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WO1990004633A1 - ACTIVATION ET CROISSANCE DE LYMPHOCYTES HUMAINS INFILTRANT LES TUMEURS AU MOYEN D'ANTICORPS ANTI-CD3 OU ANTI-TcR - Google Patents

ACTIVATION ET CROISSANCE DE LYMPHOCYTES HUMAINS INFILTRANT LES TUMEURS AU MOYEN D'ANTICORPS ANTI-CD3 OU ANTI-TcR Download PDF

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Publication number
WO1990004633A1
WO1990004633A1 PCT/US1989/004189 US8904189W WO9004633A1 WO 1990004633 A1 WO1990004633 A1 WO 1990004633A1 US 8904189 W US8904189 W US 8904189W WO 9004633 A1 WO9004633 A1 WO 9004633A1
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til
tumor
activated
cells
growth
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PCT/US1989/004189
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English (en)
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Deric D. Schoof
Timothy Eberlein
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Brigham And Women's Hospital
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Publication of WO1990004633A1 publication Critical patent/WO1990004633A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • 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

  • the present invention relates to methods of stimulating growth and activating cytokine production and anti-tumor cytotoxicity of tumor-infiltrating lymphocytes (TIL) using solid-phase monoclonal antibodies to lymphocyte surface structures CD3 or TcR.
  • TIL tumor-infiltrating lymphocytes
  • the invention also relates to the use of these TIL in the treatment of disease and the study of immunotherapy.
  • Adoptive im unotherapy is defined as the transfer to the tumor-bearing host of active immunologic agents, such as cells with antitumor reactivity, that can mediate, either directly or indirectly, antitumor effects.
  • adoptive immunotherapy represents an attractive approach to the therapy of cancer and other conditions related to immune dysfunction. It should be noted that because active immunologic agents are being transferred to the host, complete host im unocompetence may not be required. Thus, the immunosuppression generally associated with the tumor-bearing state may not represent a significant problem to this therapeutic alternative. Since adoptive transfer of immune cells may not depend on the host's immunologic contribution, adoptive immunotherapy can be combined with other therapies such as chemotherapy and radiotherapy. When the transferred agents are im unologically specific, this treatment modality is expected to be highly specific and consequently less toxic. Further, in contrast to most other therapies, no immuno ⁇ suppression is likely to result from this adoptive immuno ⁇ therapy.
  • a major obstacle to the development of successful adoptive immunotherapy has been the unavailability of appropriate cells for adoptive transfer. These cells are required in large numbers; most animal models predict that approximately 10 ⁇ immune cells will be required to treat clinically evident human malignancies. These cells should be immunologically specific for tumor and should be well tolerated when adoptively transferred.
  • the cells should preferably be autologous (derived from the subject to be treated).
  • the administered cells should be able to traffic to sites of tumor in vivo, a property which is known to be affected by their prior culture and activation.
  • An ideal cell for adoptive transfer should be capable of expanding in number (proliferating) at the tumor site either in response to direct antigenic stimulation by tumor or by the presence of additional growth factors which may be present or provided as part of the treatment. Thus, the ability to amplify the effect of the transferred cells would be a major asset in this approach.
  • IL-2 interleukin-2
  • IL-2 is a T cell growth factor which induces the proliferation and differentiation of T cells and activates other lymphocytes such as natural killer cells, and even B cells.
  • LAK cells which have been activated by IL-2 in culture are known as ly phokine activated killer (LAK) cells.
  • LAK cells The discovery of LAK cells had two significant ramifications: first, it provided a means for overcoming the immunosuppressed state of cancer patients and second, because the LAK cells were obtained by leukophoresis, large numbers of cells could be easily obtained (U.S. Patent No. 4,690,915).
  • LAK cells clearly lyse a wide variety of tumor targets in vitro, can cause regression of tumor mass in vivo, and may be activated in vivo by the parenteral administration of high doses of rlL- 2 (Grimm et al .. supra (1982)).
  • LAK cells are non ⁇ specific in their recognition and destruction of tumor cells.
  • a major and unexpected problem with the clinical use of rIL-2 alone and with rIL-2-activated LAK cells has been the toxicity of this lymphokine in the recipient.
  • serious problems with fluid balance and cardiorespiratory dynamics have been observed (Rosenberg et al .. N. Engl. J. Med. 316:889 (1987) and West et al.. N. En ⁇ l . J. Med. 316:898 (1987)).
