Publications by authors named "Terry J Fry"

91 Publications

Long-Term Follow-Up of CD19-CAR T-Cell Therapy in Children and Young Adults With B-ALL.

J Clin Oncol 2021 Mar 25:JCO2002262. Epub 2021 Mar 25.

Pediatric Oncology Branch, Center for Cancer Research (CCR), National Cancer Institute (NCI), NIH, Bethesda, MD.

Purpose: CD19 chimeric antigen receptor (CD19-CAR) T cells induce high response rates in children and young adults (CAYAs) with B-cell acute lymphoblastic leukemia (B-ALL), but relapse rates are high. The role for allogeneic hematopoietic stem-cell transplant (alloHSCT) following CD19-CAR T-cell therapy to improve long-term outcomes in CAYAs has not been examined.

Methods: We conducted a phase I trial of autologous CD19.28ζ-CAR T cells in CAYAs with relapsed or refractory B-ALL. Response and long-term clinical outcomes were assessed in relation to disease and treatment variables.

Results: Fifty CAYAs with B-ALL were treated (median age, 13.5 years; range, 4.3-30.4). Thirty-one (62.0%) patients achieved a complete remission (CR), 28 (90.3%) of whom were minimal residual disease-negative by flow cytometry. Utilization of fludarabine/cyclophosphamide-based lymphodepletion was associated with improved CR rates (29/42, 69%) compared with non-fludarabine/cyclophosphamide-based lymphodepletion (2/8, 25%; = .041). With median follow-up of 4.8 years, median overall survival was 10.5 months (95% CI, 6.3 to 29.2 months). Twenty-one of 28 (75.0%) patients achieving a minimal residual disease-negative CR proceeded to alloHSCT. For those proceeding to alloHSCT, median overall survival was 70.2 months (95% CI, 10.4 months to not estimable). The cumulative incidence of relapse after alloHSCT was 9.5% (95% CI, 1.5 to 26.8) at 24 months; 5-year EFS following alloHSCT was 61.9% (95% CI, 38.1 to 78.8).

Conclusion: We provide the longest follow-up in CAYAs with B-ALL after CD19-CAR T-cell therapy reported to date and demonstrate that sequential therapy with CD19.28ζ-CAR T cells followed by alloHSCT can mediate durable disease control in a sizable fraction of CAYAs with relapsed or refractory B-ALL (ClinicalTrials.gov identifier: NCT01593696).
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http://dx.doi.org/10.1200/JCO.20.02262DOI Listing
March 2021

Robust anti-tumor activity and low cytokine production by novel humanized anti-CD19 CAR-T cells.

Mol Cancer Ther 2021 Feb 25. Epub 2021 Feb 25.

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome (CRS) which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR-T cells by humanizing the framework region of scFv derived from a murine FMC63 mAb and combining it with CD8a transmembrane domain, 4-1BB costimulatory domain and CD3ζ signalling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics (MD) simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared to murine scFv (mCAR19). Ex vivo studies with CAR-T cells generated from healthy donors and patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable anti-tumor activity and proliferation. More importantly, h1CAR19-8BBζ-T cells produced lower levels of cytokines (IFN-g, TNF-a upon antigen encounter and reduced the induction of IL-6 cytokine from monocytes than mCAR19-8BBζ-T cells. There was a comparable proliferation of h1CAR19-8BBζ-T cells and mCAR19-8BBζ-T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ-T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0476DOI Listing
February 2021

Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immune effector cell-related adverse events.

J Immunother Cancer 2020 12;8(2)

Cancer Immunotherapy Program, Division of Oncology, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania, USA

Immune effector cell (IEC) therapies offer durable and sustained remissions in significant numbers of patients with hematological cancers. While these unique immunotherapies have improved outcomes for pediatric and adult patients in a number of disease states, as 'living drugs,' their toxicity profiles, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), differ markedly from conventional cancer therapeutics. At the time of article preparation, the US Food and Drug Administration (FDA) has approved tisagenlecleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel, all of which are IEC therapies based on genetically modified T cells engineered to express chimeric antigen receptors (CARs), and additional products are expected to reach marketing authorization soon and to enter clinical development in due course. As IEC therapies, especially CAR T cell therapies, enter more widespread clinical use, there is a need for clear, cohesive recommendations on toxicity management, motivating the Society for Immunotherapy of Cancer (SITC) to convene an expert panel to develop a clinical practice guideline. The panel discussed the recognition and management of common toxicities in the context of IEC treatment, including baseline laboratory parameters for monitoring, timing to onset, and pharmacological interventions, ultimately forming evidence- and consensus-based recommendations to assist medical professionals in decision-making and to improve outcomes for patients.
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http://dx.doi.org/10.1136/jitc-2020-001511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745688PMC
December 2020

