac ctca gaggacggag agcta cc:gggaaag gagggcacag :cagaag taactactt: gc:tatgaaa :ggag ggcc:gcaaa atgcaatac: gggagtcaga ctccca:cag :gtc gataaggatc attc cc:gaa:gcc SD 0 gctccatctc gga:gtaag: ctaattcagg tattccc:c: ctggagcaaa gcgg gatctcagc: taaaaaccca ggaaccggtc ggtaccc:aa gaccagatac atcagag:cg tcaag :gg :ggaac: accacagaaa :ggaccc aatggag:tc AOQJOAOQJOAOO LO 0 c gaggaccag gacatgg: atg:tggact tca :cacat: gctg :tcgcaact _ :gggcta tccaaaaatc SD SD r’r 0 o‘agct :agaagg: :attgcc tct:ttt:ct :atcatagg :aatcat: :ggtatc tgca :aaattaaa gcacaccaag ‘ agatg aaccgac:tg gaaagtaact caaatcctgc ’ ccaaatc t accatgctca aagaggcctc :tatg a tcac ac:ggcgccc A OF‘FSDOLQLQLQSDSDLQOF‘FSD cctaaatgct agagctcgct cccc :cccccgtgc aaat gaggaaattg :ggggtgggg caggacagca :gcggtgggc :c:atg ctt aagaaccagc cgcgcgggga ttt cgctct:ccg A ‘ ctcgg ct gtatcagctc ‘ ggtaatacg cacag ga aagaacatgt ccagcaaaa aacc gc gcgttt:tcc cccccctga tcaca OWQJWWOLQOLQOLQF’FQJO aggtggcgaa actataaag gcgt 51) Q 0 gtgcgc:ctc gc A accggatacc 0 0 ggaagcgtgg atgctcacg :gtaggtatc cgctccaagc gcacgaaccc cccgttcagc ggtaac:atc caacccgg:a agacacgact actggtaaca gtat gtaggcggtg ‘ aact tagaaggaca gtatttggta ‘aagcca gttacc:tcg tggtagctct tgatccggca LO 0 FA LO LO FA 51) Q 0 ggtggt:ttt gcagcaga:t acgcgcagaa caagaagat cctttgatct gtctgacgct cagtggaacg :aagggatt ttggtcatga aaggatct:c acctagatcc aaatgaagt tttaaa:caa atatgagtaa acttggtctg :gcttaatc agtgaggcac tc:a tttcgttcat ‘ :gactcccc gtcgtg:aga acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc ggctccagat ttatcagcaa :aaaccagcc agccggaagg gccgagcgca tgcaactt:a tccgcctcca :ccagtctat taattgttgc cgggaagcta ttcgccag:t aatagtttgc gcaacgttgt tgccattgct acaggcatcg cacg ctcgtcgttt ggtatggctt cattcagctc ccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa ttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga tatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc ctta ccgctgttga gttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg ggaa atgttgaata ctcatactct tcctttttca atattattga tatc agggttattg tctcatgagc ggatacatat gtat ttagaaaaat aaacaaatag cgcg cacatttccc cgaaaagtgc cacctgac 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, NJ, USA), with results shown in . aMOLM-14 aAPCs were y-irradiated at 100 Gy and frozen.
Exam le 4 —Ex ansion of Tumor Inflltratin L m hoc tes Usin MOLM-14 Artificial Anti en 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, ered 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 , , , and , 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 , 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 bed in detail in e 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 TlLs is through TCR activation (specific killing) or non-specific killing. Following 4 hours of incubation, luciferin was added to the wells and ted for 5 minutes. After the tion, bioluminescence ity was read using a luminometer. The percentage cytotoxicity and percentage survival were calculated using the following formula: % Survival = imental survival-minimum)/ (maximum signal-minimum signal) X 100 or % Cytotoxicity = 100 - (% Survival).
In , 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 X 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, 4 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 med 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 1L-2 on Day 4 and 7. Viable cells were counted on Day 7. shows the mean plus standard deviation (SD) of viable cell s counted on Day 11, with a e calculated by the t Z-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 (T1L:aMOLM14) with OKT-3 and IL-2 yields a similar expansion when ed to PBMC feeders with OKT-3 and IL-2 (p = 0.0598).
In , the results of expansions performed with higher ratios of TILs to MOLM- 14 aAPCs, and otherwise performed as bed above for , 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 1L-2. These results were verified using different TIL batches in repeat experiments shown in and . In ular, as seen in , 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 TILzaMOLM14 ratios of 1:200 to 1:300.
In and , TILs expanded with aMOLM14 or PBMC were compared by flow cytometry analysis to confirm that the TILs exhibited a r 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 oc/B PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28PE, CD27 APC-C7, and erCP-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 ter (Becton, Dickinson, and Co., Franklin Lakes, NJ, USA). Data was analyzed by Cytobank re 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 4 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 MOLMl4. There is no difference in the expression of CD28, CD57, and CD27 differentiation markers. onal flow cytometry data is shown in , and depicts a flow cytometry contour plot showing a memory subset (CD45RA+/—, CCR7+/—) gated on Live, TCR d/B +, CD4+ or CD8+ TILs, indicating that the memory subset ed 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 and . The color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIL3, PD1 and CD137 or CD69, CD154, KLRGl 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 4 aAPCs effectively replicate the TIL phenotypic results ed with PBMC feeders.
TILs expanded against aMOLM14 or PBMC were also analyzed for metabolic proflles.
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TILs after expansion with ated PBMC feeders or 4 aAPCs were measured using a dual ondrial-glycolytic stress test. Briefly, cells were washed in assay medium (XF Assay Medium, Agilent Technologies, Santa Clara, CA, USA), supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, at pH 7.4, and then 1 X 105 viable cells were plated onto an adhesive-coated (Cell-TakTM, Corning) XFp cell culture microplate. Plates were spun to adhere the cells to the plate, then equilibrated at 37 0C in a humidified, non-C02 incubator prior to analysis of cellular metabolism. Mitochondrial and glycolytic stress test experiments were performed using a Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, CA, USA), sequentially injecting the following compounds at specified intervals for aneous analysis of mitochondrial and glycolytic ation of the cells: 1 uM oligomycin, 0.5 uM FCCP, 50 mM 2-deoxyglucose, and 0.5 uM each of ne and antimycin A. Results were analyzed using WAVE v2.3.0 software (Agilent Technologies, Santa Clara, CA, USA) and GraphPad Prism V6.07 graphing software and are shown in and , where points represent mean :: SEM measured in triplicate. Both TILs grown with aMOLMl4 aAPCs and PBMC feeders show similar oxphos and glycolysis behavior. This data ts that aMOLMl4 does not alter the metabolic programming of TILs when compared with PBMC feeders.
Example 5 — Preparation of EM-3 Artificial Antigen Presenting Cells gaEM3 aAPCs) EM-3 cells were obtained from Creative Bioarray, Inc. (Shirley, NY, USA). To develop an EM-3 based ial APC, EM-3 cell lines were engineered with CD86, 4-1BBL, and antibody t IgG Fc region (Clone 7C12 or Clone 8B3). Human CD86 and human 4- lBBL/CDl37 genes were cloned into commercially-available PLV43OG and co-transfected with PDONR221 vectors (Invitrogen) using a iral transduction method. The gateway cloning method was used as described in Katzen, Expert Opin. Drug Disc. 2007, 4, 57 1—5 89, to clone hCD86 and hCDl37L genes onto the G 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, CA, USA) using APC-conjugated CD86 and PE- conjugated CDl37L to isolate and enrich the cells. The ed cells were checked for purity by flow cytometry. Single-chain Fv (scFv) antibody clones ated 7Cl2 and 8B3 were generated against Fc of mouse IgGl, IgG2a and IgG2b (Viva Biotech Ltd., Chicago, IL, USA).
The amino acid sequences of these scFv clones are given in Table 7 (SEQ ID N027 and SEQ ID NO:28). The generated scFv clones were ed for Fc binding efficiency against OKT-3, engineered towards pLV430lG containing eGFP as co-reporter to produce lentivirus. The 293T cell line was used for ing and lentiviral production. Engineered EM-3 (CD86/CD137L) cells were transduced using the lentiviral system and sorted using eGFP.
WO 81789 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. (geiiliigtfdn) Sequence (One-Letter Amino Acid Symbols) SEQ ID NO:27 QVQLVQSGGG LVKPGGSLRL SCAASGFNFN DQYMSWIRQA PGKGLEWVSF ISGSGGTTYY 60 (mFC77Cl2 TDSVKGRFTI DSLY LQMNSLTVED TAVYYCARGG GRGT LVTVSAGGGG 120 SCFV) SGAPDIQMTQ SPGTLSLSPG ERAILSCRAS QSVSGYLAWY QQKPGQAPRL LIYGASSRAT 180 GIPDRFSGSG SGTDFTLTIS IGTY YCKQYINAPF TFGGGTKVEI K 231 SEQ ID NO:28 QVQLQQSGAE VKKPGSSVKV GTFS SYAISWVRQA WMGW ISPYNGNTDY (mFC*8B3 SCFV) AQKVQGRVTL TTDTSTSTAY RSDD ATGG GTWYSDLWGR GTLVTVSAGG GGSGGGGSGG GGSGAPEIVL TQSPSTLSAS ITCR ASQSIGGSLA WYQQKPGKAP KLLISEASTL RFSG SGSGTDFTLT ISSLQPEDVA TYYCQKYNSV PLTFGPGTKV A non-limiting protocol for preparation of aEM3 aAPCs, which may also be adapted for use with aMOLMl4 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 ug, DONR vector (e.g., pDONR222) 50-100 ug, BR Clonase 11 (Life Technologies) 1 uL, and TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q.s. to bring volume to 5 uL). Incubate at room temperature for at least 1 hour. After incubation perform bacterial transformation either by heat shock method or oporation. To generate destination vector, the ing cocktail may be used: recombined pDONR vector (e.g., pDON222-geneX) 50-100 ug, destination vector (e.g., Gateway adapted lentivector) 50- 100 ug, LR Clonase 11 (Life Technologies) 1 uL, and TE buffer ((1 mM Tris, 0.1 mM EDTA, pH 8.0, q. s. to bring volume to 5 uL). Incubate at room temperature for at least 1 hour. After incubation, perform bacterial ormation 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 nnox or YT) with antibiotic for selection. Ensure that Recovery Medium (supplied by Lucigen, Middleton, WI, USA) is readily available at room temperature. Optionally, sterile culture tubes may be chilled on ice (e.g., 17 mm X 100 mm tubes (14 mL tube)), one tube for each transformation reaction). Remove E. cloni cells (Lucigen) from an -80 oC freezer and thaw completely on wet ice (5-15 minutes). Optionally add 40 uL of E. cloni cells to the chilled culture tube. Add 1-4 uL ofDNA sample to the 40 uL 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 oC water bath for 45 seconds. Return the 1.7 mL tube or culture tubes to ice for 2 minutes. Add 350 uL room temperature Recovery Medium to the cells or 960 uL 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. te the plates overnight at 37 oC. Transformed clones can be r grown in any rich culture medium (e.g., LB or TB).
Colonies for Miniprep (Qiagen, Inc., ia, CA, USA) may be grown as s.
After colonies have formed from plating recovered transformation reaction ofDNA 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 uL soft pipette tip (VWR 8903 1-3 52) or 10 uL 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 0C. After ght tion, 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 ds are screened and selected). m Miniprep according to the Qiagen Mini prep kit ol (Qiagen, Inc., Valencia, CA, 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 s. The following media composition is prepared: 500 mL DMEM/F12 (Sigma), 25 mL FBS Heat Inactivated (HI) (Hyclone), 10mM HEPES (Life Technologies), 1X Primocin (Invivogen), 1X Plasmocin (Invivogen), and 1X 2- mermactoethanol (Life Technologies). Harvest T75 flasks (Thermo Fisher Scientific) ning 90% confluent 293T cells. Aspirate media. Add 10 ml PBS, rinse gently and aspirate off. Add 2mL TrprE s (Life Technologies) and evenly bute it over the cell layer, let sit for 3-5 minutes at 37 oC (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 X 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 on). lly, a multiplication factor of 2.5 per day may be used where confluence is 2><105 cells/cmz. Bring volume up to 25 mL of media. To plate for titration of stocks, each well of the assay requires ><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 l.7><106 cells on the morning of transfection. (Seeding on the day of transfection may reduce the ion in ection ncy). Bring volume in flask up to mL with media. For e, 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 ug Baculo p35 plasmid (optional, only necessary if packaging a death gene), 2 ug VSVG env plasmid (e.g., pMD2.G or PCIGO VSV-G), 4.7 ug Gag/polymerase plasmid (e.g., psPAX2 or pCMV-deltaR8.9l), and 2.3 ug of the lentiviral vector described above. Determine the amount of VSV and R8.2/9.l (+/-Baculo) plasmids needed for all samples (make a mixture of these DNAs if preparing many samples). Each T75 transfection requires 90 uL LipofectAmine 2000 (Thermo Fisher Scientific) in 2 mL Opti-MEM medium o 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 uL room temperature Opti-MEM medium to a microtube and mix, and label as tube B. Add the 500 uL ofDNA from tube B to the 2 mL of the ctAmine 2000 mix in tube A and mix gently, and incubate for 20-30 minutes at room temperature. te 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. Supematants 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 , work with only 5 flasks at a time. Keep collected atants at 4 0C until pooling the next day. Cool supernatants again on the following day and pool as appropriate. Spin the supernatants at 2000 rpm for 5 s to sediment any contaminating 293T cells. Filter harvested supernatants through a 0.45 pm or 0.8 pm filter unit containing a pre-filter disc. Use a large enough filtration unit so that the ion speed is relatively fast. Store at 4 0C until ready to concentrate.
Virus may be concentrated using the PEG-it method (System Biosciences, Inc., Palo Alto, CA 94303) for longer-term storage at -80 oC. 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 0C for ght. fuge at 3500 rpm (1500 g) at 4 0C for 30 s. Remove supernatant and centrifuge at 3500 rpm at 4 0C for 5 minutes. Remove remaining supernatant. end virus in desired amount of phosphate-buffered saline (PBS) and freeze aliquots at -80 0C.
Transduction of cell line using lentivirus may be med as follows. Adjust cells to be transduced to either: l><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 l><107/mL and plate 100 uL 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/cm2 (e.g., for 293T cells, confluence = 2><105/cm2). Total volume of transduction per well should be approximately 500 uL with 3-10 ug/mL Polybrene (Hexadimethrine bromide, Sigma-Aldrich Co., St. Louis, MO, USA). The amount of concentrated virus added will depend on the M01 (multiplicity of infection) d. A typical M01 is 10:1 but this may vary depending on cell type. The transfection well should n 100 uL of standard media containing either l><106 suspension cells or 50% confluent cells. For a MOI of 10:1 (e.g., virus activity is l>< lO8 IU/mL and the target is to infect l>< 106 cells, then l>< lO7 virions or 100 uL of virus is needed). Add standard media to 500 uL. Add Polybrene to 3 ug/mL (primary cells) to ug/mL (tumor cell lines). Spin plate(s) at 1800 rpm for 1.5 to 2 hours at 30 oC. Incubate plate(s) at 37 oC/5% C02 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 s. Culture the cells in the media described above until the cell count reaches a minimum of 10-20 million. Take l>< 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 ng media (Invitrogen), to make the cell bank of the same cells.
Cells may be banked after each transduction and sorting procedure.
Nucleotide ce information for the 7Cl2 and 8B3 scFv clones (SEQ ID N029 and SEQ ID N030) and their lentiviral vectors are given in Table 8. Sequences used for generation of the pLV430lG 7Cl2 scFv mIgG hCD8 flag vector are provided as SED IQ NO:3l to SEQ ID N034 and are depicted in to . Sequences used for generation of the pLV430lG 8B3 scFv mIgG hCD8 flag vector are provided as SEQ ID NO:35 to SEQ ID N038 and are depicted in to .
TABLE 8. Nucleotide sequences for preparation of lentivirus for transduction of aAPCs.
