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Successful engraftment but high viral reactivation after reduced intensity unrelated umbilical cord blood transplantation for sickle cell disease
S8
Oral Abstracts
to integrate the X5 scFv into a CAR to genetically modify HXTC and propose that X5 CAR expressing HXTC could be a potent therapeutic for the treatment of HIVpos individuals post-SCT, targeting multiple HIV antigens while preventing infection of infused CD4+ T cells.
2 SUCCESSFUL ENGRAFTMENT BUT HIGH VIRAL REACTIVATION AFTER REDUCED INTENSITY UNRELATED UMBILICAL CORD BLOOD TRANSPLANTATION FOR SICKLE CELL DISEASE A. Abraham1, A. Cluster2, D. Jacobsohn1, D. Delgado3, M. Hulbert2, D. Kukadiya1, L. Murray2, S. Shenoy2 1 Children’s National Medical Center, Washington, District of Columbia, United States, 2Washington University School of Medicine, St. Louis, Missouri, United States, 3Indiana University School of Medicine, Indianapolis, Indiana, United States Introduction: HLA-matched sibling donor (MSD) hematopoietic stem cell transplant is curative in >92% of patients with sickle cell disease (SCD) but only 18% have MSD. Unrelated cord blood transplantation (UCBT) expands donor options but is associated with high graft rejection rates (50–62%). We hypothesized that a previously used reduced intensity conditioning (RIC) regimen when combined with thiotepa would have acceptable toxicity and support donor engraftment after UCBT for SCD. Methods: Study patients were ≤21 years with severe SCD complications. UCB units were 5–6/6 HLA-matched. The RIC regimen included oral hydroxyurea 30 mg/kg (Days −50 to −21), and intravenous alemtuzumab 3 mg, 10 mg, 15 mg and 20 mg (Days −22 to −19), fludarabine 30 mg/m2/dose (Days −8 to −4), thiotepa 4 mg/kg × 2 (Day −4) and melphalan 140 mg/m2 (Day −3). GVHD prophylaxis consisted of a calcineurin inhibitor and MMF. Results: Ten children (6 male), median age 3.5 years (range 3–10) and weight 17.8 kg (range 15.1–37.6) underwent UCBT for cerebrovascular (n = 9) or recurrent vaso-occlusive (n = 1) SCD complications. Median cell dose was 7.2 × 107 TNC/kg (range 3.9–69.5). Follow up was 2.4 years (range 0.3–4.4). Median neutrophil (ANC ≥500/cumm) and platelet (≥50,000/cumm) engraftment were 19 (range 12–38) and 48 (range 28–204) days respectively. Eight patients have sustained donor engraftment. Two had autologous recovery on Days +33 and +39. Grade 3 acute GVHD developed in three, grade 4 in one patient. Mild (n = 2) and moderate (n = 1) chronic GVHD affected skin and fascia. Four of 5 patients >2 years post UCBT have stopped systemic immunosuppression. Eight patients tested positive for CMV or adenovirus by Day+100. Of these, 2 had CMV disease, one of which had pneumonitis and subsequent graft rejection. The second had pneumonitis before Day+30 and was treated with IV ganciclovir for 5 months for persistent viremia. CMV retinitis developed upon stopping IV ganciclovir and she received third party virus-specific T cells (VSTs) with anti-CMV activity with improvement in disease. One patient with recurrent adenoviremia was treated with donor UCB-derived VSTs on Day+117 and was able to stop IV cidofovir. Overall and disease free survival rates are 100% and 80% respectively. Conclusions: This RIC regimen supported engraftment in 8/10 UCBT recipients but early viral infections are of concern. Future efforts will focus on novel targeted therapies such as VSTs to reduce these complications.
3 BALANCE OF ANTI-CD123 CHIMERIC ANTIGEN RECEPTOR (CAR) BINDING AFFINITY AND DENSITY FOR THE TARGETING OF ACUTE MYELOID LEUKEMIA S. Arcangeli1, M. Rotiroti1, M. Bardelli2, L. Simonelli2, C. Magnani1, L. Varani2, A. Biondi1, S. Tettamanti1, E. Biagi1 1 Centro Ricerca M. Tettamanti, Clinica Pediatrica, Università Milano Bicocca, Osp. San Gerardo/Fondazione MBBM, Monza, MB, Italy, 2Istituto di Ricerca in Biomedicina, Università degli Studi della Svizzera Italiana, Bellinzona, Switzerland Chimeric Antigen Receptors (CARs)-redirected T lymphocytes are a promising immunotherapeutic approach, nowadays object of accurate preclinical evaluation also for Acute Myeloid Leukemia (AML) targeting. We recently developed a CAR against the CD123 antigen, found to be over-expressed on AML blasts and leukemic stem cells (LSCs). However, the potential recognition of low CD123-positive healthy tissues, through the so called “on-target-offorgan” effect, limits the safe clinical employment of CAR-T cells.
