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WO1995032729A1 - Stimulation anti-cd3 a court terme des lymphocytes destinee a en accroitre l'activite in vivo - Google Patents

Stimulation anti-cd3 a court terme des lymphocytes destinee a en accroitre l'activite in vivo Download PDF

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WO1995032729A1
WO1995032729A1 PCT/US1995/006703 US9506703W WO9532729A1 WO 1995032729 A1 WO1995032729 A1 WO 1995032729A1 US 9506703 W US9506703 W US 9506703W WO 9532729 A1 WO9532729 A1 WO 9532729A1
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cells
lymphocytes
lymphocyte
stimulated
population
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Augusto Carlos Ochoa
Mark L. Saxton
Dan L. Longo
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    • 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
    • C12N5/0648—Splenocytes
    • 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
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/50—Colon

Definitions

  • the present invention relates to the culturing of lymphocytes in the presence of a lymphocyte surface receptor antibody so as to elicit an immune response in vivo. Specifically, it relates to the short-term stimulation of lymphocytes in vi tro with anti-CD3 monoclonal antibody followed by the reintroduction of the activated lymphocytes in vivo .
  • the lymphocytes may comprise a total lymphocyte population, or alternatively, a depleted or positively selected lymphocyte population.
  • the invention is particularly represented by improved antitumor activity.
  • T lymphocytes i.e., T cells
  • T cells are central players in the immune response by virtue of their ability to recognize antigens with a high degree of specificity, to act as effector cells in the lysis of specific target cells, and to regulate the nature and intensity of the immune response.
  • Interleukin-2 IL-2
  • helper T cells stimulates the growth of T cells and ⁇ K cells that bear IL-2 receptors, either in vivo or in vi tro .
  • lymphocytes are referred to as lymphokine activated killer (LAK) cells.
  • LAK lymphokine activated killer
  • Typical adoptive immunotherapy involves the administration of immunologically active cells to an individual for the purpose of providing a beneficial immunological effect to the individual, e.g., reduction or control of cancerous or diseased tissue.
  • immunologically active cells e.g., lymphokine-activated killer cells and tumor infiltrating cells
  • Lymphocytes are typically taken by venipuncture or leucophaeresis .
  • Tumor infiltrating cells are taken from tumors removed during surgery. The lymphocytes are cultured to increase their number and activate their antitumor activity, and infused back into the patient.
  • LAK cells plus IL-2, or IL-2 alone can be effective in mediating the regression of established metastatic cancer in selected patients. See, for example, S.A. Rosenberg, "Immunotherapy of Patients with Advanced Cancer Using Interleukin-2 Alone or in Combination With Lymphokine Activated Killer Cells" in Important Advances in Oncolocry 1988, J.B. Lippincott Co., 217, (1988) .
  • T cells cultured in the presence of IL-2 and monoclonal antibodies (MoAb) against the antigen receptor complex CD3, i.e., anti-CD3 MoAb, have been found to proliferate and demonstrate in vi tro LAK activity on a per cell basis.
  • MoAb monoclonal antibodies
  • P.M. Anderson et al. Cancer Immunol. Immunother.. 27, 82 (1988)
  • P.M. Anderson et al. Jj_ Immunol.. 142, 1383 (1989)
  • A.C. Ochoa et al. Cancer Res. , 49, 963 (1989) .
  • Anti-CD3 induces, at least in part, activation of tumoricidal T cells as well as activated NK (LAK cells) .
  • LAK cells activated NK
  • Conventional IL-2 therapy only activates LAK cells.
  • certain tumor target cells are more susceptible to anti-CD3 activated T cells than LAK cells. See, for example, J. Stankova et al . , Cell. Immunol . , 121, 13 (1989) and Yun et al . , Cancer Res. 49, 4770 (1989) .
  • lymphocytes capable of proliferation and enhanced immunotherapeutic efficacy that: (1) is not time consuming; (2) is less expensive; (3) has fewer, if any, side effects; (4) is simple and convenient; (5) does not require prolonged in vi tro culture; and (6) can activate any and all types of T cells required for immunotherapy.
  • Patients subjected to a bone marrow transplantation encounter a period of severe immune deficiency following high dose chemotherapy and/or total body irradiation
  • TBI bone marrow
  • the patient's bone marrow is replaced with a small amount of healthy bone marrow cells that proliferate in the body until enough marrow cells have been generated to achieve a repopulation of the peripheral blood by red blood cells, platelets and white blood cells of the immune system.
  • a typical bone marrow engraftment can take anywhere from 20 to 30 days to regenerate (engraft) sufficient quantities of bone marrow cells.
  • the patient's immune system is virtually non-functional, and the patient must be closely monitored to prevent the onslaught of disease.
  • These bone marrow cells can be induced to proliferate with factors such as G-GSF, GM-CSF, IL-3 and the like.
  • a method of enhancing the immunotherapeutic activity of lymphocytes by stimulating the lymphocytes in vi tro in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of a low level of IL-2, for less than about 24 hours to form stimulated lymphocytes, infusing the stimulated lymphocytes into a tumor-bearing mammal, and administering an effective amount of IL-2 to the mammal.
  • the antibody to a lymphocyte surface receptor is preferably anti-CD3.
  • the lymphocytes may comprise a total lymphocyte population, or alternatively, a depleted or positively selected lymphocyte population.
  • the depletion method for preparing a depleted lymphocyte population includes separating at least one cell subset, or subpopulation, that is capable of down-regulating the immunotherapeutic activity of an immune cell population, from that immune cell population to form a depleted immune cell population; and culturing the depleted immune cell population in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of IL-2, to form a stimulated depleted immune cell population.
  • this method reduces or eliminates a regulatory mechanism from the immune cell population which allows the remaining cells to more fully express their immune function.
  • the immunotherapeutic activity of the remaining cell population as represented by a measure of the ability of the immune cell population to reduce tumor volume, is increased when compared to a similarly treated undepleted immune cell population.
  • the positive selection method of enhancing the immunotherapeutic activity in accordance with an object of the present invention includes separating and positively selecting at least one cell subset, or subpopulation, that is capable of up-regulating the immunotherapeutic activity, or developing the immunotherapeutic activity, of an immune cell population, from that immune cell population to form an immune cell subset; and then culturing the immune cell subset, e.g.
  • CD4 + or CD8 + cells in the presence of an antibody to a lymphocyte surface receptor, to form a stimulated immune cell subset.
  • the immunotherapeutic activity of the positively selected and stimulated immune cell subset as represented by a measure of the ability of the immune cell subset to reduce tumor volume, is increased when compared to a similarly treated total lymphocyte population.
  • the total lymphocyte population is cultured in a medium in the presence of an antibody to a lymphocyte surface receptor, optionally in the presence of IL-2, for less than about 24 hours, e.g., for 12 to 18 hours, more preferably less than 4 hours, and as short as 30 minutes.
  • the antibody to a lymphocyte surface receptor is anti-CD3. If IL-2 is present in the culture medium, it is present in low doses ( ⁇ 300 units/ml) , in contrast to high doses and long exposure as was practiced in the prior art.
