scholarly journals In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

10.3791/59275 ◽  
2019 ◽  
Author(s):  
Dongrui Wang ◽  
Renate Starr ◽  
Darya Alizadeh ◽  
Xin Yang ◽  
Stephen J. Forman ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor

scholarly journals 112 Tumor-specific reactivity and effector function of chimeric antigen receptor engineered macrophages targeting MUC1

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A122-A122
Author(s):  
Seth Eisenberg ◽  
Amy Powers ◽  
Jason Lohmueller ◽  
James Luketich ◽  
Rajeev Dhupar ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cancer Vaccine ◽  
Chimeric Antigen Receptor ◽  
Effector Function ◽  
Specific Antibodies ◽  
The University

BackgroundChimeric antigen receptors (CAR) have demonstrated remarkable efficacy in licensing T cells for antitumor responses against hematopoietic malignancies but have had limited success against solid tumors. Macrophages, both archetypic phagocytes and professional antigen presenting cells, may exert profound effector functions which complement adaptive cellular immunity.1 Recently, it was shown that human macrophages engineered to express CARs (CAR-Ms) demonstrated antigen-specific phagocytosis, inhibited solid xenograph tumors, and induced an inflammatory tumor microenvironment boosting antitumor T cell responses.2 Kimura et al. previously completed the first prophylactic cancer vaccine trial based on a non-viral antigen, tumor-associated hypoglycosylated Mucin 1 (MUC1).3 A panel of fully-human affinity-matured MUC1-specific antibodies raised in healthy subjects following immunization was identified from these patients.4 Using these MUC1-specific scFv domains for CAR generation, we have now engineered MUC1-targeting CAR-Ms that may potentially possess reduced off-target specificities.MethodsLentiviral CAR expression vectors containing the scFv domains of three unique hypoglycosylated MUC1-specific antibodies or a CD20-specific antibody, the CD3zeta signaling domain, and CD28 and OX40 co-stimulatory domains were constructed. The human monocyte/macrophage U937, SC, and THP-1 lines were stably transduced and flow-sort purified to generate MUC1- or CD20-specific CAR-Ms. CAR-Ms were differentiated into macrophages via 48 hour PMA treatment, and subsequently evaluated for antigen-specific function against MUC1- and/or CD20-expressing K562, ZR-75-1, and Raji cells or cancer cells isolated from solid lung tumors or malignant pleural effusions. CAR-M phenotype was evaluated by flow cytometry following in vitro differentiation and polarization with conventional ‘M1’ and ‘M2’ stimuli. Phagocytosis and lysosomal processing of phagocytosed cargo were evaluated by fluorescence microscopy of GFP/CellTrace labeled targets or detection of pH-sensitive pHrodo expression following CAR-M and tumor cell co-culture, respectively. Antigen-specific cytokine production was determined via cytometric bead array following co-culture of CAR-Ms with MUC1- or CD20-expressing tumor cells or 100mer MUC1 peptide.ResultsDifferentiated CAR-Ms possessed an inflammatory phenotype expressing IL-8 and CD86 which was further enhanced by IFNgamma or LPS treatment and was resistant to ‘M2’ polarization with conventional stimuli. CAR-Ms exhibited phagocytosis and subsequent lysosomal processing in an antigen-specific manner, with minimal reactivity against tumor cell targets in the absence of the corresponding MUC1 or CD20 antigen. MUC1-specific CAR-Ms stimulated with MUC1 peptide or MUC1+ tumor cells secreted robust levels of pro-inflammatory IL-8, TNFa, and IL-1beta, but not immunosuppressive IL-10.ConclusionsMUC1-targeting CAR-Ms exert potent tumor-restricted effector function in vitro and may provide a novel treatment strategy either alone or in potential synergistic combination with T cell-mediated immunotherapies.AcknowledgementsThe authors would like to thank Dr. Olivera J. Finn for generously providing reagents and guidance and Dr. Michael T. Lotze for his mentorship. This study was supported by funding from the University of Pittsburgh’s Department of Cardiothoracic Surgery to ACS and RD.ReferencesWilliams CB, Yeh ES, Soloff AC. Tumor-associated macrophages: unwitting accomplices in breast cancer malignancy. Npj Breast Cancer [Internet]. Breast Cancer Research Foundation/Macmillan Publishers Limited; 2016;2:15025. Available from: http://dx.doi.org/10.1038/npjbcancer.2015.25Klichinsky M, Ruella M, Shestova O, Lu XM, Best A, Zeeman M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38:947–53.Kimura T, McKolanis JR, Dzubinski LA, Islam K, Potter DM, Salazar AM, et al. MUC1 Vaccine for Individuals with Advanced Adenoma of the Colon: A Cancer Immunoprevention Feasibility Study. Cancer Prev Res [Internet] 2013;6:18–26. Available from: http://cancerpreventionresearch.aacrjournals.org/content/6/1/18.abstractLohmueller JJ, Sato S, Popova L, Chu IM, Tucker MA, Barberena R, et al. Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential. Sci Rep 2016;6:31740.Ethics ApprovalThe study was approved by the University of Pittsburgh’s Institutional Review Board approval number CR19120172-005.

