Chimeric antigen receptors (CAR) combine an antigenbinding domain with a CD3-Z signaling motif to redirect Tcell specificity to clinically important targets. First-generation CAR, such as the CD19-specific CAR (designated CD19R), may fail to fully engage genetically modified T cells because activation is initiated by antigen-dependent signaling through chimeric CD3-Z, independent of costimulation through accessory molecules. We show that enforced expression of the fulllength costimulatory molecule CD28 in CD8 + CD19R + CD28À T cells can restore fully competent antigen-dependent T-cell activation upon binding CD19 + targets expressing CD80/CD86. Thus, to provide costimulation to T cells through a CD19-specific CAR, independent of binding to CD80/CD86, we developed a second-generation CAR (designated CD19RCD28), which includes a modified chimeric CD28 signaling domain fused to chimeric CD3-Z. CD19R + and CD19RCD28 + CD8 + T cells specifically lyse CD19 + tumor cells. However, the CD19RCD28 + CD8 + T cells proliferate in absence of exogenous recombinant human interleukin-2, produce interleukin-2, propagate, and up-regulate antiapoptotic Bcl-X L after stimulation by CD19 + tumor cells. For the first time, we show in vivo that adoptively transferred CD19RCD28 + T cells show an improved persistence and antitumor effect compared with CD19R + T cells. These data imply that modifications to the CAR can result in improved therapeutic potential of CD19-specific T cells expressing this second-generation CAR. (Cancer Res 2006; 66(22): 10995-1004)
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...
Currently, the lineage-specific cell-surface molecules CD19 and CD20 present on many B-cell malignancies are targets for both antibody-and cell-based therapies. Coupling these two treatment modalities is predicted to improve the antitumor effect, particularly for tumors resistant to single-agent biotherapies. This can be shown using an immunocytokine, composed of a CD20-specific monoclonal antibody fused to biologically active interleukin 2 (IL-2), combined with ex vivo expanded human umbilical cord blood-derived CD8 + T cells, that have been genetically modified to be CD19 specific, for adoptive transfer after allogeneic hematopoietic stem-cell transplantation. We show that a benefit of targeted delivery of recombinant IL-2 by the immunocytokine to the CD19 + CD20 + tumor microenvironment is improved in vivo persistence of the CD19-specific T cells, and this results in an augmented cell-mediated antitumor effect. Phase I trials are under way using anti-CD20-IL-2 immunocytokine and CD19-specific T cells as monotherapies, and our results warrant clinical trials using combination of these two immunotherapies.
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
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