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Inclusion of an Igg1-Fc Spacer Abrogates Efficacy of CD19 CAR T

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Inclusion of an Igg1-Fc Spacer Abrogates Efficacy of CD19 CAR T Gene Therapy (2015) 22, 391–403 © 2015 Macmillan Publishers Limited All rights reserved 0969-7128/15 www.nature.com/gt ORIGINAL ARTICLE Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model H Almåsbak1, E Walseng2,3,8, A Kristian4,5,8, MR Myhre1, EM Suso1, LA Munthe6,7, JT Andersen6,MYWang1, G Kvalheim1, G Gaudernack2 and JA Kyte1 Cancer therapy with T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical responses in recent trials, but also severe side effects. Whereas most protocols use permanently reprogrammed T cells, we have developed a platform for transient CAR expression by mRNA electroporation. This approach may be useful for safe clinical testing of novel receptors, or when a temporary treatment period is desirable. Herein, we investigated therapy with transiently redirected T cells in vitro and in a xenograft mouse model. We constructed a series of CD19-specific CARs with different spacers and co-stimulatory domains (CD28, OX40 or CD28-OX40). The CAR constructs all conferred T cells with potent CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti-leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the CH2-domain eradicated leukemia in vivo, without notable toxicity. Follow-up studies demonstrated that the CH2CH3-spacer bound soluble mouse Fcγ-receptor I and mediated off-target T-cell activation towards murine macrophages. Our findings highlight the importance of non-signalling CAR elements and of in vivo studies. Finally, the results show that transiently redirected T cells control leukemia in mice and support the rationale for developing an mRNA-CAR platform. Gene Therapy (2015) 22, 391–403; doi:10.1038/gt.2015.4; published online 5 February 2015 INTRODUCTION electroporation.21–27 The mRNA strategy represents a safer Adoptive T-cell therapy holds considerable promise as a novel alternative for first-in-man studies with novel receptors, and may concept for cancer treatment. Recent studies with T cells also be useful in patients where a limited treatment period is retargeted with chimeric antigen receptors (CARs) against CD19 desirable. We have previously reported a GMP platform for have induced remarkable clinical responses in heavily pre-treated transient redirection of T cells with mRNA encoding the CD19- patients with acute lymphocytic leukemia, chronic lymphocytic specific CAR fmc63-IgG1Fc-CD28-OX40-CD3ζ (19-IgFc-28OXζ).21 1–6 leukemia or B-cell lymphoma. CARs are recombinant receptors We demonstrated that this method resulted in efficient transfec- comprising an extracellular antigen binding domain from a tion of 490% of the cell population, highly uniform CAR monoclonal antibody and signalling domains from the T-cell expression and potent killing of CD19+ cell lines, primary leukemia 7 receptor (TCR) complex. The second generation CAR constructs cells and lymphoma cells. include a signalling domain from a co-stimulatory molecule (for Here, we report in vitro and in vivo studies of a series of fmc63- example, CD28, 41BB or OX40), and third generation CARs 8–11 based CARs, using a xenograft model of NOD-SCID gamma-c comprise two co-stimulatory domains. knockout (NSG) mice inoculated with the human leukemia cell line The striking clinical responses achieved with CD19 CAR T cells NALM-6. We unexpectedly observed that T cells transfected with have provoked a surge of interest in developing CARs and TCRs 21 our originally preferred 19-IgFc-28OXζ CAR construct exerted against new targets. Yet, serious toxicity observed in several trials only marginal effect on the tumor growth in vivo, in spite of the has highlighted the need to develop safer strategies for clinical 4,6,12–17 strong in vitro activity. Moreover, this construct caused serious testing. Most investigators have used viral vectors perma- fi nently integrating the receptor sequence into the T-cell genome. toxicity. On the basis of this surprising nding, we conducted a fl This approach may give durable anti-cancer activity after a single series of studies evaluating the in uence of alternative CAR T-cell infusion, but also represents a safety risk. As the gene- signalling domains and spacer variants. The results attribute the modified T cells persist and expand in the patient, serious adverse striking discrepancy between in vitro and in vivo activity to the events may develop.13–15,17 Insertional mutagenesis also remains a inclusion of a commonly used IgG1-based CAR spacer. Further, we possible concern, though not materializing in clinical trials so demonstrate the ability of T cells transiently expressing optimized far.18–20 To circumvent these issues, we and others are developing CD19 CARs to effectively eradicate leukemia in a mouse xenograft methods for transient CAR/TCR expression based on mRNA model, without detectable toxicity. 