In search for an optimization of this strategy, we evaluated the effect of several variables implicated in the CAR design, known to modulate CAR T-cell functional profiles in a context-dependent manner, such as CAR binding affinity, CAR expression and target antigen density. Therefore, we developed a novel integrated model for the functional screening of in silico-selected CAR affinity mutants (CAM), starting from predicted antibody binding properties, that displayed different binding affinities: CAM-H1 (High Affinity 1), CAM-H2, which maintained the same binding affinity of the wild type (wt) anti-CD123 CAR (10−9 M), CAM-M (Medium affinity) and CAM-L (Low affinity), displaying a 10- and 100-fold affinity reduction, respectively. The in vitro functional characterization of Cytokine-Induced Killer (CIK) effector cells genetically modified with the CAMs has allowed to define both “lytic” and “activation” antigen thresholds showing that, while the early cytotoxic activity is not affected neither by CAR expression nor by CAR affinity tuning, the CAR expression represents the main variable impairing later effector functions. All these variables are essential for a further clinical translation of this approach, and the lowest affinity mutant could represent the one with an affinity threshold granting a proper balance between safety and efficacy profiles, below which the antileukemic efficacy could be impaired.
4 LOW IL-2 CONCENTRATION FAVORS GENERATION OF EARLY MEMORY T CELLS OVER TERMINAL EFFECTORS DURING CAR T-CELL EXPANSION A. Luostarinen1, T. Kaartinen1, P. Maliniemi1, J. Keto2, M. Arvas2, H. Belt3, J. Koponen3, A. Loskog4, S. Mustjoki5,6, K. Porkka5, S. Ylä-Herttuala3,7, M. Korhonen1 1 Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland, 2Research & Development, Finnish Red Cross Blood Service, Helsinki, Finland, 3Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland, 4Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden, 5 Hematology Research Unit Helsinki, Biomedicum Helsinki, Department of Medicine, Division of Hematology, University of Helsinki, Helsinki, Finland, 6 Department of Clinical Chemistry and Hematology, University of Helsinki, Helsinki, Finland, 7Heart Center, Kuopio University Hospital, Kuopio, Finland Adoptive T-cell therapy offers new options for cancer treatment. The therapeutic efficacy of T-cell therapy is linked to the persistence of administrated T cells, which is dependent on T-cell memory. Hence, the need for cells possessing early memory phenotypes in T-cell products is evident. The amount of interleukin-2 (IL-2) supplementation during in vitro expansion affects T-cell proliferation and effector differentiation. Here, we present a simplified and costeffective expansion method for the production of chimeric antigen receptor (CAR) T cells with potentially improved therapeutic capacity. Lymphocytes were transduced with third generation lentiviral vectors encoding CD19-targeted CARs, and expanded using CD3/CD28 microbeads and differing IL-2 concentrations (range from 0 to 300 IU/mL). After expanding the cells for 10 to 20-days, the effects of altering IL-2 levels and duration of expansion on the phenotype and functionality of the T-cell products were assessed. High IL-2 concentrations favored the generation of more differentiated effector T-cell phenotypes and decreased the proportion of early T memory (TM) cells. TM stem cells (TSCM, CD95+CD45RO−CD45RA+CD27+), which represent the most primitive T-cell memory phenotype with possibly the highest potential to provide long-term persistence, were not present in all expansions, but their existence was independent of IL-2 and could be linked to expansion kinetics. All CD19-CAR T-cell products demonstrated in vitro response against CD19+ leukemia cell lines, and the effector function was enhanced when T cells were expanded in high IL-2 concentration. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early TM cells, provided a tenfold cell expansion, and the cells were functionally potent. The memory T cell composition of T-cell preparations can be adjusted to favor of TM cells by limiting the length of expansion and using a low level of supplemental IL-2 in T-cell expansion. This method yields early memory T cells with potentially improved in vivo effectiveness. These findings are significant for robust and cost-effective T-cell manufacturing.
Oral Abstracts
to integrate the X5 scFv into a CAR to genetically modify HXTC and propose that X5 CAR expressing HXTC could be a potent therapeutic for the treatment of HIVpos individuals post-SCT, targeting multiple HIV antigens while preventing infection of infused CD4+ T cells.
2 SUCCESSFUL ENGRAFTMENT BUT HIGH VIRAL REACTIVATION AFTER REDUCED INTENSITY UNRELATED UMBILICAL CORD BLOOD TRANSPLANTATION FOR SICKLE CELL DISEASE A. Abraham1, A. Cluster2, D. Jacobsohn1, D. Delgado3, M. Hulbert2, D. Kukadiya1, L. Murray2, S. Shenoy2 1 Children’s National Medical Center, Washington, District of Columbia, United States, 2Washington University School of Medicine, St. Louis, Missouri, United States, 3Indiana University School of Medicine, Indianapolis, Indiana, United States Introduction: HLA-matched sibling donor (MSD) hematopoietic stem cell transplant is curative in >92% of patients with sickle cell disease (SCD) but only 18% have MSD. Unrelated cord blood transplantation (UCBT) expands donor options but is associated with high graft rejection rates (50–62%). We hypothesized that a previously used reduced intensity conditioning (RIC) regimen when combined with thiotepa would have acceptable toxicity and support donor engraftment after UCBT for SCD. Methods: Study patients were ≤21 years with severe SCD complications. UCB units were 5–6/6 HLA-matched. The RIC regimen included oral hydroxyurea 30 mg/kg (Days −50 to −21), and intravenous alemtuzumab 3 mg, 10 mg, 15 mg and 20 mg (Days −22 to −19), fludarabine 30 mg/m2/dose (Days −8 to −4), thiotepa 4 mg/kg × 2 (Day −4) and melphalan 140 mg/m2 (Day −3). GVHD prophylaxis consisted of a calcineurin inhibitor and MMF. Results: Ten children (6 male), median age 3.5 years (range 3–10) and weight 17.8 kg (range 15.1–37.6) underwent UCBT for cerebrovascular (n = 9) or recurrent vaso-occlusive (n = 1) SCD complications. Median cell dose was 7.2 × 107 TNC/kg (range 3.9–69.5). Follow up was 2.4 years (range 0.3–4.4). Median neutrophil (ANC ≥500/cumm) and platelet (≥50,000/cumm) engraftment were 19 (range 12–38) and 48 (range 28–204) days respectively. Eight patients have sustained donor engraftment. Two had autologous recovery on Days +33 and +39. Grade 3 acute GVHD developed in three, grade 4 in one patient. Mild (n = 2) and moderate (n = 1) chronic GVHD affected skin and fascia. Four of 5 patients >2 years post UCBT have stopped systemic immunosuppression. Eight patients tested positive for CMV or adenovirus by Day+100. Of these, 2 had CMV disease, one of which had pneumonitis and subsequent graft rejection. The second had pneumonitis before Day+30 and was treated with IV ganciclovir for 5 months for persistent viremia. CMV retinitis developed upon stopping IV ganciclovir and she received third party virus-specific T cells (VSTs) with anti-CMV activity with improvement in disease. One patient with recurrent adenoviremia was treated with donor UCB-derived VSTs on Day+117 and was able to stop IV cidofovir. Overall and disease free survival rates are 100% and 80% respectively. Conclusions: This RIC regimen supported engraftment in 8/10 UCBT recipients but early viral infections are of concern. Future efforts will focus on novel targeted therapies such as VSTs to reduce these complications.
3 BALANCE OF ANTI-CD123 CHIMERIC ANTIGEN RECEPTOR (CAR) BINDING AFFINITY AND DENSITY FOR THE TARGETING OF ACUTE MYELOID LEUKEMIA S. Arcangeli1, M. Rotiroti1, M. Bardelli2, L. Simonelli2, C. Magnani1, L. Varani2, A. Biondi1, S. Tettamanti1, E. Biagi1 1 Centro Ricerca M. Tettamanti, Clinica Pediatrica, Università Milano Bicocca, Osp. San Gerardo/Fondazione MBBM, Monza, MB, Italy, 2Istituto di Ricerca in Biomedicina, Università degli Studi della Svizzera Italiana, Bellinzona, Switzerland Chimeric Antigen Receptors (CARs)-redirected T lymphocytes are a promising immunotherapeutic approach, nowadays object of accurate preclinical evaluation also for Acute Myeloid Leukemia (AML) targeting. We recently developed a CAR against the CD123 antigen, found to be over-expressed on AML blasts and leukemic stem cells (LSCs). However, the potential recognition of low CD123-positive healthy tissues, through the so called “on-target-offorgan” effect, limits the safe clinical employment of CAR-T cells.
In search for an optimization of this strategy, we evaluated the effect of several variables implicated in the CAR design, known to modulate CAR T-cell functional profiles in a context-dependent manner, such as CAR binding affinity, CAR expression and target antigen density. Therefore, we developed a novel integrated model for the functional screening of in silico-selected CAR affinity mutants (CAM), starting from predicted antibody binding properties, that displayed different binding affinities: CAM-H1 (High Affinity 1), CAM-H2, which maintained the same binding affinity of the wild type (wt) anti-CD123 CAR (10−9 M), CAM-M (Medium affinity) and CAM-L (Low affinity), displaying a 10- and 100-fold affinity reduction, respectively. The in vitro functional characterization of Cytokine-Induced Killer (CIK) effector cells genetically modified with the CAMs has allowed to define both “lytic” and “activation” antigen thresholds showing that, while the early cytotoxic activity is not affected neither by CAR expression nor by CAR affinity tuning, the CAR expression represents the main variable impairing later effector functions. All these variables are essential for a further clinical translation of this approach, and the lowest affinity mutant could represent the one with an affinity threshold granting a proper balance between safety and efficacy profiles, below which the antileukemic efficacy could be impaired.
4 LOW IL-2 CONCENTRATION FAVORS GENERATION OF EARLY MEMORY T CELLS OVER TERMINAL EFFECTORS DURING CAR T-CELL EXPANSION A. Luostarinen1, T. Kaartinen1, P. Maliniemi1, J. Keto2, M. Arvas2, H. Belt3, J. Koponen3, A. Loskog4, S. Mustjoki5,6, K. Porkka5, S. Ylä-Herttuala3,7, M. Korhonen1 1 Advanced Cell Therapy Centre, Finnish Red Cross Blood Service, Helsinki, Finland, 2Research & Development, Finnish Red Cross Blood Service, Helsinki, Finland, 3Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland, 4Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden, 5 Hematology Research Unit Helsinki, Biomedicum Helsinki, Department of Medicine, Division of Hematology, University of Helsinki, Helsinki, Finland, 6 Department of Clinical Chemistry and Hematology, University of Helsinki, Helsinki, Finland, 7Heart Center, Kuopio University Hospital, Kuopio, Finland Adoptive T-cell therapy offers new options for cancer treatment. The therapeutic efficacy of T-cell therapy is linked to the persistence of administrated T cells, which is dependent on T-cell memory. Hence, the need for cells possessing early memory phenotypes in T-cell products is evident. The amount of interleukin-2 (IL-2) supplementation during in vitro expansion affects T-cell proliferation and effector differentiation. Here, we present a simplified and costeffective expansion method for the production of chimeric antigen receptor (CAR) T cells with potentially improved therapeutic capacity. Lymphocytes were transduced with third generation lentiviral vectors encoding CD19-targeted CARs, and expanded using CD3/CD28 microbeads and differing IL-2 concentrations (range from 0 to 300 IU/mL). After expanding the cells for 10 to 20-days, the effects of altering IL-2 levels and duration of expansion on the phenotype and functionality of the T-cell products were assessed. High IL-2 concentrations favored the generation of more differentiated effector T-cell phenotypes and decreased the proportion of early T memory (TM) cells. TM stem cells (TSCM, CD95+CD45RO−CD45RA+CD27+), which represent the most primitive T-cell memory phenotype with possibly the highest potential to provide long-term persistence, were not present in all expansions, but their existence was independent of IL-2 and could be linked to expansion kinetics. All CD19-CAR T-cell products demonstrated in vitro response against CD19+ leukemia cell lines, and the effector function was enhanced when T cells were expanded in high IL-2 concentration. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early TM cells, provided a tenfold cell expansion, and the cells were functionally potent. The memory T cell composition of T-cell preparations can be adjusted to favor of TM cells by limiting the length of expansion and using a low level of supplemental IL-2 in T-cell expansion. This method yields early memory T cells with potentially improved in vivo effectiveness. These findings are significant for robust and cost-effective T-cell manufacturing.