Redirected, genetically-engineered t regulatory cells and their use in suppression of autoimmune and inflammatory disease
a technology of t-regulatory cells and autoimmune diseases, applied in the field of immunology and medicine, can solve the problems of inability to successfully vaccinate tumor patients with a large variety of tumor-associated antigen vaccines, inability inability to effectively vaccinate patients with ibd, etc., to suppress t-regulatory cells, suppress t-regulatory cells, and suppress t-regulatory
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Examples
example i
Phenotypic Characterization of TNP-Specific Tregs
[0280]The inventors have produced transgenic (Tg) mice expressing a TNP-specific tripartite chimeric receptor (TpCR) that serve as a source of redirected Treg cells specific for the trinitrophenyl (TNP) hapten. This hapten has served as a “classical” antigen for years in studying both antibodies and T cell-mediated immunity. A chemically reactive form of this hapten, TNBS, is a contact sensitizing agent that induces and evokes delayed-type hypersensitivity (DTH) responses as well as inducing colitis in animals, as described herein.
[0281]Generation of TNP-specific Tregs was achieved by the creation of Tg mice that express TNP-specific TpCR that comprises an scFv from the TNP-specific mAb Sp6 mAb linked to a truncated CD28 molecule which was inserted between the scFv and the cytoplasmic part of the FcRγ chain (abbreviated as y herein (see FIG. 1). This construct includes the hinge region, transmembrane region, and cytoplasmic region of ...
example ii
Isolation of Trees in which TNP-Specific TpCR are Highly Expressed
[0285]Tregs were isolated using double magnetic bead separation (Miltenyi Biotech) or by fluorescent cell sorting in which fluorescently labeled CD4+CD25+ cells were sorted using the FACSARIA cell sorting system.
[0286]Treg expression of TNP-specific TpCR was assessed by containing cells for Foxp3 (considered the “gold standard” marker of Tregs) and PE-labeled mAb specific for TNP antibody (generated in the inventors' laboratory). Controls included groups stained with the appropriate isotype controls. As is shown in FIG. 2, Tregs from TNP-Tg mice, but not from wild-type mice, expressed high levels of TNP-specific TpCR.
example iii
TNP-Tg Mice Posses Increased Numbers of Foxp3+Tree Population
[0287]Peripheral lymphocytes from the spleen as well as gut-associated lymphocytes from the lamina propria of the colon were stained. As shown in FIG. 3, a CD4+CD25+ cell population (represented as the ratio of CD4+CD25+ cells among CD4+T-cells) was elevated modestly in TNP-Tg mice in comparison to control mice (wildtype, ErbB2-Tg and TNP-CD28 null-Tg mice). In contrast, higher numbers of Foxp3+ cells were observed in TNP-Tg animals compared to the control animals in comparison to all other mouse types (FIG. 4).
[0288]To resolve what may have appeared to be an inconsistency between the highly elevated Foxp3+Treg population in TNP-Tg mice and the modestly elevated CD4+CD25+ Treg population in these mice, effector CD4+CD25− cells were isolated by cell sorting to a level of 99% purity. Isolated cells were stained for Foxp3 (FIG. 5). As expected, no positive Foxp3 staining was noted in T effector cells from wildtype, ErbB2-Tg a...
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Abstract
Description
Claims
Application Information
- IPC
- A61K35/12; C12N5/10; C07H21/04; C07K16/00; A61P29/00; A61P37/00; A61K39/00; C12N5/0783
- CPC
- A61K39/0008; C12N5/0636; A61K39/0011; A61P29/00; A61P37/00; A61K39/464406; A61K39/4621; A61K39/4611
- Inventors
- ESHHAR, ZELIG; ELINAV, ERAN