  • IL-2 is present in the culture medium during stimulation of lymphocytes in vi tro, it is an amount of IL-2 that is sufficient to maintain the viability of cells without inducing substantial lymphocyte proliferation.
  • the total lymphocyte population or alternatively, a depleted or positively selected lymphocyte population, can be administered to the patient after stimulation with the antibody against the surface receptor. These cells will then proliferate in vivo after IL-2 is administered, preferably liposomal IL-2. While not intending to be bound by any theory, it is believed that culturing the immune cell population, or alternatively, a depleted or positively selected lymphocyte population, in the presence of an antibody to a lymphokine surface receptor
  • IL-2 "primes" the cells to upregulate the production of IL-2 receptor sites on the cells.
  • the stimulated cells bind more IL-2 and a higher level of cytotoxic activity is induced.
  • These stimulated cells when bound with IL-2, then are capable of lysing tumors because the IL-2 may generate a signal cascade within the cells that results in LAK activity.
  • Immune cell populations stimulated in accordance with any of the foregoing methods are useful for the following methods of treatment.
  • a method of treating a mammal having tumors with a stimulated total lymphocyte population, or alternatively, a depleted or positively selected lymphocyte population having an enhanced immunotherapeutic activity prepared by the procedures outlined above. This method therefore includes (i) enhancing the immunotherapeutic activity of an immune cell population as described immediately above;
  • the anti-CD3 stimulated lymphocytes display enhanced immunotherapeutic efficacy in vivo as represented by a decrease in the tumor burden by at least about 20%.
  • the short-term stimulation of the lymphocytes can be effected over a period of about 12-18 hours, more preferably less than 4 hours, and as short as 30 minutes, and the tumor burden is decreased by at least about 40%, more preferably by at least about 60%, and most preferably by at least about 80%.
  • a method of stimulating the proliferation of bone marrow cells comprising the steps of first enhancing the immunotherapeutic activity of an immune cell population in accordance with any of the procedures outlined above.
  • the stimulated cells are then incubated in the presence of bone marrow, optionally in the presence of additional cytokines including granulocyte macrophage colony stimulating factor (GM- CSF) , IL-3, Kit Ligand (KL) , erythropoietin (EPO) or IL-2 to thereby increase the number of bone marrow cells.
  • the bone marrow cells, cultured in vi tro then can be administered to a mammal to stimulate the proliferation of additional bone marrow cells.
  • the stimulated lymphocytes can be administered to a mammal having a compromised bone marrow cell population to stimulate growth of bone marrow cells in vivo .
  • a method of enhancing the engraftment of bone marrow cells and/or killing residual tumor cells from bone marrow cells comprising the steps of first enhancing the immunotherapeutic activity of an immune cell population in accordance with any of the procedures outlined above.
  • the stimulated cells are then infused, optionally together with bone marrow cells, into the patient.
  • the patient is optionally treated with a lymphokine (s) such as IL-2.
  • s lymphokine
  • FIG. 1 shows the comparative increase of IL-2 receptors after 24 hours of stimulation in varying concentrations of anti-CD3.
  • Fig. 2 shows the increase in the expression of the oc chain of the IL-2 receptor on human T lymphocytes after overnight incubation with anti-CD3.
  • Fig. 3 shows human lymphocyte growth after 24 hour stimulation with anti-CD3 and subsequent culturing in media containing IL-2.
  • Fig. 4 shows the growth of lymphocytes stimulated with differing concentrations of anti-CD3 for four hours followed by culture in IL-2 containing medium.
  • Fig. 5 shows the white blood cell count from a patient whose lymphocytes were cultured overnight in anti-CD3. The lymphocytes were infused back into the patient and the patient subsequently received IL-2 treatments.
  • stimulating indicates the process whereby cells are placed in a tissue culture medium comprising nutrients to sustain the life of the cells, and other additives, such as the anti- CD3 monoclonal antibody. Stimulation can be done in the presence of IL-2 but generally IL-2 is not included. If stimulation of lymphocytes is to be done in the presence of IL-2, low doses of IL-2 ( ⁇ 300 units/ml) are used in contrast to the high and long exposure as was practiced in the prior art. This process can take place in any vessel or apparatus. The process can involve various stages of culturing and subculturing. However, typically, only one culturing or stimulating step is desirable. IL-2 activity is expressed herein in Hoffmann-LaRoche units. 2.3 Hoffmann-LaRoche units equal 6 International Units.
  • the phrase "depletion method” denotes a method of enhancing the immunotherapeutic activity of a total lymphocyte population by utilizing a "depleted lymphocyte population.
  • a depleted lymphocyte population is the portion of the total lymphocyte population remaining after at least one cell subset, or subpopulation, that is capable of down-regulating the immunotherapeutic activity of that lymphocyte population, has been separated and removed therefrom.
  • the resulting depleted lymphocyte population, when stimulated, is referred to herein as a "stimulated depleted lymphocyte population.
  • depleted lymphocyte population preferably refers to a total cell population derived from a sample of peripheral blood, spleen, lymph node, tumor, pleural effusions, in vi tro cultured lymphocytes, and the like, with at least one cell subset or subpopulation, which down-regulates the immunotherapeutic activity of the total population, removed therefrom.
  • a “depleted lymphocyte population” can be further depleted by removal of a subpopulation or subset, e.g., CD4 + from which a subset, e.g., 2H4, is removed.
  • lymphocyte subset is the portion of the lymphocyte population that has been separated and removed, or panned from the lymphocyte population. This lymphocyte subset, when stimulated, is referred to herein as a “stimulated lymphocyte subset.”
  • immuno ⁇ therapeutic activity refers to any of a variety of immune responses of immune cells. This includes antitumor activity, such as lymphokine production, specific lytic activity, or the nonspecific lytic activity of lymphokine activated killer (LAK) cells and natural killer (NK) cells.
  • LAK activity is defined as the ability of lymphocytes to lyse tumor cells, and to a lesser degree normal cells. This activity in lymphocytes is typically stimulated by lymphokines, such as IL-2.
  • NK activity is defined as the ability to lyse tumor cells, but not normal cells, which does not result from prior stimulation.
  • antiitumor activity includes a reduction in the number of tumors as well as a reduction in the size of tumors which can be collectively referred to as reduction in tumor burden.
  • immunotherapeutic activity also includes suppression of an undesirable immune response.
  • T lymphocytes can also function as suppressor cells such that they inhibit an on-going immune response. It is therefore expected that some of the T lymphocyte subsets, activated according to the present invention, will be useful in treatment of diseases where the immune response produces damage to normal tissues, e.g., autoimmune disease.
  • the increased immunotherapeutic activity of lymphocyte populations can be determined in vi tro by the percent cytotoxicity, which is a measure of the ability of immune cells to destroy a radioactively labeled tumor target. That is, the antitumor activity is determined by a comparison of the level of radioactivity released in tissue culture media from the effector/target combination to the level of radioactivity in the culture media released from the target alone.
  • increased immunotherapeutic activity, as defined herein, of immune cells is typically demonstrated by an increase in the percent cytotoxicity of the effector cells on tumor cells.
  • the tumor cell lines can be any of a variety of cell lines commercially available, including leukemia cells and fresh tumor targets. Preferably, they are leukemia cells.
