WO1990014097A1 - Improved culture of human tumor-infiltrating lymphocytes by combining gas permeable bags and a primary culture container - Google Patents

Improved culture of human tumor-infiltrating lymphocytes by combining gas permeable bags and a primary culture container Download PDF

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Publication number
WO1990014097A1
WO1990014097A1 PCT/US1990/002671 US9002671W WO9014097A1 WO 1990014097 A1 WO1990014097 A1 WO 1990014097A1 US 9002671 W US9002671 W US 9002671W WO 9014097 A1 WO9014097 A1 WO 9014097A1
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culture
primary
bulk
cells
primary culture
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PCT/US1990/002671
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French (fr)
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Ara Toonnaz Nahapetian
Jerilou Ann Wiedmeyer
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E.I. Du Pont De Nemours And Company
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]

Definitions

  • Adoptive immunotherapy involves either a) partial removal of host peripheral blood lymphocytes and activation in vitro with interleukin-2 (IL-2) for production of lymphokine activated killer cells (LAK) or b) surgery, enzymatic digestion of tumor mass, in vitro expansion of tumor infiltrating lymphocytes (TIL) cells and infusion of "pure" LAK or TIL cells with antitumor activity to the tumor-bearing host.
  • IL-2 interleukin-2
  • TIL tumor infiltrating lymphocytes
  • Both media are supplemented with 20% autologous LAK conditioned medium, 1000 U/mL recombinant IL-2 (rIL-2), 2 mM glutamine, 50 IU/mL penicillin, streptomycin, gentamicin and amphotericin 250 ng/mL and Hepes buffer 10 mM.
  • TTL cultures are distributed in 2 ml aliquots into 24-well culture plates for experimental purposes or into 175 cm 2 flasks , 850 cm 2 roller bottles or 750 cm 2 culture bags for bulk cultures. They are maintained at 37° C in a humidified 5% CO2 atmosphere.
  • the present invention relates to improvements of this conventional protocol for TIL culture.
  • a process for the expansion and maintenance of TIL cells which significantly enhances cell density, overall expansion rate and duration of active expansion.
  • Using the process of the present invention and particularly by supplementing the bulk culture with primary culture cells and/or conditioned medium it is possible to expand sufficient TIL cells for one round of therapy, about 2 x lOell cells, using a total of approximately 80 L, a significant improvement over conventional techniques which require approximately 200 L of culture medium (See: Topalian, et al, "Immunotherapy of Patients with Advanced Cancer Using Tumor- Infiltrating Lymphocytes and Recombinant Interleukin-2: A Pilot Study", Journal of Clinical
  • the invention is an improved process for the culture of human tumor-infiltrating lymphocytes (TIL).
  • TIL tumor-infiltrating lymphocytes
  • the process comprises placing a human tumor sample in a suitable primary culture container wherein the tumor sample and particularly the TIL cells derived therefrom are maintained and expanded for a period of time. After a period of time, a substantial amount of the TIL cells present in the primary culture container are transferred to a suitable bulk culture container. A portion, preferrably about 25%, of the TIL cells in the primary culture container, is retained in the primary culture container wherein it is maintained and expanded by the periodic addition of suitable culture medium and split if necessary.
  • the portion of the primary culture transferred to the bulk culture container hereinafter referred to as the bulk culture, is maintained and expanded by adding culture medium and splitting the bulk culture periodically. None of the conditioned medium from either the bulk cultures or the primary cultures is discarded throughout expansion.
  • the bulk culture is further maintained and expanded by periodically supplementing the bulk culture with primary culture cells, or primary culture conditioned medium or both, from the primary culture container to boost or activate the TIL cells present in the bulk culture. Periodically, said bulk culture is mixed, such as by shaking, and said bulk culture and primary culture containers are stored lying flat in an incubator at 37° C, in 5% CO2.
  • Tumor-derived material means cells and particularly TIL cells, tumor antigen, antigen presenting cells and conditioned medium derived from the tumor sample.
  • Constant medium means cell-free medium obtained from a culture of cells.
  • Primary culture means those TIL cells and other tumor- derived material originally placed in culture in a primary culture container such as a multiwell plate or a vessel made of ionomers, such as sodium or zinc neutralized copolymers of ethylene and acrylic or methacrylic acid or derivatives thereof, such as a Surlyn ® bag, which are continuously maintained and expanded in said original primary culture-. container.
  • a primary culture container such as a multiwell plate or a vessel made of ionomers, such as sodium or zinc neutralized copolymers of ethylene and acrylic or methacrylic acid or derivatives thereof, such as a Surlyn ® bag, which are continuously maintained and expanded in said original primary culture-. container.
  • Bulk culture means those TIL cells expanded from the original tumor sample but which are transfe ⁇ ed from the primary culture container into one or more secondary vessels such as gas permeable bags, bags made of ionomers, flasks or roller bottles wherein the TIL cells are maintained and expanded.
  • Substantial amount means about 50 to 75% and prefe ⁇ ably 75% of TIL cells present in the primary culture container.
  • Splitting means dividing the cell culture into additional bulk culture containers.
  • “Supplementing” means the adding of tumor-derived material, TIL cells and/or conditioned medium, from the primary culture into the bulk culture.
  • a human tumor sample is placed in a suitable primary culture container, such as a polystyrene multiwell plate, commercially available from Costar, Cambridge, MA, or a container made of an ionomer such as Surlyn ® , commercially available from E. I. du Pont de Nemours, Wilmington, DE.
  • a suitable primary culture container such as a polystyrene multiwell plate, commercially available from Costar, Cambridge, MA, or a container made of an ionomer such as Surlyn ® , commercially available from E. I. du Pont de Nemours, Wilmington, DE.
  • suitable primary culture container such as a polystyrene multiwell plate, commercially available from Costar, Cambridge, MA, or a container made of an ionomer such as Surlyn ® , commercially available from E. I. du Pont de Nemours, Wilmington, DE.
  • suitable culture medium preferrably supplemented with rIL-2.
  • TIL cells present in the tumor sample may be stimulated by the presence of tumor derived material, including but not limited to tumor antigen, antigen presenting cells or conditioned medium.
