Early Stage Professionals Committee proceedings from the International Society for Cell & Gene Therapy 2022 Annual Meeting.

In this Committee Proceedings, representatives from the Early Stage Professional (ESP) committee highlight the innovative discoveries and key take-aways from oral presentations at the 2022 International Society for Cell and Gene Therapy (ISCT) Annual Meeting that cover the following subject categories: Immunotherapy, Exosomes and Extracellular Vesicles, HSC/Progenitor Cells and Engineering, Mesenchymal Stromal Cells, and ISCT Late-Breaking Abstracts.

Manufacture and Characterization of Good Manufacturing Practice-Compliant SARS-COV-2 Cytotoxic T Lymphocytes.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 virus-specific cytotoxic T-cell lymphocytes (vCTLs) could provide a promising modality in COVID-19 treatment. We aimed to screen, manufacture, and characterize SARS-CoV-2-vCTLs generated from convalescent COVID-19 donors using the CliniMACS Cytokine Capture System (CCS). METHODS: Donor screening was done by stimulation of convalescent COVID-19 donor peripheral blood mononuclear cells with viral peptides and identification of interferonγ (IFN-γ)+ CD4 and CD8 T cells using flow cytometry. Clinical-grade SARS-CoV-2-vCTLs were manufactured using the CliniMACS CCS. The enriched SARS-CoV-2-vCTLs were characterized by T-cell receptor sequencing, mass cytometry, and transcriptome analysis. RESULTS: Of the convalescent donor blood samples, 93% passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that were 79% (standard error of the mean 21%) IFN-γ+ T cells. SARS-CoV-2-vCTLs displayed a highly diverse T-cell receptor repertoire with enhancement of both memory CD8 and CD4 T cells, especially in CD8 TEM, CD4 TCM, and CD4 TEMRA cell subsets. SARS-CoV-2-vCTLs were polyfunctional with increased gene expression in T-cell function, interleukin, pathogen defense, and tumor necrosis factor superfamily pathways. CONCLUSIONS: Highly functional SARS-CoV-2-vCTLs can be rapidly generated by direct cytokine enrichment (12 hours) from convalescent donors. CLINICAL TRIALS REGISTRATION: NCT04896606.

The Future of Natural Killer Cell Immunotherapy for B Cell Non-Hodgkin Lymphoma (B Cell NHL).

OPINION STATEMENT: Natural killer (NK) cells have played a critical-if largely unrecognized or ignored-role in the treatment of B cell non-Hodgkin lymphoma (NHL) since the introduction of CD20-directed immunotherapy with rituximab as a cornerstone of therapy over 25 years ago. Engagement with NK cells leading to lysis of NHL targets through antibody-dependent cellular cytotoxicity (ADCC) is a critical component of rituximab's mechanism of action. Despite this important role, the only aspect of B cell NHL therapy that has been adopted as standard therapy that even indirectly augments or restores NK cell function is the introduction of obinutuzumab, a CD20 antibody with enhanced ability to engage with NK cells. However, over the last 5 years, adoptive immunotherapy with effector lymphocytes of B cell NHL has experienced tremendous growth, with five different CAR T cell products now licensed by the FDA, four of which target CD19 and have approved indications for some subtype of B cell NHL-axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, and tisagenlecleucel. These T cell-based immunotherapies essentially mimic the recognition, activation pathway, and cytotoxic machinery of a CD19 antibody engaging NK cells and lymphoma targets. Despite their efficacy, these T cell-based immunotherapies have been difficult to implement because they require 4-6 weeks of manufacture, are costly, and have significant toxicities. This renewed interest in the potential of cellular immunity-and the manufacturing, supply chain, and administration logistics that have been addressed with these new agents-have ignited a new wave of enthusiasm for NK cell-directed therapies in NHL. With high safety profiles and proven anti-lymphoma efficacy, one or more new NK cell-directed modalities are certain to be introduced into the standard toolbox of NHL therapy within the next few years, be it function-enhancing cytokine muteins, multi-domain NK cell engagers, or adoptive therapy with expanded or genetically modified NK cells.

Natural killer cell therapy for hematologic malignancies: successes, challenges, and the future.

The adoptive transfer of natural killer (NK) cells is an emerging therapy in the field of immuno-oncology. In the last 3 decades, NK cells have been utilized to harness the anti-tumor immune response in a wide range of malignancies, most notably with early evidence of efficacy in hematologic malignancies. NK cells are dysfunctional in patients with hematologic malignancies, and their number and function are further impaired by chemotherapy, radiation, and immunosuppressants used in initial therapy and hematopoietic stem cell transplantation. Restoring this innate immune deficit may lead to improved therapeutic outcomes. NK cell adoptive transfer has proven to be a safe in these settings, even in the setting of HLA mismatch, and a deeper understanding of NK cell biology and optimized expansion techniques have improved scalability and therapeutic efficacy. Here, we review the use of NK cell therapy in hematologic malignancies and discuss strategies to further improve the efficacy of NK cells against these diseases.