Diagnostic approach to the evaluation of myeloid malignancies following CAR T-cell therapy in B-cell acute lymphoblastic leukemia.

J Immunother Cancer 2020 11;8(2)

Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland, USA

Immunotherapeutic strategies targeting B-cell acute lymphoblastic leukemia (B-ALL) effectively induce remission; however, disease recurrence remains a challenge. Due to the potential for antigen loss, antigen diminution, lineage switch or development of a secondary or treatment-related malignancy, the phenotype and manifestation of subsequent leukemia may be elusive. We report on two patients with multiply relapsed/refractory B-ALL who, following chimeric antigen receptor T-cell therapy, developed myeloid malignancies. In the first case, a myeloid sarcoma developed in a patient with a history of myelodysplastic syndrome. In the second case, two distinct events occurred. The first event represented a donor-derived myelodysplastic syndrome with monosomy 7 in a patient with a prior hematopoietic stem cell transplantation. This patient went on to present with lineage switch of her original B-ALL to ambiguous lineage T/myeloid acute leukemia. With the rapidly evolving field of novel immunotherapeutic strategies, evaluation of relapse and/or subsequent neoplasms is becoming increasingly more complex. By virtue of these uniquely complex cases, we provide a framework for the evaluation of relapse or evolution of a subsequent malignancy following antigen-targeted immunotherapy.
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http://dx.doi.org/10.1136/jitc-2020-001563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703409PMC
November 2020

Fatal capillary leak syndrome in a child with acute lymphoblastic leukemia treated with moxetumomab pasudotox for pre-transplant minimal residual disease reduction.

Pediatr Blood Cancer 2021 Jan 22;68(1):e28574. Epub 2020 Sep 22.

Pediatric Hematology-Oncology, Children's Hospital Los Angeles, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California.

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http://dx.doi.org/10.1002/pbc.28574DOI Listing
January 2021

Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients.

J Clin Invest 2020 10;130(10):5425-5443

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute (NCI), NIH.

Late-onset inflammatory toxicities resembling hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) occur after chimeric antigen receptor T cell (CAR T cell) infusion and represent a therapeutic challenge. Given the established link between perforin deficiency and primary HLH, we investigated the role of perforin in anti-CD19 CAR T cell efficacy and HLH-like toxicities in a syngeneic murine model. Perforin contributed to both CD8+ and CD4+ CAR T cell cytotoxicity but was not required for in vitro or in vivo leukemia clearance. Upon CAR-mediated in vitro activation, perforin-deficient CAR T cells produced higher amounts of proinflammatory cytokines compared with WT CAR T cells. Following in vivo clearance of leukemia, perforin-deficient CAR T cells reexpanded, resulting in splenomegaly with disruption of normal splenic architecture and the presence of hemophagocytes, which are findings reminiscent of HLH. Notably, a substantial fraction of patients who received anti-CD22 CAR T cells also experienced biphasic inflammation, with the second phase occurring after the resolution of cytokine release syndrome, resembling clinical manifestations of HLH. Elevated inflammatory cytokines such as IL-1β and IL-18 and concurrent late CAR T cell expansion characterized the HLH-like syndromes occurring in the murine model and in humans. Thus, a murine model of perforin-deficient CAR T cells recapitulated late-onset inflammatory toxicities occurring in human CAR T cell recipients, providing therapeutically relevant mechanistic insights.
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http://dx.doi.org/10.1172/JCI130059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524496PMC
October 2020

CD4/CD8 T-Cell Selection Affects Chimeric Antigen Receptor (CAR) T-Cell Potency and Toxicity: Updated Results From a Phase I Anti-CD22 CAR T-Cell Trial.

