Most Closely HLA Matched Allogeneic Virus Specific Cytotoxic T-Lymphocytes (CTL) to Treat Persistent Reactivation Or Infection With Adenovirus, CMV and EBV After Hemopoietic Stem Cell Transplantation (HSCT)

Patients may be screened for study entry when they have persistent disease despite standard therapy as defined in the inclusion criteria. At that stage a search will be done of the available lines. Lines were generated from HSCT donors who consented to the use of CTLs not required for their recipient for research or from normal donors. All donors were screened and deemed to be eligible as transplant donors. We will also manufacture additional lines with the goal of covering common HLA types and will consult with the NMDP to determine what HLA types would be desirable. Additional donors will be screened by a transplant donor center physician and must be deemed eligible before a line can be manufactured.

CTL Lines: We will use trivirus specific CTL lines generated as described previously. Generation of trivirus-specific CTL lines requires the generation of several different components from PBMC. The CTL line will be derived from donor peripheral blood T cells, by multiple stimulations with antigen-presenting cells (APCs) presenting CMV, EBV and adenovirus antigens and expansion with interleukin-2 (IL-2). The APCs used to stimulate and expand the CMV-specific T cells will be derived from patient mononuclear cells and B lymphocytes.

To initiate the trivirus-specific CTL line, PBMC will be transduced with an adenovirus vector (Ad5f35-pp65) expressing the immunodominant antigen of CMV, pp65. The monocyte fraction of PBMC expressed and presented CMV-pp65 peptide epitopes to the CMV-specific T cell fraction of the PBMC, while the virion proteins from the adenovirus vector were processed and presented to the adenovirus-specific T cell fraction.

To expand trivirus -specific T cells we used EBV-transformed B lymphoblastoid cell lines (EBV-LCLs) transduced with Ad5f35-pp65. This transduction allows the EBV-LCLs to present CMV-pp65 and adenovirus virion peptides to the T cells as well as endogenously expressed EBV antigens.

EBV-LCLs are derived from PBMC-B lymphocytes by infection with a clinical grade, laboratory strain of Epstein-Barr virus (EBV). About 5 x 10^6 PBMC, or 5 to 10 mLs of blood is required to generate the EBV-LCL