Hematopoietic Cell Transplantation for DOCK8 Deficiency: Results from a Prospective Clinical Trial.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) deficiency is a primary immunodeficiency in which allogeneic hematopoietic cell transplantation (HCT) represents the only known cure. We tested the ability of a busulfan-based regimen to achieve reliable engraftment and high levels of donor chimerism with acceptable toxicity in a prospective clinical trial in DOCK8 deficiency. OBJECTIVE: To both evaluate the ability of HCT to reverse the clinical phenotype and to correct the immunologic abnormalities by 1-year post-HCT. METHODS: We conducted a prospective HCT trial for recipients with DOCK8 deficiency. Subjects were recruited from October 5th, 2010, to December 30th, 2022. Donor sources included fully matched related (MRD) and unrelated (MUD) and haploidentical (Haplo) donors. The reduced toxicity, myeloablative conditioning regimen contained no serotherapy. Graft-versus-host disease (GVHD) prophylaxis included either a calcineurin inhibitor (CNI) with methotrexate (MTX) or post-HCT cyclophosphamide (PT/Cy) followed by tacrolimus and mycophenolate mofetil (MMF). The trial was later amended to study PT/Cy in all patients. (ClinicalTrials.gov NCT01176006). RESULTS: Thirty-six subjects, children, and adults (median age 16.4 years) underwent HCT for DOCK8 deficiency. Most patients, 33 of 36 (92%), achieved full (≥98%) donor chimerism in whole blood as early as day +30. With a median potential follow-up of 7.4 years, 29 (80.6%) were alive with no evidence of new DOCK8 deficiency-related complications. PT/Cy was effective in reducing the risk of acute GVHD in patients who had received MUDs and Haplo transplants, but it was associated with transient delays in immune-reconstitution and hemorrhagic cystitis (HC). CONCLUSION: A busulfan-based HCT regimen using PT/Cy for GVHD prophylaxis and a broad range of donor types and hematopoietic cell sources were well-tolerated, leading to the reversal of the clinical immunophenotype.

Best Practice Considerations by The American Society of Transplant and Cellular Therapy: Infection Prevention and Management After Chimeric Antigen Receptor T Cell Therapy for Hematological Malignancies.

Chimeric antigen receptor (CAR) T-cell therapy is rapidly advancing, offering promising treatments for patients with hematological malignancy. However, associated infectious complications remain a significant concern because of their contribution to patient morbidity and non-relapse mortality. Recent epidemiological insights shed light on risk factors for infections after CAR T-cell therapy. However, the available evidence is predominantly retrospective, highlighting a need for further prospective studies. Institutions are challenged with managing infections after CAR T-cell therapy but variations in the approaches taken underscore the importance of standardizing infection prevention and management protocols across different healthcare settings. Therefore, the Infectious Diseases Special Interest Group of the American Society of Transplantation and Cellular Therapy assembled an expert panel to develop best practice considerations. The aim was to guide healthcare professionals in optimizing infection prevention and management for CAR T-cell therapy recipients and advocates for early consultation of Infectious Diseases during treatment planning phases given the complexities involved. By synthesizing current evidence and expert opinion these best practice considerations provide the basis for understanding infection risk after CAR T-cell therapies and propose risk-mitigating strategies in children, adolescents, and adults. Continued research and collaboration will be essential to refining and effectively implementing these recommendations.

How I treat post-immunotherapy relapsed B-ALL.