scholarly journals Evaluation of chimeric antigen receptor T cell therapy in non-human primates infected with SHIV or SIV

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248973
Author(s):  
Nami Iwamoto ◽  
Bhavik Patel ◽  
Kaimei Song ◽  
Rosemarie Mason ◽  
Sara Bolivar-Wagers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Chimeric Antigen Receptor ◽  
Viral Suppression ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics.

Inhibition of Interleukin-10 in the tumor microenvironment can restore mesothelin chimeric antigen receptor T cell activity in pancreatic cancer in vitro

Surgery ◽  
2018 ◽  
Vol 163 (3) ◽  
pp. 627-632 ◽  
Author(s):  
Ramesh B. Batchu ◽  
Oksana V. Gruzdyn ◽  
Ebrahem M. Mahmud ◽  
Fatme Chukr ◽  
Rajesh Dachepalli ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Chimeric Antigen Receptor ◽  
Cell Activity ◽  
Antigen Receptor ◽  
Interleukin 10

scholarly journals HIV-1-Specific Chimeric Antigen Receptor T Cells Fail To Recognize and Eliminate the Follicular Dendritic Cell HIV Reservoir In Vitro

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Matthew T. Ollerton ◽  
Edward A. Berger ◽  
Elizabeth Connick ◽  
Gregory F. Burton
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Follicular Dendritic

ABSTRACT The major obstacle to a cure for HIV infection is the persistence of replication-competent viral reservoirs during antiretroviral therapy. HIV-specific chimeric antigen receptor (CAR) T cells have been developed to target latently infected CD4+ T cells that express virus either spontaneously or after intentional latency reversal. Whether HIV-specific CAR-T cells can recognize and eliminate the follicular dendritic cell (FDC) reservoir of HIV-bound immune complexes (ICs) is unknown. We created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a CAR construct that enables the expression of CD4 (domains 1 and 2) and the carbohydrate recognition domain of mannose binding lectin (MBL) to target native HIV Env (CD4-MBL CAR). We assessed CAR-T cell cytotoxicity using a carboxyfluorescein succinimidyl ester (CFSE) release assay and evaluated CAR-T cell activation through interferon gamma (IFN-γ) production and CD107a membrane accumulation by flow cytometry. CD4-MBL CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells but were ineffective at targeting FDC bearing HIV-ICs. CD4-MBL CAR-T cells were unresponsive to cell-free HIV or concentrated, immobilized HIV-ICs in cell-free experiments. Blocking intercellular adhesion molecule-1 (ICAM-1) inhibited the cytolytic response of CD4-MBL CAR-T cells to Env-expressing cell lines and HIV-infected CD4+ T cells, suggesting that factors such as adhesion molecules are necessary for the stabilization of the CAR-Env interaction to elicit a cytotoxic response. Thus, CD4-MBL CAR-T cells are unable to eliminate the FDC-associated HIV reservoir, and alternative strategies to eradicate this reservoir must be sought. IMPORTANCE Efforts to cure HIV infection have focused primarily on the elimination of latently infected CD4+ T cells. Few studies have addressed the unique reservoir of infectious HIV that exists on follicular dendritic cells (FDCs), persists in vivo during antiretroviral therapy, and likely contributes to viral rebound upon cessation of antiretroviral therapy. We assessed the efficacy of a novel HIV-specific chimeric antigen receptor (CAR) T cell to target both HIV-infected CD4+ T cells and the FDC reservoir in vitro. Although CAR-T cells eliminated CD4+ T cells that express HIV, they did not respond to or eliminate FDC bound to HIV. These findings reveal a fundamental limitation to CAR-T cell therapy to eradicate HIV.

