Chimeric antigen receptor-engineered (CAR)-T cell therapy remains limited by significant toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The optimal management of severe and/or refractory CRS/ICANS remains ill-defined. Anakinra has emerged as a promising agent based on preclinical data, but its safety and efficacy in CAR-T therapy recipients are unknown. The primary objective of this study was to evaluate the safety of anakinra to treat refractory CRS and ICANS after CAR-T therapy. The secondary objective was to evaluate the impact of key treatment-, patient-, and disease-related variables on the time to CRS/ICANS resolution and treatment-related mortality (TRM). We retrospectively analyzed the outcomes of 43 patients with B cell or plasma cell malignancies treated with anakinra for refractory CRS or ICANS at 9 institutions in the United States and Spain between 2019 and 2022. Cause-specific Cox regression was used to account for competing risks. Multivariable cause-specific Cox regression was used to estimate the effect of anakinra dose on outcomes while minimizing treatment allocation bias by including age, CAR-T product, prelymphodepletion (pre-LD) ferritin, and performance status. Indications for anakinra treatment were grade ≥2 ICANS with worsening or lack of symptom improvement despite treatment with high-dose corticosteroids (n = 40) and grade ≥2 CRS with worsening symptoms despite treatment with tocilizumab (n = 3). Anakinra treatment was feasible and safe; discontinuation of therapy because of anakinra-related side effects was reported in only 3 patients (7%). The overall response rate (ORR) to CAR-T therapy was 77%. The cumulative incidence of TRM in the whole cohort was 7% (95% confidence interval [CI], 2% to 17%) at 28 days and 23% (95% CI, 11% to 38%) at 60 days after CAR-T infusion. The cumulative incidence of TRM at day 28 after initiation of anakinra therapy was 0% in the high-dose (>200 mg/day i.v.) recipient group and 47% (95% CI, 20% to 70%) in the low-dose (100 to 200 mg/day s.c. or i.v.) recipient group. The median cumulative incidence of CRS/ICANS resolution from the time of anakinra initiation was 7 days in the high-dose group and was not reached in the low-dose group, owing to the high TRM in this group. Univariate Cox modeling suggested a shorter time to CRS/ICANS resolution in the high-dose recipients (hazard ratio [HR], 2.19; 95% CI, .94 to 5.12; P = .069). In a multivariable Cox model for TRM including age, CAR-T product, pre-LD ferritin level, and pre-LD Karnofsky Performance Status (KPS), higher anakinra dose remained associated with lower TRM (HR, .41 per 1 mg/kg/day increase; 95% CI, .17 to .96; P = .039. The sole factor independently associated with time to CRS/ICANS resolution in a multivariable Cox model including age, CAR-T product, pre-LD ferritin and anakinra dose was higher pre-LD KPS (HR, 1.05 per 10% increase; 95% CI, 1.01 to 1.09; P = .02). Anakinra treatment for refractory CRS or ICANS was safe at doses up to 12 mg/kg/day i.v. We observed an ORR of 77% after CAR-T therapy despite anakinra treatment, suggesting a limited impact of anakinra on CAR-T efficacy. Higher anakinra dose may be associated with faster CRS/ICANS resolution and was independently associated with lower TRM. Prospective comparative studies are needed to confirm our findings.

Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through the release of perforin-containing lytic granules. In the present study, we first performed probability-state modeling of differentiation and lytic granule markers on CD8⁺ T cells to enable the comparison of bona fide CTLs with NK cells. Analysis identified CD57^bright expression as a reliable phenotype of granule marker–containing CTLs. We then compared CD3⁺CD8⁺CD57^bright CTLs with NK cells. Healthy adult peripheral blood CD3⁺CD8⁺CD57^bright CTLs expressed more granzyme B but less perforin than CD3⁻CD56^dim NK cells. On stimulation, such CTLs degranulated more readily than other T-cell subsets, but had a propensity to degranulate that was similar to NK cells. Remarkably, the CTLs produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistiocytosis, CTL and NK cell degranulation were similarly impaired. Therefore, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3⁺CD8⁺CD57^bright CTLs and NK cells and suggest that analysis of CD57^bright CTL function may prove useful in the diagnosis of primary immunodeficiencies including familial hemophagocytic lymphohistiocytosis.

Purpose: NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8+ T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D–NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated in vitro and in vivo the safety and cytotoxic capacity against osteosarcoma cells of CD45RA− memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR). Experimental Design: CD45RA− cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. In vitro cytotoxicity of NKG2D-CAR+ CD45RA− T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the in vivo orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rgnull mice. Results: Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA− cells. Genetic stability was preserved in transduced cells. In vitro, NKG2D-CAR+ memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR+ memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective. Conclusions: Our results demonstrate NKG2D-4-1BB-CD3z CAR–redirected memory T cells target NKG2DL-expressing osteosarcoma cells in vivo and in vitro and could be a promising immunotherapeutic approach for patients with osteosarcoma. Clin Cancer Res; 23(19); 5824–35. ©2017 AACR.

