Interleukin-15-armored GPC3-CAR T cells for patients with solid cancers.

Interleukin-15 (IL15) promotes the survival of T lymphocytes and enhances the antitumor properties of CAR T cells in preclinical models of solid neoplasms in which CAR T cells have limited efficacy1-4. Glypican-3 (GPC3) is expressed in a group of solid cancers5-10, and here we report the first evaluation in humans of the effects of IL15 co-expression on GPC3-CAR T cells. Cohort 1 patients (NCT02905188/NCT02932956) received GPC3-CAR T cells, which were safe but produced no objective antitumor responses and reached peak expansion at two weeks. Cohort 2 patients (NCT05103631/NCT04377932) received GPC3-CAR T cells that co-expressed IL15 (15.CAR), which mediated significantly increased cell expansion and induced a disease control rate of 66% and antitumor response rate of 33%. Infusion of 15.CAR T cells was associated with increased incidence of cytokine release syndrome, which was rapidly ameliorated by activation of the inducible caspase 9 safety switch. Compared to non-responders, tumor-infiltrating 15.CAR T cells from responders showed repression of SWI/SNF epigenetic regulators and upregulation of FOS and JUN family members as well as genes related to type I interferon signaling. Collectively, these results demonstrate that IL15 increases the expansion, intratumoral survival, and antitumor activity of GPC3-CAR T cells in patients.

Autologous HER2-specific CAR T cells after lymphodepletion for advanced sarcoma: a phase 1 trial.

In this prospective, interventional phase 1 study for individuals with advanced sarcoma, we infused autologous HER2-specific chimeric antigen receptor T cells (HER2 CAR T cells) after lymphodepletion with fludarabine (Flu) ± cyclophosphamide (Cy): 1 × 108 T cells per m2 after Flu (cohort A) or Flu/Cy (cohort B) and 1 × 108 CAR+ T cells per m2 after Flu/Cy (cohort C). The primary outcome was assessment of safety of one dose of HER2 CAR T cells after lymphodepletion. Determination of antitumor responses was the secondary outcome. Thirteen individuals were treated in 14 enrollments, and seven received multiple infusions. HER2 CAR T cells expanded after 19 of 21 infusions. Nine of 12 individuals in cohorts A and B developed grade 1-2 cytokine release syndrome. Two individuals in cohort C experienced dose-limiting toxicity with grade 3-4 cytokine release syndrome. Antitumor activity was observed with clinical benefit in 50% of individuals treated. The tumor samples analyzed showed spatial heterogeneity of immune cells and clustering by sarcoma type and by treatment response. Our results affirm HER2 as a CAR T cell target and demonstrate the safety of this therapeutic approach in sarcoma. ClinicalTrials.gov registration: NCT00902044 .

Eighteen-year survival after GD2-directed Chimeric Antigen Receptor-Modified Immune Effector Cell Treatment for Neuroblastoma.

We report long-term outcomes up to 18 years of a clinical trial treating children with neuroblastoma with EBV-specific T lymphocytes and CD3-activated T cells - each expressing a first-generation chimeric antigen receptor targeting GD2 with barcoded transgenes to allow tracking of each population. Of 11 patients with active disease at infusion, three patients achieved a complete response that was sustained in 2, one for 8 years until lost to follow up and one for 18+ years. Of eight patients with a history of relapse or at high risk of recurrence, five are disease-free at their last follow-up between 10-14 years post-infusion. Intermittent low levels of transgene were detected during the follow up period with significantly greater persistence in those who were long-term survivors. In conclusion, patients with relapsed/refractory neuroblastoma achieved long-term disease control after receiving GD2 CAR-T cell therapy including one patient now in remission of relapsed disease for >18 years.

Human platelet lysate enhances in vivo activity of CAR-Vδ2 T cells by reducing cellular senescence and apoptosis.

BACKGROUND AIMS: Vγ9Vδ2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy as the result of their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vδ2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain. METHODS: Specifically, we compared the anti-tumor activity of Vδ2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium. RESULTS: We found that HPL supported greater expansion of CAR-Vδ2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vδ2 T cells. HPL-expanded CAR-Vδ2 T cells showed enhanced in vivo anti-tumor activity with longer T-cell persistence compared with FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vδ2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vδ2 T cells and increased telomerase activity. CONCLUSIONS: This study supports enhancement of therapeutic potency of CAR-Vδ2 T cells through a manufacturing improvement.

A robust quality infrastructure is key to safe and effective delivery of immune effector cells: how FACT-finding can help.