  • These problems have prompted the search for other means of activating lymphoid cells in vitro and in vivo.
  • methods of cell manipulation and therapy which result in increased specificity and cytotoxicity for autologous tumor cells over that obtainable with LAK cells would be highly desirable.
  • Tumor cells express surface antigens that serve as targets for cell-mediated immune responses (Klein, G., Ann.
  • TIL Tumor-Infiltrating Lymphocytes
  • TIL are obtained by removing a sample of a patient's tumor, isolating, and cultivating the lymphocyte cells contained within that sample.
  • Autologous cytotoxic T lymphocytes have been isolated from TIL in colorectal cancers (Werkmeister et al . , Br. J. Cancer (1979); Hutchinsoh et al .. Br. J. Cancer 44:396 (1981); Vose et al .. Br. J. Cancer 44:846 (1981)) and ovarian ascitic fluid (Ferrini et al.. Int. J. Cancer 36:337 (1985)), among other tissues.
  • TIL isolated from human tumors have been found to be enriched for specific reactivities to the autologous tumor when examined in vitro (Kurnick et al . , Clin. Immunol. Immunopath. 38:367 (1986); Vanky et al .. Cancer Immunol . Immunother. 169 (1986); Vose et al .. Int. J. Cancer £0:895 (1977)). It is also known that these lymphocytes, re-exposed to syngeneic tumor cells in vitro, can produce regression of some murine tumors (Mazumder et al .. Cancer Immunol . Immuno ⁇ ther. 15:1 (1983); Rosenstein et al .. J. Natl . Cancer Inst. 72:1161 (1984)).
  • TIL had been avoided by Grimm and her co-workers in their earlier studies of LAK induction, possibly because such cells were believed to be deficient in natural cytotoxic activity (Moy, P.M., et al.. Can. Res. 45:57-60 (1985); Vose, B.M., et al.. Int. J. Cane. 24:579-585 (1979)).
  • Vankv et al .. Cancer Immunol . Immunother. 21:69 (1986) reported an 88%
  • TIL isolated from autologous fresh tumor are cultured in medium with IL-2 (Kurnick et al ., Clin. Immunol. Immunopath. 38:367-80 (1986)) they secrete ly phokines, bind to and kill autologous and allogeneic cancer cells, and lyse K562 cells. These TIL display enhanced killing of autologous tumor cells compared to peripheral blood lymphocytes from the homologous donors.
  • TIL isolated from urine sarcomas have been expanded in IL-2 (Yron et al.. J. Immunol. 125:238 (1980)), the adoptive transfer of which has been shown to be effective in mediating the regression of a variety of murine tumors (Rosenberg et al.. Science 233:1318 (1986)).
  • lymphocyte surface structures such as cluster of differentia ⁇ tion (CD3) or T-cell antigen receptor (TcR) can mimic physiologic ligand and cause activation of T cells, as measured by lymphokine production, itogenesis, and cytotoxi ⁇ city (Perez et al.. Nature 316:354-356 (1985)).
  • CD3 cluster of differentia ⁇ tion
  • TcR T-cell antigen receptor
  • one anti-CD3 antibody (0KT3), in soluble form, has recently been found to enhance PBL proliferation and LAK generation induced by rIL-2 (Ochoa et al.. J. Immunol. 138:2728 (1987)).
  • Anti-CD3 can, by itself, cause PBL to effect lectin-dependent lysis and anti-CDS heterodimer-mediated lysis of human target cells. (Gromo et al.. Nature (London) 327:424 (1987) and Jung et al.. J. Immunol. 139:639 (1987)).
  • TIL The i munotherapeutic application of TIL requires the ability to reproducibly obtain suitable numbers of lymphocytes for adoptive transfer. In the past there has been tremendous variability in the proficiency of high concentrations of rIL-2 to either induce or sustain the growth of TIL in clinically therapeutic numbers.
  • TIL TIL-induced cell proliferation.
  • clinically useful numbers of TIL may be grown under standard conditions in rIL-2 (1000 U/ml) as currently practiced in the art, this process is successful less than 50% of the time.
  • TIL grown under these conditions are assessed for cytotoxicity, a pattern is often found that cannot be distinguished from that seen when peripheral blood mononuclear cells are cultured in rIL-2 (i.e., LAK cells). This appears to be a result of using high concentrations (1000 U/ml) of rIL-2.
