Researchers Submit Patent Application, "Method to Induce and Expand Therapeutic Alloantigen-Specific Human Regulatory T Cells in Large-Scale", for Approval
The patent's assignee is The Board Of Trustees Of The
News editors obtained the following quote from the background information supplied by the inventors: "Treatment with immunosuppressive drugs is widely accepted as an effective treatment for bone marrow and solid organ transplantation to improve the graft survival. However, chronic rejection of transplants still has a considerable impact on the long term outcome. Moreover, many immunosuppressive drugs nonspecifically target the immune response, leading to unwanted side effects, such as weakened overall immune system. Thus, the goal in transplantation is the induction of a sustained state of specific tolerance to donor alloantigens with minimization or complete withdrawal of global immunosuppression.
"CD4.sup.+CD25.sup.+Foxp3.sup.+ regulatory T cells (Treg) are negative regulators of immune responses to self- and foreign-antigens and play a critical role in maintaining immune tolerance by suppressing pathological immune responses in autoimmune diseases, transplant allograft rejection, and graft-versus-host disease (GVHD)..sup.1-3 Upon adoptive transfer in rodents, Treg were found to control experimental autoimmune diseases.sup.4 inhibit GVHD.sup.5,6 and prevent transplant allograft rejection.sup.7,8 indicating that Treg-based therapy has a great therapeutic potential for these diseases in humans.
"An important obstacle to Treg-based therapy has been the limited numbers of these cells that are available, as only about 1-2% of circulating human CD4.sup.+ T cells are Treg. Several groups have developed protocols to expand a large number of polyclonal CD4.sup.+CD25.sup.+ Treg in vitro with repeated stimulation by either CD3 and CD28 mAbs or artificial antigen-presenting cells (APC) for activation through CD3 and CD28, together with exogenous high-dose IL-2..sup.9-11 polyclonal Treg may cause global immune suppression..sup.4,7 In addition, since there are only few antigen-specific Treg in the population of the polyclonal Treg, very large numbers of non-specifically expanded Treg are required to inhibit bone-marrow allograft rejection in animal models..sup.12 All of these characteristics of polyclonal Treg hamper their clinical applications.
"In contrast, adoptive transfer of antigen-specific Treg has been shown to prevent and treat T-cell-mediated inflammatory diseases with high efficiency. In animal models, small number of antigen-specific Treg can suppress experimental autoimmune diseases.sup.13 prevent GVHD and allograft rejection in bone marrow and solid organ transplantation..sup.14,15 Importantly, the transfer of antigen-specific Treg prevented target antigen-mediated T-cell responses such as GVHD and allograft rejection but did not compromise host general immunity including the graft-versus-tumor activity and antiviral immunity..sup.5,15-17 Based on these studies, antigen-specific Treg has substantial promise for human immunotherapy.
"The reliable induction and expansion of rare antigen-specific Treg is technically challenging. Currently, several protocols for murine antigen-specific Treg induction and expansion have been reported in which either purified CD4.sup.+CD25.sup.- or CD4.sup.+CD25.sup.+ cells were co-cultured with autologous dendritic cells (DCs) pulsed with alloantigen in the presence of high-dose IL-2 or directly co-cultured with allogeneic DCs..sup.14,18-20 Similar protocol has also been reported for generation of human antigen-specific Treg recently..sup.21 In this protocol, antigen-specific CD4.sup.+l CD25.sup.+ Treg can be generated by using the co-culture of CD4.sup.+CD25.sup.- T cells with allogeneic monocyte-derived DCs. However, the large-scale in vitro expansion of alloantigen-specific Treg is difficult because of certain features of DCs. For example, DCs are relatively rare in peripheral blood and are usually derived from apheresis or marrow sources including monocytes..sup.22,23 Further, DCs are not homogeneous and include multiple subsets with different functional capacities..sup.24 Finally, there is no effective way to expand human DCs so far..sup.25 In addition, the current approaches to generate human DCs in vitro are expensive and laborious..sup.26
"Schultze et al. reported a simple and low-cost method to expand large number human CD40-activated B cells up to 10.sup.5,6-fold from human peripheral blood mononuclear cells (PBMC)..sup.27 These expanded B cells are effective as APCs and can efficiently induce antigen-specific T cells and cytotoxic T lymphocytes..sup.26,27
"However, the art lacks an effective means of generating human antigen-specific Treg on a large scale. Thus, there exists a need in the art for a method of inducing or generating human antigen-specific Treg on a large scale."
As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "The present invention provides novel methods for the induction, expansion, and/or generation of alloantigen-specific regulatory T cells. Advantageously, the induction, expansion, and/or generation of the regulatory T cells can be performed on a large scale. The subject invention further provides cells produced according to the methods set forth herein.
"In some embodiments, the subject invention provides novel protocols to induce and expand highly efficient human alloantigen-specific Treg in large-scale by co-culture of nave CD4.sup.+CD25.sup.- T cells with human allogeneic CD40-activated B cells without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO.sup.+ and CCRT memory cells, and expressed the common Treg markers (CD25 and Foxp3), as well as the lymph node homing receptor CD62L (L-selectin). They were also identifiable by a CD4.sup.high surface phenotype. The suppressive function of these CD4.sup.highCD25.sup.+Foxp3.sup.+ alloantigen-specific Treg was cell-cell contact dependent but did not involve cell-mediated cytotoxicity.
