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Modulation of anti-tumour activity of ex vivo expanded NK and GD2CAR-NK cells against neuroblastoma
S20
Poster Abstract Presentations
38 MODULATION OF ANTI-TUMOUR ACTIVITY OF EX VIVO EXPANDED NK AND GD2CAR-NK CELLS AGAINST NEUROBLASTOMA S. Shen12,3, K. Ferrell3, K. Chaudhry4, A. Vu3, R. Nordon5 & T. O'Brien1,2,3 1 Kids Cancer Centre, Sydney Children's Hospital, Randwick, New South Wales, Australia, 2Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia, 3Children's Cancer Institute for Medical Research, Sydney, New South Wales, Australia, 4Graduate School of Biomedical Engineering, University of New south wales, Sydney, New South Wales, Australia, 5University of New South Wales, Sydney, New South Wales, Australia Background & Aim: Chimeric antigen receptor engineered T cells (CAR T) have shown clinical success especially in acute lymphoblastic leukemia. Innate Natural Killer (NK) cells bear functional resemblance to T cells, but display a safer cytokine profile, immediate availability and no risk of GvHD. CAR NK are thus an attractive alternative to CAR T, especially in an allogeneic setting. Our study aims to investigate the cytolytic activity of NK and GD2CAR-NK cells against GD2 positive neuroblastoma (NB), either alone or in combination with histone deacetylase inhibitor (HDACi) and Programmed death 1 (PD-1) blocker. Methods, Results & Conclusion: We have shown NK cells expanded from mononuclear cells by co-culture with irradiated K562-mbIL15-41BBL cells exhibited robust cytotoxicity against NB cells. HDACi (selective Class I inhibitor entinostat and pan-inhibitor panobinostat) up-regulated surface expression of NKG2D ligands MIC A/B & ULBPs on NB cells and significantly increased NK-mediated killing in NB cell lines and patient-derived-xenograft (PDX) samples. Immune checkpoint blockade via neutralizing antibodies against PD1/PDL1 axis (upregulated during NB and NK co-culture), when used in conjunction with HDACi, further improved efficiency of NK-mediated killing. In NB subcutaneous and metastatic xenograft model, adoptive NK cell therapy significantly prolonged event free survival, an effect further enhanced by treatment with both HDACi and PD1 blocker. Retrovirally transduced GD2CAR-NK cells significantly increased killing against GD2positive NB cells as compared to control NK cells. Live cell imaging showed robust infiltration of GD2CAR-NK cells into 3D tumor spheroids, significantly reduced growth rate and able to destroy tumor spheroid. In a metastatic xenograft model, GD2CAR-NK treated mice showed significantly lower bioluminescence signal 11 days post tumor inoculation compared to control NK cells. However, although lower signal persisted in GD2CAR-NK group, significance was lost by day 18. Persistence of NK cells in mice was detected for one time point and no expansion observed. Taken together, our data suggest that NK cells mediate efficient killing of NB cells, enhanced by use of HDACi and PD1/PD-L1 blocker. GD2CARNK further enhance killing of target cells in vitro and although transiently, slowed tumor progression in vivo. Further investigation to improve transduction efficiency and NK/GD2CAR-NK cell expansion and persistence, with aim to enhance efficacy of NK/GD2CAR-NK cell therapy in vivo. 39 FC ENGINEERED ANTI-CD33MAB POTENTIATES CYTOTOXICITY OF MBIL-21 EXPANDED NK-CELLS AGAINST PRIMARY AML PRE-TREATED WITH DECITABINE R. Mani1, G. Rajgolikar1, J. Nunes1, K. Zapolnik1, R. Wasmuth1, X. Mo1, J. Byrd1, D.A. Lee2, N. Muthusamy1 & S. Vasu1 1 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States, 2Nationwide Children’s Hospital, Columbus, Ohio, United States Background & Aim: Natural killer (NK) cells are limited in number and require cytokines for in vivo expansion. Recipient NK cells are shown to have both qualitative and quantitative defects in acute myeloid leukemia (AML) patients, providing rationale for infusion of donor-derived NK cells. We showed that decitabine (DAC) enhances expression of NKG2D ligands in AML blasts and renders the blasts susceptible to killing by NK cells (Vasu et al. 2016). We conducted a phase I trial giving 5-day DAC in relapsed, refractory AML patients followed by haploidentical NK cells on day 0 and six doses of IL-2, where we demonstrated short term engraftment of donorderived NK cells (up to 48 hours) can be achieved by fludarabine and DAC. Here, we report preclinical studies using membrane bound IL-21 (mbIL-21) expanded NK cells, which can be given without extraneous cytokine support. Methods, Results & Conclusion: NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying mbIL-21