Identifier Sequence Descri ntion SEQ ID NO:29 caggtgcagc tggtgcagtc tgggggaggc aagc ctggagggtc cctgagactc (mFC77Cl2 tcctgtgcag cctctggatt caatttcaat taca tgagttggat ggct scFv) ccagggaagg ggctggagtg ggtttcattc attagtggta gtggtggtac cacatactac acagactctg tgaagggccg gttcaccatc tccagggaca acaccaagga gtat ttgcaaatga acagcctgac ggac acggccgtgt actactgtgc gagaggaggg aattattata cttcggtggg ccggggcacc ctggtcaccg tctcggccgg tggcggcgga gcgc cagacatcca gatgacccag tctccaggca ccctgtcttt gtctccaggg gaaagagcca cctg cagggccagt gtta gcggctacct agcctggtat aaac ctggccaggc tcccaggctc ctcatctatg gtgcatccag cagggccact ggcatcccag acaggttcag tggcagtggg tctgggacag acttcactct caccatcagc agcctgcggc ctgaagatat tggaacatat tactgtaaac agtacattaa attc actttcggcg ccaa gatc aaa SEQ ID NO:3O caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctgggtcctc ggtc (mFC78B3 scFv) tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc cc:ggacaag ggcttgagtg gatggga:gg acaatggtaa aagg tccagggcag agtcacc:tg catccacgag atggagctga tgag atccgacgac attactgtgc QQQaCCZQQt actccgatct ctggggccgt tctc 99 gtgg gtccggtggc gcgcgccaga ‘ LO 0 Ft ac cctccaccct gtctgca:ct gagtcagcat 9c gtattggtgg ggcc aaaagccagg aa tctctgaggc gtctact:ta tcccatcaag a9 ggacagat:t cactctcacc c:ga ‘ Ft LO 0 c gtcaaaaa taacagtgtc tcggccc:gg LQSDOFtLQLQOO SEQ ID NO:3l g aattcgatag catatgc:tc acatatgcta ttgaa:tag (destination t atagtata:a ctactacccg gctacccgtt taggg’‘ WV 0 $1) vector 9 ggccacga:g cgtccggcgt atgtgag:ta cctcactca pLV430lG) taca ctttatgctt tgttgtg:gg aattg:gag ttcacacagg aaacagc:at acgccaagcg cccaa’‘ F’F $1) $1) gggaacaaaa gctggagctg gtagtct:at caat $1) 0 F’F 0 caacatgg:a acga:gagtt agcaacatgc cttacaagga agaaaaag ccgattgg:g gaag:aaggt ggtacgatcg tgcctta:ta gaaggcaa gacatgga:t ggacgaacca ctgaa:tgcc gcattgcaga atat:gta agctcgatac ataaacgggt ctctc:ggt agaccagatc tgagcctgg ctaactaggg aacccactgc c:tg ccttgagtg tgtgcccgt ctgt:gtgtg ctagaga:cc ccc tggaaaa:c tctagcagtg cgaac agggact:ga aagcgaaag agctctc:c gacgcaggac :tgc gaagcgcgca cggcaagagLQLQHOLQHOOHOOHOLQ cgactggtga gtacgccaaa -gac agcggaggct agaaggagag cacagcgtca gtat:aagcg aa: agatcgcgat aaa: ccagggggaa agaaaaaata -aaaa g:at ggcaagcag cgattcgcag ttaa:cctgg -agaa acatcagaag gaca cagctacaac catcccttca atca gaagaac:ta gatcat:ata caaccctct attg:gtgca gagataaaag agga ‘atag SD ‘0 aagagcaaaa accaccgcac agcaagcggc ‘acctQ rhoSDSDSDOFtOLQFtLQLQLQOSDF’FSDLQ ggaggagata ttggagaagt gaat:a:ata gaaccattag caccaaggca aagagaagag g m gcag:gggaa gttccttggg ggag g aggaag sum gcagcgtcaa FtrtSlJSlJSDSDLQSDLQF‘FO $1)$1) ggtacaggcc agacaa:tat :ggta: aatt tattgaggcg caacagcatc gcaac: LQLQ ggca:caagc aagaatcc:g gctg:ggaaa cctaaa ctcc:gggga \ I ctctggaaaa ctca:t:gca :gctg: gctagttg tctggaacag atttggaatc gacctg gacagagaaa cacaagct:a atacac:cct :gaaga cagcaagaaa agaattat:g gaat:agata ggcaag H51)" tggt:taaca gctgtggtat :tat aatga: ttgg:agctt ttttgctg:a ctttctatag :agag: ccat H gacccacc:c ccaaccccga acccga ggaa aag H agagagagac agagacagat cca::cgat: H aaagaaaagg ggggat:ggg ggg:acagtg cag aactaaagaa :tacaaaaac tccagaaaaa ggggggaatg cgccat:t:g caaggca:gg aggaacagag agacagcaga gcca cccggc:cag ggccaagaac LQLQSDFtSDSDOFtOF’FF’FOF’FSDSDSD LQ tccc LQOLQLQF’F OSDSDOF‘F SIJ‘OF‘FFtSD gagaacca:c aga:gtt:cc LQ cccc OF’F 00 00 gaactaacca atcagttcgc cttc agcc cacaacccct SD 0 r’r 0 o‘gcgc 0a fix atat cacaagt:tg acaaaaaag tcaatata:t gatt :gcataaaa atatccag:c actatggcgg cgcattagg snsnucz rrsnsn mmrr ctcgtataat :gga:tt gagttagga taaaatggag :ca :ggatatac caccg ‘ LOO (HWY 051) agaacatt:t gtcagttgc tcaatgta 0 0 fiQJQJOQJQJOQJOkQfiOfifi ggatattacg gaccgtaaa gaaaaataag tattcaca:t gatgaatgc tcatccggaa aaga cggtgagc:g :agtgttca cccttgttac ccgttt: aaac tgaaacgt:t gagtgaata ccacgacgat :ccggcagt ttctacacat atattcgcaa :tacggtga aaacctggcc Wfif‘tf‘tmF‘FOFtSlJFtF‘FSDSDOOSDOOLQMHSDSD atttccc:a aagggtttat tgagaata:g cagccaatcc ctgggtgagt :caccag:t ttgatttaaa cgtggccaat aact tcttcgcccc cgttttcacc :gggcaaat cgca aggcgacaag gtgctgatgc cgat tcaggttcat gtgatggctt ccatgtcggc agaatgct:a atgaa:taca acagtactgc agggcggggc gtaaatggat ccggcttact aaaagccaga taacagtatg cgctgat:tt tgcggtataa gaatatatac tgata:gtat acccgaagta aggtatgcta tgaagcagcg ta:tacag:g acagt:gaca gcgacagcta aaggcatata tgatgtcaat atctccgg:c tggtaagcac aaccatgcag :cgtctgcg tgccgaacgc tggaaagcgg aaaatcagga aggg LQ ga aatgaacggc tc:tttgc:g acgagaacag gggctggtg aggtttaca cctataaaag agagagccgt ta:cg:ctgt ttgtggatg :attgaca cgcccgggcg acggatgg:g atccccctgg ccag :aaag:ct cccgtgaact ttacccgg:g gtgca:atcg g HQ)" cg atatggccag :gccgg:c tccgt:atcg cggaagaagrho cc at:aacctga :ggg A0 fifififlififiifir’rQJQJLOSDSDLOLO gccaccgcg aaaatgacataggctccc ttatacacag :9 gg:cgaccat agtg :acag:at gtct caaaa:ctaa mrromsnomsnsnsnmrr :ta LOOSDOOAOF‘FSDAOF‘FSD :a :ac gtttctcgtt gtacaaagtg ’ cccccctctc ccctaacgtt tgtgcgtttg at:t:ccacc cggaaacctg :gacgagc ggaatgcaag :cgtgaag caaacaacg :gcagg ctctgcg LQ O aaaagccacg :aag cacgtt t9 gttgga:agt aaggggctg aggatg O O O A tgcacatLQ O ttacatgtg gggacg Ff LQ A0 tttcct:tg agcggc A0 LQ SD gctccc O rt O A0 ‘agcaag thtQ O O O tcctgg A O A0 A0 ‘taaacg gcgag A0 agg F’F ctgaccc ‘ gcaag O ccg O rho c accccc gacttct LO O0A0 O O ‘ :gcccgaa ageae $1) acgacg gacccgcgcc OOWWQJQJQJQJWQJOQJLQOQJLQQJF’FQJ $1151) 0A0 AofifiAOOAOOOOAOAO LOSDOLOOOOSDOSDSDOAOSD ca:cgacttc actacaacag ccacaacgtc Ff A0 acttcaagat ccgccacaac 51) O agaacacccc cgac agtccgccct gagcaaagac tgaccgccgc cac: tagagc:agc gg:accatgc WLQQJOLQLQLQWOWOLQQJLQF’FF’FQJQJOF’F COOLQLQWOQJLQQJOLQQJF’FWF’FF’F :tcg:agg :ggg : ‘tacag :gtc :ata aacc :tat :gtc attggatgtt ‘ccacaaga SD51) SD51) SD51) SlJr’r SlJr’r :caa :tt: agaaaacttc ‘cctattgaLQOF’FF’FSD :caacgaat :ct: tttg tacacaatg ggttatcctg :gcc :gca tgtat:caat tttcacttt :cgccaactt OLO SlJr‘r SlJr‘r LOLO LOSD O O F’F F’F :aaa caatacctga ccg ’ caacggccag :ctgtgcca gacgcaaccc gggcttggtc atgggccatc gcgca:gcg tcggc:cctc tactgcggaa ctcctagccg :tgtt:tgc tctggagcaa gactgataac :ctgt:gtcc tttccatggc :g tgctgccaac atcctgc O51) LOF‘F LOO LOO SDO OLO LOO F‘FSD O51) gtcccgtcgg aatcc tgcggacgac tc:cggg A A O LQ O F’r :ggg cccct:ctcc OLQF’FOOLQQJOOLQ ‘ccg:t ccgaccgacc ‘gggcgca O F’F O F’F O :tta ccgtc:gtgc :catct ccgt gtccacttcg :tcacctct ‘ ‘ tggagaccac gtgaacgcc caccaaatat :gcccaaggt :taca:aag gactc:cagc SD :caacg accgaccttg aggca:actt caaagactgt actgggagga gggag agattaggt :aaaggtctt tgtactagga ataaa:tggt cacca caccatggc gcaatcacta gagcggggta caatgactta ‘cagct tagatctta gccac:tttt aaaagaaaag taat :cccaa cgaagacaag atctgctttt tgcttgtact ggttagacca :gagcc tgggagctct ctggc:aact agggaaccca ctcaa:aaag :tgccttga gtgcttcaag gtgc ccgtctgttg gtaac:agag :ccctcaga cccttttagt cagtg:ggaa tagc catgtcatct attattcag tatttataac ttgcaaagaa atgaatatca gag ggaac:tgtt attgcagct ggtt acaaa:aaag caatagcatc acaaa caaataaagc :ttttttca ctgcattcta gttgtggttt actc atcaa cttatcatgt :ggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc ttcc gcccattctc cgccccatgg ctgac:aatt ttttttattt atgcagaggc cgaggccgga tcccttgagt catc ctggagcaga ct:tgcag:c ctgc acat tgccttt tg:aactctt ggctgaagct ct:acaccaa thZQQQQQa catgtacc:c ccaggggOQJfifiQJQJfiQJfifiLQfiOQJ aggaagacta cgggaggcta caccaacg:c aatcagaggg gcctg:gtag OSDOSDOOSDSDLQSDSDF’FSDSDO :accga gcggaccctc aagagggcat tagcaatagt gtt:ataagg ccccc:tg:t $1) F’F F’F 0 F’F F’F gacgaaaggg cctcgtgata cgcctatt tatagg:taa tgtca:ga:a :aatgg cgtc aggtggcact g atg:gcgcgg aaccccta:t LQ F? F? FA 51) FA tc:aaataca tatg tgagacaata accctgataa :gcttc aa:attgaaa aaggaagagt aacatt:ccg tgtcgccc:t :tccct ttgcggcatt ttgccttcct acccagaaac ctgg:gaaa :aaaag ctgaaga:ca gttgggtgca aca:cgaac: gatc:caac tccttgagag ttt:cgcccc ttccaa:ga: agcac:t:t ta:gtggcgHSDHSDHSDOHSDHHHSDO ggtattatcc :gttg ccgggcaaga caac:cggt ac:attc:c gaa:gacttg gt:gagtOSDSDLQLQOF‘FF‘FLQ 0 F’F caccag:cac agaaaagcat gcatgacag aagagaatta tgcagtg cca:aacca: gagtga:aac ac:tac::c gacaacgatc ggaggaccga aggagc:aac cgctt::t:g gggatca:g aac:cgcctt ga:cgttggg aaccggagc: A0 aatgaagcc acgagcg:g caccacgatg cc:gcagcaa tggcaacaac A0 :gcgcaaa : tac:ctagct tcccggcaac aat:aa:aga 0 gga:ggag ttgcagg act:ctgcgc tcggcccttc cggctggctg A0 :ta::gct gagccgg gcg:gggtct cgcggtatca cac: ggccagat taagccct cccgta: agt:atctac anaCQQQQa gtcaggcaac :gga:gaa aaatagac agatcgc gataggtgcc tcactgatta agcattggta :gtcagac caag:ttact catata: ttagattgat ttaaaacttc atttttaat: FfSlJF’FAO atc tagg:gaaga tccttt: catg accaaaatcc cttaacgtga ttc aaaggatctt cttgagatcc fiAO cactgagcgt cagaccc agaaaagatc cgcg:aatct gctgct: aacaaaaaaa ctac cagcggtgg: fi gatcaagagc c ttt:ccgaag gtaactggct tcagcagagc aatactgtcc ttctag: gccgtagtta ggccaccact actc fiAO tacatacc tcgctc:gc aatcctgtta ccagtggctg ctgccagtgg Q ggttggactc aagacgatag ttaccggata aggcgcagcg cacaca gcccagcttg gagcgaacga cctacaccga on ttcaga aagcgccacg cttcccgaag ggagaaaggc m SD 0 SD LO 0 aacagg cgcacgaggg agcttccagg A0 C9 cacctctgac ttgagcgtcg SD FALQLQOFALQLQOFAF’FF’FQJOQJLQF’FF’FF’F aacgccagca acgcggcctt tggt cgtcatctac ccgccggaag gaat SEQ ID NO:32 attcgcgtta aatttttgtt aaatcagctc (donor vector aatcccttat aaatcaaaag aa:agaccga 1, pMK 7cl2 cccattcgcc attcaggctg cgcaactgtt anti mFC scFV ctattacgcc agctgg agggggatgt COOp ECORV F’F F’F gggttttccc agtcacgacg ttgtaaaacg SacII LlRS) ‘ ‘ ‘acg actatagggc gaattgaagg aaggccgtca aataatg SD F’r tgatagtgac ctgttcgttg caacaaattg cttttttat tgtacaaaaa acga cagccattc ctggcagtgc tgcagggcgt tgcagtctg gtgaaacctg gcggcagcct gcggcttca cgac atga gctggatccg tggaatggg gtccttcatc agcggcagcg gcggcaccac agggccggt caccatcag cgggacaaca ccaaggacag gcctgaccg gccgtgtact actgcgccag gcgtgggca LQSD 051) SDLQ 051) Off 051) (*0 gtgt CthtQQCQQ gatcagggg ggagcacccg atatccagat tgtctctga agagccatcc tgagctgcag agccagccag gatacctgg cagaagcccg gccaggcccc cagactgctg gca atccccgata gattcagcgg cagc tga ctgcggccc aggacatcgg cacctactat acatcaacg ttcggcg gcaccaaggt ggaaatcaag ccgcge actttgtat caaaag acgagaaac taaaatgata taaatatcaa tagattttgOfififiOQJOOLQOLQLQLQHHHSDLQHSDHAA ataaaa 0 gactacataa tactgtaaaa cacaacatat ccagtcacta tgaatcaac acttag tattagtgac ctgtactggg cctcatgggc ttca ctgcccgct tccagt aaacctgtcg ctgc attaacatgg tcatagctgt ttccttgcg attggg 0 ctccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cgggtaaag ctgggg 0 gcaa aaggccagca aaaggccagg aaccgtaaaa cgtt gctggc ttccataggc cccc tgacgagcat cacaaaaatc gacgctcaag tcagag cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgt cgaccctgcc gcttaccgga tccg cctttctccc ttcggg OOLQFAOFALQLQSDSDOOOLQSDOOOO tctcctgttcgtggcgcttt gctc acgctgtagg tatc:cagtt cggtgtaggt ctcc aagctgggc: gtg:gcacga cgtt cagcccgacc gctgcgcctt atccggtaac :atcgtcttg agtccaaccc ggtaagacac gact:atcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aac:acggc: acactagaag aacagtattt tgcg ctctgctgaa gccagttacc aaaa gag:tggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt :tt:ttgtt: gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atc:tttcta cggggtctga gtgg aacgaaaact cacgttaagg ggtc atgagatta: caaaaaggat cttcacctag atcc:tttaa aatg aagttttaaa :caatctaaa gta:atatga gtaaacttgg tctgacagtt aaaa t:catccagc agacgataaa acgcaatacg ctggctatcc ggtgccgcaa tgccatacag caccagaaaa cga:ccgccc attcgccgcc cagttcttcc gcaa:atcac gggtggccag cgcaatatcc :ga:aacga: