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Targeting leukemia using an inducible universal chimeric antigen receptor (UniCAR) T cell technology
Oral Abstracts
5 TARGETING LEUKEMIA USING AN INDUCIBLE UNIVERSAL CHIMERIC ANTIGEN RECEPTOR (UNICAR) T CELL TECHNOLOGY J. Dietrich1, S. Loff2, J. Meyer1, M. von Bonin3, M. Hetzenecker4, J. Spehr1, C. Gründer2, J. Riewaldt1, G. Ehninger3, M.P. Bachmann4,5, A. Ehninger2, M. Cartellieri1 1 Cellex Patient Treatment GmbH, Dresden, Germany, 2Gemoab Monoclonals GmbH, Dresden, Germany, 3Medical Clinic and Policlinic I, University Hospital Carl Gustav Carus, Dresden, Germany, 4 Tumorimmunology, University Cancer Center (UCC), Medical Faculty Carl Gustav Carus, TU Dresden, Dresden, Germany, 5Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany T cells genetically engineered to express chimeric antigen receptors (CARs) represent a promising approach in cancer immunotherapy. CARs consist of an extracellular binding moiety providing antigen specificity and an intracellular signaling domain derived from an activating immune receptor. Both are linked via a hinge and transmembrane domain. Introducing co-stimulatory domains, such as CD28 or CD137, can enhance signaling. Although, CAR T cells showed encouraging clinical results in patients with B cell malignancies, major drawbacks include on-target off-tumor effects or severe cytokine release syndrome (sCRS). A universal CAR (UniCAR) platform recently introduced by us is a novel approach to gain clinical safety whilst, at the same time, maintaining efficacy of CAR T cell therapy. The new CAR technology splits antigen-binding specificity and signaling aspects in two separate entities. Antigen-specificity is provided by soluble peptide linkers (targeting modules, TMs), consisting of an antigenspecific binding moiety fused to a non-immunogenic peptide motif, not present on healthy human tissues, and specifically recognized by UniCAR engineered T cells. Our results demonstrate that UniCAR driven T cell reactivity against tumor cells is strictly dependent on the presence of antigen-specific TMs and the corresponding antigen on target cells. Applying TMs binding CD19 and CD123 we can demonstrate high efficient anti-leukemic effects of UniCAR T cells both in vitro and in vivo including primary AML and ALL samples. Moreover, architecture of the receptor framework plays an important role for efficacy. Hence, T cells engineered with CD28-derived hinge and signaling domain showed superior function compared to T cells engrafted with UniCARs composite of CD8α hinge with CD137 co-stimulation. In conclusion, the modular composition of the UniCAR maintains high anti-tumor specificity of CAR T cells while adding better control mechanisms and interchangeable target flexibility.
6 DIRECT COMPARISON OF IN VIVO FATE OF SECOND AND THIRD-GENERATION CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELLS IN PATIENTS WITH B CELL NONHODGKIN LYMPHOMA (B-NHL): REVERSAL OF TOXICITY FROM TONIC SIGNALING D. Gomes da Silva1, M. Mukherjee1, S. Madhuwanti1, O. Dakhova1, H. Liu1, B. Grilley1, A. Gee1, S.S. Neelapu2, C. Rooney1, H. Heslop1, B. Savoldo1, G. Dotti1, M. Brenner1, M. Mamonkin1, C.A. Ramos1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital and Texas Children’s Hospital, Houston, Texas, United States, 2Department of Lymphoma/Myeloma, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States Although 2nd-generation (2G) CD19-specific CARs containing CD28 or 4-1BB costimulatory endodomains (CE) show remarkable efficacy against B-NHL, the optimal choice of CEs in these and other CARs remains controversial. Individual CEs, such as CD28 may be associated with deleterious ligandindependent tonic signaling in the transduced T cell, but it is unclear if tonic 4-1BB signaling may have such consequences as well, and if such effects can be reversed. We therefore modeled tonic CAR signaling in T cells by transducing them with gammaretroviral vectors expressing 2G CD19.CAR constructs containing CD3-ζ and either the CD28 or 4-1BB CEs. 4-1BB CD19.CAR-T cells (CARTs) expanded 70% slower, which was coupled with a 4-fold increase in apoptosis and a gradual downregulation of CAR expression. Because of the toxicity of 4-1BB in our CAR construct, we could not directly compare the in vivo fate of 4-1BB CD19.CARTs with that of CD28 CD19.CARTs. We found,