  • rIL-2 peripheral blood mononuclear cells
  • TIL grown in high concentrations of IL-2 also demonstrate non-specific anti-tumor cytotoxicity. Tumor specificity is, of course, a major objective in devising useful immunotherapy parameters.
  • the cytoto ⁇ xicity of most TIL in bulk cultures eventually subsides to undetectable levels.
  • the use of lymphocytes in conjunction with high doses of rIL-2 in part to overcome this refractory problem, is often highly toxic to the recipient.
  • TIL isolating and expanding TIL
  • Another approach to isolating and expanding TIL has been to permit the lymphocytes to migrate out of a small chunk of tumor in vitro. TIL can then be expanded by growth in low concentrations of IL-2 and/or stimulated periodically by the
  • TIL tumor cell suspen ⁇ sions or tumor fragments under the influence of IL-2.
  • a more reliable strategy is required because of several problems attendant with the expansion of the lymphocyte population to clinically useful numbers.
  • First, not all clinical samples of tumor (known by microscopic examination to contain TIL) respond to high concentrations of rIL-2 with the desired growth of TIL. If, during the first few weeks of the culture, TIL fail to proliferate, it is unlikely that such a sample will be of future clinical value to the patient.
  • mitogen (PHA) stimulation of human TIL requires the use of an accessory feeder cell population. This is undesirable because the ability of TIL to respond to mitogen (by proliferation) will depend on variables such as the batch of PHA and the (biologic) condition of the feeder cells. This adds to the complexity and cost of TIL expansion.
  • the use of soluble monoclonal antibodies such as anti-CD3 or anti-TcR to activate TIL would also require the use of feeder cells.
  • the present invention is directed toward methods of activating and growing TIL useful in the treatment of disease and the study of immunotherapy.
  • the present invention is also directed toward a method for activating the proliferative growth, cytotoxicity, and lymphokine production of tumor infiltrating lymphocytes comprising: a. contacting TIL in culture media with a solid phase support having either an anti-CD3 or anti-TcR antibody immobilized thereon; and b. incubating said TIL obtained in step (a) under conditions which permit the activation of said TIL.
  • the present invention further relates to the above method wherein the activity of said activated TIL obtained in step (b) is maintained by the addition of a lymphokine in an amount sufficient to maintain the proliferative growth, cytotoxicity, or lymphokine production of said activated TIL obtained in step (b).
  • the present invention is also directed to a method for mediating the suppression of the growth or development of a tumor in an animal which comprises: a. providing TIL prepared by the above-described methods; and b. providing said activated TIL to an animal in a manner sufficient to suppress the growth or development of said tumor.
  • the present invention also relates to a method of treating lymphopenia, immunosuppression, and for expanding peripheral T lymphocytes (for example, for tissue graft therapy) comprising administering to an animal in need of such treatment or expansion the activated TIL obtained by the above-described methods.
  • the inventors have observed that human TIL obtained from freshly resected tumor, incubated with solid-phase bound anti- CD3 antibody or anti-TcR antibody in the absence of exogenous recombinant IL-2 (rIL-2), followed by culture in reduced concentrations of rIL-2, can be activated to greater cell growth, lymphokine production, and anti-tumor cytotoxicity beyond than that which is achievable according to the teaching of the current art.
  • rIL-2 exogenous recombinant IL-2
  • the present invention offers distinct and important advantages over known methodologies. These advantages are important because they provide a method for reproduciblv stimulating cell proliferation and anti-tumor cytotoxicity, factors which are of critical importance for the subsequent adoptive transfer of lymphocytes into patients with cancer or other immune dysfunction conditions and diseases, as well as for the study of immune responses to tumors and tumor antigens in general.
  • one of the objectives of the present invention is improved cellular immunotherapy using tumor-derived TIL as a replacement to current treatment modalities, such as the use of LAK cells incubated with large quantities of rIL-2.
  • TIL which have become unresponsive in IL-2-induced proliferation can be stimulated into mitogenesis by activation with solid-phase bound anti-CD3 or anti-TcR antibodies and that a significant increase in numbers of TIL from the very beginning of cultivation may be achieved using the methods of the present invention.
  • TIL need no longer be grown in high concentrations of IL-2.
  • the loss of antigen specificity associated with culture in high concentrations of rIL-2 may now be avoided.
  • TIL activated with solid-phase anti-CD3 or anti-TcR, according to the present invention demonstrate specificity (and potent anti-tumor cytotoxicity) for autologous tumor cells, whereas TIL cultured under standard conditions demon ⁇ strate little or no such behavior.
  • the present invention teaches that perturbation of CD3 or TcR is a useful approach to sustain the proliferation of TIL in bulk cultures beyond the period in which they become refractory to rIL-2-induced expansion when cultivated by known methodologies.
  • solid-phase antibody activation of anti ⁇ tumor cytotoxicity by TIL will avert the need for periodic stimulation by a combination of feeder cells plus mitogen, prolonged culture in high concentrations of rIL-2, and will help alleviate the problem of an often limited supply of autologous tumor.
  • SUBSTITUTESHEET increase our understanding of immune response to tumors and tumor antigens.
  • TIL in vitro expansion of TIL
  • methods of in vitro expansion of TIL include their use with patients who are, for various reasons, lymphopenic and immunosuppressed. Since this form of activation will cause all CD3-bearing cells to proliferate, the methods of this invention can also be used to expand peripheral blood lymphocytes before a patient was exposed to bone marrow depletion, for example.
  • the infiltrating cells involved in tissue rejection can be more thoroughly studied and cells may be grown in large numbers for tissue grafting, etc.
  • the present invention will also be useful in the study and therapy of organ transplant therapies.
  • FIG. 1 An alternative method of evaluating TIL cytotoxicity: The ability of human TIL to kill tumor declines with the age of the bulk culture. With time, the ability of TIL to kill Daudi, K562, autologous tumor, and heterologous tumor becomes extinct. For example, see day 80. Untreated TIL grown for 80 days in high-dose rIL-2 fail to kill Daudi, K562, and allogeneic tumor. Modest autologous tumor killing is observed. However, these cells are not fully activated to cytotoxicity since addition of 250 ng/ml soluble anti-TcR to
  • the present invention is directed toward methods of isolating, activating and growing TIL useful in the treatment of disease and the study of immunotherapy.
  • the pathological conditions which are treatable in accordance with the present invention include, but are not limited to, cancer, tumors, immune disease or dysfunction, ly phopenia, bone marrow depletion, and tissue graft rejec ⁇ tion.
  • a "tumor-infiltrating lymphocyte” refers to a mononuclear cell which infiltrates solid animal tumors. They are tumor- specific and generally more potent than LAK ceT-ls in mediating immunotherapy of established micrometastases. Although such cells may be isolated and purified through a variety of techniques, it is preferable to employ the techniques disclosed by Rosenberg et al .. Science 233:1318 (1986).
  • treatment is included the prevention, elimination and attenuation or amelioration of the conditions of pathology described above.
  • immunosuppression is meant the suppression of immunologic, response in an animal, often with reference to grafts, organ transplants, tumor growth, etc., by the use of physical or immunologic agents, or by a disease process, and are well-known to those of skill in the art.
  • lympho ⁇ a reduction, rela ⁇ tively or absolutely, in the number of circulating lympho ⁇ cytes. This condition is often associated with diseases and disease states such as Hodgkin's disease, lymphosarcoma, leukemia, and macroglobulinemia, for example, and is associ ⁇ ated with susceptibility to infections due to the decreased
  • antibody refers to monoclonal or monospecific antibodies which have a substantially homogeneous population of molecules. Monoclonal antibodies directed toward CDS or TcR may be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256:495- 497 (1975) and U.S. Patent No. 4,376,110. These publications, as well as those mentioned hereunder, are incorporated herein by reference. Unless defined otherwise, various terms used herein have the same meaning as is well understood in the art to which the invention belongs.
  • antibody is also meant as well to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding to antigen.
  • solid phase support any support capable of binding antibodies.
  • supports include but are not limited to nitrocellulose, diazocellulose, icrotiter plates, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, affinity support gels such as Sepharose or agar, starch, and nylon.
  • a preferred support is poly ⁇ styrene.
  • activation is meant the stimulation of subject cells to behave in a certain manner, i.e., to proliferatively grow, to become cytotoxic, or to produce a substance, such as a lymphokine.
  • animal By the term “animal” is intended all animals in which TIL may be obtained from tumor tissue and for which the reduction of tumor growth and other uses of the present invention, described below, have a beneficial effect. Foremost among such animals are humans; however, the invention is not intended to be so limiting, it being within the contemplation of the invention to treat any and all animals which may experience the beneficial effects of the invention.