Despite significant advancements in single-antigen targeted therapies for B-cell acute lymphoblastic leukemia (B-ALL), non-response and relapse persist as major challenges. Antigen escape following blinatumomab or CD19-directed chimeric antigen receptor T cells (CD19-CAR), as CD19-negative B-ALL or lineage switch (LS) to acute myeloid leukemia, present diagnostic and treatment complexities. Given the poor outcomes for patients experiencing a post-infusion relapse, particularly those with loss of the target antigen, a strategic approach to diagnosis and treatment is imperative. In this discussion, we outline a systematic approach to managing post-immunotherapy events, categorized by CD19-positive relapse, CD19-negative relapse, and LS. We explore treatment modalities including CD19-CAR re-infusions, humanized CAR constructs, combinatorial strategies, and alternative antigen-targeted therapies, such as blinatumomab and inotuzumab. Challenges in diagnosis, particularly with antigen-escape, are addressed, highlighting the role of next-generation sequencing and multiparameter flow cytometry for myeloid marker monitoring.

Diagnostic pitfalls in assessment of ferritin following CAR-T-cell therapy: Understanding the hook effect.

The hook effect is a well-described but clinically underappreciated immunoassay interference, where a falsely lowered result is caused by analyte excess. We describe a situation in which ferritin immunoassay results from a 27-year-old female with immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome were more than 1000 times lower at a reference laboratory than those determined in-house after dilution. This case underscores the importance for clinical care providers to be aware of the impact of the hook effect on ferritin measurements, and to promptly communicate with the laboratory when there are discrepancies between clinical symptoms and test results.

Optimizing early phase clinical trial washout periods: a report from the therapeutic advances in childhood leukemia and lymphoma (TACL) consortium.

PURPOSE: The National Cancer Institute (NCI) issued a 2021 memorandum adopting the American Society of Clinical Oncology (ASCO) and Friends of Cancer Research (Friends) task force recommendations to broaden clinical study eligibility criteria. They recommended that washout periods be eliminated for most prior cancer therapy and when required, to utilize evidence/rationale-based criteria. The Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) consortium responded to this guidance. PROCESS: A TACL task force reviewed the consortium's research portfolio, the relevant literature and guidance documents from ASCO-Friends, NCI, and US Food and Drug Administration (FDA) to make expert consensus and evidence-based recommendations for modernizing, broadening and codifying TACL-study washout periods while ensuring consistency with pediatric ethics and federal regulations. TACL's screening log was reviewed to estimate the impact that updated washout periods would have on patient inclusivity and recruitment. RESULTS: Over a 19-year period, 42 patients (14.6% of all screened ineligible (n = 287) patients), were identified as excluded from TACL early-phase studies exclusively due to not meeting washout criteria. An additional six (2.1%) did not meet washout and at least one other exclusion criterion. A new TACL washout guidance document was developed/adopted for use. Where washout criteria were not eliminated, rationale/evidenced-based criteria were established with citation. CONCLUSION: In an effort to reduce unnecessary exclusion from clinical trials, TACL created rationale/evidenced-based washout period standards largely following guidance from the NCI/ASCO-Friends recommendations. These new, expanded eligibility criteria are expected to increase access to TACL clinical trials while maintaining safety and scientific excellence.

T Cell Activators Exhibit Distinct Downstream Effects on Chimeric Antigen Receptor T Cell Phenotype and Function.

T cell activation is an essential step in chimeric Ag receptor (CAR) T (CAR T) cell manufacturing and is accomplished by the addition of activator reagents that trigger the TCR and provide costimulation. We explore several T cell activation reagents and examine their effects on key attributes of CAR T cell cultures, such as activation/exhaustion markers, cell expansion, gene expression, and transduction efficiency. Four distinct activators were examined, all using anti-CD3 and anti-CD28, but incorporating different mechanisms of delivery: Dynabeads (magnetic microspheres), TransAct (polymeric nanomatrix), Cloudz (alginate hydrogel), and Microbubbles (lipid membrane containing perfluorocarbon gas). Clinical-grade lentiviral vector was used to transduce cells with a bivalent CD19/CD22 CAR, and cell counts and flow cytometry were used to monitor the cells throughout the culture. We observed differences in CD4/CD8 ratio when stimulating with the Cloudz activator, where there was a significant skewing toward CD8 T cells. The naive T cell subset expressing CD62L+CCR7+CD45RA+ was the highest in all donors when stimulating with Dynabeads, whereas effector/effector memory cells were highest when using the Cloudz. Functional assays demonstrated differences in killing of target cells and proinflammatory cytokine secretion, with the highest killing from the Cloudz-stimulated cells among all donors. This study demonstrates that the means by which these stimulatory Abs are presented to T cells contribute to the activation, resulting in differing effects on CAR T cell function. These studies highlight important differences in the final product that should be considered when manufacturing CAR T cells for patients in the clinic.

