Booth, Natalie

Sickle cell disease (SCD) is a chronic hematologic disorder which causes progressive cerebral arteriopathy beginning in childhood. As a result, arterial ischemic stroke is a major cause of morbidity and mortality in SCD, and SCD is a leading cause of childhood stroke worldwide. Allogenic hematopoietic stem cell transplant (HSCT) may be curative for individuals with SCD. Long-term outcomes and effects are currently being studied. In this report, we describe a child with SCD who presented with arterial ischemic stroke at 6 years of age and was found to have a severe form of cerebral large vessel arteriopathy by catheter-directed angiography. The patient initially underwent revascularization surgery by indirect superficial temporal artery to middle cerebral artery bypass, and 1 year later, he underwent curative HSCT. Approximately 3 years after HSCT, repeat catheter-directed angiography revealed a striking reversal of cerebral large vessel arteriopathy. This article reveals a previously unrecognized and potentially beneficial effect of HSCT that may ameliorate cerebral large vessel arteriopathy and improve cerebrovascular health for children with SCD.

Abstract Purpose:The incidence of severe combined immunodeficiency (SCID) in the United States was reported as 1 in 58,000 live births. In Arizona, it was anticipated that newborn screening would identify two to four cases of SCID per year. This estimate did not consider ethnic nuances in Arizona, with higher percentages of American Indian and Hispanic populations compared to national percentages. The true incidence of SCID and non SCID T-cell lymphopenia have not previously been reported in Arizona. Methods:A retrospective chart review was performed of abnormal SCID Newborn Screening (NBS) tests from January 1, 2018 to December 31, 2019, using data from the Arizona Department of Health Service and the Phoenix Children’s Hospital’s electronic medical record [IRB# 20-025]. Results: Seven infants were diagnosed with SCID, yielding an incidence of 1 in 22,819 live births. Four of these infants had Artemis type SCID. Eighteen infants were identified with an abnormal initial NBS which ultimately did not lead to a diagnosis of SCID. Four of these infants were diagnosed with congenital syndromes associated with T-cell lymphopenia. Infants of Hispanic ethnicity were over-represented in this cohort. Conclusion: NBS in Arizona confirmed an incidence more than 2.5 times that reported nationally. This increased incidence is likely reflective of Arizona’s unique population profile, with a higher percentage of American Indian population. The findings in our non-SCID cohort are in alignment with previously published data, except for a higher than anticipated number of infants of Hispanic/Latino ethnicity, likely related to Arizona’s higher percentage of Hispanic/Latino population.

Abstract Introduction: CAR T-cell therapy has revolutionized the treatment of patients with relapsed/refractory (R/R) acute leukemia, NHL, and multiple myeloma. However, there are still areas of improvement in their clinical activity, source of the effector cells, prevention, and management of adverse events that require particular attention. Because of those reasons, NK cells appear as a viable effector cell alternative that can help address these challenges. NK cells offer a profile of activation, expansion, persistence, and cytotoxicity that is different from T cells and, when modified to bear CAR constructs, may provide significant advantages. However, the preclinical development of NK-CARs is challenging mainly because of the difficulty of generating large quantities of cells for testing and well-established pathways for CAR optimization before in vivo evaluation. Therefore, we developed a CAR optimization platform using the NK-92 cell line. NK-92 cells conserve their cytotoxic ability and can be easily expanded in vitro and used for functional and phenotypical evaluations of novel CAR-NK constructs. Here we present a rechallenge cytotoxic assay that mimics repetitive in vivo effector interactions with the target cells and its use for optimization, comparison, and development of NK-based cellular therapies. Methods: We generated lentivirus transduced CD19 CARs (FMC63-41BB-z) using T cells from healthy donors and NK-92 cells for comparison.T cells were expanded for 12 days, and a 