Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
• • 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
  • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
• • 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
• • 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
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • 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
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
• • 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
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/515—CD3, T-cell receptor complex

Definitions

• Adoptive immunotherapy is a form of immunotherapy where ex vivo processed cells are introduced into the body.
• Pre-clinical studies suggested anti-tumor activity could be enhanced by using interleukin-2 together with ex vivo activated and expanded autologous lymphocytes.
• the first objective responses with high-dose bolus interleukin-2 therapy were noted in patients receiving interleukin-2 together with LAK cells prepared through in vitro activation of autologous peripheral blood lymphocytes that were harvested by lymphopheresis, and initially, it appeared that the combination of interleukin-2/LAK was more active than interleukin-2 alone.
• IL-2 is a cytokine produced by Thl helper cells. Interest in the use of adoptive immunotherapy declined after clinical studies failed to demonstrate that the addition of activated LAK or TIL cells with IL-2 is any more effective than IL-2 alone.
• CD4+ T (Th) cells are crucial for the activation and regulation of most aspects of the host defense against infections and for adequate function of cytotoxic CD8+ lymphocytes (CTL).
• CTL cytotoxic CD8+ lymphocytes
• This invention discloses a method for producing clinically-relevant numbers of CD4+ cells that also contain the cytolytic granules granzyme B and perforin and function both as Thl cells (produce IFN-gamma and not IL-4) and have NK-like activity (recognize and kill tumor cells and not normal cells) for use in therapeutic cancer vaccine and adoptive immune cell therapy protocols.
• Fig. 1A and Fig. IB are flow cytometry plots from immunostaining of CD4 surface antigen demonstrating the cell phenotype before and after CD4+ selection, respectively.
• Fig. 2A is a series of flow cytometry plots demonstrating the phenotype of source cells having as resting CD4 naive (CD4, CD45RA) cells.
• Fig. 6B is a plot of expression of Granzyme B by CFB.
• Fig. 7 is a graph of perforin-Granzyme B activity with and without EGTA. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
• the present invention includes methods to cause the differentiation and expansion of this novel cell type to clinically-relevant numbers for adoptive immunotherapy.
• Methods for deriving the novel cells can include isolating T-cells, specifically CD4+ T-cells from the peripheral blood or other sources of T-cells. These CD4+ T-cells can be differentiated and expanded according to the methods described herein to clinically-relevant numbers and used for immunotherapy.
• the CD4+ T-cells prepared according to these methods can exhibit cytolytic activity similar to the cytolytic activity generally found in NK/CTL cells and also exhibit Thl characteristics and activity.
• T-helper cells do not directly exhibit cytolytic activity but produce cytokines and other factors that then elicit other cells such as NK cells and CTLs to inactivate or kill the diseased cells.
• the CD4+ T-cells in the present compositions can directly kill the diseased cells as well as produce cytokines to stimulate other cells such as NK cells that then carry out the cytolytic activity.
• the cytolytic activity of these CD4+ T-cells is mediated through Granzyme B and perforin resulting in the killing of the tumor or infected cells.
• the CD4+ cells isolated from the buffy coat and used as source material for production of Thl/killer cells can be characterized by expression of a number of cell surface markers.
• the population of CD4+ cells purified from the buffy coat of the peripheral blood can have a mixed population of CD45RA/CD45RO markers.
• the CD4+ T-cells are generally predominantly CD25- .
• the source CD4+ cells are mostly naive (express CD45RA and not CD45RO).
• Source CD4+ T-cells can also be evaluated for CD62L expression and CD40L expression.
• the source CD4+ T-cells express very low amounts of CD40L.
• Expression of CD62L can be a bimodal (high and low) mean fluorescent intensity (MFI) peak.
• Source CD4+ cells preferably have a high CD62L expression.
• the cells with the activating agent attached is formulated in a device, such as a syringe, suitable for infusion or injection.
• a device such as a syringe, suitable for infusion or injection.
• activation refers to both binding of the cell surface molecules by agents such as monoclonal antibodies and cross-linking of these agents by cross- linking agents. This type of activation is, for example, described in U.S. Patent No. 7,435,592 and U.S. Patent No. 7,402,431, both of which are incorporated herein by reference.
• the CD4+ T-cells may be activated in a variety of ways including by the use of immobilized monoclonal antibodies specific for T-cell surface molecules. Suitable activated T- cells are, for example, described in U.S. Patent No. 7,435,592.
• the cells preferably have cell surface moieties that are bound by monoclonal antibodies or other binding agents that are then cross-linked. These monoclonal antibodies and/or binding agents are preferably cross-linked by an agent, for example, immobilized on a solid surface in order to activate the T-cells. These are referred to herein as cells activated in culture (CAC). These ex vivo prepared CAC can be frozen for future use or formulated for infusion.
• CAC cells activated in culture
• the CAC may be aliquoted and frozen in the gas phase of Liquid Nitrogen tanks.
• the CAC are capable of consistently meeting predefined identity and functional release criteria as described below. In process and final quality control tests are established to assure sterility and endotoxin-free status.
• the CD4+ T-cells are cultured for 9 days in the presence of CD3/CD28 ClinEx Vivo Dynal Beads obtained from Invitrogen, Carlsbad, CA.
• the cells are grown in Life Cell Flasks (Baxter Scientific, Deerfield, IL) at 37°C and 5% C0 2 and re- stimulated with the beads at days 3 and 6 of culture. After 9 days in culture, the beads can be removed and the cells can be referred to as CAC.
• Other methods of culturing cells for activation are also within the scope of the invention.
• the Thl/killer cells in the composition can exhibit a novel combination of functional characteristics. These functions include Thl characteristics and cytolytic functions that are similar to NK functions.
• the Thl/killer cells generally produce IFN-gamma and have substantially diminished or no IL-4 production. Preferably, the IL-4 production is below 50pg/10 6 cells and IFN-gamma production greater than lOOOpg/10 6 cells.