  • tumor derived material including but not limited to tumor antigen, antigen presenting cells or conditioned medium.
  • a substantial amount of the primary culture is transferred to a bulk culture container, prefe ⁇ ably a gas permeable culture vessel such as a SteriCellTM bag, commercially available from E.I. du Pont de Nemours, Inc., Wilmington, DE.
  • the bulk culture is maintained and expanded by feeding the culture with suitable culture medium such as that used in the Rosenberg protocol (AIM-V supplemented with rIL-2) and periodically splitting the bulk culture into additional bulk culture containers.
  • suitable culture medium such as that used in the Rosenberg protocol (AIM-V supplemented with rIL-2)
  • the bulk culture is supplemented with primary culture cells and/or primary culture conditioned medium.
  • the TIL cells in the bulk culture are stimulated by the addition of these primary culture
  • a prefe ⁇ ed embodiment of this invention comprises the process as described herein utilizing either a polystyrene multiwell plate or a Surlyn® bag (Du Pont) as the primary culture container and a gas permeable culture vessel such as the SteriCellTM bag (Du Pont), as the bulk culture container.
  • Most prefe ⁇ ed is the combination of the Surlyn® bag with the SteriCellTM bag because of the convenience associated with use of bags compared to multiwell plates and because this combination renders a completely closed system for cell culture.
  • gas permeable nature of the prefe ⁇ ed SteriCellTM bulk culture container allows CO2 and O2 to freely pass through the bag into and out of the culture medium, this reduces the risk that the lymphocytes will have limited expansion due to lack of O 2 .
  • cell culture bags such as SteriCellTM provide an increased surface area on which the cells expand, this increased surface area prevents aggregation or clumping of cells which may be undesirable because it often results in cells being O2 starved.
  • gas permeable vessels are the prefe ⁇ ed bulk culture containers, other vessels such as T-flasks or roller bottles can be used in the present process.
  • a fresh tumor sample is placed in the primary culture container on the day of surgery or shortly thereafter.
  • a cryopreserved tumor sample may be used by being placed in the primary culture container upon being thawed.
  • a substantial amount of the primary culture about 50-75% and prefe ⁇ ably about 75%, is transfe ⁇ ed to the bulk culture container.
  • the pH and cell density need not be measured, but rather may be determined by observation, such as by color of the culture medium or by estimation of cell number.
  • the color of the culture medium is usually orange-yellow where phenol red is the pH indicator.
  • a portion of the primary culture about 25-50% and prefe ⁇ ably about 25%, is retained in the primary culture container wherein it is maintained and expanded in cell number by the addition of suitable culture medium.
  • the bulk culture is also maintained and expanded in cell number by the periodic addition of suitable culture medium.
  • the addition of such culture medium to the bulk culture is based on the needs of the cultures, for example, the pH of the bulk culture, such that when the pH declines to 7.0 or lower the volume in the bulk culture is doubled with fresh medium.
  • the bulk culture is split into additional bulk culture vessels and maintained and expanded in all additional bulk culture containers such that none of the culture medium is ever discarded or wasted.
  • Such feeding and splitting procedures are known to those skilled in the art of cell culturing.
  • the bulk culture is supplemented with cells and/or conditioned medium from the primary culture container.
  • Primary culture cells are the most prefe ⁇ ed source of supplementation although primary culture conditioned medium or a combination of primary culture cells and conditioned medium are also effective.
  • the bulk culture is maintained and expanded in cell number until 2 x lOel l TIL cells, the desirable dose for reintroduction into a patient, have been obtained.
  • TIL cells can then be suspended in a suitable pharmaceutical carrier such as saline, saline containing 5% normal human serum albumin or Hank's balanced salt solution to provide a composition which also prefe ⁇ ably includes rIL-2, which can be infused into a patient inflicted with a tumor.
  • a suitable pharmaceutical carrier such as saline, saline containing 5% normal human serum albumin or Hank's balanced salt solution to provide a composition which also prefe ⁇ ably includes rIL-2, which can be infused into a patient inflicted with a tumor.
  • the TIL cells obtained using the prefe ⁇ ed process of the present invention demonstrate cytotoxic activity greater than or equal to that of cells cultured by the conventional method.
  • the culture medium used in the present invention may be of a kind known in the art, such as RPMI-1640 or AIM-V both prefe ⁇ ably supplemented with LAK supernatant, antibiotics and rIL-2, as discussed above.
  • a further prefe ⁇ ed embodiment of this invention provides for mixing of the bulk culture containers about once daily during expansion of the TIL cells. This mixing can be by vigorous shaking in a horizontal left and right direction for a few seconds if the bulk culture device is a gas permeable vessel such as a SteriCellTM culture vessel. Such mixing breaks up aggregated cells which often form in clump or cluster formations which cause the cells in the center of the clump or cluster to become O 2 starved.
  • Another embodiment of this invention provides an improved single step process for TIL cell expansion and maintenance wherein the human tumor sample is placed in a primary culture container wherein the sample is maintained and expanded throughout the process such that no culture medium is discarded or wasted.
  • the term primary culture container is interchangeable with bulk culture container as it is used in previous embodiments of this invention, and is prefe ⁇ ably a Surlyn ® bag (DuPont). Since one small Surlyn ® bag (100cm 2 ) can substitute for two multiwell plates and renders a closed system for cell culture, the bags are prefe ⁇ ed over multiwell plates for this embodiment.
  • the cells expanded solely in the Surlyn ® bag differ morphologically from those expanded solely in the multiwell plates. Cells expanded in the Surlyn ® bags appear more healthy, plump and bright, whereas cells expanded in the multiwell plates tend to form aggregates, appear smaller and less bright and start to disintegrate toward the end of the expansion period.
  • the tumor sample and particularly the TIL cells therein, placed in the primary culture container is maintained by periodic feeding and splitting whereby each additional Surlyn® bag has cells and conditioned medium from the original culture.
  • the expansion of ⁇ L cells using this process is exemplified in Example 2, showing expansion of cells in both multiwell plates only, and expansion of cells in Surlyn® bags only.
