Activation of Cell-Intrinsic Signaling in CAR-T Cells via a Chimeric IL7R Domain.

Chimeric antigen receptor (CAR) T cells can effectively treat leukemias, but sustained antitumor responses can be hindered by a lack of CAR T-cell persistence. Cytotoxic effector T cells are short-lived, and establishment of CAR-T cells with memory to ensure immune surveillance is important. Memory T cells depend on cytokine support, with IL7 activation of the IL7 receptor (IL7R) being critical. However, IL7R surface expression is negatively regulated by exposure to IL7. We aimed to support CAR T-cell persistence by equipping CAR-T cells with a sustained IL7Rα signal. We engineered T cells to constitutively secrete IL7 or to express an anti-acute myeloid leukemia-targeted IL7Rα-chimeric cytokine receptor (CCR) and characterized the phenotype of these cell types. Canonical downstream signaling was activated in CCR-T cells with IL7R activation. When coexpressed with a cytotoxic CAR, functionality of both the CCR and CAR was maintained. We designed hybrid CAR-CCR and noted membrane proximity of the intracellular domains as vital for signaling. These data show cell-intrinsic cytokine support with canonical signaling, and functionality can be provided via expression of an IL7Rα domain whether independently expressed or incorporated into a cytotoxic CAR for use in anticancer therapy. SIGNIFICANCE: To improve the phenotype of tumor-directed T-cell therapy, we show that provision of cell-intrinsic IL7R-mediated signaling is preferable to activation of cells with exogenous IL7. We engineer this signaling via independent receptor engineering and incorporation into a CAR and validate maintained antigen-specific cytotoxic activity.

Return to Play After Arthroscopic Management of Rotator Cuff Tears in Professional Athletes of Contact Sports.

Background: Rotator cuff tears are rare injuries in professional athletes who participate in contact sports, and limited data exist to guide players and team physicians regarding outcomes after surgical management. Purpose: To report the outcomes and return-to-play rates of professional contact sport athletes who underwent arthroscopic management of rotator cuff tears. Study Design: Case series; Level of evidence, 4. Methods: All professional athletes of contact sports who underwent arthroscopic management of a rotator cuff tear between 2002 and 2019 at the institution were identified. Patient information collected were age, sport, position, date of injury, date of surgery, and time to return to play; surgical data included tear size, acuity, pattern, and procedure performed. The primary outcome measure was the ability to return to play and the number of games played after surgery. Results: Overall, 10 rotator cuff tears in 9 professional athletes (8 tears in football players and 2 tears in hockey players) were identified; 9 of the tears were full-thickness tears that underwent arthroscopic single-row repair, while 1 was a partial tear that was debrided. Of the 9 athletes, 8 were able to return to play at the same level, at a mean time of 9.5 ± 4.3 months. The mean playing time after surgery was 32 ± 25 games (2.7 ± 2 seasons) for the football players and 22 games (1 season) for the hockey player who returned. Postoperative imaging was available in 8 of the 10 tears, and 7 of 8 (88%) demonstrated a healed repair. One football player and 1 hockey player with large (>3 cm) full-thickness tears did not return to play. The mean age of these players was 34.5 years and both had >10 years of professional playing experience.≥. Conclusion: The study findings demonstrated that the majority (80%; n = 8) of the professional athletes in contact sports in this series were able to return to play at the same level after arthroscopic management of a symptomatic rotator cuff tear.

Return to play after rotator cuff tear in National Football League athletes.