and 4-1BBL (CSTX002) to derive mbIL-21 NK cells. These cells have minimal senescence despite robust proliferation with the feasibility to infuse multiple NK cell doses of 1 £ 108/kg (Denman et al. 2012). We investigated if mbIL-21 NK cells exhibit natural killer activity alone or in the presence of Fcengineered anti-CD33 antibody [antibody-dependent cellular cytotoxicity (ADCC)] against primary AML samples from patients who received DAC, pre-NK cell infusion (day-1) and at 24 days post haplo-NK infusion (Post-NK infusion). Upon incubation with primary AML blasts, mbIL-21 NK cells displayed an activation profile as evidenced by surface CD107a induction (p=0.0051) and variable donor-dependent intra-cellular IFNg production, which was further increased with CD33mAb coated AML cells (p=0.0018). ADCC assays using chromium-51 (Cr51) labelled primary AML blasts revealed mbIL-21 NK cells lysed primary AML blasts alone which was significantly enhanced with CD33mAb coated target cells (p=0.0004). Importantly, CD33mAb dependent enhanced-cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post DAC treatment. Further, in-vivo infusion of mbIL-21 NK cells in AML patient derived xenograft (AML PDX) mice, treated with CD33mAb, reduced the tumor burden. These data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of Fc engineered anti-CD33 antibody in AML.
40 A SINGLE DOSE OF DONOR LYMPHOCYTES DEPLETED OF ANTI-HOST REACTIVE T CELLS (ATIR101) FOLLOWING TCELL-DEPLETED HAPLOIDENTICAL HSCT IS SAFE AND EFFICACEOUS D. Roy12, I. Walker3, J. Maertens4, P. Lewalle5, E. Olavarria6, Y. Beguin7, D. Selleslag8, E. Wagner13, H. B€ onig11,12 & S. Mielke9,10 1 Centre de recherche Hopital Maisonneuve-Rosemont, Montreal, Quebec, Canada, 2Universite de Montreal, Montreal, Quebec, Canada, 3Juravinski Hospital and Cancer Centre, Hamilton, Ontario, Canada, 4University Hospital Gasthuisberg, Leuven, Belgium, 5Institut Jules Bordet, Brussels, Belgium, 6Hammersmith Hospital, London, United Kingdom, 7Centre Hospitalier Universitaire de Liege, Liege, Belgium, 8Algemeen Ziekenhuis Sint-Jan, Bruges, Belgium, 9W€ urzburg University Medical Center, W€ urzburg, Germany, 10Karolinska Institute and University Hospital, Stockholm, Sweden, 11 German Red Cross Blood Service BaW€ uHe, Frankfurt, Germany, 12 University of Washington School of Medicine, Seattle, Washington, United States, 13University Medical Center, Mainz, Germany Background & Aim Introduction: Ex vivo photodepletion using dibromorhodamine (ATIR101, Kiadis Pharma) enables the administration of donor lymphocytes after haploidentical allogeneic hematopoietic stem cell transplantation (haplo-HSCT), in the absence of immune suppression, to promote antiinfection and anti-leukemia activity. The safety and efficacy of ATIR101 are presented in a pooled analysis of 2 phase II trials (CR-AIR-007, NCT01794299; CR-AIR-008, NCT02500550). Methods, Results & Conclusion Methods: A pooled analysis of 2 trials with similar protocols, in 37 adult patients. N=32 patients received a single dose of ATIR101 (2.0 £ 106 cells/kg); n=5 discontinued after HSCT. Of the 37 patients, 65% had acute myeloid leukemia, 27% had acute lymphoblastic leukemia, and 8% had myelodysplastic syndromes; patients had intermediate (57%) and high (43%) disease risk index. All underwent myeloablative conditioning followed by a CD34+ selected stem cell graft from a haploidentical family donor. ATIR101 cells were given at a median of 28 days post HSCT without prophylactic immunosuppression. Outcomes were compared with a