"The methods of the subject invention for in vitro induction and expansion of alloantigen-specific Treg should facilitate the development of Treg-based clinical immunotherapy. For example, adoptive transfer of alloantigen-specific regulatory T cells can be used according to the subject invention for inhibiting allogeneic immune responses, e.g. GVHD, and preventing transplant allograft rejection. Additionally, methods of the present invention can be used to generate human alloantigen-specific Treg that can be used to control autoimmune diseases.
"The methods of the subject invention are unique in their use of CD40-activated B cells as APCs rather than allogeneic monocyte-derived DCs or PBMC. CD40-activated B cells have an important advantage for this purpose in that they can be readily expanded in vitro to a relatively large numbers, while, in contrast, monocytes differentiating in vitro into dendritic cells do not undergo cell division. Cryopreserved CD40-activated B cells also retain their APC function upon thawing, and are relatively cost-effective to produce. In addition, because B cells stimulated with t-CD40-L cells or recombinant sCD40-L were equally effective at generating alloantigen-specific Treg, the use of sCD40-L significantly improves the clinical applicability of the procedure.
BRIEF DESCRIPTION OF THE FIGURES
"The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
"FIG. 1A-C shows that CD40 activation is highly effective in generating large numbers of CD40-activated B cells that express high levels of MHC and co-stimulatory molecules. (1A) shows an overall expansion of CD40-activated B cells from 8 different individuals. CD40-activated B cells were generated by the co-culture of PBMC from 5 ml of peripheral blood with CD40L-transfected NIFI3T3 (t-CD40-L) cells. (1B) shows sCD40-L is as efficient as t-CD40-L cells at expanding human B cells in culture. CD40-activated B cells were generated by means of t-CD40-L cells or different concentrations of soluble hexameric CD40-L. The results shown are representative of three independent experiments. (IC) shows expression of CD80, CD86, and MHC class I and II on the CD40-activated B cells cultured for 8 days (solid histograms). The filled histograms were obtained with relevant isotype controls. Data shown here are representative of B-cell populations obtained from 8 different healthy adult donors.
"FIG. 2A-C shows Human alloreactive CD4.sup.high cells induced by CD40-activated B cells are Treg. (2A) CD4 expression in CD4.sup.+CD25.sup.- T cells stimulated with allogeneic B cells for 5 days (top panels), and its relationship with cell proliferation based on the loss of CFSE label and CD45RA expression. Top panels represented the T cells gated on CD4. The percentage of CD4.sup.+ T cells in each gate is indicated. For the bottom panel, open histograms indicate the CFSE fluorescence intensities of the unstimulated control T cells, and the filled histograms represent the CFSE fluorescence intensities of the allostimulated T cells. The numbers in each histogram represent the percentage of cells that have undergone mitosis from each cell subset. (2B) CD4.sup.high cells express both CD25 and Foxp3. The dot plot on the left shows CD25 expression after 5 days of allostimulation. Open histograms on the right show the Foxp3 expression, and filled histograms indicate the isotype controls. The results shown are representative of four different experiments. (2C) CD4.sup.highCD25.sup.+ Treg generated from CD4.sup.+CD25.sup.- T cells potently suppressed MLR in an antigen-nonspecific manner. Freshly purified CD4.sup.+CD25.sup.- T cells were co-cultured with CD40-activated allogeneic B cells for 7 days. The sorted CD4.sup.highCD25.sup.+ (black squares) and CD4.sup.mediumCD25.sup.- (open squares) cells were added into MLR culture system as described in Materials and Methods. Proliferation (y-axis) is shown for 3 days of MLR. The results shown are representative of five different experiments.
"FIG. 3A-B shows Human CD4.sup.high Treg induced from nave CD4.sup.+CD25.sup.- T cells by CD40-activated allogeneic B cells are alloantigen-specific Treg. (3A) Characteristics of CD4.sup.high Treg induced from naive CD4.sup.+CD25.sup.- T cells. Freshly purified naive CD4.sup.+CD25.sup.- T cells were labeled with CFSE and co-cultured with CD40-activated allogeneic B cells for 7 days. Representative data of CD4 and CD25 expression (left panel), CFSE dilution (right top panel) and Foxp3 expression (right bottom panel) from 6 independent experiments are shown. Open histograms show the CFSE fluorescence intensity (right top panel) and Foxp3 expression (right bottom panel) of CD4.sup.mediumCD25.sup.- cells. Filled histograms represent the CFSE fluorescence intensity (right top panel) and Foxp3 expression (right bottom panel) of CD4.sup.highCD25.sup.+ cells. (3B) CD4.sup.highCD25.sup.+ Treg generated from naive CD4.sup.+CD25.sup.- T cells potently suppressed MLR in an alloantigen-specific manner, and unsorted CD4.sup.+ T cells generated from nave CD4.sup.+CD25.sup.- T cells had similar suppressor capacities in MLR. Freshly purified CD4.sup.+CD45RA.sup.+CD25.sup.- T cells were co-cultured with CD40-activated allogeneic B cells for 7 days. The sorted CD4.sup.highCD25.sup.+ (black squares) and CD4.sup.meediumCD25.sup.- (open squares), and unsorted CD4.sup.+ T cells (crosses) were added into MLR culture system as described in Materials and Methods. Proliferation (y-axis) was shown for day 3 of MLR. The results shown are representative of 8 independent experiments.