ABSTRACT: Immune effector cells (IECs) include a broad range of immune cells capable of modulating several disease states, including malignant and nonmalignant conditions. The growth in the use of IECs as both investigational and commercially available products requires medical institutions to develop workflows/processes to safely implement and deliver transformative therapy. Adding to the complexity of this therapy are the variety of targets, diseases, sources, and unique toxicities that a patient experiences following IEC therapy. For over 25 years, the Foundation for the Accreditation of Cellular Therapy (FACT) has established a standard for the use of cellular therapy, initially with hematopoietic cell transplantation (HCT), and more recently, with the development of standards to encompass IEC products such as chimeric antigen receptor (CAR)-T cells. To date, IEC therapy has challenged the bandwidth and infrastructure of the institutions offering this therapy. To address these challenges, FACT has established a programmatic framework to improve the delivery of IEC therapy. In this study, we outline the current state of IEC program development, accreditation, and solutions to the challenges that programs face as they expand their application to novel IEC therapy.

Antitumor efficacy and safety of unedited autologous CD5.CAR T cells in relapsed/refractory mature T-cell lymphomas.

ABSTRACT: Despite newer targeted therapies, patients with primary refractory or relapsed (r/r) T-cell lymphoma have a poor prognosis. The development of chimeric antigen receptor (CAR) T-cell platforms to treat T-cell malignancies often requires additional gene modifications to overcome fratricide because of shared T-cell antigens on normal and malignant T cells. We developed a CD5-directed CAR that produces minimal fratricide by downmodulating CD5 protein levels in transduced T cells while retaining strong cytotoxicity against CD5+ malignant cells. In our first-in-human phase 1 study (NCT0308190), second-generation autologous CD5.CAR T cells were manufactured from patients with r/r T-cell malignancies. Here, we report safety and efficacy data from a cohort of patients with mature T-cell lymphoma (TCL). Among the 17 patients with TCL enrolled, CD5 CAR T cells were successfully manufactured for 13 out of 14 attempted lines (93%) and administered to 9 (69%) patients. The overall response rate (complete remission or partial response) was 44%, with complete responses observed in 2 patients. The most common grade 3 or higher adverse events were cytopenias. No grade 3 or higher cytokine release syndrome or neurologic events occurred. Two patients died during the immediate toxicity evaluation period due to rapidly progressive disease. These results demonstrated that CD5.CAR T cells are safe and can induce clinical responses in patients with r/r CD5-expressing TCLs without eliminating endogenous T cells or increasing infectious complications. More patients and longer follow-up are needed for validation. This trial was registered at www.clinicaltrials.gov as #NCT0308190.

Incipient clonal hematopoiesis is accelerated following CD30.CAR-T therapy.

Chimeric antigen receptor (CAR) T-cells are an emerging therapy for refractory lymphomas. Clonal hematopoiesis (CH), the preferential outgrowth of mutated bone marrow progenitors, is enriched in lymphoma patients receiving CAR-T cells. CAR-T therapy requires conditioning chemotherapy and often induces systemic inflammatory reactions, both of which have been shown to promote expansion of CH clones. Thus, we hypothesized that pre-existing CH clones could expand during CAR-T cell treatment. We measured CH at 154 timepoints longitudinally sampled from 26 patients receiving CD30.CAR-T therapy for CD30+ lymphomas on an investigational protocol (NCT02917083). Pre-treatment CH was present in 54% of individuals and did not correlate with survival outcomes or inflammatory toxicities. Longitudinal tracking of single clones in individual patients revealed distinct clone growth dynamics. Initially small clones, defined as VAF <1%, expanded following CAR-T administration, compared with relatively muted expansions of larger clones (3.37-fold vs. 1.20-fold, P = 0.0014). Matched clones were present at low magnitude in the infused CD30.CAR-T product for all CH cases but did not affect the product's immunophenotype or transduction efficiency. As cellular immunotherapies expand to become frontline treatments for hematological malignancies, our data indicates CAR-T recipients could be enriched for CH, and further longitudinal studies centered on CH complications in this population are warranted.

Moving CAR-Ts to the outpatient clinic.

As centres obtain more experience with commercial CARs, there has been increasing interest in trying to move as much as the procedure as possible to the outpatient clinic to reduce costs, maximize reimbursement and increase patient satisfaction. The report by Ly et al. details how their centre implemented outpatient CAR therapy and were able to reduce admission time without affecting outcomes. Commentary on: Ly et al. Outpatient CD19-directed CAR T-cell therapy is feasible in patients of all ages. Br J Haematol 2023;203:688-692.

Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy.