Development of human NKG2D-CD3ε chimeric antigen receptor (CAR) for T-cell-mediated cancer immunotherapy.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 150-150
Author(s):  
Sergei Kusmartsev ◽  
Johaness Vieweg ◽  
Victor Prima
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Adaptor Protein ◽  
T Cell Subsets ◽  
Human T Cells

150 Background: NKG2D is a lectin-like type 2 transmembrane receptor that expressed by natural killer cells and some T cell subsets. Stimulation of NKG2D receptor with specific agonistic ligands produces activating signals through signaling adaptor protein DAP10 leading to the enhanced cytokine production, proliferation, and cytotoxicity against tumor cells. There is strong evidence that NKG2D ligands are expressed in many human tumors, including melanoma, leukemia, myeloma, glioma, and carcinomas of the prostate, breast, lung, and colon. Recent studies also demonstrated that T cells bearing chimeric antigen receptor (CAR) NKG2D linked to CD3ζ (zeta) chain produce marked in vitro and in vivo anti-tumor effects. The aim of current study was to determine whether human T cells bearing chimeric antigen receptor (CAR) NKGD2 linked to CD3ε (epsilon) chain could be activated by the NKG2D-specific stimulation and able to kill human cancer cells. Given the important role of CD3ε in activation and survival of T cells, we hypothesized that NKG2D-CDε-bearing T cells could exert strong in vitro and in vivo anti-tumor effects. Methods: NKG2D CAR was produced by linking human NKG2D to DAP10 and the cytoplasmic portion of the CD3ε chain. Original full-length human cDNA clones were obtained from NIH Mammalian Gene Collection (MGC). Functional domain analysis and oligonucleotide design in the in-Fusion system of DNA cloning (Clontech) was used to generate the retroviral expression constructs. Results: Human PBMC-derived T cells were retrovirally transduced with newly generated NKG2D-CD3ε CAR DNA construct. These NKG2D CAR-expressing human T cells responded to NKG2D-specific activation by producing IFN-γ and exhibited significant cellular cytotoxicity against human tumor cells in vitro. In vivo studies demonstrated that NKG2D-CD3ε-bearing cells are capable of inhibiting growth of DU-145 human prostate cancer in the immunodeficient mice. Conclusions: Collectively, our data indicate the feasibility of developing chimeric antigen receptor NKG2D-CD3ε for T cells and suggest that adoptive transfer of T cells bearing NKG2D-CD3ε CAR could be potentially effective for immunotherapy of cancer patients.

Novel media formulations to enhance Chimeric Antigen Receptor (CAR) T-cell potency and anti-tumor cell function for adoptive immunotherapy

Cytotherapy ◽  
2020 ◽  
Vol 22 (5) ◽  
pp. S133
Author(s):  
S. Ghassemi ◽  
F. Martinez-Becerra ◽  
A. Master ◽  
S.A. Richman ◽  
D. Heo ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Cell ◽  
Chimeric Antigen Receptor ◽  
Adoptive Immunotherapy ◽  
Cell Function ◽  
Antigen Receptor ◽  
Car T Cell ◽  
Car T

scholarly journals In Vitro-Transcribed mRNA Chimeric Antigen Receptor T Cell (IVT mRNA CAR T) Therapy in Hematologic and Solid Tumor Management: A Preclinical Update

2020 ◽  
Vol 21 (18) ◽  
pp. 6514
Author(s):  
Thangavelu Soundara Rajan ◽  
Agnese Gugliandolo ◽  
Placido Bramanti ◽  
Emanuela Mazzon
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Chimeric Antigen Receptor ◽  
Solid Tumor ◽  
Antigen Receptor ◽  
Hematologic Malignancies ◽  
Car T ◽  
Tumor Management

Adoptive T cell immunotherapy has received considerable interest in the treatment of cancer. In recent years, chimeric antigen receptor T cell (CAR T) therapy has emerged as a promising therapy in cancer treatment. In CAR T therapy, T cells from the patients are collected, reprogrammed genetically against tumor antigens, and reintroduced into the patients to trigger an immense immune response against cancer cells. CAR T therapy is successful in hematologic malignancies; however, in solid tumors, CAR T therapy faces multiple challenges, including the on-target off-tumor phenomenon, as most of the tumor-associated antigens are expressed in normal cells as well. Consequently, a transient in vitro-transcribed anti-mRNA-based CAR T cell (IVT mRNA CAR T) approach has been investigated to produce controlled cytotoxicity for a limited duration to avoid any undesirable effects in patients. In vitro and in vivo studies demonstrated the therapeutic ability of mRNA-engineered T cells in solid tumors, including melanoma, neuroblastoma and ovarian cancer; however, very few clinical trials are registered. In the present review, we discuss the effect of IVT mRNA CAR T therapy in preclinical studies related to hematologic malignancies and solid tumor management. In addition, we discuss the clinical trial studies based on IVT mRNA CAR T therapy in cancer.

