Relapse of B-lineage acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem cell transplantation (HSCT) commonly results from the failure of a graft-versus-leukemia (GVL) effect to eradicate minimal residual disease. Augmenting the GVL effect by the adoptive transfer of donor-derived B-ALL-specific T-cell clones is a conceptually attractive strategy to decrease relapse rates without exacerbating graft-versus-host disease (GVHD). Toward this end, we investigated whether a genetic engineering approach could render CD8 ؉ cytotoxic T lymphocytes ( 1 (LFA-1), and LFA-3. We observed that recognition of B-lineage tumor lines by CD19-specific CTLs was not impaired by low levels of ICAM-1, LFA-1, and LFA-3 cell surface expression, a functional attribute that is likely a consequence of our high-affinity CD19-specific chimeric immunoreceptor. Furthermore, the CD19-specific CTLs could lyse primary B-ALL blasts. These preclinical observations form the basis for implementing clinical trials using donor-derived CD19-specific T-cell clones to treat or prevent relapse of B-ALL after alloge-

IntroductionAdoptive transfer of ex vivo-expanded T cells specific for immunodominant viral epitopes into immunocompromised hosts can reconstitute protective antiviral immunity and can result in the long-term persistence of transferred cells. [1][2][3][4][5] In contrast, the application of adoptive T-cell transfer to the successful cellular immunotherapy of malignancy has proved to be significantly more challenging, in part because of the difficulty of isolating high-affinity, tumor-specific T cells that can mediate effective antitumor in vivo effector functions and the potential for tumors to evade immunologic clearance through a variety of escape mechanisms, including the down-regulation of restricting HLA molecules. [6][7][8] Several groups, including ours, are developing alternative strategies for targeting tumors using genetically modified T cells that are endowed with redirected antigen specificity through the expression of chimeric antigen receptors (CARs), such as a CD19-specific chimeric immunoreceptor. These chimeras typically use HLA-independent, highaffinity antigen recognition domains consisting of extracellular singlechain immunoglobulin variable fragments (scFvs) linked to cytoplasmic T-cell activation domain(s), such as CD3-. [9][10][11][12][13][14][15][16][17][18][19] Strategies to enhance the antitumor activity of adoptively transferred CAR ϩ cytotoxic T lymphocytes (CTLs) and to overcome the potentially deleterious impact of in vivo recycling of these cells solely through CAR-redirected engagement of tumor cells will likely be critical for achieving therapeutic efficacy. Because CAR-redirected T cells retain the specificity and function of their endogenous T-cell antigen receptor (TcR), expressing CARs on virus-specific T cells, such as commonly acquired latent viruses (Epstein-Barr virus [EBV] and cytomegalovirus [CMV]), is a potential approach to maintain persistence in vivo through re-encounter of these bispecific T cells with viral antigen presented by professional antigen-presenting cells (APCs). 9,20,21 Although the timing and magnitude of latent virus reactivation makes the in vivo restimulation of bispecific T cells difficult to control, we hypothesize that the grafting of antitumor CARs to T cells specific for common nonlatent viruses and the delivery of a viral antigen vaccine boost(s) after adoptive transfer (transfer-boost strategy) is an approach amenable to iatrogenic regulation.Here we describe the usefulness of ex vivo-expanded CD8 ϩ and CD4 ϩ T cells to function as APCs by their genetic modification to express a model viral antigen (influenza A MP1) for eliciting the in vitro expansion of MP1-specific CTLs and for augmenting the clearance of CD19 ϩ Daudi lymphoma in vivo, by CD19ϫMP1-bispecific CTLs by post-transfer boosting. Our finding that human Materials and methods Plasmid expression vectorsThe pMG expression vector (InvivoGen, San Diego, CA) was modified by site-directed mutagenesis to remove a PacI restriction enzyme (RE) site at position 307 to generate pMGP ac ( Fig...

Disease relapse is a barrier to achieving therapeutic success after unrelated umbilical cord-blood transplantation (UCBT) for B-lineage acute lymphoblastic leukemia (B-ALL). While adoptive transfer of donor-derived tumor-specific T cells is a conceptually attractive approach to eliminating residual disease after allogeneic hematopoietic stem cell transplantation, adoptive immunotherapy after UCBT is constrained by the difficulty of generating antigen-specific T cells from functionally naive umbilical cord-blood (UCB)-derived T cells. Therefore, to generate T cells that recognize B-ALL, we have developed a chimeric immunoreceptor to redirect the specificity of T cells for CD19, a B-lineage antigen, and expressed this transgene in UCB-derived T cells. An ex vivo process, which is compliant with current good manufacturing practice for T-cell trials, has been developed to genetically modify and numerically expand UCB-derived T cells into CD19-specific effector cells. These are capable of CD19-restricted cytokine production and cytolysis in vitro, as well as mediating regression of CD19 ؉ tumor and being selectively eliminated in vivo. Moreover, time-lapse microscopy of the genetically modified T-cell clones revealed an ability to lyse CD19 ؉ tumor cells specifically and repetitively. These data provide the rationale for infusing UCB-derived CD19-specific T cells after UCBT to reduce the incidence of CD19 ؉ B-ALL relapse. IntroductionBanked unrelated umbilical cord blood (UCB) is source of hematopoietic stem cells for patients with B-lineage acute lymphoblastic leukemia (B-ALL). 1 However, despite maximally intensive preparative regimens, disease-relapse remains a significant cause of mortality after umbilical cord-blood transplantation (UCBT).Adoptive therapy after allogeneic hematopoietic stem cell transplantation (HSCT) with ex vivo-expanded donor-derived tumor-specific T cells might be used to augment the graft-versusleukemia (GVL)-effect, thereby reducing the incidence of leukemic relapse, without exacerbating graft-versus-host disease (GVHD). 2,3 While the feasibility of isolating, expanding, and infusing antigen-specific ␣␤ T-cell receptor (TCR) ϩ T cells from peripheral blood (PB) has been validated in animals and humans, 4-10 the naive function of neonatal T cells precludes a priori identification of resident tumor-specific T cells. Redirecting the specificity of T cells through enforced expression of antigenspecific immunoreceptors and differentiating UCB-derived T cells into cytotoxic T lymphocytes (CTLs) is one approach to overcoming this lack of endogenous tumor-specific T cells specific for desired targets. 11,12 We and others are developing adoptive immunotherapy platforms using single-chain chimeric immunoreceptors to redirect the specificity of primary human T cells and NK cells for cell-surface proteins expressed on tumor targets, such as the B-lineage-specific antigen CD19, a molecule expressed on normal and neoplastic B cells. [13][14][15][16][17][18][19][20][21][22] These chimeric immunoreceptor...