BACKGROUND: The wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons for and solutions to this failure, taking as our model the preparation of Epstein-Barr virus (EBV)-specific T cells (EBVSTs) for the treatment of EBV-positive lymphoma. EBVSTs could not be manufactured from almost one-third of patients, either because they failed to expand, or they expanded, but lacked EBV specificity. We identified an underlying cause of this problem and established a clinically feasible approach to overcome it. METHODS: CD45RO+CD45RA- memory compartment residing antigen-specific T cells were enriched by depleting CD45RA positive (+) peripheral blood mononuclear cells (PBMCs) that include naïve T cells, among other subsets, prior to EBV antigen stimulation. We then compared the phenotype, specificity, function and T-cell receptor (TCR) Vß repertoire of EBVSTs expanded from unfractionated whole (W)-PBMCs and CD45RA-depleted (RAD)-PBMCs on day 16. To identify the CD45RA component that inhibited EBVST outgrowth, isolated CD45RA+ subsets were added back to RAD-PBMCs followed by expansion and characterization. The in vivo potency of W-EBVSTs and RAD-EBVSTs was compared in a murine xenograft model of autologous EBV+ lymphoma. RESULTS: Depletion of CD45RA+ PBMCs before antigen stimulation increased EBVST expansion, antigen-specificity and potency in vitro and in vivo. TCR sequencing revealed a selective outgrowth in RAD-EBVSTs of clonotypes that expanded poorly in W-EBVSTs. Inhibition of antigen-stimulated T cells by CD45RA+ PBMCs could be reproduced only by the naïve T-cell fraction, while CD45RA+ regulatory T cells, natural killer cells, stem cell memory and effector memory subsets lacked inhibitory activity. Crucially, CD45RA depletion of PBMCs from patients with lymphoma enabled the outgrowth of EBVSTs that failed to expand from W-PBMCs. This enhanced specificity extended to T cells specific for other viruses. CONCLUSION: Our findings suggest that naïve T cells inhibit the outgrowth of antigen-stimulated memory T cells, highlighting the profound effects of intra-T-cell subset interactions. Having overcome our inability to generate EBVSTs from many patients with lymphoma, we have introduced CD45RA depletion into three clinical trials: NCT01555892 and NCT04288726 using autologous and allogeneic EBVSTs to treat lymphoma and NCT04013802 using multivirus-specific T cells to treat viral infections after hematopoietic stem cell transplantation.

Posoleucel, an Allogeneic, Off-the-Shelf Multivirus-Specific T-Cell Therapy, for the Treatment of Refractory Viral Infections in the Post-HCT Setting.

PURPOSE: Viral infections are a major cause of morbidity and mortality following allogeneic hematopoietic cell transplantation (allo-HCT). In the absence of safe and effective antiviral treatments, virus-specific T cells have emerged as a promising therapeutic option. Posoleucel is a multivirus-specific T-cell therapy for off-the-shelf use against six viral infections that commonly occur in allo-HCT recipients: adenovirus, BK virus (BKV), cytomegalovirus, Epstein-Barr virus, human herpes virus-6, and JC virus. PATIENTS AND METHODS: We conducted an open-label, phase II trial to determine the feasibility and safety of posoleucel in allo-HCT recipients infected with one or more of these viruses. Infections were either unresponsive to or patients were unable to tolerate standard antiviral therapies. Fifty-eight adult and pediatric patients were enrolled and treated. RESULTS: Posoleucel was well tolerated, with no cytokine release syndrome or other infusion-related toxicities; two patients (3.4%) developed Grade 2 and one patient (1.7%) Grade 3 GvHD during the trial. The overall response rate 6 weeks after the first posoleucel infusion was 95%, with a median plasma viral load reduction of 97%. Of the 12 patients who had two or more target viral infections identified at study entry, 10 (83%) had a clinical response for all evaluable viruses. Of the 23 patients treated for refractory BKV-associated hemorrhagic cystitis, 74% had resolution of symptoms and macroscopic hematuria by 6 weeks post-infusion. CONCLUSIONS: In this open-label trial, treatment of refractory viral infections/disease in allo-HCT recipients with posoleucel was feasible, safe, and effective.

Paving the Road for Chimeric Antigen Receptor T Cells: American Society for Transplantation and Cellular Therapy 80/20 Task Force Consensus on Challenges and Solutions to Improving Efficiency of Clinical Center Certification and Maintenance of Operations for Commercially Approved Immune Effector Cell Therapies.