ccgccacgcc gccg caatcaataa agccgctaaa acggccattt :ccaccataa tgt:cggcag gcacgcatca ccatgggtca ccaccagatc t:cgccatcc ggcatgctcg ctt:cagacg cgcaaacagc tctgccgg cctg a:gttcttca :ccagatca: cctgatccac caggcccgct tcca tacgcgcacg t:caatacga :gt:tcgcc: gatgatcaaa cggacagg:c gggtatgcag acgacgcatg gca:ccgcca taa:gctcac tttttctgcc ggctagacag cagatcctga cccggcact: cgcccagcag cagccaatca cggtcaccac a:ccagcacc gccgcacacg gaacaccggt ggtggccagc gcgccgc -c a:cctgcagc :cg:tcagcg caccgctcag atcggttt:c ccggacg cc c:gcgcgctc agacgaaaca ccgccgca:c agagcagcca gcgccca :c aaac :cca cccacgctgc cgggctaccc catcctg :c aatcatactc at:g aagcatttat c atacata tttagaaaaa taaacaaa:a c aaaagtg SEQ ID NO:33 t:ttgttaaa aatttttg:t aaatcagc:c (donor vector aaatcggcaa aaatcaaaag aatagaccga 2, pMK hCDBa cgct attcaggc:g tg:t scaffold TN L5 ggcctcttcg agctggcgaa agggggatgt ‘ ggtaacgcca agtcacgacg ttgtaaaacg aatacgactc gaattgaagg gtca ttattttgac ctgttcgt:g caacaaat:g atg atgcccaact aagtggcccg acaa cccctgcccc accccagccc cagccagcct ctgagcctg ggcccgaggc :agacct ggcg caccagagga ctggatttc cctgcgacat ctacatctgg gcccctctgg tggcgtgc:g ctgctgagc :cgtga:cac cctgtactgc ggctccacca caa cccggc tctggcg SD ccag cggcgactac aaggacgacg ata gata:c rho ‘ ggttcag :cttgtacaa agt:ggcatt aagc aat tgttgc aacgaac 5110 m :cacta:cag tcaaaataaa atcattattt tgg ccttcc tttcactLO 0 cgctttccag aacc :g:cgtgcca cat tca:a t00 :gcgta:tgg gcgctctccg ct:cctcgct ctg ctcgg g LQ A0 aaagcc:ggg gtgcc:aatg agcaaaaggc cca ‘ aaccg g LO 0 gcgttgctgg ccgc agc cacaa aaatcga 0 A0 :caagtcaga ggtg cccgacagga acc gcg: H tccccct agctccctcg tgcg :tccgacc ccg atacc H gtccgcc ctccct:cgg gaag :t:ctcat gta gtatc H cagttcgchrmcz fir’rto :aggtcgttc gctc gc :g ccgttcagc0 cgaccgctgOLQfiQJOOfifiOLQfiLQOLQQJ gcctta:ccg gtaa :tgagtcc aC’I atcgccactg gcagcagcca ctgg :tagcaga taggcggtg tacagagttc :tgaag:ggt gc:acact tatttggta ctgcgctctg ctgaagccag aaaaagag:t gatccggca acaaaccacc gctggtagcc :g:t:gcaag cgcgcagaa aaaaggatct caagaagatc :tctacgggg agtggaacg aaactcacgt :aagggattt at:a:caaaa cctagatcc tttaaattaa aaatgaagtt ctaaagta:a cttggtctg cagttattag aaaaattcat ccag ataaaacgca tatccggtg cgcaatgcca acca gaaaacga cgcccattcg ‘ cttccgcaa atcacgggtg gccagcgcaa tatcc:ga acga:ccgcc ggccgcaat aataaagccg ctaaaacggc cattt:ccac cataatgt:c catcaccat ggtcaccacc agatcttcgc catccggcat gctcgctt:c agacgcgcaa ctg cggtgccagg tgtt cttca:ccag atca:cctga tccaccaggc ccgcttcca acgggtacgc gcacgttcaa tacga:gt:t cgcc:gatga tcaaacggac aggtcgccg gtccagggta tgcagacgac gcatggca:c aa:g ctcacttttt ctgccggcg cagatggcta gacagcagat cctgacccgg gccc agcagcagcc aatcacggc cgcttcggtc accacatcca gcaccgccgc acacggaaca ccggtggtgg ccagccagc cagacgcgcc gcttcatcct gcagc:cg:t cagcgcaccg ctcagatcgg rt rt rt rt 0 $1) 0 $1) $1) cagcaccgga cgaccctgcg cgctcagacg aaacaccgcc gagc agccaatggfiWfiOOLQHOLQOHOSDHSDSDSDHOH ctgctgcgcc caatcatagc caaacagacg ttccacccac gctgccgggc tacccgcatg caggccatcc :gttcaatca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcgga: acatatttga atgtatttag aaac aaataggggt tccgcgcaca cgaa aagtgccac SEQ ID NO:34 ataaccc: aattcgatag catatgcttc ccgttgggta acatatgcta tag (Final vector 4tctgg atagtatata cccg ggaagcata: gctacccgtt tagggttca used for ‘atgcc gatg cgtccggcgt agaggatcta atgtgag:ta tca lentiviral :aggcacccc aggctttaca ctttatgctt ccggc:cgta g:gg aattgtgag production, ataacaa: ttcacacagg ctat gacca:gat: acgccaagcg cgcaattaa pLV430lG 7Cl2 gggaacaaaa gctggagctg caagc:taa: gtagtct:at gcaatactc scFV mIgG hCDB ‘ ‘:cttg ggta acga:gagtt agcaacatgc agga gagaaaaag flag) accgtgcatg ggtg gaag:aaggt ggtacgatcg tgcctta:ta ggaaggcaa agacgggtc: gacatggatt ggacgaacca tgcc caga gatattgta :taag:gcc: agctcgatac ataaacgggt ctctc:ggt: gatc tgagcctgg ctc:ctgg ctaactaggg aacccactgc cctca ataaagc:tg ccttgagtg :tcaagtag: tgtgcccgt ctgt:gtgtg ‘gtaa ctagaga:cc ctcagaccc :ttac:cag: tggaaaatc tctagcagtg cgaac agggact:ga aagcgaaag gaaaccagag agctctctc gacgcaggac :tgc: gaagcgcgca cggcaagagLQLQIIOLQIIOOIIOOIIOLQ CQaQQQQng cgactggtga gtacgccaaa :gac: agcggaggct agag a9 IIIIIILQLQIIOOSDIISDLQSD :gggtgc cacagcgtca agcg aa:: agatcgcgat gggaaaaaa: LQ :aagg ccagggggaa agaaaaaata :aaaa catatag:at gggcaagcag SDLQ :agaa cgattcgcag ttaa:cctgg :agaa acatcagaag gctg:agaca :ggga cagctacaac catcccttca atca gaagaac:ta gatcat:ata IIIIII cagta gcaaccctct attg:gtgca gagataaaag acaccaagga :agac ‘atag SD m aagagcaaaa accaccgcac agcaagcggc gatct: ‘acctQ ggaggagata ttggagaagt gaat:a:ata OOIISD :aaag- fiIO gaaccattag caccaaggca aagagaagag gcagag In gcag:gggaa I‘II‘ISDSDSDSDLQSDLQI‘IO $1)$1) gttccttggg ggag aggaag mIo gcagcgtcaa ggtacaggcc agacaa:tat :ggta: cagaacaatt LQLQ tattgaggcg caacagcatc gcaac: ggca:caagc aagaatcc:g gaaa cctaaa ctcc:gggga I I ctctggaaaa :gca :gctg: gctagttg tctggaacag atttggaatc gacctg I gacagagaaa I cacaagct:a atacac:cct :gaaga cagcaagaaa agaattat:g gaat:agata ggcaag tggt:taaca IISD gctgtggtat ataaaa:tat aatga: ttgg:ag ttttgctg:a ctttctatag :agag: tattcac0 II gacccacc:c ccaaccccga acccga ggaa:ag51) Q II agagagagac agat :cgat: SDSDSDOI‘IOLQI‘ILQLQLQOSDI‘ISDLQ tctcg I SD 0 m I II aaagaaaagg ggggat:ggg ggg:acagtg aatag fl SD m 0 cagacataca aactaaagaa :tacaaaaac aattc $1) $1) $1) :ttatcga ttatttag:c tccagaaaaa ggggggaatg cctgt :ggcaagc gtaa cgccat:ttg caaggca:gg actga mIo gagaagt: gatcaagg:t aggaacagag agacagcaga aacag fi ctgtggta cagttcctgc cccggc:cag ggccaagaac cagatLQLQLQSD cccgccc gcagtttc:a gagaaccatc agatgtt:cc aaggacOOSDSDIO fiko SDIIII tgccttat:t gaactaacca atcagttcgc ‘cttc tgttcgc Io LO ccgagctcaa taaaagagcc cacaacccct 49C9 gccagtc0 0 cgtcgcccgg gtaccgatat caccaac:tt acaaaaa gctgaacgSDI‘IOLQLQSDI‘II‘ISDSDLQSDSDI‘I II tgggcagcac agccattctg gccctgc:gc cagtg0 gcagggcgtg tggt gcagtctggc ggcggac:cg aaacctg cggcagcctg ctgccgccag cggcttcaac ttcaacgacc tacatga ctggatccgg gcaag gact ggaatgggtg tccttca:ca gcagcg cggcaccacc atagcgtgaa gggccggttc accatcagcc acaaca caaggacagc acag cctgaccgtg tgtact actacaccag acagtgFtWLQLQWQJOQJOLQLQLQF’FQJO aagataccg ctgcgccaga cgtgggcaga :cg tgctggcgga ccggcggagg atcaggggga gcggaagcg ‘cacccg tatccagatg accct gtctctgagc cctggcgaaa ‘ccatcc gagctgcaga tccgg atacctggct tggtatcagc aagcccg ccaggccccc gcgc cagcagcaga gccacaggca cccgata attcagcggc ‘gcagcg ‘actt caccctgaca atcagctccc cggcccg ggacatcggc acctactatt ‘cagta cgcc cccttcacct tCQQngaQQ caccaaggtg gaaatcaagc ccaa ctttgtatac aaaagtggcc ‘cggacaac aacccctgcc cccagacctc ccctacaatt cccagccagc ctctgagcct gaggcccgag gcttgtagac cggagccgtg cacaccagag gactggattt cgcctgcgac atctacatct ggccggcaca tgtggcgtgc tgctgctgag cctcgtgatc accctgtact cagcggctcc cgcaagcccg gcga gggctccacc agcggcgact acaagg cgatgacaag taataggata tcggttcagc tttcttgtac ggga ttcgag ttaagttaac caattccccc cctctccctc ccccccccct actg cccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt :ccacca cttt tggcaatg:g agggcccgga aacctggccc cttg acgagca ctaggggtct tc:c gccaaaggaa tgcaaggtct gttgaatgtc gtgaag cagttcctct ggaagcttct tgaagacaaa ctgt agcgaccctt :gcagg ggaacccccc acctggcgac aggtgcctct gc gccaaaa gccacgtgta :aag ctgcaaaggc ggcacaaccc cagtgccacg tt g ‘ttgtg OWF’F LQQJOLQOOOQJOQJQJOLQQJLQLQLQF’FOLQQJOLQ H aatggctctc ctcaagcg:a ttcaacaagg ggctgaagga cccagaag gtatgggatc tgatctgggg cctcg catgctttac tttag tcta ggccccccga cgtggttttc rho $1) atatggccac aaccatggga gcggaggctc gcaagggcga ggagctgt:c tcct LQSD $110 00 051: LQF’F 0m $1) 0 ‘omo‘om taaacggcca caagttcagc agggcgagg SD ‘0 tgaccctgaa gttcatctgc ‘ctgcccg (‘HQ 0 0 ‘0 ccaccctgac ctacggcg:g acc $1) 0 acttcttcaa gtccgcca:g cccgaag ctccaggOF’FLQSDSDLQOSDLQF’FLQSDSDSDSDOF’FLQ $1151) on man acgacggcaa ctacaagacc cgcgccgagg aagttcg gcatcgagct caagggca:c‘ aagg gacggca H A0 agtacaacta caacagccac aacgtctata tcatggccg H 0 aggtgaactt caagatccgc cacaacatcg acggca $1) 0 accagcagaa caccccca:c ggcgacggcc t9 gcacccagtc cgccctgagc aaagacccca a9 $110 SlJr‘r LQLQ 00 AOAO agttcgtgac cgccgccggg atcac:ctcg gcatggacg cgggtctaga gg:a cg tcga 0mm" F‘FO COOLQfiWLQOLQOOfiWWWW‘OWO‘OWWW gtgccat:tg ttcagtgg:t tcccccact atgatgtggt at:gggggcc agcatcttg ccaattt:ct tt:gtctt:g taaacccta actctctaaa tt:tatgggt gatgttatg aaaa aa:caaagaa aacttccta aagtatg:ca acgaattg:g atcctgcgtt ga:gcctt:g taa caacttacaa ggcctttc:g cctgaacct ggccagg:ct gtgccaag:g t aacccccac gccatcagcg :gcgtgga a gcc gcggaact0 tagccgc:tg a A0 cat :atcggg gataac:cFf t:gtcctatc a atggctgct aggc:gtgct gccaac:gg cctgcgcgg 9 gtcggcgct caatcctgcg gacgaccct :cgggg:cg t 0H :ct cccg:tccc‘ acg gcgcacctc g actccccgt OfiOSDSDLQLQSDLQOLQOfiOOLQOOSDF‘FLQ :tttgctg :gtgcc:tc :catctg 0 Q gaccgtgtg cttcgc:tc mmfiomfimfiom OQJfifiOLQSDLQOSDLQOLQSD gga accaccgtg aacgccc $1) 0 aaatat:gc aaggtc:ta :cttggact :cagcaatg :caacga 0 0 accttgagg :acttcaaa :aaagac:g LO 51) LO LO 51) LG :tg ggggagg SD ttaggt:aaa gtctttgta taa H F’r LO Q U :gc gcaccag 0 mm catggcgcaa aat acttacaag gcagctgLQF'F $1151) atcttagcca OSDF’FSDSDLQLQOLQOfiLQSDOSDLQOF‘F cactagagc:ttttaaaa actggaagg 0 Ff $1) $1) :tcac :cccaac gacaagatct ctttttgct :ctctgg:t gaccagatc :gagcct agctctctg :aactaggg :aagcctca :aaagc:tg cc:tgagFfLQ mm ttcaagtag :gtgcccgt ctctggtaa :agaga:cc cagaccc cag :ggaaaatc :agttca:g :tatt :cagtat ttg aagaaa:ga $1)me :gagaggaa :tatt agcttat aca :aaagcaat :ttcacaaa :tt :tcactg 9 :cc attctagtt :a :gg \QJOHLQSDO \HHHOHH ctagctat cct ctccgcc :ccgccc :ctccgcc ccatggctg :aattt : ccgag gccggatccc cagtggct ttcatcctg agcagac: caaca caaca:tgcc MHMOOLQM ataagcggOUSDLQHLQLQH :atgtgta actcttggc gaagctc: aca:g tacc ccccagga agactacgg aggctaca ggcct gtg accctcaag gggcatta gcccc cttg :gaagacg aaagggcct gtgatacg :c atga ctttctta ‘ gacgtcagg ggcactt: ‘gaacc ccta :ttttcta aatacattc aatatgta acaataaccc caataata ttgaaaaagLQQJF’FOQJLQF’FLQQJQJLQQJOF'FF'FLQ aagagta: tg:c :tttttgc ggcattttg 0 cttcctg: agaaacgc:g acatgctga agatcagttg ggtgcacg‘ cgaactggat :aagatcct tgagagtttt cgccccga aatgatgagc :ctgctatg ggta ttatcccg:g gcaagagcaa gcatacacta ttctcagaat gacttgg::g agtcacagaa aagca:c:ta cggatggcat gacagtaaga gaattatgca : aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct ttt::gcaca acatggggga tcatgtaact cgccttga:c gttgggaacc ggagctgaat gaagcca:ac caaacgacga gcgtgacacc acgatgcc:g cagcaatggc aacaacgUHHLQLQOMLQLQOMMMSDOSDLQF’Ffifi cgcaaactat taactggcga ac:acttact ctagcttccc ggcaacaatt aatagac atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctgg attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc g ccagatggta cccg ta:cgtagtt atctacacga cggggagtca t gatgaacgaa atagacagat cgctgagata ggtgcctcac agca ttggtaa tcagaccaag :ttactcata ta:actttag attgatttaa aacttcattt ttaattt agg :gaagatcct tt:tgataat ctcatgacca aaatccctta acgtgag gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcct :aatctgctg ct:gcaaaca aaaaaaccac ‘ctaccag ggtggtt SDLQ LQF’F aagagctacc tttt ccgaaggtaa O F’F LO LO 0 F’F F’F 0 $1) cagagcg ctgtccttct :gtagccg ggcc caactct catacctcgc :gctaatc ctgttaccag cagtggc SD F’F :taccg‘gtt actcaaga cgatagttac gg g ggggttcgtg acacagccc agcttggagc gaacg $1) 0 0 Ft caccgaa tgagaaagc gccacgcttc ccgaaggga aaaggcg g:cggaaca ggagagcgca cgagggagc tccaggg cctgtcggg tttcgccacc tctgacttg cgtcga cta tggaaaaacg ccagcaacg gccttt cctttttga agctgtccct gatggtcgt atctacc cgcgggcatc ccgatgccgc cgaLQOOSDFtLQSDOOSDLQO