GMP production and testing of Xcellerated T Cells for the treatment of patients with CLL.

BACKGROUND Pre-clinical studies suggest Xcellerated T Cells have the potential to produce a potent anti-tumor effect, restore broad immune function and reduce the risk of infectious complications in patients with CLL. Unlike other cancer settings, T cells constitute only a small fraction of CLL patients' PBMC. To generate large numbers of Xcellerated T Cells of high purity from CLL patients' PBMC, a reproducible, streamlined and cost-effective good manufacturing process (GMP) is required. METHODS The 10-L volume Wave Bioreactor-based Xcellerate III Process using Xcyte Dynabeads in a single custom 20-L Cellbag container was adapted, qualified and implemented for GMP operations. RESULTS For n=17 CLL patients, starting with approximately 1.34 x 10(9) CD3+ T cells at 6.8+/-7.5% purity in the PBMC leukapheresis products, using the 10-L volume Wave Bioreactor-based Xcellerate III Process, it was feasible to manufacture 137.0+/-34.3 x 10(9) Xcellerated T Cells at 98.5+/-1.0% CD3+ T-cell purity. An average 400-fold clearance of malignant B cells was documented during the manufacturing process. The Xcellerated T Cells produced from the Xcellerate III Process exhibited high in vitro biologic activity and have their T-cell receptor repertoire restored to a normal diversity. In-process T-cell activation was reproducibly robust, as measured by increase in cell size, up-regulation of CD25 and CD154 expression and the secretion of IL-2, IFN-gamma and tumor necrosis factor (TNF)-alpha. DISCUSSION A low-volume, high-yield bioreactor-based process has been developed, qualified and implemented for the reproducible, GMP manufacture of high purity, biologically active Xcellerated T Cells for the treatment of CLL patients in clinical trials.

[1]  K. Smith,et al.  Functional and molecular characteristics of T-cell growth factor. , 1980, Molecular immunology.

[2]  Kendall A. Smith,et al.  T‐Cell Growth Factor , 1980, Immunological reviews.

[3]  L. Old,et al.  Tumor necrosis factor (TNF). , 1985, Science.

[4]  P. Linsley,et al.  Role of the CD28 receptor in T-cell activation. , 1990, Immunology today.

[5]  B. Kissela,et al.  Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. Correlation with immune status. , 1994, Journal of immunology.

[6]  L. Wen,et al.  Analysis of the peripheral T-cell receptor V beta repertoire in newly diagnosed patients with type I diabetes. , 1994, Autoimmunity.

[7]  H. Everaus,et al.  Active and indolent chronic lymphocytic leukaemia – immune and hormonal peculiarities , 1997, Cancer Immunology, Immunotherapy.

[8]  D. Neuberg,et al.  Characterization of T cell repertoire in patients with graft-versus-leukemia after donor lymphocyte infusion. , 1997, The Journal of clinical investigation.

[9]  R. Flavell,et al.  CD40 and CD154 in cell-mediated immunity. , 1998, Annual review of immunology.

[10]  J. V. van Dongen,et al.  Correction of abnormal T-cell receptor repertoire during interferon-alpha therapy in patients with hairy cell leukemia. , 1998, Blood.

[11]  C. Katlama,et al.  Perturbation of CD4+ and CD8+ T-cell repertoires during progression to AIDS and regulation of the CD4+ repertoire during antiviral therapy , 1998, Nature Medicine.

[12]  E. Robinet,et al.  Retrovirus-mediated gene transfer in primary T lymphocytes: influence of the transduction/selection process and of ex vivo expansion on the T cell receptor beta chain hypervariable region repertoire. , 2000, Human gene therapy.

[13]  C. Siegrist,et al.  Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity. , 2000, Blood.

[14]  M. Boccadoro,et al.  Severe and long‐lasting disruption of T‐cell receptor diversity in human myeloma after high‐dose chemotherapy and autologous peripheral blood progenitor cell infusion , 2001, British journal of haematology.

[15]  E. Sercarz,et al.  Molecular characterization of the T cell repertoire using immunoscope analysis and its possible implementation in clinical practice. , 2001, Current molecular medicine.

[16]  G. Pellicanò,et al.  Short‐term evolution of autoreactive T cell repertoire in multiple sclerosis , 2001, Journal of neuroscience research.

[17]  V. Kuchroo,et al.  T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and regulating the autopathogenic T cell repertoire. , 2002, Annual review of immunology.

[18]  T. Klingebiel,et al.  Distinct contributions of CD4(+) and CD8(+) naive and memory T-cell subsets to overall T-cell-receptor repertoire complexity following transplantation of T-cell-depleted CD34-selected hematopoietic progenitor cells from unrelated donors. , 2002, Blood.

[19]  Stewart Craig,et al.  Comparison of a Static Process and a Bioreactor-based Process for the GMP Manufacture of Autologous Xcellerated T Cells for Clinical Trials , 2003 .

[20]  S. Mackinnon,et al.  Assessing diversity: immune reconstitution and T‐cell receptor BV spectratype analysis following stem cell transplantation , 2003, British journal of haematology.

[21]  M. Connors,et al.  Immune reconstitution following autologous transfers of CD3/CD28 stimulated CD4(+) T cells to HIV-infected persons. , 2004, Clinical immunology.

[22]  T. Kipps,et al.  A Phase I/II Trial of Xcellerated T Cells™ in Patients with Chronic Lymphocytic Leukemia. , 2004 .