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Apoptotic cells therapy for the prevention of cytokine release syndrome (CRS) in CAR T-cell therapy
S10
Oral Abstracts
into CAR T cell persistence and anti-CAR responses will be vital to improve durable remission rates in this highly refractory population.
8 DIFFERING CO-STIMULATORY, LINKER AND SPACER DOMAINS PRODUCE VARIATIONS IN CD4 AND CD8 CELL COMPOSITION AND CYTOTOXIC POTENTIAL IN CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR (CAR19) T CELLS GENERATED WITH THE PIGGYBAC TRANSPOSASE D. Bishop1,2,3, N. Xu4,5, S. Shen4,5, T. O’Brien4,5, D. Gottlieb1,2,3, A. Dolnikov4,5, K. Micklethwaite1,2,3 1 Westmead Institute for Medical Research, Sydney, New South Wales, Australia, 2Haematology Department, Westmead Hospital, Sydney, New South Wales, Australia, 3Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia, 4Cord & Marrow Transplant Facility, Sydney Children’s Hospital, Sydney, New South Wales, Australia, 5Children’s Cancer Institute, University of New South Wales, Sydney, New South Wales, Australia Aim: CAR19 T cells have efficacy against B cell malignancies, however data regarding the relationship of CAR structure, product CD4/CD8 composition and killing activity is limited. We examined these parameters whilst optimising low-cost piggyBac transposase-generated CAR19 T cells for clinical use. Methods: We designed two new 2 nd -generation CD19-specific CARs (CAR19h28z and CAR19h41BBz) to replace the previously described CAR1928z (Table I) that interacts with cells expressing FcgR in vivo, leading to poor CAR T cell efficacy and persistence. CAR19 T cells were generated by electroporation of piggyBac transposon and transposase plasmids and were expanded over 22 days via CD19 stimulation with IL-15 support. The new CARs were compared to CAR1928z in vitro and their in vivo activity was evaluated over 12 weeks in NSG mice xenografted with B-ALL. Results: PiggyBac CAR19 T cell cultures expanded robustly (minimum 120 fold) and >80% of T cells in final products expressed CAR. CAR19h28z T cell products were CD4 predominant (mean CD4+ 85%), while CAR1928z and CAR19h41BBz T cell products were CD8 predominant (mean CD8+ 71% and 62%, respectively). CD8+ CAR19 T cell proliferation was reduced in CAR19h28z cultures at day 8, but there was no difference between products in CD4+ CAR19 T cell proliferation or proportions of apoptotic CD4+ and CD8+ CAR T cells. IFN-γ production in both CD4+ and CD8+ CAR19 T cells from all products was specific to CD19+ targets. CD19 specific dosedependent cytotoxicity was seen with all products, although to a significantly lower degree with CAR19h28z. A single dose of either CAR19h28z or CAR19h41BBz T cells administered to NSG mice 1 week after B-ALL injection prolonged EFS (defined as >25% human CD19+ leukemic blasts in peripheral blood) compared to untreated mice (median EFS: 42 days for untreated vs >82 days [median not reached], P < 0.05). Conclusions: In piggyBac-generated CAR19 T cells, substitution of the CAR spacer IgG1 Fc region with a (G4S)3 linker led to reduced CD8+ CAR19 T cell proliferation, CD4+ CAR19 T cell predominance and reduced in vitro cytotoxicity in CD28 but not 4.1BB containing constructs. Despite this, CAR T cells expressing either optimised CAR construct displayed potent preclinical in vivo activity against B-ALL. We selected CAR19h41BBz to proceed to a first in man clinical trial of piggyBac-generated CAR19 T cells, and predict the low cost and simplicity of manufacture will improve access to CAR19 T cells.
