Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1998-06-05
2001-11-06
Bansal, Geetha P. (Department: 1642)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S130100, C424S144100, C424S809000, C530S388220, C530S388700, C530S388750, C530S388800, C530S388850
Reexamination Certificate
active
06312691
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to compositions and methods useful for activating lymphotoxin-&bgr; receptor signaling, which in turn elicits potent anti-proliferative effects on tumor cells. More particularly, this invention relates to lymphotoxin heteromeric complexes formed between lymphotoxin-&agr; and multiple subunits of lymphotoxin-&bgr;, which induce cytotoxic effects on tumor cells in the presence of lymphotoxin-&bgr; receptor activating agents. Also within the scope of this invention are antibodies directed against the lymphotoxin-&bgr; receptor which act as lymphotoxin-&bgr; receptor activating agents alone or in combination with other lymphotoxin-&bgr; receptor activating agents either in the presence or absence of lymphotoxin-&agr;/&bgr; complexes. A screening method for selecting such antibodies is provided. This invention also relates to compositions and methods using cross-linked anti-lymphotoxin-&bgr; receptor antibodies in the presence of other lymphotoxin-&bgr; receptor activating agents to potentiate tumor cell cytotoxicity.
BACKGROUND OF THE INVENTION
The tumor necrosis factor (TNF) receptor family has several members whose signaling can induce tumor cell death by necrosis or apoptosis (programmed cell death). The ligands TNF and lymphotoxin-&agr; (LT-&agr;; formerly called TNF-&bgr;) bind to and activate TNF receptors (p60 and p80; herein called “TNF-R”). TNF-R signaling initiates general immune responses to infection or stress in normal cells, but is cytotoxic to cells with transformed phenotypes or to tumor cells. TNF-R signaling can selectively lyse tumor cells and virus-infected cells. The cytotoxic effects of TNF-R signaling on tumor cells are enhanced by interferon-&ggr;) and by a variety of conventional chemotheropeutic agents.
It would be useful to take advantage of the anti-proliferative or cytotoxic activities induced by TNF-R signaling in tumor cells for therapeutic purposes. However, TNF-R activation has pleiotropic effects on a variety of immunoregulatory responses including the initiation of proinflammatory cascades. Thus it has not been possible to direct the cytotoxic effects of TNF-R signaling to tumor cells without co-stimulating inflammatory responses which lead to general toxicity in humans.
Similarly, stimulation of another TNF-related receptor called the Fas receptor (FasR) can trigger cytotoxicity by programmed cell death in a variety of both tumor and non-tumor cell types. However, FasR activation has been shown to cause rapid liver necrosis, thus precluding its therapeutic application in humans.
Recently, another receptor in the TNF family called the LT-&bgr; receptor (LT-&bgr;-R) was identified (Crowe et al.,
Science,
264, pp. 707-10 (1994). The LT-&bgr;-R binds heteromeric lymphotoxin complexes (LT-&agr;/&bgr;) which comprise LT-&agr; subunits in association with another TNF-related polypeptide called lymphotoxin-&bgr; (LT-&bgr;). These LT-&agr;/&bgr; complexes are membrane-associated and most have a LT-&agr;1/&bgr;2 stoichiometry (Browning et al.,
Cell,
72, pp. 847-56 (1993); Browning et al.,
J. Immunol.,
154, pp. 33-46 (1995)).
By analogy to TNF-R and other TNF-like receptors, the activation of LT-&bgr;-R signaling is thought to occur when multiple receptors on the cell surface are brought into close proximity (Crowe et al.,
Science,
264, pp. 707-10 (1994)). This process is referred to as receptor clustering. The TNF and LT ligands are multivalent complexes which can simultaneously bind to and thus aggregate more than one receptor. Receptor clustering as a means for receptor activation in other systems has been well-documented, especially for receptor tyrosine kinases (Ullrich and Schlessinger,
Cell,
61, pp. 203-212 (1990); Kolanus et al.,
Cell,
74, pp. 171-83 (1993)). Accordingly, administering LT-&agr;1/&bgr;2 ligands and/or LT-&bgr;-R activating agents which can induce the clustering and downstream signaling of LT-&bgr;-R molecules on the surface of target tumor cells would be useful for directly stimulating the LT-&bgr;-R pathway in these cells.