CD19 CAR T cells for multiple sclerosis: Forging further into the new frontier.

The efficacy of CD19 chimeric antigen receptor (CAR) T cells in B cell malignancies has generated recent interest in their application to other B cell-related pathologies, such as autoimmune diseases. Fischbach et al.1 report on the use of CD19 CAR T cells in two patients with progressive multiple sclerosis, demonstrating feasibility and safety for the first time in this disease process.

Pediatric Cancer Immunotherapy and Potential for Impact on Fertility: A Need for Evidence-Based Guidance.

The use of immunotherapies for the treatment of cancer in children, adolescents, and young adults has become common. As the use of immunotherapy has expanded, including in earlier lines of therapy, it has become evident that several aspects of how these immunotherapies impact longer-term outcomes among survivors are understudied. Traditional cancer therapies like alkylating and platin agents carry the greatest risk of infertility, but little is known about the impact of novel immunotherapies on fertility. This topic is of great interest to patients, patient advocates, and clinicians. In this article, we review immunotherapeutic agents used to treat childhood and young adult cancers and discuss potential mechanisms by which they may impact fertility based on the known interplay between the immune system and reproductive organs. We highlight the relative paucity of high-quality literature examining these late effects. We discuss interventions to optimize fertility preservation (FP) for our patients. Conducting longitudinal, collaborative, and prospective research on the fertility outcomes of pediatric and young adult patients with cancer who receive immunotherapy is critical to learn how to effectively counsel our patients on long-term fertility outcomes and indications for FP procedures. Collection of patient-level data will be necessary to draft evidence-based guidelines on which providers can make therapy recommendations.

CAR T-cell toxicities: from bedside to bench, how novel toxicities inform laboratory investigations.

ABSTRACT: Multiple chimeric antigen receptor (CAR) T-cell therapies are US Food and Drug Administration-approved, and several are under development. Although effective for some cancers, toxicities remain a limitation. The most common toxicities, that is, cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, are well described. With increasing utilization, providers worldwide are reporting other emergent and often complicated toxicities. Given the evolving toxicity profiles and urgent need to catalog these emerging and emergent CAR T-cell toxicities and describe management approaches, the American Society of Hematology Subcommittee on Emerging Gene and Cell Therapies organized the first scientific workshop on CAR T-cell toxicities during the annual society meeting. The workshop functioned to (1) aggregate reports of CAR T-cell emergent toxicities, including movement disorders after B-cell maturation antigen CAR T cell, coagulation abnormalities, and prolonged cytopenia; (2) disseminate bedside-to-bench efforts elucidating pathophysiological mechanisms of CAR T-cell toxicities, including the intestinal microbiota and systemic immune dysregulation; and (3) highlight gaps in the availability of clinical tests, such as cytokine measurements, which could be used to expand our knowledge around the monitoring of toxicities. Key themes emerged. First, although clinical manifestations may develop before the pathophysiologic mechanisms are understood, they must be studied to aid in the detection and prevention of such toxicities. Second, systemic immune dysregulation appears to be central to these emergent toxicities, and research is needed to elucidate the links between tumors, CAR T cells, and microbiota. Finally, there was a consensus around the urgency to create a repository to capture emergent CAR T-cell toxicities and the real-world management.

Treatment strategies for progressive immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome: case series.

Not available.

Immune effector cell-associated haematotoxicity after CAR T-cell therapy: from mechanism to management.