aagaatca ‘gggaagg tcgcgtcg SEQ ID NO:35 ‘ataacc aattcgatag ccgttgggta acatatgcta ttgaattag (destination cttagtc:gg atagtatata ggaagcata: gctacccgtt tagggttca , ccgtgatgcc ggccacgatg agaggatcta atgtgag:ta LO 0 F‘F 0 $1) 0 F‘F 0 $1) lG) -aggcacccc aggctttaca ccggc:cgta tgttgtg:gg aattgtgag ggataacaa: ttcacacagg aaacag gacca:gat: acgccaagcg cgcaattaa cctcactaaa gggaacaaaa gctggag caagc:taa: gtagtct:at gcaatactc :gtag:cttg caacatggta acga:gac accaacatgc cttacaagga gagaaaaag accgtgcatg ccgattggtg gaag’‘ $1) $1) gctacgatcg tgcctta:ta ggaaggcaa gtc: gacatggatt ggacgaa0m ctgaa:tgcc gcattgcaga gatattgta :taag:gcc: agctcgatac ataaacg ctctc:ggt agaccagatc tgagcctgg ctc:ctgg ctaactaggg aacccac rho ataaagc:tg ccttgagtg :tcaagtag: tgtgcccgt ctgt’I Q rt mm a:cc ctcagaccc :cag: tggaaaatc tctagca cgaac t:ga aagcgaaag agag agctctctc gacgcag A0 :tgc: gaagcgcgca cggcaagagLQLQHOLQHOOHOOHOLQ ggcgg cgactggtga gtacgccaaa :gac: agcggaggct agaaggagag HUHLQLQHOOSDHSDLQSD ggtgc gtca gtat:aagcg aa:: agatcgcgat gggaaaaaa: LQ :aagg ccagggggaa agaaaaaata -aaaa catatag:at gggcaagcag SDLQ :agaa cgattcgcag ttaa:cctgg -agaa acatcagaag gaca :ggga caac catcccttca atca gaagaac:ta :ata mum cagta ctct attg:gtgca gagataaaag acaccaagga :agac ‘atag SD ‘0 aagagcaaaa accaccgcac cggc gatct: ‘acctQ rho ggaggagata ttggagaagt gaat:a:ata 00MB) :aaag- gaaccattag caccaaggca aagagaagag gcagag m gcag:gggaa gttccttggg ttct:gggag aggaag ggtacaggcc agacaa:tat :ggta: sum cagaacaatt tattgaggcg caacagcatc gcaac: SDSDSDOFtOLQFtLQLQLQOSDF’FSDLQ tcaa firtSlJSlJSDSDLQSDLQF’FO $1)$1) LQLQ ggca:caagc aagaatcc:g gctg:ggaaa cctaaa ctcc:gggga x I ctctggaaaa ctca:t:gca :gctg: gctagttg tctggaacag atttggaatc gacctg \ gacagagaaa I cacaagct:a atacac:cct :gaaga cagcaagaaa agaattat:g gaat:agata ggcaag tggt:taaca H51) gctgtggtat ataaaa:tat aatga: ttgg:agctt ttttgctg:a ctttctatag :agag: tattcaccat H gacccacc:c ccaaccccga acccga gaag H agagagagac agagacagat :cgat: tctcgaccgt H aaagaaaagg :ggg ggg:acagtg aatagtagac cagacataca aactaaagaa :tacaaaaac aattcaaaat :ttatcga ttatttag:c tccagaaaaa ggggggaatg ‘acccca cctg:aggtt :ggcaagc gcttaagtaa cgccat:ttg atgg aaaatacata actgagaata gagaagt: gatcaagg:t aggaacagag agacagcaga atatg gcca atat ctgtggta cagttcctgc :cag ggccaagaac agatg tccc caga:gcggt cccgccc: gcagtttc:a catc agatgtttcc aggg ‘cccc aaggacctga aatgaccc tgccttat:t gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgc: ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagac cgtcgcccgg gtaccgatat cacaagtttg tacaaaaaag ctgaacgaga aacgtaaa gatataaa:a tcaatatatt aaattagatt ttgcataaaa aacagactac ataatac- aaaacacaac atatccagtc actatggcgg ccgcattagg caccccaggc tttacac: atgcttccgg ctcgtataat tgagttagga tccgtcgaga ttttcaggag ctaaggaagc taaaatggag ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacattt: agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg agaccgtaaa gaaaaataag tt:t atccggcctt tattcacat: tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgtt: ggagtgaata cgat ttccggcagt ttctacacat atattcgcaa gttacggtga aaacctggcc ta:ttccc:a aagggtttat tgagaatatg cagccaatcc ctgggtgagt ttcaccag:t taaa cgtggccaa: tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag cgctggcgat tcat g :t gtgatggctt ccatgtcggc atgaa:taca acagtactgc ga:g 51) Q Ff agggcggggc gtaaatgga: aagccaga taacagtatg cg:attt cgctgat:tt cggtataa tgata:gtat acccgaagta tg:c $1) 51) 51; aggtatgcta aagcagcg acagt:gaca ccgacagcta tcagF’F F’F m tata atgtcaa: tggtaagcac aaccatgcag aa:gSIJOF’FSD D Q‘ormomm QJOQJfiLQfiOOOSDLQLQF’F HIx051)00SDOLQ QJfifiOOOLQLQO fifififlififiifigfiifiiflfiifiiflfl:cgtctgcg ccgaacgc aaaatcagga aggg gagg aatgaacggc acag aa SDLQ :ttat:gaggtttaca cctataaaag ctgt ac SD" :attgaca cgcccgggcg atccccctgg ccag tc :aaag:ct cccgtgaact gtgca:atcg \ c Igaccaccg atatggccag tccgt:atcg cggaagaag OfiWfiSLQOOSDLQH F’r gccaccgcg aaaatgacat at:aacctga 1999 $1) aggctccc ttatacacag gg:cgaccat agtg H :acag tatgtagtct caaaa:ctaa ‘orr :ta H cattt gtttctcgtt gtacaaagtg SDacgaa cccccctctc cgtt :aaggccg tgtgcgtttg at:t:ccacc ‘OfiO‘OQJO‘OQJQJQJ LQLQLQLQOQJOO‘OF’FQJ LQ :gagg cggaaacctg :gacgagc OOOLQSDLQLQOOOSDSDOLQ ggaatgcaag :cgtgaag caaacaacg :gcagg ctctgc aaaagccacg :aag cacgtt ‘o‘o fiLQ m0 gttgga:agt aagggg 0 F’F aggatg 0 0 0OSDSDSDSDSDSDF’FSDOSDLQOSDLQSlJfiSD tgcaca cggacg agcggc A0 ‘agcaag ‘ tggtg A0 ‘taaacg gcgag c ggcaag accaccc ttcagc C acttct :gcccgaa ggctac acg gacccgcgcc gaggtg caccctgg ca:cgacttc aaggag ggggcacaac actacaacag ccacaacgtc tatatcW‘OQJKOOOAOQAOfifi gaagaacgc acttcaagat ccgccacaac atcgag SDF‘FSDSDFfLQF‘FLQOLQLQLQ QAOQAOQQAOQQQJAOQAO gc:cgccg SDmfifimomooo‘ agaacacccc :cggcgac ggcccc F’F cta agtccgccct cccaac ca:ggtcct tgaccgccgc ctcggc caagtaacg tagagc:agc attacg F‘FLQSDOLQLQLQF‘FOF‘FOLQSDLQF‘FF‘FQJQJOF’F cta :tgttcagt cccc tt:cagtta LO LO F? 51) F? :ggg gtacagcatc tt:taccgc :gtc catttaaacc atg :tat attggatgtt gccacaaga :caa agaaaacttc gcctattga ttggg:tttg tacacaatg ggttatcctg tgtat:caat tttcacttt Ofififli :cgccaactt caatacctga cgttgcccgg caacggccag gacgcaaccc -ggc gggcttggtc atgggccatc tcggc:cctc tgccgatcca tactgcggaa ctcctagccg gcaa tcgg gactgataac :ctgttgtcc atatacatcg tttccatggc gc:g tgctgccaac :ggatcctgc ct:tgt:tac gtcccgtcgg cgctgaatcc tgcggacgac ccttctcggg ac:ctc:cg: cccct:ctcc gtctgccg:t ccgaccgacc acggggcgca ccgtc:gtgc cgcggactcc cttc:catct ccgt gtgcacttcg tggagaccac gcacgtcgca cgtgaacgcc :gcccaaggt OOOLQLQF‘FOSIJLQQJOLQQJF‘FF‘FfiF‘F caccaaatat aggactcttg gactc:cagc aatg:caacg accgaccttg aggcatactt caaagactgt ttgtttaaag agga gttgggggag cagattaggt :aaaggtctt tgtactagga ggctgtaggc ataaa:tggt ctgcgcacca gcaccatggc gcaatcacta ggta cctttaagac caatgactta WO 81789 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactg aagggctaa: tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctc gg:tagacca gatctgagcc tgggagctct ctg gctaact ccca ctgcttaag ctcaataaag cttgcc:tga gtgcttcaag tac tgtgtgc ccgtctgt:g tgtgactct gtaactagag atccc:caga tagt cac tgtggaa aatctctagc agtagtagt ca:gtca:c: tatta::cag tatttataac ttg caaagaa ca gagagtgag ggaacttgt: tattgc tataatggtt acaaataaag SD 51) rt 51) LG 0 $1) :c acaaatttc caaataaagc attt: m ctgcattcta gtt9t9 gttt ccaaac:c atcaatgta a:g: ctggc m cgcc cctaactccg 0catcccgc ccc:aactc t:cc gccca m cgccccatgg ctg actaatt [A :t agg cgaggccgga tccc:[A tSDSDFtSDF‘FFtLQF’FOSDSD 99ctttcatc ctc gag caga H :tgcag:c tgtggactg aacacaaca: tgcc:AA m tg tt ggctgaagct H :acaccaa t99199999 ca:gtacc ccagggon aggaagacta c99 gag gcta SD51) ccaacg:c aatcagagg g:ag SDOSDOOSDSDLQSDSDF‘FSDSDO -accg gcggaccctc aac agc gcat gcaatagt gtt:ataag ccccc:tg 51) m gacgaaaggg cctcgtgata LQ cctatt tatagg nS ga HAO ct :agacgtc a99 t99 cact 19999 atg:gcgcgLQQJLQLQQJOOOF’FQJQJF’FLQOF’FLQ aaccccta tc :aaataca ttcaaatatg SD :cch tgagacaata accctga:aa A0‘AQ aa :attgaaa aag gaagagt [A gagta aacatt:ccg tg:cgccc:: ttgcggcatt ttg ccttcct acccagaaac c:gg:gaaa ct gtt999 tgca fiLQOLQOLQQJfifiOfiOOOF’FOLQOSD MQH DLQ agtgg OSDSDLQLQOF’FF’FLQ gaaga:ca aca:cgaac: aac A tccttgagag ttt :cg cccc agaac ttccaa:ga: agcact::: 0 ta 19t99C9 ggtattatcc LQ :gttg ccgggcaaga caac:cgg: :attc:c :gacttg A ac gaa A :gagt 0 F’F caccac:cac agaaaagca: gcatgacag aagagaatta cagtg cca:aacca_ gagtgataac ac :tac::c gacaacgatc ggaggaccga aggagc:aac cgctt:t::g 99gatca:g aac :cgcctt ga 199tt999 aaccggagc: gaatgaagcc acgagcg:g caccacgatg cc :gcagcaa tggcaacaac A0 :gcgcaaa :ac tac :ctagct tcccggcaac aat:aa:aga 0 LO LO 511 :ggag gac act :ctgcgc tcggcccttc cggctggctg :ta:tgct ga9999919 9C9 :gggtct cgcggtatca ttgcagcac: ggccagat ct cccgta:cg :atctac ac gtcaggcaac fiAOA0 :ggatgaa agt gacgggga aaatagac agatcgc:gHSDHSDHSDOHSDHHHSDO gataggtgcc tcactgatta agcattggta SD :gtcagac caag:ttact :ac tgat ttaaaacttc atttttaat: rt 51) $1) 51) LG 0 atc tagg:gaaga tccttt::g taa :ctcatg caaaatcc cttaacgtga A F’r F’r ac fiAO A 0 0 ttc cactgagcgt cagaccccg agaaaagatc aaaggatctt cttgagatcc cgcg:aatct gctgct:gca aacaaaaaaa ccaccgctac cagcggtgg: fi A ccg gatcaagagc taccaac:c: ttt :ccgaag gtaactggct tcagcagagc agatacca aatactgtcc ttctag:gta gccgtagtta ggccaccact tcaagaactc fiA0 tcgctc:gc: aatcctgtta ccagtggctg ctgccagtgg Q 99 ttggactc aagacgatag ttaccggata aggcgcagcg QJAO 0 LO LO LO 0 cacaca gcccagcttg gagcgaacga cctacaccga ttcaga aagcgccacg cttcccgaag ggagaaaggc A0 aacaggagag cgcacgaggg agcttccagg A0 A0 cacctctgac ttgagcgtcg SD fiLQLQOfiLQLQOSfifiSDOSDLQfififi F’F F’F F’F 0 Ft fiAO aacgccagca acgcggcctt 5110 A0 tggt cgtcatctac CCgccggaag cgagaagaat SEQ ID NO:36 attcgcgtta aatttttgtt aaatcagctc (donor vector aatcccttat aaatcaaaag aa:agaccga 1, pMK 8B3 cccattcgcc attcaggctg tgtt anti mFC scFV cgcc agctggcgaa agggggatgt COOp ECORV 99 gttttccc agtcacgacg ttgtaaaacg SacII LlRS) actatagggc gaattgaagg aaggccgtca t9 atagtgac ctgttcgttg caacaaattg t9 tacaaaaa agctgaacga tatcgccacc ctggcagtgc t99399999t gtcagctcag gg aagaaacccg gcagcagcgt gaaggtgtcc gcggcggcac tacgccattt Ctt99911999 ccaggcccct t9 gaatgga: a9 cccctaca acggcaacac cgactacgcc a9 ggcagag: accgacacca gcacctccac cgcctacatg gcctgagaag gccgtgtact actgtgccac aggcggcgga gcgatctgtg accctcgtga cagtgtctgc tggcggcgga gaag 99 ag cacctgagat cgtgctgacc gcacactgag 99 cgacagag tgtccatcac ctgtagagcc tcggaggcag tatcagcaga agcctggcaa caag ctgaggccag caccctg a9 aggcgtgc ccagcagatt ttccggcagc ctctgg ccgacttcac cctgacaatc ac cagcctgc agcccgagga cgtggccacc tactactg agaagtacaa cagcgtgccc ctgaccttcg gccctggcac caaggtggaa atcaagccgc gggccaactt tgtatacaaa gaaacgtaaa atgatataaa tatcaatata ttaaattaga aca:aatact gtaaaacaca acatatccag tgaa ngfifiOLQOLQOLQF‘FQJOOF‘FF‘FOF‘FF‘F:ttgcataa 0 $1) $1) 0 F’F $1) 0 F’F F’F agtgacctg actgggcc:c atgggccttc ctgc cgctttcca agctgcat:a acatggtcat agctgtttcc ttg tcactgac:c gctgcgctcg gtcgttcggg aaagcctgg caaaag ccagcaaaag gccaggaacc gtaaaaaggc gcgttgctg ggctcc cccccctgac gagcatcaca aaaatcgacg :caagtcag F‘FLQSDLQLQF‘FOOSDSDLQLQO 08:g:cgtgc LQAAOAASDO OLQOSDLQF’F :cctcg cgacag aaga taccaggcgt :tccccctgg agctccctc :tccga cctgccg :t accggatacc :gtccgcctt ctcccttcg :t:ctc tagct 0 51) Q 0 tgtaggtatc :cagttcggt :aggtcgtt gcacg $1) $1) cccgttcagc ccgaccgctg tcc m 0 caac 0 0 on m SD agacacgact :atcgccact gcagcagcc LQ agcg LO 51) FA ctacagagtt :tgaagtg :ctgcgctct ctgaagcc 0 ccac gctggtag aaaaaggatc caagaaga 0H0 F‘FF’FF‘F aaaactcacg :aagggat F’FLQQJF’FQJF'FLQOOO O0 $1)0A 0 :tttaaatta aaatgaag $1) $1) gtctg acagttatta $1) $1) $1) 51) W W 0 IAHOOOHSDLQSDAAOLQOHSDOLQLQLQOOF’F SDSDOLQOOOSDSDSDFALQLQSDOSDF‘FFAF‘FOSDLQ O fir? ctatccggtg ccgcaatgcc :acagcac ccgcccatt 0 SD on tcttccgcaa :atcacgggt ggccagcgc aacga:ccg 0 SD m caat caataaagcc gctaaaacg ccataatgt cggcag ccat gggtcaccac cagatcttc tgctcgctt cagacg aacagctctg ccggtgccag gccctgatg gatca:cctg atccac tcca :acgggtacg cgcacgttc tcgcc:gatg atcaaa caggtcgccg ggtccagggt atgcagacg ccgccataa: gctcac tctgccggcg ggct agacagcag gcact:cgcc cagcag caatcacggc ccgcttcggt caccacatc cacacggaac accggt gccagccagc :cagacgcgc cgcttcatc tcagcgcacc gctcag gttttcacaa acagcaccgg acgaccctg gaaacaccgc cgcatcWWLQOF’FOOO‘O AQF‘HQSDF’HQSDAQO m0mwofiAOAOQm cagccaatgg gcgc ccaatcata gttccaccca cgctgc 0Q A0 gcat gcaggccatc ctgttcaat aa:a ttattg$1) 51) A0 atttatcagg :attg ’ catgagcgg aatgtatt:a gaaaaataaWOLQLQLQLQOF'FQJLQQJOQJF’FLQ caaatagggg ccg SEQ ID NO:37 ctaaattgta ttttgttaaa aatttttg:t aaatcagct (donor vector attttttaac aaatcggcaa aaatcaaaag aatagaccg 2, pMK hCDBa gatagggttg acagggcgct attcaggc:g cgcaactgt scaffold TN L5 ggcg ggcctcttcg agctggcgaa agggggatg L2) gctgcaaggc ggtaacgcca agtcacgacg ttgtaaaacg acggccagtg aatacgactc OSDSDLQOLQOF‘FF’FSDLQOOSDSDSDF‘FSD aagg aaggccgtca aggccgcata ttattttgac ctgttcgt:g caacaaattg atgagcaatg atgcccaact cccg cggacaacaa cccctgcccc accccagccc cagccagcct ctgagcctg ggcccgaggc gctgctggcg A0 caccagagga ctggatttc cctgcgacat gcccctctgg 0 A0 tggcgtgc:g ctgctgagc :cgtga:cac ggctccacca 0 A0 caagcccggc tctggcg gctccaccag cggcgactac aaggacgacg [A ata gata:c ggttcag :cttgtacaa ag:tggcat: ataagaaagc H fiAO aat tgttgc aacgaac :cacta:cag tcaaaataaa atcattattt H ooggcc tgg ccttcc tttcact cgctttccag tcgggaaacc gcca gctgca: cat tca:a gctgtttOLQSDOSD vorer :gcgta:tgg gcgctctccg cgct cactgac:cg ctg ctcgg tcgttcg aaagcc:ggg gtgcc:aatg aggc cagcaaaagg cca aaccg taaaaag ctgg :aggctccgc ccccctgacg agca cacaa a caga ggtg cccgacag ctataaagat acc gcg: tccccct ctcg tgcg ctgccgc:ta ccg atacc gtccgcc ctccct:cgg gaag agctcacgct gtaggtatc cagttcgLQFALQOLQLQ rtrfAOAOQAOQ :aggtcgttc gctc cacgaacccc ccgttcagcc cgaccgctgOLQFASDOOFAF’FOLQF’FLQOLQSD gcctta:ccg gta aacccgg:aa gacacgac:: atcgccactg gcagcagcca Cth gcgaggtatg taQQngth tacagagttc :tgaag:ggt ggcc agaagaacag tatttggta: ctgcgctctg ctgaagccag ttac ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtgg cagcaga:ta cgcgcagaaa aaaaggatct caagaagatc tctgacgctc agtggaacga aaactcacgt :aagggattt aggatct:ca cctagatcc: tttaaattaa aaatgaagtt ctaaagtata tatgagtaaa cttggtctga cagttattag aaaaattcat ccag ataaaacgca atacgctggc tatccggtgc cgcaatgcca :acagcacca gaaaacgatc cgcccattcg ccgcccagtt cttccgcaa: atcacgggtg gccagcgcaa tatcc:gata acgatccgcc acgcccagac ggccgcaatc aataaagccg ctaaaacggc cat:ttccac cataatgttc catcaccatg ggtcaccacc agatcttc catccggcat gctcgctttc acagctctgc cggtgccagg ccctgatg cttcatccag atcatcctga ccgcttcca: acgggtacgc gcacgttc tacgatgttt cgcctgatga agg:cgccgg gtccagggta tgcagacg gca:ggcatc cgccataatg ctgccggcgc cagatggcta gacagcag ccgg cacttcgccc aa :cacgg cgcttcggtc accacatc gcaccgccgc acacggaaca ccagccag cagacgcgcc gcttcatc gcagctcgtt accg tt ::caca cagcaccgga cgc:cagacg cgcc tg ctgctgcgcc caaacagacg ttccacccac ta C caggccatcc tac:cttcct ttttcaatat tt ttattgtctc aca:atttga atgtatttag aaa tccgcgcaca SEQ ID NO:38 aat:cgatag catatgcttc ttgaattag (Final vector atagtatata ctactacccg tagggttca used for ggccacgatg cgtccggcgt LO 0 F‘F 0 $1) 0 F‘F 0 $1) lentiviral aggctttaca ctttatgctt aattgtgag production, ttcacacagg aaacagctat 19 cgcaattaa lG 8B3 gggaacaaaa gctggagctg :taa LO 0 $1) $1) F’F $1) 0 F’F 0 scFV mIgG hCDB caacatggta acga:gagtt agcaacatgc -acaag gagaaaaag flag) ccgattggtg gaag:aaggt ggtacgatcg tgcctta ggaaggcaa gacatggatt ggacgaacca ctgaa:tgCC gcattgcaga gatattgta agctcgatac ataaacgggt ctctc:ggt agaccagatc tgagcctgg ctaactaggg aacccactgc ataaagc:tg ccttgagtg tgtgcccgt ctgt:gtgtg ctagaga:cc ctcagaccc tggaaaatc tctagcagtg cgaac agggact:ga aagcgaaag agctctctc gacgcaggac :tg C gaagcgcgca cggcaagagLQLQHOLQHOOHOOHOLQ cgactggtga gtacgccaaa :gaC: agcggaggct agag HUHLQLQHOOSDHSDLQSD gtca agcg aa agatcgcgat gggaaaaaa: ccagggggaa agaaaaaata :aaaa catatag:at gggcaagcag SDLQ cgattcgcag ttaa:cctgg -agaa acatcagaag gctg:agaca caac catcccttca atca gaagaac:ta gatcat:ata mum gcaaccctct attg:gtgca gagataaaag agga ‘atag SD ‘0 SDSDSDOFtOLQFtLQLQLQOSDF’FSDLQ aaaa accaccgcac agcaagcggc ‘acctQm ggaggagata ttggagaagt gaat:a:ata SD F? gaaccattag caccaaggca aagagaagag 00H gcagag K) gcag:gggaa fififiiflimgflfiiflfifi gttccttggg ttct:gggag aggaag m gcagcgtcaa ggtacaggcc agacaa:tat :ggta: SD cagaacaatt tattgaggcg caacagcatc ‘gcaac: ggca:caagc LQLQ aagaatcc:g gctg:ggaaa SD cctaaa ctcc:gggga ‘ ctctggaaaa ctca:t:gca :gctg: gctagttg tctggaacag atttggaatc gacctg \ gacagagaaa ‘ cacaagct:a atacac:cct :gaaga cagcaagaaa "51) agaattat:g gaat:agata ggcaag tggt:taaca gctgtggtat ataaaa:tat aatga: ttgg:ag SD ttttgctg:a atag :agag: tattcac0 m gacccacc:c ccaaccccga acccga ggaa:ag51) Q m agagagagac agagacagat :cgat: tctcg SD 0 ‘0 H m aaagaaaagg ggggat:ggg ggg:acagtg aatagr’r SD ‘0 0 cagacataca aactaaagaa aaac aattc 51) $1) $1) m :ttatcga ttatttag:c tccagaaaaa ggggggaatg ‘ ‘acccca cctg ‘ SD ‘0 ‘0 m gc gcttaagtaa cgccat:ttg caaggcatgg cata actg51) L0 $1) s1; gagaagt: gatcaagg:t aggaacagag agacagcaga atatg gcca aacag LO 511 Ft ctgtggta cagttcctgc cccggc:cag ggccaagaac agatg tccc caga cccgccc: gcagtttc:a gagaaccatc agatgtttcc aggg ‘cccc aaggSlim CLO 00 AOFT’AO flirtOLQLQSDSF’rSDSDLQSDSDF’VHI‘SD aatgaccc :g t9 ccttat:t gaactaacca atcagttcgc ttctcgcttc tgttc FtLQ 0 LG c:cc ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccag 0 0 0 cgatagac :g C9 gg atat cttt gtacaaaaaa gctg \ SD QB) 00 mm ‘ atcgccacca t9 ggcagcac agccattctg gccctgctgc tggcagtgct gcagg r’r LQ tcagctcagg t9 cagctgca gcagtctggc gccgaagtga agaaacccgg cagcagcgtg aaggtgtcct ctag cggcggcacc ttcagcagct acgccatttc ttgggtgcgc caggcccc :g gcct ggaatggatg ggctggatca gcccctacaa cacc gactacgccc a9 aaagtgca gggcagagtg accctgacca ccgacaccag cacctccacc gcctacatgg aactgcggag cctgagaagc gacgacaccg ccgtgtacta ctgtgccaca ggcggcggaa cctggtacag cgatctgtgg ggcagaggca ccctcgtgac agtgtctgct ggcggcggag gatctggcgg aggcggaagt ggcgggggag gagc acctgagatc accc a9 agccctag cacactgagc gccagcgtgg gcgacagagt gtccatcacc tgtagagcca gccagagcat cagc ctggcctggt atcagcagaa gcctggcaag gcccccaagC t9 ctgatctc tgaggccagc accctggaaa gaggcg:gcc Cagcagattt tccggcagcg gctctggcac cgacttcacc ctgacaatca gcagcc:gca gcccgaggac gtggccacct actactgcca caac agcgtgcccc :cgg ccctggcacc aagg:ggaaa tcaagccgcg ggccaacttt gtatacaaaa gtggcccgcg cctgccccca gacctcc:ac cccagcccct acaattgcca ctct cccgaggctt c:gc tgctggcgga gccgtgcaca gact tgcgacatct acatctgggc ccctctggcc tgtg ccgtgc:gct accc tgtactgcgg ctccaccagc ggctccggca agcccggctc tccaccagcg gcgactacaa ggacgacgat gacaagtaat aggata:cgg ttgtacaaag ttgggat:cg agttaattaa gttaacgaat tccccccctc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt ttat:t:cca :attgcc gtcttttggc aatg:gaggg cccggaaacc ttct:gacga :tcc:ag gggtctttcc gcca aaggaa:gca aatg:cgtga aagcagt tcctctggaa gcttcttgaa gacaaacaac accc:t:gca agcggaa ccccccacct ggcgacaggt gcctctgc fi'Q cgtg:a:aag QJAO§DAOQ fiAOAOQQ WW cacc:gc aaaggcggca caaccccagt gccacg wfi 9tt919 :caaatg gctctcctca agcg:attca acaagggg0A0 fi cagaag 0A0 0W attg:at gggatctgat ctggggcctc ggtgca Wfi ¢2 ctttag A0 :aaaaaa cgtctaggcc ccccgaacca CgQQQa fiQ gaaaaacacg W fiAO A0fi0AOQJOA0 ataa:at aacc atgggaggcg aagcg 0A0 Q cgaggcacca fi A0 A0 :gagcaa gggcgaggag ctgt:caccg thQQ 0 cagc:ggacg A0 0 A0 acgtaaa cggccacaag gtgt ‘gcg A0 Q gccacc:acg fiAO 00 W W0A ctgac gttc atctgcacca 0fi tggcccaccc "3 AA accac ctac ggcg:gcagt QJAO§DAOQ NWQ caca:gaagc W 0W actt gtcc gcca:gcccg acca:c:tct fi 0A0 W W acg W cggcaactac aagacccgcg W gacaccctgg fi A0 S) c 0W ff cgagctgaag ggca:cgact W ctggggcaca W A0 0 Wa r’r Q) caactacaac agccacaacg fi cagaagaacg A0 0 W Wa A0 ff gaacttcaag atccgccaca W cagc:cgccg W 0 (1 W 0 0W gcagaacacc ccca:cggcg fi cact W 0 0 51: 0 cgcc ctgagcaaag W caca:ggtcc 0 ff cgtgaccgcc gccgggatca 0 :acaagtaac U2 tctagagc:a :aat:aagct WAOF‘r 0A0 $2 gcgg:accat Q fiAO 0 0 cat:tgttca gtgg:tcg:a Q gctt:cagtt W W W :g aagt cttt:taccg 0fl W :g $2 tctt:ggg fi'O acatttaaa acaaagagat 0 0 0 atgggtta fi 0WfifiQQWflE :tgccacaag ff tg fi :agaaaact aggcctattg ff attg:ggg fi :ttggg:tt :ttacacaat ta 0atgtat:ca gctttcactt tg Wacaatacct cccgttgccc ggcc caag:gt fi'O :gacgcaac ccccaC’ :ggggcttgg tcatgggcca C9 :tcggc:cc tctgccgatc catactgcgg aactcctagc Qgtctggagc aaacat:atc gggactgata ac:ctgt:gt 0gtttccatg aggc :gtgctgcca ac:ggatcct Wcgtcccgtc ggcgctgaat cctgcggacg acccttc:cg Q :cccct:ct ccg:ctgccg :tccgaccga ccacggggcg tacgcgg ccccgtc:gt gcc:tc:cat ctgccggacc gtgtgcactt ctgcacg’ catggagacc accgtgaacg cccaccaaat at:gcccaag agaggac:ct tggactc:ca gcaatg:caa cgaccgacct tgaggca:ac ttcaaagact gtttgtt:aa agac1gggag gag:tggggg aggagattag gt:aaaggtc tttgtactag gaggctg:ag fi'Q :c:gcccac cagcaccatg gcgcaatcac tagagcgggg :acctttaag W 0aagccag ctgtagatct tagccac:tt ttaaaagaaa aQQQQ aCt W Hfl cac:ccc aacgaagaca agatctgctt tttgcttgta ctggg :ct OWQatc:gag cctgggagct ctctggc:aa ctagggaacc cactg :aa WQ O :tgcc:t gagtgcttca g:gt gcccgtctgt tc WQW :ccctca gaccctttta gtcagtg:gg aaaatctcta :ag agtatttata aaatgaatat acttgcaaag :ga fi'O :tattat:c cag atgg ttacaaa:aa agcaatagca :tt Q 0a:tttt:t cactgcattc tagttgtggt ttgtccaaac :gt Q :c:ggctct agctatcccg cccctaactc cgcccatccc gcccctaact 0cgcccat:c tccgccccat ggctgac:aa ttttttttat :tatgcagag Qatcccttga gtggctttca tcctggagca gactttgcag :ctgtggact ctcttacacc aatgctgggg fi'W :tgcctt:a tgtgtaactc ttggctgaag cccaggggc ccaggaagac tacgggaggc tacaccaacg :caatcagag fi'W gctaccgat aagcggaccc tcaagagggc attagcaata gtgtttataa :aattct:g aagacgaaag gtga tacgcctatt ggtt fiaataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ccta fi :tgttta:t aata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc cctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaaga: cagttgggtg cacgagtggg ttacatcgaa ‘gatctca acagcggtaa tgag agttt:cgcc ccgaagaacg ttttccaatg ttct gctatgtggc gcggtattat cccgtgttga agcac:t cgccgggcaa LQOLQWWQJOF’FLQQJOQJQJQJLQQJO caactcg gtcgccgcat acactattc: cagaa:gact tggttgagta ctcaccagtc agc atcttacgga tggcatgaca :aagagaat tatgcagtgc agtga:a acactgcggc 0A0 tgccataacc A A 0A0 caacttac: :gacaacga tcggaggacc gaaggagcta m gcc ttgatcgttg ggaaccggag 0H0 AOAo gcttt:t tgcacaacat gggggatcaaatgaag ccataccaaa cgacgagcg :gcgca 0A0 acaccacga tgcctgcagc aatggcaaca A‘A‘AALQLQOIALQLQOHHHQJOQJLQN’N’N’ aactattaac tggcgaac:a :tac:ctag cttcccggca acaattaata LQSD m0 ggatgg aggcggataa agttgcagga Qcact:ctgc gctcggccct tccggctggc :tat:g ctgataaatc tggagccgg: agcg:gggt ctcgcggtat cattgcagca m ggccag atggtaagcc ctcccgta:c Avo :agt:atct acacgacggg gagtcaggca sun snrm aacgaaatag acagatcgc: cctcactgat ttgg :tta 0A0 agataggtg ta:a :ttagattg atttaaaact tcatttttaa tctagg:gaa gatccttt:: m ataa:ctca tgaccaaaat cccttaacgt tccactgagc gtcagacccc OfiOOAO :aatcctgt:agaaaaga tcaaaggatc ttcttgagat tgcgcg:aat ctgctgct:g aaccaccgct accagcggtg cggatcaaga gctaccaac aggtaactgg caga caaatactgt ccttctag taggccacca cttcaagaac cgcctacata cctcgc:c taccagtggc tgctgccagt cgtgtc:tac cgggttggac cgat agttaccgga taaggcgcag gaacgggggg ca cagcccagct tggagcgaac gacctacacc acctacagcg tgagca:t cca cgcttcccga agggagaaag SD 0 SD LO 0 A atccgg:aag cggcaggg gag agcgcacgag ggagcttcca cctg ttatag:cct :ttc gccacctc:g acttgagcg HLQ FASD FASD "SD 0 HLQLQHHLQHHHHOOOOF’FSD gatg aggggggcgg agccta:g aaaacgccag caacgcggc tcct ttgctggcct aac gtccctga gtcgtcatc acag ctgcaacgcg c tgccgccg agcgagaag ggaa ccagCC’ SEQ ID NO:39 gtcgacg LO 511 OWWOLQQJQJLQF’FQJF’FF’FQJOQJ n A0 cccg :cccc atctgctc[A g (pLentiiciMyci atgccgcat 0 Q tatctgctcc gctgagta A0 t DDK OX40L) gcgcg 0 $1) [A acaacaaggc atgaagaaAA c tht 99 AALQ fifiO [A cttc cgcgttga0a ttga:tatt $1) [A aatag:aatc atagccca a tatgg F’F F’F 0 $1) [A aacttacggt cgcccaac9 a ccccc 0 $1) $11 