8 research outputs found
CD133-directed CAR T-cells for MLL Leukemia: On-Target, Off-Tumor Myeloablative Toxicity
Acknowledgements: We thank the Interfant treatment protocol and local physicians for contributing patient samples: Dr. Ronald W Stam (Princess Maxima Centre, Utrech), Dr. Mireia Camos and Dr. Jose Luis Fuster (Spanish Society of Pediatric Hematoncology), Dr. Paola Ballerini (A. Trousseau Hospital, Paris). We also thank Prof. Paresh Vyas (Oxford Univeristy, UK) and Prof. Kajsa Paulsson (Lund University, Sweden) for facilitating access to their RNA-seq database. This work has been supported by the European Research Council (CoG-2014-646903, PoC-2018-811220) to PM, the Spanish Ministry of Economy and Competitiveness (MINECO, SAF-SAF2016-80481-R, BIO2017-85364-R) to PM and EE, the Generalitat de Catalunya (SGR330, SGR102 and PERIS) to PM and EE, the Spanish Association against cancer (AECC-CI-2015) to CB, and the Health Institute Carlos III (ISCIII/FEDER, PI14-01191) to CB. PM also acknowledges financial support from the Obra Social La Caixa-Fundaciò Josep Carreras. SRZ and TV are supported by a Marie Curie fellowships. OM is supported by the Catalan Government through a Beatriu de Pinos fellowship. MB is supported by MINECO through a PhD scholarship. PM is an investigator of the Spanish Cell Therapy cooperative network (TERCEL)
Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope
Altres ajuts: Funding This work was supported by the Obra Social La Caixa (LCF/PR/HR19/52160011), the Spanish Cancer Association and Leo Messi Foundation to PM.Background There are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19 - either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22 + CD19 - B-ALL relapses and CD19 - preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied. Methods Here, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays. Results Conformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy. Conclusions We report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22 high and CD22 low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22-CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL
Bone Marrow Clonogenic Myeloid Progenitors from NPM1-Mutated AML Patients Do Not Harbor the NPM1 Mutation: Implication for the Cell-Of-Origin of NPM1+ AML
The cell-of-origin of NPM1- and FLT3-mutated acute myeloid leukemia (AML) is still a matter of debate. Here, we combined in vitro clonogenic assays with targeted sequencing to gain further insights into the cell-of-origin of NPM1 and FLT3-ITD-mutated AML in diagnostic bone marrow (BM) from nine NPM1+/FLT3-ITD (+/-) AMLs. We reasoned that individually plucked colony forming units (CFUs) are clonal and reflect the progeny of a single stem/progenitor cell. NPM1 and FLT3-ITD mutations seen in the diagnostic blasts were found in only 2/95 and 1/57 individually plucked CFUs, suggesting that BM clonogenic myeloid progenitors in NPM1-mutated and NPM1/FLT3-ITD-mutated AML patients do not harbor such molecular lesions. This supports previous studies on NPM1 mutations as secondary mutations in AML, likely acquired in an expanded pool of committed myeloid progenitors, perhaps CD34-, in line with the CD34-/low phenotype of NPM1-mutated AMLs. This study has important implications on the cell-of-origin of NPM1+ AML, and reinforces that therapeutic targeting of either NPM1 or FLT3-ITD mutations might only have a transient clinical benefit in debulking the leukemia, but is unlikely to be curative since will not target the AML-initiating/preleukemic cells. The absence of NPM1 and FLT3-ITD mutations in normal clonogenic myeloid progenitors is in line with their absence in clonal hematopoiesis of indeterminate potential.We thank CERCA/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for their institutional support. Financial support for this work was obtained from the Generalitat de Catalunya (SGR330) to P.M., the Spanish Ministry of Economy and Competitiveness (SAF2016-80481-R to P.M. and SAF2016-76758-R to I.V.), the Fundación Uno entre Cienmil, the Obra Social La Caixa (ID 100010434, under agreement LCF/PR/HR19/52160011), the Josep Carreras Foundation, the Leo Messi Foundation, and the Banco Santander Foundation to P.M.; and the Spanish Association against cancer (AECC-CI-2015) to C.B. E.A. acknowledges support form “Fundación Hay Esperanza”. P.M. is an investigator of the Spanish Cell Therapy cooperative network (TERCEL)
Innovative CAR T-cell based immunotherapy for the treatment of Relapsed/Refractory Acute Leukemias
[eng] Resistance to standard therapy and relapses are observed in several cases of both myeloid and lymphoid cell malignancies. Such phenomenon poses unquestionable limits to the utility of current therapeutic approaches, and remains a critical endeavor for the clinical management of patients with advanced cancers. To counteract resistance and to prevent relapse, the deployment of a wide portfolio of agents was put into action, providing at times only marginal benefits with large trade off in toxicity and tolerability. With the advent of immunotherapy huge improvements in patients’ management and overall survival was achieved, particularly in the field of CAR T-cells. Engineering T-cells to redirect them against a tumor antigen have granted impressive responses in the treatment of B-cell malignancies. On the other hand, the clinical implementation of CARTs for other blood cancers is lagging behind. Major reasons lay within the