Mehrdad Hefazi - Academia.edu (original) (raw)

Papers by Mehrdad Hefazi

Research paper thumbnail of Efficacy of Donor Lymphocyte Infusions in Achieving Disease Control Among Myeloid Malignancy Patients Treated with an Allogeneic Hematopoietic Stem Cell Transplantation

Transplantation and Cellular Therapy

Research paper thumbnail of Elevated Pre-Transplant Ferritin Is Associated with Development of Liver Graft-Vs-Host Disease in Myelodysplastic Syndromes Following Allogeneic Stem Cell Transplantation

Transplantation and Cellular Therapy, 2022

Research paper thumbnail of Multi-institutional study evaluating clinical outcome with allogeneic hematopoietic stem cell transplantation after blinatumomab in patients with B-cell acute lymphoblastic leukemia: real-world data

Bone Marrow Transplantation, 2021

Safety and efficacy of allogeneic hematopoietic stem cell transplantation (alloHCT) consolidation... more Safety and efficacy of allogeneic hematopoietic stem cell transplantation (alloHCT) consolidation after blinatumomab is largely undetermined. To address this issue, we assembled multi-center data of relapsed refractory (RR) acute lymphocytic leukemia (ALL) patients who received alloHCT after blinatumomab. From December 2014 to May 2019, 223 patients who received blinatumomab for RR ALL outside clinical trials were identified. Among them, 106 (47%) patients transplanted post blinatumomab were evaluated for response and toxicity. Ninety-two (87%) patients received alloHCT after achieving CR, while remaining received subsequent salvage prior to undergoing alloHCT. Progression free survival (PFS) and overall survival (OS) at 2 years post alloHCT was 48% (95% CI: 36–59%) and 58% (95% CI: 45–69%), respectively. The cumulative incidence of GIII–IV aGVHD at 3 months was 9.9% (95% CI: 5.0–16.6%). Similarly, cumulative incidence of moderate to severe cGVHD at 2 years was 34.4% (95% CI: 23.7–45.3%). The overall survival at 2 years was not significantly different in patient who achieved CR with MRD negative (68.4% [95% CI: 28.5–89.1%]) compared to CR with MRD positive (63.4% [95% CI: 47.8–75.4%]) prior to alloHCT (p = 0.8). Our real-world analysis suggests that alloHCT is feasible and effective post blinatumomab in patients with RR ALL.

Research paper thumbnail of Clinical Outcome with Allogeneic Hematopoietic Stem Cell Transplantation after Blinatumomab or Inotuzumab Ozogamicin in Patients with B-Cell Acute Lymphoblastic Leukemia: Real World Experience

Biology of Blood and Marrow Transplantation, 2020

Research paper thumbnail of Low-Dose Total Body Irradiation (TBI) Use As Part of Pre-Transplant Conditioning Regimen Is Associated with Worse Outcomes in Patients with Severe Aplastic Anemia (SAA) Treated with Allogeneic Hematopoietic Stem Cell Transplantation (HCT)

Biology of Blood and Marrow Transplantation, 2020

to 43 £ 10^6/kg). The median myeloid engraftment was day +15 (range 11-30 days), and median plate... more to 43 £ 10^6/kg). The median myeloid engraftment was day +15 (range 11-30 days), and median platelet engraftment was day +16 (range 12-30 days). In eight of the nine patients, a sustained graft was achieved. One patient never achieved platelet engraftment and died because of cytomegalovirus infection. There was not treatment related toxicity reported. None of the patients had GVHD. The overall survival up to date is 88.8%. The eight surviving patients remain free of transfusions. Conclusions: Haplo-SCT could be established as first line treatment when there is no matched related or unrelated donor. According to this short sample and previous reports, PBSC is a feasible option to be effectively used as only source of stem cells.

Research paper thumbnail of Abstract 4082: Impact of immunosuppressive monocytes on CART19 cell effector functions and outcomes

Cancer Research

CD19 directed chimeric antigen receptor T (CART19) cell therapy has resulted in remarkable outcom... more CD19 directed chimeric antigen receptor T (CART19) cell therapy has resulted in remarkable outcomes in B cell malignancies and was FDA approved in multiple indications. However, durable remissions are limited to 40% of treated patients. Inhibitory myeloid cells in tumor microenvironment have been found to suppress T cell expansion and contribute to failure of CART19 cell therapy. In this study, we aimed to unravel the interactions between monocytes, CART19 cells and tumor cells to understand how monocytes-CART19 cell interactions impact CART19 cell effector functions and clinical outcomes. Two sets of experiments were conducted, 1) use of healthy CART19 cells, CD19+ tumor cells, and healthy monocytes; 2) use of brexu-cel products from ZUMA-2 clinical trial treating mantle cell lymphoma (MCL), patient-matched monocytes and circulating MCL tumor cells (n = 11; 6 durable responders, 2 relapsed after initial response and 3 non-responders). CD28 costimulated CART19 (CART19-28ζ) cells gen...

