GENERATION OF CHIMERIC ANTIGEN RECEPTOR - MODIFIED MEMORY STEM CELL CD8+ T LYMPHOCYTES FROM NAIVE PRECURSORS BY MODULATION OF WNT/BETA-CATENIN PATHWAY OR INHIBITION OF AKT-SIGNALING
(Abstract release date: 05/19/16)
EHA Library. Hartwig U. 06/11/16; 135271; S515
Assoc. Prof. Udo Hartwig
Contributions
Contributions
Abstract
Abstract: S515
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C14
Background
Adoptive cellular therapy (ACT) of chimeric antigen receptor (CAR)-reprogrammed T cells has advanced as a personalized and effective immunotherapy for leukemia and solid tumors. However, ACT, especially to solid tumors is often hampered by limited T cell engraftment and limited capability of terminally differentiated, high avidity effector T cells (TEFF) to establish sustained antitumor immunity. Recently, long-living stem cell memory T cells (TSCM) with an enhanced capacity for self-renewal and plasticity to differentiate into central memory (TCM), effector memory (TEM) and TEFF could be shown to elicit potent antitumor responses, prolonged survival and memory. Moreover, modulating the Wnt/ß-catenin or PI3K-Akt-mTOR signaling pathways in T cells using inhibitors of glycogen-sythase-kinase-3β (TWS119) or Akt (inhibitor VIII) have emerged as promising approaches to block CD8+ effector T cell differentiation and to facilitate the in vitro generation of TSCM and TCM.
Aims
In the present proof of concept study, we therefore investigated the generation of CD19 CAR expressing CD8+ TSCM from naive CD8+ T lymphocytes by modulating T-cell differentiation using TWS119 or Akt inhibitor VIII to be used for ACT.
Methods
Naive CD8+CD45RA+ T cells isolated from PBMC by MACS® were polyclonally stimulated with CD3/CD28 Dynabeads in the presence of various cytokines and retrovirally transduced with a second generation CD19 CAR three days after activation. To promote a TSCM/CM phenotype transduced cells were either polyclonally restimulated or co-cultured with CD19+ B-ALL (NALM16) together with TWS119 or Akt inhibitor VIII. CD19 CAR expression was determined by flow cytometry. IFN-γ ELISPOT and 51chromium-release cytotoxicity assays were used to analyze effector functions of CD19 CAR expressing TSCM and TCM. Adoptive transfer studies of redirected T cells into NALM-16 engrafted NSG mice were performed to evaluate antileukemic responses in vivo.
Results
Upon repetitive restimulation and TWS119/Akt inhibitor VIII treatment we obtained strong expansion of T cells with a TSCM/CM phenotype. In contrast, this effect was less pronounced by naive T cells cultured in the sole presence of interleukin (IL)-12 followed by IL-2, IL-7, IL-15, and IL-21, confirming that both Wnt/ß-catenin and PI3K-Akt-mTOR pathways play a key role in T cell differentiation. In addition, CD8+CD45RA+CD45RO-CD95+CD62L+CCR7+ TSCM/CM showed high expression levels of CD19 CAR, elicited strong IFN-γ release and cytolytic activity to CD19+ NALM-16 cells when compared to mock transduced controls. This effect was also seen in CD19 CAR positive total CD8+ T cells although less pronounced.First studies to evaluate the therapeutic efficacy of CD19-CAR redirected TSCM/CM revealed that upon adoptive transfer of 1–5x106 T cells into NSG mice engrafted with NALM-16 B-ALL significant regression of leukemia could be observed in spleen and bone marrow. Again, antileukemic responses were also seen with CD19 CAR transduced TEM and TEFF but TSCM/CM appeared to be more effective when applied at lower numbers (1 x106 cells/mouse). Further studies are in progress to elucidate the functional properties of CD19 CAR TSCM/CM in more detail.
Conclusion
In conclusion, these proof of concept studies demonstrate that ex vivo generated TSCM/CM redirected to acute leukemia by retroviral transfer of optimized leukemia- or tumor-reactive CARs may be a promising approach to improve ACT.
Session topic: Gene therapy, cellular immunotherapy and vaccination
Keyword(s): Acute leukemia, Cancer immunotherapy, Cellular therapy, Memory T cells
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C14
Background
Adoptive cellular therapy (ACT) of chimeric antigen receptor (CAR)-reprogrammed T cells has advanced as a personalized and effective immunotherapy for leukemia and solid tumors. However, ACT, especially to solid tumors is often hampered by limited T cell engraftment and limited capability of terminally differentiated, high avidity effector T cells (TEFF) to establish sustained antitumor immunity. Recently, long-living stem cell memory T cells (TSCM) with an enhanced capacity for self-renewal and plasticity to differentiate into central memory (TCM), effector memory (TEM) and TEFF could be shown to elicit potent antitumor responses, prolonged survival and memory. Moreover, modulating the Wnt/ß-catenin or PI3K-Akt-mTOR signaling pathways in T cells using inhibitors of glycogen-sythase-kinase-3β (TWS119) or Akt (inhibitor VIII) have emerged as promising approaches to block CD8+ effector T cell differentiation and to facilitate the in vitro generation of TSCM and TCM.
