Genetically Targeted T Cells Eradicate Systemic Acute Lymphoblastic Leukemia Xenografts

Abstract

Purpose: HumanTcells targeted to the B cell^specific CD19 antigen through retroviral-mediated transfer of a chimeric antigen receptor (CAR), termed 19z1, have shown significant but partial in vivo antitumor efficacy in a severe combined immunodeficient (SCID)-Beige systemic human acute lymphoblastic leukemia (NALM-6) tumor model. Here, we investigate the etiologies of treatment failure in this model and design approaches to enhance the efficacy of this adoptive strategy. Experimental Design: A panel of modified CD19-targeted CARs designed to deliver combined activating and costimulatory signals to theT cell was generated and tested in vitro to identify an optimal second-generation CAR. Antitumor efficacy of Tcells expressing this optimal costimulatory CAR, 19-28z, was analyzed in mice bearing systemic costimulatory ligand-deficient NALM-6 tumors. Results: Expression of the 19-28z CAR, containing the signaling domain of the CD28 receptor, enhanced systemic T-cell antitumor activity when compared with 19z1 in treated mice. A treatment schedule of 4 weekly T-cell injections, designed to prolong in vivo T-cell function, further improved long-term survival. Bioluminescent imaging of tumor in treated mice failed to identify a conserved site of tumor relapse, consistent with successful homing by tumor-specific T cells to systemic sites of tumor involvement. Conclusions: Both in vivo costimulation and repeated administration enhance eradication of systemic tumor by genetically targeted T cells. The finding that modifications in CAR design as well asT-cell dosing allowed for the complete eradication of systemic disease affects the design of clinical trials using this treatment strategy.

Ligation of the CD2 co-stimulatory receptor enhances IL-2 production from first-generation chimeric antigen receptor T cells

“…This reduction in IL-2 production was even more pronounced when CD58 was blocked on the Nalm-6 cell line (Figure 2d) potentially due to the lack of CD80/CD86 co-stimulatory molecules expressed by this tumour line. 12 In contrast to mouse T cells, the addition of the CD28-signalling domain into the CAR induced an enhanced level of IL-2 production as compared with aCD19z T --cells, which could be partially reduced through CD58 blockade (Figure 2e). To extend these observations, aCD19z and aCD19CD28z CAR T cells from five donors were co-cultured with Raji or Nalm-6 cell lines in the presence of anti-CD58 or isotype control antibodies.…”

Cytotoxic T cells transduced with chimeric anti-CD19 receptors prevent engraftment of primary lymphoblastic leukemia in vivo

“…4 In chronic lymphocytic leukemia, a disease that can be subdivided into two subgroups based on a SHM threshold of 2%, AICDA expression is found in both subgroups, although more frequently in cases with o2% SHM. 5 We have recently shown that the IGH gene repertoires and SHM patterns of HCL-variant are more similar to that of SMZL than of typical HCL. 6 There has been only one report so far on AICDA mRNA expression in HCL, which found eight of nine cases to express this gene.…”

“…CARs against CD19 have now been generated by various groups and have been demonstrated to mediate potent T-cell effector functions against CD19-expressing malignant cells in vitro and in vivo. 5 Specifically, human T cells endowed with CD19-specific CARs have been shown to effectively eradicate human CD19 þ tumor xenografts in immuno-deficient mouse models. 5 So far, in vivo evidence for the antileukemia activity of CD19-specific T cells has been restricted to established tumor cell lines.…”

“…5 Specifically, human T cells endowed with CD19-specific CARs have been shown to effectively eradicate human CD19 þ tumor xenografts in immuno-deficient mouse models. 5 So far, in vivo evidence for the antileukemia activity of CD19-specific T cells has been restricted to established tumor cell lines. Primary leukemia cells more closely mimic the heterogeneity of the disease in humans and allow to differentiate between individual subgroups with variable biological risk profiles.…”

“…CD19z was generated by cloning the variable domains of the heavy and light chains of the monoclonal anti-CD19 antibody FMC63 (provided by H Zola, Adelaide, South Australia, Australia) in frame with a membranespanning domain consisting of human immunoglobulin constant domains CH2 and CH3, and the transmembrane and cytoplasmic domains of the TCR z chain. Costimulatory domains found to enhance the systemic antitumor activity of non-specifically stimulated, CAR-modified T cells 5 were not included, as previous studies have shown that CAR activity in virus-specific CTLs does not benefit from additional signaling elements. 8 For control experiments, an analogous CAR directed against the neuroectodermal antigen G D2 (14.G2az) was used.…”