  • lymphokine refers to a compound derived from lymphoid cells capable of activating TIL to attack tumor cells. Lymphokines are, in general, proteins or glycoproteins having a molecular weight of 10-100 kDa. Examples of lymphokines are disclosed by Stephenson (In: Animal Cell Biotechnology. Vol. 2, Spier, R.E. et al .. eds., Academic Press, pp. 41-45 (1985)). Although any compound capable enhancing TIL proliferation, mitogenesis, and/or cytotoxicity may be employed in accordance with the present invention, it is preferable to employ a lymphokine, and it is especially preferable to employ the lymphokine, interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • Interleukin-2 is a 15 kDa glycoprotein (Gillis, S., et al.. J. Exoer. Med. 152:1709 (1980)) which is secreted by activated T lymphocytes (Bonnard, et al . , J. Immunol. 123:2704 (1979)). This molecule is believed to interact with a membrane receptor present on activated lymphocytes, thereby inducing a cascade phenomena which leads to the proliferation of the activated cells (Robb, J.R., Immunol . Today 7:203 (1984)). Procedures for the preparation of lymphokines and IL-2 from natural tissue sources are well known in the art (Gillis, S., et al.. J. Exoer.
  • rIL-2 during the TIL culture period at a concentration of between 5 U/ml-1000 U/ml, and most preferably to employ rIL-2 at a concentration of approximately 1-180 ng/ml.
  • rIL-2 should contain approximately 5-1000 half-maximum units/ml although other concentrations can be useful, as will be obvious to those of skill in the art.
  • Both control and rIL-2-containing wells may be monitored regularly by light microscopy for growth of lymphocytes or contaminating tumor cells over the subsequent weeks. Cultures should be fed RPMI-1640, serum, and IL-2 as needed. When proliferation is noted in wells, cells can be transferred into additional wells and eventually into tissue culture flasks as needed.
  • the cell density and volume are _ relatively unimportant.
  • a 48 hour incubation on the solid-phase is useful, because if TIL are left on the solid-phase too long (approximately 96 hours or more) TIL cultures rapidly die.
  • other solid-phase incubation periods will be effective, for example, 24 hours, or any other incubation period sufficient to activate the TIL population in accordance with this invention, as will be obvious to those of skill in the art.
  • the term “harvest” means to collect subject cells of interest from a larger cell pool or culture medium.
  • Harvesting techniques that are useful for the present invention are well known by those of skill in the art.
  • TIL may be collected by sterile pipette, followed by washing the solid-phase with culture medium twice. Cells may be pelleted by centrifugation.
  • culture medium includes those compositions which promote the growth of the subject cells.
  • a common culture medium useful in the present invention is RPMI- 1640 plus either 10% fetal calf serum or 10% human type AB plasma, supplemented with 1000 U/ml rIL-2 and 25 mM HEPES buffer.
  • Such media may also include antibiotics such as penicillin or streptomycin, and a ino acids such as 1- gluta ine, etc.
  • Other appropriate culture media will be apparent to those of skill in the art, including serum-free media formulations.
  • TIL cytotoxicity in vitro may be determined by any well-known quantitative in vitro assay.
  • the techniques of Brunner et al .. Immunology 14:181 (1968) or Simpson et al .. Eur. J. Immunol . 5:451 (1975), based on the release of 51 Cr from -labeled target cells may be modified in a variety of ways which will be apparent to those of skill in the art (see, for example, Canty et al .. J. Nat. Cancer Inst. 5:761 (1970).
  • cytotoxicity' assay be quantitated using radioactive isotopes, but the present invention also contemplates the use of fluorescent compounds, fluorescent emitting metals, chemiluminescent compounds, bioluminescent compounds, enzymes, and the like, which alternate compounds will be obvious to those of skill in the art.
  • compositions comprising TIL when administered in a suitable pharmaceutically acceptable (sterile and non- toxic) carrier to humans suffering from immune-dysfunction conditions or cancer.
  • the preparations and compositions of the present invention are said to "mediate suppression" of tumor growth if, when provided to an animal, their administration results in either a decrease in the proliferation of tumor growth, and inhibition of the formation of metastases, a
  • compositions and preparations of the present invention are to be provided to an animal in amounts and at frequencies capable of successfully mediating the suppression of said tumor growth.
  • compositions within the scope of this invention include all compositions wherein each of the components thereof is contained in an amount effective to achieve its intended purpose.
  • the binding activity of a given lot of antibody directed toward TIL may be determined according to methods well known by those of skill in the art.