COVID-19 in immunocompromised patients: A systematic review of cancer, hematopoietic cell and solid organ transplant patients.

BACKGROUND: The clinical impact of severe coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in immunocompromised patients has not been systematically evaluated. METHODS: We reviewed current literature reporting on COVID-19 in cancer (CA), hematopoietic cell (HCT), and solid organ transplant (SOT) patients and compared their clinical data and outcomes to the general population. For adult CA, HCT and SOT patients, an extensive search strategy retrieved all articles published until July 20, 2020 by combining the terms coronavirus, coronavirus infection, COVID-19, and SARS-CoV-2 in PubMed, Cochrane, and Web of Science, and following the Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines. For the pediatric CA cohort, a global COVID-19 registry was used. For the general population cohort, a large meta-analysis was used to compare pooled prevalence estimates, and two large meta-analyses were utilized to serve as pooled comparators for hospitalized COVID-19 patients. FINDINGS: Compared to the general population, adult CA and SOT patients with COVID-19 had higher comorbidities, greater levels of inflammatory markers at diagnosis, and higher rates of intensive care and hospital mortality. Pediatric CA patients and HCT patients with COVID-19 tended to have clinical presentations and outcomes similar to the general population. INTERPRETATION: To our knowledge, this is the first systematic review evaluating COVID-19 phenotype and outcomes in immunocompromised patients and comparing them to the general population, which shows that hospital outcomes appear to be worse in adult CA and SOT patients, potentially due to their higher co-morbidity burden. FUNDING: None.

Renal Tumors in Children and Young Adults Older Than 5 Years of Age.

Renal masses are most common in children between ages 1 to 3 years, with less known about renal tumors in older children and young adults. The aim of this study was to review the presentation, demographics, histology, and outcomes in patients over 5 years of age with renal tumors compared with younger children. 111 renal tumors were diagnosed in patients 5 years of age and older (median, 7 y; range, 5 to 31 y) between 1950 and 2017 at a single institution. Wilms tumor (WT) was the most common histology in 84 patients (75%), followed by renal cell carcinoma in 12 patients (10.7%). Abdominal pain was the most common presenting symptom (46%) followed by hematuria (28.8%), and a palpable abdominal mass (24.3%). For WT, older children more commonly presented with advanced-stage disease (stages 3 and 4) than younger children (57.7% vs. 11.5%; P<0.001). Event-free survival (EFS) and overall survival (OS) for favorable histology WT were not different between younger and older children (OS, P=0.43; EFS, P=0.46). In this cohort, older children more frequently present with variable signs and symptoms, less common histopathologies although WT was still most frequent, and more advanced-stage disease compared with younger cohorts, but without differences in EFS or OS.

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins.

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGFß. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Renal Tumors in Children Younger Than 12 Months of Age: A 65-Year Single Institution Review.

Wilms tumor (WT) is the most prevalent pediatric renal tumor and most commonly occurs between ages 1 and 5 years. Data are lacking on children younger than 12 months with renal tumors. The cancer registry at the authors' institution was queried to identify patients 12 months and younger with renal masses. Demographics, clinical presentation, histopathology, stage, and survival outcomes were reviewed. The most common presenting symptoms included an asymptomatic abdominal mass (73%) and hematuria (9%). Histopathology revealed WT in 73% of patients, mesoblastic nephroma in 20%. Of those infants younger than 1 month of age, mesoblastic nephroma was the most common histopathology (68%). The 5-year overall survival (OS) was 93%, and 5-year event-free survival (EFS) was 93% for the entire group. For patients with WT, 5-year OS was 88% and 5-year EFS was 83%. Outcomes for congenital mesoblastic nephroma were excellent with 5-year OS and EFS of 100%. Reasons for good prognosis may be multifactorial and may include frequent well child checks in the first year of life and favorable histology. Patients in this age group are more likely to be classified as very low risk and may be treated with surgical resection alone.

EMS helicopter crashes: what influences fatal outcome?