1Section for Cell Therapy, Department of Oncology, Oslo University Hospital Radiumhospitalet, Oslo, Norway; 2Department of Immunology, Oslo University Hospital Radiumhospitalet, Oslo, Norway; 3Department of Cancer Biology, Scripps Research Institute, Jupiter, FL, USA; 4Department of Tumor Biology, Oslo University Hospital Radiumhospitalet, Oslo, Norway; 5KG Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, University of Oslo, Oslo, Norway; 6Centre for Immune Regulation and Department of Immunology, Oslo University Hospital Rikshospitalet, Oslo, Norway and 7Institute of Clinical Medicine, University of Oslo, Oslo, Norway. Correspondence: Dr JA Kyte, Section for Cell Therapy, Department of Oncology, Oslo University Hospital Radiumhospitalet, Mail Box 4950 Nydalen, Oslo, 0424, Norway. E-mail: [email protected] 8These authors contributed equally to this work. Received 28 June 2014; revised 3 November 2014; accepted 6 January 2015; published online 5 February 2015 IgG1 spacer abrogates CAR efficacy in vivo H Almåsbak et al 392 RESULTS differences between the IL-2 +/ − groups (P = 0.43, t-test). Three days CAR19-IgFc-28OXζ T cells failed to control leukemia in vivo and after T-cell transfer, the study animals started to regain weight. The caused serious toxicity results suggested that there was a considerable component of off- On the basis of our promising in vitro studies of a third generation target toxicity and that this toxicity could not be avoided by 19-IgFc-28OXζ CAR,21 we conducted an in vivo study evaluating omitting adjuvant IL-2. this CAR construct in NSG mice challenged with the NALM-6 cell line. Transiently redirected T cells (mRNA electroporated) were Superior in vivo functionality of a second generation CAR with compared with constitutively redirected T cells (retroviral trans- short spacer duction). Separate series with high or low tumor load were set up We next asked whether T cells expressing lower densities of the to model advanced disease and minimal residual disease (MRD), 19-IgFc-28OXζ CAR could retain killing capacity while causing less respectively (Figures 1a and b). Control groups of mice were toxicity. The mRNA technology allows for controlling CAR treated with mock-transfected T cells (mock T cells) and IL-2. Flow expression levels by adjusting the concentration of mRNA at cytometry analysis showed CAR expression in 68% of the stably electroporation.21 We compared T cells expressing high (CARhigh; transduced T cells and 495% of the mRNA-transfected T cells 100 μg mRNA ml − 1) and low (CARlow;40μg mRNA ml − 1) CAR (data not shown). surface levels. Further, we compared with a second generation In the advanced disease model, bioluminescence imaging on CD19 CAR without OX40 and with a minimal spacer replacing the day 4 (before T-cell injection) indicated similar leukemia progres- IgG1Fc domain (19-short-28ζ) (Supplementary Figure 1a). The sion in the CAR T-cell-treated groups (transient or stable CAR surface CAR expression correlated well with the mRNA concentra- expression) (Figure 1c). Strikingly, the bioluminescence signals one tions at electroporation (Supplementary Figure 1b). In vitro day after T cell injection were significantly lower in control mice functionality assays showed that T cells expressing the different receiving mock T cells, compared with the animals treated with CAR constructs (19-IgFc-28OXζ or 19-short-28ζ) responded with CAR-expressing cells (P = 0.002, analysis of variance/Student New- similar levels of interferon (IFN)-γ production and degranulation man Keuls (ANOVA/SNK)). Three days after T cell injection, we (Supplementary Figure 1c). This applied both to CD4+ and CD8+ could readily detect T cells in mice receiving mock T cells, whereas T cells. Further, we observed that a fraction of the mock- no remaining T cells could be detected in mice treated with CAR transfected T cells displayed activity against the ff-Luc-NALM-6 T cells (data not shown). All CAR T-cell-treated mice developed (Supplementary Figure 1c). ⩾ 15% decrease in body weight and signs of distress, including We injected 24 NSG mice with 1 × 106 ff-Luc-NALM-6 cells at day ruffled fur, shivering and lethargy. This required euthanasia 0 (Figure 2a). Subsequently, three mouse groups were treated with according to internal guidelines. By contrast, the mice treated CAR T cells expressing either (i) 19-IgFc-28OXζ (H), (ii) 19-IgFc-28OXζ with mock T cells appeared unaffected. (L) or (iii) 19-short-28ζ (H). Leukemic control mice were treated with The transfer of CAR T cells was also accompanied by severe mock-transfected T cells or no T cells. Further, healthy mice received toxicity in the MRD model. However, after an initial weight loss, 19-IgFc-28OXζ (H) or 19-short-28ζ (H) CAR T cells. The biolumines- four out of five mice treated with CAR mRNA-redirected T cells cence data showed that mock T cells exerted a moderate anti- recovered and survived (Figure 1d). In contrast, all mice treated leukemia activity, compared with the leukemic controls not treated with virally transduced T cells had to be killed within 1 week with T cells (Figures 2b and c; Po0.05, ANOVA/SNK).
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