Production of leukemia-reactive T-cells via a good manufacturing practice procedure for treatment of patients with acute or chronic B-cell malignancies after allogeneic stem cell transplantation

      Donor T-cells recognizing malignant cells of the recipient can mediate a potent therapeutic graft-versus-leukemia (GvL) effect after allogeneic stem cell transplantation (alloSCT). However, in a significant number of patients no adequate GvL response is mounted. Adoptive transfer of in-vitro generated leukemia-reactive T-cells may be an effective strategy to boost the GvL effect. In this study, we developed a Good-Manufacturing-Practice (GMP) procedure to generate potent leukemia-reactive T-cells for adoptive transfer. Primary malignant B-cells harvested from peripheral blood or bone marrow of patients with chronic lymphocytic leukemia, acute lymphoblastic leukemia, or mantle cell lymphoma were transformed into antigen-presenting cells (APC) by crosslinking of CD40 using a 2-step strategy consisting of CpG/IL4 pre-activation followed by co-culture with irradiated CD3/28 activated donor T-cells expressing CD40-ligand. Next, donor PBMC depleted of CD14+ monocytes and CD25+ regulatory T-cells were stimulated with the malignant APC and cultured for 14 days. After secondary stimulation with the malignant APC leukemia-reactive T-cells were isolated based on expression of the activation marker CD137 using clinical grade CD137-biotin, anti-biotin beads and cliniMACS isolation (Miltenyi Biotec). After 7–10 days of post-isolation culture functional reactivity of the isolated T-cells was tested. Sufficient numbers of phenotypical appropriate malignant APCs expressing CD40, CD80 and CD86 could be reproducibly generated from primary malignant B-cells both harvested at diagnosis or at a later stage after initial treatment. Isolated leukemia-reactive T-cells displayed an oligoclonal T-cell receptor repertoire, and showed profound cytotoxic activity against the primary malignant B-cells as well as against the malignant APC and no reactivity against donor cells. All T-cell products produced in the large-scale testruns under GMP conditions met the pre-defined criteria for release for clinical application. A clinical phase I/II study is initiated to demonstrate feasibility and safety of administration of these leukemia-reactive T-cells to patients with recurrent or persistent mature B-cell neoplasms after alloSCT.
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