  • TIL cells expanded using the process of the present invention have higher cell density, improved expansion rate and longer duration of active expansion.
  • TIL cell expansion via the present invention requires considerably less culture medium than conventional expansion techniques, approximately 120 L less per therapeutic dose, thereby conserving space in the laboratory as well as reducing the cost of expansion of TIL cells.
  • the TIL cells maintain cytotoxicity equal to or greater than those TIL cells expanded using the conventional method.
  • Feeding and splitting of the cultures were based on need. During the entire course of the study, when either the culture pH was found to be lower than 7 (by visual examination/pH determination) or viable cell number was found to be higher than 1 x 10e6 cells/mL, culture volume was doubled with the basal medium described above and it was split into additional experimental vessels.
  • SteriCell- 1 and SteriCell-2 were fed by addition of AIM-V as discussed above. Due to the increase in volume of the bulk cultures with the addition of AIM-V during feeding, the bulk cultures, designated SteriCell-1 and SteriCell-2, were split such that all bulk cultures which originated from the MWP primary culture and SteriCell- 1 bulk culture container were divided into bulk culture containers designated SteriCell-3 and SteriCell-5. The bulk cultures originating from the Surlyn® bag primary culture container and SteriCell-2 bulk culture container were split into bulk culture containers designated SteriCell-4 and SteriCell-6.
  • splitting of the culture was done based on color and/or cell number as discussed above. Such splitting is known to those skilled in the art of cell culturing. It is noted that when the cell cultures are split, the conditioned medium is not removed from the cells and none of the conditioned medium is discarded. Generally, the culture volume was doubled with AIM-V in a SteriCellTM vessel and then about 50% of the volume was transfe ⁇ ed to an additional SteriCellTM vessel such that splitting of SteriCell- 1 (150cm 2 ) resulted in transfers of about 50% of the volume to SteriCell-3 (150cm 2 ) and to SteriCell-5 (150cm 2 ). The bulk culture in SteriCell-5 was later transfe ⁇ ed into a 700 cm 2 SteriCellTM vessel and ultimately into four 700 cm 2 SteriCellTM vessels, still designated SteriCell-5.
  • SteriCell-1 As with SteriCell-1, SteriCell-3 and SteriCell-5, splitting of SteriCell-2 (150cm 2 ) resulted in transfers of approximately 50% of the volume, to SteriCell-4 (150cm 2 ) and SteriCell-6 (150cm 2 ).
  • SteriCell-6 The bulk culture of SteriCell-6 was later transfe ⁇ ed into a 700cm 2 SteriCellTM vessel, and ultimately into four 700cm 2 SteriCellTM vessels, still designated SteriCell-6.
  • the primary cultures in the multiwell plates and Surlyn ® bags were fed and about 50 - 75% of the primary cultures were added to their respective bulk culture SteriCellTM vessels such that only primary culture cells and/or conditioned medium from the multiwell plates were used to supplement SteriCells-1, 3 and 5 while only primary cultures cells and/or conditioned medium from the Surlyn ® bags were used to supplement SteriCells-2, 4 and 6.
  • TIL 805 Cells Total Viable Cell Number
  • TIL 805 Cells Total Viable Cell Number
  • SteriCell-4 1 9 1.68 X 10e7 SteriCell-4 29 4.96 X 10e8
  • SteriCell-6 2 1 2.35 X 10e7 SteriCell-6 32 1.70 X lOelO
  • TIL 805 Cells Percent Cell Viability of Cells Present in the MWP Primary Culture
  • TIL 805 Cells Percent Cell Viability of Cell Present in the Surlyn® Bag Primary ult re
  • TIL 805 cells being supplemented from the multiwell plate primary culture containers were expanded from 2.80 X 10e7 mixed tumor and TIL cells to 1.66 X lOelO TIL cells (593 fold expansion) with about 87% viability. This expansion required a total of about 8 L of AIM-V medium and took 30 days.
  • TIL 805 cells were studied, the expansion in this example focuses on the TIL 805 cells which were maintained and expanded throughout the study in the original Surlyn® bag primary culture container. The material and methods overlap those in Example 1.
  • Example 1 On day two following surgery, 28mL of the culture of Example 1 (containing about 1.40 x 10e7 cells) was transfe ⁇ ed to a Surlyn ® bag (90cm 2 , Du Pont). The cells were maintained and expanded in this Surlyn® bag throughout the study and were fed and split as follows:
  • a Surlyn ® bag is much more convenient than a multiwell plate and is particularly more convenient in the single step process exemplified in Example 2.
  • ImL per each well 24 wells/plate
  • bags are more easily stored than plates and conserve space and provide a closed system for culturing cells.
  • the process for the expansion and maintenance of TIL cells of the present invention is commercially and practically advantageous over conventional systems and may be therapeutically advantageous. The process yields a higher density of cells which maintain a high specificity of cytotoxicity.

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Abstract

There is disclosed a process for the enhanced expansion and maintenance of human tumor-infiltrating lymphocytes (TIL). The process comprises placing a human tumor sample in a primary culture container and maintaining the sample in the primary culture container for a period of time before transferring a substantial amount of the primary culture to a bulk culture container. The primary culture cells and/or conditioned medium retained in the primary culture container are periodically used to supplement the bulk culture. Both primary and bulk cultures are maintained and expanded by periodic addition of a suitable culture medium as needed.