Research paper thumbnail of Supplemental Figure Video S2 from Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity

Supplemental Figure Video

Research paper thumbnail of Supplemental Figure Video S1 from Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity

Supplemental Figure Video

Research paper thumbnail of GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity

Leukemia

Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen rec... more Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigenspecific activation of GM-CSF KO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSF KO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models. Despite the remarkable activity of CD19-directed chimeric antigen receptor T cell (CART19) therapy in treating B-cell hematologic malignancies, 1, 2 limitations include 1) the development of potential life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS) 3, 4 and 2) lack of durable response. 5-8 Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CART cell toxicities and contribute to CART inhibition. 9-12 Specifically, and of relevance to the work presented in this manuscript, granulocyte-macrophage colony-stimulating factor (GM-CSF) is implicated in the development of NT and CRS after CART19 therapy based on correlative studies from pivotal clinical trials. 13, 14 Cox et al.

Research paper thumbnail of The Impact of Prior Treatment with a CD19 Targeting Monoclonal Antibody on Subsequent Treatment with CD19 Targeting CART Cell Therapy in Preclinical Models

Transplantation and Cellular Therapy, 2022

Research paper thumbnail of Favorable Modulation of Chimeric Antigen Receptor T Cells Safety and Efficacy By the Non-Covalent BTK Inhibitor Vecabrutinib

Blood, 2021

CD19-targeted chimeric antigen receptor T cell (CART19) therapy has been remarkably successful in... more CD19-targeted chimeric antigen receptor T cell (CART19) therapy has been remarkably successful in treating a subset of patients with hematological malignancies. However, it is associated with significant toxicities, including cytokine release syndrome (CRS) and neurotoxicity (NT). Furthermore, the rate of durable responses after CART19 therapy is low, and most patients develop resistance to the therapy. One of the predominant mechanisms for CART cell resistance is intrinsic T cell dysfunction which leads to a suboptimal CART product. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to favorably modulate CART phenotype and function through downregulation of inhibitory receptors and enhancement of CART cell efficacy in preclinical models and early clinical studies. However, ibrutinib is an irreversible receptor tyrosine kinase (RTK) inhibitor, and thus there are concerns that it inhibits CART cell proliferation and expansion. In contrast to ibrutinib and other...

Research paper thumbnail of Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity

Cancer Immunology Research, 2021

Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain ... more Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detect...

Research paper thumbnail of Efficient Gene Editing of CART Cells with CRISPR-Cas12a for Enhanced Antitumor Efficacy

Blood, 2020

Chimeric antigen receptor T (CART) cells are engineered with an artificial receptor which redirec... more Chimeric antigen receptor T (CART) cells are engineered with an artificial receptor which redirects T cells to recognize cancer cells expressing a particular surface antigen. CART cell therapy has been astonishingly successful at eradicating certain B cell malignancies, but relapse is common, and efficacy is lacking in many cancers. Gene editing of CART cells is being investigated to enhance efficacy and safety and to develop off-the-shelf products. Currently, genome engineering tools used to modify CART cells include zinc finger nucleases, transposons, TALENs, and CRISPR-Cas9. CRISPR-Cas9 uses a trans-activating (tracrRNA): CRISPR RNA (crRNA) duplex to trigger imprecise DNA repair through targeted double stranded breaks, causing indels and often resulting in loss of protein function. Gene-edited CART cells have entered the clinic to provide an allogeneic cell source (TALEN TCRα knockout), safer treatment (CRISPR-Cas9 GM-CSF knockout), and resistance to exhaustion (CRISPR-Cas9 PD-1 ...