9 APOPTOTIC CELLS THERAPY FOR THE PREVENTION OF CYTOKINE RELEASE SYNDROME (CRS) IN CAR T-CELL THERAPY D. Mevorach Medicine, Hadassah-Hebrew University, Jerusalem, Israel Allocetra™ (Enlivex Therapeutics Ltd.) is a cell-based therapy composed of donor mononuclear-enriched leukocytes, containing early apoptotic cells with no necrotic cells. It was shown as safe and potentially efficacious for the prevention of aGVHD in patients with hematologic malignancies (Mevorach et al. BBMT 2014). Chimeric antigen receptor (CAR)-modified T cells with specificity against CD19 have shown promise in highly refractory hematologic malignancies, albeit with significant toxicity. 30% of patients with acute lymphoblastic leukemia develop severe forms of CRS and possibly related neurotoxicity. CRS resembles macrophage-activating syndrome and hemophagocytosis in response to CAR T-secreting IFN-g and possibly added cytokines. We tested the effects of early apoptotic cells on CRS and CAR T cell cytotoxicity. Methods: CD19-expressing HeLa (in vitro) and Raji (in vivo) were used in vivo for leukemia induction. 2nd generation, with TM CD28, CD19-specific CARmodified cells were used (ProMab) for anti-tumor effect against CD19bearing cells. Cytotoxicity assay was examined in vivo and in vitro. 25 mouse cytokines and 32 human cytokines were evaluated (Luminex technology). IFN-g effect was evaluated (STAT1 phosphorylation and biological products). Early apoptotic cells were produced as shown in the GVHD clinical trial. Results: Apoptotic cells had no negative effect in vitro or in vivo on CARmodified T cells with specificity against CD19. There were comparable E/T ratios for CAR T in the presence/absence of apoptotic cells in vitro, and comparable survival curves in vivo. Significant downregulation (p < 0.01) of proinflammatory cytokines, including IL-6, IP-10, TNF-a, MIP-1a, MIP-1ß, was documented. IFN-g was not downregulated, but its effect on macrophages and dendritic cells was inhibited at the level of phosphorylated STAT1. IFN-ginduced expression of CXCL10 and CXCL9 was reduced. Conclusion: CRS evolves from several factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and as a response to the CAR T cell effect and expansion. Apoptotic cells decrease pro-inflammatory cytokines that originate from innate immunity and inhibit the IFN-g effect on monocyte/macrophages/ dendritic cells without harming IFN-g levels or CAR-T cytotoxicity. Apototic cells are safe to use for the prevention of CRS in clinical studies using CAR-T cell therapy.
10 AUTOLOGOUS MESENCHYMAL STEM CELL THERAPY IN BEHCET DISEASE H. Gabr1, W. Abo Elkheir2, Y. Abo Elkheir2 1 Clinical Pathology, Cairo University, Cairo, Egypt, 2Egyptian Society for Progenitor Stem Cell Research, Cairo, Se, Egypt Background: Behcet’s disease is a rare form of autoimmune vasculitis characterized by a triad of recurrent oral ulcers, genital ulcers and uveitis. Aim of the Work: The aim of the present trial is to evaluate the efficacy of double-armed autologous mesenchymal stem cell (MSCs) therapy: systemic MSCs for immunomodulation followed by retrobulbar and subtenon MSCs for ocular regeneration. This trial was approved by the IRB and aims to recruit 20 patients with active Behcet’s disease.
Table I. Structure of CD19-specific CAR constructs. CAR construct CAR1928z CAR19h28z CAR19h41BBz
Signal peptide
scFv
scFv heavy/light chain linkers
Spacer domain
Transmembrane domain
Costimulatory domain
Stimulatory domain
IgG heavy chain CD8a CD8a
FMC63 FMC63 FMC63
(G4S)4 (G4S)3 (G4S)3
IgG1h + CH2CH3 (G4S)3-IgG1h (G4S)3-IgG1h
CD28 CD28 CD28
CD28 CD28 4.1BB
CD3z CD3z CD3z
CAR = chimeric antigen receptor, scFv = single chain variable fragment, FMC63 = CD19-specific monoclonal antibody clone FMC63, (G4S)4 or (G4S)3 = glycine-glycine-glycine-glycine-serine flexible linker, number after parentheses indicates number of sequence repeats used, IgG1h = IgG1 hinge region, CH2CH3 = IgG1 Fc region constant heavy domains 2 and 3.