Description

TITLE
IMPROVED CULTURE OF HUMAN TUMOR-INFILTRATING
LYMPHOCYTES BY COMBINING GAS PERMEABLE BAGS AND A PRIMARY CULTURE
CONTAINER
BACKGROUND OF THE INVENTION
Because of the relative ineffectiveness of conventional chemotherapy, new approaches to cancer therapy have been developed, one such new approach is adoptive cellular immunotherapy. Adoptive immunotherapy involves either a) partial removal of host peripheral blood lymphocytes and activation in vitro with interleukin-2 (IL-2) for production of lymphokine activated killer cells (LAK) or b) surgery, enzymatic digestion of tumor mass, in vitro expansion of tumor infiltrating lymphocytes (TIL) cells and infusion of "pure" LAK or TIL cells with antitumor activity to the tumor-bearing host. Clinical trials at the National
Cancer Institute (NCI) using LAK cells and recombinant interleukin-2 (rIL-2) in the adoptive immunotherapy of patients with advanced cancers have been under way for some time. These studies have demonstrated tumor regression in selected patients and also the feasibility of administering adoptive immunotherapy regimens containing IL-2. Further studies identified TIL cells as tumoricidal cells that are more therapeutic in vivo than LAK cells, and therefore useful in clinical trials. These findings led to a pilot study of the use of TIL cells for Immunotherapy. See: Topalian et al,'Tmmunotherapy of Patients with Advanced Cancer Using Tumor-Infiltrating Lymphocytes and Recombinant Interleukin-2: A Pilot Study", Journal of Clinical Oncology, Vol. 6, No 5 (May), 1988: pp839-853 and Rosenberg et al, "Immunotherapy of Patients with Metastatic Melanoma Using Tumor Infiltrating Lymphocytes and Interleukin-2: Preliminary Report", at the National Cancer Institute. According to the Rosenberg protocol, the standard protocol in the field of immunotherapy with TIL cells, tumor and TIL cell mixed cultures are initiated following an enzymatic digestion from freshly resected or cryopreserved tumor mass. Single-cell tumor suspensions are diluted in culture medium at a concentration of 2.5- 5.0 x 105 viable cells/mL. Culture media used, are either a serum- free medium (such as AIM-V) or RPMI-1640 supplemented with 10% heat inactivated human AB serum. Both media are supplemented with 20% autologous LAK conditioned medium, 1000 U/mL recombinant IL-2 (rIL-2), 2 mM glutamine, 50 IU/mL penicillin, streptomycin, gentamicin and amphotericin 250 ng/mL and Hepes buffer 10 mM. TTL cultures are distributed in 2 ml aliquots into 24-well culture plates for experimental purposes or into 175 cm2 flasks , 850 cm2 roller bottles or 750 cm2 culture bags for bulk cultures. They are maintained at 37° C in a humidified 5% CO2 atmosphere. Cultures are harvested, pelleted and resuspended in fresh medium with fresh rIL-2 on a weekly basis, or as dictated by the growth rate of the culture. Viable cell concentrations are returned to 2.5 x 105 cells/ml at each passage. See Topalian, S. et al., "Expansion of Human Tumor Infiltrating Lymphocytes For Use in Immunotherapy Trials," Journal of Immunological Methods, 102 (1987) 127-141.
The present invention relates to improvements of this conventional protocol for TIL culture. There is provided a process for the expansion and maintenance of TIL cells which significantly enhances cell density, overall expansion rate and duration of active expansion. Using the process of the present invention and particularly by supplementing the bulk culture with primary culture cells and/or conditioned medium, it is possible to expand sufficient TIL cells for one round of therapy, about 2 x lOell cells, using a total of approximately 80 L, a significant improvement over conventional techniques which require approximately 200 L of culture medium (See: Topalian, et al, "Immunotherapy of Patients with Advanced Cancer Using Tumor- Infiltrating Lymphocytes and Recombinant Interleukin-2: A Pilot Study", Journal of Clinical
Oncology, Vol. 6, No 5 (May), 1988: pρ839-853) thereby reducing cost significantly as well as conserving space in the laboratory. In addition, the expanded cells maintain their cytotoxicity equal to or greater than TIL cells expanded in conventional systems.
SUMMAR Y OF THE INVENTION The invention is an improved process for the culture of human tumor-infiltrating lymphocytes (TIL). The process comprises placing a human tumor sample in a suitable primary culture container wherein the tumor sample and particularly the TIL cells derived therefrom are maintained and expanded for a period of time. After a period of time, a substantial amount of the TIL cells present in the primary culture container are transferred to a suitable bulk culture container. A portion, preferrably about 25%, of the TIL cells in the primary culture container, is retained in the primary culture container wherein it is maintained and expanded by the periodic addition of suitable culture medium and split if necessary. The portion of the primary culture transferred to the bulk culture container, hereinafter referred to as the bulk culture, is maintained and expanded by adding culture medium and splitting the bulk culture periodically. None of the conditioned medium from either the bulk cultures or the primary cultures is discarded throughout expansion. The bulk culture is further maintained and expanded by periodically supplementing the bulk culture with primary culture cells, or primary culture conditioned medium or both, from the primary culture container to boost or activate the TIL cells present in the bulk culture. Periodically, said bulk culture is mixed, such as by shaking, and said bulk culture and primary culture containers are stored lying flat in an incubator at 37° C, in 5% CO2.
DETAILED DESCRIPTION OF THE INVENTION As used herein:
"Tumor-derived material" means cells and particularly TIL cells, tumor antigen, antigen presenting cells and conditioned medium derived from the tumor sample.
"Conditioned medium" means cell-free medium obtained from a culture of cells.
"Primary culture" means those TIL cells and other tumor- derived material originally placed in culture in a primary culture container such as a multiwell plate or a vessel made of ionomers, such as sodium or zinc neutralized copolymers of ethylene and acrylic or methacrylic acid or derivatives thereof, such as a Surlyn® bag, which are continuously maintained and expanded in said original primary culture-. container.
"Bulk culture" means those TIL cells expanded from the original tumor sample but which are transfeπed from the primary culture container into one or more secondary vessels such as gas permeable bags, bags made of ionomers, flasks or roller bottles wherein the TIL cells are maintained and expanded.
"Substantial amount" means about 50 to 75% and prefeπably 75% of TIL cells present in the primary culture container.
"Splitting" means dividing the cell culture into additional bulk culture containers.
"Supplementing" means the adding of tumor-derived material, TIL cells and/or conditioned medium, from the primary culture into the bulk culture.