  • cytolytic activity can be measured quantitatively using the cell-mediated lympholysis assay (CML) as described in S.-L. Wee et al. , Hu . Immunol.. 3_, 45 (1981), which is incorporated herein by reference.
  • CML cell-mediated lympholysis assay
  • lymphocytes include T cells, such as CD3 + T cells, including CD4 + and CD8 + cells. It is understood that this includes tumor infiltrating lymphocytes (TIL cells) .
  • TIL cells tumor infiltrating lymphocytes
  • the lymphocytes are T cells, or T cell subsets, including, but not limited to CD4 + , or any CD4 + subsets such as 2H4 or 4B4, CD8 + , or any of its subsets.
  • lymphocytes can be stimulated in vi tro with an antibody to a lymphocyte surface receptor, without the presence of IL-2, for short periods of time, i.e., for less than about 24 hours, e.g., 12-18 hours and as short as 30 minutes. These cells have a high therapeutic efficacy upon in vivo injection.
  • Anti-CD3 stimulated cells are less toxic and are generally smaller in size than cells cultured only in IL-2 for several days. Reduced pulmonary toxicity may be due to the smaller size of anti-CD3 stimulated cells which presumably circulate more freely through the vascular system than larger LAK cells.
  • the antibody to a lymphocyte surface receptor can be any of a variety of monoclonal antibodies (MoAb) against a surface antigen receptor complex having mitogenic potential.
  • MoAb monoclonal antibodies
  • it can be an anti-CD3, anti-CD2, anti-CD4, anti-CD5, anti-CD28, anti-CDllb, etc., MoAb. It is preferably an anti-CD3 MoAb.
  • the antibodies can be used alone or in various combinations with other antibodies.
  • anti-CD3 can be used in combination with anti-CD2, anti-CD4, anti-CD5, anti-CD28 or anti-CDllb, for effective results.
  • the anti-CD3 MoAb used to stimulate human T cells can be, but is not limited to, OKT3, WT32, Leu-4, SPV-T3c, RIV9, 64.1, etc.
  • the anti-CD3 MoAb used to stimulate murine T cells is more preferably the anti-murine CD3 MoAb 145-2C11, which has been identified by 0. Leo et al . , Proc. Na l . Acad. Sci. USA, 84, 1374-1378 (1978) , and is available from American Type Culture Collection (ATCC) .
  • ATCC American Type Culture Collection
  • Mouse anti- human 0KT3 is available from the Ortho Division of Johnson and Johnson.
  • Cells treated with anti-CD3 MoAb for less than about 24 hours are preferably treated with a total dose of about 0.1 ng/ml to 10 ng/ml anti-CD3 antibody, in stimulating one whole leucophaeresis.
  • a "leucophaeresis” is defined as the peripheral blood lymphocytes (PBLs) enriched for mononuclear cells removed during the course of a standard phaeresis procedure. More preferably, anti-CD3 is used in a total dose of about 10 ng/ml used in stimulating one whole leucophaeresis.
  • the anti-CD3 stimulation induces the expression of the IL-2 receptor.
  • These cells are then collected and placed in the body of an organism, preferably a mammal, such as a mouse or a human, where they develop immunotherapeutic activity, e.g., cytotoxic activity or lymphokine production, upon the administration of IL-2. More preferably the cells are placed in the body of a human for immunotherapeutic treatment.
  • mice of large numbers > 50 x 10 6
  • IL-2, or IL-2 and anti-CD3 can produce acute respiratory problems in mammals due to sludging, i.e., stasis or blocking the lung capillaries, by these large cells in pulmonary capillaries.
  • stimulation of cells with anti-CD3 alone or anti-CD3 + IL-2 for less than 24 hours does not generally produce an increase in the size of the cells.
  • the cells display an enhanced proliferation and antitumor activity.
  • the administration of IL-2 preferably occurs over a period of about 7 days.
  • Lymphokines e.g., IL-2
  • the amount of IL-2 effective for enhancing cell proliferation and antitumor activity in vivo depends on the mammal being treated and also on the form in which it is administered. For example, the effective dose of IL-2 is expected to be lower when administered as liposomal IL-2 than when administered as free IL-2.
  • IL-2 About 10,000-70,000 units/day of IL-2, preferably about 50,000 units/day of IL-2, are administered to mice, and about 1 x 10 4 to 1.8 x 10 7 International Units/m/day are administered to humans.
  • Certain stimulated depleted immune populations or stimulated lymphocyte subsets may exhibit enhanced proliferation and antitumor activity in vivo without having to administer lymphokines, such as IL-2, or with administration of only low doses of IL-2 to the patient.
  • lymphokines such as IL-2
  • it is preferred that the cells are initially stimulated for less than 24 hours with anti-CD3 MoAb.
  • IL-2 is present in an amount of less than about 30% of the amount of IL-2 typically used in culturing procedures.
  • lymphocytes When lymphocytes are cultured optionally in the presence of IL-2, this typically will mean that if IL-2 is used at all, it is used in a "relatively minor amount.” For example, if about 1,000 units of IL-2 typically are used in culturing a specific lymphocyte population, then less than about 300 units would be used in the present invention if culturing with IL-2 is optional, and IL-2 is utilized.
  • lymphokines in addition to, or in place of IL-2, in the initial culture with an antibody to the lymphocyte surface receptor.
  • lymphokines include IL-1, IL-4, IL-6, IL-12, interferon, etc. It is envisioned that they can be used alone, in sequence, or in combination with low dose IL-2 as an adjunct to simulation with an antibody to a lymphocyte surface receptor.
  • lymphokines can be administered to the patient following infusion of the lymphocytes stimulated with an antibody to a surface receptor.
  • the lymphokines can be administered alone, in sequence, or in combination with IL-2. It is contemplated that administration of different lymphokines will play a role in selecting or supplementing the immune response and effector mechanisms.
  • the CD4 + subset appear to control and modulate the development of immune responses .
  • TH cells play a major role in determining which epitopes become targets for the immune response and selection of effector mechanisms.
  • the TH cells select and activate appropriate effector cells including B cells that produce antibody and modulate the actions of other effector cells, Tc cells, NK cells, macrophages, granulocytes and antibody dependent cytotoxic (K) cells.
  • TH-1 cells secrete IL-2 and IFN-7 which tend to activate macrophages and cytotoxic cells.
  • TH-2 cells secrete IL-4, IL-5, IL-6 and IL-10 and tend to increase production of eosinophils and mast cells and to enhance production of antibody including IgE and decrease the function of cytotoxic cells. It is therefore contemplated that under certain circumstances it may be desirable to administer one or more of the lymphokines secreted by TH-1 cells in order to mimic a TH-1-type response. On the other hand, it may be desirable to administer the lymphokines secreted by TH-2 cells in order to mimic a TH-2-type response.
  • lymphocytes pre- or at the same time as, lymphocytes are stimulated with an antibody to a lymphocyte surface receptor.
  • antigens Any of a number of different antigens can be used including tumoral, microbiological, etc.
  • the antitumor activity of the total lymphocyte population stimulated in vi tro with anti-CD3 is increased in vivo upon the administration of IL-2 by about 20%, preferably by about 50%, more preferably by about 60%, and most preferably by about 80%.