  • Suitable pharmaceutically acceptable carriers may comprise excipients and auxiliaries which facilitate process ⁇ ing of the active compounds into preparations which can be used pharmaceutically. These will be readily apparent to those of skill in the art.
  • the resulting cells are capable of lysing fresh tumor cells without affecting normal cells.
  • These activated cells are reinfused, preferably by systemic administration, into the autologous patient.
  • an "effective amount of a cell prepara ⁇ tion containing TIL" is an amount capable of mediating the suppression of tumor growth in an animal. It is preferable that such an effective amount comprise the administration of between 1-5 x 10 15 TIL per patient per administration; it is more preferable, however, to employ as many TIL per patient per administration as possible. Depending on the condition of the patient, of course, more or less TIL cells could be administered. The interval between the time of initiating cultures from the biopsy and the use of cells for therapeutic infusions may vary between 4-20 weeks or more.
  • the dosage of TIL to be administered will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if
  • SUBSTITUTESHEET any, frequency of the treatment, the nature of the effect desired, and the number of TIL grown in vitro.
  • compositions comprising the present invention may be administered by any manner known to the art. Typical modes of administration include by injection, infusion, or implanta ⁇ tion. As necessary to successfully suppress the tumor of the subject animal or human patient, additional effective amounts of TIL may be provided to the subject. The therapeutic regime is maintained or altered as necessary to cause the suppression of the tumor or cancer.
  • Suitable formulations for infusion of the compositions of the present invention include aqueous solutions of the active compounds in water-soluble form such as water- soluble salts, or normal saline plus albumin.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipo- philic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • the preparations contain from about 0.1 to 99 percent, preferably from about 25-85 percent of active com ⁇ pound ⁇ ), together with the excipient.
  • TIL cells may be administered intravenously, for example, through a central venous catheter or into a large peripheral vein, by direct infusion into the hepatic artery via a percutaneous catheter. An initial infusion of approximately 10 10 cells may be performed followed five minutes later by the remainder of the cells over approximately 20 minutes. No filters need be used in the infusion line. The infusion bag should be gently mixed every five minutes during the infusion.
  • the activated cells in accordance with the present invention can be employed for the treatment of cancers, viral and other infective diseases, autoimmune diseases, for the correction of immune-deficiency diseases and the like. They may also be used to study immune deficiencies and to expand peripheral blood lymphocytes, for example.
  • Fresh human tumor was obtained from the Department of Surgery at the Brigham and Women's Hospital in Boston, MA. It was finely minced with scissors and enzymatically dissociated using collagenase, hyaluronidase, and DNase at room tempera ⁇ ture for 3 hours. (Rosenberg et al ., Science 233:1318 (1986)).
  • Tumor cell lines Daudi and K562 were obtained from the American Type Culture Collection, Maryland. Cultures were seeded into six-well plates at a density of 10 * ⁇ tumor cells/ml in RPMI-1640 + 10% human type AB plasma supplemented with 1000 U/ml rIL-2.
  • Anti-CD3 anti-0KT3, Ortho Pharmaceutical Corporation
  • anti-TcR anti-WT31, Becton-Dickinson
  • the excess antibody was removed by washing the plate or flask 3 times with a 3-fold volume of Hank's Balanced Salt Solution (HBSS) for plates and a 5.0 ml volume of the same solution for flasks.
  • HBSS Hank's Balanced Salt Solution
  • the TIL population was re-suspended in RPMI-1640, supplemented with penicillin/streptomycin, 2 mM 1- glutamine, and 10% type AB human plasma.
  • the concentration of cells to be activated depends on the number of non-lymphocytes contaminating the cell suspension. For example, fresh tumor suspensions contain anywhere from 1- 50% lymphocytes; the remainder are tumor cells. Since nonlymphoid cells compete for space on the antibody-coated matrix, 5 X 10 6 cells/T-25 flask may be used (10 5 cells/ml; 3 ml/plate).
  • the flask was then incubated for 24-48 hours at 37°C.
  • Cells were harvested using sterile pipettes and the cells were then pelleted by centrifugation.
  • the matrix was thoroughly washed 2-3 times with HBSS, resuspended in RPMI-1640 (as described above), supplemented with 50 U/ml IL-2, and incubated at 37°C (see, for example, harvesting techniques in In Vitro Methods in Cell-Mediated and Tumor Immunity, Bloom and David, eds., Academic Press, N.Y. (1976)).
  • rIL-2 Amgen Corp., California
  • the rIL-2 used had a specific activity of 3.7 X l ⁇ 6 units/ml.
  • Each vial of rIL-2 contained approxi- ately .380 mg/ml rIL-2, 10 M Na acetate, 4.9% glucose, the remaining volume consisting of water.
  • IL-2 from non-recombinant sources can, of course, also be used.
  • TIL were cultured without rIL-2 in T-25 tissue culture flasks previously coated with anti-CDS (1 ug/ml in 0.05 molar borate buffer, pH 8.6) or anti-TcR.
  • Table One compares anti-CD3-induced TIL growth with standard conditions two weeks following culture initiation:
  • TIL which have become refractory to rIL-2-induced growth (Standard culture; less than 100% yield) are capable of growing after treatment with
  • TIL were sampled from bulk cultures and maintained as described above approximately 1-2 weeks after subculture.
  • TIL activated by solid-phase anti-CD3 antibodies retained their cytotoxicity for autologous tumor compared to those cultured under standard conditions (p ⁇ .05), yet did not substantially cross-react with heterologous fresh tumor or the cell lines Daudi, sensitive target cells for the antigen- nonspecific cytotoxicity characteristic of LAK and natural killer cells.
  • Daudi sensitive target cells for the antigen- nonspecific cytotoxicity characteristic of LAK and natural killer cells.
  • cell proliferation assessed by counts of viable cells using Trypan blue exclusion four days following anti-CD3 activation, was 2-4 fold higher than that of TIL cultured exclusively in rIL-2 (p ⁇ .05) (Wilcoxon rank sum).
  • TIL incubated on a solid-phase matrix coated with MoAbs to CD3 and TcR can be specifically and significantly activated in terms of cell growth, lymphokine production, and anti-tumor cytotoxicity.
  • this invention provides a reproducible stimulus for cell proliferation and anti-tumor cytotoxicity, and for the expansion of cloned lymphocytes, important for the adoptive transfer of autologous lymphocytes into cancer
  • the observed benefits of this invention include the production of a high yield of TIL from the very beginning of the culture period and the maintenance of a high rate of growth of TIL when, using other methods, TIL populations would become refractory to IL-2 and stop growing.
  • a primary benefit of the present invention is the capability of activating TIL in the absence of rIL-2. Subsequent to activation, cells are expanded in reduced concentrations of rIL-2.
  • this use of solid-phase MoAbs to CD3 and TcR to isolate, activate, and induce TIL proliferation and cytotoxicity paves the way for the implementation of effective human immunotherapy programs.
  • Variables which up until now were not controllable i.e., cell proliferation, lymphokine production, and cytotoxi ⁇ city, are now more controllable and can be easily manipulated according to the teaching of the present invention.
  • lymphocytes Uses for the in vitro expansion of these lymphocytes include improved cellular immunotherapy with tumor-derived TIL as a replacement to current treatment modalities, including the use of LAK cells incubated with large quantities of rIL-2. It also provides a method for the immunotherapy of patients who are, for various reasons, ly phopenic and immuno ⁇ suppressed.
  • the method of the present invention could also be used to study infiltrating cells involved in tissue-graft rejection and would also be useful for organ-transplant therapies.

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Abstract

On décrit des procédés d'activation et de prolifération de lymphocytes infiltrants les tumeurs (TIL), ainsi que l'utilisation de telles lymphocytes dans le traitement de maladies et les recherches en immunothérapie.