Background: Rotator cuff tears are rare injuries in National Football League (NFL) athletes and there are limited data to help guide players and team physicians. The purpose of this study was to assess return to play (RTP) rates, performance levels, and career length following a rotator cuff tear during their playing career. Methods: Using publicly available data, we identified players who sustained a rotator cuff tear between 2000 and 2019. Demographic information, treatment (operative vs. nonoperative), RTP rate, preinjury and postinjury performance score, position, and career length were entered into the analysis. Results: Twenty-nine athletes with a mean age of 27.4 years (±3.1) at the time of injury were included in this study. Forty-eight percent were offensive and 52% defensive players. 79.3% (23/29) were able to RTP at the same professional level for an average of 2.8 ± 3.4 years. The average time to RTP after injury was 198.4 ± 125.3 days. The average age of players who RTP was 26.7 ± 2.5 years compared to those who did not (30.3 ± 3.7, P = .02). Similarly, the preinjury NFL career length was 4.0 ± 2.2 in players who RTP compared to those who did not (7.5 ± 2.7, P = .01). Most injuries (82.2%) were treated surgically; however, there was no significant difference (P > .05) in RTP rates, performance score, or career longevity between operative and nonoperative cohorts. Conclusion: Overall RTP rates for NFL athletes following a rotator cuff injury are promising with approximately 80% returning at the same performance level regardless of treatment type. Older, veteran players particularly those over the age of 30 were significantly less likely to RTP and should be counseled accordingly.

Impact of High Disease Burden on Survival in Pediatric Patients with B-ALL Treated with Tisagenlecleucel.

CD19-specific chimeric antigen receptor (CAR) T-cell therapies, including the FDA-approved tisagenlecleucel, induce high rates of remission in pediatric patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, post-treatment relapse remains an issue. Optimal management of B-ALL after tisagenlecleucel treatment remains elusive, and continued tracking of outcomes is necessary to establish a standard of care for this population. We sought to evaluate outcomes on the real-world use of tisagenlecleucel in a contemporary pediatric patient population and to identify risk factors influencing event-free survival (EFS) and overall survival (OS). Additionally, we aimed to describe post-tisagenlecleucel management strategies, including use of allogeneic hematopoietic cell transplantation (AlloHCT) or repeat CAR T-cell infusions. We report on 31 pediatric and adolescent and young adult patients (AYA) with B-ALL, treated with lymphodepleting chemotherapy followed by tisagenlecleucel. Patients were treated at Johns Hopkins Hospital and St. Jude Children's Research Hospital between March 2018 and November 2020. Data on patient, disease, and treatment characteristics were collected retrospectively from medical records and described. EFS and OS were estimated by the Kaplan-Meier method and compared by the log-rank test. Single-factor and multiple-factor analysis of EFS and OS were performed by fitting Cox regression models. Of the 30 evaluable patients, 25 (83.3%) experienced a complete response, with 21 having negative minimal residual disease. Treatment was well tolerated, with expected rates of cytokine release syndrome (61.3%) and immune effector cell-associated neurotoxicity (29%). After initial complete response, 12 patients (48%) had subsequent disease recurrence, with CD19-negative relapse (n = 6) occurring sooner than CD19-positive relapse (P = .0125). With a median follow-up time of 386 days (range 11-1187 days), the EFS for the entire cohort (n = 31) at 6 and 12 months after infusion was 47% (95% confidence interval [CI], 28.4%-63.4%) and 35.2% (95% CI, 18.4%-52.5%), respectively. In multivariate analysis, high pretreatment leukemic burden (≥5% bone marrow blasts) was an independent risk factor for inferior EFS (HR 5.98 [95% CI, 1.1-32.4], P = .0380) and OS (HR 4.2 [95% CI, 1.33-13.39], P = .0148). Tisagenlecleucel induced high initial response rates in a contemporary cohort of pediatric and AYA patients with B-ALL. However, 48% of patients experienced subsequent disease relapse, including 6 with antigen-escape variants. This highlights a considerable limitation of single-agent autologous CD19-CAR T-cell therapy. Pretreatment leukemic disease burden of ≥5% blasts was significantly associated with worse outcomes in this study, including lower EFS and OS. Our findings suggest that reducing preinfusion leukemic burden is a viable treatment strategy to improve outcomes of CAR T-cell therapy.

Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities.

BACKGROUND: The prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence. METHODS: We characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models. RESULTS: CARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model. CONCLUSION: Transgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

Glycoprotein Targeted CAR-NK Cells for the Treatment of SARS-CoV-2 Infection.

H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19.