scholarly journals Bigger, Stronger, Faster: Chimeric Antigen Receptor T Cells Are Olympic Killers

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 814-814 ◽  
Author(s):  
Paul J Neeson ◽  
Alexander James Davenport ◽  
Joseph A Trapani ◽  
Michael Kershaw ◽  
Ryan Cross ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Time Lapse ◽  
Immune Synapse ◽  
Car T Cells ◽  
Car T

Abstract Chimeric antigen receptor T cells (CAR T) re-directed to CD19, have induced remarkable responses in clinical trials for patients with B-cell malignancies. Patients have responded to therapy with a CAR T dose which is a fraction of the pre-existing tumor burden. Explanations for this observation include studies which show the proliferative potential of the CAR T cells (Kalos M et al Sci Transl Med 2011), as well as our recent study which showed that individual CAR T cells can serial kill tumor cells (Davenport AJ et al CIR 2015, Figure 6). Using our dual antigen receptor model (OT-I T cell receptor and 2nd generation HER-2 CAR in the same T cell, termed CAR.OTI cells), we also observed a reproducible and significantly shorter time from CAR- vs TCR-mediated activation to detachment from dying tumor cells (Davenport AJ et al CIR 2015, Figure 4D)suggesting that CAR-mediated individual killing events are actually faster. To explore how this may occur, we examined the immune synapse structure at 20 minutes of CAR.OTI CTL co-culture with tumor cells expressing the cognate antigen for either the CAR or TCR. At this timepoint, CAR.OTI CTL, activated via the TCR, formed a conventional bull's eye immune synapse with accumulation of LCK and actin clearance (Figure 1). Surprisingly, CAR.OTI CTL activated via their CAR had an immune synapse with no or diffuse LCK and small actin rings (Figure 1). At the same timepoint, CAR-activated CAR.OTI CTL conjugates with tumor cells were characterized by a microtubule organizing center (MTOC) distant from the immune synapse LCK accumulation. In contrast TCR activated CAR.OTI CTL conjugates consistently had the MTOC proximal to the LCK accumulation (Figure 2A). Despite this, CAR-mediated CAR.OTI CTL killing of tumor targets was inhibited by a protein kinase C zeta inhibitor and is, therefore, MTOC dependent (Figure 2B). The MTOC circumnavigates the activated CTL nucleus and moves to the immune synapse, bringing cytotoxic granules with it. Using time lapse live video (TLLV) microscopy, we compared CAR.OTI CTL cytotoxic granule movement when the CAR.OTI were activated via the TCR vs CAR. We showed that following CAR vs TCR activation, CAR.OTI cytotoxic granules moved with a significantly higher velocity, and had a shorter time lapse to reach the immune synapse following activation (Ca2+ flux), and a significantly shorter time to detachment from the dying tumor cell (Figure 2C). We then re-examined immune synapse formation at an earlier timepoint, to explore whether the data from Figures 1-2 could be explained by a more rapid CTL response following CAR-mediated signaling. In contrast to our observations at 20 minutes, at five minutes we showed CAR-activated CAR.OTI CTL formed conjugates with tumor targets and the immune synapse showed distinct LCK accumulation and actin clearance (Figure 3A). Finally, we explored CAR.OTI CTL signaling and showed that CAR-mediated activation induced a significantly lower number of Ca2+ fluxes, however each Ca2+ flux amplitude was not different (Figure 3B). We also examined changes in proximal (phospho-LCK, pLCK) and distal (phospho-ERK, pERK) signals in CAR- versus TCR- activated CAR.OTI cells, and showed that CAR-mediated activation induced more rapid proximal and distal activation signals (Figure 3C). In conclusion, this study showed that compared to activation by TCR ligation, T cells respond to CAR ligation with faster phospho-protein signaling, Ca2+ flux, formation of an immune synapse and a more rapid movement of the MTOC and delivery of the cytotoxic granules to kill the tumor cells. Furthermore, LFA-1 did not accumulate at the immune synapse following CAR activation, therefore, reduced adhesion may facilitate the observed rapid detachment from the dying tumor cell, and enable the CAR T to rapidly move onto the next tumor target for 'bigger, stronger, faster' killing. Disclosures No relevant conflicts of interest to declare.

