A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD

Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willibrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. Here we developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167±257 mU/ml) but the VWF was completely incapable of binding FVIII, resulting in 53±23 mU/ml of plasma FVIII levels that were similar to those in VWF deficient (VWF-/-) mice. When wild-type human or mouse VWF was infused into VWF2N/2N mice, endogenous plasma FVIII was restored, peaking at 4-6 hours post-infusion, demonstrating that FVIII expressed in VWF2N mice is viable, but short-lived unprotected in plasma due to dysfunctional 2N-VWF. The whole blood clotting time and thrombin generation were impaired in VWF2N/2N but not in VWF-/- mice. The bleeding time and blood loss in VWF2N/2N mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF2N/2N, but not VWF-/- mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that there are other alternative pathway(s) that can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.

Casirivimab-imdevimab neutralizing SARS-CoV-2: post-infusion clinical events and their risk factors

Background Casirivimab-imdevimab has been developed to neutralize SARS-CoV-2. The global clinical trials in outpatients documented several adverse effects (AE), which mandate caution in Japan where part of patients return home. To investigate post-infusion clinical events and their risk factors, we attempted a retrospective study. Main body Subjects were a consecutive series of inpatients with COVID-19 undergoing an infusion of casirivimab-imdevimab in our institute. The criteria for administration were in accordance with previous clinical trials, e.g., exclusion of patients necessitating oxygen supply. In Japan, however, SARS-CoV-2 vaccinees were eligible. Methods were review of background factors of status, imaging, and laboratory findings for the outcome of post-infusion events such as temperature increase (Temp+), pulse oximetry below 94%, and other events. Also, we documented the drug efficacy. Of a total of 96 patients with a median follow-up of 54 days, one (1.0%) died who alone was an exception demanding oxygen supply. Other 95 patients (99.0%) recovered from fever and hypoxia by Day 4 and later had no worsening of COVID-19. Median increase of body temperature was 1.0 degrees Celsius, which was used for computation of Temp+. Multivariate analysis showed that for Temp+ (n = 47), white blood cell counts more than 4.3 × 103/microliter (Odds Ratio [OR] 2.593, 95% Confidence Interval [CI] 1.060–6.338, P = 0.037) was at risk, whereas 2-time vaccination for SARS-CoV-2 (OR 0.128, 95% CI 0.026–0.636, P = 0.012) was a preventing factor. Likewise for lowered oximetry (n = 21), CT showing bilateral ground glass attenuation (OR 5.544, CI 1.599–19.228, P = 0.007) was a significant risk factor. Two patients (2.1%) showed bradycardia (asymptomatic, intervention not indicated) on Day 3 and recovery on Day 5. Limitations for this study included the difficulty distinguishing AE from worsening of COVID-19, thus we documented as clinical events. Conclusions For 24 h after infusion of casirivimab-imdevimab, COVID-19 patients with increased white blood cell counts may be predisposed to temperature elevation more than 1.0 degrees centigrade, as may bilateral ground glass opacity to lowered oximetry. Thus, patients with leukocytosis and bilateral ground glass attenuation may need precaution for transient fever and hypoxia, respectively.

Common trajectories of highly effective CD19-specific CAR T cells identified by endogenous T cell receptor lineages

Current chimeric antigen receptor-modified (CAR) T cell therapy products are evaluated in bulk, without assessment of the possible heterogeneity in effector potential between cells. Conceivably, only a subset of the pre-infusion product differentiates into optimal effectors. We generated a comprehensive single-cell gene expression and T cell receptor (TCR) sequencing dataset using both pre- and post-infusion CD19-CAR T cells from peripheral blood and bone marrow of pediatric patients with B cell acute lymphoblastic leukemia (B-ALL). We identified potent effector post-infusion cells with identical TCRs to a subset of pre-infusion CAR T cells. Effector precursor CAR T cells exhibited a unique transcriptional profile compared to other pre-infusion cells, and the number of effector precursor cells infused correlated with peak CAR T cell expansion. Additionally, we identified an unexpected cell surface phenotype (TIGIT+, CD62Llo, CD27-), conventionally associated with inhibiting effective T cell responses, that we used to successfully enrich for subsequent effector potential. Collectively, these results demonstrate that highly diverse effector potentials are present among cells in pre-infusion cell products, which can be exploited for diagnostic and therapeutic applications. Furthermore, we provide an integrative experimental and analytical framework for elucidating the biological mechanisms underlying effector development in other CAR T cell therapy products.