As the number and type of regulatory authority-approved cellular therapies grow, clinical treatment centers face a heavy burden of duplicative documentation around initial qualification, ongoing auditing, and reporting, with overlapping requirements from each manufacturer to ensure safe use of their specific product, which in the United States are stipulated under individual Food and Drug Administration (FDA) Biologic License Applications. The American Society for Transplantation and Cellular Therapy (ASTCT) convened the 80/20 Task Force to consider challenges and potential solutions to these issues. The Task Force proposed that 80% of manufacturers' requirements for onboarding and ongoing operations of commercially available products could be standardized and streamlined. Task Force members interviewed dozens of stakeholders, including clinicians at large academic medical centers already using commercial and investigational immune effector cell (IEC) products, regulators, members of accrediting bodies and professional cellular therapy societies, and manufacturers of IEC therapies for oncologic indications. In November 2021, the Task Force organized and led virtual discussions in a public forum and at a private ASTCT 80/20 Workshop at the online AcCELLerate Forum, a cellular-therapy stakeholders' meeting organized by the ASTCT, National Marrow Donor Program (NMDP), and Center for International Blood and Marrow Transplant Research (CIBMTR). At the workshop, approximately 60 stakeholders worked to identify and prioritize common challenges in onboarding and maintenance of operations at clinical sites for commercial FDA-approved and future IEC therapies and ways to streamline the process. It was agreed that standardization would improve efficiency of onboarding, allowing more cost-effective, sustainable growth of approved IEC therapies at treatment centers, and facilitate wider access while maintaining safety and clinical success. This early but extensive survey of stakeholders resulted in 5 overarching suggestions for both established and emerging treatment centers: (1) eliminate duplication in accreditation and auditing of clinical sites; (2) define expectations for the education about and management of CAR-T therapy toxicities to potentially replace product-specific REMS programs; (3) streamline current REMS education, testing, and data reporting; (4) standardize information technology (IT) platforms supporting enrollment, clinical site-manufacturer communication, and logistics of maintaining chain of identity/chain of custody across multiple transportation steps; and (5) encourage the use of universal nomenclature by cell therapy manufacturers. Future discussions need to engage a broader range of stakeholders, including administrators, pharmacists, nurses, data coordinators, surgeons, pathologists, and those developing promising cellular therapies for solid tumors, as well as teams from smaller academic or community cancer center settings. Continued collaboration with stakeholders outside of clinical sites will include accrediting bodies/auditors, established and emerging cell therapy companies, software developers, professional societies, and the patients who receive these therapies. Active dialog with government regulators remains essential. Such joint efforts are critical as the number of IEC therapies for myriad oncologic and nononcologic indications grows.

Allogeneic, off-the-shelf, SARS-CoV-2-specific T cells (ALVR109) for the treatment of COVID-19 in high-risk patients.

Defects in T-cell immunity to SARS-CoV-2 have been linked to an increased risk of severe COVID-19 (even after vaccination), persistent viral shedding and the emergence of more virulent viral variants. To address this T-cell deficit, we sought to prepare and cryopreserve banks of virus-specific T cells, which would be available as a partially HLA-matched, off-the-shelf product for immediate therapeutic use. By interrogating the peripheral blood of healthy convalescent donors, we identified immunodominant and protective T-cell target antigens, and generated and characterized polyclonal virus-specific T-cell lines with activity against multiple clinically important SARS-CoV-2 variants (including 'delta' and 'omicron'). The feasibility of making and safely utilizing such virus-specific T cells clinically was assessed by administering partially HLA-matched, third-party, cryopreserved SARS-CoV-2-specific T cells (ALVR109) in combination with other antiviral agents to four individuals who were hospitalized with COVID-19. This study establishes the feasibility of preparing and delivering off-the-shelf, SARS-CoV-2-directed, virus-specific T cells to patients with COVID-19 and supports the clinical use of these products outside of the profoundly immune compromised setting (ClinicalTrials.gov number, NCT04401410).

Cord blood transplantation for nonmalignant disorders: early functional immunity and high survival.

There is no consensus on the best donor for children with nonmalignant disorders and immune deficiencies in the absence of a matched related donor (MRD). We evaluated the 2-year overall survival (OS) after umbilical cord blood transplantation (UCBT) in patients with nonmalignant disorders from 2009 to 2020 enrolled in a prospective clinical trial using either 5/6 or 6/6 UCB as the cell source. Patients receive a fully ablative busulfan, cyclophosphamide, and fludarabine without serotherapy. Fifty-five children were enrolled, median age 5 months (range, 1-111 months); primary immune deficiency (45), metabolic (5), hemophagocytic lymphohistiocytosis (1), and hematologic disorders (4). Twenty-six patients had persistent infections before transplant. Nineteen of them (34%) were 6/6 matched, and 36 (66%) were 5/6 human leukocyte antigen-matched. The OS at 2 years was 91% (95% cumulative incidence, 79-96), with a median follow-up of 4.3 years. The median time to neutrophil and platelet recovery were 17 days (range, 5-39 days) and 37 days (range, 20-92 days), respectively. All but one evaluable patient achieved full donor chimerism. The cumulative incidence of acute GVHD grades 2-4 on day 100 was 16% (n = 9). All patients with viral infections at the time of transplant cleared the infection at a median time of 54 days (range, 44-91 days). All evaluable patients underwent correction of their immune or metabolic defects. We conclude that in the absence of MRD, UCBT following myeloablative conditioning without serotherapy is an excellent curative option in young children with nonmalignant disorders. This trial has been registered at www.clinicaltrials.gov as NCT00950846.