"FIG. 4A-B shows characteristics of CD4.sup.highCD25.sup.+ alloantigen-specific Treg. Freshly purified naive CD4.sup.+CD25.sup.- T cells were co-cultured with CD40-activated allogeneic B cells for the indicated time. The expression of cell surface markers (4A) and intracellular cytokines (4B) were determined and analyzed by FACS as described in Materials and Methods. The percentage of positive cells for each cell surface marker or intracellular cytokine within the CD4.sup.highCD25.sup.+ and CD4.sup.mediumCD25.sup.- subsets are indicated. The results shown are representative of four independent experiments.
"FIG. 5A-C shows CD4.sup.highCD25.sup.+ alloantigen-specific Treg have no cytotoxic capacity and their suppressor function is dependent on cell-cell contact and partially relies on CTLA-4 expression. CD4.sup.highCD25.sup.+ Treg or CD4.sup.mediumCD25.sup.- T cells were sorted after 7 days of allostimulation as shown in FIG. 3B. (5A) Cytotoxic capacity of induced CD4.sup.highCD25.sup.+ Treg. (5B) The alloantigen-specific suppressor function of CD4.sup.highCD25.sup.+ Treg is cell-cell contact dependent. (5C) Neutralizing anti-CTLA-4 mAb partially reverses the alloantigen-specific suppression mediated by CD4.sup.highCD25.sup.+ Treg, but neutralizing mAbs to IL-4, IL-10, TGF-.beta. and GITR fail to reverse that suppression. Responder ® CD4.sup.+CD25.sup.- and gamma-irradiated stimulator PBMC (S) were co-cultured with or without sorted CD4.sup.highCD25.sup.+ Treg or CD4.sup.mediumCD25.sup.- T cells. The cytotoxic activities (A) of human IL-2activated NK cells against K562 cells were set as positive controls (PC). Stimulator (S) or responder ® cells alone were set as controls. For transwell experiments (B), the same amount of responder ® and stimulator (S) cells were plated in the bottom wells of a transwell system. The top well insert was inoculated with same amount of sorted CD4.sup.highCD25.sup.+ Treg. For the blocking experiments (C), the neutralization mAbs (open bars) and their relevant isotype controls (filled bars) were added in the co-culture system. Proliferation (y-axis) is shown for day 3 of cultures. Data for four different experiments are shown (n=4). The two-tailed unpaired Student's t tests were used for comparison. * indicate p
"FIG. 6A-E shows CD4.sup.highCD25.sup.+ alloantigen-specific Treg can be continuously expanded by CD40-activated B cells in large-scale without loss of function, and exogenous IL-2 does not enhance this cell expansion. Freshly purified nave CD4.sup.+CD25.sup.-T cells were co-cultured with CD40-activated allogeneic B cells for the indicated time. (6A) The percentages of CD4.sup.highCD25.sup.+ and CD4.sup.mediumCD25.sup.- cells in the cultures (n=10). (6B) Expansion of CD4.sup.highCD25.sup.- alloantigen-specific Treg from 10 different individuals. The expansion was normalized for the CD4.sup.highCD25+ cells, and the fold increase of the CD4.sup.highCD25- was shown. (6C) Naive CD4.sup.+CD25.sup.- were co-cultured with CD40-activated allogeneic B cells with or without IL-2. The expansion was normalized for the CD4.sup.highCD25.sup.+ cells, and the fold increase of the CD4.sup.highCD25.sup.+ is shown (n=4). (6D) Absolute numbers of CD4.sup.highCD25+ alloantigen-specific Treg generated from 1.times.10.sup.6 nave CD4.sup.+CD25.sup.- T cells (n=10). (6E) CD4.sup.highCD25.sup.+ alloantigen-specific Treg induced and expanded by CD40- activated B cells for 21 days remain functional. Freshly purified naive CD4.sup.+CD25.sup.- T cells (responder) were co-cultured with CD40-activated allogeneic B cells (target antigen) to induce and expand CD4.sup.highCD25.sup.+ Treg for 21 days with replacement of B cells every 7 days. The sorted CD4.sup.highCD25.sup.+ and CD4.sup.mediumCD25.sup.- cells were added into the MLR culture system as described in Materials and Methods. Data shown here are representative of three independent experiments."
For additional information on this patent application, see: Tu, Wenwei; Lau, Yu-Lung; Lewis,
Keywords for this news article include: Biomedical Engineering, Biomedicine, Monocytes, Immunology, Blood Cells, CD Antigens, CD3 Antigens, CD4 Antigens, Legal Issues, Therapeutics,
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