• Thl/killer cells can include for example, expression of functional molecules such as CD40L, FasL, perforin and granzymeB, co- stimulatory molecules 4-1BBL, CD28, CTLA4, and TNF-related activation-induced cytokine (TRANCE), TWEAK, PD-1, B7 family, adhesion molecules such as the integrins, the cadherins, and the selectins and secretion of a variety of cytokines and chemokines and expression of receptors for these cytokines and chemokines.
• the Thl/killer cells can produce and secrete large amounts of cytokines.
• the cytokines produced can be, for example, IFNcc, GM-CSF and TNF-cc, with only residual IL4 secretion
• Thl/killer cells of the present invention can express the molecules Granzyme B and Perforin.
• Granzyme B and Perforin are generally not expressed in CD4+ T-cells, whether naive or after maturation by differentiation, activation and the like.
• the amount of Granzyme B and Perforin in the activated Thl cells can vary. Generally, the amount of Granzyme B and Peforin expressed is sufficient to destroy diseased cells.
• the present invention includes compositions that include Thl/killer cells.
• Thl/killer cells can inactivate a variety of tumor cells including the myeloma tumor cell lines such as ARH77 cells.
• the inactivation of the diseased cells can be through a mechanism sensitive to EGTA.
• the effector cell (Thl/killer cells) to target cell (diseased cell) ratio can vary.
• the effector to target cell ratio can be at least 1:9, preferably at least 1:5 and more preferably at least 1: 1.
• the Thl/killer cells can be administered to the patient by a variety of routes including intradermally, intravenously, intraperitoneally and the like.
• the patient may be administered one dose or multiple doses.
• the number of cells administered to the patient can vary and may depend on the specific disease, the patient, the method of activation and other factors.
• the patient is administered at least 10 7 of activated Thl cells, preferably at least 108 and more preferably at least about 10 9 cells.
• the present invention also includes a method of treating a patient by administering a composition that includes Thl/killer cells.
• the patient may receive one or more doses of the Thl/killer cells.
• the amount and the frequency of the doses can vary and can depend on the specific disease.
• the Thl/killer cells are allogeneic to the patient and administration of the Thl/killer cells to the patient induces a host versus tumor effect along with a host versus graft effect without graft versus host disease.
• PE-conjugated CD40L was purchased from Beckman Coulter, Brea, CA. 7- Amino-Actinomycin D (7-AAD) (lOOOx) was purchased from Cayman Chemical Co., Ann Arbor, MI. PlasmaLyte A was purchased from Baxter Scientific, Deerfield, IL. Human serum albumin (HSA) was purchased from McKesson, San Francisco, California. FcR Binding Inhibitor was purchased from eBioscience, San Diego, CA. Dynabeads ClinExVivoTM was purchased from Invitrogen, Carlsbad, CA.
• CAC cells were resuspended at a concentration of 1 x 10 7 cells/ml. Reactivation was done at a live cell concentration of 1 x 10 cells/ml. Reactivation was done in a 24 well plate, 6 well plate or a 75 cm 3 flask depending on the volume. Dynabeads ClinExVivo XM CD3/CD28 were added to reactivate the cells and incubated at 36-38°C and 5% C0 2 for 4 hours.
• CFB were resuspended in FFB at a concentration 10 7 cells per ml.
• the CFB were resuspended for ID, IT or IV administration.
• 1ml of the cell suspension was added to a 3ml syringe as an ID formulation.
• IT and IV formulation were 3 ml and 5 ml, respectively.
• the syringes with the appropriate formulations were stored in refrigeration with an average temperature of about 4°C.
• Flow cytometry (CD40L and 7-AAD)- 50ul cell suspension were transferred from above (150 ul) into 3 eppendorf tubes, labeled as unstained, CD40L and 7-AAD, respectively.
• the unstained tube was incubated on ice for 20 min.
• the cells were pre- incubated with FcR Binding inhibitor according to the instructions of the manufacturer for 20 minutes on ice.
• 40ul staining buffer (PBS+1%FBS) and lOul PE-CD40L antibody was added into the cell suspension and incubated for additional 20 min on ice in the dark.
• CD40L Expression Pattern The density of CD40L expression on CAC was consistently increased on the surface of CAC after a 4 hour activation with CD3/CD28 beads.
• Fig. 4 shows the change in the arithmetic mean and percentage of CD40L expression of a representative batch comparing CD4+ at day 0 (Fig. 4A), day 9 harvested CAC before final activation (Fig. 4B) and after 4 hours activation, CFB (Fig. 4C).
• Example 2-Direct Killing by activated Thl/killer cells To test if CFB may have direct effect on tumor cells, the ability of the cells to kill the ARH77 cell line was tested. This cell line is an established myeloma cell line. The ARH77 cells were stained in CFSE to differentiate them from the CFB. CFB were mixed with the stained ARH77 cells at different Effector (CFB) to Target (ARH77) for 18 hours. After that time, 7AAD was added and cells were acquired using the FC500MPL flow cytometer. This experiment was repeated with different CAC batches and with fresh and thawed CFB. ARH77 alone, with DynaBeads, CD44- cells or with CAC had no significant death (less than 10%) data not shown.
• Fig. 5 The results shown in Fig. 5 indicate that CFB, but not CAC, CD44- cells or beads, have direct killing effect on the ARH77 cell line.
• the nature of the direct killing effect was examined to see if the effect was due to the Perforin-Granzyme pathway. Both internal staining and western blot techniques were used to stain for Perforin and Granzyme B.
• Fig. 6A shows that CAC express very little Granzyme B, as can be seen.
• Fig. 6B shows that CFB express significant amount of Granzyme B.
• Fig. 6C shows that Perforin can be detected only by the use of western blot.
• Lane 1 is the extract of activated PBMCs, as a positive control.
• Lane 2&4 are of CAC from different batches, and lanes 3&5 are of the CFB from the same batches.
• Inhibition assays were also performed to elucidate the nature of the direct killing mechanism.
• CFB were mixed with ARH77 cells as described above and different inhibitory agents were added as shown below Table 2.
• Antibodies against CD40L and/or FAS-L were added to the CFB:ARH77 mix. Table 2 shows the even the highest concentration did not prevent the killing profile.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• General Health & Medical Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Cell Biology (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Hematology (AREA)
• Medicinal Chemistry (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Virology (AREA)
• Veterinary Medicine (AREA)
• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Developmental Biology & Embryology (AREA)
• Epidemiology (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Molecular Biology (AREA)
• Tropical Medicine & Parasitology (AREA)
• AIDS & HIV (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Compounds Of Unknown Constitution (AREA)