41.9% CAR+ expression was achieved (CART19). Transduced NK-92 cells were sorted by FACS to obtain a population of 98.3 % CAR+ cells (CARNK19) and subsequently expanded for 12 days. JeKo-1 cells were used as CD19+ targets and BxPC3 cells as CD19 neg control (both cell types were GFP-Luc-PuroR). We developed a Luciferase-based rechallenge cytotoxicity assay. For this, we diluted the effector to target (E/T) ratio to obtain a logarithmic trendline of the cells' cytotoxicity. E/T ratio to get viability of 50% (IC50) measured at 4h (for CARNK19) and 24h (for CART19) was used as a proxy of the product's potency. Both CAR Immune Effector Cells (IECs) were co-cultured with their targets at an E/T ratio to obtain 70% cytotoxicity. After 24 hours with the target, we estimated the remaining IEC amount in the culture using GFP exclusion in flow analysis (IEC cells/mL = total cells/mL x GFP neg%). We repeated the plating of E/T ratio dilutions to perform daily IC50 curves using this rechallenge strategy for a total of 5 days. CAR and PD1 expression were measured on Day 0 and Day 5 by flow cytometry. Results: CART19 showed a higher IC50 than CARNK19 at baseline, 1.7 vs. 0.19 (Figure 1A). The IC50 trend of both IECs over time showed an uptrend that suggests progressive functional exhaustion (Figure 1B). At 5 days of rechallenge, it was 29 times higher in T cells than in NK-92 (12.07 vs. 0.42) and with a slope 265 times higher (10.6 vs. 0.04). Furthermore, we observed that when comparing the levels of CAR expression on Day 0 vs. Day 5, CART19 showed a decrease in CAR expression that was not present in CARNK19 (41.9 to 10.9% vs. 98.3 to 95.5%) (Figure 1C). In addition, there was a higher increase in PD1 expression in CART19 cells than CARNK19 cells from Day 0 to Day 5 of the in vitro rechallenge (9.9 to 46.8% vs. 0.88 to 8.88%) (Figure 1D). Conclusion: Our data shows the use of NK-92 cells as a tool for optimization and preclinical development of NK cell-based cellular therapies. We demonstrated that it is feasible to set up repetitive cytotoxic challenges that mimic closer in vivo E/T engagement. Moreover, using the cytotoxic IC50 calculated with this platform, we show increased cytotoxicity, less functional exhaustion, and less expression of PD1 in CARNK19 than in its T cell counterpart. Overall, the NK-92 rechallenge cytotoxicity assay platform constitutes a helpful tool for research, development, and optimization of cellular therapies based on NK cell effector function. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Background: Chimeric Antigen Receptor (CAR) T cell therapy is arguably one of the most significant breakthroughs in cancer treatment. There are currently five FDA-approved products that are commercially available. However, despite their success, these CAR T-cell therapies cannot induce long-term durable responses in approximately 50% of leukemia or lymphoma-treated patients. Similarly, the results of CAR T-cells in solid tumors have been somewhat disappointing. Therefore, there is an urgent need to design and develop novel CAR T cells with improved efficacy in hematologic malignancies and solid tumors. ROR1 is a carcinoembryonic antigen expressed in different cancers and is associated with tumor stemness, proliferation, metastatic transformation, and treatment resistance. In this project, we optimize an anti-ROR1 CAR using a humanized single-chain variable fragment (scFv) with second (2G) or third-generation (3G) costimulatory domains. Methods: Several optimization steps in silico were performed using a selected scFv binding domain that targets ROR1. Those included codon optimizations, positional arrangement of heavy-light chains, evaluation of the ideal length of linkers based on tridimensional modeling of the docking between the antibody-like paratope with the target antigen (Figure 1A). After this initial scFv optimization process, we constructed a lentiviral vector that encodes CARs using the selected scFv linked to a transmembrane domain CD28 and different signaling endodomains for 2G and 3G variants (CD28, 41BB, ICOS, OX40), each linked to the T cell receptor CD3z domain. The cytotoxic activity of these constructs was evaluated using an in vitro rechallenge luciferase assay in ROR1 expressing JeKo-1 cells and ROR1(negative) controls. Results: The 2G 41BB-z construct with V H-V L scFv orientation and