Oral Abstracts
into CAR T cell persistence and anti-CAR responses will be vital to improve durable remission rates in this highly refractory population.
8 DIFFERING CO-STIMULATORY, LINKER AND SPACER DOMAINS PRODUCE VARIATIONS IN CD4 AND CD8 CELL COMPOSITION AND CYTOTOXIC POTENTIAL IN CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR (CAR19) T CELLS GENERATED WITH THE PIGGYBAC TRANSPOSASE D. Bishop1,2,3, N. Xu4,5, S. Shen4,5, T. O’Brien4,5, D. Gottlieb1,2,3, A. Dolnikov4,5, K. Micklethwaite1,2,3 1 Westmead Institute for Medical Research, Sydney, New South Wales, Australia, 2Haematology Department, Westmead Hospital, Sydney, New South Wales, Australia, 3Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia, 4Cord & Marrow Transplant Facility, Sydney Children’s Hospital, Sydney, New South Wales, Australia, 5Children’s Cancer Institute, University of New South Wales, Sydney, New South Wales, Australia Aim: CAR19 T cells have efficacy against B cell malignancies, however data regarding the relationship of CAR structure, product CD4/CD8 composition and killing activity is limited. We examined these parameters whilst optimising low-cost piggyBac transposase-generated CAR19 T cells for clinical use. Methods: We designed two new 2 nd -generation CD19-specific CARs (CAR19h28z and CAR19h41BBz) to replace the previously described CAR1928z (Table I) that interacts with cells expressing FcgR in vivo, leading to poor CAR T cell efficacy and persistence. CAR19 T cells were generated by electroporation of piggyBac transposon and transposase plasmids and were expanded over 22 days via CD19 stimulation with IL-15 support. The new CARs were compared to CAR1928z in vitro and their in vivo activity was evaluated over 12 weeks in NSG mice xenografted with B-ALL. Results: PiggyBac CAR19 T cell cultures expanded robustly (minimum 120 fold) and >80% of T cells in final products expressed CAR. CAR19h28z T cell products were CD4 predominant (mean CD4+ 85%), while CAR1928z and CAR19h41BBz T cell products were CD8 predominant (mean CD8+ 71% and 62%, respectively). CD8+ CAR19 T cell proliferation was reduced in CAR19h28z cultures at day 8, but there was no difference between products in CD4+ CAR19 T cell proliferation or proportions of apoptotic CD4+ and CD8+ CAR T cells. IFN-γ production in both CD4+ and CD8+ CAR19 T cells from all products was specific to CD19+ targets. CD19 specific dosedependent cytotoxicity was seen with all products, although to a significantly lower degree with CAR19h28z. A single dose of either CAR19h28z or CAR19h41BBz T cells administered to NSG mice 1 week after B-ALL injection prolonged EFS (defined as >25% human CD19+ leukemic blasts in peripheral blood) compared to untreated mice (median EFS: 42 days for untreated vs >82 days [median not reached], P < 0.05). Conclusions: In piggyBac-generated CAR19 T cells, substitution of the CAR spacer IgG1 Fc region with a (G4S)3 linker led to reduced CD8+ CAR19 T cell proliferation, CD4+ CAR19 T cell predominance and reduced in vitro cytotoxicity in CD28 but not 4.1BB containing constructs. Despite this, CAR T cells expressing either optimised CAR construct displayed potent preclinical in vivo activity against B-ALL. We selected CAR19h41BBz to proceed to a first in man clinical trial of piggyBac-generated CAR19 T cells, and predict the low cost and simplicity of manufacture will improve access to CAR19 T cells.
