Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation

a technology for autoimmunity and hematopoietic stem cells, applied in the field of compositions and methods for eliminating undesired subpopulations of t cells in patients with immunological defects, can solve the problems of inability to regenerate the supply of accessory cells, cells may contaminate the entire t cell population during long-term culture, and the increased robustness and ease of t cell preparation remain less than ideal

Inactive Publication Date: 2005-04-21
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] One aspect of the present invention provides for a method for eliminating at least a substantial portion of a clonal T cell population from a mixed population of T cells from an individual, comprising, providing a population of cells wherein at least a portion thereof comprises T cells; exposing the population of cells ex vivo to one or more pro-apoptotic compositions wherein said exposure induces apoptosis in at least a portion of the T cells; thereby eliminating at least a substantial portion of said clonal T cells from the mixed population.
[0024] In certain embodiments, the patient requires a hematopoietic stem cell transplant. In a related embodiment, the composition that sensitizes recipient PBMCs that have been treated such that they are unable to continue dividing and the population of cells comprises donor T cells. The present invention also provides for populations of T cells generated according to the above methods. The present invention also provides methods for reducing the risk of, or the severity of, an adverse GVHD effect in a patient who is undergoing a hematopoietic stem cell transplant, comprising administering to said patient the population of T cells according to the methods described herein.
[0025] In certain embodiments, the patients to receive the cells of the present invention require an organ transplant. In a related embodiment the composition that sensitizes comprises irradiated donor cells and the population of cells comprises recipient T cells. The present invention also provides for a population of cells generated according to this method. In one embodiment, these cells are administered to a patient receiving an organ transplant to reduce the risk of organ rejection. In a related embodiment, the organ transplant patient is treated with a T cell ablative therapy prior to administration of the population of T cells.
[0035] The present invention provides a method for eliminating at least a substantial portion of a clonal T cell subpopulation from a mixed population of T cells from an individual, comprising, exposing a population of cells, wherein at least a portion thereof comprises T cells, to one or more pro-apoptotic compositions wherein said exposure induces apoptosis in at least a substantial portion of at least one clonal T cell population present in the mixed population of T cells thereby eliminating at least a substantial portion of said clonal T cell population from the mixed population of T cells.

Problems solved by technology

The requirement for MHC-matched APCs as accessory cells presents a significant problem for long-term culture systems because APCs are relatively short-lived.
The necessity for a renewable supply of accessory cells is problematic for treatment of immunodeficiencies in which accessory cells are affected.
In addition, when treating viral infection, if accessory cells carry the virus, the cells may contaminate the entire T cell population during long-term culture.
While these methods are capable of achieving therapeutically useful T cell populations, increased robustness and ease of T cell preparation remain less than ideal.
In addition, the methods currently available in the art have not focused on short-term expansion of T cells or obtaining a more robust population of T cells and the beneficial results thereof.
None of these methods has described using such or similar methods to eliminate an undesired clonal or oligoclonal T cell population from a T cell population nor the beneficial results thereof.
Moreover, the methods previously available tend to further skew the clonality of the T cell population rather than eliminate undesired reactive clones from a T cell population, and restore a normal immune repertoire.
When the immunosuppressive agents are stopped, disease recurs often concomitant with reappearance of disease causing T cells that re-emerge in these patients.
The major problem in hematopoietic stem cell transplantation is graft-versus-host disease (GVHD), which is caused by alloreactive T cells present in the infused hematopoietic stem cell preparation.

Method used

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  • Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation
  • Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation
  • Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation

Examples

Experimental program
Comparison scheme
Effect test

example 1

Deletion of Antigen-Specific T Cells Following Restimulation with CD3 / CD28 XCELLERATE™ Beads

[0208] This example describes the elimination of antigen-specific T cells from a mixed population of cells by restimulation with anti CD3 / CD28 XCELLERATE™ beads (3×28 beads). The generation of XCELLERATED T cells™ using the processes described herein is essentially as described in U.S. patent application Ser. No. 10 / 133,236.