J Clin Oncol 2020 06 14;38(17):1938-1950. Epub 2020 Apr 14.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.

Purpose: Patients with B-cell acute lymphoblastic leukemia who experience relapse after or are resistant to CD19-targeted immunotherapies have limited treatment options. Targeting CD22, an alternative B-cell antigen, represents an alternate strategy. We report outcomes on the largest patient cohort treated with CD22 chimeric antigen receptor (CAR) T cells.

Patients And Methods: We conducted a single-center, phase I, 3 + 3 dose-escalation trial with a large expansion cohort that tested CD22-targeted CAR T cells for children and young adults with relapsed/refractory CD22 malignancies. Primary objectives were to assess the safety, toxicity, and feasibility. Secondary objectives included efficacy, CD22 CAR T-cell persistence, and cytokine profiling.

Results: Fifty-eight participants were infused; 51 (87.9%) after prior CD19-targeted therapy. Cytokine release syndrome occurred in 50 participants (86.2%) and was grade 1-2 in 45 (90%). Symptoms of neurotoxicity were minimal and transient. Hemophagocytic lymphohistiocytosis-like manifestations were seen in 19/58 (32.8%) of subjects, prompting utilization of anakinra. CD4/CD8 T-cell selection of the apheresis product improved CAR T-cell manufacturing feasibility as well as heightened inflammatory toxicities, leading to dose de-escalation. The complete remission rate was 70%. The median overall survival was 13.4 months (95% CI, 7.7 to 20.3 months). Among those who achieved a complete response, the median relapse-free survival was 6.0 months (95% CI, 4.1 to 6.5 months). Thirteen participants proceeded to stem-cell transplantation.

Conclusion: In the largest experience of CD22 CAR T-cells to our knowledge, we provide novel information on the impact of manufacturing changes on clinical outcomes and report on unique CD22 CAR T-cell toxicities and toxicity mitigation strategies. The remission induction rate supports further development of CD22 CAR T cells as a therapeutic option in patients resistant to CD19-targeted immunotherapy.
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http://dx.doi.org/10.1200/JCO.19.03279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280047PMC
June 2020

Using erythrocyte tools to enhance CAR T cells.

Blood 2020 02;135(9):595-596

University of Colorado Anschutz; Children's Hospital Colorado.

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http://dx.doi.org/10.1182/blood.2019003944DOI Listing
February 2020

Serial evaluation of CD19 surface expression in pediatric B-cell malignancies following CD19-targeted therapy.

Leukemia 2020 11 26;34(11):3064-3069. Epub 2020 Feb 26.

Pediatric Oncology Branch, Center for Cancer Research (CCR), National Cancer Institute (NCI), NIH, Bethesda, MD, USA.

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http://dx.doi.org/10.1038/s41375-020-0760-xDOI Listing
November 2020

Disease detection methodologies in relapsed B-cell acute lymphoblastic leukemia: Opportunities for improvement.

Pediatr Blood Cancer 2020 04 25;67(4):e28149. Epub 2020 Jan 25.

Pediatric Oncology Branch, Center for Cancer Research (CCR), National Cancer Institute (NCI), NIH, Bethesda, Maryland.

Background: Accurate disease detection is integral to risk stratification in B-cell acute lymphoblastic leukemia (ALL). The gold standard used to evaluate response in the United States includes morphologic evaluation and minimal residual disease (MRD) testing of aspirated bone marrow (BM) by flow cytometry (FC). This MRD assessment is usually made on a single aspirate sample that is subject to variability in collection techniques and sampling error. Additionally, central nervous system (CNS) assessments for ALL include evaluations of cytopathology and cell counts, which can miss subclinical involvement.

Procedure: We retrospectively compared BM biopsy, aspirate, and FC samples obtained from children and young adults with relapsed/refractory ALL to identify the frequency and degree of disease discrepancies in this population. We also compared CNS FC and cytopathology techniques.

Results: Sixty of 410 (14.6%) BM samples had discrepant results, 41 (10%) of which were clinically relevant as they resulted in a change in the assignment of marrow status. Discrepant BM results were found in 28 of 89 (31.5%) patients evaluated. Additionally, cerebrospinal fluid (CSF) FC identified disease in 9.7% of cases where cytopathology was negative.