Dual Transgenesis of T Cells with a Novel CD44v6-Specific Chimeric Antigen Receptor and a Suicide Gene for Safe and Effective Targeting of Chemoresistance in Hematopoietic Tumors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3125-3125
Author(s):  
Monica Casucci ◽  
Laura Falcone ◽  
Benedetta Nicolis di Robilant ◽  
Barbara Camisa ◽  
Pietro Genovese ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antitumor Effect ◽  
Antigen Receptor ◽  
Suicide Gene ◽  
Nsg Mice ◽  
Donor T Cells

Abstract Abstract 3125 Background: We have previously demonstrated that the genetic induction of a conditional suicidal phenotype in donor T cells allows for an operational dissociation of the GVL effect from GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, leukemia often escapes the immunological pressure of alloreactive donor T cells by losing “passenger” mismatched HLAs. Conversely, redirecting T cells against a non HLA-restricted antigen critically involved in the neoplastic phenotype may circumvent tumor escape due to the emergence of antigen-loss variants. The isoform variant 6 of CD44 is expressed by different epithelial and hematological cancers, and is possibly involved in tumor-cell survival and proliferation. Clinical experience with chemo-conjugated CD44v6-specific mAbs in epithelial tumors showed substantial efficacy, which was however limited by skin toxicity due to background expression of CD44v6 on keratinocytes Aim: By analogy with our experience in HSCT, we reasoned that CD44v6 targeting with suicide gene-modified T cells would provide a major therapeutic effect against hematological tumors, while granting a safety switch in case of toxicity. To this aim, we designed a novel CD44v6-specific chimeric antigen receptor (CAR) and developed a strategy for its co-expression with a suicide gene Results: CD44v6 expression by FACS was observed at high levels in 6/17 (37%) cases of acute myeloid leukemia (AML), but did not associate with enhanced leukemia initiation after infusion into NSG mice (83% vs 88%). In all cases, however, AML cells isolated from the bone marrow (BM) of engrafting mice were brightly positive for CD44v6, suggesting in vivo regulation by microenvironmental factors. In vitro, co-culturing primary AML cells with human BM-derived mesenchymal stromal cells (MSCs) caused a selective up-regulation of CD44v6 (P<0.01). The phenomenon was causally linked to MSC-induced acquisition of resistance to the chemotherapeutic agents daunorubicin and ara-c, as demonstrated by lentiviral vector (LV)-assisted short-hairpin (sh) RNA interference. At difference with AML, CD44v6 was constitutively expressed in 9/11 (81%) cases of multiple myeloma (MM). Knocking-down CD44v6 by shRNA interference significantly increased baseline sensitivity of MM cells to bortezomib. After validating CD44v6 as a common target of chemoresistant AML and MM cells, we generated a 2G CAR by cloning the scFv of a humanized CD44v6-specific mAb in a CD28/TCR zeta chain backbone and expressed it along with the Herpes simplex virus thymidine kinase (HSV-tk) suicide gene by means of a LV carrying a bi-directional promoter. After LV transduction, primary T cells concomitantly acquired CD44v6-specific in vitro cytotoxicty against autologous AML and MM cells, and a selective sensitivity to the suicide gene-activating prodrug ganciclovir. CD44v6-specific recognition associated with T-cell proliferation, IL-2 and IFN-gamma production, and complete clearance of AML cells in a BM-niche model with MSCs at very low E :T ratios (1:5–1:10). Interestingly, in the same model CD34+CD38- healthy cells were not eliminated by CD44v6-redirected T cells, consistently with stable lack of CD44v6 surface expression on healthy HSC. Once infused into NSG mice, CD44v6-redirected T cells had a major antitumor effect against previously engrafted CD44v6-positive AML and MM cell lines (THP1, P<0.001 and MM1.S, P<0.05 vs control CAR, respectively) and against autologous primary AML cells (P<0.005). Since the performance of the suicide gene is felt to be critical for controlling possible toxicities of CAR-redirected T cells, we also evaluated and inducible form of caspase 9 (icasp9) as a possible alternative to HSV-tk. Casp9 activation by its prodrug (the AP1903 dimerizer) permitted efficient CD44v6-redirected T-cell elimination even in the absence of cell proliferation and with a much faster kinetics than HSV-tk (>90% elimination: 18 hrs vs 112 hrs average, P<0.005) Conclusions: We demonstrated that LV-mediated dual transgenesis of primary human T cells with a novel CD44v6-specific CAR and a suicide gene is feasible, results into a powerful antitumor effect against chemoresistant AML and MM cells, and enables effective T-cell ablation in case of toxicity. The premise that suicide gene-modified CAR-redirected T cells can widen the therapeutic index of CD44v6 targeting awaits clinical confirmation Disclosures: Bordignon: Molmed SpA: Employment. Bonini:MolMed: Consultancy.

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