Research paper thumbnail of Vesicular Stomatitis Virus (VSV) Engineered to Express CD19 Stimulates Anti-CD19 Chimeric Antigen Receptor Modified T Cells and Promotes Their Anti-Tumor Effects

Transplantation and Cellular Therapy, 2021

Research paper thumbnail of Circulating Extracellular Vesicles Induce Chimeric Antigen Receptor T Cell Dysfunction in Chronic Lymphocytic Leukemia (CLL)

Biology of Blood and Marrow Transplantation, 2020

vector (LVV). Patients received myeloablative busulfan conditioning, were infused with LentiGlobi... more vector (LVV). Patients received myeloablative busulfan conditioning, were infused with LentiGlobin drug product (DP) and monitored for adverse events (AEs), Hb fractions, and other parameters. LVV presence in transduced cells (%LVV+) was measured by qPCR of individual colonies from colony-forming unit assays from pre-infusion DP) and post-infusion from CD34+ bone marrow (BM) HSCs and peripheral blood mononuclear cells (PBMCs). Data are shown as median (min-max). Results: As of 7 March 2019, 13 Group C patients received DP, with follow-up of 9.0 (1.0-15.2) months. All but 1 patient had neutrophil and platelet engraftment as of the data cut date. Median HbS was 50% of total Hb in those with 6 months follow-up (n=8; Figure 1). Total unsupported Hb at last visit in patients with 6 months of follow-up was 11.5 (10.2-15.0) g/ dL, with HbA T87Q levels of 5.3 (4.5-8.8) g/dL. Six of these 8 patients had a history of VOCs or ACS; the annualized VOC+ACS rate decreased from 5.3 (3-14) pre-treatment to 0 (0-2) posttreatment (Figure 2). A decrease in hemolysis markers was also seen post-DP. Most common non-hematologic Grade 3 AEs were febrile neutropenia (n=10) and stomatitis (n=7). Serious AEs occurred in 6 patients; the most frequent were nausea and vomiting. To date, there have been no cases of DP-related AEs, graft failure, vector-mediated replication competent lentivirus, or clonal dominance. The %LVV+ colonies from PBMCs at 9 months and BM at 12 months post-DP infusion (n=5) were 79.2 (67.0-88.4) % and 81.5 (60.6-88.1) %, respectively, indicating stable engraftment of transduced cells from DP (%LVV+ was 80 [71-88] %). Conclusions: Patients in HGB-206 Group C show stable Lenti-Globin engraftment, with median total Hb >10 g/dL and median HbS 50% of total Hb in those with 6 months followup. The decrease in SCD-related complications and hemolysis in this cohort demonstrate a strong therapeutic benefit of Len-tiGlobin in patients with SCD.

Research paper thumbnail of Development of a Sensitive and Efficient Reporter Platform for the Detection of Chimeric Antigen Receptor T Cell Expansion, Trafficking, and Toxicity

Biology of Blood and Marrow Transplantation, 2020

Hematopoietic stem cell (HSC) transplantation is a widely successful treatment, but the risk of m... more Hematopoietic stem cell (HSC) transplantation is a widely successful treatment, but the risk of mortality remains. Preclinical modeling is critical to uncover the biological components that underlie poor patient outcomes. Zebrafish is a vertebrate animal with high genetic and cellular conservation with human hematopoiesis with many advantages to discover regulators of HSC transplant biology, such as high fecundity and short generation time. We developed a novel HSC transplant model using bloodless runx1 W84X mutant zebrafish which are devoid of endogenous HSCs. Most embryos die 8-12 days post fertilization (dpf) due to the absence of definitive hematopoiesis. We hypothesized that the empty HSC niche and lack of an immune system would prevent graft rejection, making robust HSC engraftment possible. We transplanted donor marrow ubiquitously expressing green fluorescent protein (ubi:GFP) via intravascular injection into runx1 homozygous mutants and heterozygotes at 2dpf. Sham-injected and uninjected embryos served as negative controls. We transplanted an average of 100-200 recipients per experimental day. Survival was significantly improved in transplanted runx1 mutants, with 64% surviving in the transplanted cohort compared to 5% in the sham-injected controls, suggesting HSC transplantation likely supplied these fish with a functional hematopoietic compartment critical for survival. Chimerism was quantified by flow cytometry 8 weeks posttransplant. Successful engraftment was defined as >5% GFP+ myeloid cells. Over 99% of animals meeting this criterion also showed robust multi-lineage engraftment. Over 70% of runx1 mutant recipients were engrafted compared to only 3% of the runx1 heterozygotes. The myeloid chimerism of engrafted mutant fish was 84% (+/-25%), while the engrafted heterozygous control had only 21% myeloid chimerism. Transplanted fish remain robustly engrafted >6 months post-transplant. The runx1 mutants supported HSC self-renewal, as the GFP+ marrow cells from primary recipients were able to robustly engraft secondary runx1 mutant hosts. We also demonstrated that competitive transplantation in runx1 mutants can be used to measure HSC frequency, a critical feature needed to functionally and quantitatively assess HSC potential following genetic or pharmacological perturbations. These data demonstrate that the runx1 mutant zebrafish is an advantageous HSC transplant host that allows quantification of long-term serially-repopulating true HSCs. The advantages of our zebrafish transplant model allow the real-time visualization of stem cell trafficking and homing in a healthy, uninjured niche and the ability to perform large-scale screens to identify drugs that modify engraftment. This will provide unprecedented insight into both the donor and host factors needed for robust HSC engraftment that will be helpful in improving human HSC graft outcomes.

Research paper thumbnail of Relapses after Achieving EFS24 in Patients with Diffuse Large B-Cell Lymphoma in the Rituximab Era

Blood, 2018

Introduction: The addition of Rituximab to chemotherapy has significantly improved the outcome of... more Introduction: The addition of Rituximab to chemotherapy has significantly improved the outcome of patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL). Patients treated with immunochemotherapy for DLBCL who achieve EFS24 (event-free for 2 years after diagnosis) have an overall survival equivalent to that of the age- and sex-matched general population. Relapses after achieving EFS24 have been considered to be unusual but have been understudied. We sought to define the rate, clinical characteristics, treatment pattern, and outcomes of such relapses. Methods: 1448 patients with newly diagnosed DLBCL from March 2002 to June 2015 were included. Patients were enrolled in the Molecular Epidemiology Resource (MER) of the University of Iowa/Mayo Clinic Lymphoma SPORE, treated per physician choice (predominantly R-CHOP immunochemotherapy) and followed prospectively. An event was defined as progression or relapse, unplanned re-treatment after initial therapy, or death from any c...

Research paper thumbnail of Non-internal tandem duplication (ITD), non-tyrosine kinase domain (TKD) FLT3 mutations in myeloid malignancies: A brief report of 10 patients

Research paper thumbnail of Real World Outcomes of Adult B-Cell Acute Lymphocytic Leukemia Patients Treated with Blinatumomab

Blood, 2019

Background Blinatumomab is a bispecific T cell antibody construct, that binds and allows CD-3 pos... more Background Blinatumomab is a bispecific T cell antibody construct, that binds and allows CD-3 positive cytotoxic T cells to recognize and eradicate CD19-positive acute lymphocytic leukemia (ALL) blasts. The drug is approved for patient (pts) with relapsed/ refractory (RR) B-cell ALL and those with minimal residual disease (MRD) based on efficacy in clinical trials. We sought to evaluate the safety and efficacy of blinatumomab in the "real world" setting. Methods We conducted a retrospective multicenter study in collaboration with 11 U.S. academic institutions, and evaluated the outcome of RR B-cell ALL pts, who received blinatumomab outside of clinical trials. These pts were evaluated for response, duration of response (DOR), overall survival (OS) from the time of blinatumomab initiation and toxicity. DOR was estimated among pts who had achieved complete remission (CR)/CR with incomplete count recovery (CRi). Survival curves were estimated using the Kaplan-Meier method and...

Research paper thumbnail of Fludarabine Busulfan Compared to Fludarabine Melphalan Is Associated with Increased Relapse Risk in Reduced Intensity Conditioning Transplant Despite Pharmacokinetic Dosing

Blood, 2015

Background: Fludarabine plus busulfan (FB) and fludarabine plus melphalan (FM) are two commonly u... more Background: Fludarabine plus busulfan (FB) and fludarabine plus melphalan (FM) are two commonly used reduced intensity conditioning (RIC) regimens for allogeneic hematopoietic stem cell transplantation (SCT). A recent analysis showed that both regimens had similar overall survival (OS) but relapse incidence (RI) was significantly higher with FB with a trend towards higher non-relapse mortality (NRM) in FM (Baron et al., Cancer 2015). However, that cohort included patients treated with oral and intravenous busulfan without pharmakokinetic targeting of intravenous which may impact anti-leukemic activity. Aim: Compare transplant related outcomes of FB vs. FM using intravenous (IV) busulfan targeted to the area under the curve (AUC). Methods: After due IRB approval, patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS) receiving RIC-SCT from 2008-2014 were identified. All baseline and laboratory features were retrospectively extracted. Busulfan dose in FB wa...

Research paper thumbnail of Efficacy of Donor Lymphocyte Infusions in Achieving Disease Control Among Myeloid Malignancy Patients Treated with an Allogeneic Hematopoietic Stem Cell Transplantation

Transplantation and Cellular Therapy

Research paper thumbnail of Elevated Pre-Transplant Ferritin Is Associated with Development of Liver Graft-Vs-Host Disease in Myelodysplastic Syndromes Following Allogeneic Stem Cell Transplantation

Transplantation and Cellular Therapy, 2022

Research paper thumbnail of Multi-institutional study evaluating clinical outcome with allogeneic hematopoietic stem cell transplantation after blinatumomab in patients with B-cell acute lymphoblastic leukemia: real-world data

Bone Marrow Transplantation, 2021

Safety and efficacy of allogeneic hematopoietic stem cell transplantation (alloHCT) consolidation... more Safety and efficacy of allogeneic hematopoietic stem cell transplantation (alloHCT) consolidation after blinatumomab is largely undetermined. To address this issue, we assembled multi-center data of relapsed refractory (RR) acute lymphocytic leukemia (ALL) patients who received alloHCT after blinatumomab. From December 2014 to May 2019, 223 patients who received blinatumomab for RR ALL outside clinical trials were identified. Among them, 106 (47%) patients transplanted post blinatumomab were evaluated for response and toxicity. Ninety-two (87%) patients received alloHCT after achieving CR, while remaining received subsequent salvage prior to undergoing alloHCT. Progression free survival (PFS) and overall survival (OS) at 2 years post alloHCT was 48% (95% CI: 36–59%) and 58% (95% CI: 45–69%), respectively. The cumulative incidence of GIII–IV aGVHD at 3 months was 9.9% (95% CI: 5.0–16.6%). Similarly, cumulative incidence of moderate to severe cGVHD at 2 years was 34.4% (95% CI: 23.7–45.3%). The overall survival at 2 years was not significantly different in patient who achieved CR with MRD negative (68.4% [95% CI: 28.5–89.1%]) compared to CR with MRD positive (63.4% [95% CI: 47.8–75.4%]) prior to alloHCT (p = 0.8). Our real-world analysis suggests that alloHCT is feasible and effective post blinatumomab in patients with RR ALL.

Research paper thumbnail of Clinical Outcome with Allogeneic Hematopoietic Stem Cell Transplantation after Blinatumomab or Inotuzumab Ozogamicin in Patients with B-Cell Acute Lymphoblastic Leukemia: Real World Experience

Biology of Blood and Marrow Transplantation, 2020

Research paper thumbnail of Low-Dose Total Body Irradiation (TBI) Use As Part of Pre-Transplant Conditioning Regimen Is Associated with Worse Outcomes in Patients with Severe Aplastic Anemia (SAA) Treated with Allogeneic Hematopoietic Stem Cell Transplantation (HCT)

Biology of Blood and Marrow Transplantation, 2020

to 43 £ 10^6/kg). The median myeloid engraftment was day +15 (range 11-30 days), and median plate... more to 43 £ 10^6/kg). The median myeloid engraftment was day +15 (range 11-30 days), and median platelet engraftment was day +16 (range 12-30 days). In eight of the nine patients, a sustained graft was achieved. One patient never achieved platelet engraftment and died because of cytomegalovirus infection. There was not treatment related toxicity reported. None of the patients had GVHD. The overall survival up to date is 88.8%. The eight surviving patients remain free of transfusions. Conclusions: Haplo-SCT could be established as first line treatment when there is no matched related or unrelated donor. According to this short sample and previous reports, PBSC is a feasible option to be effectively used as only source of stem cells.

Research paper thumbnail of Abstract 4082: Impact of immunosuppressive monocytes on CART19 cell effector functions and outcomes

Cancer Research

CD19 directed chimeric antigen receptor T (CART19) cell therapy has resulted in remarkable outcom... more CD19 directed chimeric antigen receptor T (CART19) cell therapy has resulted in remarkable outcomes in B cell malignancies and was FDA approved in multiple indications. However, durable remissions are limited to 40% of treated patients. Inhibitory myeloid cells in tumor microenvironment have been found to suppress T cell expansion and contribute to failure of CART19 cell therapy. In this study, we aimed to unravel the interactions between monocytes, CART19 cells and tumor cells to understand how monocytes-CART19 cell interactions impact CART19 cell effector functions and clinical outcomes. Two sets of experiments were conducted, 1) use of healthy CART19 cells, CD19+ tumor cells, and healthy monocytes; 2) use of brexu-cel products from ZUMA-2 clinical trial treating mantle cell lymphoma (MCL), patient-matched monocytes and circulating MCL tumor cells (n = 11; 6 durable responders, 2 relapsed after initial response and 3 non-responders). CD28 costimulated CART19 (CART19-28ζ) cells gen...

Research paper thumbnail of Supplemental Figure Video S2 from Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity

Supplemental Figure Video

Research paper thumbnail of Supplemental Figure Video S1 from Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity

Supplemental Figure Video

Research paper thumbnail of GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity

Leukemia

Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen rec... more Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigenspecific activation of GM-CSF KO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSF KO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models. Despite the remarkable activity of CD19-directed chimeric antigen receptor T cell (CART19) therapy in treating B-cell hematologic malignancies, 1, 2 limitations include 1) the development of potential life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS) 3, 4 and 2) lack of durable response. 5-8 Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CART cell toxicities and contribute to CART inhibition. 9-12 Specifically, and of relevance to the work presented in this manuscript, granulocyte-macrophage colony-stimulating factor (GM-CSF) is implicated in the development of NT and CRS after CART19 therapy based on correlative studies from pivotal clinical trials. 13, 14 Cox et al.

Research paper thumbnail of The Impact of Prior Treatment with a CD19 Targeting Monoclonal Antibody on Subsequent Treatment with CD19 Targeting CART Cell Therapy in Preclinical Models

Transplantation and Cellular Therapy, 2022

Research paper thumbnail of Favorable Modulation of Chimeric Antigen Receptor T Cells Safety and Efficacy By the Non-Covalent BTK Inhibitor Vecabrutinib

Blood, 2021

CD19-targeted chimeric antigen receptor T cell (CART19) therapy has been remarkably successful in... more CD19-targeted chimeric antigen receptor T cell (CART19) therapy has been remarkably successful in treating a subset of patients with hematological malignancies. However, it is associated with significant toxicities, including cytokine release syndrome (CRS) and neurotoxicity (NT). Furthermore, the rate of durable responses after CART19 therapy is low, and most patients develop resistance to the therapy. One of the predominant mechanisms for CART cell resistance is intrinsic T cell dysfunction which leads to a suboptimal CART product. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to favorably modulate CART phenotype and function through downregulation of inhibitory receptors and enhancement of CART cell efficacy in preclinical models and early clinical studies. However, ibrutinib is an irreversible receptor tyrosine kinase (RTK) inhibitor, and thus there are concerns that it inhibits CART cell proliferation and expansion. In contrast to ibrutinib and other...

Research paper thumbnail of Development of a Clinically Relevant Reporter for Chimeric Antigen Receptor T-cell Expansion, Trafficking, and Toxicity

Cancer Immunology Research, 2021

Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain ... more Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detect...

Research paper thumbnail of Efficient Gene Editing of CART Cells with CRISPR-Cas12a for Enhanced Antitumor Efficacy

Blood, 2020

Chimeric antigen receptor T (CART) cells are engineered with an artificial receptor which redirec... more Chimeric antigen receptor T (CART) cells are engineered with an artificial receptor which redirects T cells to recognize cancer cells expressing a particular surface antigen. CART cell therapy has been astonishingly successful at eradicating certain B cell malignancies, but relapse is common, and efficacy is lacking in many cancers. Gene editing of CART cells is being investigated to enhance efficacy and safety and to develop off-the-shelf products. Currently, genome engineering tools used to modify CART cells include zinc finger nucleases, transposons, TALENs, and CRISPR-Cas9. CRISPR-Cas9 uses a trans-activating (tracrRNA): CRISPR RNA (crRNA) duplex to trigger imprecise DNA repair through targeted double stranded breaks, causing indels and often resulting in loss of protein function. Gene-edited CART cells have entered the clinic to provide an allogeneic cell source (TALEN TCRα knockout), safer treatment (CRISPR-Cas9 GM-CSF knockout), and resistance to exhaustion (CRISPR-Cas9 PD-1 ...

Research paper thumbnail of Vesicular Stomatitis Virus (VSV) Engineered to Express CD19 Stimulates Anti-CD19 Chimeric Antigen Receptor Modified T Cells and Promotes Their Anti-Tumor Effects

Transplantation and Cellular Therapy, 2021

Research paper thumbnail of Circulating Extracellular Vesicles Induce Chimeric Antigen Receptor T Cell Dysfunction in Chronic Lymphocytic Leukemia (CLL)

Biology of Blood and Marrow Transplantation, 2020

vector (LVV). Patients received myeloablative busulfan conditioning, were infused with LentiGlobi... more vector (LVV). Patients received myeloablative busulfan conditioning, were infused with LentiGlobin drug product (DP) and monitored for adverse events (AEs), Hb fractions, and other parameters. LVV presence in transduced cells (%LVV+) was measured by qPCR of individual colonies from colony-forming unit assays from pre-infusion DP) and post-infusion from CD34+ bone marrow (BM) HSCs and peripheral blood mononuclear cells (PBMCs). Data are shown as median (min-max). Results: As of 7 March 2019, 13 Group C patients received DP, with follow-up of 9.0 (1.0-15.2) months. All but 1 patient had neutrophil and platelet engraftment as of the data cut date. Median HbS was 50% of total Hb in those with 6 months follow-up (n=8; Figure 1). Total unsupported Hb at last visit in patients with 6 months of follow-up was 11.5 (10.2-15.0) g/ dL, with HbA T87Q levels of 5.3 (4.5-8.8) g/dL. Six of these 8 patients had a history of VOCs or ACS; the annualized VOC+ACS rate decreased from 5.3 (3-14) pre-treatment to 0 (0-2) posttreatment (Figure 2). A decrease in hemolysis markers was also seen post-DP. Most common non-hematologic Grade 3 AEs were febrile neutropenia (n=10) and stomatitis (n=7). Serious AEs occurred in 6 patients; the most frequent were nausea and vomiting. To date, there have been no cases of DP-related AEs, graft failure, vector-mediated replication competent lentivirus, or clonal dominance. The %LVV+ colonies from PBMCs at 9 months and BM at 12 months post-DP infusion (n=5) were 79.2 (67.0-88.4) % and 81.5 (60.6-88.1) %, respectively, indicating stable engraftment of transduced cells from DP (%LVV+ was 80 [71-88] %). Conclusions: Patients in HGB-206 Group C show stable Lenti-Globin engraftment, with median total Hb >10 g/dL and median HbS 50% of total Hb in those with 6 months followup. The decrease in SCD-related complications and hemolysis in this cohort demonstrate a strong therapeutic benefit of Len-tiGlobin in patients with SCD.

Research paper thumbnail of Development of a Sensitive and Efficient Reporter Platform for the Detection of Chimeric Antigen Receptor T Cell Expansion, Trafficking, and Toxicity

Biology of Blood and Marrow Transplantation, 2020

Hematopoietic stem cell (HSC) transplantation is a widely successful treatment, but the risk of m... more Hematopoietic stem cell (HSC) transplantation is a widely successful treatment, but the risk of mortality remains. Preclinical modeling is critical to uncover the biological components that underlie poor patient outcomes. Zebrafish is a vertebrate animal with high genetic and cellular conservation with human hematopoiesis with many advantages to discover regulators of HSC transplant biology, such as high fecundity and short generation time. We developed a novel HSC transplant model using bloodless runx1 W84X mutant zebrafish which are devoid of endogenous HSCs. Most embryos die 8-12 days post fertilization (dpf) due to the absence of definitive hematopoiesis. We hypothesized that the empty HSC niche and lack of an immune system would prevent graft rejection, making robust HSC engraftment possible. We transplanted donor marrow ubiquitously expressing green fluorescent protein (ubi:GFP) via intravascular injection into runx1 homozygous mutants and heterozygotes at 2dpf. Sham-injected and uninjected embryos served as negative controls. We transplanted an average of 100-200 recipients per experimental day. Survival was significantly improved in transplanted runx1 mutants, with 64% surviving in the transplanted cohort compared to 5% in the sham-injected controls, suggesting HSC transplantation likely supplied these fish with a functional hematopoietic compartment critical for survival. Chimerism was quantified by flow cytometry 8 weeks posttransplant. Successful engraftment was defined as >5% GFP+ myeloid cells. Over 99% of animals meeting this criterion also showed robust multi-lineage engraftment. Over 70% of runx1 mutant recipients were engrafted compared to only 3% of the runx1 heterozygotes. The myeloid chimerism of engrafted mutant fish was 84% (+/-25%), while the engrafted heterozygous control had only 21% myeloid chimerism. Transplanted fish remain robustly engrafted >6 months post-transplant. The runx1 mutants supported HSC self-renewal, as the GFP+ marrow cells from primary recipients were able to robustly engraft secondary runx1 mutant hosts. We also demonstrated that competitive transplantation in runx1 mutants can be used to measure HSC frequency, a critical feature needed to functionally and quantitatively assess HSC potential following genetic or pharmacological perturbations. These data demonstrate that the runx1 mutant zebrafish is an advantageous HSC transplant host that allows quantification of long-term serially-repopulating true HSCs. The advantages of our zebrafish transplant model allow the real-time visualization of stem cell trafficking and homing in a healthy, uninjured niche and the ability to perform large-scale screens to identify drugs that modify engraftment. This will provide unprecedented insight into both the donor and host factors needed for robust HSC engraftment that will be helpful in improving human HSC graft outcomes.

Research paper thumbnail of Relapses after Achieving EFS24 in Patients with Diffuse Large B-Cell Lymphoma in the Rituximab Era

Blood, 2018

Introduction: The addition of Rituximab to chemotherapy has significantly improved the outcome of... more Introduction: The addition of Rituximab to chemotherapy has significantly improved the outcome of patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL). Patients treated with immunochemotherapy for DLBCL who achieve EFS24 (event-free for 2 years after diagnosis) have an overall survival equivalent to that of the age- and sex-matched general population. Relapses after achieving EFS24 have been considered to be unusual but have been understudied. We sought to define the rate, clinical characteristics, treatment pattern, and outcomes of such relapses. Methods: 1448 patients with newly diagnosed DLBCL from March 2002 to June 2015 were included. Patients were enrolled in the Molecular Epidemiology Resource (MER) of the University of Iowa/Mayo Clinic Lymphoma SPORE, treated per physician choice (predominantly R-CHOP immunochemotherapy) and followed prospectively. An event was defined as progression or relapse, unplanned re-treatment after initial therapy, or death from any c...

Research paper thumbnail of Non-internal tandem duplication (ITD), non-tyrosine kinase domain (TKD) FLT3 mutations in myeloid malignancies: A brief report of 10 patients

Research paper thumbnail of Real World Outcomes of Adult B-Cell Acute Lymphocytic Leukemia Patients Treated with Blinatumomab

Blood, 2019

Background Blinatumomab is a bispecific T cell antibody construct, that binds and allows CD-3 pos... more Background Blinatumomab is a bispecific T cell antibody construct, that binds and allows CD-3 positive cytotoxic T cells to recognize and eradicate CD19-positive acute lymphocytic leukemia (ALL) blasts. The drug is approved for patient (pts) with relapsed/ refractory (RR) B-cell ALL and those with minimal residual disease (MRD) based on efficacy in clinical trials. We sought to evaluate the safety and efficacy of blinatumomab in the "real world" setting. Methods We conducted a retrospective multicenter study in collaboration with 11 U.S. academic institutions, and evaluated the outcome of RR B-cell ALL pts, who received blinatumomab outside of clinical trials. These pts were evaluated for response, duration of response (DOR), overall survival (OS) from the time of blinatumomab initiation and toxicity. DOR was estimated among pts who had achieved complete remission (CR)/CR with incomplete count recovery (CRi). Survival curves were estimated using the Kaplan-Meier method and...

Research paper thumbnail of Fludarabine Busulfan Compared to Fludarabine Melphalan Is Associated with Increased Relapse Risk in Reduced Intensity Conditioning Transplant Despite Pharmacokinetic Dosing

Blood, 2015

Background: Fludarabine plus busulfan (FB) and fludarabine plus melphalan (FM) are two commonly u... more Background: Fludarabine plus busulfan (FB) and fludarabine plus melphalan (FM) are two commonly used reduced intensity conditioning (RIC) regimens for allogeneic hematopoietic stem cell transplantation (SCT). A recent analysis showed that both regimens had similar overall survival (OS) but relapse incidence (RI) was significantly higher with FB with a trend towards higher non-relapse mortality (NRM) in FM (Baron et al., Cancer 2015). However, that cohort included patients treated with oral and intravenous busulfan without pharmakokinetic targeting of intravenous which may impact anti-leukemic activity. Aim: Compare transplant related outcomes of FB vs. FM using intravenous (IV) busulfan targeted to the area under the curve (AUC). Methods: After due IRB approval, patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS) receiving RIC-SCT from 2008-2014 were identified. All baseline and laboratory features were retrospectively extracted. Busulfan dose in FB wa...