taatgacgta :agggact t ccat OOQOQOmfifiK-QLQQJLQLQON’QJLQOQJQJHOKQ agtat:tacg :acatcaa9 t gtatc 0 A0 cccctat:ga gca ttatg H0 A00 tatgggactt m t catcg tgcgg:t:tg A0 t tgaC’ 0 $11 gtctccaccc A0 a ccaaa caaaa:g:cg 0A0 0A g C99 aggtc:a:at [A tctgg gcctgggag 0 m [A agcc A 0 $1) $1) F’r c:t m 0 [A fiQJgQJQJQJOF’FQJQJLQSDSDSDSDLQSDLQF’FLQOO g A $1) $1) 0 F’r agaccct:t aaaatct cgaaaggga :ctcgac HAO LQLQ caagaggcg ctggtgag:a A aggagagag agcgtcag:a @ t:c gggggaaaga [Am SD caagcaggg :tcgcagt:a 0 [A FALQSDSDOLQOOOF’FOSDSDSDLQFA 0 51) LO 511 51) LO LO 0 aaa ctacaaccat 0 cattata:a accctcta:t [AAO $1) $1) 0 aag atagaggaag 0 $1) taagacc ccgcacag aagcggccg :cagacctgg agggaca :tggagaa tgaatta:a :agtaaaaat HAO c caccaagg aaagagaag gagaaaaaag 51151)" ‘agctt HAO gttcttggg gcactatggg acgctga LQLQ LQ F’F $1) 0 51) LG 0ttccttg cagacaa:t A A :agtgcagca gcagaacaSDOLQFASDSDSSDOSDLQSDOOSDA ctgaggg :attgagg gcaacagca :cacagtctg gggcatcaag agctccagg cct ggctgtgga aggatcaaca gctcctgggg :ttggggtt 0 F’F 0 F’F LO LG 51) $1) $1) actcatt:g :gccttggaa tgctagt:gg aaat :ctggaaca gatttggaaHOSDAASDSDSDAALQOSDMSDLQSDSDSDF’FO ggatggagtg ggacagagaa :taacaatt cacaagctt aatacac:cc aatcgcaaaa ccagcaagaa gaatgaac agaattatt ggaattaga: gtttgtggaa ttggtttaac :aacaaatt gctgtggta tataaaa:ta :agtaggagg cttggtaggt :aagaatag :tttgctgt tata :taggcaggg atattcacca :tatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg cagagagaga cagagacaga tccattcga: tagtgaacgg atcggcactg cgtgcgccaa :tctgcagac aaatggcagt :tcatccac aattttaaaa gaaaaggggg ggggg -acagtgcag gggaaagaat atagcaacag aaac ‘aatta caaaaacaaa :tacaaaaat cgggtttat: acagggacag :cca ctttggttag gccc A AASDAAOAAAA SD tgtccaata: gaccgccatg :tat :gacta aata acggggtca: tagttcatag cagt -ccgcg :tacataact ggcccgcctg gc:gaccgcc cgcccattga cgtcaa:aat cccatagtaa cgccaatagg -gacctcaat ggg:ggagta AOQQJQAOAOFT’FT’AOFT’AO OLQLQQJQJQJOSLQF'FLQQJQJQJ SD actgcccac tggcagtaca cata -gccaa cccc AA aatgacggta aa:ggcccgc ccccagtaca :gacct:acg acttggcag- acgt ccta :tacca :gg:ga:gcg SD A OMAALQAAOSDAAAAAASDAA tacaccaatc ggcgtggata -cacggggat :tccaagtct A gacgtcaatg tgtt aatcaacggg act:tccaaa 51) aaccccg cg:tgacgca gtac ggtgggaggt tagtgaaccg ctaa -acgac :agg tggatccggt :ctgccgccg cga:cgccat A0 aagagaatgt cccaggccaa gat:cgagag cc:ctgtaat Qggc :gctcc :gtgct:cac 0A0 ctgctcttca cggtatcctc gaa:tcaaag aa:ataagaa :tca :cctca cttcccaaaa tgcag aa atcaactgtg atgggt:t:a ac:tctccca attagccttc attaccagaa aactgaagaa gtcaactcct tga:gg:ggc aagtctactt actgacaata cctccc:gga cagaactgat caaaatcctg gtgaat:c:g ctcgagca tcagaagagg atc:ggcagc ga:gacg taaacggccg gccgcggtct ‘gacctaa agca :acatt atacgaag:t ttccacta atatcaagct :aat ttct :aacta tgt:gc:cct g A atgc :at:gc ttcccg:a:g tctcctcctt ctct :ta:ga ggagttgtgg ggcaacgtgg ctgacgcaac ccccac:ggt ccaccacctg tcgc :ttccc cctccc:a:t aactcatcgc ggacaggggc tcggctgt:g attccgtggt cctt :cc:tg gctgctcgcc cctggattct acgtccc:tc caat ttccttcccg ccggctctgc ggcc :ct:cc :cgc agacgagtcg tgggccgcc ccccgca:cg ataccg:cga cctcgatcga gacctagaaa aatcacaag agcaa:acag ccaa ctgattgt gcctggctag ggaggag gtggg:t:tc cacc aggtacct ttaagaccaa ggcagct gatc _:agcc actttt:aaa aaaagggg gaa A0 a agacaagata tccttgatct ‘atctac cacacacaa tgattg A0 cagggccagg ‘atat ccactgacc rho ctacaa A0 gtaccag:tg agaa gccaatgaa A0 A0 caccc:g:ga gcctgcatgg ccggagagag AOAo AAOAASDLQOAAAAAAOOOLQ LO 51) acac gacagccgcc tagcatttca cgagagctgc ctctggt:ag accagatctg ctctctggc aagcC tgcc caagtagtg tC’ agagatccct tagtcagtg gccagcaaaa cgtaaaaagg LQLQ $1) 0 51) A0 gcccccctga aaaaatcgac gactataaag tttccccctg LG 0 A0 0 AA I ccctgccgct ctgtccgcc: F‘FSD 051) F‘FLQ 00 51) fig) 0A0 ctcagttcgg tgtag 0 WQ 511AA LQLQ OH fifiO LQOO fifiLQ 0051) atagctcacg tgcacgaacc cccgaccgc: gcgccfik-QofimfiQJOK-QLQHQJHH F’r $1) 51) AA SD51) 0AA ccaacccggt ccac cagc gagcgaggta gctacagag: tcttgaag:g ctagaagaac atctgcgctc tgctgaagcc ttggtagctc acca ccgctggtag agcagcagat aaaaaagga: ctcaagaaga acgc gaaaactcac gttaagggat gccctgccac actgttgtaa ttcattaagc atcacaaacg cctgaatcgc cagcggcatc ataatatttg aaacgggggc gaagaagttg atcaaaactg cccagggatt ggctgagacg aaaaaca :caataaa ccctttaggg ggttttcacc gtaacacgcc tgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc aacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata cggaactccg gatgagcatt catcaggcgg atgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata gatt tttttctcca tcct ttttcaatat tattgaagca aggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt cccgcgcaca tttccccgaa aagtgccacc tgac 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, NJ, USA), with results shown in . aEM3 aAPCs were diated 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-lBBL), as described above, were genetically engineered with a co-stimulatory human OX-4OL using a similar lentiviral transduction approach. To generate lentivirus containing human OX-4OL, pLenti-C-Myc-DDK OX4OL (PS100064, Origene, SEQ ID NO:39, ) vector together with the VSV-G envelope plasmid (pCIGO- VSVG) 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-lOO membrane. aEM-3 cells were then infected with concentrated lentivirus and r ed for five days.
The cells were stained with PE-conjugated anti-human OX4OL, Brilliant Violet 421-conjugated anti-human CDl37L (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, OX4OL, CDl37L (when included), and CD86 using a S3e Cell Sorter (Bio-Rad, Inc., es, CA, USA). The purity of sorted cells was further validated using flow cytometry. The enriched cells were checked for purity by flow cytometry.
Example 6 — ion of Tumor rating Lymphocytes Using EM-3 Artif1cial 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 and . 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 , where the bar graph shows cell numbers determined on Day illustrates the results of TIL expansions with EM-3 aAPCs (aEM3) at different TlL:aAPC ratios. The s show that aEM3 aAPCs perform ably to and in some cases better than PBMCs, particularly at ratios of 1:200 at longer e times (14 days). illustrates the low variability in cell counts from TIL expansions with EM-3 aAPCs (aEM3) in comparison to PBMC feeders. TILs (2 X 104) were co-cultured with five different PBMC feeder lots or aEM3 (in triplicate) at 1:100 ratio with IL-2 (3 000 IU/mL) in a G- Rex 24 well plate. The graph shows viable cell numbers (mean) with 95% confidence interval counted on Day 14. 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 X 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 ed 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 oc/B PE-Cy7, CD4 FITC, CD8 PB, CD56 APC, CD28 PE, CD27 7, and erCP-Cy5.5 for differentiation panel 1, CD45RA , 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, KLRGl 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 ter. Data was analyzed using Cytobank re (Cytobank, Inc., Santa Clara, CA, 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 d 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 and . shows that TILs expanded against aEM3 aAPCs maintained the CD8+ phenotype when compared to the same TILs ed against PBMC s. shows the results of a second batch of TILs from a different patient expanded against aEM3 aAPCs, where a clear se of CD8+ cells ) is seen in ison to the results from expansion using PBMC s (25%).
The CD4 and CD8 SPADE tree of TILs expanded with aEM3 aAPCs or PBMC feeders using CD3+ cells is shown in and . The color gradient is proportional to the mean fluorescence intensity (MFI) of LAG3, TIL3, PDl and CD137 or CD69, CDl54, KLRGl 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 ed with PBMC feeders.
Spare atory capacity (SRC) and glycolytic reserve were also evaluated for TILs expanded with aEM3 aAPCs in comparison to PBMC feeders, with results shown in and . The Seahorse XF Cell Mito Stress Test measures mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells, using tors 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 tochondrial 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 ates 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 ne potential. As a result, electron flow through the electron transport chain is uninhibited and oxygen is maximally consumed by complex IV. The timulated OCR can then be used to calculate spare atory capacity, defined as the difference between maximal respiration and basal respiration. Spare atory capacity (SRC) is a e 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. 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-l4 gaMOLMl4] and EM-3 [aEM3] aAPCs TILs expanded with PBMC feeders and aMOLMl4 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 and , and show that TILs expanded with aAPCs have similar functional properties (and expected clinical efficacy) to those expanded with PBMC feeders.
IFN-y release and Granzyme B release from TILs expanded with PBMC s and aMOLMl4 and aEM3 aAPCs as bed above was also assessed following overnight stimulation with microbeads coated with anti-CD3/CD28/4-1BB. The IFN—v release results are shown in and , and the Granzyme B release results are shown in and . Significant and surprising ses in IFN—v release and Granzyme B release were ed for TILs ed with aEM3 aAPCs relative to those expanded with PBMC feeders, but not for TILs expanded by aMOLMl4 aAPCs. t being bound by theory, this suggests that TILs ed with aEM3 aAPCs may be more active in vivo as a cancer y. Most other differences observed were not statistically significant.
The s of TIL expansions with aEM3 and aMOLMl4 aAPCs are summarized in Table 9.
TABLE 9. Summary of TIL ion results with aAPCs.
CD8 (%) Relative aAPC TIL# aAPC expansion PBMC aAPC aMOLM14 M1032-T2 092 LII W 65 M1033-T6 091 LII O 57 M1021T-5 099 \o ,_‘ 82 n-— M1030T-4 860 853 \D \o I; 78 08 00 I --— M1021T-1 087 I --— M1032T-1 085 I --— 133 00 8 -n— 0 99 g 87 aEM3 M1054 0 .80 \D 00 96 2 .10 N LII 66 M1021T-1 0.94 \l 75 0.78 --— Exam le 8 — Pre n 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 d Eagle Medium DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA), 50 mL fetal bovine serum (FB S) Heat Inactivated (HI) (Hyclone), 10 mM 4-(2-hydroxyethyl) piperazineethanesulfonic acid (HEPES buffer) (Life logies), 1X Primocin (Invivogen), 1X Plasmocin (Invivogen), and 1X 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 O.5-1><1O6 cells/mL in 24-well plates, and maintaining at about O.5-1.5><1O6 cells/mL, with incubation at 37 oC and 5% C02.
Further steps that may be ed 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 terization (HLA high resolution typing); cytokine release testing; testing of human serum to replace PBS to grow aAPC; testing freezing media to freeze aAPCs; master cell banking (including raw material testing and stability testing); standardization of irradiation ding 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); y; ; and identity g; mycoplasma and ity 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 ed 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 ing TILs is depicted in . 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 4 aAPCs); is shown in .