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however, that the 4-1BB toxicity could be overcome in a 3rd-generation (3G) CD19.CAR vector containing both CD28 and 4-1BB. We thus compared the fate of that 3G vector with the 2G vector containing CD28 alone. To determine whether CD28 alone was optimal (which would suggest 4-1BB is antagonistic) or whether 4-1BB had an additive or synergistic effect contributing to superior expansion of the CD28-41BB combination, 8 relapsed/refractory B-NHL patients were simultaneously infused with 1–20 × 106 of both 2G and 3G CARTs/m2 48–72 hours after lymphodepletion with cyclophosphamide (500 mg/m2/d) and fludarabine (30 mg/m2/d) × 3. Persistence of infused T cells was assessed in blood by qPCR assays specific for each CAR. Molecular signals peaked approximately 2 weeks post infusion, remaining detectable for up to 6 months. The 3G CARTs had a mean 23-fold (range 1.1 to 109-fold) higher expansion than 2G CARTs and longer persistence. Two patients had grade 2 cytokine release syndrome at the time of peak expansion. Of the 6 patients evaluable for response, 2 entered complete remission (the longest ongoing for 1 year), 1 has had continued complete remission after ASCT, 1 had a partial response, and 2 progressed. In conclusion, our data indicate that infusion of T cells carrying a CD19.CAR containing CD28 and 4-1BB endodomains is safe and can have efficacy at every dose level tested. Additionally, in a side-by-side comparison, the 3G vector produced greater in vivo expansion than an otherwise identical CART population with CD28 alone.
7 HUMANIZED CHIMERIC ANTIGEN RECEPTOR (CAR)-MODIFIED T CELLS TARGETING CD19 INDUCE REMISSIONS IN CHILDREN AND YOUNG ADULTS WITH RELAPSED/REFRACTORY LYMPHOBLASTIC LEUKEMIA/LYMPHOMA G.E. Hucks1, D. Barrett1, S.R. Rheingold1, R. Aplenc1, D.T. Teachey1, C. Callahan1, D. Baniewicz1, C. White1, M. Talekar1, P. Shaw2, J.L. Brogdon3, R. Young2, J. Scholler2, K. Marcucci2, A. Chew2, B.L. Levine2, N. Frey2, D. Porter2, S. Lacey2, J. Melenhorst2, C. June2, S. Grupp1, S. Maude1 1 Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States, 2University of Pennsylvania, Philadelphia, Pennsylvania, United States, 3Novartis Institute for Biomedical Research, Cambridge, Massachusetts, United States Background: Chimeric antigen receptor (CAR)-modified T cells targeting CD19 can produce potent and sustained responses in children with relapsed/ refractory (r/r) acute lymphoblastic leukemia (ALL). However, limited persistence, seen in a subset of patients, can increase the risk of relapse. Anti-mouse reactivity is one potential cause of poor persistence that may be overcome by fully human or humanized CAR designs. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19-directed CAR T cells (CTL119). Design: A pilot/phase 1 study of CTL119 enrolled children and young adults with r/r B lymphoblastic leukemia/lymphoma with or without prior exposure to a CAR T cell product. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of CD3ζ, 4-1BB, and humanized anti-CD19 scFv domains. Patients received lymphodepletion with cyclophosphamide and fludarabine 1 week prior to infusion. Results: 37 children and young adults aged 29 mo–24 yr were infused with CTL119. At assessment 1 month after infusion, 31 patients (84%) were in CR, defined as morphologic remission with B cell aplasia, including 9/15 (60%) patients with prior CD19 CAR T cell exposure and 22/22 (100%) patients with no prior CAR T cell therapy. In responding patients, relapsefree survival at 6 months was 76% in the retreatment cohort (median followup 10 mo) and 83% in the CAR-naïve cohort (median follow-up 7 mo), with 3 patients proceeding to allogeneic stem cell transplant in remission. B cell aplasia, a functional marker of CD19-targeted CAR T cell persistence, continued for 3 months or more in 2/8 retreatment and 17/19 CARnaïve patients with adequate follow-up. Cytokine release syndrome (CRS) was observed in 33/37 patients and mild in most (27 grade 1/2, 5 grade 3, 1 grade 4). Severe CRS requiring vasopressor or ventilator support was successfully managed with tocilizumab in 3 patients. Conclusion: In the first study of humanized anti-CD19 CAR T cells, CTL119 induced remissions in patients with r/r B lymphoblastic leukemia/lymphoma, with an MRD-negative CR rate of 100% in CAR-naïve patients. While retreatment with murine CARs has produced mixed results, the CR rate with humanized CTL119 was 60% in the retreatment setting. Further investigation
5 TARGETING LEUKEMIA USING AN INDUCIBLE UNIVERSAL CHIMERIC ANTIGEN RECEPTOR (UNICAR) T CELL TECHNOLOGY J. Dietrich1, S. Loff2, J. Meyer1, M. von Bonin3, M. Hetzenecker4, J. Spehr1, C. Gründer2, J. Riewaldt1, G. Ehninger3, M.P. Bachmann4,5, A. Ehninger2, M. Cartellieri1 1 Cellex Patient Treatment GmbH, Dresden, Germany, 2Gemoab Monoclonals GmbH, Dresden, Germany, 3Medical Clinic and Policlinic I, University Hospital Carl Gustav Carus, Dresden, Germany, 4 Tumorimmunology, University Cancer Center (UCC), Medical Faculty Carl Gustav Carus, TU Dresden, Dresden, Germany, 5Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany T cells genetically engineered to express chimeric antigen receptors (CARs) represent a promising approach in cancer immunotherapy. CARs consist of an extracellular binding moiety providing antigen specificity and an intracellular signaling domain derived from an activating immune receptor. Both