  • the anti-CD3 stimulated lymphocytes display enhanced immunotherapeutic activity, e.g., cytotoxicity or lymphokine production, in vivo as represented by a decrease in the tumor burden by at least about 20%, preferably by at least about 40%, more preferably by at least about 60%, and most preferably by at least about 80%.
  • untreated animals will usually develop greater than about 250 tumors, while animals treated with anti-CD3 stimulated cells from a total lymphocyte population and IL-2 will usually develop less than about 40 tumors.
  • the immunotherapeutic activity of a lymphocyte population first is enhanced in accordance with any of the methods described above.
  • the stimulated total lymphocyte population, depleted lymphocyte population or stimulated lymphocyte subset then is administered to a mammal having tumors.
  • the mammal is pretreated with an immunosuppressant that preferably also is chemotherapeutic. While not intending to be bound by any theory, the use of an immunosuppressant may serve to suppress the activity of other immune cells thereby permitting the stimulated lymphocytes to function more effectively upon administration to the mammal or the immunosuppressant may serve to diminish the total volume of the tumor.
  • immunosuppressants such as doxorubicin or cyclophosphamide (cytoxan) are used, although those skilled in the art readily recognize that other immunosuppressants can be used in accordance with the present invention such as corticosteroids.
  • the tumors which have shown a reduction upon treatment with cells stimulated by the method of the present invention and IL-2 include MCA-38 (colon carcinoma) , RENCA (renal cell carcinoma) , MCA-102 (sarcoma) and 38C (lymphoma) which are all tumors having different embryonic origins.
  • the colon carcinoma is derived from cells which embryologically were derived from the ectoderm (the origin of the lining of all organs as well as the skin)
  • the renal cell carcinoma is derived from cells which embryologically were derived from the neural crest (origin of nervous tissue, gonads and adrenal glands)
  • the sarcoma is derived from cells which embryologically were derived from the connective tissue (bone, ligaments) . Therefore, it is expected that tumors derived from cells that embryologically are derived from ectoderm, endoderm and mesoderm will respond to therapy.
  • T lymphocytes can also function as suppressor cells, i.e., they have regulatory mechanisms by which they can stop an on-going immune response.
  • T lymphocytes activated during the preparation of T-AK cells will be useful in the treatment of diseases where the immune response produces damage to normal tissues, e.g., autoimmune diseases such as Lupus erythematosus, multiple sclerosis, rheumatological conditions (rheumatoid arthritis) , allergic conditions, and the rejection of transplanted organs such as kidney, liver, heart, lung, or bone marrow (autologous or allogeneic) .
  • autoimmune diseases such as Lupus erythematosus, multiple sclerosis, rheumatological conditions (rheumatoid arthritis) , allergic conditions, and the rejection of transplanted organs such as kidney, liver, heart, lung, or bone marrow (autologous or allogeneic) .
  • the immunotherapeutic activity or cytotoxicity of the lymphocytes can be enhanced by separating at least one cell subset, or subpopulation, from the lymphocytes to form a depleted lymphocyte preparation wherein the separated cell subset down-regulates the immunotherapeutic activity or cytotoxicity of the lymphocytes.
  • this method reduces or eliminates a regulatory mechanism from the lymphocytes, which allows the remaining cells to more fully express their immune function.
  • the immunotherapeutic activity of the remaining lymphocytes as represented by a measure of the cytotoxicity or antitumor activity of the cells, can be increased by a factor of at least 1.2, preferably by a factor of at least about 2.0.
  • the depleted lymphocytes are preferably cultured in a first medium with an antibody to a lymphocyte surface receptor, optionally in the presence of low doses of IL-2 ( ⁇ 300 units/ml) for less than about 24 hours.
  • this method involves the depletion of T lymphocyte populations, such as PBL populations, before the initiation of culture with an antibody to a lymphocyte surface receptor.
  • the separated cells are CD4 + or CD8 + lymphocytes, or more specific subsets of each of these populations.
  • the cytotoxicity, or antitumor activity can be specific or nonspecific lytic activity.
  • it is nonspecific lytic or LAK activity.
  • the lymphocytes, and more preferably peripheral blood mononuclear lymphocytes can be depleted of specific T cell subsets by any method.
  • the PBLs are depleted of specific subsets by negative depletion using magnetic beads, immunoaffinity columns, or antibody coated polystyrene plates.
  • this involves the labelling of the PBLs with an antibody to a lymphocyte surface receptor for the T cells that are to be removed from the total PBL population.
  • This mixture of labelled and unlabelled cells are then mixed with goat an i-mouse IgG-coated magnetic beads.
  • a complex of the beads and the labelled T cells, i.e., those cells complexed with the surface receptor antibody, is formed.
  • the beads/labelled T cell complexes are then separated from the mixture using a magnetic separator. In this way, a specific T cell subset, or portion thereof, can be removed from the PBL mixture.
  • the specific immune cell subset removed can be any that down-regulates the immunotherapeutic activity, preferably the cytotoxic activity, of the total immune cell population.
  • This can include: CD4 + , or any of its subsets such as 2H4 or 4B4; CD8 + , or any of its subsets; NK cells, or any of its subsets; macrophages; B cells; and the like.
  • the immune cell subsets removed are T cell subsets, and more preferably they are CD4 + or CD8 + cells.
  • a typical sample of PBLs from a sample of human whole blood contains about 20-30% CD8 + cells and about 30-50% CD4 + cells.
  • the cells that inhibit or down-regulate the immunotherapeutic activity of the population need only be removed until an increase in the immunotherapeutic activity, as represented in vi tro by cytotoxicity, by a factor of about 1.2 is observed in the remaining cell population.
  • the number of CD4 + or CD8 + cells are reduced in the depleted immune cell populations by at least about 75%, more preferably by at least about 90%.
  • a "substantially completely depleted" immune cell population e.g., PBL population
  • a "substantially completely CD4 + -depleted immune cell population” contains less than about 5% CD4 + cells.
  • the method of the present invention includes separating at least about 50% preferably at least about 75%, and more preferably at least about 90% of the CD4 + or CD8 + cells from PBLs to increase the immunotherapeutic activity, e.g., antitumor activity, of the remaining "depleted" cell population.
  • a method has been developed to enhance the cytotoxicity of immune cells by first culturing an immune cell population to form a cultured immune cell population; separating or positively selecting a cell subset or subpopulation, that is capable of developing immunotherapeutic activity or cytotoxicity; and separately culturing or subculturing the selected cells in a second medium in the presence of a monoclonal antibody to a lymphocyte cell surface antigen, optionally in the presence of low doses of IL-2.
  • the immunotherapeutic activity, as represented in vi tro by the cytotoxicity or cytokine production, of this subpopulation can be increased by a factor of at least about 1.2, preferably by a factor of at least about 2.0.
  • the separated cells are CD4 + or CD8 + lymphocytes, or subsets of each of these populations .
  • Specific T lymphocyte subsets or subpopulations can be selected or separated from an unseparated or total lymphocyte population, preferably PBLs, by any method.
  • the specific cell subpopulations are separated from the total populations by positive selection using fluorescence-labelled monoclonal antibodies.