Safety and Efficacy of Repeat Doses of Allogeneic Mesenchymal Stem Cells in Patients with Age Related Frailty

Age-related frailty is a common geriatric condition characterized by a decline in physical and immunological capacity that is associated with depletion of endogenous stem cells and leads to increased vulnerability for adverse health outcomes. Allogeneic mesenchymal stem cells (allo-MSCs) exert immunomodulatory effects and promote tissue repair, which may be able to impede the negative effects of the aging process. The objective of this study was to explore the safety and efficacy of repeated infusions of allo-MSCs in subjects with aging-frailty. Mean age at time of first and second infusions was 75.5 and 77 years of age, respectively. In this open-label clinical trial, 24 participants received two intravenous infusions of allo-MSCs with an average interval of 17.6 months between doses. Safety endpoints included incidence of treatment-emergent serious adverse events (TE-SAEs) within 1-month post-infusion and increase in Panel Reactive Antibodies (PRAs) at 6-months post-infusion. Primary efficacy endpoint was change in 6-minute walk test (6MWT) distance at 6-months post-infusion. No TE-SAEs occurred within 1-month post-infusion. PRAs remained stable throughout the study, indicating no evidence of immune rejection. 6MWT increased by 42 meters after the first infusion (P=0.018). Eighteen months later elevation persisted (P=0.026), but did not increase further after the second infusion. In summary, repeated intravenous infusions of allo-MSCs were safe in participants with age-related frailty and showed remarkable improvement in physical performance. Given the excellent safety and efficacy profiles demonstrated in this study, larger clinical trials are warranted to further quantify the efficacy of repeated dosing of allo-MSCs in this multisystem disorder.

A Phase 1 Study of Safety, Tolerability and Pharmacokinetics of Single Ascending Doses of a First in Human Engineered Cationic Peptide, PLG0206, Intravenously Administered in Healthy Subjects

Background : In this first in human study, PLG0206, a novel engineered cationic antimicrobial peptide was evaluated for safety, tolerability and pharmacokinetics when intravenously administered as a single dose to healthy subjects. Methods : Six cohorts of 8 subjects received escalating single IV infusions of PLG0206 at 0.05, 0.125, 0.25, 0.5, or 1 mg/kg dose or placebo over 1-to-4-hours. Subjects were randomized to receive either PLG0206 (6 per cohort) or placebo (2 per cohort). Serial pharmacokinetic samples were taken prior to infusion and up to 48 hours post infusion. Safety and tolerability were assessed throughout the study. Results : The demographic characteristics of subjects were comparable between those treated with PLG0206 and placebo and between dose groups. The incidence of treatment emergent adverse events (TEAE) related to PLG0206 was low and most events were mild in severity and were similar between the PLG0206 treatment and placebo groups. The most common adverse events reported for PLG0206 were infusion related reactions, which were mitigated with increasing infusion time and volume. There were no serious adverse events (SAE), life-threatening events, or deaths throughout the study. IV PLG0206 exhibited linear pharmacokinetics over the dose range of 0.05 to 1.0 mg/kg. The median terminal half-life (t ½ ) ranged from 7.37 to 19.97 hours. Conclusion : Following a single IV infusion to healthy subjects, PLG0206 was safe and well tolerated and exhibited linear PK at doses ranging from 0.05 to 1 mg/kg. These findings support the ongoing development of IV PLG0206 as an antimicrobial agent.

A Phase II Trial of Anakinra for the Prevention of CAR-T Cell Mediated Neurotoxicity