a long linker (V H-L-V L) showed optimal cytotoxicity with a CAR expression level in T cells of 36% (Range 28-49% for other constructs, Figure 1B-C). The V H-L-V L 41BB-z construct was evaluated comparatively using a rechallenge cytotoxic assay with 3G constructs that expressed CD28, ICOS, or OX40 signaling domains using JeKo-1 and ROR1(negative) target cells as controls. All the tested constructs showed specific ROR1 medicated cytotoxicity. CD28-41BB-z and ICOS-41BB-z showed the lowest cytotoxicity levels during the Day 1 of the repetitive rechallenge. However, the cytotoxicity levels of those constructs gradually increased during the 7 days of rechallenge and were closed to the levels induced by the 2G- 41BB-z construct (>80% of cytotoxicity). There were no significant differences in CAR T cells subsets generated by the different constructs during the 7 days of rechallenge with a predominance of effector memory phenotype (CCR7-, CD45RA-) and no difference in PD1 expression. Conclusions: Our results demonstrate that optimization of the CAR constructs enhances T-cell effector function and cytotoxicity against ROR1+ target cells. In previous studies, 3G CARs have shown longer persistence of the transduced T cells in peripheral blood, sustained and regulated cellular activation, improved solid tumor infiltration, and positive modulation of the tumor microenvironment. Our preclinical in vitro optimization demonstrates strategies to generate 3G constructs with a progressive and modulated cytotoxic profile that may confer benefits when tested in vivo in terms of enhanced persistence and lower adverse events profile. Additional experiments in vivo will be presented during the meeting to corroborate our findings. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

e14516 Background: IEC are arguably the most promising therapy in oncology, and FDA approvals for CAR T cells are a testament to their real potential. This has led to a great growth of clinical trials (CT), making the evaluation of their associated data increasingly challenging. To facilitate this process, we performed a systematic review of ClinicalTrials.gov focused on IEC therapies in oncology. We performed an analysis of the CT data and forecast trends for 2025. Methods: CT registries were retrieved from ClinicalTrials.gov with a systematic search query. We included CT registered between 1993-2020, which used IEC-based therapies in oncology. Statistical analysis using descriptive and inferential methods allowed us to identify trends and establish forecasts. Results: We identified 938 registrations of IEC-CTs in oncology, and 51% of those were active. The most common IEC type was CAR T (51%), followed by NK (15%), TCR T (8%), TIL (8%), and CIK (3%). 69% of IEC-CTs were aimed at a specific target vs. 31% that lack that specificity. The number of annual CAR T-CTs continue to increase, and we forecast 320 registrations during 2025. Since 2018, the number of CTs using unmodified T cells and TIL has increased (largely due to metastatic melanoma studies). NK cells CTs represent 8% of all registrations, and by 2025 we anticipate that >150 NK-based CTs will be registered per year, a number similar to CAR T-CTs registered during 2020. IEC-CTs based on allogeneic sources represent 18% of all registrations and are expected to grow 2.5 fold in the next 5 years. In solid tumors, the most common IEC-CTs are based on Non-CAR studies, while in hem-malignancies 76% of CTs are CAR T. Only one solid tumor, CAR T-CT, has reached phase 2/3, while 7 CIK-CTs were used to target various organ tumors. Conclusions: IEC-CTs continue to grow exponentially and represent an active field of clinical research in oncology. Allogeneic and NK-CTs increase rapidly and are among the most promising IEC-CTs. A rising proportion of CTs in solid tumors are using CIK and TIL rather than CAR T-cells, which are more popular for treating hem-malignancies. This is a direct reflection of the challenges associated with CAR T infiltration and persistence in solid tumors. Our analyses indicate that annual IEC-CTs registrations will double by 2025. We anticipate the increase in cellular therapy options will demand an organized response, including adequate logistical planning and policy implementation in response to the landscape changes in clinical oncology during the upcoming years.[Table: see text]