Example 10 — P815 Bioluminescent Redirected Lysis Assay ] In this example; the pment 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 or 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-CD38 (OKT-3); ing 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-specif1c (lymphokine activated killing, LAK) respectively. Following 4 hours of incubation, firefly luciferin ((4S)(6-hydroxy-1,3- benzothiazolyl)-4,5-dihydrothiazolecarboxylic acid, commercially available from multiple sources) was added to the wells and incubated for 5 minutes. Bioluminescence intensity was read using a meter. Percent cytotoxicity and survival were ated using the following formula: % Survival = (experimental survival - minimum)/(maximum signal -minimum signal) X 100, % Cytotoxicity = 100 - (% Survival). Interferon gamma release in the media supernatant of co-cultured TILs was analyzed by ELISA, and LAMPl (CD107a, clone eBioH4A3) expression on TILs was analyzed on a flow cytometer to te the cytotoxic potency of TILs.
Results are shown in to . illustrates percent toxicity of TIL batch M1033T-1 tured with P815 Clone G6 (with and without anti-CD3) at individual effector:target ratios by BRLA. illustrates enzyme-linked sorbent assay (ELISA) data g the amount of IFN—y released against different ratios of effector to target cells. illustrates LAMPl (%) 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 or to target cells for 4 hours and 24 hours co-culture.
The results were confirmed using a second TIL batch as shown in , which illustrates BRLA for TIL batch M1030. The cytotoxicity (measured as LU50/1 X 106 TIL) by BRLA is 26 :: 16. illustrates the results of a standard chromium release assay for TIL batch M1030. The cytotoxicity (measured as LU50/1 X 106 TIL) by chromium release assay is ] Results were r med using a third TIL batch. illustrates BRLA results for TIL batch M1053, showing lytic units of the TILs by BRLA as 70 :: 17. illustrates the results of a rd chromium release assay for TIL batch M1053, showing lytic unit of the TILs by chromium assay as 14 :: 5. Comparison of two assay s shows the comparable performance of the BRLA result to the chromium release assay . illustrates the linear relationship between IFN—y release and cytotoxic potential of TILs. illustrates ELISpot results for IFN—y. illustrates enzymatic IFN—y release for TIL batch M1053. illustrates enzymatic IFN—y release for TIL batch M1030. illustrates ELISpot data showing Granzyme B release by M1053T and M1030T. illustrates enzymatic Granzyme B release for TIL batch M1053. illustrates tic Granzyme B release for TIL batch M1030. illustrates ELISpot data showing TNF-d release by M1053T and M1030T. illustrates tic TNF-d release for TIL batch M1053. illustrates enzymatic TNF-d release for TIL batch M1030. The data in to confirms the potency of these s 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 ment of Lampl expression on TILs at individual time points demonstrates degranulation of cytotoxic T cells relative to the y 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 ility of BRLA. BRLA has a linear relationship with IFNv release by TILs. e assay of IFN—v, TNFOL and Granzyme B by ELISpot is consistent with the cytotoxic efficiency of the TILs evaluated by BRLA.
Example 11 — s 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 , aEM3 cells were successfully weaned off of FBS to hAB serum.
Example 12 — Freezing Media Formulation Op_timization ] To cryobank EM3 cells cultured as described herein, methods were freezing media formulation were optimized. As shown in , three freezing media were used and their effect on cell numbers were counted. The cell media utilized included CryStor 10 (Biolife Solutions (CSlO)) (A), hAB [90%] and DMSO [10%] (B), and hAB [20%] with DMSO [10%] and cDMEM2 [70%] (C). 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 ed in gas permeable cell culture flasks (1'.e., GREX flasks (Wilson Wolf Manufacturing)) and the effect on cell ng time was observed over an 8 day time course. As shown in , 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 ing to the processes described herein, a flow panel is was used to determine the purity of aEM3 aAPCs. The results of such analysis are described in FIGS. 79 and 80. As shown in , 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 (). e 15 — aEM3 Feeder Cells as an Alternative to PBMC Feeders ] As described , 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 sion, PBMCs and aEM3 cells were stimulated by treatment with OKT-3. As shown in , aEM3 cells displayed a comparatively different cytokine expression profile as compared to PBMCs. Surprisingly, the aEM3 cells of the present invention provide efflcacious TILs (as shown herein) without reproducing the same cytokine ion 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 CMl 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 zer (ThermoFisher), and Xvivo-20 (Lonza). As shown in , TIL expansion (PreREP) with CTS provided increased cell numbers as compared to CMl.
Additionally, tissue fragments were cultured with CMl 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 and , when counting cells at Days 11 and Day 22, respectively, TIL expansion (PreREP) with Prime CDM provided increased cell numbers as compared to CMl.
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 e of serum, aEM3 cells were cultured using various serum free media. aEM3 cells were cultured in 24 well plates at l X 106 cells per well for 3 days using general cell culture ols 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 ed with serum free media. The serum free media utilized for the study included CTS r oFisher), Xvivo 20 (Lonza), Prime-TCDM (Irvine), and XFSM (MesenCult) media. Cells were then counted on Day 3.
As shown in , CTS Omeizer and Prime-TCDM serum free media provided cell growth that was comparable to serum-based media (1'.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 ed for the preparation and preservation of aAPCs. Specifically, aEM3 cells from a cell line designated 3 were propagated and cryopreserved.
Thawing and recovery of aEM3 cells may be accomplished using the following nonlimiting procedure. eserved aEM3 cells are warmed slowly in pre-warmed media (37 0C) that is ed from CTS Omeizer Basal Media (Thermo Fisher), CTS Omeizer Cell Supplement o Fisher), Gentamicin (Lonza), and Glutamax (Life logies). The suspended cells are then centrifuged at 1500 rpm for 5 minutes at 4 oC. The resulting supernatant is discarded and the remaining aEM3 cells are resuspended in the foregoing media and plated (5 X 106 cells / 10 mL per well of a 6 well plate). ] ation of aEM3 cells may be accomplished using the following non-limiting procedure. Aliquots of the ing media are prepared in gas permeable cell culture flasks (i.e., GREX lO flasks (Wilson Wolf Manufacturing)). The plated aEM3 cells are washed by centrifugation (i.e., 1500 rpm for 5 minutes at 4 0C), resuspended in media, and added to the GREX flasks at cell density of l-2 X 106 cells/mL. The aEM3 cell suspension was diluted with mL of media and the GREX flasks were then incubated for 3-4 days at 37 0C under C02.
After 3-4 days, the GREX flasks were removed from the incubator and placed in a biological safety cabinet (B SC). The cultured aEM3 cells are carefully extracted from the GREX flasks by pipette and the resulting extraction is centrifuged to provide the sed number of aEM3 cells, which may be resuspended at a cell density of 10-20 X 106 cells per GREX lO flask.
An ative cryopreservation of aEM3 cells may be accomplished using the following non-limiting procedure. The foregoing GREX lO 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 sed number of aEM3 cells, which is resuspended in a volume of CryStor lO (Biolife Solutions) to provide a concentration of 10-100 X 106 cells/vial in cryovials. The aEM3 cell suspensions may be placed in a freezing container and transferred to a -80 0C freezer.
Example 19 — Demonstration of Rapid fl of aEM3 Cells Following Cflopreservation aEM3 cells from the TIL-R3 cell line (1-2 X 106 cells) were eserved according to the procedure set forth in Example 18 using CS-lO 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 (1'.e., Post-Thaw Recovery). As shown in and , the total live cell counts recovered y post thaw in two separate experiments.
TIL-R3 cells (l X 106 cells) were thawed (Day 3 post-thaw) and plated at a density of 0.5 X 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 X 106 cm2 or 0.5 X 106 cells/cmz. At the end of the first e, a cell count was performed. The resulting cell counts are shown in , which demonstrate both a recovery phase post-thaw and a growth phase.
Furthermore, TIL-R3 cells (20 X 106 cells) were cultured at a density of 2 X 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 , 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 X 106 cells/cmz.
Example 20 — CD8 Skewness, Expansion Performance, and CD3 Contamination of TILs Cultured with aEM3 aAPCs Fifteen different PreREP TIL lines (0.4 X 105 cells) were co-cultured with either aEM3 aAPCs (as described herein) or PBMC feeders (10 X 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 ed by flow try. The relative ratio of CD8+ cells was ated by the formula (CD8% aEM3)/(CD8% PBMC). The results shown in indicate that TILs cultured with aEM3 cells surprisingly promote CD8+ skewing and and an ed TIL product.
Additional results of these experiments are shown in , , and , where the results shown that TILs cultured with aEM3 aAPCs displayed able ion and less 3+ 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, ei al., Leukemia, 2013, 27, 6. 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 obin (hgb) PCR reaction and the primers used were as follows: Tel-1b primer (CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT) (SEQ ID NO:40), Tel-2b primer (GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT) (SEQ ID NOz4l); hgb1 primer (GCT TCT GAC ACA ACT GTG TTC ACT AGC) (SEQ ID N042); and hgb2 primer (CAC CAA CTT CAT CCA CGT TCA CC) (SEQ ID NO:43).
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 int standard curve from 0.08 ng to 250 ng using genomic DNA isolated from the 1301 human T-cell ia cell line able 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 . 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.
Aspects of the ion are as follows: 11. The aAPC of any one of Claims 1 to 10, wherein the nucleic acid encoding CD86 comprises SEQ ID NO:19. 12. The aAPC of any one of Claims 1 to 11, wherein the one or more costimulatory molecules comprises a 4-1BBL protein. 13. The aAPC of Claim 12, wherein the 4-1BBL protein comprises a sequence as set forth in SEQ ID N09, or a sequence comprising one or more conservative amino acid substitutions thereof. 14. The aAPC of Claim 12, wherein the one or more nucleic acids encoding the 4-1BBL protein comprises SEQ ID NO: 16.
. The aAPC of any one of Claims 1 to 14, wherein the one or more costimulatory molecules comprises an OX4OL protein. 16. The aAPC of Claim 15, wherein the OX4OL protein comprises a sequence as set forth in SEQ ID NO: 10, or a sequence comprising one or more conservative amino acid substitutions thereof. 17. The aAPC of any one of Claims 1 to 16, wherein the aAPC has been grown in a serum free media. 18. A method of expanding tumor ating lymphocytes (TILs), the method sing the step of contacting a population of TILs with an aAPC according to any of Claims 1 to 17, wherein the population of TILs is expanded. 19. A method of expanding a population of tumor infiltrating lymphocytes (TILs), the method comprising the steps of: (a) ucing 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 se a nucleic acid encoding CD86 and one or more nucleic acids ng one or more costimulatory molecules and wherein the myeloid cell expresses a CD86 protein and one or more costimulatory molecules, and (b) contacting the tion of TlLs with the tion of aAPCs in a cell culture medium.
. The method of Claim 19, wherein the cell culture medium further comprises 1L-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an l concentration of about ng/mL. 21. The method of any one of Claims 19 to 20, wherein the population of APCs expands the population of TILs by at least 50-fold over a period of 7 days in a cell e medium. 22. The method of any one of Claims 19 to 21, wherein the myeloid cell endogenously expresses HLA-A/B/C, ICOS-L, and CD58. 23. The method of any one of Claims 19 to 22, wherein the myeloid cell is a MOLM-14 cell. 24. The method of any one of Claims 19 to 23, n the myeloid cell is a EM-3 cell.
. The method of Claim 24, wherein the EM-3 cell is further transduced to express a single chain fragment variable (scFv) binding domain capable of binding the Fc domain of OKT-3 antibody. 26. The method of Claim 25, wherein the scFv binding domain comprises clone 7C12 (SEQ ID NO:27) or clone 8B3 (SEQ ID , or a sequence comprising one or more conservative amino acid substitutions thereof. 27. The method of any one of Claims 19 to 26, wherein the CD86 protein comprises SEQ ID N08, or a sequence comprising one or more conservative amino acid substitutions thereof. 28. The method of any one of Claims 18 to 26, wherein the nucleic acid encoding CD86 comprises SEQ ID NO: 19. 29. The method of any one of Claims 18 to 28, wherein the one or more costimulatory molecules comprises a 4-1BBL protein.
. The method of Claim 29, wherein the 4-1BBL protein comprises a ce as set forth in SEQ ID N09, or a sequence comprising one or more conservative amino acid substitutions thereof. 31. The method of Claim 29, wherein the one or more nucleic acids encoding the 4-1BBL n ses SEQ ID NO: 16. 32. The method of any one of Claims 18 to 31, wherein the one or more costimulatory molecules WO 81789 comprises an OX4OL protein. 33. The method of Claim 32, wherein the OX4OL protein comprises a sequence as set forth in SEQ ID NO: 10, or a sequence comprising one or more conservative amino acid substitutions thereof. 34. The method of any one of Claims 18 to 33, wherein the expansion is performed using a gas permeable ner.