Introduction: Chimeric antigen receptor (CAR)-T cell therapy is limited in most cases to inpatient use due to risk of severe treatment-related toxicities. The two primary toxicities observed with CAR-T therapy, cytokine release syndrome (CRS) and neurotoxicity, are associated with increased circulating inflammatory cytokines such as IL-6 and IL-1. Targeting IL-6 with tocilizumab is effective for treating CRS but not neurotoxicity. Anakinra is an FDA-approved recombinant IL-1 receptor antagonist that competitively inhibits IL-1 receptor signaling and therefore blocks downstream production of inflammatory cytokines including IL-6. Leveraging support from Kite Pharma, we opened an investigator-initiated clinical trial (NCT04150913) with the hypothesis that anakinra could be administered prophylactically to prevent severe CRS and neurologic events (NE) in patients receiving axicabtagene ciloleucel (axi-cel). Here we report preliminary outcomes of this study. Study Design and Methods: This is a phase II single center, open-label study for patients ≥18 years old with relapsed or refractory large cell lymphoma. Patients must have progressed after ≥2 lines of systemic therapy but could not have CNS disease or have been previously treated with CAR-T therapy. Following leukapheresis and manufacturing, patients received 3 days of lymphodepleting chemotherapy (LDC, cyclophosphamide 500mg/m 2 and fludarabine 30 mg/m 2) and 200 mg of subcutaneously administered anakinra starting 4 hours prior to axi-cel infusion and daily thereafter for a total of 7 days. CRS and NE were graded based on the Lee 2013 criteria and the CTCAE 4.03 criteria, respectively, to enable direct comparison to the pivotal Zuma-1 cohorts. The primary endpoint is the rate and severity of NE within the first 30 days of infusion; secondary endpoints include the incidence and severity of CRS and disease response. CAR-T cell expansion, serum cytokines, and circulating biomarkers of toxicity were measured at baseline, day 3, 7, 14, 21, and 28 post CAR-T cell infusion. Results: Interim analysis of the first 6 patients demonstrated a median age of 68 (range 59-72). Patients included a diverse group of histologies including double-hit lymphoma (n=2), transformed indolent NHL (n=3), and DLBCL NOS (n=1). Two patients were considered primary refractory at time of enrollment. Pre-LDC baseline characteristics included a median SPD of 2819 mm 2 (range 1063-5802), median LDH of 415 (range 147-497) which were comparable to the pivotal ZUMA-1 cohorts. Baseline ferritin, CRP, SAA and IL-15 were similar to the pivotal ZUMA-1 cohorts. While low-grade CRS was observed in 5/6 patients, no patients experienced severe CRS and median onset occurred on day +8 (range 1-8). Four patients did not experience any NE, while two patients experienced grade 3 NE on days +6 till +9 (somnolence) and +12 (global aphasia only, for one day) respectively. With a median follow-up of 4 months, the day +28 overall response rate was 100% (4 CRs, 2 PRs), with 4/6 patients having an ongoing complete response at last disease assessment. One patient was re-infused at progression and remains in a CR 3 months from re-infusion. Responses were seen despite varying CAR-T peak level with most patients demonstrating expansion in the lower quartile of the historic ZUMA-1 cohort. Median post-infusion peak of CRP, ferritin, IL-2, GM-CSF, IFNγ, IL-10, IL-6 and SAA were lower than that observed in the pivotal ZUMA-1 cohorts. All patients remain alive at time of data analysis. Conclusions: With a limited number of patients analyzed thus far, anakinra appears to provide benefit to the toxicity profile of axi-cel, presenting reduced and/or delayed CRS and NE and a decrease in post-infusion inflammatory analytes, when compared to ZUMA-1 pivotal cohorts. No severe CRS was observed in this initial analysis and 2/6 patients experienced grade 3 NE (somnolence and global aphasia) after day 6. Despite CAR-T expansion in the lower quartile of that of ZUMA-1, we observed a 100% ORR with 4 patients remaining in CR at a median follow-up of 4 months. Additional subjects will be assessed to investigate the role of prophylactic anakinra in the management of CRS and NE, which has potential for making axi-cel treatment an outpatient therapy. Disclosures Frigault: BMS: Consultancy; Editas: Consultancy; Iovance: Consultancy; Arcellx: Consultancy; Takeda: Consultancy; Kite: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Wehrli: CSL Behring: Patents & Royalties; Nestle: Current equity holder in publicly-traded company; Novartis: Current equity holder in publicly-traded company. Chou: Kite Pharma: Current Employment. Shen: Atara: Current Employment, Current equity holder in publicly-traded company, Other: Leadership role, Patents & Royalties; Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment, Other: Leadership role, Patents & Royalties. Filosto: Kite, a Gilead Company: Current Employment; Gilead Sciences: Other: stock or other ownership ; Tusk Therapeutics: Patents & Royalties: or other intellecular property. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Maus: Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; AstraZeneca: Consultancy; Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company.

Total Car-T Cost of Care Beyond the Price of Car-T Cell Therapy in Patients with Multiple Myeloma

Background: Chimeric antigen receptor T (CAR-T) cell therapy is a recent treatment option for triple-class exposed relapsed refractory multiple myeloma (RRMM) patients, who have previously received proteasome inhibitors, immunomodulatory agents, and monoclonal antibodies. In addition to CAR-T cell therapy acquisition costs, there are expected CAR-T healthcare costs separate from the cost of the CAR-T cell therapy itself. Identification and quantification of these costs have not previously been fully investigated. Healthcare payers, hospitals, and physicians need this important cost information in order to make informed healthcare decisions. Objective: The objective of this study was to quantify CAR-T therapy associated healthcare costs, i.e., costs aside from CAR-T therapy acquisition costs, associated with use of CAR-T therapy in RRMM patients. Methods: RRMM CAR-T clinical trial data and published literature, including peer-reviewed publications and conference presentations, were used for identification of these additional healthcare cost components. Inclusion/exclusion of additional cost components was based on previously published oncology pharmacoeconomic studies and input from key opinion leaders (KOLs) with expertise in the treatment of RRMM patients. The study categorized costs for RRMM patients who receive the CAR-T infusion. Costs were categorized as pre-infusion, peri-infusion (excluding CAR-T therapy acquisition costs), and post-infusion costs. Pre-infusion costs included evaluation costs, apheresis costs, bridging therapy costs, and conditioning therapy costs. Peri-infusion costs included either inpatient or outpatient infusion costs. Post-infusion costs included 100-day post-infusion monitoring costs, additional infusion monitoring costs in the first year, and management of serious adverse event (AE) costs associated with the CAR-T therapy. Serious AEs included AEs that require or prolong hospitalization or result in death. CAR-T acquisition costs are known and readily accessible, but this is not the case for other costs associated with CAR-T therapy, i.e., the pre-, peri-, and post-infusion costs. The costs must be individually calculated utilizing CAR-T clinical trial data, medical resource costs and utilization, and AE costs and rates. Based on a targeted literature review, there are sufficient data in the public domain to quantify the component additional costs, however there are no publications quantifying the entirety of these costs specifically in RRMM patients receiving CAR-T. Methodology was developed for derivation of these costs, which comprise an important and consequential portion of the total CAR-T therapy costs. Results: The RRMM patient pre- and peri-infusion CAR-T healthcare cost, aside from CAR-T therapy acquisition costs, was estimated to be $15,478. The components of this cost were apheresis ($112), bridging therapy ($8,570), conditioning therapy ($3,435), and a single day of inpatient infusion ($3,362). The post-infusion costs (including additional inpatient hospital days, AE management, and monitoring costs) are dependent on the types and rates of AEs associated with CAR-T therapy. These results will be presented at the ASH 2021 meeting. Conclusions: This study developed a methodology for the categorization, quantification, and calculation of the important healthcare costs, aside from CAR-T therapy acquisition costs, associated with the use of CAR-T in triple-class exposed RRMM patients. This information has been lacking in the literature and the analysis provides valuable, holistic information that key stakeholders require to make informed decisions. Disclosures Jagannath: Bristol Myers Squibb: Consultancy; Janssen Pharmaceuticals: Consultancy; Karyopharm Therapeutics: Consultancy; Legend Biotech: Consultancy; Takeda: Consultancy; Sanofi: Consultancy. Joseph: Johnson and Johnson: Current Employment, Current equity holder in publicly-traded company. Crivera: Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Jackson: Janssen: Current Employment; Memorial Sloan Kettering Cancer Center: Consultancy. Valluri: Janssen: Current Employment, Current equity holder in publicly-traded company. Cost: Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Phelps: Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Slowik: Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Klein: Medical Decision Modeling Inc.: Current Employment. Yu: Medical Decision Modeling Inc.: Current Employment. Smolen: Medical Decision Modeling Inc.: Current Employment. Cohen: GlaxoSmithKline: Consultancy, Research Funding; Janssen: Consultancy; Takeda: Consultancy; Oncopeptides: Consultancy; BMS/Celgene: Consultancy; AstraZeneca: Consultancy; Genentech/Roche: Consultancy; Novartis: Research Funding.