Priority Applications (13)

Applications Claiming Priority (4)

Publications (2)

Family

ID=45594250

Family Applications (1)

Country Status (19)

Cited By (1)

Families Citing this family (6)

Citations (2)

Family Cites Families (7)

• 2011
  • 2011-08-22 ES ES11818884.6T patent/ES2660577T3/es active Active
  • 2011-08-22 DK DK11818884.6T patent/DK2606125T3/en active
  • 2011-08-22 JP JP2013526060A patent/JP6408219B2/ja active Active
  • 2011-08-22 BR BR112013003989-2A patent/BR112013003989B1/pt active IP Right Grant
  • 2011-08-22 SG SG2013011069A patent/SG187846A1/en unknown
  • 2011-08-22 EP EP11818884.6A patent/EP2606125B1/en active Active
  • 2011-08-22 AU AU2011291477A patent/AU2011291477B2/en active Active
  • 2011-08-22 CN CN201810168834.1A patent/CN108245673A/zh active Pending
  • 2011-08-22 KR KR1020137004186A patent/KR101900807B1/ko active Active
  • 2011-08-22 CA CA2808873A patent/CA2808873C/en active Active
  • 2011-08-22 NO NO11818884A patent/NO2606125T3/no unknown
  • 2011-08-22 CN CN2011800402324A patent/CN103068973A/zh active Pending
  • 2011-08-22 PH PH1/2013/500519A patent/PH12013500519B1/en unknown
  • 2011-08-22 TR TR2018/02334T patent/TR201802334T4/tr unknown
  • 2011-08-22 HU HUE11818884A patent/HUE036094T2/hu unknown
  • 2011-08-22 US US13/214,534 patent/US20120045423A1/en not_active Abandoned
  • 2011-08-22 PT PT118188846T patent/PT2606125T/pt unknown
  • 2011-08-22 WO PCT/US2011/048578 patent/WO2012024666A2/en not_active Ceased
  • 2011-08-22 SG SG10201510524TA patent/SG10201510524TA/en unknown
  • 2011-08-22 PL PL11818884T patent/PL2606125T3/pl unknown
• 2013
  • 2013-02-19 IL IL224813A patent/IL224813B/en active IP Right Grant
  • 2013-03-13 US US13/800,066 patent/US20130196428A1/en not_active Abandoned
  • 2013-03-13 US US13/800,193 patent/US10385315B2/en active Active
• 2016
  • 2016-12-02 JP JP2016235386A patent/JP6525946B2/ja active Active

Patent Citations (2)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events