Genetically engineered chimeric antigen receptor (CAR) T cells have become an effective treatment option for several advanced B-cell malignancies. Haematological side-effects, classified in 2023 as immune effector cell-associated haematotoxicity (ICAHT), are very common and can predispose for clinically relevant infections. As haematopoietic reconstitution after CAR T-cell therapy differs from chemotherapy-associated myelosuppression, a novel classification system for early and late ICAHT has been introduced. Furthermore, a risk stratification score named CAR-HEMATOTOX has been developed to identify candidates at high risk of ICAHT, thereby enabling risk-based interventional strategies. Therapeutically, growth factor support with granulocyte colony-stimulating factor (G-CSF) is the mainstay of treatment, with haematopoietic stem cell (HSC) boosts available for patients who are refractory to G-CSF (if available). Although the underlying pathophysiology remains poorly understood, translational studies from the past 3 years suggest that CAR T-cell-induced inflammation and baseline haematopoietic function are key contributors to prolonged cytopenia. In this Review, we provide an overview of the spectrum of haematological toxicities after CAR T-cell therapy and offer perspectives on future translational and clinical developments.

Overcoming the challenges of primary resistance and relapse after CAR-T cell therapy.

INTRODUCTION: While CAR T-cell therapy has led to remarkable responses in relapsed B-cell hematologic malignancies, only 50% of patients ultimately have a complete, sustained response. Understanding the mechanisms of resistance and relapse after CAR T-cell therapy is crucial to future development and improving outcomes. AREAS COVERED: We review reasons for both primary resistance and relapse after CAR T-cell therapies. Reasons for primary failure include CAR T-cell manufacturing problems, suboptimal fitness of autologous T-cells themselves, and intrinsic features of the underlying cancer and tumor microenvironment. Relapse after initial response to CAR T-cell therapy may be antigen-positive, due to CAR T-cell exhaustion or limited persistence, or antigen-negative, due to antigen-modulation on the target cells. Finally, we discuss ongoing efforts to overcome resistance to CAR T-cell therapy with enhanced CAR constructs, manufacturing methods, alternate cell types, combinatorial strategies, and optimization of both pre-infusion conditioning regimens and post-infusion consolidative strategies. EXPERT OPINION: There is a continued need for novel approaches to CAR T-cell therapy for both hematologic and solid malignancies to obtain sustained remissions. Opportunities for improvement include development of new targets, optimally combining existing CAR T-cell therapies, and defining the role for adjunctive immune modulators and stem cell transplant in enhancing long-term survival.

ACT To Sustain: Adoptive Cell Therapy To Sustain Access to Non-Commercialized Genetically Modified Cell Therapies.

Genetically modified cell therapies (GMCT), particularly immune effector cells (IEC) such as chimeric receptor antigen (CAR) T cells, have shown promise in curing cancer and rare diseases after a single treatment course. Following close behind CAR T approvals are GMCT based on hematopoietic stem cells, such as products developed for hemoglobinopathies and other disorders. Academically sponsored GMCT products, often developed in academic centers without industry involvement, face challenges in sustaining access after completion of early phase studies when there is no commercial partner invested in completing registration trials for marketing applications. The American Society for Transplantation and Cellular Therapy (ASTCT) formed a task force named ACT To Sustain (Adoptive Cell Therapy to Sustain) to address the "valley of death" of academic GMCT products. This paper presents the task force's findings and considerations regarding financial sustainability of academically sponsored GMCT products in the absence of commercial development. We outline case scenarios illustrating barriers to maintaining access to promising GMCT developed by academic centers. The paper also delves into the current state of GMCT development, commercialization, and reimbursement, citing examples of abandoned products, cost estimates associated with GMCT manufacturing and real-world use of cost recovery. We propose potential solutions to address the financial, regulatory, and logistical challenges associated with sustaining access to academically sponsored GMCT products and to ensure that products with promising results do not languish in a "valley of death" due to financial or implementational barriers. The suggestions include aligning US Food and Drug Administration (FDA) designations with benefit coverage, allowing for cost recovery of certain products as a covered benefit, and engaging with regulators and policy makers to discuss alternative pathways for academic centers to provide access. We stress the importance of sustainable access to GMCT and call for collaborative efforts to develop regulatory pathways that support access to academically sponsored GMCT products.

