Methods for the production of TcR&ggr;&dgr;+ T cells

Details Methods for the production of TcR&ggr;&dgr;+ T cells Methods for the production of TcR&ggr;&dgr;+ T cells

2001-07-30

2003-03-25

Fredman, Jeffrey (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Culture medium, per se

C435S325000, C435S041000, C435S373000, C435S383000, C435S384000

Reexamination Certificate

active

06537812

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to novel culture methods for the ex vivo expansion of TcR&ggr;&dgr;
+
T cells.
BACKGROUND OF THE INVENTION
TcR&ggr;&dgr;
+
cells are a small subset of circulating T lymphocytes that are distinct from conventional TcR&agr;&bgr;
+
T cells which recognize, with fine specificity, foreign peptide antigens in the context of classical class I or class II major histocompatibility complex (MHC) restriction elements. By contrast, TcR&ggr;&dgr;
+
T cells are able to recognize both peptide and non-peptide antigens which may be derived from either foreign microorganisms or endogenous cellular products induced by stress such as viral infection or transformation. Moreover, unlike antigen recognition by TcR&agr;&bgr;
+
T cells, antigen recognition by TcR&ggr;&dgr;
+
T cells is not MHC-restricted.
The T cell receptors of TcR&agr;&bgr;
+
and TcR&ggr;&dgr;
+
T cells are distinguished by the different genetic elements that encode them. The majority of TcR&ggr;&dgr;
+
T cells are classified into two main subsets, V&dgr;1
+
and V&dgr;2
+
, based on the genes that encode their &dgr; chain. The major subset of TcR&ggr;&dgr;
+
T cells in human peripheral blood expresses V&dgr;2 in combination with V&ggr;9, while most of the remainder express V&dgr;1 in combination with V&ggr;2, V&ggr;3, V&ggr;4, V&ggr;5 or V&ggr;8 (Salerno, A. and Dieli, F., 1998).
Since TcR&ggr;&dgr;
+
T cells lack the fine specificity characteristics of TcR&agr;&bgr;
+
T cells, it has been proposed that they represent a more primitive immune mechanism that provides a first-line surveillance function against infection and tumours (Boismenu, R. et al., 1997). Several studies have documented the response of TcR&ggr;&dgr;
+
T cells to various viruses, bacteria and parasites (Bukowski, J. F. et al., 1994; Wallace, M. et al., 1995; Lang, F. et al., 1995; Elloso, M. M. et al., 1996) as well as their ability to mediate lysis of tumour cells of various origins (Zocchi, M. R. et al., 1990; Kitayama, J. et al., 1993; Choudhary, A. et al., 1995). Hematopoietic tumours may be particularly susceptible to the lytic effects of TcR&ggr;&dgr;
+
T cells, since transgenic mice expressing the V&ggr;1.1 transgene display spontaneous resistance to injected T cell leukemias, and TcR&ggr;&dgr;
+
T cell hybridomas derived from these mice preferentially respond to hematopoietic malignant cells over non-hematopoietic tumour cells (Penninger, J. et al., 1995). Moreover, human TcR&ggr;&dgr;
+
T cells clones derived from patient peripheral blood and bone marrow have been shown to lyse autologous leukemic cells in acute lymphoblastic leukemia and acute myeloid leukemia, respectively (Bensussan, A. et al., 1989; Jahn, B. et al., 1995). Furthermore, improved disease-free survival in leukemia patients after allogeneic bone marrow transplantation has been shown to be associated with an increase in the number and percentage of TcR&ggr;&dgr;
+
T cells in peripheral blood (Lamb, L. S. et al., 1996). Collectively, these results suggest that TcR&ggr;&dgr;
+
T cells may have therapeutic potential in the treatment of cancer and infectious diseases.
Many of the published methods describing the ex vivo expansion of TcR&ggr;&dgr;
+
T cells require the presence of antigen. Virus-infected or transformed cells or cell lines, bacteria and parasites have been shown to stimulate TcR&ggr;&dgr;
+
T cell expansion ex vivo, as have established tumour cell lines. For example, herpes simplex virus (HSV)-infected cells were used to stimulate the expansion of V&dgr;2
+
cells (Bukowski, J. F. et al., 1994), while Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines were used to stimulate the expansion of V&dgr;1
+
