Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1998-09-29
2002-06-04
Crouch, Deborah (Department: 1632)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S320100, C435S325000, C530S387300, C530S387900, C530S388800, C530S388850, C530S391100, C530S350000
Reexamination Certificate
active
06399054
ABSTRACT:
The invention is concerned with a method for the production of activated marked tumor-specific T cells by co-cultivating tumor cells from a patient with T cells from that patient, a therapeutic composition containing such activated T cells as well as the use thereof in tumor therapy.
Tumor-specific T lymphocytes recognize peptides derived from proteins synthesized by tumor cells and presented on their cell surface by MHC molecules (Lurquin et al., Cell 58 (1989) 293 and Hellström, K. E., et al., The Biologic Therapy of Cancer, J. B. Lippincot Co., Philadelphia (1991) p. 35). However, T cells require two activating signals to express full effector functions (Mueller, D. L., et al., Annu. Rev. Immunol. 7 (1989) 445). Signal
1
is generated when the T cell receptor (TCR) interacts with the MHC peptide complex. Signal
2
is provided by costimulatory molecules expressed by professional antigen-presenting cells (APC). Many tumors, particularly those of non-hematopoietic origin, do not express costimulatory molecules and thus fail to activate tumor-specific T lymphocytes (Chen, L., et al., Immunol. Today 14 (1993) 483). This finding has provided a rationale for the introduction of genes encoding costimulatory molecules into tumor cells to increase their immunogenicity and vaccination potential.
Among the different costimulatory molecules, B7 proteins (e.g. B7-1, B7-2 and B7-3) are of particular interest since they are expressed on professional APC (Vandenberghe, P., et al., Int. Immunol. 3 (1993) 229; Guinan, E. C., et al., Blood 84 (1994) 3261-3282; WO 95/03408). These costimulatory molecules interact with CD28 and CTLA4 counter-receptors expressed on most T cells leading to a marked increase of IL-2 production, proliferation and acquisition of effector function in both CD4
+
and CD8
+
T cells (Azuma, M., et al., J. Immunol. 115 (1993) 2091). Blocking the ligation of B7 with a soluble CTLA4-Ig chimeric molecule provokes unresponsiveness in vitro, which has dramatic suppressive effects on the humoral response and graft rejection in vivo. In addition, it has been shown that the transfection of the B7-1 gene into different mouse tumor lines can lead, in some cases, to both their primary rejection and the establishment of a protective immunity (Chen, L., et al., J. Exp. Med. 179 (1994) 523 and Ramarathinam, L., et al., J. Exp. Med. 179 (1994) 1205). However, these studies have revealed a limited efficiency of B7-1 activity on T cell-dependent tumor immunity.
The efficiency of B7 costimulation of anti-tumor T cells is enhanced by cooperation between B7 and ICAM-1, whereby an efficient tumor-specific immune response is stimulated. This effect is dependent on the recruitment of a potent inflammatory reaction (Cavallo, F., et al., Eur. J. Immunol. 25 (1995) 1154-1162).
Molecules of the B7 family are CD28 counter-receptors expressed on APCs. B7-1 was characterized and sequenced in Freeman, G. J., et al., J. Immunol. 143 (1989) 2714-2722. B7-2 and B7-3 were characterized and sequenced in Freeman, G. J., Science 262 (1993) 909-911 and WO 95/03408. The B7 molecules are members of the Ig supergene family with two Ig-like domains (IgV and IgC) and a transmembrane domain. It is suggested that the B7 molecules exist as a monomer or a homodimer on the cell surface, but little, if any, evidence suggests that it can form a heterodimer with CD28 (Lindsten, T., et al., J. Immunol. 151 (1993) 3489). The B7 molecules have a higher affinity for CTLA-4 than for CD28. The genes of the B7-1 and B7-2 molecules have been localized to chromosomal region 3q13.3-3q21. Though these molecules were not highly homologous at the DNA level, they share the identical Ig supergene family structure and the ability to bind to CD28 and CTLA-4, as mentioned above.
