Contributions
Abstract: S250
Type: Oral Presentation
Session title: Cellular immunotherapy and gene therapy - Experimental
Background
Prognosis in relapsed/refractory AML is extremely poor. Despite recent advances with small-molecule therapeutics, the only curative option to date is allogeneic stem cell transplantation, which is often associated with toxicity and therapeutic failure. Therefore, novel therapies with potential for high efficacy yet tolerable safety profile are highly needed. CAR therapy has been clinically validated for CD19-positive B cell malignancies and is currently being evaluated for its application in other malignancies, however suitable CAR targets in AML still need to be identified. Integrating proteomic and transcriptomic target expression data we have previously discovered n=24 CAR candidate targets with a favorable profile in AML and normal tissues and provided a rationale for several combinatorial CAR approaches in AML (Perna et al. Cancer Cell 2017).
Aims
We hypothesized that rational combinatorial CAR design targeting both ADGRE2 and CLEC12A enhances anti-leukemic efficacy without cumulating potential on-target/off-tumor toxicity.
Methods
We validated favorable target pairs among our 24 candidates, based on a multimodal in-depth analysis including multiparameter spectral flow cytometry of primary r/r AML and normal bone marrow samples, along with normal tissue immunohistochemistry studies and mass-spectrometry. To provide a platform for identification of the ideal combinatorial CAR design, we established in-vitro and in-vivo models based on a human AML cell line with up- or down-regulated antigen levels of ADGRE2 and CLEC12A to mimic both AML antigen-low escape and toxicity to normal cells. In addition, we established a humanized mouse model that allowed in-vivo assessment of hematologic toxicities in the context of anti-leukemic CAR efficacy.
Results
We focus here on ADGRE2 and CLEC12A, two cell surface molecules highly co-expressed in AML but with largely non-overlapping expression profiles in normal tissues. Using a bicistronic gamma-retroviral vector, we screened different combinatorial CAR formats targeting ADGRE2 and CLEC12A. Specific to the combined target expression profile in malignant versus normal cells, we fine-tuned both scFv affinities considering total avidity, eventually achieving thresholds for mediating cytolysis in the context of optimized CD3 zeta signaling. In addition, we further optimized the chimeric receptor combination by evaluating different hinge/transmembrane and costimulatory domains. Ultimately, using therapeutically relevant T cell doses we were able to identify a combinatorial CAR format - ADCLEC.syn1 - that allowed complete and durable AML remission across relevant target levels while maintaining a favorable safety profile.
Conclusion
We developed a novel combinatorial CAR platform – ADCLEC.syn1 – and demonstrated in pre-clinical models its high potential for safe and efficient application in r/r AML.
Keyword(s): Acute myeloid leukemia, CAR-T, Cellular therapy, Immunotherapy
Abstract: S250
Type: Oral Presentation
Session title: Cellular immunotherapy and gene therapy - Experimental
Background
Prognosis in relapsed/refractory AML is extremely poor. Despite recent advances with small-molecule therapeutics, the only curative option to date is allogeneic stem cell transplantation, which is often associated with toxicity and therapeutic failure. Therefore, novel therapies with potential for high efficacy yet tolerable safety profile are highly needed. CAR therapy has been clinically validated for CD19-positive B cell malignancies and is currently being evaluated for its application in other malignancies, however suitable CAR targets in AML still need to be identified. Integrating proteomic and transcriptomic target expression data we have previously discovered n=24 CAR candidate targets with a favorable profile in AML and normal tissues and provided a rationale for several combinatorial CAR approaches in AML (Perna et al. Cancer Cell 2017).
Aims
We hypothesized that rational combinatorial CAR design targeting both ADGRE2 and CLEC12A enhances anti-leukemic efficacy without cumulating potential on-target/off-tumor toxicity.
Methods
We validated favorable target pairs among our 24 candidates, based on a multimodal in-depth analysis including multiparameter spectral flow cytometry of primary r/r AML and normal bone marrow samples, along with normal tissue immunohistochemistry studies and mass-spectrometry. To provide a platform for identification of the ideal combinatorial CAR design, we established in-vitro and in-vivo models based on a human AML cell line with up- or down-regulated antigen levels of ADGRE2 and CLEC12A to mimic both AML antigen-low escape and toxicity to normal cells. In addition, we established a humanized mouse model that allowed in-vivo assessment of hematologic toxicities in the context of anti-leukemic CAR efficacy.
Results
We focus here on ADGRE2 and CLEC12A, two cell surface molecules highly co-expressed in AML but with largely non-overlapping expression profiles in normal tissues. Using a bicistronic gamma-retroviral vector, we screened different combinatorial CAR formats targeting ADGRE2 and CLEC12A. Specific to the combined target expression profile in malignant versus normal cells, we fine-tuned both scFv affinities considering total avidity, eventually achieving thresholds for mediating cytolysis in the context of optimized CD3 zeta signaling. In addition, we further optimized the chimeric receptor combination by evaluating different hinge/transmembrane and costimulatory domains. Ultimately, using therapeutically relevant T cell doses we were able to identify a combinatorial CAR format - ADCLEC.syn1 - that allowed complete and durable AML remission across relevant target levels while maintaining a favorable safety profile.
Conclusion
We developed a novel combinatorial CAR platform – ADCLEC.syn1 – and demonstrated in pre-clinical models its high potential for safe and efficient application in r/r AML.
Keyword(s): Acute myeloid leukemia, CAR-T, Cellular therapy, Immunotherapy