Conclusions: These results support further investigation of the role of concurrent BM biopsy, with aspirate and FC evaluations, and the addition of FC to CSF evaluations, to fully assess disease status and response, particularly in patients with relapsed/refractory ALL. Prospective studies incorporating more comprehensive analysis to evaluate the impact on clinical outcomes are warranted.
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http://dx.doi.org/10.1002/pbc.28149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036332PMC
April 2020

Introduction: Immunological Reviews volume 290.

Authors:
Terry J Fry

Immunol Rev 2019 07;290(1):4-5

Human Immunology and Immunotherapy Initiative, and Director of Cancer Immunotherapy, University of Colorado School of Medicine, Aurora, Colorado.

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http://dx.doi.org/10.1111/imr.12797DOI Listing
July 2019

Multi-Specific CAR Targeting to Prevent Antigen Escape.

Curr Hematol Malig Rep 2019 10;14(5):451-459

Center for Cancer and Blood Disorders, Children's Hospital Colorado, University of Colorado Anschutz Medical Campus, 13123 East 16th Avenue, Box B115, Aurora, CO, 80045, USA.

Purpose Of Review: Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable remission induction rates for relapsed/refractory B cell malignancies. However, loss of the CAR-targeted antigen, known as antigen escape, accounts for a substantial percentage of relapses following CAR therapy and is a major barrier to durable remission. Here, we discuss mechanisms for antigen escape and strategies to prevent this pattern of relapse, including the use of multi-specific CARs, which recognize and target multiple tumor-associated antigens simultaneously.

Recent Findings: Preclinical and early clinical trial data indicates that multi-specific CAR therapy for B cell malignancies is both safe and effective. Optimal combinations of target antigens, as well as different multi-specific CAR formats, are currently being evaluated. Although still in early stages of development, multi-specific CAR therapy represents a promising approach to mitigate antigen loss-related relapses and improve durability of remission in patients with refractory B cell malignancies, and may be applicable to other types of cancer.
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http://dx.doi.org/10.1007/s11899-019-00537-5DOI Listing
October 2019

Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics.

Mol Ther 2019 07 30;27(7):1275-1285. Epub 2019 May 30.

Center for Cellular Engineering, NIH Clinical Center, Bethesda, MD, USA.

As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating cryopreservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservation-related subtle micro-cellular damage needs further study.
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http://dx.doi.org/10.1016/j.ymthe.2019.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612799PMC
July 2019

Introduction by the Guest Editor, Terry J. Fry.

Authors:
Terry J Fry

Cancer J 2019 May/Jun;25(3):178

From the University of Colorado Anschutz Medical Campus, Aurora, CO.

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http://dx.doi.org/10.1097/PPO.0000000000000382DOI Listing
July 2020

Modulation of Target Antigen Density Improves CAR T-cell Functionality and Persistence.

Clin Cancer Res 2019 Sep 20;25(17):5329-5341. Epub 2019 May 20.

Pediatric Oncology Branch, National Cancer Institute/Center for Cancer Research, National Institutes of Health, Bethesda, Maryland.

Purpose: Chimeric antigen receptor T-cell (CART) therapy targeting CD22 induces remission in 70% of patients with relapsed/refractory acute lymphoblastic leukemia (ALL). However, the majority of post-CD22 CART remissions are short and associated with reduction in CD22 expression. We evaluate the implications of low antigen density on the activity of CD22 CART and propose mechanisms to overcome antigen escape.

Experimental Design: Using ALL cell lines with variable CD22 expression, we evaluate the cytokine profile, cytotoxicity, and CART functionality in the setting of low CD22 expression. We develop a high-affinity CD22 chimeric antigen receptor (CAR) as an approach to improve CAR sensitivity. We also assess Bryostatin1, a therapeutically relevant agent, to upregulate CD22 and improve CAR functionality.

Results: We demonstrate that low CD22 expression negatively impacts and CD22 CART functionality and impairs CART persistence. Moreover, low antigen expression on leukemic cells increases naïve phenotype of persisting CART. Increasing CAR affinity does not improve response to low-antigen leukemia. Bryostatin1 upregulates CD22 on leukemia and lymphoma cell lines for 1 week following single-dose exposure, and improves CART functionality and persistence. While Bryostatin1 attenuates IFNγ production by CART, overall and CART cytotoxicity is not adversely affected. Finally, administration of Bryostain1 with CD22 CAR results in longer duration of response.

Conclusions: We demonstrate that target antigen modulation is a promising strategy to improve CD22 CAR efficacy and remission durability in patients with leukemia and lymphoma..
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3784DOI Listing
September 2019

Chimeric Antigen Receptors Incorporating D Domains Targeting CD123 Direct Potent Mono- and Bi-specific Antitumor Activity of T Cells.

Mol Ther 2019 07 17;27(7):1262-1274. Epub 2019 Apr 17.

Arcellx, Inc., Germantown, MD 20876, USA.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive initial response rates in hematologic malignancies. However, relapse rates are significant, and robust efficacies in other indications, such as solid tumors, will likely require novel therapeutic strategies and CAR designs. To that end, we sought to develop simple, highly selective targeting domains (D domains) that could be incorporated into complex, multifunctional therapeutics. Herein, we describe the identification and characterization of D domains specific for CD123, a therapeutic target for hematologic malignancies, including acute myelogenous leukemia (AML). CARs comprised of these D domains mediate potent T cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We describe a strategy of engineering less immunogenic D domains through the identification and removal of putative T cell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal.
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http://dx.doi.org/10.1016/j.ymthe.2019.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612629PMC
July 2019

Mechanisms of resistance to CAR T cell therapy.

Nat Rev Clin Oncol 2019 06;16(6):372-385

Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

The successes with chimeric antigen receptor (CAR) T cell therapy in early clinical trials involving patients with pre-B cell acute lymphoblastic leukaemia (ALL) or B cell lymphomas have revolutionized anticancer therapy, providing a potentially curative option for patients who are refractory to standard treatments. These trials resulted in rapid FDA approvals of anti-CD19 CAR T cell products for both ALL and certain types of B cell lymphoma - the first approved gene therapies in the USA. However, growing experience with these agents has revealed that remissions will be brief in a substantial number of patients owing to poor CAR T cell persistence and/or cancer cell resistance resulting from antigen loss or modulation. Furthermore, the initial experience with CAR T cells has highlighted challenges associated with manufacturing a patient-specific therapy. Understanding the limitations of CAR T cell therapy will be critical to realizing the full potential of this novel treatment approach. Herein, we discuss the factors that can preclude durable remissions following CAR T cell therapy, with a primary focus on the resistance mechanisms that underlie disease relapse. We also provide an overview of potential strategies to overcome these obstacles in an effort to more effectively incorporate this unique therapeutic strategy into standard treatment paradigms.
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http://dx.doi.org/10.1038/s41571-019-0184-6DOI Listing
June 2019

T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy.

J Clin Invest 2019 02 25;129(4):1551-1565. Epub 2019 Feb 25.

Parker Institute for Cancer Immunotherapy, New York, New York, USA.

Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.
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http://dx.doi.org/10.1172/JCI121491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436880PMC
February 2019

Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22.

Mol Ther Oncolytics 2018 Dec 6;11:127-137. Epub 2018 Nov 6.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, USA.

Despite high remission rates following CAR-T cell therapy in B-ALL, relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may reduce the likelihood of antigen loss, thus improving sustained remission rates. A systematic approach to the generation of CAR constructs incorporating two target-binding domains led to several novel CD19/CD22 bivalent CAR constructs. Importantly, we demonstrate the challenges associated with the construction of a bivalent CAR format that preserves bifunctionality against both CD19 and CD22. Using the most active bivalent CAR constructs, we found similar transduction efficiency compared to that of either CD19 or CD22 single CARs alone. When expressed on human T cells, the optimized CD19/CD22 CAR construct induced comparable interferon γ and interleukin-2 compared to single CARs against dual-antigen-expressing as well as single-antigen-expressing cell lines. Finally, the T cells expressing CD19/CD22 CAR eradicated ALL cell line xenografts and patient-derived xenografts (PDX), including a PDX generated from a patient with CD19 relapse following CD19-directed CAR therapy. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce risk of antigen loss.
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http://dx.doi.org/10.1016/j.omto.2018.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300726PMC
December 2018

Cytomegalovirus Infection Incidence and Risk Factors Across Diverse Hematopoietic Cell Transplantation Platforms Using a Standardized Monitoring and Treatment Approach: A Comprehensive Evaluation from a Single Institution.

Biol Blood Marrow Transplant 2019 03 18;25(3):577-586. Epub 2018 Oct 18.

National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Electronic address:

Human cytomegalovirus (CMV) infection and disease remains a significant cause of morbidity and mortality for hematopoietic cell transplantation (HCT) recipients. Disruption of or weak reconstitution of virus-specific cellular immune function, such as with certain HCT approaches, poses significant risk for CMV-related complications. The incidence of and risk factors for CMV infection and the nature of CMV disease were evaluated retrospectively among 356 consecutive HCT recipients transplanted at the National Institutes of Health using all graft sources, including bone marrow, peripheral blood stem cell (PBSC), and umbilical cord blood (UCB), and a range of in vivo and ex vivo approaches for graft-versus-host disease (GVHD) prophylaxis. The cumulative incidence of CMV infection was higher for CMV-seropositive recipients at 33%, regardless of donor CMV serostatus. Patients transplanted with CMV-seropositive donors had a significantly shorter duration of antiviral therapy. Among graft sources UCB was associated with the highest cumulative incidence of CMV infection at 65% and significantly longer treatment duration at a median of 36days, whereas PBSC HCT was associated with the lowest incidence at 26% and the shortest CMV treatment duration at a median of 21days. There were significant differences in the cumulative incidence of CMV infection by T cell manipulation strategy when systemic steroids were included as a risk-modifying event. Over one-third of CMV infections occurred in the setting of systemic steroid administration. CMV disease occurred in 5% of HCT recipients, with 70% of cases in the setting of treatment for GVHD. Although factors related to serostatus, graft source, and GVHD prophylaxis were associated with varied CMV infection incidence, unplanned post-HCT corticosteroid therapy contributed greatly to the incidence of both CMV infection and disease across HCT approaches, highlighting this post-HCT intervention as a key time to potentially tailor the approach to monitoring, preemptive therapy, and even prophylaxis.
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http://dx.doi.org/10.1016/j.bbmt.2018.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445771PMC
March 2019

Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell.

Nat Med 2018 10 1;24(10):1499-1503. Epub 2018 Oct 1.

Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19 leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.
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http://dx.doi.org/10.1038/s41591-018-0201-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511988PMC
October 2018

Murine pre-B-cell ALL induces T-cell dysfunction not fully reversed by introduction of a chimeric antigen receptor.

Blood 2018 11 12;132(18):1899-1910. Epub 2018 Sep 12.

Hematologic Malignancies Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.

Adoptive transfer of patient-derived T cells modified to express chimeric antigen receptors (CARTs) has demonstrated dramatic success in relapsed/refractory pre-B-cell acute lymphoblastic leukemia (ALL), but response and durability of remission requires exponential CART expansion and persistence. Tumors are known to affect T-cell function, but this has not been well studied in ALL and in the context of chimeric antigen receptor (CAR) expression. Using TCF3/PBX1 and MLL-AF4-driven murine ALL models, we assessed the impact of progressive ALL on T-cell function in vivo. Vaccines protect against TCF3/PBX1.3 but were ineffective when administered after leukemia injection, suggesting immunosuppression induced early during ALL progression. T cells from leukemia-bearing mice exhibited increased expression of inhibitory receptors, including PD1, Tim3, and LAG3, and were dysfunctional following adoptive transfer in a model of T-cell receptor (TCR)-dependent leukemia clearance. Although expression of inhibitory receptors has been linked to TCR signaling, pre-B-cell ALL induced inhibitory receptor expression, at least in part, in a TCR-independent manner. Finally, introduction of a CAR into T cells generated from leukemia-bearing mice failed to fully reverse poor in vivo function.
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http://dx.doi.org/10.1182/blood-2017-12-815548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213322PMC
November 2018

Chimeric Antigen Receptor T-Cell Therapy.