are linked via a hinge and transmembrane domain. Introducing co-stimulatory domains, such as CD28 or CD137, can enhance signaling. Although, CAR T cells showed encouraging clinical results in patients with B cell malignancies, major drawbacks include on-target off-tumor effects or severe cytokine release syndrome (sCRS). A universal CAR (UniCAR) platform recently introduced by us is a novel approach to gain clinical safety whilst, at the same time, maintaining efficacy of CAR T cell therapy. The new CAR technology splits antigen-binding specificity and signaling aspects in two separate entities. Antigen-specificity is provided by soluble peptide linkers (targeting modules, TMs), consisting of an antigenspecific binding moiety fused to a non-immunogenic peptide motif, not present on healthy human tissues, and specifically recognized by UniCAR engineered T cells. Our results demonstrate that UniCAR driven T cell reactivity against tumor cells is strictly dependent on the presence of antigen-specific TMs and the corresponding antigen on target cells. Applying TMs binding CD19 and CD123 we can demonstrate high efficient anti-leukemic effects of UniCAR T cells both in vitro and in vivo including primary AML and ALL samples. Moreover, architecture of the receptor framework plays an important role for efficacy. Hence, T cells engineered with CD28-derived hinge and signaling domain showed superior function compared to T cells engrafted with UniCARs composite of CD8α hinge with CD137 co-stimulation. In conclusion, the modular composition of the UniCAR maintains high anti-tumor specificity of CAR T cells while adding better control mechanisms and interchangeable target flexibility.
6 DIRECT COMPARISON OF IN VIVO FATE OF SECOND AND THIRD-GENERATION CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELLS IN PATIENTS WITH B CELL NONHODGKIN LYMPHOMA (B-NHL): REVERSAL OF TOXICITY FROM TONIC SIGNALING D. Gomes da Silva1, M. Mukherjee1, S. Madhuwanti1, O. Dakhova1, H. Liu1, B. Grilley1, A. Gee1, S.S. Neelapu2, C. Rooney1, H. Heslop1, B. Savoldo1, G. Dotti1, M. Brenner1, M. Mamonkin1, C.A. Ramos1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital and Texas Children’s Hospital, Houston, Texas, United States, 2Department of Lymphoma/Myeloma, University of Texas M. D. Anderson Cancer Center, Houston, Texas, United States Although 2nd-generation (2G) CD19-specific CARs containing CD28 or 4-1BB costimulatory endodomains (CE) show remarkable efficacy against B-NHL, the optimal choice of CEs in these and other CARs remains controversial. Individual CEs, such as CD28 may be associated with deleterious ligandindependent tonic signaling in the transduced T cell, but it is unclear if tonic 4-1BB signaling may have such consequences as well, and if such effects can be reversed. We therefore modeled tonic CAR signaling in T cells by transducing them with gammaretroviral vectors expressing 2G CD19.CAR constructs containing CD3-ζ and either the CD28 or 4-1BB CEs. 4-1BB CD19.CAR-T cells (CARTs) expanded 70% slower, which was coupled with a 4-fold increase in apoptosis and a gradual downregulation of CAR expression. Because of the toxicity of 4-1BB in our CAR construct, we could not directly compare the in vivo fate of 4-1BB CD19.CARTs with that of CD28 CD19.CARTs. We found,
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however, that the 4-1BB toxicity could be overcome in a 3rd-generation (3G) CD19.CAR vector containing both CD28 and 4-1BB. We thus compared the fate of that 3G vector with the 2G vector containing CD28 alone. To determine whether CD28 alone was optimal (which would suggest 4-1BB is antagonistic) or whether 4-1BB had an additive or synergistic effect contributing to superior expansion of the CD28-41BB combination, 8 relapsed/refractory B-NHL patients were simultaneously infused with 1–20 × 106 of both 2G and 3G CARTs/m2 48–72 hours after lymphodepletion with cyclophosphamide (500 mg/m2/d) and fludarabine (30 mg/m2/d) × 3. Persistence of infused T cells was assessed in blood by qPCR assays specific for each CAR. Molecular signals peaked approximately 2 weeks post infusion, remaining detectable for up to 6 months. The 3G CARTs had a mean 23-fold (range 1.1 to 109-fold) higher expansion than 2G CARTs and longer persistence. Two patients had grade 2 cytokine release syndrome at the time of peak expansion. Of the 6 patients evaluable for response, 2 entered complete remission (the longest ongoing for 1 year), 1 has had continued complete remission after ASCT, 1 had a partial response, and 2 progressed. In conclusion, our data indicate that infusion of T cells carrying a CD19.CAR containing CD28 and 4-1BB endodomains is safe and can have efficacy at every dose level tested. Additionally, in a side-by-side comparison, the 3G vector produced greater in vivo expansion than an otherwise identical CART population with CD28 alone.