  • fluorescence-labelled monoclonal antibodies typically, this involves adding a fluorescein isothiocyanate-conjugated MoAb or a phycoerythrin-conjugated MoAb to a cultured lymphocyte population, incubating the cells with the conjugate for 30 minutes at 4°C, washing the cells, and sorting or selecting out the labelled cells using a fluorescence- activated cell sorter.
  • the monoclonal antibody OKT8 can be used
  • CD4 + cells the monoclonal antibody OKT4 can be used, both of which are available from the Ortho Division of Johnson & Johnson.
  • the culturing process involves stimulating the depleted lymphocytes with an antibody to a lymphocyte surface receptor for less than about 24 hours, optionally in the presence of IL-2.
  • the antibody to a lymphocyte surface receptor is preferably an anti-CD3 MoAb which is an antibody against the antigen receptor complex CD3 , such as OKT3.
  • the antibody to a lymphocyte surface receptor can be any of a variety of monoclonal antibodies against a surface antigen receptor complex having mitogenic potential including, but not limited to, anti- CD2, anti-CD4, anti-CD28 or anti-CDllb.
  • the stimulated depleted lymphocytes are infused into the tumor-bearing mammal, and an effective amount of IL-2 is administered to the mammal .
  • the interleukin-2 is a commercially available T cell growth factor. It can be a naturally occurring IL-2, such as might be derived from cultured rat splenocytes, or it can be recombinant IL-2. It is believed that other lymphokines can also be used in the present invention either in low doses together with anti-CD3 in vi tro or administered to the patient following infusion of anti- CD3 stimulated lymphocytes. These include IL-1, IL-4, IL-6, IL-12, interferon, etc. It is envisioned that they can be used alone, in sequence, or in combination with IL-2 as an adjunct to primary anti-CD3 stimulation.
  • the IL-2 is preferably incorporated into liposomes as a delivery system.
  • These phospholipid vesicles can contain varying amounts of IL-2, or other cytokines or bioactive compounds, depending on the type of interaction between the solute and the phospholipid.
  • Many methods have been proposed for the preparation of liposomes . Most of these methods involve a form of aqueous hydration of the lipid, which may be either in a powdered form or as a dried film.
  • One of the most widely used techniques is known as the film method. Briefly, lipids of the desired composition in solution with an organic solvent are dried in the form of a thin film on the walls of a round-bottomed flask.
  • a bioactive compound can be included in the film at this stage.
  • the dry film is hydrated by adding a suitable aqueous phase and gently swirling the flask. With a hydrophilic bioactive compound, an aqueous solution is used for hydration.
  • the liposomes formed by this procedure generally have a number of concentric bilayers and are called multilammelar vesicles (MLVs) .
  • the liposomal IL-2 used in the present invention is preferably prepared using dimyristyl phosphatidyl choline
  • Liposomes are formulated utilizing a freeze/thaw and bath sonication technique, according to the method described by P.M. Anderson et al . , Cancer Research, 50, 1853-1856 (1990) .
  • routes of administration can be used for the administration of liposomes, for example, intravenous, intratumoral, subcutaneous, intraperitoneal, and oral delivery.
  • An important advantage of liposomal delivery is the change in tissue distribution and binding properties as compared to the free forms of the bioactive ingredient, resulting in enhanced therapeutic index and decreased toxicity.
  • the stimulated lymphocytes are added to the mammal with liposomal IL-2. More advantageously, the stimulated lymphocytes are administered intravenously whereas the liposomal IL-2 is administered intraperitoneally. Usually, liposomal IL-2 is added periodically after the initial administration of stimulated lymphocytes. In addition, the liposomal IL-2 and the stimulated lymphocytes can be administered to the mammal a second time after the initial treatment. Preferably, the second treatment takes place about 1 week after the initial treatment, although a second treatment any time after about 4 days is effective in substantially eradicating the tumor. As used herein, the phrase "substantial eradication of tumor" denotes a reduction in tumor volume to a point where the tumor either is completely destroyed or is so small that it is not readily recognizable by conventional methods .
  • Mammals undergoing bone marrow engraftment have a compromised bone marrow cell population for a period of about 15 to about 25 days.
  • the term "compromised bone marrow cell population” denotes an incomplete or depleted bone marrow cell population when compared to the mammal's normal bone marrow cell population.
  • a compromised bone marrow cell population can include a bone marrow cell population that is insufficient to achieve homeostasis in the mammal's immune system.
  • the present invention therefore advantageously further provides a method of stimulating the proliferation of bone marrow cells to generate bone marrow cells more readily in a compromised bone marrow cell population.
  • the stimulated lymphocytes provide an anti-tumor effect against residual tumor cells in the bone marrow upon introduction into the patient, for example, by means of intravenous infusion.
  • the immunotherapeutic activity of an immune cell population is enhanced in accordance with one of the methods described above.
  • the immunotherapeutic activity is enhanced by culturing positively selected CD4 + cells in the presence of anti-CD3 for a period of less than about 24 hours.
  • the resulting stimulated CD4 + cells are capable of stimulating the proliferation of bone marrow cells as defined, for example, by an increase in the number of colony forming units.
  • bone marrow cells can be extracted and purified from a mammal using techniques well known in the art, and then incubated with the stimulated lymphocytes for a period of time sufficient to generate more bone marrow cells. Preferably, the cells are incubated for a period of about 10 to about 20 days and most preferably, about 14 days at about 37°C.
  • cytokines such as GM-CSF, IL-3, KL and EPO can be added to the bone marrow during culturing.
  • the bone marrow cells produced in accordance with this method then can be infused into a mammal having a compromised bone marrow cell population using known techniques, in particular, intravenous injection.
  • the bone marrow cells may also be injected at the same time as the stimulated lymphocytes during a bone marrow transplant such that the stimulated lymphocytes stimulate the proliferation of additional bone marrow cells.
  • the stimulated lymphocytes can be administered to a mammal having a compromised bone marrow population using known techniques, e.g., intravenous injection.
  • the mammal is pretreated with an immunosuppressant such as cytoxan, that preferably also is chemotherapeutic.
  • an immunosuppressant such as cytoxan, that preferably also is chemotherapeutic.
  • the stimulated depleted immune cell population or the stimulated immune cell subset is administered periodically to the mammal, for example every four or five days. More advantageously, the stimulated depleted immune cell population or stimulated immune cell subset is administered with an additional cytokine such as IL-2, IL-3, GM-CSF, KL, EPO and the like.
  • the administration of the stimulated lymphocytes serves to stimulate the proliferation of additional bone marrow cells in a compromised bone marrow cell population of the treated mammal to generate bone marrow cells more rapidly when compared to proliferation of an unstimulated compromised bone marrow cell population.
  • the stimulated lymphocytes provide an anti-tumor effect against residual tumor cells in the bone marrow.
  • Example 1 Tumor Preparation and Tumor Cell Lines
  • the murine tumor cell line, MCA-38 a weakly immunogenic murine colon adenocarcinoma
  • a MCA-38 tumor cell line was established in tissue culture media (TCM) with 10% fetal calf serum (FCS, available from GIBCO Laboratories, Grand Island, NY) and used as a target for cytotoxicity assays.
