Method to induce and expand therapeutic alloantigen-specific human regulatory T cells in large-scale

An allogeneic antigen, allogeneic technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problem of lack of human antigen-specific Treg effective means, and achieve the effect of preventing rejection

Active Publication Date: 2011-06-01
VERSITECH LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, the art lacks effective means for large-scale production of human antigen-specific Tregs

Method used

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  • Method to induce and expand therapeutic alloantigen-specific human regulatory T cells in large-scale
  • Method to induce and expand therapeutic alloantigen-specific human regulatory T cells in large-scale
  • Method to induce and expand therapeutic alloantigen-specific human regulatory T cells in large-scale

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: CD40-activated B cells expanded by incubation with CD40-ligand transfected cells or soluble hexameric CD40-ligand express high levels of MHC and co-stimulatory molecules

[0071] as previously reported 27 , can be expanded from circulating B cells contained in PBMCs by treating NIH3T3 (t-CD40-L) cells transfected with CD40-ligand (CD40-L), IL-4, and low concentrations of cyclosporin A. Increases untransformed CD40-activated B cells. At day 8, CD19 + CD3 - The purity of B cells was at least 83% and over 95% at day 12. At 28-32 days in culture, more than 99% of cells are CD19 + CD3 - B cells. To evaluate the rate of expansion of B cells, we monitored CD19 production from 5.0 ml of peripheral blood from 8 unselected healthy adult donors + CD3 - Absolute number of cells. We found that after 32 days in culture, 8.1-54.3 x 10 7 CD40-activated B cells ( figure 1 A). We next determined whether soluble hexameric CD40-ligand (sCD40-L) could replace t...

Embodiment 2

[0072] Example 2: Human alloreactive CD4 induced by CD40-activated B cells 高 cells are Treg

[0073] To determine whether allogeneic CD40-activated B cells could be converted from CD4 + CD25 - T cell induction of Treg, purified circulating CD4 stimulated with allogeneic CD40-activated B cells + CD25 - T cells (purity >99%) for 7 days. Surprisingly, after 5 days of allogeneic stimulation, a new subset of cells with significantly upregulated levels of CD4 surface expression was induced, and these CD4 高 Most of the cells lost CD45RA expression ( figure 2 A), and CD45RO expression was obtained (data not shown). In addition, these CD4 高 Most of the cells also lost CFSE staining, while CD4 中 Cells still maintain their CFSE content ( figure 2 A), showing the induction of CD4 高 The cells are proliferating alloreactive cells. These putative alloreactive CD4 高 Cells express CD25 and Foxp3, while CD4 中 Cells do not express these two Treg markers ( figure 2 B). Togethe...

Embodiment 3

[0075] Example 3: CD40-activated B cells can be derived from naive CD4 + CD25 - alloantigen-specific CD4 高 CD25 + Tregs

[0076] We next determined whether, by co-culture with allogeneic CD40-activated B cells, + CD25 - cells (CD4 + CD45RA + CD45RO - CD25 + ) produces alloantigen-specific Tregs. As unisolated CD4 + CD25 - In the case of T cells, naive CD4 cells expanded by co-culture with CD40-activated B cells ± CD25 - Cells also acquired CD4 after 7 days in culture 高 , CD25 + and Foxp3 + Phenotype ( image 3 A). In addition, these CD4 高 CD25 + Foxp3 + Treg underwent 7-8 cell divisions during allogeneic stimulation for 7 days ( image 3 A). Instead, CD4 中 Cells neither divide nor express CD25 and Foxp3 ( image 3 A).

[0077] We further examined the induction of CD4 from naive precursors 高 CD25 + Treg suppressive capacity and alloantigen specificity. These CD4 高 CD25 + Treg significantly suppressed the original target alloantigen-induced p...

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Abstract

Methods for inducing, expanding, and / or generating alloantigen-specific regulatory T cells. Alloantigen-specific regulatory T cells can be induced, expanded, and / or generated from naive CD4+CD25' T cells by using CD40- activated B cells. The regulatory T cells can be human T cells. In one embodiment, the alloantigen-specific human regulatory T cells can be CD4hIghCD25+-Foxp3+ regulatory T cells.

Description

[0001] Statement Regarding Federally Funded Research or Development [0002] This invention was made with US Government support under Grant No. AI050153 of the National Institutes of Health. The US Government has certain rights in this invention. [0003] Cross References to Related Applications [0004] This application claims the benefit of priority to U.S. Provisional Application Serial No. 61 / 133,643, filed June 30, 2008, the entire contents of which are incorporated herein by reference. Background technique [0005] Treatment with immunosuppressive drugs is widely accepted as an effective treatment for bone marrow and solid organ transplantation to improve graft survival. However, chronic rejection of grafts still has a significant impact on long-term outcome. Furthermore, many immunosuppressive drugs target the immune response non-specifically, leading to undesired side effects such as a weakened overall immune system. Thus, the goal of transplantation is to induce a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/14A61P37/00A61P37/06A61K35/12A61K35/17
CPCC12N2501/52A61K2035/122C12N2501/23C12N5/0636C12N2502/11A61K35/17A61P37/00A61P37/06
Inventor 涂文伟刘宇隆B.D.刘易斯
Owner VERSITECH LTD
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