Aims
In the present proof of concept study, we therefore investigated the generation of CD19 CAR expressing CD8+ TSCM from naive CD8+ T lymphocytes by modulating T-cell differentiation using TWS119 or Akt inhibitor VIII to be used for ACT.
Methods
Naive CD8+CD45RA+ T cells isolated from PBMC by MACS® were polyclonally stimulated with CD3/CD28 Dynabeads in the presence of various cytokines and retrovirally transduced with a second generation CD19 CAR three days after activation. To promote a TSCM/CM phenotype transduced cells were either polyclonally restimulated or co-cultured with CD19+ B-ALL (NALM16) together with TWS119 or Akt inhibitor VIII. CD19 CAR expression was determined by flow cytometry. IFN-γ ELISPOT and 51chromium-release cytotoxicity assays were used to analyze effector functions of CD19 CAR expressing TSCM and TCM. Adoptive transfer studies of redirected T cells into NALM-16 engrafted NSG mice were performed to evaluate antileukemic responses in vivo.
Results
Upon repetitive restimulation and TWS119/Akt inhibitor VIII treatment we obtained strong expansion of T cells with a TSCM/CM phenotype. In contrast, this effect was less pronounced by naive T cells cultured in the sole presence of interleukin (IL)-12 followed by IL-2, IL-7, IL-15, and IL-21, confirming that both Wnt/ß-catenin and PI3K-Akt-mTOR pathways play a key role in T cell differentiation. In addition, CD8+CD45RA+CD45RO-CD95+CD62L+CCR7+ TSCM/CM showed high expression levels of CD19 CAR, elicited strong IFN-γ release and cytolytic activity to CD19+ NALM-16 cells when compared to mock transduced controls. This effect was also seen in CD19 CAR positive total CD8+ T cells although less pronounced.First studies to evaluate the therapeutic efficacy of CD19-CAR redirected TSCM/CM revealed that upon adoptive transfer of 1–5x106 T cells into NSG mice engrafted with NALM-16 B-ALL significant regression of leukemia could be observed in spleen and bone marrow. Again, antileukemic responses were also seen with CD19 CAR transduced TEM and TEFF but TSCM/CM appeared to be more effective when applied at lower numbers (1 x106 cells/mouse). Further studies are in progress to elucidate the functional properties of CD19 CAR TSCM/CM in more detail.
Conclusion
In conclusion, these proof of concept studies demonstrate that ex vivo generated TSCM/CM redirected to acute leukemia by retroviral transfer of optimized leukemia- or tumor-reactive CARs may be a promising approach to improve ACT.
Session topic: Gene therapy, cellular immunotherapy and vaccination
Keyword(s): Acute leukemia, Cancer immunotherapy, Cellular therapy, Memory T cells
Abstract: S515
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C14
Background
Adoptive cellular therapy (ACT) of chimeric antigen receptor (CAR)-reprogrammed T cells has advanced as a personalized and effective immunotherapy for leukemia and solid tumors. However, ACT, especially to solid tumors is often hampered by limited T cell engraftment and limited capability of terminally differentiated, high avidity effector T cells (TEFF) to establish sustained antitumor immunity. Recently, long-living stem cell memory T cells (TSCM) with an enhanced capacity for self-renewal and plasticity to differentiate into central memory (TCM), effector memory (TEM) and TEFF could be shown to elicit potent antitumor responses, prolonged survival and memory. Moreover, modulating the Wnt/ß-catenin or PI3K-Akt-mTOR signaling pathways in T cells using inhibitors of glycogen-sythase-kinase-3β (TWS119) or Akt (inhibitor VIII) have emerged as promising approaches to block CD8+ effector T cell differentiation and to facilitate the in vitro generation of TSCM and TCM.
Aims
In the present proof of concept study, we therefore investigated the generation of CD19 CAR expressing CD8+ TSCM from naive CD8+ T lymphocytes by modulating T-cell differentiation using TWS119 or Akt inhibitor VIII to be used for ACT.
Methods
Naive CD8+CD45RA+ T cells isolated from PBMC by MACS® were polyclonally stimulated with CD3/CD28 Dynabeads in the presence of various cytokines and retrovirally transduced with a second generation CD19 CAR three days after activation. To promote a TSCM/CM phenotype transduced cells were either polyclonally restimulated or co-cultured with CD19+ B-ALL (NALM16) together with TWS119 or Akt inhibitor VIII. CD19 CAR expression was determined by flow cytometry. IFN-γ ELISPOT and 51chromium-release cytotoxicity assays were used to analyze effector functions of CD19 CAR expressing TSCM and TCM. Adoptive transfer studies of redirected T cells into NALM-16 engrafted NSG mice were performed to evaluate antileukemic responses in vivo.