[0209] Human PBMC were screened for HLA-A2 CMVpp65 positivity by flow cytometry using HLA-A2 tetramers loaded with CMVpp65 peptide (HLA-A2-CMVpp65). Approximately 3% of the CD3+ CD8+T cells in the donor selected expressed TCR specific for HLA-A2-CMVpp65 (FIG. 1).

[0210] PBMC from the donor (donor 2) and control donor (donor 1) were activated with CMV antigen coated onto paramagnetic beads and by day 10 of culture, many cells were shown by flow cytometric analysis to be CD25 (IL-2R) positive, and all of the HLA-A2 CMVpp65+T cells expressed high levels of CD25, indicating ...

example 2

Determination of Apoptosis

[0213] This example describes an illustrative assay for measuring apoptosis.

[0214] DNA Fragmentation Assay: Cells are lysed in 50 μl of lysis buffer (10 mM EDTA, 50 mM Tris pH 8, 0.5% sodium dodecyl sulfate, 0.5 mg / ml proteinase K). RNAse A (0.5 mg / ml) is added and lysates are incubated for 1 hr. at 37° C. Two phenol extraction (equal volumes) are performed, followed by one chloroform extraction. DNA is precipitated with two volumes of ice-cold ethanol and incubated at −80° C. for 1 hr. DNA is pelleted by centrifugation at 14,000 rpm for 10 minutes at 4° C. Pellets are air-dried for 30 minutes, resuspended in 50 μl of Tris-EDTA pH 8. DNA is electrophoresed in a 1.8% agarose gel in 1×TBE running buffer (0.05 M Tris base, 0.05 M boric acid, 1 mM disodium EDTA), according to the methods of Preston, et al., Cancer Res., 1994, 54, 4214-4223.

example 3

Induction of Apoptosis in B-Cells by Coculture with XCELLERATED T Cells™

[0215] This example describes the deletion of leukemic B-cells in B-CLL patient samples by co-culture with XCELLERATED T cells™.

[0216] XCELLERATED T cells™, generated essentially as described in U.S. patent application Ser. No. 10 / 133,236, were co-cultured with unmanipulated autologous leukemic cells from B-CLL patients. Cell surface markers for CD54, CD80, CD95 (FAS) and CD86, and Annexin / PI (apoptosis) were measured at 24 and 48 hours by flow cytometry. XCELLERATED T cells™ were shown to drive up expression of CD95 (FAS) on leukemic B cells (FIG. 4). After 48 hours of co-culture with day 12 XCELLERATED T cells™, autologous leukemic B cells show increased expression of CD95 and sensitivity to anti-FAS as measured by flow cytometry (FIG. 5). As shown in FIG. 5, addition of anti-FAS antibody to co-cultured T:B cells led to increased apoptosis in the leukemic B-cells. In an additional study, it was shown that T c...

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Abstract

The present invention relates generally to methods for stimulating T cells, and more particularly, to methods to eliminate undesired (e.g. autoreactive, alloreactive, pathogenic) subpopulations of T cells from a mixed population of T cells, thereby restoring the normal immune repertoire of said T cells. The present invention also relates to compositions of cells, including stimulated T cells having restored immune repertoire and uses thereof.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The present invention relates generally to methods for stimulating T cells to restore normal immune repertoire. The present disclosure includes methods to eliminate undesired (e.g. autoreactive, alloreactive, pathogenic) subpopulations of T cells from a mixed population of T cells, thereby restoring the normal immune repertoire of said T cells. The present invention also relates to compositions of cells, including stimulated T cells having restored immune repertoire and uses thereof. [0003] 2. Description of the Related Art [0004] The ability of T cells to recognize the universe of antigens associated with various cancers or infectious organisms is conferred by its T cell antigen receptor (TCR), which is made up of both an α (alpha) chain and a β (beta) chain or a γ (gamma) and a δ (delta) chain. The proteins which make up these chains are encoded by DNA, which employs a unique mechanism for generating the tremendou...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/14A61K35/17C12N5/00C12N5/0783
CPCA61K35/17C12N5/0636C12N5/0087A61P37/00Y02A50/30C12N2501/48C12N2502/1114
Inventor BERENSON, RONALD J.BONYHADI, MARKKALAMASZ, DALE
Owner LIFE TECH CORP
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