Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1998-11-12
2003-01-28
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S372300, C435S384000, C435S386000
Reexamination Certificate
active
06511848
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention concerns a process for producing and multiplying lymphocytes as well as a composition which is suitable as a culture medium for lymphocytes.
2. Description of the Related Art
The production and multiplication of lymphocytes is problematic. Some types of lymphocytes cannot be cultured at all in vitro or are very difficult to culture in vitro. Native B lymphocytes can for example only be cultured for a short period and T lymphocytes require difficult culture conditions for longer culture such as a combination of growth factor(s) and “feeder cells” (“nurse cells”) or the use of unphysiological and potentially dangerous substances such as tumour promoters (phorbol esters) in combination with ionophoric substances (e.g. ionomycin) or plant lectins (e.g. phytohaemagglutin, PHA). This puts severe constraints on possibilities for the long-term culture and multiplication of T lymphocytes and other lymphocytes, and the production of lymphocytes in significant amounts for diagnostic or therapeutic purposes is very limited or impossible.
However, there is manifold interest in the ability to produce, culture and multiply lymphocytes in vitro e.g. B lymphocytes as producers of specific antibodies or cytotoxic T lymphocytes (CTL) to treat infections or tumours, in addition regulator T lymphocytes (helper or suppressor T lymphocytes) for the diagnosis and treatment of autoimmune diseases or “natural killer (NK) lymphocytes for treating malignant growths.
Hybridoma cells which are formed from a fusion of B lymphocytes or T lymphocytes with malignant, lympoid cells (e.g. myeloma cells) can be cultured and multiplied without difficulty. Such hybridoma cells have the immunological function of the original lymphocytes as well as the essentially unlimited ability to proliferate of the malignant fusion partner. However, the application of the hybridoma technique is limited to a few animal species (mouse, rat) and essentially fails in other mammalian species and also in particular in humans.
A process for the production of proliferating CD4+ lymphocytes is described in WO90/10059. According to this peripheral mononuclear blood cells (PBNMC) are treated with alkyl esters to remove monocytes and granulocytes and subsequently cultured in a culture medium which contains a T cell stimulant and/or IL-2. Mitogens such as PHA are used as the T cell stimulant. However, the use of IL-2 alone only leads to a low proliferation of the cells. The addition of mitogenic substances during the culture of cells which are subsequently to be implanted into a patient is critical.
A process for the culture of T cells in the presence of interleukin-2 is also described in EP-A 0 203 403. A disadvantage of this process is also that the T cells can only be proliferated to a slight extent by this means.
A process for culturing and multiplying tumoricidal T lymphocytes is described in WO94/23014 by co-culturing lymphocytes with a cell line (stimulator cells) while avoiding an allogenic stimulation and without addition of interleukin-2. In this process resting T lymphocytes are activated to effector cells which recognize and kill tumour cells or inhibit their growth. A considerable amount of fermentation is required to provide such stimulator cells for the mass proliferation of for example tumoricidal killer T cells. In addition the killer T cells must be separated in a sterile manner from these stimulator cells or their cell debris before use (reinfusion into the patient).
V. Kutnik et al., Period. Biol. 92 (1990) 48 describe that, in the allogenic mixed lymphocyte reaction of mouse spleen lymphocytes, the addition of IL2 restores the cyclosporin A-induced inhibition of the proliferation of the responder cells and increases their alloreactivity.
Pierson, B. A. et al., Blood 87 (1996) 180-189 describe that isolated human NK cells (CD56+, CD3−) can be multiplied to a slight extent in culture by adding IL2. The addition of supernatants of irradiated mononuclear cells from blood increases the multiplication of the NK cells. Thrombospondin was identified as the active principle of this effect which does not act directly but rather indirectly by activation of latent TGF&bgr;. TGF&bgr; activated in this manner inhibits the proliferation in the early phase of the culture and increases the further proliferation. A repeated addition of TGF&bgr; when the medium is changed inhibits the growth of the cells and in this case suppresses the proliferation of the NK cells. In Immunological Invest. 25 (1996) 129-151 I. A. Ayoub investigates the effect of human TGF&bgr; on a bovine CD4+ lymphoblastoid T cell line (BLTC) which grows autonomously in IL2-containing medium. The cell line is arrested in medium without IL2. The addition of TGF&bgr; to the arrested BLTC drives them rapidly into apoptosis. The simultaneous addition of IL2 abolishes the arrest and prevents the induction of apoptosis. The addition of TGF&bgr; to suboptimal concentrations of IL2 co-stimulates the proliferation of the BLTC.
In Intern. Immunol. 6 (1994) 631-638 R. de Jong describes the effect of TGF&bgr;1 on the proliferation of isolated subpopulations of human CD4+ T lymphocytes. TGF&bgr;1 amplifies the proliferation of CD4 cells (CD45 RA+) by antibodies in the presence of IL2, but the proliferation of pre-activated T cells (CD45 RO+) is inhibited by the addition of TGF&bgr;1. However, it turned out that the induced proliferation of the CD45 RA+ cells is inhibited after five days by addition of TGF&bgr;1.
A. Cerwenka describes in J. Immunol. 156 (1996) 459-464 that the presence of TGF&bgr;1 during the primary stimulation of human T lymphocytes increases their ability to survive in secondary cultures and reduces their susceptibility to apoptosis-inducing anti-Fas antibodies. The addition of TGF&bgr;1 also reduces the apoptosis susceptibility of primary activated T lymphocytes to secondary activation. A survival of the T cells over a long period is ensured in the presence of IL2 and TGF&bgr;1.
T. H. Inne et al., J. Immunol. 148 (1992) 3847-3856 describe that the proliferation of CTLL-2 cells (murine T cell line) in IL2-containing medium is inhibited by addition of TGF&bgr;1. In addition to the inhibition of proliferation, TGF&bgr;1 induces a change in the cell morphology and induces the expression of the surface molecule CD8 in CTLL-2. A combination of IL2 and TGF&bgr; also induces an increased expression of CD8 in murine thymocytes which have been activated by phorbol dibutyrate and ionomycin. In this case the addition of TGF&bgr;1 also reduces the proliferation rate.
However, TGF&bgr; is not a substance that is readily and cheaply available in adequate amounts. TGF&bgr; is usually either isolated from natural sources or produced recombinantly. The main object of the invention was to provide an effective and cheap means for culturing and multiplying lymphocytes as well as a process for the production of pancytotoxic T cells. A further object of the present invention is to provide a process which enables lymphocytes to be produced, cultured and/or multiplied in a simple manner on a large scale.
SUMMARY OF THE INVENTION
The subject matter of the invention is a process for culturing and/or multiplying lymphocytes in a cell culture medium which contains a lymphocyte growth factor and additionally aurintricarboxylic acid, cyclosporin and/or ascomycin.
Surprisingly the process according to the invention enables lymphocytes with special properties to be produced in a simple manner, to be cultured over a long period and to be multiplied on a considerable scale. The process according to the invention is particularly suitable for culturing and multiplying T lymphocytes and NK lymphocytes.
Furthermore it has turned out that the process according to the invention particularly advantageously enables lymphocytes to be cultured over a long period (more than 14 days) and to be multiplied on a large scale (factor of 100, 1000 or more). In addition a combination of a lymphocyte growth fa
Albert Winfried
Barchet Heinrich
Jungfer Herbert
Afremova Vera
Arent Fox Kintner & Plotkin & Kahn, PLLC
Marx Irene
Winfried Albert
LandOfFree
Process for producing and multiplying lymphocytes does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for producing and multiplying lymphocytes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for producing and multiplying lymphocytes will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3072717