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity. METHODS: This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products. RESULTS: A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response. CONCLUSIONS: pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

Need for standardization of cytokine profiling in CAR T cell therapy.

With expansion of chimeric antigen receptor (CAR) T cell therapy and broader utilization of anti-cytokine directed therapeutics for toxicity mitigation, the routine assessment of cytokines may enhance understanding of toxicity profiles, guide therapeutic interventions, and facilitate cross-trial comparisons. As specific cytokine elevations can correlate with and provide insights into CAR T cell toxicity, mitigation strategies, and response, we explored the reporting of cytokine detection methods and assessed for the correlation of cytokines to cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) across clinical trials. In this analysis, we reviewed 21 clinical trials across 60 manuscripts that featured a US Food and Drug Administration-approved CAR T cell construct or one of its predecessors. We highlight substantial variability and limited reporting of cytokine measurement platforms and panels used across CAR T cell clinical trials. Specifically, across 60 publications, 28 (46.7%) did not report any cytokine data, representing 6 of 21 (28.6%) clinical trials. In the 15 trials reporting cytokine data, at least 4 different platforms were used. Furthermore, correlation of cytokines with ICANS, CRS, and CRS severity was limited. Considering the fundamental role of cytokines in CAR T cell toxicity, our manuscript supports the need to establish standardization of cytokine measurements as a key biomarker essential to improving outcomes of CAR T cell therapy.

Immunotherapy-resistant acute lymphoblastic leukemia cells exhibit reduced CD19 and CD22 expression and BTK pathway dependency.

While therapies targeting CD19 by antibodies, chimeric antigen receptor T cells (CAR-T), and T cell engagers have improved the response rates in B cell malignancies, the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19 immunotoxin. Single-cell RNA-Seq (scRNA-Seq) showed an increase in transcriptionally distinct CD19lo populations among resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19lo-resistant cells. An assay for transposase-accessible chromatin with sequencing (ATAC-Seq) showed decreased chromatin accessibility at promoters of both CD19 and CD22 in the resistant cell populations. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with B cell acute lymphoblastic leukemia (B-ALL) that relapsed after CD19 CAR-T-targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19lo resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both Bruton's tyrosine kinase (BTK) and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.

Manufacture of CD22 CAR T cells following positive versus negative selection results in distinct cytokine secretion profiles and γδ T cell output.

Chimeric antigen receptor T cells (CART) have demonstrated curative potential for hematological malignancies, but the optimal manufacturing has not yet been determined and may differ across products. The first step, T cell selection, removes contaminating cell types that can potentially suppress T cell expansion and transduction. While positive selection of CD4/CD8 T cells after leukapheresis is often used in clinical trials, it may modulate signaling cascades downstream of these co-receptors; indeed, the addition of a CD4/CD8-positive selection step altered CD22 CART potency and toxicity in patients. While negative selection may avoid this drawback, it is virtually absent from good manufacturing practices. Here, we performed both CD4/CD8-positive and -negative clinical scale selections of mononuclear cell apheresis products and generated CD22 CARTs per our ongoing clinical trial (NCT02315612NCT02315612). While the selection process did not yield differences in CART expansion or transduction, positively selected CART exhibited a significantly higher in vitro interferon-γ and IL-2 secretion but a lower in vitro tumor killing rate. Notably, though, CD22 CART generated from both selection protocols efficiently eradicated leukemia in NSG mice, with negatively selected cells exhibiting a significant enrichment in γδ CD22 CART. Thus, our study demonstrates the importance of the initial T cell selection process in clinical CART manufacturing.