9 APOPTOTIC CELLS THERAPY FOR THE PREVENTION OF CYTOKINE RELEASE SYNDROME (CRS) IN CAR T-CELL THERAPY D. Mevorach Medicine, Hadassah-Hebrew University, Jerusalem, Israel Allocetra™ (Enlivex Therapeutics Ltd.) is a cell-based therapy composed of donor mononuclear-enriched leukocytes, containing early apoptotic cells with no necrotic cells. It was shown as safe and potentially efficacious for the prevention of aGVHD in patients with hematologic malignancies (Mevorach et al. BBMT 2014). Chimeric antigen receptor (CAR)-modified T cells with specificity against CD19 have shown promise in highly refractory hematologic malignancies, albeit with significant toxicity. 30% of patients with acute lymphoblastic leukemia develop severe forms of CRS and possibly related neurotoxicity. CRS resembles macrophage-activating syndrome and hemophagocytosis in response to CAR T-secreting IFN-g and possibly added cytokines. We tested the effects of early apoptotic cells on CRS and CAR T cell cytotoxicity. Methods: CD19-expressing HeLa (in vitro) and Raji (in vivo) were used in vivo for leukemia induction. 2nd generation, with TM CD28, CD19-specific CARmodified cells were used (ProMab) for anti-tumor effect against CD19bearing cells. Cytotoxicity assay was examined in vivo and in vitro. 25 mouse cytokines and 32 human cytokines were evaluated (Luminex technology). IFN-g effect was evaluated (STAT1 phosphorylation and biological products). Early apoptotic cells were produced as shown in the GVHD clinical trial. Results: Apoptotic cells had no negative effect in vitro or in vivo on CARmodified T cells with specificity against CD19. There were comparable E/T ratios for CAR T in the presence/absence of apoptotic cells in vitro, and comparable survival curves in vivo. Significant downregulation (p < 0.01) of proinflammatory cytokines, including IL-6, IP-10, TNF-a, MIP-1a, MIP-1ß, was documented. IFN-g was not downregulated, but its effect on macrophages and dendritic cells was inhibited at the level of phosphorylated STAT1. IFN-ginduced expression of CXCL10 and CXCL9 was reduced. Conclusion: CRS evolves from several factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and as a response to the CAR T cell effect and expansion. Apoptotic cells decrease pro-inflammatory cytokines that originate from innate immunity and inhibit the IFN-g effect on monocyte/macrophages/ dendritic cells without harming IFN-g levels or CAR-T cytotoxicity. Apototic cells are safe to use for the prevention of CRS in clinical studies using CAR-T cell therapy.
10 AUTOLOGOUS MESENCHYMAL STEM CELL THERAPY IN BEHCET DISEASE H. Gabr1, W. Abo Elkheir2, Y. Abo Elkheir2 1 Clinical Pathology, Cairo University, Cairo, Egypt, 2Egyptian Society for Progenitor Stem Cell Research, Cairo, Se, Egypt Background: Behcet’s disease is a rare form of autoimmune vasculitis characterized by a triad of recurrent oral ulcers, genital ulcers and uveitis. Aim of the Work: The aim of the present trial is to evaluate the efficacy of double-armed autologous mesenchymal stem cell (MSCs) therapy: systemic MSCs for immunomodulation followed by retrobulbar and subtenon MSCs for ocular regeneration. This trial was approved by the IRB and aims to recruit 20 patients with active Behcet’s disease.
Table I. Structure of CD19-specific CAR constructs. CAR construct CAR1928z CAR19h28z CAR19h41BBz
Signal peptide
scFv
scFv heavy/light chain linkers
Spacer domain
Transmembrane domain
Costimulatory domain
Stimulatory domain
IgG heavy chain CD8a CD8a
FMC63 FMC63 FMC63
(G4S)4 (G4S)3 (G4S)3
IgG1h + CH2CH3 (G4S)3-IgG1h (G4S)3-IgG1h
CD28 CD28 CD28
CD28 CD28 4.1BB
CD3z CD3z CD3z
CAR = chimeric antigen receptor, scFv = single chain variable fragment, FMC63 = CD19-specific monoclonal antibody clone FMC63, (G4S)4 or (G4S)3 = glycine-glycine-glycine-glycine-serine flexible linker, number after parentheses indicates number of sequence repeats used, IgG1h = IgG1 hinge region, CH2CH3 = IgG1 Fc region constant heavy domains 2 and 3.