In vitro test system for predicting patient tolerability of therapeutic agents
AssigneeChiron Corporation
InventorsYing Cao, Kimberly Denis-Mize, Susan Wilson
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Abstract
Methods for predicting patient tolerability to therapeutic agents, such as cytokines, lymphokines and immunotoxins, are disclosed. The methods utilize an in vitro model of vascular leak syndrome (VLS) to assess the effect of the agent in question on the permeability of large proteins across confluent monolayers of endothelial cells (EC).
Claims19
An in vitro method for predicting tolerability or intolerability by a patient to a selected therapeutic agent, said method comprising:
- (a) providing a confluent monolayer of endothelial cells attached to an adherence substrate;
- (b) contacting said monolayer with
- (i) said selected therapeutic agent, or a preparation of lymphokine-activated killer (LAK) cells wherein said LAK cells are produced by activating peripheral blood mononuclear cells using said therapeutic agent, or the supernatant from said LAK cells, and
- (ii) a detectably labeled macromolecule, wherein said detectably labeled macromolecule is substantially retained by said confluent monolayer when said monolayer is intact;
- (c) incubating said monolayer from step (b) for a period of time and under conditions that allow for said detectably labeled macromolecule to pass through said confluent monolayer and said adherence substrate if the integrity of said monolayer is disrupted;
- and
- (d) detecting macromolecule that passes through said confluent monolayer and said adherence substrate as an indication of tolerability or intolerability by a patient to said therapeutic agent.
An in vitro method for predicting tolerability or intolerability by a patient to an interleukin-2 (IL-2) mutein, said method comprising:
- (a) providing a confluent monolayer of endothelial cells attached to an adherence substrate;
- (b) contacting said monolayer with
- (i) a preparation of lymphokine-activated killer (LAK) cells, wherein said LAK cells are produced by activating peripheral blood mononuclear cells using said IL-2 mutein, and
- (ii) a detectably labeled macromolecule, wherein said detectably labeled macromolecule is substantially retained by said confluent monolayer when said monolayer is intact;
- (c) incubating said monolayer from step (b) for a period of time and under conditions that allow for said detectably labeled macromolecule to pass through said confluent monolayer and said adherence substrate if the integrity of said monolayer is disrupted;
- and
- (d) detecting macromolecule that passes through said confluent monolayer and said adherence substrate as an indication of tolerability or intolerability by a patient to said IL-2 mutein.
An in vitro method for predicting tolerability or intolerability by a patient to an interleukin-2 (IL-2) mutein, said method comprising:
- (a) providing a confluent monolayer of human umbilical vein endothelial cells (HUVEC) attached to an adherence substrate comprising a collagen matrix;
- (b) contacting said monolayer with
- (i) a preparation of lymphokine-activated killer (LAK) cells, wherein said LAK cells are produced by activating peripheral blood mononuclear cells using said IL-2 mutein, and
- (ii) a fluorescently labeled albumin;
- (c) incubating said monolayer from step (b) for a period of time and under conditions that allow for said fluorescently labeled albumin to pass through said confluent monolayer and said adherence substrate if the integrity of said monolayer is disrupted;
- and
- (d) detecting fluorescently labeled albumin that passes through said confluent monolayer as an indication of tolerability or intolerability by a patient to said IL-2 mutein.
Dependencies are inferred from the claim text.