Poster Abstract Presentations
38 MODULATION OF ANTI-TUMOUR ACTIVITY OF EX VIVO EXPANDED NK AND GD2CAR-NK CELLS AGAINST NEUROBLASTOMA S. Shen12,3, K. Ferrell3, K. Chaudhry4, A. Vu3, R. Nordon5 & T. O'Brien1,2,3 1 Kids Cancer Centre, Sydney Children's Hospital, Randwick, New South Wales, Australia, 2Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia, 3Children's Cancer Institute for Medical Research, Sydney, New South Wales, Australia, 4Graduate School of Biomedical Engineering, University of New south wales, Sydney, New South Wales, Australia, 5University of New South Wales, Sydney, New South Wales, Australia Background & Aim: Chimeric antigen receptor engineered T cells (CAR T) have shown clinical success especially in acute lymphoblastic leukemia. Innate Natural Killer (NK) cells bear functional resemblance to T cells, but display a safer cytokine profile, immediate availability and no risk of GvHD. CAR NK are thus an attractive alternative to CAR T, especially in an allogeneic setting. Our study aims to investigate the cytolytic activity of NK and GD2CAR-NK cells against GD2 positive neuroblastoma (NB), either alone or in combination with histone deacetylase inhibitor (HDACi) and Programmed death 1 (PD-1) blocker. Methods, Results & Conclusion: We have shown NK cells expanded from mononuclear cells by co-culture with irradiated K562-mbIL15-41BBL cells exhibited robust cytotoxicity against NB cells. HDACi (selective Class I inhibitor entinostat and pan-inhibitor panobinostat) up-regulated surface expression of NKG2D ligands MIC A/B & ULBPs on NB cells and significantly increased NK-mediated killing in NB cell lines and patient-derived-xenograft (PDX) samples. Immune checkpoint blockade via neutralizing antibodies against PD1/PDL1 axis (upregulated during NB and NK co-culture), when used in conjunction with HDACi, further improved efficiency of NK-mediated killing. In NB subcutaneous and metastatic xenograft model, adoptive NK cell therapy significantly prolonged event free survival, an effect further enhanced by treatment with both HDACi and PD1 blocker. Retrovirally transduced GD2CAR-NK cells significantly increased killing against GD2positive NB cells as compared to control NK cells. Live cell imaging showed robust infiltration of GD2CAR-NK cells into 3D tumor spheroids, significantly reduced growth rate and able to destroy tumor spheroid. In a metastatic xenograft model, GD2CAR-NK treated mice showed significantly lower bioluminescence signal 11 days post tumor inoculation compared to control NK cells. However, although lower signal persisted in GD2CAR-NK group, significance was lost by day 18. Persistence of NK cells in mice was detected for one time point and no expansion observed. Taken together, our data suggest that NK cells mediate efficient killing of NB cells, enhanced by use of HDACi and PD1/PD-L1 blocker. GD2CARNK further enhance killing of target cells in vitro and although transiently, slowed tumor progression in vivo. Further investigation to improve transduction efficiency and NK/GD2CAR-NK cell expansion and persistence, with aim to enhance efficacy of NK/GD2CAR-NK cell therapy in vivo. 39 FC ENGINEERED ANTI-CD33MAB POTENTIATES CYTOTOXICITY OF MBIL-21 EXPANDED NK-CELLS AGAINST PRIMARY AML PRE-TREATED WITH DECITABINE R. Mani1, G. Rajgolikar1, J. Nunes1, K. Zapolnik1, R. Wasmuth1, X. Mo1, J. Byrd1, D.A. Lee2, N. Muthusamy1 & S. Vasu1 1 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States, 2Nationwide Children’s Hospital, Columbus, Ohio, United States Background & Aim: Natural killer (NK) cells are limited in number and require cytokines for in vivo expansion. Recipient NK cells are shown to have both qualitative and quantitative defects in acute myeloid leukemia (AML) patients, providing rationale for infusion of donor-derived NK cells. We showed that decitabine (DAC) enhances expression of NKG2D ligands in AML blasts and renders the blasts susceptible to killing by NK cells (Vasu et al. 2016). We conducted a phase I trial giving 5-day DAC in relapsed, refractory AML patients followed by haploidentical NK cells on day 0 and six doses of IL-2, where we demonstrated short term engraftment of donorderived NK cells (up to 48 hours) can be achieved by fludarabine and DAC. Here, we report preclinical studies using membrane bound IL-21 (mbIL-21) expanded NK cells, which can be given without extraneous cytokine support. Methods, Results & Conclusion: NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying mbIL-21