An optimized environment for the expansion and maintenance of TIL cells is provided by the process of the present invention. A human tumor sample, either fresh or cryopreserved, is placed in a suitable primary culture container, such as a polystyrene multiwell plate, commercially available from Costar, Cambridge, MA, or a container made of an ionomer such as Surlyn® , commercially available from E. I. du Pont de Nemours, Wilmington, DE. The sample is maintained and expanded in this primary culture container for a period of time by the addition of suitable culture medium, preferrably supplemented with rIL-2. While in the primary culture container, TIL cells present in the tumor sample may be stimulated by the presence of tumor derived material, including but not limited to tumor antigen, antigen presenting cells or conditioned medium. Following initial expansion of the primary culture, a substantial amount of the primary culture is transferred to a bulk culture container, prefeπably a gas permeable culture vessel such as a SteriCell™ bag, commercially available from E.I. du Pont de Nemours, Inc., Wilmington, DE. The bulk culture is maintained and expanded by feeding the culture with suitable culture medium such as that used in the Rosenberg protocol (AIM-V supplemented with rIL-2) and periodically splitting the bulk culture into additional bulk culture containers. Periodically the bulk culture is supplemented with primary culture cells and/or primary culture conditioned medium. The TIL cells in the bulk culture are stimulated by the addition of these primary culture cells and/or primary culture conditioned medium.
A prefeπed embodiment of this invention comprises the process as described herein utilizing either a polystyrene multiwell plate or a Surlyn® bag (Du Pont) as the primary culture container and a gas permeable culture vessel such as the SteriCell™ bag (Du Pont), as the bulk culture container. Most prefeπed is the combination of the Surlyn® bag with the SteriCell™ bag because of the convenience associated with use of bags compared to multiwell plates and because this combination renders a completely closed system for cell culture. The gas permeable nature of the prefeπed SteriCell™ bulk culture container allows CO2 and O2 to freely pass through the bag into and out of the culture medium, this reduces the risk that the lymphocytes will have limited expansion due to lack of O2. In addition, cell culture bags such as SteriCell™ provide an increased surface area on which the cells expand, this increased surface area prevents aggregation or clumping of cells which may be undesirable because it often results in cells being O2 starved. Although, gas permeable vessels are the prefeπed bulk culture containers, other vessels such as T-flasks or roller bottles can be used in the present process. In a prefeπed embodiment of this invention, a fresh tumor sample is placed in the primary culture container on the day of surgery or shortly thereafter. Alternatively, a cryopreserved tumor sample may be used by being placed in the primary culture container upon being thawed. After having been maintained in the primary culture container until the pH of the primary culture falls below 7 and/or the cell density of the primary culture is greater than or equal to 1 x 106 cells /mL, a substantial amount of the primary culture, about 50-75% and prefeπably about 75%, is transfeπed to the bulk culture container. The pH and cell density need not be measured, but rather may be determined by observation, such as by color of the culture medium or by estimation of cell number. These observations are known to those skilled in the art of cell culturing, for example, when the pH of a culture drops below 7.0, the color of the culture medium is usually orange-yellow where phenol red is the pH indicator. A portion of the primary culture about 25-50% and prefeπably about 25%, is retained in the primary culture container wherein it is maintained and expanded in cell number by the addition of suitable culture medium. The bulk culture is also maintained and expanded in cell number by the periodic addition of suitable culture medium. Preferably, the addition of such culture medium to the bulk culture is based on the needs of the cultures, for example, the pH of the bulk culture, such that when the pH declines to 7.0 or lower the volume in the bulk culture is doubled with fresh medium. Following such feeding and depending on the volume of the bulk culture and cell density, the bulk culture is split into additional bulk culture vessels and maintained and expanded in all additional bulk culture containers such that none of the culture medium is ever discarded or wasted. Such feeding and splitting procedures are known to those skilled in the art of cell culturing. Periodically, when the pH of the primary culture falls below 7 and/or the cell denisty of the primary culture is greater than or equal to 1 x 106 cells /mL, the bulk culture is supplemented with cells and/or conditioned medium from the primary culture container. Primary culture cells are the most prefeπed source of supplementation although primary culture conditioned medium or a combination of primary culture cells and conditioned medium are also effective.
In the prefeπed embodiment of this invention, the bulk culture is maintained and expanded in cell number until 2 x lOel l TIL cells, the desirable dose for reintroduction into a patient, have been obtained. These TIL cells can then be suspended in a suitable pharmaceutical carrier such as saline, saline containing 5% normal human serum albumin or Hank's balanced salt solution to provide a composition which also prefeπably includes rIL-2, which can be infused into a patient inflicted with a tumor. Using the prefeπed process of the present invention, it is possible to expand sufficient TIL cells for one round of therapy (2 X lOell) using a total of approximately 80 L of medium. Whereas using the conventional protocol, approximately 200 L of medium are required to expand sufficient ΗL cells for one round of therapy. In addition to the economic benefits of this improved process for expansion of TIL cells, the TIL cells obtained using the prefeπed process of the present invention demonstrate cytotoxic activity greater than or equal to that of cells cultured by the conventional method.
The culture medium used in the present invention may be of a kind known in the art, such as RPMI-1640 or AIM-V both prefeπably supplemented with LAK supernatant, antibiotics and rIL-2, as discussed above.
A further prefeπed embodiment of this invention provides for mixing of the bulk culture containers about once daily during expansion of the TIL cells. This mixing can be by vigorous shaking in a horizontal left and right direction for a few seconds if the bulk culture device is a gas permeable vessel such as a SteriCell™ culture vessel. Such mixing breaks up aggregated cells which often form in clump or cluster formations which cause the cells in the center of the clump or cluster to become O2 starved. Another embodiment of this invention provides an improved single step process for TIL cell expansion and maintenance wherein the human tumor sample is placed in a primary culture container wherein the sample is maintained and expanded throughout the process such that no culture medium is discarded or wasted. In this embodiment, the term primary culture container is interchangeable with bulk culture container as it is used in previous embodiments of this invention, and is prefeπably a Surlyn® bag (DuPont). Since one small Surlyn® bag (100cm2) can substitute for two multiwell plates and renders a closed system for cell culture, the bags are prefeπed over multiwell plates for this embodiment. In addition the cells expanded solely in the Surlyn® bag differ morphologically from those expanded solely in the multiwell plates. Cells expanded in the Surlyn® bags appear more healthy, plump and bright, whereas cells expanded in the multiwell plates tend to form aggregates, appear smaller and less bright and start to disintegrate toward the end of the expansion period. In this embodiment, the tumor sample and particularly the TIL cells therein, placed in the primary culture container is maintained by periodic feeding and splitting whereby each additional Surlyn® bag has cells and conditioned medium from the original culture. The expansion of ΗL cells using this process is exemplified in Example 2, showing expansion of cells in both multiwell plates only, and expansion of cells in Surlyn® bags only.
