Preclinical development of adoptive cellular immunotherapeutic protocols for the outcome improvement of onco-hematologic patients after allogeneic hematopoietic stem cell transplantation

Although important advances have been made in the treatment of hematological malignancies using chemotherapy, and more recently with targeted therapies, curative treatments often require allogeneic hematopoietic stem cell transplantation (HSCT). One of the major challenges in HSCT is to find a balance between the harmful induction of graft-versus-host disease (GVHD) and the beneficial aspects of tissue disparity, namely graft-versus-leukemia (GVL). Unfortunately, even this intensive treatment fails to prevent relapse in 10%-60% of cases, depending on whether the disease was successfully treated early or was relapsing or refractory at the time of transplantation. Relapse remains the major cause of treatment failure after HSCT for patients with high-risk hematological malignancies. In addition, strong immunosuppression absolutely essential for the prevention of severe GVHD, leads to infectious complications. To overcome these issues, cellular based-immunotherapeutic approaches represent a promising tool for the graft recipient patients, while maintaining the GVL effect. The so-called GVL effect is a major component of the effectiveness of HSCT for hematological malignancies and is due primarily to immunologic recognition and elimination of recipient leukemia cells by donor T cells: improvements in exploiting the GVL effect to prevent relapse in high-risk leukemias while minimizing toxicity have focused on the use of targeted antileukemic immunotherapy. These strategies include methods to boost the GVL effect with donor lymphocyte infusion (DLI) but the limited spectrum of activity and high risk of GVHD remain major limitations of this approach. Recent clinical studies with cytokine-induced killer (CIK) cells explore the antitumor and alloreactive potentials of this heterogeneous population of polyclonal T lymphocytes, that might favourably affect the balance between GVL and GVHD. After HSCT, a prolonged and severe immune deficiency often leads to infectious complications: despite prophylactic or pre-emptive antiviral treatment viral reactivations consist in about 30% of death after HSCT. Human cytomegalovirus (CMV) infection is one of such life-threatening complication. Rapid selection strategies to isolate donor-derived CMV-specific T cells (CMV-T) are currently being tested in the clinical setting and recent advances in the manufacturing of T-cell therapy for viral infections have greatly simplified the production. Several approaches of adoptive cellular therapies are now being evaluated in late-phase or licensing studies, raising the prospect that adoptive transfer of CMV-T may become a standard of care after HSCT. To overcome the impossibility to produce T cells against CMV from CMV-seronegative donors, new strategies able to induce pathogen-specific responses should be developed. These approaches relies on recipient dendritic cells (DCs) vaccination, third-party adoptive T-cell transfer and on CMV-reactive Vδ2neg γδT cells expansion. Among these, vaccination with antigen-loaded DCs has been shown to be a potent and versatile immunostimulatory approach and some clinical trials results are already available. This study is aimed at the development of adoptive cellular immunotherapeutic protocols for the production of Advanced Therapy Medicinal Products (ATMPs) for the outcome improvement of patients after HSCT. In particular, we provide standard operating procedures (SOPs) to obtain fully effective and characterized CIK cells ready to use in a clinical setting under good manufacturing practice (GMP)-like conditions. Indeed, the established protocol can be used to compile the Investigational Medicinal Product Dossier (IMPD), the basis for the approval of a clinical trial with investigational new drugs by the competent authorities in the European Union. We report preliminary data on manufacturing of CMV-T and CMV-loaded DCs: we set up experimental procedures to isolate, expand and characterize the immune cells prior to infusion. The main goals of these pre-clinical studies are to determine the safety and potency profiles of the cellular product for its translation to the clinic, in the control of CMV reactivation. This part of the project was largely developed in collaboration with the Department of Internal Medicine II of the University Hospital of Würzburg (Germany), the first in Europe to use cellular therapy for treatment of infections after HSCT. The overall project was lead by the Advanced Cellular Therapy Laboratory (LTCA), part of the Cell Therapy and Hematology Department of the S. Bortolo Hospital, in collaboration with the Bone Marrow Transplant Center and with the Hematology Project Foundation.