  • TCM tissue culture media
  • FCS 10% fetal calf serum
  • the MCA-38 was maintained by subcutaneous (s.c.) passage in C57BL.6 mice.
  • the TCM consisted of Rosewell Park Memorial Institute (RPMI) 1640 medium (obtained from GIBCO) , supplemented with 25 mM HEPES [N- (2-hydroxyethyl)piperazine-N' - (2-ethanesulfonic acid) (available from GIBCO Laboratories) , 2 mM L- glutamine (available from GIBCO Laboratories) , and 100 units/ml penicillin (GIBCO) , 100 ⁇ g/ml streptomycin
  • the cell line was maintained in tissue culture media.
  • IL-2 specific activity 1.5 x 10 7 units/mg Hoffmann-LaRoche, Inc., Nutley, NJ
  • IL-2 containing 25 mg human serum albumin per 1 x 10 6 units IL-2 was diluted in Hanks' Balanced Salt Solution (HBSS, obtained from GIBCO Laboratories, Grand Island, NY, also available from SIGMA Chemical Co., St. Louis, MO) and added to dimyristyl phosphatidyl choline (DMPC, Avanti Polar Lipids, Pelham, AL) .
  • HBSS Hanks' Balanced Salt Solution
  • DMPC dimyristyl phosphatidyl choline
  • Liposomes were formulated utilizing a freeze/thaw and bath sonication technique, according to the method described by P.M. Anderson et al., Cancer Research, 50, 1853-1856 (1990) , which is incorporated herein by reference.
  • IL-2 liposomes were synthesized utilizing a constant ratio of lipid: IL-2 solution of 15 mg DMPC: 0.2 ml IL-2 solution. Mice treated with IL-2 liposomes received 5.0 x 10 4 units IL-2 liposomes.
  • Example 3 Cells Stimulated with Anti-CD3 In Vi tro and Their Effect on Hepatic Tumors
  • Peripheral blood lymphocytes, or splenocytes from C57BL.6 mice were obtained by extracting the spleen and crushing it with a glass stopper in a petri dish with HBSS. A single suspension was created by repeated pipetting.
  • the splenocytes were purified over Ficoll- Hypaque, (Pharmacia, Piscataway, NJ) , washed twice in phosphate buffered saline (PBS, pH 7.4 available from GIBCO, Grand Island, NY) and placed in culture in RPMI 1640 with 5% fetal calf serum, and 2% by volume of anti- CD3 supernatant 145-2C11 at a concentration of 2 x 10 6 cells/ml without IL-2 (anti-murine CD3 MoAb 145-2C11, which has been identified by 0. Leo et al. , Proc. Natl . Acad. Sci. USA, 84. 1374-1378 (1978), is available from ATCC) .
  • the concentration of anti-CD3 in the supernatant of 145-2C11 hybridoma is approximately 1- 2 micrograms/ml.
  • splenocytes were stimulated with anti-CD3 (145-2C11) and IL-2 (100-300 units/ml) in the following manner.
  • Cells were separated by Ficoll- Hypaque, washed 3 times in PBS, and placed at 1-2 x 10° cells/ml in RPMI 1640 + 5% fetal calf serum + 2% by volume anti-CD3 (145-2C11) .
  • the cells were diluted every 48-72 hours such that the concentration of cells was maintained at approximately 1 x 10° cells/ml.
  • the cells injected into mice were harvested on day 3 and 5 of culture.
  • Lymphocytes obtained from murine spleens, as described above in Example 3 were stimulated with the anti-CD3 monoclonal antibody 145-2C11 overnight, i.e., for approximately 12-18 hours as described above in Example 3.
  • mice Approximately 15 million of these cells were injected intravenously (i.v.) into C57BL.6 mice bearing hepatic tumors of MCA-38 colon adenocarcinoma. The mice also received IL-2 in liposomes (50,000 units/day) intraperitoneal (i.p.) for five days. The number of hepatic tumors were determined on day 14 as described above in Example 3.
  • mice C57BL.6 mice injected with tumor cells in a similar manner as the treated mice; however, these mice were treated with IL-2 liposomes (50,000 units/day) only.
  • the results are listed below in Table 2. These results demonstrate that with as little as 15 million cells stimulated for 24 hours with anti-CD3, there is a significant reduction in the number of hepatic tumors.
  • Splenocytes obtained as described above in Example 3, were stimulated with OKT3 and IL-2 as described above in Example 3.
  • mice Nine week old C57BL.6 female mice (Harlan) bearing hepatic tumors of MCA-38 colon adenocarcinoma were divided into seven groups.
  • the mice in the three groups intravenously received 5 million, 10 million, or 20 million cells stimulated with anti-CD3 and cultured in the presence of 100 units/ml IL-2 on days 3 and 5. All mice, including a control group, which did not receive adoptively transferred cells, received 50,000 units IL-2 liposomes intraperitoneal qd., i.e. everyday, on days 3- 7.
  • the nu: ;r of hepatic tumors were determined on day 14 as described above in Example 3. The results are presented below in Table 3. These results demonstrate that there is a distinct correlation between the quantity of cells administered and the number of tumors the mice develop. The mice receiving more cells developed fewer tumors.
  • C57BL.6 murine splenocytes were harvested and activated with anti-murine CD3 MoAb 145-2C11 and interleukin-2 (IL-2,) as described by P.M. Anderson et al., J. Immunol . , 142, 1383-1394 (1989) , which is incorporated herein by reference.
  • the IL-2 used had a specific activity of 1.5 x 10 7 units/mg, and is available from Hoffmann-LaRoche.
  • Fresh splenocytes were incubated in culture flasks at a concentration of 1.0 x 10 6 cells/ml of tissue culture media (TCM) .
  • TCM tissue culture media
  • T-activated killer cells i.e., T-AK cells
  • 2 ⁇ g of 145-2C11 MoAb was added per one ml of TCM.
  • IL-2 was added to the TCM at a concentration of 100 units/ml.
  • Anti-CD3 MoAb was added only at the start of the culturing process . All cultures were supplemented with fresh TCM containing IL-2 to produce a cell density of 0.2-0.5 x 10° cells/ml every two to three days. Cultures were sampled on days 3, 5, 7, and 9; cell viability and increases in cell number were determined by the commonly used trypan blue exclusion procedure. Cells utilized in the adoptively transferred T cell infiltration experiment were from day 5, 7, and 9 of culture.
  • Example 7 Adoptively Transferred T Cells Infiltrate MCA-38 Hepatic Tumors
  • T-AK cells adoptively transferred T cells with cytolytic activity
  • T-AK cells were generated from splenocytes of C57BL.6 mice that express Thy 1.2 on their lymphocytes, as described in Example 8.
  • mice of the congenic strain B6:PL-Thy- l a /Cy (available from Frederick Cancer Research Center and Development) that express Thy 1.1 on their T cells were inoculated with MCA-38 and then treated with T-AK cells from Thyl.2 mice (C57BL.6) , IL-2 liposomes prepared as described above in Example 2, or a combination of IL-2 liposomes and T-AK cells.
  • Liver tissue was collected every 48 hours beginning the day after therapy was started (day 4 post tumor inoculation) , and evaluated for the number of Thy 1.2 + cells, Lyt.2.2 + cells, L3T4 + cells, and Mac 1 + cells infiltrating the hepatic tumors.
  • Ethidium bromide was added to facilitate identification of the early micrometastases within the liver, according to the method of J.L. Platt et al . described in J ⁇ Exp. Med.. 157, 17 (1982) .
  • Tetraethylrhodamine isothiocyanate conjugated rabbit antiserum to human basement membrane (TRIC) was used to allow differentiation of intravascular cells from infiltrating cells, according to the method of J.L. Platt et al. described in J. Exp. Med. , 158, 1227 (1983) .
  • Monoclonal antibodies were purified from ascites utilizing saturated ammonium sulfate, washed twice with 50% ammonium sulfate, dialyzed against PBS, and adjusted to a final concentration of 500 ⁇ g/ml in PBS.
  • the antibody recognizing the cell marker Lyt.2 is 2.43 (ATCC Cat. No. TIB 210) , and that recognizing the cell marker L3T4 is GK1.5 (ATCC Cat. No. TIB 207) .
  • Rat anti-Thy 1.2 FITC was obtained from Becton Dickinson (Mountain View, CA) and anti-MAC-1 from Boehringer Mannheim (Indianapolis, IN) .
  • Mac 1 + cells (including both PMNs and monocytes) also infiltrated the tumors of these mice. Rare L3T4 + cells were also noticed. T-AK cells (Thyl.2 + ) infused without IL-2 liposomes into Thy 1.1 + mice were observed in the tumors two days after initial injection. Rare Mac 1 + cells were also observed, however, no L3T4 + cells were observed.
  • T-activated killer cells T-AK
  • C57BL.6 mice were injected intravenously with 15 x 10° anti-CD3 activated cells obtained from B6:PL Thy-l a /Cy mice.
  • C57BL.6 mice express the LY5.2 allele
  • B6:PL Thy-l a mice express the Ly5.1 allele.
  • Splenocytes from LY5.1 (B6:PL Thy-l a ) mice were activated as previously described. Briefly, purified splenocytes were cultured in tissue culture media (TMC) containing 3% volume:volume of the supernatant from the 145-2C11 hybridoma (Hamster anti-mouse anti-CD3 MoAb) for a period of 18 hours. The activated cells were then injected intravenously into LY5.2 (C57BL.6) mice. These mice also received intraperitoneal (i.p.) IL-2-liposomes
  • TAC + Percent T cells expressing the IL-2 receptor
  • TAC + Percent T cells expressing the IL-2 receptor
  • LY5.1 cells in the spleen 4. The number of white blood cells in the peripheral blood and spleen.
  • CD4 and CD8 On day 0 (after stimulation of LY5.1 cells with anti-CD3, but prior to infusion) the T cells (CD4 and CD8) in the stimulated culture expressed an increased level of IL-2 receptor (78 and 92%) (Table 4) . This is an increase from baseline which usually is ⁇ 10% in a normal unstimulated animal. As observed, the percentage of CD8 + cells increased in vivo over time while the CD4 + cells decreased.
  • the number of LY5.1 cells in the spleen of the LY5.2 recipient animal increased over time until day 10 where 34% of the spleen cells were of .LY5.1 origin, confirming their expansion in vivo .
  • the number of white blood cells in the peripheral blood increased dramatically to 88,000/mm 3 .
  • the data confirm the in vivo proliferation of cells activated in vi tro with anti-CD3.
  • mice receiving IL-2- liposomes with T-AK cells had tumor progressively grow while nine out of ten had complete tumor regression. All the mice in the groups receiving no therapy and 50% of the mice receiving IL-2 liposomes + cytoxan developed tumors which progressively increased in size.
  • IL-2-LIPOSOMES 50 K/D X 5D
  • Example 10 Increase in IL-2 Receptor Expression After Stimulation With Anti-CD3 IL-2 receptor (p55,CD25) is upregulated on human T lymphocytes after a 12 hour incubation with anti-CD3 (OKT3-Ortho) ( Figure 1) . Lymphocytes were cultured as described above and the expression of the IL-2 receptor was measured by flow cytometry using a fluorescein- labeled anti-CD25 MoAb against the p55 or ⁇ chain of the IL-2 receptor (Becton-Dickinson, CA) .
  • Human T lymphocytes also increase their expression of the chain (p75) of the IL-2 receptor after overnight incubation with anti-CD3 ( Figure 2) . It is noted that the receptor itself has a high affinity for IL-2, as does the cell because it has so many receptors on it. The increased expression of both the oc and ⁇ chains makes the IL-2 receptor expressed by these T cells a receptor with very high affinity of IL-2.
  • TMC tissue culture media
  • RPMI 1640 or AIM-V supplemented with 25mM HEPES, 2mM L-glutamine, 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 5% heat inactivated human AB serum (GIBCO) and 30, 100, 300, or
  • Example 12 0KT3 Induced Cell Proliferation Lymphocytes stimulated with anti-CD3 for as short as 4 hours will proliferate adequately in the presence of IL-2. Lymphocytes were cultured with no 0KT3, or 3 or 10 ng/ml of 0KT3 for 4 hours. Lymphocytes were washed twice with HBSS and then were cultured in tissue culture media containing 100 u/ml of IL-2. As shown in Figure 4, either concentration of OKT3 tested induced cell proliferation.
  • Human T lymphocytes proliferate in vivo in cancer patients after receiving T-AK cells and IL-2.
  • Patient lymphocytes were obtained by leucophaeresis one day prior to the initiation of treatment (day-1) .
  • the cells were purified over Ficoll-Hypaque and cultured overnight at a concentration of 3 X 10° cells/ml in AIM-V media with 10 ng/ml of OKT3. After overnight culture, the cells were washed 3 times in HBSS, resuspended in reinfusion medium
  • saline solution (Abott Laboratories) containing 25% human serum albumin (American Red Cross) and 30 U/ml of IL-2) and given back to the patients by intravenous infusion over 30 minutes.
  • the patients then received IL- 2 intravenously (3 million U/m2 continuous infusion and 1.5 million U/m2 bolus for a total of 7 days (proliferation has also been observed with lower doses of IL-2 being administered) .
  • Samples of peripheral blood were obtained from the patient on the days present in Figure 5 and a WBC count and differential counts were done. There was a marked increase in the WBC after 6-8 days in therapy. This increase was on the average 4-6 fold over the baseline number of WBC. The WBC ranged up to 86,000 mm 3 in one patient.
  • CD8 + cells infiltrate the site of tumor.
  • a significant lymphocytic infiltration was observed as compared with the pre-treatment biopsy. Further analysis using well-established, routine techniques of immunohistology revealed that these cells were mostly CD8 + cells.
  • Depleted Lymphocytes T-cells are purified from peripheral blood, lymph nodes, or any other source that provides T-cells, using methods well known to the skilled artisan.
  • peripheral blood lymphocytes PBLs
  • PBLs peripheral blood lymphocytes
  • Isolated mononuclear cells are washed three times with phosphate buffered saline (PBS, pH 7.4) (GIBCO Laboratories, Grand Island, NY) and counted.