PCT/US1989/004189 1988-10-21 1989-09-27 ACTIVATION ET CROISSANCE DE LYMPHOCYTES HUMAINS INFILTRANT LES TUMEURS AU MOYEN D'ANTICORPS ANTI-CD3 OU ANTI-TcR WO1990004633A1 (fr)

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EP0421380A1 (fr) * 1989-10-03 1991-04-10 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Inducteur de cellules de lyse de tumeurs, méthode et appareillage pour induire des cellules de lyse de tumeurs
GR900100551A (fr) * 1989-07-21 1991-12-10 Ortho Pharma Corp
EP0495639A1 (fr) * 1991-01-16 1992-07-22 Schering Corporation Utilisation de l'interleukine-10 dans l'immunothérapie adoptive du cancer
WO1993000918A1 (fr) * 1991-07-10 1993-01-21 Ochoa Augusto C Stimulation anti-cd3 a court terme de lymphocytes afin d'augmenter leur activite in vivo
US5527713A (en) * 1993-06-11 1996-06-18 Coulter Corporation Anti-CD3 antibody-aminodextran conjugates for induction of T-cell activation and proliferation
US5641677A (en) * 1991-04-05 1997-06-24 Regents Of The University Of Minnesota Method of enhancing the immunotherapeutic activity of immune cells by depletion of CD8+ T cells
US5725855A (en) * 1991-04-05 1998-03-10 The United States Of America As Represented By The Department Of Health And Human Services Method of treating tumors with CD8+ -depleted or CD4+ T cell subpopulations
US11111493B2 (en) 2018-03-15 2021-09-07 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy

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US4808151A (en) * 1987-04-27 1989-02-28 E. I. Du Pont De Nemours And Company Simplified method for the preparation of human lymphokine activated killer cells

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CANCER RESEARCH, Vol. 48, 15 February 1988; "Synergy of Tumor Necrosis Factor and Interleukin 2 in the Activation of Human Cytotoxic Lymphocytes", (OWEN-SCHAUB), See pages 788-92. *
CANCER RESEARCH, Vol. 48, January 1988; "Interleukin 2 Expanded Tumor-Infiltrating Lymphocytes in Human Renal Cell Cancer", (BELLDEGRUN), See pages 206-14. *
JOURNAL IMMUNOLOGY, Vol. 138, April 1987; "Long-Term Growth of Lymphokine-Activated Killer (LAK) Cells: Role of Anti-CD3 beta IL-1, Interferon-alpha and beta", (OCHOA), See pages 2728-33. *
JOURNAL IMMUNOLOGY, Vol. 138, March 1987, "Lysis of Tumor Cell by CD3+4-8-16+ T Cell Receptor alpha beta-Clones, Regulated Via CD3 and CD16 Activation Sites, Recombinant Interleukin 2, and Interferon beta", (GRIEND), See pages 1627-33. *
JOURNAL IMMUNOLOGY, Vol. 140, June 1988; "Lymphocytes Infiltrating Human Ovarian Tumors", (HEO), See pages 4042-49. *
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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR900100551A (fr) * 1989-07-21 1991-12-10 Ortho Pharma Corp
EP0409655A3 (en) * 1989-07-21 1992-04-15 Ortho Pharmaceutical Corporation Method for stimulating proliferation of peripheral blood lymphocytes
EP0421380A1 (fr) * 1989-10-03 1991-04-10 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Inducteur de cellules de lyse de tumeurs, méthode et appareillage pour induire des cellules de lyse de tumeurs
EP0495639A1 (fr) * 1991-01-16 1992-07-22 Schering Corporation Utilisation de l'interleukine-10 dans l'immunothérapie adoptive du cancer
WO1992012726A1 (fr) * 1991-01-16 1992-08-06 Schering Corporation Emploi de l'interleukine-10 dans l'immunotherapie adoptive du cancer
US5776451A (en) * 1991-01-16 1998-07-07 Schering Corporation Use of interleukin-10 in adoptive immunotherapy of cancer
US5641677A (en) * 1991-04-05 1997-06-24 Regents Of The University Of Minnesota Method of enhancing the immunotherapeutic activity of immune cells by depletion of CD8+ T cells
US5725855A (en) * 1991-04-05 1998-03-10 The United States Of America As Represented By The Department Of Health And Human Services Method of treating tumors with CD8+ -depleted or CD4+ T cell subpopulations
AU674128B2 (en) * 1991-07-10 1996-12-12 Cynthia Loeffler Short-term anti-CD3 stimulation of lymphocytes to increase their in vivo activity
US5316763A (en) * 1991-07-10 1994-05-31 The United States Of America As Represented By The Department Of Health And Human Services Short-term anti-CD3 stimulation of lymphocytes to increase their in vivo acitivity
WO1993000918A1 (fr) * 1991-07-10 1993-01-21 Ochoa Augusto C Stimulation anti-cd3 a court terme de lymphocytes afin d'augmenter leur activite in vivo
US5527713A (en) * 1993-06-11 1996-06-18 Coulter Corporation Anti-CD3 antibody-aminodextran conjugates for induction of T-cell activation and proliferation
US11111493B2 (en) 2018-03-15 2021-09-07 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11421228B2 (en) 2018-03-15 2022-08-23 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
US11608500B2 (en) 2018-03-15 2023-03-21 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy

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