7 HUMANIZED CHIMERIC ANTIGEN RECEPTOR (CAR)-MODIFIED T CELLS TARGETING CD19 INDUCE REMISSIONS IN CHILDREN AND YOUNG ADULTS WITH RELAPSED/REFRACTORY LYMPHOBLASTIC LEUKEMIA/LYMPHOMA G.E. Hucks1, D. Barrett1, S.R. Rheingold1, R. Aplenc1, D.T. Teachey1, C. Callahan1, D. Baniewicz1, C. White1, M. Talekar1, P. Shaw2, J.L. Brogdon3, R. Young2, J. Scholler2, K. Marcucci2, A. Chew2, B.L. Levine2, N. Frey2, D. Porter2, S. Lacey2, J. Melenhorst2, C. June2, S. Grupp1, S. Maude1 1 Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States, 2University of Pennsylvania, Philadelphia, Pennsylvania, United States, 3Novartis Institute for Biomedical Research, Cambridge, Massachusetts, United States Background: Chimeric antigen receptor (CAR)-modified T cells targeting CD19 can produce potent and sustained responses in children with relapsed/ refractory (r/r) acute lymphoblastic leukemia (ALL). However, limited persistence, seen in a subset of patients, can increase the risk of relapse. Anti-mouse reactivity is one potential cause of poor persistence that may be overcome by fully human or humanized CAR designs. We developed a humanized anti-CD19 scFv domain and now report on treatment with humanized CD19-directed CAR T cells (CTL119). Design: A pilot/phase 1 study of CTL119 enrolled children and young adults with r/r B lymphoblastic leukemia/lymphoma with or without prior exposure to a CAR T cell product. Patient-derived T cells were transduced ex vivo with a lentiviral vector encoding a CAR composed of CD3ζ, 4-1BB, and humanized anti-CD19 scFv domains. Patients received lymphodepletion with cyclophosphamide and fludarabine 1 week prior to infusion. Results: 37 children and young adults aged 29 mo–24 yr were infused with CTL119. At assessment 1 month after infusion, 31 patients (84%) were in CR, defined as morphologic remission with B cell aplasia, including 9/15 (60%) patients with prior CD19 CAR T cell exposure and 22/22 (100%) patients with no prior CAR T cell therapy. In responding patients, relapsefree survival at 6 months was 76% in the retreatment cohort (median followup 10 mo) and 83% in the CAR-naïve cohort (median follow-up 7 mo), with 3 patients proceeding to allogeneic stem cell transplant in remission. B cell aplasia, a functional marker of CD19-targeted CAR T cell persistence, continued for 3 months or more in 2/8 retreatment and 17/19 CARnaïve patients with adequate follow-up. Cytokine release syndrome (CRS) was observed in 33/37 patients and mild in most (27 grade 1/2, 5 grade 3, 1 grade 4). Severe CRS requiring vasopressor or ventilator support was successfully managed with tocilizumab in 3 patients. Conclusion: In the first study of humanized anti-CD19 CAR T cells, CTL119 induced remissions in patients with r/r B lymphoblastic leukemia/lymphoma, with an MRD-negative CR rate of 100% in CAR-naïve patients. While retreatment with murine CARs has produced mixed results, the CR rate with humanized CTL119 was 60% in the retreatment setting. Further investigation