  • CD4 + and CD8 + enriched cultures are obtained by negative depletion using magnetic beads (obtained from Baxter Healthcare Corporation, Deerfield, IL; also available from Advanced Magnetics, Massachusetts; or Dynal Corp., Norway) .
  • PBLs are labelled by incubation with either of the monoclonal antibodies 0KT4 or 0KT8 (Ortho, Raritan, NJ) for 30 minutes on ice.
  • the cells are then washed twice with cold PBS and mixed with goat anti-mouse IgG-coated magnetic beads (obtained from Baxter Healthcare; also available from Dynal Corp., Norway) at a bead:cell ratio of 10:1.
  • the bead/cell mixture is incubated for 30 minutes at 4°C while rotating at 5-6 rpm. At the end of the incubation, the bead/cell suspension was diluted two ⁇ fold with cold PBS.
  • the beads are allowed to collect against the side of the test tube for five minutes .
  • the supernatant containing unbound cells is then collected and transferred to a new tube. This process is repeated three times to completely remove the beads and bead-bound cells.
  • the cells that remain in suspension (CD4 + -depleted or CD8 + -depleted PBLs) are washed and counted.
  • the depleted lymphocyte population (5 x 10 6 cells) are cultured in 25 cm 2 flasks (Corning, Corning, NY) in 10 ml tissue culture medium (TCM) .
  • TCM tissue culture medium
  • the TCM consists of Rosewell Park Memorial Institute (RPMI) 1640 medium (available from GIBCO, Grand Island, NY) supplemented with 25 mM Hepes [N- (2-hydroxyethyl)piperazine-N' - (2- ethanesulfonic acid)] , 2 mM L-glutamine, 100 units/ml penicillin , 100 ⁇ g/ml streptomycin
  • the cultures are supplemented with 10 ng/ml of the anti- CD3 MoAb OKT3 (Ortho Division, Johnson & Johnson, Raritan, NJ) .
  • the cultures are optionally supplemented with low doses of IL-2, for example ⁇ 300 units/ml of highly purified recombinant human IL-2 from E. coli (Hoffman-LaRoche, Nutley, NJ) .
  • IL-2 for example ⁇ 300 units/ml of highly purified recombinant human IL-2 from E. coli (Hoffman-LaRoche, Nutley, NJ) .
  • Cultures are incubated at 37°C in a humidified atmosphere of 5% C0 2 for less than about 24 hours, preferably 12 to 18 hours, and for as little as 4 hours or 30 minutes.
  • the lymphocytes are incubated in roller bottles, hollow fibers, gas permeable bags, or regular tissue culture flask.
  • the lymphocytes are washed with PBS and placed in a suspension of saline solution, human serum and IL-2 (75 U.ml) .
  • the stimulated and depleted T lymphocyte preparation is administered to the patient, preferably with IL-2, most preferably with IL-2 liposomes .
  • Example 16 Treatment of Cancer Patients with a Short-term Stimulated Positively-Selected T Lymphocyte Subset
  • T-cells are purified from a known source, such as from PBLs, as described above.
  • the cells are resuspended in tissue culture medium and plated onto polystyrene plates previously coated with anti-CD4 or anti-CD8.
  • the lymphocytes are incubated at 4°C for 45 to 60 min.
  • the unbound cells are washed off by rinsing the plates with PBS.
  • the bound cells are enriched for CD4 + or CD8 + lymphocytes .
  • the positively selected lymphocyte subset (5 x 10 6 cells) is cultured in RPMI 1640 medium supplemented with 25 mM Hepes [N- (2-hydroxyethyl)piperazine-N' - (2- ethanesulfonic acid)] , 2 mM L-glutamine, 100 units/ml penicillin , 100 ⁇ g/ml streptomycin (penicillin/streptomycin mix available from GIBCO, Grand Island, NY) , and 5% pooled heat-inactivated human serum.
  • the cultures are supplemented with 10 ng/ml of the anti- CD3 MoAb OKT3 (Ortho Division, Johnson & Johnson, Raritan, NJ) .
  • the cultures are optionally supplemented with low doses of IL-2, for example ⁇ 300 units/ml of highly purified recombinant human IL-2 from E. coli (Hoffman-LaRoche, Nutley, NJ) .
  • IL-2 for example ⁇ 300 units/ml of highly purified recombinant human IL-2 from E. coli (Hoffman-LaRoche, Nutley, NJ) .
  • Hoffman-LaRoche, Nutley, NJ See, A. Wang et al . , Science, 224 , 1431 (1984) ; and S.A. Rosenberg et al . , Science, 223, 1412 (1984) , supra.
  • Cultures are incubated at 37°C in a humidified atmosphere of 5% C0 2 for less than about 24 hours, preferably 12 to 18 hours, and for as little as 4 hours or 30 minutes.
  • the lymphocyte subset is incubated in roller bottles, hollow fibers, gas permeable bags, or regular tissue culture flask.
  • the lymphocyte subset is washed with PBS and placed in a suspension of saline solution, human serum and IL-2 (75 U.ml) .
  • the stimulated lymphocyte subset is administered to the patient, preferably with IL-2, most preferably with IL-2 liposomes.

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Abstract

Un mammifère porteur de tumeur est traité par stimulation des lymphocytes, y compris les sous-ensembles de lymphocytes T. Cette stimulation s'effectue in vitro en présence d'un anticorps caractéristique d'un récepteur de surface d'anticorps pendant moins d'environ 24 heures, et facultativement en présence d'un IL-2. Cette opération donne des lymphocytes stimulés. Le procédé consiste ensuite à perfuser les lymphocytes ainsi stimulés chez un mammifère porteur de tumeur et administrer audit mammifère une ou plusieurs lymphokines. Les lymphocytes ainsi stimulés font preuve d'une activité immunothérapeutique accrue, et notamment d'une cytotoxicité accrue ou d'une capacité de production in vivo de lymphokines accrue, ce qui provoque une régression de l'invasion tumorale d'environ 20 % au moins.
PCT/US1995/006703 1994-05-31 1995-05-31 Stimulation anti-cd3 a court terme des lymphocytes destinee a en accroitre l'activite in vivo Ceased WO1995032729A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
US7541184B2 (en) 2000-02-24 2009-06-02 Invitrogen Corporation Activation and expansion of cells
US7572631B2 (en) 2000-02-24 2009-08-11 Invitrogen Corporation Activation and expansion of T cells

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EP0409655A2 (fr) * 1989-07-21 1991-01-23 Ortho Pharmaceutical Corporation Procédé de stimulation de la prolifération des lymphocytes sanguins
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

Patent Citations (2)

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EP0409655A2 (fr) * 1989-07-21 1991-01-23 Ortho Pharmaceutical Corporation Procédé de stimulation de la prolifération des lymphocytes sanguins
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

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Title
JOURNAL OF IMMUNOLOGY, Volume 146, issued 15 May 1991, R.L. GELLER, "Generation of Lymphokine-Activated Killer Activity in T Cells", pages 3280-3288. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7541184B2 (en) 2000-02-24 2009-06-02 Invitrogen Corporation Activation and expansion of cells
US7572631B2 (en) 2000-02-24 2009-08-11 Invitrogen Corporation Activation and expansion of T cells

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