Decreased Chimeric Antigen Receptor T-Cell Efficacy with Severe or Prolonged Post-Infusion Cytopenias

INTRODUCTION Prolonged cytopenias following Chimeric Antigen Receptor (CAR) T-cell therapy are common. The cytopenias are mainly due to the genotoxic effects of conditioning chemotherapy and systemic inflammatory insult to hematopoietic stem and progenitor cells from CAR T-cell activity. Tocilizumab, an anti-IL6 receptor antibody, reduces cytokine release syndrome (CRS), a clinical manifestation of CAR T inflammatory toxicity. We sought to determine if prophylactic tocilizumab can decrease the risk and/or severity of CRS. We secondarily hypothesized that decreasing the inflammatory injury to hematopoietic stem cells could improve bone marrow function after CAR T. Lastly, we analyzed the association between cytopenias and CAR T efficacy generally. METHODS In this single-institution, retrospective cohort study, we examined all patients who had received axicabtagene ciloleucel for relapsed/refractory diffuse large B cell lymphoma (DLBCL) between March 2018 and April 2021 (Table 1). Each patient was evaluated for prolonged cytopenias, defined as requiring granulocyte-colony stimulating factor (G-CSF), red blood cell or platelet transfusion beyond day 28 after CAR T infusion. After CAR T infusion, no patient was given any anti-lymphoma therapy prior to collection of blood count data. We compared response to CAR T 6 weeks post-infusion, progression-free survival (PFS), and overall survival (OS) for patients with and without prolonged cytopenias. A univariate regression analysis was performed, and between group differences were calculated using Chi-squared, Fisher's exact tests, and Cox Proportional hazard regression model. Kaplan-Meier and log-rank analyses were used to evaluate the PFS and OS. We also compared patients who did and did not receive prophylactic tocilizumab 36 hours after CAR T infusion for differences in prolonged cytopenias, PFS, OS, and development and severity of CRS and neurotoxicity. CRS and neurotoxicity were graded using the ASTCT consensus grading system. RESULTS The study included 54 patients, 31 (57.4%) met criteria for prolonged cytopenias. 1 (1.9%) patient died of progressive disease prior to day 28 and was excluded from analysis. All patients who died in this study did so on the basis of progressive lymphoma. Likelihood of no response to CAR T at 6 weeks was higher in patients who needed G-CSF support past day 28 (HR 2.20, p = 0.048), had a hemoglobin nadir less than 8 g/dL (HR 2.61, p = 0.031), were still requiring platelet transfusions after day 28 (HR 2.26, p = 0.038), or had a platelet nadir less than 25,000 per µl during the first 28 days (HR 4.01, p = 0.013, Table 2). For patients who had neutrophil counts below the median at day 28, the median PFS was 4.5 months vs. 17.9 months (HR 2.07, p = 0.035) compared to those above the median. In patients with a platelet nadir less than 25,000 per µl or below the median at day 28 (Figure 1), the median PFS was 4.5 months vs. not reached (HR 3.12, p = 0.003) and 3.9 months vs. not reached (HR 3.85, p = 0.0004) respectively. Median OS was 9.44 months in patients who required G-CSF support past day 28 (HR 2.79, p = 0.022) vs. not reached in those who didn't receive G-CSF past day 28. For patients with an ANC below the median on day 28, median OS was 19.87 months vs not reached (HR 2.65, p = 0.022). In patients with a platelet nadir less than 25,000 per µl or below the median at day 28, the median OS were 7.8 months vs not reached (HR 3.72, p = 0.006) and 7.79 months vs not reached (HR 3.39, p = 0.007) respectively. Among patients who received prophylactic tocilizumab, 12 (54.5%) had prolonged bone marrow failure compared to 19 (57.6%) among those who did not (p = 0.82). PFS, OS, and rates and severity of CRS and neurotoxicity did not differ between the prophylactic tocilizumab groups. CONCLUSIONS In this study of patients with relapsed/refractory aggressive B-cell lymphomas receiving a CD28 domain-containing CAR T product, severe short-term or prolonged cytopenias were associated with an increased risk of lymphoma progression and death. This effect was not ameliorated by administration of prophylactic tocilizumab, which also did not affect the development or severity of CAR T inflammatory toxicities. Further study is required to determine the mechanistic nature of the relationship between post-infusion cytopenias and CAR T efficacy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

CD45RO+ T-Cell Add Back and Prophylactic Blinatumomab Administration Post Tcrαβ/CD19-Depleted Haploidentical Transplantation in Pediatric Patients with High Risk Acute Leukemia