STUDY OBJECTIVE: In recent years, air transport of patients has been associated with disproportionate increases in crashes and deaths. We identify factors related to fatal outcome in air medical helicopter crashes and suggest preventive measures. METHODS: This was a retrospective study using National Transportation Safety Board records for helicopter emergency medical services (EMS) crashes between January 1, 1983, and April 30, 2005. The main outcome measure was the percentage of air medical crashes resulting in 1 or more deaths. RESULTS: There were 182 helicopter EMS crashes during the 22.3-year study period; 39% were fatal. One hundred eighty-four occupants died: 45% of the 44 patients and 32% of the 513 crewmembers. Fifty-six percent of crashes in darkness were fatal compared with 24% of crashes not in darkness. Seventy-seven percent of crashes in instrument meteorological conditions were fatal compared with 31% in visual conditions. Thirty-nine percent of all deaths occurred in crashes with postcrash fires; 76% of crashes with postcrash fire were fatal compared with 29% of other crashes. Multivariate logistic regression revealed that controlling for other factors, the odds of fatal outcome was increased by postcrash fire (odds ratio [OR] 16.1; 95% confidence interval [CI] 5.0 to 51.5], bad weather (OR 8.0; 95% CI 2.4 to 26.0), and darkness (OR 3.2; 95% CI 1.3 to 7.9). CONCLUSION: Fatalities after helicopter EMS crashes are associated especially with postcrash fire and with crashes that occur in darkness or bad weather and can be addressed with improved crashworthiness and measures to reduce flights in hazardous conditions. Further studies will be necessary to determine which changes will decrease the fatal crash rate and which are cost effective.

Characteristics of alcohol-related fatal general aviation crashes.

BACKGROUND: The effects of alcohol on piloting performance have been studied extensively. Information describing alcohol-related aviation crashes, however, is scant. METHODS: We developed a data system for fatally injured pilots in Maryland, New Mexico, and North Carolina by linking autopsy data from the state medical examiner offices and crash investigation reports from the National Transportation Safety Board. Alcohol-related crashes are defined as those in which the pilot had a blood alcohol concentration of 20 mg/dL or greater. Differences between alcohol- and non-alcohol-related crashes were assessed with regard to pilot characteristics, crash circumstances, and human factors. RESULTS: The National Transportation Safety Board recorded 313 general aviation crashes fatal to the pilot in the three states between 1985 and 2000. Of these crashes, 255 (81%) were matched successfully with medical examiner records. Alcohol testing results were available for 233 of the fatally injured pilots. Of those tested for alcohol, 25 (11%) had blood alcohol concentrations > or =20 mg/dL (mean=75 +/- 64 mg/dL). The majority of alcohol-related crashes (52%) occurred at night (7p.m. to 6a.m.), compared with 28% of other crashes (P < 0.01). Alcohol-related crashes were significantly more likely than other crashes to have involved continued flight under visual flight rules (VFR) into instrument meteorological conditions (IMC) (32% versus 12%, P < 0.01), and flawed decisions (64% versus 41%, P = 0.03). CONCLUSIONS: Distinctive epidemiological patterns are exhibited in alcohol-related fatal general aviation crashes. Alcohol appears to play a particularly important role in crashes involving flight under VFR into IMC.

Human factors in aviation crashes involving older pilots.

BACKGROUND: Pilot errors are recognized as a contributing factor in as many as 80% of aviation crashes. Experimental studies using flight simulators indicate that due to decreased working memory capacity, older pilots are outperformed by their younger counterparts in communication tasks and flight summary scores. OBJECTIVE: This study examines age-related differences in crash circumstances and pilot errors in a sample of pilots who flew commuter aircraft or air taxis and who were involved in airplane or helicopter crashes. METHODS: A historical cohort of 3306 pilots who in 1987 flew commuter aircraft or air taxis and were 45-54 yr of age was constructed using the Federal Aviation Administration's airmen information system. Crash records of the study subjects for the years 1983-1997 were obtained from the National Transportation Safety Board (NTSB) by matching name and date of birth. NTSB's investigation reports were reviewed to identify pilot errors and other contributing factors. Comparisons of crash circumstances and human factors were made between pilots aged 40-49 yr and pilots aged 50-63 yr. RESULTS: A total of 165 crash records were studied, with 52% of these crashes involving pilots aged 50-63 yr. Crash circumstances, such as time and location of crash, type and phase of flight, and weather conditions, were similar between the two age groups. Pilot error was a contributing factor in 73% of the crashes involving younger pilots and in 69% of the crashes involving older pilots (p = 0.50). Age-related differences in the pattern of pilot errors were statistically insignificant. Overall, 23% of pilot errors were attributable to inattentiveness, 20% to flawed decisions, 18% to mishandled aircraft kinetics, and 18% to mishandled wind/runway conditions. CONCLUSIONS: Neither crash circumstances nor the prevalence and patterns of pilot errors appear to change significantly as age increases from the 40s to the 50s and early 60s.