J Natl Compr Canc Netw 2018 09;16(9):1092-1106

Patients with relapsed or refractory (R/R) cancers have a poor prognosis and limited treatment options. The recent approval of 2 chimeric antigen receptor (CAR) autologous T-cell products for R/R B-cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma treatment is setting the stage for what is possible in other diseases. However, there are important factors that must be considered, including patient selection, toxicity management, and costs associated with CAR T-cell therapy. To begin to address these issues, NCCN organized a task force consisting of a multidisciplinary panel of experts in oncology, cancer center administration, and health policy, which met for the first time in March 2018. This report describes the current state of CAR T-cell therapy and future strategies that should be considered as the application of this novel immunotherapy expands and evolves.
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http://dx.doi.org/10.6004/jnccn.2018.0073DOI Listing
September 2018

Systematic Evaluation of Neurotoxicity in Children and Young Adults Undergoing CD22 Chimeric Antigen Receptor T-Cell Therapy.

J Immunother 2018 Sep;41(7):350-358

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda.

Neurotoxicity associated with CAR-T cell therapy can be life-threatening. With rapid development of CAR-T therapies, a systematic method is needed to identify and monitor symptoms of neurotoxicity, elucidate potential etiologies, and compare toxicity across trials. This paper presents a systematic evaluation developed and used to prospectively assess neurotoxicity in our phase I anti-CD22 CAR-T-cell trial and describes the symptoms of neurotoxicity identified using this methodology. Central nervous system (CNS) studies included routine lumbar punctures performed for disease evaluation pretherapy and posttherapy and a baseline brain MRI. Brief cognitive evaluations, assessing 4 domains (attention, working memory, cognitive flexibility, and processing speed), were administered preinfusion and postinfusion. A newly developed CAR-T-specific neurological symptom checklist (NSC) was completed by caregivers at 3 designated time-points. Serial serum cytokine levels were compared with neurotoxicity symptoms and severity. The majority of the first 22 consecutively treated subjects (ages, 7-30) demonstrated stable or improved cognitive test scores following therapy and no irreversible neurotoxicity, despite CAR-T-related antileukemic response, cytokine release syndrome, and trafficking of CAR-T cells to the CSF. The NSC allowed us to document the type and timing of symptoms and explore the etiology of neurotoxicity associated with CD22 CAR-T therapy. Cytokine profiling demonstrated that more concerning symptoms of neurotoxicity, such as hallucination and disorientation, were significantly associated with higher serum cytokine levels, supporting the hypothesis of inflammation-driven neurotoxicity. Systematic assessments of neurotoxicity were feasible in acutely ill children and young adults and served to characterize and monitor the symptoms associated with CAR-T therapy. We recommend these evaluations be incorporated into future immunotherapy protocols.
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http://dx.doi.org/10.1097/CJI.0000000000000241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086728PMC
September 2018

Outcomes of Measurable Residual Disease in Pediatric Acute Myeloid Leukemia before and after Hematopoietic Stem Cell Transplant: Validation of Difference from Normal Flow Cytometry with Chimerism Studies and Wilms Tumor 1 Gene Expression.

Biol Blood Marrow Transplant 2018 10 19;24(10):2040-2046. Epub 2018 Jun 19.

Division of Hematology, Oncology, and Blood and Marrow Transplantation, Children's Hospital Los Angeles, USC Keck School of Medicine, Los Angeles, CA, USA.

We enrolled 150 patients in a prospective multicenter study of children with acute myeloid leukemia undergoing hematopoietic stem cell transplantation (HSCT) to compare the detection of measurable residual disease (MRD) by a "difference from normal" flow cytometry (ΔN) approach with assessment of Wilms tumor 1 (WT1) gene expression without access to the diagnostic specimen. Prospective analysis of the specimens using this approach showed that 23% of patients screened for HSCT had detectable residual disease by ΔN (.04% to 53%). Of those patients who proceeded to transplant as being in morphologic remission, 10 had detectable disease (.04% to 14%) by ΔN. The disease-free survival of this group was 10% (0 to 35%) compared with 55% (46% to 64%, P < .001) for those without disease. The ΔN assay was validated using the post-HSCT specimen by sorting abnormal or suspicious cells to confirm recipient or donor origin by chimerism studies. All 15 patients who had confirmation of tumor detection relapsed, whereas the 2 patients with suspicious phenotype cells lacking this confirmation did not. The phenotype of the relapse specimen was then used retrospectively to assess the pre-HSCT specimen, allowing identification of additional samples with low levels of MRD involvement that were previously undetected. Quantitative assessment of WT1 gene expression was not predictive of relapse or other outcomes in either pre- or post-transplant specimens. MRD detected by ΔN was highly specific, but did not identify most relapsing patients. The application of the assay was limited by poor quality among one-third of the specimens and lack of a diagnostic phenotype for comparison.
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http://dx.doi.org/10.1016/j.bbmt.2018.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239928PMC
October 2018

Progenitor B-1 B-cell acute lymphoblastic leukemia is associated with collaborative mutations in 3 critical pathways.

Blood Adv 2017 Sep 8;1(20):1749-1759. Epub 2017 Sep 8.

Genetics Branch and.

B-1 and B-2 lymphocytes are derived from distinct developmental pathways and represent layered arms of the innate and adaptive immune systems, respectively. In contrast to a majority of murine B-cell malignancies, which stain positive with the B220 antibody, we discovered a novel form of B-cell leukemia in - () transgenic mice. The immunophenotype (Lin B220 CD19 AA4.1) was identical to that of progenitor (pro) B-1 cells, and V gene usage was skewed toward 3' V regions, similar to murine fetal liver B cells. Moreover, the gene expression profile of these leukemias was most similar to that of fetal liver pro-B fraction BC, a known source of B-1 B cells, further supporting a pro-B-1 origin of these leukemias. The pro-B-1 acute lymphoblastic leukemias (ALLs) acquired spontaneous mutations in both and Janus kinase () pathway (// and ) genes, supporting a hypothesis that mutations in 3 critical pathways (stem-cell self-renewal, B-cell differentiation, and cytokine signaling) collaborate to induce B-cell precursor (BCP) ALL. Finally, the thymic stromal lymphopoietin (Tslp) cytokine is required for murine B-1 development, and chromosomal rearrangements resulting in overexpression of the TSLP receptor (CRLF2) are present in some patients with high-risk BCP-ALL (referred to as r ALL). Gene expression profiles of pro-B-1 ALL were more similar to that of r ALL than non-r ALL, and analysis of V gene usage from patients with ALL demonstrated preferential usage of V regions used by human B-1 B cells, leading to the suggestion that this subset of patients with BCP-ALL has a malignancy of B-1, rather than B-2, B-cell origin.
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http://dx.doi.org/10.1182/bloodadvances.2017009837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728343PMC
September 2017

TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance.

Sci Transl Med 2017 Nov;9(417)

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Chimeric antigen receptor (CAR)-expressing T cells induce durable remissions in patients with relapsed/refractory B cell malignancies. CARs are synthetic constructs that, when introduced into mature T cells, confer a second, non-major histocompatibility complex-restricted specificity in addition to the endogenous T cell receptor (TCR). The implications of TCR activation on CAR T cell efficacy has not been well defined. Using an immunocompetent, syngeneic murine model of CD19-targeted CAR T cell therapy for pre-B cell acute lymphoblastic leukemia in which the CAR is introduced into T cells with known TCR specificity, we demonstrate loss of CD8 CAR T cell efficacy associated with T cell exhaustion and apoptosis when TCR antigen is present. CD4 CAR T cells demonstrate equivalent cytotoxicity to CD8 CAR T cells and, in contrast, retain in vivo efficacy despite TCR stimulation. Gene expression profiles confirm increased exhaustion and apoptosis of CD8 CAR T cells upon dual receptor stimulation compared to CD4 CAR T cells and indicate inherent differences between CD4 and CD8 CAR T cells in the use of T cell-associated signaling pathways. These results provide insights into important aspects of CAR T cell immune biology and indicate opportunities to rationally design CAR constructs to optimize clinical efficacy.
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http://dx.doi.org/10.1126/scitranslmed.aag1209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944272PMC
November 2017

CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy.

Nat Med 2018 01 20;24(1):20-28. Epub 2017 Nov 20.

Department of Pediatrics and Standford Cancer Center, Stanford University, Stanford, California, USA.

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 10 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19 or CD19 B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22 cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.
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http://dx.doi.org/10.1038/nm.4441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774642PMC
January 2018