However, it was found that B7-1 and B7-2 differ in their appearance after B cell activation. B7-2 appears on the cell surface within 24 hours of B cell activation and B7-1 appears later (Boussiotis, V. A., et al., Proc. Natl. Acad. Sci. USA 19 (1993) 11059). It was further found that in unstimulated human monocytes B7-2 is constitutively expressed whereas B7-1 expression is induced after activation (Azuma, M., et al., Nature 366 (1993) 76). B7-3 is also described in Boussiotis et al. B7-3 has not yet been molecularly cloned.
In WO 95/03408 it is suggested to modify a tumor cell to express B7-2 and/or B7-3 by a transfection of the tumor cell with the nucleic acid encoding B7 in a form suitable for expression of B7 on the tumor cell surface. Alternatively, the tumor cell is modified by contact with an agent which induces or increases the expression of B7 on the tumor cell surface. It is further suggested to couple B7-2 and/or B7-3 to the surface of the tumor cell to produce a modified tumor cell. The term “coupling” as used in WO 95/03408 refers to a chemical, enzymatic or other means (e.g. antibody) by which B7-2 and/or B7-3 is linked to a tumor cell such that the costimulatory molecule (B7) is present on the surface of the tumor cell and is capable of triggering a costimulatory signal in T cells. It is further suggested to cross-link B7 chemically to the tumor surface, using commercially available cross-linking reagents. Another approach would be to couple B7-2 and/or B7-3 to a tumor cell by a B7-specific antibody which binds to both the costimulatory molecule B7 and a cell surface molecule on the tumor cell.
The production of activated tumor-specific T cells may be accomplished by co-cultivating tumor cells from a patient, which tumor cells carry, on their surface, such a costimulatory molecule, with T cells from that patient. Modifying such tumor cells with B7 according to the known method involves a number of drawbacks, however, and is rather unsuitable for routine therapy. Transfecting the tumor cells with the nucleic acid encoding a costimulatory molecule usually is not very effective. In addition to this, it is necessary that the transfected and non-transfected cells should be separated, in a laborious procedure, prior to co-cultivation with the activated T cells. McHugh, R. S., et al., Proc. Natl. Acad. Sci. USA 92 (1995) 8059-8063 suggest to introduce B7-1 onto the surface of tumor cells by using a purified GPI (glycosyl-phosphatidyl-innositol) anchored B7-1 molecule (GPI-B-7) which is able to bind its cognate ligand CD28 and incorporate itself into tumor cell membranes after a short incubation. However, the stability of the GPI-B-7 on the surfaces of irradiated tumor cells is limited and the cells do retain only minimal presentation of B7 capable of effective binding to CD28.
Coupling of B7 to a tumor cell by using a B7-specific antibody which binds both the costimulatory molecule and the cell surface molecule of the tumor has also severe disadvantages. B7 antibodies which are described in the state of the art bind to B7 unfortunately in such a way that the binding of B7 to CD28 decreases dramatically or is completely inhibited. The reason for this is that all known anti-B7-1 and anti-B7-2 monoclonal antibodies interact with CD28 and thus inhibit the T cell response (Azuma, M., et al., J. Exp. Med. 175 (1992) 353-360; Azuma, M., et al., J. Immunol. 149 (1992) 1115; Azuma, M., et al., J. Exp. Med. 177 (1993) 845; Caux, C., et al., J. Exp. Med. 180 (1995) 1841-1847).
It is therefore the object of the present invention to provide a method for the production of activated tumor-specific T cells which can be carried out in a simple manner and exhibits a high efficacy.
The subject-matter of the invention is a method for the production of activated tumor-specific T cells by co-cultivating, ex vivo, tumor cells from a patient with T cells from that patient, comprising the steps of
i) incubating the tumor cells with a first fusion protein obtained from a B7 protein and one partner of a biological binding pair and a second fusion protein obtained from an antibody against a cell surface antigen and the other partner of the biological binding pair;
ii) inhibiting the proliferation of the tumor cells prior to or after that incubation;
iii) co-cultivating th
Casorati Giulia
Dellabona Paolo
Arent Fox Kintner & Plotkin & Kahn, PLLC
Beckerleg Anne Marie S.
Crouch Deborah
Science Park RAF S.p.A.
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