. The method of any one of Claims 18 to 34, wherein the ratio of the population of TILs to the population of aAPCs is between 1 to 200 and l to 400. 36. The method of Claim 35, wherein the ratio of the population of TILs to the population of aAPCs is about 1 to 300. 37. 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), n the myeloid aAPC comprises CD86 and at least one co-stimulatory ligand that specifically binds with at least one co-stimulatory molecule on the TILs, wherein binding of the co-stimulatory molecule with the co-stimulatory ligand induces proliferation of the TILs, y specifically expanding TILs, and wherein the at least one co-stimulatory ligand comprises 4-lBBL. 38. A method of expanding tumor infiltrating lymphocytes (TILs), the method sing ting a population of TILs comprising a population of TILs with a myeloid artificial n presenting cell (aAPC), wherein the myeloid aAPC comprises CD86 and at least one co-stimulatory ligand that specifically binds with at least one co-stimulatory molecule on the TILs, n binding of the co-stimulatory le with the co-stimulatory ligand induces proliferation of the TILs, thereby specifically expanding TILs, and wherein the at least one co-stimulatory ligand comprises OX4OL. 39. A method of expanding tumor infiltrating lymphocytes (TILs), the method comprising contacting a population of TILs comprising a population of TILs with a d artificial antigen presenting cell (aAPC), wherein the myeloid aAPC comprises CD86 and at least two co-stimulatory ligands that specifically binds with at least two co-stimulatory molecules on the TILs; wherein binding of the co-stimulatory molecules with the mulatory ligands induces proliferation of the TILs; thereby specifically expanding TILs; and wherein the at least two co-stimulatory ligand comprises 4-lBBL and OX4OL. 40. 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 tion 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-A/B/C; ICOS—L; and CD58; and wherein the myeloid aAPCs are transduced to express a CD86 protein and a 4-1BBL protein. 41. The method of Claim 40; wherein the myeloid aAPCs comprise MOLM-l4 cells transduced with one or more viral vectors; wherein the one or more viral s comprise a nucleic acid encoding CD86 and a nucleic acid encoding ; and wherein the 4 cells express a CD86 n and a 4-lBBL protein. 42. The method of Claim 40; wherein the d aAPCs comprise EM-3 cells uced with one or more viral vectors; wherein the one or more viral s comprise a nucleic acid encoding CD86 and a nucleic acid encoding 4-lBBL; and wherein the EM-3 cells express a CD86 protein and a 4-lBBL protein. 43. The method of Claim 42; wherein the EM-3 cells are further transduced to s a single chain fragment variable (scFv) binding domain capable of binding the Fc domain of OKT-3 44. The method of Claim 43; wherein the scFv binding domain comprises clone 7C12 (SEQ ID NO:27) or clone 8B3 (SEQ ID NO:28), or conservative amino acid substitutions thereof. 45. The method of any one of Claims 40 to 44, wherein the rapid expansion is performed over a period not greater than 14 days. 46. The method of any one of Claims 40 to 45, wherein the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/mL and OKT-3 dy at an initial concentration of about 30 ng/mL. 47. The method of any one of Claims 40 to 46, wherein the expansion is performed using a gas permeable container. 48. The method of any one of Claims 40 to 47, wherein the ratio of the second population of TlLs to the population of aAPCs is n 1 to 200 and l to 400. 49. The method of Claim 48, wherein the ratio of the second population of TILs to the population of aAPCs is about 1 to 300. 50. The method of any one of Claims 40 to 49, 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 , renal cell carcinoma, pancreatic , and glioblastoma. 51. The method of any one of Claims 40 to 50, further comprising the step of treating the t with a non-myeloablative lymphodepletion regimen prior to administering the second population of TlLs to the patient. 52. The method of Claim 51, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of cyclophosphamide at a dose of 60 mg/mZ/day for two days followed by administration of fludarabine at a dose of 25 day for five days. 53. The method of any one of Claims 40 to 52, further comprising the step of treating the patient with a high-dose IL-2 regimen starting on the day after administration of the second population of TlLs to the patient. 54. The method of Claim 53, n the ose IL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin, or a biosimilar or t thereof, administered as a 15-minute bolus WO 81789 intravenous infusion every eight hours until tolerance. 55. A method of treating a cancer with a population of tumor infiltrating lymphocytes (TILs) comprising the steps of: (a) ing 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 1L-2, (c) performing a rapid expansion of the second population of TILs using a population of myeloid artificial antigen presenting cells (myeloid aAPCs) in a second cell culture medium to obtain a third population of TlLs, wherein the third population of TILs is at least 50-fold greater in number than the second population of TlLs after 7 days from the start of the rapid expansion; and wherein the second cell e medium comprises IL-2 and OKT-3, (d) administering a therapeutically effective portion of the third population of TlLs to a t with the cancer. 56. The method of Claim 55, wherein the myeloid aAPCs comprise MOLM-l4 cells transduced with one or more viral vectors, wherein the one or more viral vectors se a nucleic acid encoding CD86 and a nucleic acid encoding 4-1BBL, and wherein the MOLM-l4 cells express a CD86 n and a 4-1BBL protein. 57. The method of Claim 55, 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 ng CD86 and a nucleic acid ng 4-lBBL, and wherein the EM-3 cells express a CD86 protein and a 4-lBBL protein. 58. The method of Claim 57, wherein the EM-3 cells are further transduced to express a single chain fragment le (scFv) binding domain capable of binding the Fc domain of OKT-3 antibody. 59. The method of Claim 58, wherein the scFv binding domain comprises clone 7C12 (SEQ ID NO:27) or clone 8B3 (SEQ ID NO:28), or conservative amino acid substitutions thereof. 60. The method of any one of Claims 55 to 59, wherein 1L-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 e medium. 61. The method of any one of Claims 55 to 60, wherein the rapid expansion is performed over a period not greater than 14 days. 62. The method of any one of Claims 55 to 61, wherein the initial expansion is performed using a gas permeable container. 63. The method of any one of Claims 55 to 62, wherein the rapid expansion is med using a gas permeable container. 64. The method of any one of Claims 55 to 63, wherein the ratio of the second population of TlLs to the population of aAPCs in the rapid ion is between 1 to 80 and l to 400. 65. The method of Claim 64, wherein the ratio of the second population of TILs to the population of aAPCs in the rapid expansion is about 1 to 300. 66. The method of any one of Claims 55 to 65, wherein the cancer is selected from the group consisting of ma, 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. 67. The method of any one of Claims 55 to 65, further comprising the step of treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TlLs to the patient. 68. The method of Claim 67, wherein the non-myeloablative lymphodepletion regimen comprises the steps of administration of hosphamide at a dose of 60 mg/mZ/day for two days ed by administration of fludarabine at a dose of 25 mg/mZ/day for five days. 69. The method of any one of Claims 55 to 68, further comprising the step of treating the patient with a high-dose IL-2 regimen ng on the day after administration of the third population of TlLs to the patient. 70. The method of Claim 69, n the high-dose IL-2 regimen ses 600,000 or 720,000 IU/kg of aldesleukin, or a biosimilar or variant f, administered as a 15-minute bolus intravenous infusion every eight hours until tolerance. 71. The method of any one of Claims 55 to 70, wherein the first cell culture medium further comprises a second population of myeloid aAPCs, and wherein the second population of myeloid aAPCs comprises MOLM-l4 or EM-3 cells transduced with one or more viral vectors, wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and one or more nucleic acids encoding a co-stimulatory molecule selected from the group ting of 4-1BBL, OX4OL, or combinations f, and wherein the myeloid aAPCs s a CD86 protein and a co-stimulatory le. 72. A kit for specifically inducing proliferation of a tumor infiltrating lymphocyte expressing a known co-stimulatory molecule, the kit comprising an ive amount of an aAPC, wherein said aAPC comprises a MOLM-l4 cell or a EM-3 cell uced using a lentiviral vector (LV), wherein the LV comprises a c 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. 73. A method for assessing the potency of tumor infiltrating lymphocytes (TlLs) against cancer cells comprising the steps of: (a) providing a plurality of mouse mastocytoma P815 cells expressing the nous CD16 Fc receptor, wherein the P815 cells are uced with a lentiviral vector based on enhanced green fluorescent protein (GFP) and firefly luciferase, (b) co-culturing the plurality of P815 cells and TILs with OKT-3 to assess T cell receptor (TCR) activation for specific killing, and without OKT-3 to assess lymphokine activated killing (LAK) for non-specific killing, (c) incubating the co-culture for four hours, (d) adding luciferin and incubating for 5 minutes, (e) reading bioluminescence intensity from the co-culture using a luminometer, and (f) calculating percent cytotoxicity and survival. 74. A population of tumor infiltrating lymphocytes (TILs) for use in treating a cancer, wherein the population of tumor infiltrating lymphocytes is a first population of TILs and is able by a method comprising the steps of: (a) performing a rapid ion 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, n the first population of TILs are preViously obtained from a tumor resected from a patient, and further 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-A/B/C, ICOS—L, and CD58, and wherein the myeloid aAPCs are transduced to express a CD86 protein and a co- stimulatory molecule ed from the group consisting of a 4-lBBL protein, an OX40L protein, and a ation thereof. 75. The population of TILs for use of Claim 74, wherein the rapid expansion is performed over a period not greater than 14 days. 76. The population of TILs for use of Claim 74 or Claim 75, wherein the cell culture medium r comprises 1L-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an initial concentration of about 30 ng/mL. 77. The population of TILs for use of any of Claims 74 to 76, wherein the expansion is performed using a gas permeable container. 78. The population of TILs for use of any of Claims 74 to 77, wherein the ratio of the second tion of TILs to the population of aAPCs is n 1 to 200 and l to 400. 79. The population of TILs for use of Claim 78, wherein the ratio of the second population of TILs to the population of aAPCs is about 1 to 300. 80. A population of tumor infiltrating lymphocytes (TILs) for use in ng a cancer in a patient, wherein the population of tumor infiltrating lymphocytes is a third population of TILs and is 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 the second population of TILs, wherein the first population of TILs are obtainable from a tumor ed from a patient, and futher 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 1L-2, (b) performing a rapid expansion of the second population of TILs using a tion of myeloid artificial antigen ting cells (myeloid 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 e medium comprises IL-2 and OKT-3. 81. The population of TILs for use of Claim 80, wherein the rapid expansion is performed over a period not greater than 14 days. 82. The population of TILs for use of Claim 80 or Claim 81, n the cell culture medium further comprises 1L-2 at an initial concentration of about 3000 IU/mL and OKT-3 antibody at an l concentration of about 30 ng/mL. 83. The population of TILs for use of any of Claims 80 to 82, 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. 84. The population of TILs for use of any of Claims 80 to 83, wherein the initial expansion is performed over a period not greater than 14 days. 85. The population of TILs for use of any of Claims 80 to 84, n the initial expansion and/or rapid expansion is performed using a gas permeable container. 86. The population of TILs for use of any of Claims 80 to 85, wherein the d aAPCs comprise MOLM-l4 cells transduced to express a CD86 protein and a co-stimulatory molecule selected from the group consisting of 4-1BBL protein, OX40L protein, and a ation thereof. 87. The population of TILs for use of any of Claims 80 to 86, wherein the myeloid aAPCs comprise EM-3 cells transduced to express a CD86 protein and a co-stimulatory molecule selected from the group consisting of 4-lBBL protein, OX40L protein, and a combination thereof. 88. The population of TILs for use of Claim 87, wherein the EM-3 cells are further transduced to express a single chain fragment variable (scFv) binding domain capable of binding the Fc domain of OKT-3 antibody. 89. The population of TILs for use of Claim 87, wherein the scFv binding domain comprises clone 7C12 (SEQ ID NO:27) or clone 8B3 (SEQ ID NO:28), or vative amino acid substitutions thereof. 90. The population of TILs for use of any of Claims 80 to 89, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer (NSCLC), lung , bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck , renal cancer, renal cell carcinoma, pancreatic cancer, and glioblastoma. 91. The population of TILs for use of any of Claims 80 to 90, which is for administration to a patient subsequent to the patient undergoing a non-myeloablative lymphodepletion regimen. 92. The population of TILs for use of Claim 91, which is for administration uent to administration of cyclophosphamide at a dose of 60 mg/mZ/day for two days followed by administration of fludarabine at a dose of 25 day for five days. 93. The population of TILs for use of any of Claims 80 to 92, which is for administration a day prior to a high-dose lL-2 regimen. 94. The tion of TILs for use of Claim 93, wherein the high-dose IL-2 regimen comprises 0 or 720,000 IU/kg of aldesleukin, or a biosimilar or variant thereof, administered as a ute bolus intravenous infusion every eight hours until tolerance. 95. A combination of (l) the population of TILs for use of any of Claims 80 to 94, (2) cyclophosphamide and (3) fludarabine, n cycolphoshamide in for administration at a dose of 60 mg/ mZ/day for two days followed by fludarabine which is for itration at a dose of 25 mg/ mZ/day for five days. 96. A combination of the population of TILs for use of any of Claims 80 to 92 and a high-dose- IL-2 regime, wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kg of aldesleukin, or a biosimilar or variant f, for administration as a 15-minute bolus intravenous on every eight hours until tolerance. 97. An artificial antigen presenting cell (aAPC) comprising a myeloid cell cally engineered to express CD86 and one or more costimulatory molecules selected from the group consisting of 4-1BBL, OX4OL, and a combination thereof. 98. The aAPC of Claim 97, wherein the myeloid cell is an EM-3 cell, and wherein the myeloid cells are further genetically engineered to eXpress a single chain nt variable (scFv) binding domain capable of binding the Fc domain of OKT-3 antibody. 99. The aAPC of Claim 98, wherein the EM-3 cell is genetically engineered to express 4-lBBL and OX4OL.
We

Claims (29)

claim:
1. An cial antigen presenting cell (aAPC) comprising a myeloid cell transduced with one or more viral s, n the one or more viral vectors comprise a nucleic acid encoding CD86 and one or more nucleic acids encoding a costimulatory molecule, and wherein the myeloid cell expresses a CD86 protein and one or more costimulatory molecules.
2. The aAPC of Claim 1, wherein the aAPC can stimulate and expand tumor infiltrating lymphocytes (TILs) contacted with the aAPC.
3. The aAPC of any one of Claims 1 to 2, 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.
4. The aAPC of any one of Claims 1 to 2, wherein the aAPC can stimulate and expand a T cell contacted with the aAPC.
5. The aAPC of any one of Claims 1 to 4, wherein the myeloid cell endogenously ses HLA-A/B/C, ICOS-L, and CD58.
6. The aAPC of any one of Claims 1 to 5, wherein the d cell is a MOLM-14 cell.
7. The aAPC of any one of Claims 1 to 5, wherein the myeloid cell is a EM-3 cell.
8. The aAPC of Claim 7, wherein the EM-3 cell is further transduced to express a single chain fragment variable (scFv) binding domain capable of binding the Fc domain of OKT-3 antibody.
9. The aAPC of Claim 8, wherein the scFv g domain comprises clone 7C12 (SEQ ID NO:27) or clone 8B3 (SEQ ID NO:28), or a sequence comprising one or more conservative amino acid substitutions thereof.
10. The aAPC of any one of Claims 1 to 9, wherein the CD86 protein comprises a sequence as set forth in SEQ ID NO:8, or a sequence sing one or more conservative amino acid substitutions thereof.
11. The aAPC of any one of Claims 1 to 10, wherein the nucleic acid ng CD86 comprises SEQ ID NO: 19.
12. The aAPC of any one of Claims 1 to 11, wherein the one or more costimulatory molecules comprises a 4-lBBL protein.
13. The aAPC of Claim 12, wherein the 4-lBBL protein comprises a ce as set forth in SEQ ID NO:9, or a sequence comprising one or more conservative amino acid substitutions thereof.
14. The aAPC of Claim 12, wherein the one or more c acids encoding the 4-lBBL n comprises SEQ ID NO: 16.
15. The aAPC of any one of Claims 1 to 14, wherein the one or more costimulatory molecules comprises an OX40L protein.
16. The aAPC of Claim 15, wherein the OX40L n comprises a sequence as set forth in SEQ ID NO: 10, or a sequence comprising one or more conservative amino acid substitutions thereof.
17. The aAPC of any one of Claims 1 to 16, wherein the aAPC has been grown in a serum free media.
18. A method of expanding tumor infiltrating lymphocytes (TILs), the method comprising the step of contacting a population of TILs with an aAPC according to any of Claims 1 to 17, wherein the population of TILs is expanded.
19. 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 tion of artificial antigen presenting cells (aAPCs), wherein the one or more viral vectors comprise a nucleic acid encoding CD86 and one or more nucleic acids encoding one or more costimulatory molecules and wherein the myeloid cell expresses a CD86 protein and one or more costimulatory molecules, and (b) contacting the population of TILs with the population of aAPCs in a cell culture medium.
20. The method of Claim 19, 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.
21. The method of any one of Claims 19 to 20, n the tion of APCs expands the population of TILs by at least 50-fold over a period of 7 days in a cell culture medium.
22. The method of any one of Claims 19 to 21, wherein the myeloid cell endogenously ses HLA-A/B/C, ICOS-L, and CD58.
23. The method of any one of Claims 19 to 22, wherein the d cell is a MOLM-14 cell.
24. The method of any one of Claims 19 to 23, wherein the myeloid cell is a EM-3 cell.
25. The method of Claim 24, wherein the EM-3 cell is further transduced to s a single chain fragment variable (scFv) binding domain e of binding the Fc domain of OKT-3 antibody.
26. The method of Claim 25, wherein the scFv binding domain comprises clone 7C12 (SEQ ID NO:27) or clone 8B3 (SEQ ID NO:28), or a sequence comprising one or more conservative amino acid substitutions thereof.
27. The method of any one of Claims 19 to 26, wherein the CD86 protein comprises SEQ ID NO:8, or a sequence comprising one or more conservative amino acid substitutions thereof.
28. The method of any one of Claims 18 to 26, n the nucleic acid encoding CD86 comprises SEQ ID NO: 19.
29. The method of any one of Claims 18 to 28, wherein the one or more costimulatory molecules ses a 4-1BBL protein. 300-- g £00 C) 4.;m M1615? mmm HG. '1 SUBSTITUTE SHEET (RULE 26) RSV U3 regicm ~ LTR - ~~ . HEVR regicn ampR Hiv Psi packaging Signai i EBNA? . HEV CPPT MSCV LTR HEV SEN ASCV LTR 406 seq F Hiv R region HESV PRE PEG. 2 SUBSTITUTE SHEET (RULE 26) kozac: B‘s 41BBL F 2 frame B2 41BBL R afi81p g 4133 0009 p 41BBL 131 82 FOR? 892 hp F¥(§.3 SUBSTITUTE SHEET (RULE 26) v «w‘ HiVRregion ampR f ‘ R IV RR” pLV43CIG hem-36 7776 hp """ Haw '>r‘a' HEW RRE’ €986 (Coon) frame PEG. 4 SUBSTITUTE SHEET (RULE 26) CD86 COOP B1 82 PCRP g 1039 hp HG. 5 SUBSTITUTE SHEET (RULE 26) mHWMnmmmam pU-C: Ori rmBfltmmmMfimflmmmam ’.. wn3 o40>fi3nward pfinfier ‘ M13 (-20) ferward primer {200% R221 hCDSE‘3 a } f frame 1 3526 hp ; ‘ i} ‘kozac 9986 (GOOD) frame T7 primer T7 pramoter PEG. 6 SUBSTITUTE SHEET (RULE 26) rrrrB T2 transcription ‘rerrrrirreter err-C: Orr .. rrrrB transcription terminator M13 (~40) ‘r’erward primer 2‘ “““ M13 (-20) forward primer weer. £3on frame 3 are. 1 r7 premerer 1 T7 primer M13 reverse primer PEG. 7 SUBSTITUTE SHEET (RULE 26) RSV U3- regian /:—« :R,.¥ LTR / ': REV R regian ...... .. g . Hi‘v' PS; pagkaging Sigma; i EBNA ’2 RN 332an ’MSCVLTR HEV SEN LTR ”K;MSCVLTR4oaseqF HIV R region ~B: Fifi. 8 SUBSTITUTE SHEET (RULE 26) rrrrB T2 irarrscripiimr terrriirraiar mm H transcripiicri terminator 5 M13 (40) forward primer . M13 (-20) forward primer M13- reverse primer r7 primer :~ T7 ier “ Fifi. 9 SUBSTITUTE SHEET (RULE 26) WO 81789
NZ793899A 2016-10-31 2017-10-31 Engineered artificial antigen presenting cells for tumor infiltrating lymphocyte expansion NZ793899A (en)

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US62/438,600 2016-12-23
US62/475,053 2017-03-22
US62/481,831 2017-04-05

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