The purpose of this controlled trial was to assess the effect of an approximately 3-wk intrahospital exercise intervention performed during inpatient hospitalization for pediatric allogeneic hematopoietic stem cell transplant (HSCT) on (i) immune cell recovery and (ii) body composition. Immune (i.e., blood counts of leukocytes, monocytes, lymphocytes, and lymphocyte subpopulations) and anthropometric variables (i.e., body mass, body mass index, and estimated fat-free mass) were measured before and after (+15 and 30 d) HSCT. Seven children (5 boys and 2 girls; age (mean +/- SD) = 8 +/- 4 yr) with high-risk cancer performed an individualized training program (aerobic + resistance exercises) in their isolated hospital rooms. We also assessed a control group (n = 13; 9 boys and 4 girls; age = 7 +/- 3 yr) with similar medical conditions and following the same transplant protocol. In both groups, the dendritic cell count decreased from pre-HSCT to +15 d post-HSCT and thereafter (up to +30 d) remained stabile; however, the posttransplant decrease was more abrupt in the control group than that in the intervention group (-87% vs -63%, respectively, from pre-HSCT to +15 d). The rest of the immune cell parameters measured showed a similar response from pre-HSCT to post-HSCT in both groups. We found a significant effect of the interaction group x time for all anthropometric variables (weight, body mass index, body fat, and fat-free mass), indicating an increase over the hospitalization period only in the intervention group, for example, body mass increased from 32.9 +/- 18.7 kg pre-HSCT to 35.4 +/- 18.6 kg at +30 d in the intervention group versus a decrease from 30.2 +/- 16.6 to 29.3 +/- 6.3 kg in the control group. Our findings support the feasibility of exercise training interventions during hospitalization, including immunocompromised children.

The randomized controlled trial “Physical Activity in Pediatric Cancer” determined the effects of an inhospital exercise intervention combining aerobic and muscle strength training on pediatric cancer patients with solid tumors undergoing neoadjuvant chemotherapy. Participants were allocated to an exercise (n = 24, 17 boys; mean ± SEM age, 10 ± 1 yr) or control group (n = 25, 18 boys; 11 ± 1 yr). Training included three sessions per week for 19 ± 2 wk. Participants were assessed at treatment initiation, termination, and 2 months after end treatment. The primary endpoint was muscle strength (as assessed by upper and lower-body five-repetition-maximum tests). Secondary endpoints included cardiorespiratory fitness, functional capacity during daily life activities, physical activity, body mass and body mass index, and quality of life. Most sessions were performed in the hospital’s gymnasium. Adherence to the program averaged 68% ± 4% and no major adverse events or health issues were noted. A significant interaction (group–time) effect was found for all five-repetition maximum tests (leg/bench press and lateral row; all P < 0.001). Performance significantly increased after training (leg press: 40% [95% confidence interval [CI], 15–41 kg); bench press: 24% [95% CI, 6–14 kg]; lateral row 25% [95% CI, 6–15 kg]), whereas an opposite trend was found in controls. Two-month post values tended to be higher than baseline for leg (P = 0.017) and bench press (P = 0.014). In contrast, no significant interaction effect was found for any of the secondary endpoints. An inhospital exercise program for pediatric cancer patients with solid tumors undergoing neoadjuvant treatment increases muscle strength despite the aggressiveness of such therapy.

This proof-of-concept single-arm open-label phase I clinical trial (NCT02481934) studied the safety and efficacy of multiple infusions of activated and expanded natural killer (NKAE) cells in combination with anti-myeloma drugs in multiple myeloma patients. It included five patients with relapsed or refractory MM who had received two to seven prior lines of therapy; NK cells were expanded for 3 weeks with K562-mb15-41BBL cells. Patients received four cycles of pharmacological treatment with two infusions of 7.5 × 10⁶ NKAEs/kg per cycle. NKAE generation, expansion, and NK monitoring was assessed using flow cytometry. Eighteen clinical-grade NKAE cell GMP-grade products were generated to obtain 627 × 10⁶ NKAEs (range: 315–919 × 10⁶) for the first infusion and 943 × 10⁶ (range: 471–1481 × 10⁶) for the second infusion with 90% (±7%) purity. Neutropenia grade II occurred in two patients and was related to chemotherapy. Of the five patients, four showed disease stabilization before the end of NKAE treatment, and two showed a 50% reduction in bone marrow infiltration and a long-term (>1 y) response. The NKAE cells had a highly cytotoxic phenotype and high cytotoxicity in vitro. Infused NKAE cells were detected in bone marrow and peripheral blood after infusions. Ex vivo expansion of autologous NK cells is feasible, NKAE cells are clinically active and the multiple infusions are well tolerated in patients with relapsed or refractory myeloma.