biology, expression and localization of the antigen to be targeted, as it can play vital roles in cell regulation and development in healthy tissues. The present work have provided extensive contribution to the field of CAR T-cells in two different manners: i) by elucidating with robust in vitro and in vivo comparative experiments whether CD123 selective targeting qualifies as myeloablative or not ii) by validating CD1a as a feasible therapeutic antigen for T-ALL and by creating a novel anti CD1a CAR, which showed robust antileukemic activity while preserving safety towards healthy T-cells and T-cells precursors. The development of innovative therapeutic tools that permit to tackle refractory-relapsed cancer with otherwise no further therapeutic options, opens the doors for important advances that will enable society to win the battle against blood cancers
Innovative CAR T-cell based immunotherapy for the treatment of Relapsed/Refractory Acute Leukemias
Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut contra la Leucèmia Fundació Josep Carreras[eng] Resistance to standard therapy and relapses are observed in several cases of both myeloid and lymphoid cell malignancies. Such phenomenon poses unquestionable limits to the utility of current therapeutic approaches, and remains a critical endeavor for the clinical management of patients with advanced cancers. To counteract resistance and to prevent relapse, the deployment of a wide portfolio of agents was put into action, providing at times only marginal benefits with large trade off in toxicity and tolerability. With the advent of immunotherapy huge improvements in patients’ management and overall survival was achieved, particularly in the field of CAR T-cells. Engineering T-cells to redirect them against a tumor antigen have granted impressive responses in the treatment of B-cell malignancies. On the other hand, the clinical implementation of CARTs for other blood cancers is lagging behind. Major reasons lay within the biology, expression and localization of the antigen to be targeted, as it can play vital roles in cell regulation and development in healthy tissues. The present work have provided extensive contribution to the field of CAR T-cells in two different manners: i) by elucidating with robust in vitro and in vivo comparative experiments whether CD123 selective targeting qualifies as myeloablative or not ii) by validating CD1a as a feasible therapeutic antigen for T-ALL and by creating a novel anti CD1a CAR, which showed robust antileukemic activity while preserving safety towards healthy T-cells and T-cells precursors. The development of innovative therapeutic tools that permit to tackle refractory-relapsed cancer with otherwise no further therapeutic options, opens the doors for important advances that will enable society to win the battle against blood cancers
Pericentriolar material analyses in normal esophageal mucosa, Barrett’s metaplasia and adenocarcinoma
Barrett’s esophagus metaplasia is a pre-cancerous condition caused by chronic esophagitis. Chromosomal instability (CIN) is common in Barrett’s cells: therefore, we investigated the possible presence of centrosomal aberrations (a main cause of CIN) by centrosomal protein immunostaining in paraffined esophageal samples of patients who developed a Barrett’s adenocarcinoma. In most (55%) patients, alterations of the pericentriolar material (PCM) signals were evident and consistently marked the transition between normal epithelium to metaplasia. The alterations could even be found in adjacent native squamous epithelium, Barrett’s mucosa and submucosal gland cells, as well as in the basal/epibasal layers of the mucosa and submucosal gland duct, which are the regions hosting esophageal stem and progenitor cells. These findings strongly support the hypothesis that the three esophageal histotypes (one being pathological) can have a common progenitor. Surprisingly, PCM defective signal eventually decreased with neoplastic progression, possibly to enhance the genome stability of advanced cancer cells. Importantly, PCM altered signals in Barrett’s mucosa and their apparent evolution in successive histopathological steps were correlated to adenocarcinoma aggressiveness, suggesting PCM as a possible prognostic marker for tumor relapse. Extending our observations in a prospective study might help in the development of new prevention protocols for adenocarcinoma patient
41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo.
Background Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative. Methods We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs. Results Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs. Conclusions This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML
41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo
Altres ajuts: We thank CERCA/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for their institutional support. PM acknowledges financial support from theSpanish Cancer Research Association (AECC-Semilla19), the Fundación Uno entre Cienmil, the Obra Social La Caixa (LCF/PR/HR19/52160011), the Leo Messi Foundation, the Banco Santander Foundation and the "Heroes hasta la médula" initiative.BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative. METHODS: We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs. RESULTS: Here, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs. CONCLUSIONS: This study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML