Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1997-03-03
2001-11-13
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S325000, C435S343200, C435S372000, C435S373000, C435S375000, C435S383000, C435S384000, C435S386000, C530S351000, C530S388850, C530S389600
Reexamination Certificate
active
06316257
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to improved methods for culturing T lymphocytes, including human antigen-specific cytolytic and helper T lymphocytes. The methods of the present invention result in the very rapid and efficient expansion of T cells which are useful, for example, in cellular immunotherapy.
BACKGROUND
T lymphocytes are formed in the bone marrow, migrate to and mature in the thymus and then enter the peripheral blood and lymphatic circulation. T lymphocytes can be phenotypically subdivided into several distinct types of cells including: helper T cells, suppressor T cells, and cytotoxic T cells. T lymphocytes, unlike B lymphocytes, do not produce antibody molecules, but express a heterodimeric cell surface receptor that can recognize peptide fragments of antigenic proteins that are attached to proteins of the major histocompatibility complex (MHC) expressed on the surfaces of target cells; see, e.g., Abbas, A. K., Lichtman, A. H., and Pober, J. S.,
Cellular and Molecular Immunology,
1991, esp. pages 15-16.
T lymphocytes that can be expanded according to the present invention are of particular interest in the context of cellular “immunotherapy”. As used herein, cellular immunotherapy refers to any of a variety of techniques involving the introduction of cells of the immune system, especially T lymphocytes, into a patient to achieve a therapeutic benefit. Such techniques can include, by way of illustration, “immuno-restorative” techniques (involving, e.g., the administration of T cells to a patient having a compromised immune system); “immuno-enhancing” techniques (involving, e.g., the administration of T cells to a patient in order to enhance the ability of that patient's immune system to avoid or combat a cancer or a pathogen such as a virus or bacterial pathogen); and “immuno-modulating” techniques (involving, e.g., the administration of T cells to a patient in order to modulate the activity of other cells of the patient's immune system, such as in a patient affected by an autoimmune condition).
Cytotoxic T lymphocytes (CTLs) are typically of the CD3+, CD4−, CD8+ phenotype and lyse cells that display fragments of foreign antigens associated with class I MHC molecules on their cell surfaces. CTLs that are CD3+, CD4+, CD8− have also been identified. Target cells for CTL recognition include normal cells expressing antigens after infection by viruses or other pathogens; and tumor cells that have undergone transformation and are expressing mutated proteins or are over-expressing normal proteins.
Most “helper” T cells are CD3+, CD4+, CD8−. Helper T cells recognize fragments of antigens presented in association with class II MHC molecules, and primarily function to produce cytokines that amplify antigen-specific T and B cell responses and activate accessory immune cells such as monocytes or macrophages. See, e.g., Abbas, A. K., et al., supra. Helper T cells can also participate in and/or augment cytolytic activites.
In addition to conventional helper T cells and cytolytic or “killer” T cells, it will also be useful to be able to rapidly expand other T cell populations. For example, T cells expressing the gamma/delta T cell receptor represent a relatively small portion of the human T cell population, but are suspected to play a role in reactivity to viral and bacterial pathogens as well as to tumor cells (see, e.g., W. Haas et al. 1993. Annu. Rev. Immunol. 11:637). Another T cell population of potential clinical importance is the population of CD1-restricted T cells. CD1 is an MHC-like molecule that shows limited polymorphism and, unlike classical MHC molecules which “present” antigenic peptides, CD molecules bind lipoglycans and appear to be important in the recognition of microbial antigens (see, e.g., P. A. Sieling et al. 1995. Science 269:227; and E. M. Beckman et al. 1994. Nature 372:691).
T lymphocytes are thus key components of the host immune response to viruses, bacterial pathogens and to tumors. The significance of properly functioning T cells is made quite clear by individuals with congenital, acquired or iatrogenic T cell immunodeficiency conditions (e.g., SCID, BMT, AIDS, etc.) which can result in the development of a wide variety of life-threatening infections or malignancies. Persons with diseases that are related to a deficiency of immunologically-competent T lymphocytes, or persons with conditions that can be improved by administering additional T lymphocytes, can thus be benefited by cellular immunotherapies, as referred to above. T cells for use in such therapies can be derived from the immunodeficient host, or from another source (preferably a compatible donor). The latter source is of course especially important in situations in which an immunodeficient host has an insufficient number of T cells, or has T cells that are insufficiently effective. In either case, it is difficult to obtain sufficient numbers of T cells for effective administration; and thus target T cells must first be grown to large numbers in vitro before administration to a host.
After undergoing such cellular immunotherapy, hosts that previously exhibited, e.g., inadequate or absent responses to antigens expressed by pathogens or tumors, can express sufficient immune responses to become resistant or immune to the pathogen or tumor.
Adoptive transfer of antigen-specific T cells to establish immunity has been demonstrated to be an effective therapy for viral infections and tumors in animal models (reviewed in Greenberg, P. D.,
Advances in Immunology
(1992)). For adoptive immunotherapy to be effective, antigen-specific T cells usually need to be isolated and expanded in numbers by in vitro culture, and following adoptive transfer such cultured T cells must persist and function in vivo. For treatment of some human diseases, the use in immunotherapy of cloned antigen-specific T cells which represent the progeny of single cells, offers significant advantages because the specificity and function of these cells can be rigorously defined and precise dose:response effects readily evaluated. Riddell et al. were the first to adoptively transfer human antigen-specific T cell clones to restore deficient immunity in humans. Riddell, S. R. et al., “Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones”,
Science
257:238-240 (1992). In that study, Riddell et al. used adoptive immunotherapy to restore deficient immunity to cytomegalovirus in allogeneic bone marrow transplant recipients. Cytomegalovirus-specific CD8+ cytotoxic T cell clones were isolated from three CMV seropositive bone marrow donors, propagated in vitro for 5 to 12 weeks to achieve numerical expansion of effector T cells, and then administered intravenously to the respective bone marrow transplant (BMT) recipients. The BMT recipients were deficient in CMV-specific immunity due to ablation of host T cell responses by the pre-transplant chemoradiotherapy and the delay in recovery of donor immunity commonly observed after allogeneic bone marrow transplant (Reusser et al.
Blood,
78:1373-1380, 1991). Riddell et al. found that no toxicity was encountered and that the transferred T cell clones provided these immunodeficient hosts with rapid and persistent reconstitution of CD8+ cytomegalovirus-specific CTL responses.
Riddell et al. (
J. Immunology,
146:2795-2804, 1991) used the following procedure for isolating and culturing the CD8+ CMV-specific T cell clones: peripheral blood mononuclear cells (PBMCs) derived from the bone marrow donor were first cultured with autologous cytomegalovirus-infected fibroblasts to activate CMV-specific CTL precursors. Cultured T cells were then restimulated with CMV-infected fibroblasts and the cultures supplemented with &ggr;-irradiated PBMCs. 2-5 U/ml of interleukin-2 (IL-2) in suitable culture media was added on days 2 and 4 after restimulation to promote expansion of CD8+ CTL (Riddell et al.,
J. Immunol.,
146:2795-2804, 1991). To isolate T cell clones, the polycl
Clary Kim W.
Flyer David C.
Guzo David
Leffers, Jr. Gerald G.
Morrison & Foerster / LLP
Targeted Genetics Corporation
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