cells (Orsini, D. L. M. et al., 1993). Extracts of
Mycobacterium tuberculosis
and blood-stage
Plasmodium falciparum
malarial antigens have been shown to stimulate proliferation of TcR&ggr;&dgr;
+
T cells (Constant, P. et al., 1994; Elloso, M. M. et al., 1996). Daudi, an immortalized human Burkitt's lymphoma cell line, can also stimulate the proliferation of TcR&ggr;&dgr;
+
T cells (Kaur, I. et al., 1993). In addition, well-characterized, non-peptidyl antigens of the prenyl phosphate family, for example, isopentenyl pyrophosphate, have been shown to stimulate the ex vivo expansion of TcR&ggr;&dgr;
+
T cells (Garcia, V. E. et al., 1998). In some of these systems, the antigen-stimulated cultures of TcR&ggr;&dgr;
+
T cells were supplemented with IL-2, IL-4 or other cytokines.
TcR&ggr;&dgr;
+
T cells have also been expanded ex vivo from populations of tumour infiltrating lymphocytes (TIL) by culture with IL-2 (Zocchi, M. R. et al., 1990) or IL-2 in combination with immobilized anti-CD3 antibody (Kitayama, J. et al., 1993) or anti-TcR&ggr;&dgr; antibody (Yu, S. et al., 1999). In these systems, selective stimulation of the TcR&ggr;&dgr;
30
T cells by the tumour antigens is presumed to have occurred in vivo prior to isolation of T cells from the cancerous tissue.
In another system, TcR&ggr;&dgr;
+
T cells were expanded from the peripheral blood of glioblastoma patients using a solid-phase, immobilized anti-CD3 antibody in combination with IL-2 followed by culture in IL-2 alone (Yamaguchi, T., et al, 1997). These authors reported that the subsequently purified TcR&ggr;&dgr;
+
T cells did not proliferate for more than one week in the presence of IL-2 alone and therefore, they concluded, that this method would be applicable only to short term studies. They further showed that the method resulted in the expansion and enrichment of both TcR&ggr;&dgr;
+
and TcR&agr;&bgr;
+
T cells, achieving TcR&ggr;&dgr;
+
T cell purities on the order of 28%. In a subsequent report, the same authors demonstrated that this method selectively expanded the V&dgr;2
+
subset (Suzuki, Y., et al, 1999).
Thus, there are limitations to cell proliferation and/or requirements for antigen stimulation in the existing methods for ex vivo culture and expansion of TcR&ggr;&dgr;
+
T cells. Furthermore, while many papers report the expansion of the V&dgr;2
+
cell subset, few papers report the expansion of the V&dgr;1
+
cell subset and none report the simultaneous expansion of both the V&dgr;2
+
and V&dgr;1
+
T cell subsets in a single culture.
In view of the foregoing, there is a need in the art for a method to selectively culture large amounts of TcR&ggr;&dgr;
+
cells in vitro.
SUMMARY OF THE INVENTION
The present invention provides novel methods for expanding TcR&ggr;&dgr;
+
T cells in culture in the absence of exogenous antigen. Accordingly, the present invention provides a method for expanding TcR&ggr;&dgr;
+
T cells in a starting sample comprising:
(1) culturing cells in the starting sample in a first culture medium comprising a T cell mitogen and at least two cytokines; and
(2) culturing the cells obtained in step (1) in a second culture medium comprising at least two cytokines to expand TcR&ggr;&dgr;
+
T cells.
In one embodiment, the present invention provides a method for expanding TcR&ggr;&dgr;
+
T cells in a starting sample comprising:
(1) culturing cells in the starting sample in a first culture medium comprising (a) a T cell mitogen, (b) interleukin-2 and (c) interleukin-4; and
(2) culturing the cells obtained in step (1) in a second culture medium comprising (i) interleukin-2 and (ii) interleukin-4 to expand TcR&ggr;&dgr;
+
T cells.
In another embodiment, the present invention provides a method for expanding TcR&ggr;&dgr;
+
T cells in a starting sample comprising:
(1) obtaining low density mononuclear cells (LDMNC) from the starting sample;
(2) culturing the cells obtained in step (1) in a first culture medium comprising (a) a T cell mitogen, (b) interleukin-2 and (c) interleukin-4; and
(3) culturing the cells obtained in step (2) in a second culture medium comprisi

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