Signaling by LT-&bgr;-R, like TNF-R, can activate pathways that lead to cytotoxicity and cell death in tumor cells. Importantly, LT-&agr;1/&bgr;2 ligands do not bind to TNF-R with any significant affinity. For this reason, directed LT-&bgr;-R activation in tumor cells would trigger cytotoxicity in those cells without stimulating the inflammatory pathways associated with TNF-R activation. Treatment with LT-&agr;1/&bgr;2 and/or other LT-&bgr;-R activating agents would thus be useful for treating or reducing the advancement, severity of effects of tumorigenic cells (neoplasia) while overcoming the potent side effect problems which have been encountered when TNF-R or FasR activation has been tried as an anti-tumor treatment.
SUMMARY OF THE INVENTION
The present invention solves the problems referred to above by providing pharmaceutical compositions and methods for treating tumor cells by stimulating LT-&bgr;-R signaling without co-stimulating TNF-R-associated inflammatory responses. In one embodiment, lymphotoxin complexes formed between LT-&agr; and multiple subunits of LT-&bgr; are provided (LT-&agr;/&bgr; heteromeric complexes) which induce cytotoxic effects on cells bearing the LT-&bgr;-R in the presence of a LT-&bgr;-R activating agent. The preferred compositions and methods of this embodiment comprise LT-&agr;1/&bgr;2 complexes in the presence of a LT-&bgr;-R activating agent. More preferably, the LT-&agr;1/&bgr;2 complexes are in a soluble rather than a membrane-bound form, and the LT-&bgr;-R activating agent is IFN-&ggr;.
In another embodiment of the invention, at least one antibody directed against LT-&bgr;-R (anti-LT-&bgr;-R Ab) is used as a second LT-&bgr;-R activating agent in conjunction with the LT-&agr;/&bgr; heteromeric complex. The preferred compositions and methods of this embodiment are characterized by LT-&agr;1/&bgr;2 in the presence of IFN-&ggr; as a first activating agent, and at least one anti-LT-&bgr;-R Ab as a second LT-&bgr;-R activating agent. More preferably, the LT-&agr;1/&bgr;2 complexes are soluble and the antibody is a monoclonal antibody (anti-LT-&bgr;-R mAb).
In another embodiment of the invention, at least one anti-LT-&bgr;-R Ab in the presence or absence of a second LT-&bgr;-R activating agent is used without an exogenous LT-&agr;/&bgr; heteromeric complex. The preferred compositions and methods of this embodiment comprise at least two anti-LT-&bgr;-R monoclonal antibodies (anti-LT-&bgr;-R mAbs) which recognize non-overlapping epitopes of LT-&bgr;-R in combination with IFN-&ggr;.
In a further embodiment, this invention provides pharmaceutical compositions and methods for potentiating tumor cell cytotoxicity characterized by cross-linked anti-LT-&bgr;-R Abs used conjunction with a second LT-&bgr;-R activating agent. In one preferred embodiment, individual anti-LT-&bgr;-R Abs are immobilized by cross-linking them onto a surface. In another preferred embodiment, the anti-LT-&bgr;-R Abs are cross-linked in solution. More preferably, the anti-LT-&bgr;-R Abs are monoclonal antibodies and the second LT-&bgr;-R activating agent is IFN-&ggr;.
This invention further provides a novel screening process for selecting LT-&bgr;-R activating agents, such as anti-&bgr;-R Abs, that function in combination with LT-&agr;/&bgr; heteromeric complexes to promote tumor cell death. The assay makes use of the increased sensitivity of human adenocarcinoma cells to LT-&agr;/&bgr; heteromeric complexes in the presence of LT-&bgr;-R activating agents in a cytotoxicity assay. The procedure used to test putative LT-&bgr;-R activating agents is exemplified for the case of anti-LT-&bgr;-R antibodies, and comprises the following steps:
1) Tumor cells (e.g. HT29 human adenocarcinoma cells) are cultured for several days in media containing IFN-&ggr; and purified LT-&agr;1/&bgr;2 in the presence or absence of the particular anti-LT-&bgr;-R Ab being assayed;
2) The cells are treated with a dye that stains living cells; and
3) The number of stained cel
Benjamin Christopher D.
Browning Jeffrey L.
Meier Werner
Bansal Geetha P.
Biogen, Inc.
Cox Niki D.
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