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Methods and compositions for expanding T regulatory cells

A composition and cell technology, applied in receptors/cell surface antigens/cell surface determinants, pharmaceutical formulations, animal/human proteins, etc., can solve problems such as defects

Inactive Publication Date: 2009-03-04
UNIV OF LOUISVILLE RES FOUND INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although insulin therapy and islet transplantation are currently the most effective treatment options, both approaches have serious drawbacks

Method used

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  • Methods and compositions for expanding T regulatory cells
  • Methods and compositions for expanding T regulatory cells
  • Methods and compositions for expanding T regulatory cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0207] Example 1: Cloning and expression of CSA-4-1BBL conjugate

[0208] Total RNA was isolated from mouse splenocytes stimulated with LPS (5 μg / ml) for 2 days using TRI reagent (Molecular Research Center, Cincinnati, OH). Two micrograms of this RNA were used to generate first-strand cDNA, which was used as a template for PCR to amplify the extracellular domain of 4-1BBL (aa 104-309) using sense (5'-ATC GAA TTC CGC ACC GAGCCT CGG CCA GCG-3') and antisense (5'-GGA CTC GAG CAT AGC AGCTTG AGG ACT TAG C-3') primers. Primers were engineered to include EcoRI and XhoI sites to facilitate directional and in-frame cloning into a DES expression vector (Invitrogen, San Diego, CA). The PCR products were cloned into the PCR2.1 TOPO vector and several positive clones were subjected to DNA sequencing. Individual clones containing the correct 4-1BBL sequence were digested with EcoRI and XhoI and subcloned into the pMT / BiP / V5-His expression vector containing a 6×His tag and core streptavidi...

Embodiment 2

[0214] Example 2: Treg Expansion Using CSA-4-BBL Conjugate

[0215] Isolation of CD4 from spleens of BALB / c mice using fluorescence-activated cell sorting (FACS) + CD25 + Treg cells ( FIG. 9A ) were activated with anti-CD3 antibody (0.5 μg / ml), CSA-4-BBL (1 μg / ml) and IL-2 (25 IU / ml) in the presence of syngeneic APC. Cells were then plated at ~1 x 10 6 cells / ml with IL-2 supplemented medium every 3 days for 10-12 days. Cultures were then subjected to another round of activation followed by maintenance with IL-2. As shown in Figure 9, this protocol resulted in a 55- to 110-fold expansion of Treg cells in 18-24 days and a 110-fold expansion in 25 days (Figure 9B). Treg cells maintained under the same conditions but without the CSA-4-1BBL conjugate gave only minimal expansion. Expanded Treg cells form CD4 + CD25 亮 Conglomerate of cells (Fig. 9A, lower right panel) expressing high levels of FoxP3 protein (measured by RT-PCR or anti-FoxP3 antibody) as well as Fas, CD62L, GIT...

Embodiment 3

[0216] Example 3: Expanded Treg cells prolong survival of islet allografts

[0217] To test the therapeutic effect of polyclonal expanded Treg cells, chemically induced (with streptozotocin) diabetic BALS / c mice were acquired by transferring 5-10 × 10 6 Treg cells expanded in culture as described above for 20-25 days, and then 24 hours later, were given a transplant of fully mismatched C57BL / 6 allogeneic islets. Monitor the animal's blood glucose level three times a week. All Treg-treated animals (o) had prolonged survival (MST=68.7±10 days), with more than 1 / 3 (66%) not rejecting the graft within ~85 days of the observation period. Figure 11 . In marked contrast, all control animals (•) not treated with Treg cells strongly rejected the graft (MST=16.6±2.7 days).

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Abstract

The present invention provides methods and compositions for expanding Treg cells ex vivo or in vivo using one or more conjugates comprising a costimulatory moiety that stimulates at least one of three signals involved in Treg cell development and / or using dendritic cells pulsed with antigens and modified to display TGF-beta, or hematopoetic stem cells or bone marrow cells modified to display TGF-beta. The methods and compositions are useful, for example, in the treatment and prevention of autoimmune disease, including Type 1 diabetes and in preventing foreign graft rejection, as well as to establish mixed chimerism, induce tolerance to autoantigens, alloantigens or xenoantigens, beta cell regeneration, prevention of foreign graft rejection, and treatment of a genetically inherited hematopoietic disorder.

Description

[0001] NIH Grant Fund [0002] This work was supported in part by the NIH (R21 DK61333, R01 AI47864, R21 AI057903, R21 HL080108), the Pediatric Diabetes Research Foundation (1-2001-328), the American Diabetes Association (1-05-JF-56) and the Commonwealth of Kentucky Research Support provided by ChallengeTrust Fund. [0003] Cross References to Related Applications [0004] This application, pursuant to 35 U.S.C. § 199(e), claims the benefit of the filing date of the following U.S. provisional applications: 60 / 748,177 (filed December 8, 2005); 60 / 758,391 (filed January 12, 2006); 60 / 799,642 (filed December 12, 2006); and 60 / 799,643 (filed May 12, 2006). Each of the foregoing applications is hereby incorporated by reference in its entirety. field of invention [0005] The present invention relates generally to the field of immunotherapy. In particular, the invention provides methods and compositions for expanding T regulatory cells. The methods and compositions are useful,...

Claims

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Application Information

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IPC IPC(8): A61K47/48C07K14/705A61K38/18C07K14/495A61K47/62A61K47/68
Inventor H·什尔万
Owner UNIV OF LOUISVILLE RES FOUND INC
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