and 4-1BBL (CSTX002) to derive mbIL-21 NK cells. These cells have minimal senescence despite robust proliferation with the feasibility to infuse multiple NK cell doses of 1 £ 108/kg (Denman et al. 2012). We investigated if mbIL-21 NK cells exhibit natural killer activity alone or in the presence of Fcengineered anti-CD33 antibody [antibody-dependent cellular cytotoxicity (ADCC)] against primary AML samples from patients who received DAC, pre-NK cell infusion (day-1) and at 24 days post haplo-NK infusion (Post-NK infusion). Upon incubation with primary AML blasts, mbIL-21 NK cells displayed an activation profile as evidenced by surface CD107a induction (p=0.0051) and variable donor-dependent intra-cellular IFNg production, which was further increased with CD33mAb coated AML cells (p=0.0018). ADCC assays using chromium-51 (Cr51) labelled primary AML blasts revealed mbIL-21 NK cells lysed primary AML blasts alone which was significantly enhanced with CD33mAb coated target cells (p=0.0004). Importantly, CD33mAb dependent enhanced-cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post DAC treatment. Further, in-vivo infusion of mbIL-21 NK cells in AML patient derived xenograft (AML PDX) mice, treated with CD33mAb, reduced the tumor burden. These data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of Fc engineered anti-CD33 antibody in AML.
40 A SINGLE DOSE OF DONOR LYMPHOCYTES DEPLETED OF ANTI-HOST REACTIVE T CELLS (ATIR101) FOLLOWING TCELL-DEPLETED HAPLOIDENTICAL HSCT IS SAFE AND EFFICACEOUS D. Roy12, I. Walker3, J. Maertens4, P. Lewalle5, E. Olavarria6, Y. Beguin7, D. Selleslag8, E. Wagner13, H. B€ onig11,12 & S. Mielke9,10 1 Centre de recherche Hopital Maisonneuve-Rosemont, Montreal, Quebec, Canada, 2Universite de Montreal, Montreal, Quebec, Canada, 3Juravinski Hospital and Cancer Centre, Hamilton, Ontario, Canada, 4University Hospital Gasthuisberg, Leuven, Belgium, 5Institut Jules Bordet, Brussels, Belgium, 6Hammersmith Hospital, London, United Kingdom, 7Centre Hospitalier Universitaire de Liege, Liege, Belgium, 8Algemeen Ziekenhuis Sint-Jan, Bruges, Belgium, 9W€ urzburg University Medical Center, W€ urzburg, Germany, 10Karolinska Institute and University Hospital, Stockholm, Sweden, 11 German Red Cross Blood Service BaW€ uHe, Frankfurt, Germany, 12 University of Washington School of Medicine, Seattle, Washington, United States, 13University Medical Center, Mainz, Germany Background & Aim Introduction: Ex vivo photodepletion using dibromorhodamine (ATIR101, Kiadis Pharma) enables the administration of donor lymphocytes after haploidentical allogeneic hematopoietic stem cell transplantation (haplo-HSCT), in the absence of immune suppression, to promote antiinfection and anti-leukemia activity. The safety and efficacy of ATIR101 are presented in a pooled analysis of 2 phase II trials (CR-AIR-007, NCT01794299; CR-AIR-008, NCT02500550). Methods, Results & Conclusion Methods: A pooled analysis of 2 trials with similar protocols, in 37 adult patients. N=32 patients received a single dose of ATIR101 (2.0 £ 106 cells/kg); n=5 discontinued after HSCT. Of the 37 patients, 65% had acute myeloid leukemia, 27% had acute lymphoblastic leukemia, and 8% had myelodysplastic syndromes; patients had intermediate (57%) and high (43%) disease risk index. All underwent myeloablative conditioning followed by a CD34+ selected stem cell graft from a haploidentical family donor. ATIR101 cells were given at a median of 28 days post HSCT without prophylactic immunosuppression. Outcomes were compared with a