Results
Upon repetitive restimulation and TWS119/Akt inhibitor VIII treatment we obtained strong expansion of T cells with a TSCM/CM phenotype. In contrast, this effect was less pronounced by naive T cells cultured in the sole presence of interleukin (IL)-12 followed by IL-2, IL-7, IL-15, and IL-21, confirming that both Wnt/ß-catenin and PI3K-Akt-mTOR pathways play a key role in T cell differentiation. In addition, CD8+CD45RA+CD45RO-CD95+CD62L+CCR7+ TSCM/CM showed high expression levels of CD19 CAR, elicited strong IFN-γ release and cytolytic activity to CD19+ NALM-16 cells when compared to mock transduced controls. This effect was also seen in CD19 CAR positive total CD8+ T cells although less pronounced.First studies to evaluate the therapeutic efficacy of CD19-CAR redirected TSCM/CM revealed that upon adoptive transfer of 1–5x106 T cells into NSG mice engrafted with NALM-16 B-ALL significant regression of leukemia could be observed in spleen and bone marrow. Again, antileukemic responses were also seen with CD19 CAR transduced TEM and TEFF but TSCM/CM appeared to be more effective when applied at lower numbers (1 x106 cells/mouse). Further studies are in progress to elucidate the functional properties of CD19 CAR TSCM/CM in more detail.
Conclusion
In conclusion, these proof of concept studies demonstrate that ex vivo generated TSCM/CM redirected to acute leukemia by retroviral transfer of optimized leukemia- or tumor-reactive CARs may be a promising approach to improve ACT.
Session topic: Gene therapy, cellular immunotherapy and vaccination
Keyword(s): Acute leukemia, Cancer immunotherapy, Cellular therapy, Memory T cells
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C14
Background
Adoptive cellular therapy (ACT) of chimeric antigen receptor (CAR)-reprogrammed T cells has advanced as a personalized and effective immunotherapy for leukemia and solid tumors. However, ACT, especially to solid tumors is often hampered by limited T cell engraftment and limited capability of terminally differentiated, high avidity effector T cells (TEFF) to establish sustained antitumor immunity. Recently, long-living stem cell memory T cells (TSCM) with an enhanced capacity for self-renewal and plasticity to differentiate into central memory (TCM), effector memory (TEM) and TEFF could be shown to elicit potent antitumor responses, prolonged survival and memory. Moreover, modulating the Wnt/ß-catenin or PI3K-Akt-mTOR signaling pathways in T cells using inhibitors of glycogen-sythase-kinase-3β (TWS119) or Akt (inhibitor VIII) have emerged as promising approaches to block CD8+ effector T cell differentiation and to facilitate the in vitro generation of TSCM and TCM.
Aims
In the present proof of concept study, we therefore investigated the generation of CD19 CAR expressing CD8+ TSCM from naive CD8+ T lymphocytes by modulating T-cell differentiation using TWS119 or Akt inhibitor VIII to be used for ACT.
Methods
Naive CD8+CD45RA+ T cells isolated from PBMC by MACS® were polyclonally stimulated with CD3/CD28 Dynabeads in the presence of various cytokines and retrovirally transduced with a second generation CD19 CAR three days after activation. To promote a TSCM/CM phenotype transduced cells were either polyclonally restimulated or co-cultured with CD19+ B-ALL (NALM16) together with TWS119 or Akt inhibitor VIII. CD19 CAR expression was determined by flow cytometry. IFN-γ ELISPOT and 51chromium-release cytotoxicity assays were used to analyze effector functions of CD19 CAR expressing TSCM and TCM. Adoptive transfer studies of redirected T cells into NALM-16 engrafted NSG mice were performed to evaluate antileukemic responses in vivo.
Results
Upon repetitive restimulation and TWS119/Akt inhibitor VIII treatment we obtained strong expansion of T cells with a TSCM/CM phenotype. In contrast, this effect was less pronounced by naive T cells cultured in the sole presence of interleukin (IL)-12 followed by IL-2, IL-7, IL-15, and IL-21, confirming that both Wnt/ß-catenin and PI3K-Akt-mTOR pathways play a key role in T cell differentiation. In addition, CD8+CD45RA+CD45RO-CD95+CD62L+CCR7+ TSCM/CM showed high expression levels of CD19 CAR, elicited strong IFN-γ release and cytolytic activity to CD19+ NALM-16 cells when compared to mock transduced controls. This effect was also seen in CD19 CAR positive total CD8+ T cells although less pronounced.First studies to evaluate the therapeutic efficacy of CD19-CAR redirected TSCM/CM revealed that upon adoptive transfer of 1–5x106 T cells into NSG mice engrafted with NALM-16 B-ALL significant regression of leukemia could be observed in spleen and bone marrow. Again, antileukemic responses were also seen with CD19 CAR transduced TEM and TEFF but TSCM/CM appeared to be more effective when applied at lower numbers (1 x106 cells/mouse). Further studies are in progress to elucidate the functional properties of CD19 CAR TSCM/CM in more detail.
Conclusion
In conclusion, these proof of concept studies demonstrate that ex vivo generated TSCM/CM redirected to acute leukemia by retroviral transfer of optimized leukemia- or tumor-reactive CARs may be a promising approach to improve ACT.
Session topic: Gene therapy, cellular immunotherapy and vaccination
Keyword(s): Acute leukemia, Cancer immunotherapy, Cellular therapy, Memory T cells
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