TIL cells expanded using the process of the present invention have higher cell density, improved expansion rate and longer duration of active expansion. In addition, TIL cell expansion via the present invention requires considerably less culture medium than conventional expansion techniques, approximately 120 L less per therapeutic dose, thereby conserving space in the laboratory as well as reducing the cost of expansion of TIL cells. Furthermore, the TIL cells maintain cytotoxicity equal to or greater than those TIL cells expanded using the conventional method.
Although the process of the present invention has been described with specific reference to TIL cells, it is also useful for preparing LAK. The following examples further illustrate the usefulness of the present invention for the maintenance and expansion of TIL cells.
EXAMPLE 1 TIL 805 Cell Study
Expansion of ΗL Cells with Primary Culture Container in Combination with Gas Permeable Bag Bulk Culture Containers
Surgery was performed to remove a melanoma tumor from a normal donor. On day 2 following surgery, 86 mL of enzymatically digested melanoma tumor and TIL mixed culture which was dispensed in AIM-V serum free medium (GIB CO) supplemented with 20% LAK conditioned medium, 1000 BRMP u/mL r-IL-2 (Du Pont), 2 mM L-glutamine, 50 IU/mL penicillin, 50 ug/mL streptomicin, 50 ug/mL gentamicin, and 250 ng/mL amphotericin, was divided into three equal portions in a 100cm2 Surlyn® bag (DuPont) and two 24 well multiwell plates (Costar). Feeding and splitting of the cultures were based on need. During the entire course of the study, when either the culture pH was found to be lower than 7 (by visual examination/pH determination) or viable cell number was found to be higher than 1 x 10e6 cells/mL, culture volume was doubled with the basal medium described above and it was split into additional experimental vessels.
Early in the study, which lasted 32 days, the cultures in both the multiwell plates (MWP) and the Surlyn® bag were sampled for cell number. After sampling, about 50% of the multiwell plate primary culture was transfeπed into a 150 cm2 SteriCell™ vessel for bulk culture (designated SteriCell-1). Likewise after sampling, about 50% of the Surlyn® bag primary culture was transfeπed into a fresh 150 cm2 SteriCell™ vessel for bulk culture (designated SteriCell-2). As the study continued, both the primary cultures (MWP and
Surlyn® bag) and the bulk cultures designated SteriCell- 1 and SteriCell-2 were fed by addition of AIM-V as discussed above. Due to the increase in volume of the bulk cultures with the addition of AIM-V during feeding, the bulk cultures, designated SteriCell-1 and SteriCell-2, were split such that all bulk cultures which originated from the MWP primary culture and SteriCell- 1 bulk culture container were divided into bulk culture containers designated SteriCell-3 and SteriCell-5. The bulk cultures originating from the Surlyn® bag primary culture container and SteriCell-2 bulk culture container were split into bulk culture containers designated SteriCell-4 and SteriCell-6. Throughout the study, splitting of the culture was done based on color and/or cell number as discussed above. Such splitting is known to those skilled in the art of cell culturing. It is noted that when the cell cultures are split, the conditioned medium is not removed from the cells and none of the conditioned medium is discarded. Generally, the culture volume was doubled with AIM-V in a SteriCell™ vessel and then about 50% of the volume was transfeπed to an additional SteriCell™ vessel such that splitting of SteriCell- 1 (150cm2) resulted in transfers of about 50% of the volume to SteriCell-3 (150cm2) and to SteriCell-5 (150cm2). The bulk culture in SteriCell-5 was later transfeπed into a 700 cm2 SteriCell™ vessel and ultimately into four 700 cm2 SteriCell™ vessels, still designated SteriCell-5.
As with SteriCell-1, SteriCell-3 and SteriCell-5, splitting of SteriCell-2 (150cm2) resulted in transfers of approximately 50% of the volume, to SteriCell-4 (150cm2) and SteriCell-6 (150cm2). The bulk culture of SteriCell-6 was later transfeπed into a 700cm2 SteriCell™ vessel, and ultimately into four 700cm2 SteriCell™ vessels, still designated SteriCell-6.
Periodically, about every 2 to 3 days, the primary cultures in the multiwell plates and Surlyn® bags were fed and about 50 - 75% of the primary cultures were added to their respective bulk culture SteriCell™ vessels such that only primary culture cells and/or conditioned medium from the multiwell plates were used to supplement SteriCells-1, 3 and 5 while only primary cultures cells and/or conditioned medium from the Surlyn® bags were used to supplement SteriCells-2, 4 and 6.
