ALBERT

Description: The safety and feasibility of adoptive immunotherapy using ex vivo-expanded differentiated human effector T cells that express tumor-specific chimeric receptors are being evaluated in clinical trials. Typically, these T cells are CCR7neg and bear a T-cell receptor of unknown specificity. To improve the therapeutic potential of genetically engineered T cells in general, and CD19-specific T cells in particular, strategies are needed to improve their ability traffic to sites of residual/macroscopic disease where infused T cells can be specifically activated for proliferation, cytokine secretion, and tumor-lysis. To accomplish these goals we have generated a selection process that uses genetically modified T cells, expressing influenza A matrix protein 1 (MP1) or CMV pp65, to act as antigen presenting cells (T-APC) in order to expand autologous viral-specific T cells in vitro and in vivo. The viral-specific effector T cells can then be genetically modified with a CD19-specific chimeric immunoreceptor (CD19R), which recognizes CD19 on malignant B cells, independent of MHC. By using these viral-specific T cells as a platform for the introduction of CD19R, we now demonstrate that bi-specific T cells express the chemokine receptor CCR7, which is implicated in the trafficking of T cells to lymph nodes. We demonstrate that this chemokine receptor functions to directionally chemotax the genetically modified bi-specific T-cells along concentration gradients of CCL19 or CCL21. We further demonstrate that both the endogenous and introduced chimeric immunoreceptor continue to function in CCR7+ bi-specific T cells. Indeed, the bi-specific T cells are capable of augmented cytokine production and proliferation upon docking with both CD19 and MP1 antigens, compared with these same T cells interacting with either CD19 or MP1 alone. This enhanced activation is an explanation for the enhanced in vivo anti-tumor activity demonstrated by bi-specific T-cells when stimulated with MP1+ T-APC in treating CD19+ lymphoma in NOD/scid mice. An advantage of this methodology is that the CCR7+ bi-specific T cells and T-APC can be genetically modified and expanded in compliance with current good manufacturing practice (cGMP) for 2nd generation Phase I/II clinical trials to test their ability to traffic to sites of lymphoma providing potent regional/local T-cell activation. Legend: (A) CCR7+ viral- and CD19-bi-specific T cells migrate along recombinant CCL19 and CCL21 concentration gradients, whereas CCR7neg CD19-specific T cells do not. (B) Stimulation of both introduced chimeric immunoreceptor and endogenous T-cell receptor on CD19- and MP1- bi-specific T-cells, using artificial APC, results in augmented cytokine production. Figure Figure

Description: Relapse of B-lineage (CD19+) acute lymphoblastic leukemia (ALL) remains a major impediment to the therapeutic success of allogeneic umbilical cord blood transplant (UCBT). The adoptive transfer of donor-derived tumor-specific T-cells is a conceptually attractive means to improve the graft-versus-leukemia-effect at the time of minimal residual disease to improve relapse-rates without exacerbating graft-versus-host-disease. However, adoptive immunotherapy after banked UCBT has been limited by the functional naïveté of neonatal T cells and difficulty obtaining T cells from the unrelated donor. These hurdles can now be overcome by genetically rendering cord blood-derived T cells to be specific for CD19 and expanding the T cells ex vivo from small numbers of cord blood cells, in compliance with current good manufacturing practices for phase I/II trials. To generate T cells that target CD19+ malignant cells, we have used non-viral gene transfer to introduce a DNA plasmid to express a CD19-specific chimeric immunoreceptor, designated CD19R, which binds to cell-surface CD19 via an scFv, independent of MHC, and triggers T-cell activation through CD3- ζ. To safeguard the safety of recipients of adoptive immunotherapy, the DNA plasmid co-expresses the bi-functional hygromycin phosphotransferase/HSV-1 thymidine kinase (HyTK) selection/suicide gene. To assess in vivo the fate of adoptively transferred T cells in mice; a novel tri-functional gene linking firefly luciferase (ffLuc) with HyTK (ffLucHyTK) was generated. The process ex vivo to expand cord blood-derived T cells, which is currently employed at COH in human trials, uses reiterative 14-day additions of OKT3, rhIL-2, cytocidal concentrations of hygromycin, and irradiated peripheral blood mononuclear cells (PBMC) and LCL as feeder cells. The expanded genetically manipulated cord-blood derived T cells express cell-surface markers of differentiated effector cells, similar to the phenotype of CD19-specific T cells derived from PBMC. In vitro the CD19R+HyTK+ cord blood-derived T cells are activated for cytolysis and cytokine production by CD19+ tumor cells. In vivo these genetically modified T cells can be used to eradicate established CD19+ tumors and undergo ganciclovir-mediated ablation, as demonstrated by non-invasive serial imaging of luciferase-mediated bioluminescence (see Figure). These data support a clinical trial to test the safety and feasibility of adoptive transfer of CD19-specific umbilical cord-blood derived T-cells for patients with high risk B-lineage ALL undergoing UCBT. Legend: Non-invasive in vivo biophotonic imaging demonstrates that (A) CD19+ tumor expressing ffLuc gene are eliminated by CD19R+HyTK+ cord-blood derived T cells, and (B) CD19R+ffLuc+HyTK+ cord-blood derived T cells are ablated by ganciclovir. Top row: prior to adoptive immunotherapy or ganciclovir treatment. Bottom row: after adoptive immunotherapy or ganciclovir treatment. Two representative mice are shown. Figure Figure