For patients receiving haploidentical donor (haplo) hematopoietic cell transplant (HCT), depletion of TCRαβ T cells from the haplo-graft allows for excellent outcomes and low rates of graft-versus-host disease (GVHD), albeit with a significant delay in immune reconstitution (IR). The adoptive transfer of donor T-cells has been used to optimize IR but increases the risk of GVHD. CD45RA-depleted (memory) T-cells are associated with low rates of alloreactivity and thereby GVHD but retain specificity for leukemic and viral antigens. We implemented a prospective trial utilizing escalating doses of CD45RA-depeleted T-cell as addback following TCRαβ/CD19-depleted haploHCT to improve IR. Patients with acute lymphoblastic leukemia (ALL) also received prophylactic Blinatumomab (Blina) following infusion of CD45RA-depeleted T-cell to overcome the risk immune escape secondary to HLA-loss and relapse (NCT03849651). Between 2019 to 2020, 30 pediatric patients (9 males, 21 females) with high-risk acute leukemia were enrolled. Median age at HCT was 8.7 years (range 0.9-18.8). Nineteen patients had ALL, 11 patients had AML. Ten patients were in CR1, 13 in CR2 and 7 in CR3/>. Five patients received prior CD19-CAR T-cell therapy. The donors used were mothers (n=15), fathers (n=13), or sibling/others (n=2). All patients received a reduced intensity preparative regimen consisting of Fludarabine, Melphalan, Cyclophosphamide and Thiotepa. ATG was given on days -5, -4 and -3. Mobilized peripheral blood graft were infused on day 0 with a median number of CD34+ cells, γδ+ T-cells, αβ+ T cells, and B cells of 14.8, 22.7, 0, and 0.09x 10 6/kg, respectively. No GVHD prophylaxis was used post-HCT. Two weeks following engraftment, patients received CD45RA-depleted T-cell addback in 3 escalating doses (DL1: 1x10 5cells/kg, DL2: 1x10 6 cells/kg, DL3:1x10 7 cells/kg). All 30 patients engrafted with a median time for neutrophil and platelet engraftment of 10 (range: 9-11) and 15 (range: 13-20) days, respectively. Infusion of escalating doses of CD45RA-depleted T-cells was well tolerated. One month post infusion, there was a significant increase in the median number of CD3 T-cells, including CD8 and CD45RO+ T-cell subsets (**p<0.01, ***p<0.001, Fig 1A). There was also significant expansion of virus-specific T-cells (VSTs) directed towards Cytomegalovirus (CMV), Adenovirus (AdV), BK, or HHV-6 as shown by Elispot assays (**p<0.01, Fig. 1B). TCR repertoire, as assessed by Vb spectratyping, was broad and comparable to the donor by month 6 post-HCT. The incidence of CMV, AdV, and HHV-6 viremia was 60%, 6.7%, and 16.7% respectively. The median duration of viremia was 4 weeks for CMV (range: 1-13), 3 weeks for AdV (range: 2-13) and 2.5 weeks for HHV-6 (range: 2-20). There were 11 episodes of viral disease (7 colitis, 3 pneumonitis, 1 lymphadenitis). All episodes of viral disease resolved, except 2 that were ongoing at the time of death. The incidence of acute GVHD within 28 days post-infusion after dose level 1, 2 and 3 was 0%, 20% and 10% respectively (p=NS). The cumulative incidence of aGVHD and grade III-IV aGVHD for the entire cohort was 26.7% (12.4-43.3%) and 13.3% (4.1-28.1%) respectively. There was no chronic GVHD; however, follow up is short. Sixteen patients received and tolerated prophylactic Blina infusions. The median time to receiving Blina after CD45RO+ infusion was 29 days (range 15-56). Four of the 16 patients who received Blina relapsed: 3 with CD19+ disease and 1 with CD19-negative disease. With a median follow up of 12.7 months (range 3.5-24.5), 1 year OS and LFS for the cohort was 86.3% (74.6-99.7%) and 69.8% (55.2-88.4%) respectively. The cumulative incidence of relapse was 31.8% (15.3-49.8%) and of non-relapse mortality was 3.3% (0.2-14.8%) respectively. In this interim analysis, addback of CD45RA- depleted T-cells following TCRαβ/CD19-depleted haplo HCT was safe and led to enhanced functional immune reconstitution. Prophylactic infusion of Blina is well tolerated and its use post-transplant warrants further investigation. Analyses into the effect of ATG on immune reconstitution are underway. Figure 1 Figure 1. Disclosures Sharma: CRISPR Therapeutics: Other, Research Funding; Novartis: Other: Salary support paid to institution; Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Spotlight Therapeutics: Consultancy; Medexus Inc: Consultancy; Vindico Medical Education: Honoraria. Gottschalk: Catamaran Bio: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Novartis: Consultancy; Tidal: Consultancy; Tessa Therapeutics: Consultancy. Triplett: Miltenyi: Other: Travel, meeting registration.