During the study, the SteriCell cultures were shaken vigorously for a few seconds once daily. Periodically, the cultures were sampled for determinations of cell number, cell phenotype and cell viability. Thirty-two days following surgery, the study was completed and the following data shown in Tables 1-1 to 1-6 were obtained:
Table 1-1
TIL 805 Cells Total Viable Cell Number
Expanded Using a Multiwell Plate (MWP. Primary Container and SteriCell™Bag Bulk Culture Container
Day (Following Cell Number
Surgery)
2 2.80 X 10e7
1 1 1.29 X 10e7
1 9 8.10 X 10e7
21 3.33 X 10e8
23 9.32 X 10e8
25 2.43 X 10e9
27 5.49 X 10e9
29 1.21 X lOelO
30 1.37 X lOelO
32 1.66 X lOelO
Table 1-2
TIL 805 Cells Total Viable Cell Number
Expanded using a Surlyn® Bag Primary Culture and SteriCell™ Bag
Bulk Culture Vessels
Daχ_(Following Cell Number
Surgery)
2 1.40 X 10e7
1 1 2.20 X 10e6
1 9 3.90 X 10e7
23 1.13 X 10e8
25 3.27 X 10e8
27 8.96 X 10e8
29 2.80 X 10e9
32 1.72 X lOelO
Table 1-3
Total Viable Cell Number
Expanded using a Multiwell Plate Primary Culture Container and
SteriCellTM Ba Bulk Culture Vessels
SteriCellTM Vessel Pay (following surgery. CellNumber
SteriCell- 1 1 1 6.45 X 10e6
SteriCell- 1 1 9 1.14 X 10e7
SteriCell- 1 27 1.79 X 10e6
SteriCell-3 1 9 4.4 X 10e7
SteriCell-3 29 2.58 X 10e8
SteriCell-5 21 1.94 X 10e8
SteriCell-5 32 1.66 X lOelO
Table 1-4
Total Viable Cell Number
Expanded using Surlyn® Bag Primary Culture Container and
SteriCellTM Bag Bulk Culture Vessels
SteriCell™ Vessel Day (following surgery. CeUNvm.ber
SteriCell-2 1 1 1.10 X 10e6 SteriCell-2 1 9 5.50 X 10e6 SteriCell-2 27 7.59 X 10e7
SteriCell-4 1 9 1.68 X 10e7 SteriCell-4 29 4.96 X 10e8
SteriCell-6 2 1 2.35 X 10e7 SteriCell-6 32 1.70 X lOelO
Table 1-5
TIL 805 Cells Percent Cell Viability of Cells Present in the MWP Primary Culture
Day (Following % Viability surgery)
1 1 26.79
1 9 66.67
2 1 86.67
23 90.70
25 87.43
27 86.16 Table 1-6
TIL 805 Cells Percent Cell Viability of Cell Present in the Surlyn® Bag Primary ult re
Pay (Following % Viability
Surgery)
1 1 9.76
1 9 78.82
2 1 81.31
23 93.53
25 87.25
27 90.66
By using the process of the present invention, exemplified above, TIL 805 cells, being supplemented from the multiwell plate primary culture containers were expanded from 2.80 X 10e7 mixed tumor and TIL cells to 1.66 X lOelO TIL cells (593 fold expansion) with about 87% viability. This expansion required a total of about 8 L of AIM-V medium and took 30 days. TIL 805 cells being supplemented from the prefeπed primary culture containers, Surlyn® bags, were expanded from 1.40 X 10e7 mixed tumor and TIL cells to 1.70 X lOelO ΗL cells (1229 fold expansion) with about 90% viability. This expansion required a total of about 8 L of AIM-V medium and took 30 days.
Comparison of the data in Table 1-1 and Table 1-2 indicates that the TIL cells expanded using the Surlyn® vessel are expanding at a faster rate and for a longer period of time. This result suggests that the Surlyn® primary culture container may provide a greater fold expansion over time periods greater than about 29 days post surgery, than can be obtained using a multiwell plate primary culture container since substantial expansion stopped as of day 29 in the MWP fed cultures whereas substantial expansion continued upto and including day 32 in the Surlyn® fed cultures.
Comparison of the data in Table 1-3 and 1-4 also suggests an advantage of using Surlyn® as primary culture containers as compared with the multiwell plate. TIL 805 cells which originated from the Surlyn® primary vessel could immediately expand upon transfer to the SteriCell™ bulk culture container on day 11, while TIL 805 cells which originated from the MWP primary vessels could not immediately expand. [See Table 1-3, cell number diminished after transfer on day 11]. This result indicates that TIL cell cultures can be transfeπed from the Surlyn® primary culture container to the SteriCell™ bag bulk culture container at an earlier time following surgery than can ΗL cell cultures in the multiwell plate primary culture container. Multiple supplementations of the bulk culture containers with cells originating from either the MWP or the Surlyn® primary culture container resulted in high expansions (593X/1229X) of the primary cells in the SteriCell™ bulk culture containers.
Example 2
TIL 805 Cells Expansion in Primary Culture Containers Only
As in Example 1, TIL 805 cells were studied, the expansion in this example focuses on the TIL 805 cells which were maintained and expanded throughout the study in the original Surlyn® bag primary culture container. The material and methods overlap those in Example 1.
On day two following surgery, 28mL of the culture of Example 1 (containing about 1.40 x 10e7 cells) was transfeπed to a Surlyn® bag (90cm2, Du Pont). The cells were maintained and expanded in this Surlyn® bag throughout the study and were fed and split as follows:
On days 6 and 11 following surgery the culture volume in the Surlyn® bag was doubled with AIM-V (GIBCO) supplemented with rIL-2 (Du Pont). On day 11, after culture volume in the Surlyn® bag was doubled with supplemented AIM-V, about 50% of the Surlyn® bag culture was transfeπed to another vessel (see Example 1) while about 50% was maintained in the Surlyn® bag. The same procedure was followed on days 19, 23 and 25 following surgery.
On days 26 and 30 the Surlyn® bag culture volume was doubled with supplemented AIM-V. On day 32 the study was completed and the following data were collected from the Surlyn® bag primary culture container. It is not the specific day of splitting or feeding that is important in this example, but rather that upon observation of the pH dropping below 7 the cultures are fed and split. This example is merely reflecting those observations in terms of the day post surgery on which they occuπed. The data is shown in comparison to that obtained using multiwell plates as primary culture containers.
Table 2-1
TIL 805 Cells Total Viable Cell Number Expanded
In Each Primary Culture Container
Day Multiwell Plate Surlyn Vessel
2 2.80 X 10e7 1.40 X 10e7
1 1 1.29 X 10e7 2.20 X 10e6
1 9 6.96 X 10e7 3.35 X 10e7
27 1.25 X 10e8 2.10 X 10e8
Table 2-2
TIL 805 Cells Percent Viability of Cells
Expanded in Each Primary Container
Day Multiwell Plate Surlvn Vessel
1 1 26.79 9.76 1 9 66.67 78.82
27 86.16 90.66
A Surlyn® bag is much more convenient than a multiwell plate and is particularly more convenient in the single step process exemplified in Example 2. In order to feed a multiwell plate, ImL per each well (24 wells/plate) must be added, this is much more time consuming and involved than doubling the culture volume in a bag. Additionally, bags are more easily stored than plates and conserve space and provide a closed system for culturing cells. The process for the expansion and maintenance of TIL cells of the present invention is commercially and practically advantageous over conventional systems and may be therapeutically advantageous. The process yields a higher density of cells which maintain a high specificity of cytotoxicity. In addition, in order to generate 2 X lOel l TIL cells only approximately 80 L of culture medium are needed whereas the conventional systems use approximately 200 L of medium. This leads to a significant reduction in the cost of the treatment and also reduces the laboratory time and space for expansion.

Claims

WHAT IS CLAIMED IS:
1. In a process wherein lymphocytes from tumor-tissue are cultured to produce a population of cells which are cytotoxic for natural killer cell resistant tumor cells, the improvement comprising: feeding and splitting the culture based on need of the lymphocytes such that cells or conditioned medium or both from the lymphocyte cell culture is used to supplement bulk expansion of the culture and no conditioned medium is discarded.
2. A process of Claim 1 wherein the lymphocytes are human cells.
3. A process of Claim 2, further comprising: a) placing a human tumor sample in a suitable primary culture container; b) maintaining and expanding the sample and tumor derived material, including tumor infiltrating lymphocytes, in the primary culture container for a period of time; c) transferring a substantial amount of the sample and tumor derived material, particularly tumor infiltrating lymphocytes, from the primary culture container to one or more suitable bulk culture containers while retaining a portion of the primary culture in the primary culture container; d) maintaining and expanding the bulk culture by adding suitable culture medium and splitting the bulk culture periodically; e) supplementing the bulk culture with primary culture cells, or primary culture conditioned medium or both; f) maintaining and expanding the primary culture by adding a suitable culture medium periodically; g) storing the primary and bulk culture containers in an incubator at 37°C; and h) optionally mixing the bulk culture periodically.
4. A process of Claim 3 wherein the primary culture container is a vessel made of an ionomer.
5. A process of Claim 3 wherein the primary culture container is a polystyrene multiwell plate.
6. A process of Claim 3 wherein the bulk culture container is a gas permeable vessel.
7. A process of Claim 3 wherein the bulk culture container is a flask.
8. A process of Claim 3 wherein the primary culture container is a vessel made of an ionomer and the bulk culture container is a gas permeable vessel.
9. A process of Claim 3 wherein culture medium is added to the bulk culture and the bulk culture is split based on the pH of the bulk culture.
10. A process of Claim 9 wherein the culture medium is added to the bulk culture when the pH of the bulk culture is less than approximately 7.0.
11. A process of Claim 3 wherein the bulk culture is supplemented with primary culture tumor infiltrating lymphocyte cells.
12. A process of Claim 3 wherein the bulk culture is supplemented with primary culture conditioned medium.
13. A process of Claim 3 wherein the bulk culture is supplemented with both primary culture tumor infiltrating lymphocyte cells and conditioned medium.
14. A process of Claim 3 wherein the bulk culture is supplemented with primary culture tumor infiltrating lymphocyte cells, or primary .culture conditioned medium or both when the pH of the primary culture is less than 7.0 and/or the cell density of the primary culture is greater than or equal to 1 x 106 cells/mL.
15. A process of Claim 3 wherein the primary culture is maintained in the primary culture container for a period of time until the pH of the primary culture is less than 7.0 and/or the cell denisty of the primary culture is greater than or equal to 1 x 106 cells /mL before a substantial amount of the primary culture is transfeπed to the bulk culture container.
16. A process of Claim 15 wherein the substantial amount of the primary culture transfeπed to the bulk culture container is about 50 to 75% of the tumor infiltrating lymphocytes in the primary culture.
17. A process of Claim 16 wherein the substantial amount is about 75%.
18. A process of Claim 3 wherein the portion of primary culture retained in the primary culture container is approximately 25% of the tumor infiltrating lymphocytes in the primary culture.
19. A composition consisting essentially of a suitable pharmaceutical caπier, recombinant interleukin-2 and tumor infiltrating lymphocytes expanded to at least 2 X lOell cells, using the process of Claim 1.
20. A composition consisting essentially of a suitable pharmaceutical caπier, recombinant interleukin-2 and tumor infiltrating lymphocytes expanded to at least 2 X lOell cells, using the process of Claim 3.
21. A method of treating a mammal inflicted with a tumor comprising administering to the mammal an effective amount of a composition of Claim 19.
22. A method of treating a mammal inflicted with a tumor comprising administering to the mammal an effective amount of a composition of Claim 20.
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EP0491851A1 (en) * 1989-09-14 1992-07-01 Cellco, Inc. Method for the production of in vitro expanded lymphoid cells for use in adoptive immunotherapy
FR2911346A1 (en) * 2007-01-12 2008-07-18 Chu Nantes In vitro culture of T-lymphocytes, preferably (non)modified T-lymphocytes penetrating tumors from tumor/blood sample, comprises emergence stage of T-lymphocytes, stimulation of the T-lymphocytes and amplification of the T lymphocytes

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0491851A1 (en) * 1989-09-14 1992-07-01 Cellco, Inc. Method for the production of in vitro expanded lymphoid cells for use in adoptive immunotherapy
EP0491851A4 (en) * 1989-09-14 1993-02-17 Cellco, Inc. Method for the production of in vitro expanded lymphoid cells for use in adoptive immunotherapy
FR2911346A1 (en) * 2007-01-12 2008-07-18 Chu Nantes In vitro culture of T-lymphocytes, preferably (non)modified T-lymphocytes penetrating tumors from tumor/blood sample, comprises emergence stage of T-lymphocytes, stimulation of the T-lymphocytes and amplification of the T lymphocytes
WO2008099088A2 (en) * 2007-01-12 2008-08-21 Centre Hospitalier Universitaire De Nantes Method for the in vitro culture of t cells, in particular t cells infiltrating the so-called til tumours
WO2008099088A3 (en) * 2007-01-12 2008-11-13 Chu Nantes Method for the in vitro culture of t cells, in particular t cells infiltrating the so-called til tumours

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