Feasibility and preclinical efficacy of CD7-unedited CD7 CAR T cells for T cell malignancies.

Chimeric antigen receptor (CAR)-mediated targeting of T lineage antigens for the therapy of blood malignancies is frequently complicated by self-targeting of CAR T cells or their excessive differentiation driven by constant CAR signaling. Expression of CARs targeting CD7, a pan-T cell antigen highly expressed in T cell malignancies and some myeloid leukemias, produces robust fratricide and often requires additional mitigation strategies, such as CD7 gene editing. In this study, we show fratricide of CD7 CAR T cells can be fully prevented using ibrutinib and dasatinib, the pharmacologic inhibitors of key CAR/CD3ζ signaling kinases. Supplementation with ibrutinib and dasatinib rescued the ex vivo expansion of unedited CD7 CAR T cells and allowed regaining full CAR-mediated cytotoxicity in vitro and in vivo on withdrawal of the inhibitors. The unedited CD7 CAR T cells persisted long term and mediated sustained anti-leukemic activity in two mouse xenograft models of human T cell acute lymphoblastic leukemia (T-ALL) by self-selecting for CD7-, fratricide-resistant CD7 CAR T cells that were transcriptionally similar to control CD7-edited CD7 CAR T cells. Finally, we showed feasibility of cGMP manufacturing of unedited autologous CD7 CAR T cells for patients with CD7+ malignancies and initiated a phase I clinical trial (ClinicalTrials.gov: NCT03690011) using this approach. These results indicate pharmacologic inhibition of CAR signaling enables generating functional CD7 CAR T cells without additional engineering.

Engineering T cells to suppress acute GVHD and leukemia relapse after allogeneic hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) limits the therapeutic benefit of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and requires immunosuppressive prophylaxis that compromises antitumor and antipathogen immunity. OX40 is a costimulatory receptor upregulated on circulating T cells in aGVHD and plays a central role in driving the expansion of alloreactive T cells. Here, we show that OX40 is also upregulated on T cells infiltrating GVHD target organs in a rhesus macaque model, supporting the hypothesis that targeted ablation of OX40+ T cells will mitigate GVHD pathogenesis. We thus created an OX40-specific cytotoxic receptor that, when expressed on human T cells, enables selective elimination of OX40+ T cells. Because OX40 is primarily upregulated on CD4+ T cells upon activation, engineered OX40-specific T cells mediated potent cytotoxicity against activated CD4+ T cells and suppressed alloreactive T-cell expansion in a mixed lymphocyte reaction model. OX40 targeting did not inhibit antiviral activity of memory T cells specific to Epstein-Barr virus, cytomegalovirus, and adenoviral antigens. Systemic administration of OX40-targeting T cells fully protected mice from fatal xenogeneic GVHD mediated by human peripheral blood mononuclear cells. Furthermore, combining OX40 targeting with a leukemia-specific chimeric antigen receptor in a single T cell product provides simultaneous protection against leukemia and aGVHD in a mouse xenograft model of residual disease posttransplant. These results underscore the central role of OX40+ T cells in mediating aGVHD pathogenesis and support the feasibility of a bifunctional engineered T-cell product derived from the stem cell donor to suppress both disease relapse and aGVHD following allo-HSCT.

Should all CAR-T therapy for acute lymphoblastic leukemia Be consolidated with allogeneic stem cell transplant?

Autologous T cells genetically modified with a CD19 chimeric antigen receptor are an effective therapy for children and adults with relapsed or refractory acute lymphoblastic leukemia with initial response rates ranging from 70 to 85%. Unfortunately, about half of these responding patients will subsequently relapse raising the question of whether allogeneic hemopoietic stem cell transplant should be considered as a consolidative therapy. Currently efforts are focused on defining risk factors for relapse to try and develop algorithms predicting which patients may benefit from allogenic transplant.