Pre-Infusion Neurofilament Light Chain (NfL) Levels Predict the Development of Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) - a Multicenter Retrospective Study

Chimeric antigen receptor (CAR) T-cell therapy is often associated with neurological complications termed immune effector cell-associated neurotoxicity syndrome (ICANS). There remains a critical need to identify patients most at risk for ICANS. Yet a biomarker for the development of ICANS is lacking. This retrospective multicenter study evaluates pre-infusion levels of plasma neurofilament light chain (NfL), a marker of neurodegeneration, as a predictive biomarker the development of ICANS. Study inclusion criteria included available pre-infusion (up to 4 weeks prior to lymphodepletion) plasma from patients treated with a CAR T cell therapy (n = 30, 36% with ICANS, ASTCT consensus ICANS grade range 1-4). Exclusion criteria included confounding diagnoses known to elevate NfL levels (e.g. dementia, recent stroke). Plasma NfL was assayed using a Simoa HD-X kit (Quanterix TM). Demographic (age, sex), oncologic (primary, stage, mean tumor volume (MTV), and history of central nervous system (CNS) involvement), and medical history (history of non-oncologic CNS disease or neuropathy) were obtained from the medical record. MTV was derived from total lesion burden on pre-infusion positron emission tomography (PET) scans using a 41% maximum standard uptake value (SUV) threshold. Pre-infusion (i.e. during lymphodepletion) and Post-infusion Day 1 (D1) platelet count, C-reactive protein (CRP), fibrinogen, lactate dehydrogenase (LDH), and ferritin levels were also obtained from the medical record. Group comparisons used log-rank testing, followed by receiver operating characteristic (ROC) curve classification and hierarchical clustering. Validation testing used a 10,000 fold cross-validation on 80% of the data. Finally, demographic and clinical characteristics correlated with pre-infusion biomarkers using point-biserial and Spearman (rank) correlation. Our results demonstrated that individuals who would go on to develop ICANS had elevations in pre-infusion NfL ([87.6 v 29.4 pg/ml; Fig 1A], p = 0.00004) with excellent classification accuracy for the development of ICANS (AUC 0.96; Fig 1B), sensitivity (0.91) and specificity (0.95). NfL further correlated with ICANS development (r = 0.74, p < 0.0001; Fig 1C). Among known post-infusion risk factors, D1 ferritin had the highest classification accuracy, but was inferior to baseline NfL (p < 0.05; Fig 1B). Both baseline NfL and D1 ferritin elevations clustered with ICANS grade (Fig 1D). Our findings show that pre-infusion plasma NfL levels are a robust early marker for the development of ICANS that exceeds known post-infusion markers. This suggests the risk of developing ICANS reflects pre-existing latent neuroaxonal injury. Predictive identification of patients at risk of developing ICANS prior to cellular infusion would permit early, preemptive or prophylactic ICANS-directed therapies, thereby improving patient outcomes. Figure 1: Baseline (pre-infusion) NfL Levels in ICANS (A) Baseline (pre-infusion) levels of NfL in patients who develop Grade 0 ICANS, Grade 1-2 ICANS, and Grade 3+ ICANS. (B) Receiver operating characteristic curve classification of patients who developed any grade ICANS (1+) vs grade 0 for baseline NfL and post-infusion day 1 (D1) markers. (C) Correlation between pre-treatment factors and pre-infusion biomarkers. All significant relationships after correction for multiple comparisons using false discovery rate (FDR) are outlined (*). (D) Hierarchical clustering of age, baseline NfL, and D1 markers. Clusters associating with ICANS are labeled in red, while those associating with cytokine release syndrome (CRS) are in blue. Figure 1 Figure 1. Disclosures Caimi: Verastem: Consultancy; ADC Theraputics: Consultancy, Research Funding; Genentech: Research Funding; XaTek: Patents & Royalties: Royalties from patents (wife); Kite Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Amgen Therapeutics.: Consultancy; TG Therapeutics: Honoraria. de Lima: BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding. Campian: AbbVie, Inc.: Speakers Bureau; NeoImmuneTech: Research Funding. Ghobadi: Amgen: Consultancy, Research Funding; Atara: Consultancy; Wugen: Consultancy; Celgene: Consultancy; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding.