Kenderian, Saad S.

Abstract With the development of various strategies of anti-CD19 immunotherapy for treatment of B cell malignancies, it remains to be elucidated whether CD19 targeting with a monoclonal antibody impairs subsequent CD19 targeted chimeric antigen receptor T cell (CART19) therapy. In this study, we evaluated the potential interference between the CD19 targeting monoclonal antibody tafasitamab and CART19 treatment (using a construct similar to the FDA approved therapy, FMC63-41BBζ, tisagenlecleucel) in preclinical lymphoma and leukemia models. For in vitro assays, the CD19 + cell lines JeKo-1 (mantle cell lymphoma), Nalm6 (acute lymphoblastic leukemia), and OCI-Ly7 (diffuse large B cell lymphoma) were used. When CART19 were co-cultured with CD19 + tumor cells in the presence of increasing concentrations of tafasitamab (10-400 µg/mL) or the isotype control, an impairment of CART19 functions at the highest concentration of tafasitamab was observed. However, when tafasitamab was cocultured with tumor cells overnight and removed from the culture by washing, there was no impairment of CART19 anti-tumor activity or effector functions compared to a pre-culture with isotype control. We then aimed to validate these findings in xenograft mouse models by determining whether prior tafasitamab treatment impairs subsequent CART cell function. First, a two-step JeKo-1 xenograft model was created through inoculation of 1x10 6 luciferase + JeKo-1 cells to immunocompromised NOD-SCID-γ -/- (NSG) mice via tail vein injection. One week later, tumor burden was assessed by bioluminescence imaging (BLI) and mice were randomized to receive either 1) PBS (Group 1) or 2) 10 mg/kg of tafasitamab (Group 2) (Fig. 1A). Treatment was performed until the mice reached the endpoint due to the high tumor load. When reaching the endpoint, mice were euthanized and spleen was harvested. Splenic cells were then injected to naïve NSG mice. Two weeks after the inoculation of splenic cells (PBS or tafasitamab treated), mice were imaged with BLI and randomized according to their tumor burden to receive 2x10 6 of 1) untransduced T cells (UTD) or 2) CART19 (i.v.) (Fig. 1B). Mice, treated with CART19 demonstrated potent anti-tumor effects and showed prolonged survival compared to UTD control mice. Interestingly, CART19-treated mice inoculated with Group 2 splenic cells (pretreated with tafasitamab) showed a significantly better tumor control and overall survival compared with mice engrafted Group 1 splenic cells (PBS pretreatment) (Fig. 1C). This finding led us to further investigate the effects of prior tafasitamab therapy on subsequent CART19 treatment. In this context, we studied the sequential treatment with tafasitamab and CART19 in an additional JeKo-1 xenograft model (Fig. 1D). In brief, NSG mice were inoculated with luciferase + JeKo-1 on day -14. On day -8, mice were imaged and randomized to the PBS (Group I) or tafasitamab (10 mg/kg) group. On day -1 tafasitamab treated mice were imaged again, randomized to the tafasitamab discontinuation (Group II) or continuation (Group III). Treatment with PBS was continued in Group I. All groups were administered with 1x10 6 of CART19 on day 0. Cotreatment of JeKo-1 xenografts with tafasitamab and CART19 led to impaired anti-tumor activity presumably as a result of CD19 binding competition between tafasitamab and CART19 (Group III). Mice treated with tafasitamab followed by CART19 had improved tumor control compared to mice treated with PBS followed by CART19 (Fig. 1E-F). Serial peripheral blood analysis demonstrated that mice from Group I showed the highest CART19 expansion on day 10. Strikingly, Group II exhibited weak CART19 expansion at day 10, but strongest expansion on day 23 (Fig. 1G). We therefore hypothesized that prior treatment with tafasitamab results in modulation of CART19 cell activation. To test this hypothesis, spleens of satellite mice were harvested 24 hours after CART19 administration. Flow cytometric analysis revealed that the mice from Group II showed significantly lower CD69 and HLA-DR expression on CART19 cells compared with Group I, and reduced apoptosis as measured by the Annexin assay (Fig. 1H-I). In summary, concomitant treatment of tafasitamab and CART19 led to impaired anti-tumor activity while, in contrast, sequential treatment of tafasitamab and CART19 did not inhibit CART19 anti-tumor activity but rather promoted anti-tumor effects in xenograft models. Figure 1 Figure 1. Disclosures Sakemura: Humanigen: Patents & Royalties. Augsberger: MorphoSys AG: Current Employment. Schanzer: MorphoSys AG: Current Employment. Patra-Kneuer: MorphoSys AG: Current Employment. Heitmüller: MorphoSys AG: Current Employment. Steidl: MorphoSys AG: Current Employment. Endell: MorphoSys AG: Current Employment. Ding: DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Acerta Pharma: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Sunesis: Research Funding. Nowakowski: Celgene, NanoString Technologies, MorphoSys: Research Funding; Celgene, MorphoSys, Genentech, Selvita, Debiopharm Group, Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Abstract Introduction: Chimeric antigen receptor T-cell (CAR-T) therapy is an effective treatment that has transformed hematologic malignancy management. However, its significant short-term toxicities, mainly cytokine release syndrome (CRS) and neurotoxicity, require frequent monitoring almost universally achieved via prolonged inpatient admissions. We have reported our institution's specialized, multi-disciplinary hospital-based outpatient (HBO) CAR-T service and experience (Bansal et.al, ASCO 2021). To enhance the HBO practice, we implemented a remote patient monitoring (RPM) program, comprised of in-home, electronic health record-integrated technology to monitor vital signs and neurologic symptoms for 30 days post CAR-T infusion. The RPM program's triaging algorithm generates alerts based on predetermined parameters to guide escalation of care if needed. In this report, we describe the characteristics of RPM program patients (pts), focusing on the alerts received within 48 hours (h) of first hospitalization in the post-CAR-T setting. Methods: Pts undergoing commercial CAR-T cell therapy at Mayo Clinic Rochester were enrolled in the RPM program as part of our standard of care from 06/2020 to 05/2021. Pts with detailed RPM data available were included in this analysis. Patient characteristics, health care utilization, inpatient interventions, and RPM alert data were reviewed. Wilcoxon test was used for continuous variables, chi-squared test was used for categorical variables. Results: Baseline characteristics are summarized in Table 1. Of 20 pts in this analysis, 13 had RPM alerts in the 48h preceding the first hospitalization post CAR-T. For the remaining 7 pts without RPM alerts, 3 were hospitalized within 24h of CAR-T infusion without significant RPM data, 1 patient hospitalized with acute pancreatitis (day +8 post CAR-T infusion), 1 for doubling of CRP (day +10), 1 for acute pain crisis (day +5), and 1 with neutropenic fever (day +5) with not reporting RPM data. The median RPM program duration was 18 days (range 3-32) which accounts for interruptions in the RPM monitoring while pts are hospitalized. The median number of data points per patient was 231 (range 22-532), with a median of 20 alerts per patient (range 2-81). The median missing data points per patient were 20 (10%) (range 0-95). Of the 13 pts with RPM alerts within 48h of first hospitalization, alerts met our definition of urgent criteria in 6 pts [temperature (temp) > 100F (n=3), temp > 100F + tachycardia (n=1), temp > 100F + hypoxia (n=1), and hypoxia (n=1)]. In the remaining 7 pts, the RPM alerts met our routine criteria definition [temp 99-100 (n=2), tachycardia + hypertension + weight loss (n=2), hypertension (n=2), new incontinence (n=1)]. CRS was reported in 12 pts [grade 1-2 (n=11), grade 3-4 (n=1)] and neurotoxicity in 8 pts [grade 1-2 (n=7), grade 3-4 (n=1)]. The median post CAR-T first hospitalization day was day +4 (range day +2-10), Figure 1. The median time from the last RPM alert to hospitalization was 5.8h for pts meeting urgent criteria vs 10.8h for those meeting routine criteria (p=0.13). Tocilizumab was used in 5 pts during the first hospitalization [3 (50%) in the urgent criteria group and 2 (28%) in the routine criteria group], with a median time from admission to tocilizumab of 12h in the urgent criteria group vs 43h in the routine criteria group (p=0.05). Early hospitalization (≤ 3 days) post CAR-T infusion was associated with later need for tocilizumab [median 51h (range 30-57) vs. 11.5h (range 6-33) from admission, p=0.03] than late hospitalization (> 4 days post CAR-T). Upon admission, oxygen supplementation was started on both pts with hypoxia noted in the RPM alerts. Only 1 patient (routine criteria group) required use of vasopressors while hospitalized, initiated more than 48h after admission. Conclusion: A CAR-T RPM program can identify adverse symptoms and vital sign changes in CAR-T pts managed in the outpatient setting. In this pilot implementation, the CAR-T RPM triaging algorithm identified pts requiring expedited hospitalization and facilitated early initiation of tocilizumab. The intermittent monitoring and proportion of missing data are the main limitations of the RPM in the acute care setting post-CAR-T infusion. Incorporation of wearable devices for continuous remote monitoring is being explored as a mitigating strategy to these limitations. Figure 1 Figure 1. Disclosures Paludo: Karyopharm: Research Funding. Dingli: Apellis: Consultancy; Janssen: Consultancy; GSK: Consultancy; Novartis: Research Funding; Sanofi: Consultancy; Alexion: Consultancy. Kapoor: Glaxo SmithKline: Research Funding; Sanofi: Consultancy; Amgen: Research Funding; AbbVie: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Ichnos Sciences: Research Funding; Pharmacyclics: Consultancy; Takeda: Research Funding; Regeneron Pharmaceuticals: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy. Gertz: Aurora Biopharma: Other: Stock option; Ionis Pharmaceuticals: Other: Advisory Board; Akcea Therapeutics, Alnylam Pharmaceuticals Inc, Prothena: Consultancy; AbbVie Inc, Celgene Corporation: Other: Data Safetly & Monitoring; Akcea Therapeutics, Ambry Genetics, Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Karyopharm Therapeutics, Pfizer Inc (to Institution), Sanofi Genzyme: Honoraria. Wang: InnoCare: Research Funding; LOXO Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; MorphoSys: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Genentech: Research Funding; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding. Kumar: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Carsgen: Research Funding; Antengene: Consultancy, Honoraria; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Bluebird Bio: Consultancy; Beigene: Consultancy; Tenebio: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Bennani: Purdue Pharma: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Vividion: Other: Advisory Board; Kymera: Other: Advisory Board; Verastem: Other: Advisory Board. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Lin: Merck: Research Funding; Vineti: Consultancy; Gamida Cell: Consultancy; Juno: Consultancy; Bluebird Bio: Consultancy, Research Funding; Legend: Consultancy; Celgene: Consultancy, Research Funding; Sorrento: Consultancy; Takeda: Research Funding; Janssen: Consultancy, Research Funding; Novartis: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding.

Abstract INTRODUCTION Sparse data exist regarding I+V in an off-trial setting, particularly in patients (pts) with progression of disease on either I or V as single agent treatment. The 1-year overall survival (OS) rates with V monotherapy following I range from 75%-91% (Eyre et al, BJH 2019; Jones et al, Lancet Oncol 2018); however, longer-term follow-up of these or other patient cohorts treated with V after I has not yet been reported. Effective options in double-refractory pts are lacking when a clinical trial is not feasible. Here, we report our experience with I+V in these patient groups. METHODS After IRB approval, we reviewed the records of pts seen at Mayo Clinic (between 4/2012-1/2021) who received I+V therapy off-trial for CLL progression. Pts treated with I+V for Richter's transformation were excluded. Time to next treatment (TTNT) was measured from I+V treatment start date to the date of next treatment start or last known treatment status with death as a competing risk. OS was measured from I+V start date until date of death or when the patient was last known alive; OS was analyzed using the Kaplan-Meier method and Cox proportional hazards model. RESULTS Thirty-three pts with progressive CLL were treated with I+V (median duration 13.3 months [95% CI: 7.1 months-not estimable (NE)]). The median age was 70 years (range 47-86), 6 (18%) were women, and 20 (65%) had del(17p) or TP53 mutation. The median number of prior lines of therapy was 3 (range 1-15). Additional characteristics at the time of I+V start are shown in Table 1. Median follow-up from start of I+V was 23.8 months. Among 22 pts with prior progression on I only (V-naïve), the median duration of I+V was 14.8 months (95% CI 7.6 months-NE). The median duration on either I or V from the start of combination was 20.3 months (95% CI 8.3 months-NE). The median TTNT was 25.1 months (Figure 1A). Four pts proceeded to cellular therapy (alloSCT [n=3], CAR-T [n=1]) while on I+V. Next therapy (n=9) consisted of Richter transformation treatment (n=2), chemotherapy plus continued I+V (n=1), anti-CD20 plus continued I+V (n=1), duvelisib + I (n=1), TP-0903 + I (n=1), reintroduction of I at disease progression after previously stopping I during I+V therapy (n=1), and reintroduction of I (n=1) or V (n=1) at disease progression after previously stopping I+V. Of 9 pts who did not receive subsequent therapy, 4 pts remain on I+V (median duration 16.4 months), 4 pts continued V after discontinuing I (median duration on V from start of I+V was 21.5 months), and 1 pt discontinued I+V following a second cancer diagnosis and died without receiving additional CLL-directed treatment. Eleven pts received I+V in the double-refractory setting; 9 pts had prior disease progression on sequential I then V and 2 pts had prior disease progression on sequential V then I. The median duration of I+V was 7.5 months (95% CI 4.3 months-NE), and the median duration of either agent from I+V start was 10.8 months (95% CI 7.1 months-NE). The median TTNT was 14.0 months (Figure 1A). Eight pts required additional treatment, consisting of PI3K inhibitor (n=2), anti-CD20 added to I+V (n=1), CAR-T (n=1), anti-CD20 with (n=1) or without (n=1) bendamustine added to continued I, and Richter transformation treatment (n=2). One pt continues I+V (17.4 months ongoing) without further treatment. Two pts died from disease progression without receiving additional treatment. The median OS of all 33 pts was 27.4 months (95% CI 25.9 months-NE; Figure 1B). The median OS of the 22 pts with prior progression on I (V-naïve) was 47.1 months (95% CI 27.7 months-NE), and the median OS of the 11 pts with prior progression on I and V separately was 27.0 months (95% CI 15.5 months-NE). In univariate analyses, prior progression on V was a predictor of shorter OS (HR=3.9; 95% CI 1.4-11.2; p=0.01). CONCLUSIONS Combination I+V therapy in heavily pre-treated CLL pts who have disease progression after I provides durable benefit with a median OS approaching 4 years. In the double-refractory setting, repurposing both drugs as combination I+V therapy can be a useful strategy, despite the short-term disease control, for pts where clinical trial options are limited or not available. Further examination of these approaches in larger cohorts and understanding the operative resistance mechanisms in pts with double-refractory disease are key next studies. Figure 1 Figure 1. Disclosures Ding: Octapharma: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Chanan-Khan: BeiGene, Jansen, Ascentage: Honoraria; Ascentage, Starton, Cellectar, NonoDev, Alpha2 Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Alpha2 Pharmaceuticals: Patents & Royalties: Tabi; Ascentage: Research Funding; Cellectar: Current equity holder in publicly-traded company; Alpha2 Pharmaceuticals, NonoDev, Starton: Current holder of stock options in a privately-held company; BieGene, Jansen, Ascentage: Consultancy. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding. Wang: TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; MorphoSys: Research Funding; Novartis: Research Funding; LOXO Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; InnoCare: Research Funding; Eli Lilly: Membership on an entity's Board of Directors or advisory committees. Ailawadhi: Pharmacyclics: Consultancy, Research Funding; Xencor: Research Funding; Janssen: Consultancy, Research Funding; Cellectar: Research Funding; Sanofi: Consultancy; BMS: Consultancy, Research Funding; GSK: Consultancy, Research Funding; Ascentage: Research Funding; Medimmune: Research Funding; Karyopharm: Consultancy; Amgen: Consultancy, Research Funding; Beigene: Consultancy; AbbVie: Consultancy; Takeda: Consultancy; Genentech: Consultancy. Koehler: AbbVie: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees. Kay: AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Combination ibrutinib and venetoclax in patients with relapsed CLL.

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive sub-type of non-Hodgkin's lymphoma(Liu, et al. Am J Hematol 2019). All three approved CD19-directed CAR-T therapies (axicabtagene ciloleucel, tisagenlecleucel, lisocabtagene maraleucel) are associated with toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) that can be severe, resulting in non-relapse mortality, ICU admission, and significant non-drug related health resource utilization which represent barriers to access and adoption (Nabhan, et al. J Clin. Pathway 2017). Studies have shown that early elevation of granulocyte-macrophage colony-stimulating factor (GM-CSF) levels 1-day post CAR-T infusion correlates with severe ICANS (Rossi, et al. EMA Workshop 2016), which is a negative prognostic factor for overall survival (Karschnia, et al. Blood 2019). It has been proposed that upon contact with the tumor, CAR-Ts produce GM-CSF, which serves as a communication conduit between the specific immune response of CAR-T and the off-target inflammatory cascade produced by myeloid lineage cells, causing myeloid cells to expand and promote the production of other downstream proinflammatory chemokines (MCP-1, IL-8, IP-10), cytokines (IL-1, IL-6), and other markers of systemic inflammation (CRP, Ferritin) (Sterner, et al. Blood 2019). Moreover, IL-6 is predominately released by tumor cells in a contact-independent manner (Barrett et al. Blood 2016), which helps explain why the prophylactic administration of tocilizumab is not effective in reducing the overall incidence of CRS or ICANS, as this cytokine is downstream in the inflammatory cascade. Further, the prophylactic use of tocilizumab has been shown to increase the incidence of all-grades and grade >3 ICANS (Locke, et al. Blood 2017). Lenzilumab is a novel Humaneered ® monoclonal antibody that neutralizes GM-CSF and has demonstrated potential to reduce the hyper-immune mediated cytokine storm induced by SARS-CoV-2 infection and significantly improve the likelihood of survival without ventilation in hospitalized COVID-19 patients, as reported in the LIVE-AIR phase 3 study (Temesgen, et al. medRxiv 2021). Methods: Eligible patients are adults (≥ 18 y) with relapsed or refractory DLBCL or are chemorefractory. Prior therapy must have included an anti-CD20 monoclonal antibody and an anthracycline-containing regimen. Patients will undergo leukapheresis and may receive optional corticosteroid bridging therapy. Patients will then receive lymphodepleting chemotherapy on Days ‒3 to ‒5 followed by infusion of lenzilumab on Day 0, 6-hrs prior to CAR-T infusion. Approximately 40 accredited sites across the U.S. certified to administer the three commercially available CAR-Ts have been engaged to participate in this 2-part study. In Part 1, all patients will receive lenzilumab 1800mg via a single 2-hour infusion prior to CAR-T administration. The objective of Part 1 is to evaluate the optimal regimen and assess whether a second dose of lenzilumab post-CAR-T infusion is required. A translational assessment of GM-CSF axis suppression, levels of CAR-T cells in blood, other inflammatory markers and lenzilumab PK/PD will be evaluated, along with the incidence and severity of CRS and ICANS, objective response rates (ORR) and rates of complete response (CR) by Day 28 to select the optimal regimen to carry forward into Part 2. The objective of Part 2 is to confirm whether lenzilumab can improve the toxicity and tolerance of CAR-T while maintaining or improving efficacy and durability of response. Up to 250 patients will be randomized 1:1 to receive lenzilumab or placebo with CAR-T per standard of care. The primary endpoint of the study is incidence of grade >2 CRS and/or ICANS by Day 28, with a key secondary endpoint of CR at 6-months in patients without grade ≥ 2 CRS and/or ICANS at Day 28 (Toxicity-free CR). This design and sample size yields 90% power to detect a 50% reduction in the primary outcome measure. Secondary endpoints include incidence of all grades and grade >3 CRS and/or ICANS, respectively; ORR and CR at 1, 3, 6, 12 months; durability of CR; progression-free survival, overall survival and health related quality of life using validated patient reported outcome measures. In addition, the study will explore the CRS and ICANS grading criteria that have been utilized with each of the approved CAR-Ts. Disclosures Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding. Durrant: Humanigen, Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Chappell: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Ahmed: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company. Kilcoyne: Humanigen, Inc.: Current Employment, Current equity holder in publicly-traded company.

Abstract CD19-targeted chimeric antigen receptor T cell (CART19) therapy has been remarkably successful in treating a subset of patients with hematological malignancies. However, it is associated with significant toxicities, including cytokine release syndrome (CRS) and neurotoxicity (NT). Furthermore, the rate of durable responses after CART19 therapy is low, and most patients develop resistance to the therapy. One of the predominant mechanisms for CART cell resistance is intrinsic T cell dysfunction which leads to a suboptimal CART product. The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to favorably modulate CART phenotype and function through downregulation of inhibitory receptors and enhancement of CART cell efficacy in preclinical models and early clinical studies. However, ibrutinib is an irreversible receptor tyrosine kinase (RTK) inhibitor, and thus there are concerns that it inhibits CART cell proliferation and expansion. In contrast to ibrutinib and other BTK inhibitors, vecabrutinib has recently emerged as a potent, reversible, non-covalent BTK inhibitor that can block the activity of both wild type and C481 mutant BTK as well as interleukin-2-inducible T-cell kinase (ITK). We hypothesized that treatment with vecabrutinib improves CART19 antitumor efficacy through the reversible inhibition of BTK/ITK, independent of its antitumor effect. To test this hypothesis, we assessed whether vecabrutinib potentiates CART19 cytotoxicity against CD19 + tumor cells. Killing of the CD19 + mantle cell lymphoma cell line, JeKo-1, was significantly increased when CART19 were combined with vecabrutinib (Fig. 1A). Of note, vecabrutinib did not induce antitumor activity in this model as a single agent (Fig. 1A). In addition, vecabrutinib (10 µM) treatment of CART19 resulted in enhanced antigen-specific proliferation (Fig. 1B). Next, we aimed to study the direct impact of vecabrutinib on CART19 cytokine production. We measured the levels of multiple pro-inflammatory cytokines in the supernatant of CART19 co-cultured for 3 days with JeKo-1 cells and vecabrutinib. Vecabrutinib did not impair CART19 degranulation, as measured by flow cytometric assessment of CD107a expression (Fig. 1C). Cytokines known to play a crucial part in the development of CRS, such as IL-6, IL-10, and MIP-1β were significantly downregulated in cocultures with vecabrutinib (Fig. 1D-F). We then investigated whether the addition of vecabrutinib to CART19 caused a synergistic antitumor effect in vivo. We used our established JeKo-1 lymphoma xenograft models. Here NSG mice were engrafted with CD19 + luciferase + JeKo-1 cells. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) CART19 with vehicle, or 3) CART19 with vecabrutinib 50 mg/kg BID administered via oral gavage over a period of 4 weeks. Vecabrutinib combination with CART19 treatment led to sustained antitumor activity (Fig.1G) and increased CART19 proliferation (Fig.1H). Encouragingly, mice treated with vecabrutinib better maintained their body weight following CART19 infusion, suggesting possibly dampened toxicity (Fig.1I). We also measured serum cytokine levels from patients on a phase 1 clinical trial (NCT03037645) testing vecabrutinib in B cell malignancies at baseline and four weeks after treatment. We found that vecabrutinib significantly reduces the levels of multiple pro-inflammatory cytokines linked to CART cell toxicity, including MIP-1β, IP-10, and TNF-a (Fig. 1J-L), further validating our preclinical findings. Finally, we interrogated the transcriptome of CART19 co-cultured with JeKo-1 with or without vecabrutinib. Total RNA sequencing of activated CART19 highlighted a significant enhanced expression of multiple genes involved in the PI3K/AKT and Th1 pathways compared to antigen stimulated CART19 alone (Fig.1M, N). These results could explain the enhanced proliferation and cytotoxicity induced by vecabrutinib. In summary, we demonstrate for the first time that using the reversible BTK inhibitor, vecabrutinib, is a potent and a novel strategy to favorably modulate CART19 function by increasing their efficacy and decreasing toxicity. Further studies are currently underway to compare the effect of reversible versus irreversible BTK/ITK inhibition on CART functions and to further validate the mechanisms responsible for the observed effects. Figure 1 Figure 1. Disclosures Sakemura: Humanigen: Patents & Royalties. Cox: Humanigen: Patents & Royalties. Fox: Sunesis Pharmaceuticals: Current Employment. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: MEI Pharma: Research Funding; Sunesis: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Abstract Introduction: It has become increasingly apparent that chimeric antigen receptor T (CART) cell activation and differentiation level is an important determinant of CART cell fate and response to therapy. In this study, we aimed to 1) measure levels of activation-induced surface death receptors and ligands on CART cells; 2) investigate how CART cell activation could impact their fitness and clinical responses, and 3) identify cell-based targets to modulate CART cell activation, apoptosis, and cytotoxicity to improve anti-tumor activity. Methods: We performed flow cytometric studies on ex-vivo stimulated, clinically annotated CART products of patients with large B cell lymphoma from the pivotal ZUMA-1 clinical trial that led to FDA-approved Axicabtagene ciloleucel (Axi-Cel). We investigated possible correlations of a number of surface death receptors and ligands with T cell differentiation status and post-infusion CART cell expansion, utilizing samples from ZUMA-1 patients who achieved a complete response as a best outcome ('responders') vs patients who achieved stable or progressive disease('non-responders'). CART cell effector functions in vitro were measured, and CART apoptosis was assessed using Annexin V. For in vitro and in vivo functional studies, we used CART19 generated from healthy donors (HD CART19) as indicated in the specific experiment. CRISPR/Cas9 was employed during CART cell production to disrupt specific genes. A xenograft model of lymphoma was used to investigate the in vivo antitumor activity of CART19. Results: Following an ex vivo stimulation of Axi-Cel products with CD19 + target cells, we observed upregulation of death receptors and ligands in CART19 from non-responders, compared to responders. We also observed a possible association between such upregulated surface markers with CART cell differentiation as measured by CCR7 expression. In an extended in vitro co-culture assay, where HD CART19 cells were repeatedly stimulated through the CAR, we found that tumor necrosis factor α receptor 2 (TNFR2), unlike other death receptors and ligands, was persistently elevated, suggesting a possible role for TNFR2 in long-term antigen-dependent CART19 dysfunction (Figure 1A). We further found that HD CART19 upregulate TNFR2, but not TNFR1, upon CAR stimulation (Figure 1B). While non-specific TCR activation (CD3 stimulation) of HD CART19 cells protected them from activation-induced apoptosis, antigen-specific activation through the CAR resulted in significant initiation of apoptosis within 2 hours of stimulation (Figure 1C). Having identified a possible association between TNFR2 and CART19 dysfunction, we aimed to study the impact of TNFR2 knockout on HD CART19 functions. Using CRISPR/Cas9 during CART cell manufacturing, we generated TNFR2 k/o HD CART19 cells with a knockout efficiency of around 50%, where the expression levels of TNFR2 in activated CART19 cells were reduced, compared to control CART19 cells (with non-targeting gRNA CRISPR/Cas9, Figure 1D). TNFR2 k/o CART19 cells demonstrated reduced early activation surface markers compared to control CART19, as measured by CD25 and CD69 surface expression (Figure 1E), reduced apoptosis initiation as measured by the Annexin V assay (Figure 1F), and enhanced antigen-specific proliferation and cytotoxicity (Figure 1G). Finally, in an in vivo xenograft model of CD19 + lymphoma, TNFR2 k/o CART19 resulted in enhanced CART cell expansion and anti-tumor activity (Figure 1H). Conclusions: Our results indicate that TNFR2 plays a role in early activation and apoptosis initiation of CART19 following CAR stimulation with CD19 + target cells and present TNFR2 knockout as a strategy to enhance CART19 anti-tumor activity. Figure 1 Figure 1. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura: Humanigen: Patents & Royalties. Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Genentech: Research Funding; Behring: Membership on an entity's Board of Directors or advisory committees; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; TG Therapeutics: Research Funding; Tolero Pharmaceuticals: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Scholler: Kite: Current Employment. Bot: Kite, a Gilead Company: Current Employment; Gilead Sciences: Consultancy, Current equity holder in publicly-traded company, Other: Travel support. Mattie: Kite: Current Employment. Kim: Gilead Sciences: Current equity holder in publicly-traded company; Kite, a Gilead Company: Current Employment. Filosto: Kite, a Gilead Company: Current Employment; Tusk Therapeutics: Patents & Royalties: or other intellecular property; Gilead Sciences: Other: stock or other ownership . Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

Abstract Anti-CD19 chimeric antigen receptor T (CART19) cell therapy has resulted in unprecedented outcomes in patients with relapsed/refractory B-cell malignancies, which led to the FDA approval for several indications. However, CART19 cell therapy is limited by the development of severe life-threatening toxicities, as well as by the limited rates of durable response. It has become apparent that myeloid cells contribute to the development of both CART cell toxicity and also to the inhibitory tumor microenvironment. We and others have identified that granulocyte-monocyte-colony-stimulating factor (GM-CSF) depletion results in decreased myeloid activation, reduced toxicities, and enhancement of CART19 cell therapy efficacy in pre-clinical models. Furthermore, we observed that GM-CSF knockout (GM-CSF k/o) in CART19 cells resulted in the improvement of their functions (in vitro and in vivo). These findings suggest that there is also a direct effect of GM-CSF on CART19 cells, which is independent of the GM-CSF impact on myeloid cell activation. To further evaluate this, we first examined GM-CSF receptor alpha (GM-CSFRα) expression by flow cytometry on resting and activated CART19 cells (using FMC63-41BBζ). When CART19 cells were stimulated with either anti-CD3/CD28 beads or lethally irradiated (120 Gy) CD19 + Nalm6 cells (B cell acute lymphoblastic leukemia cancer cell line), GM-CSFRα expression was upregulated upon both T cell receptor (TCR) (data not shown) and CAR stimulation (Figure 1A). Having demonstrated that GM-CSFRα is significantly upregulated on stimulated CART19 cells, we aimed to determine the impact of GM-CSF neutralization (clinical-grade anti-GM-CSF antibody, lenzilumab, 10 µg/mL) versus GM-CSFRα blockade (research-grade antibody, 10 µg/mL) on CART19 cell function and CART cell-monocyte interactions. An IgG isotype antibody was used as a control antibody. Neither the GM-CSF neutralizing antibody, nor GM-CSFRα blocking antibody, had any impact on CART19 cell antigen-specific killing against the CD19 + JeKo-1 cells (mantle cell lymphoma cancer cell line), in the presence or absence of CD14 + monocytes (ratio 1:1:1) isolated by magnetic beads from healthy donors (Figures 1B-C). Next, we compared the effects of GM-CSF neutralization versus GM-CSFRα blockade on CART19 cell antigen-specific proliferation. Here, CART19 cells were co-cultured with lethally irradiated CD19 + cell line JeKo-1 at 1:1 ratio in the presence of 10 µg/mL of the GM-CSF neutralizing antibody, increasing doses of the GM-CSFRα blocking antibody (10-100 µg/mL), or an IgG control. The absolute number of CART cells was measured by flow cytometry on day 5. GM-CSF neutralization did not affect CART19 cell proliferation, but GM-CSFRα blocking antibody significantly inhibited CART19 cell proliferation in a dose-dependent manner. Then, we assessed the effects of GM-CSF neutralizing antibody (20 µg/mL) versus GM-CSFRα blocking antibody (20 µg/mL) on CART19 cell antigen-specific proliferation in the presence of healthy donor monocytes (ratio 1:1:0.5) on day 3. Flow cytometric analysis revealed that GM-CSF neutralization, but not GM-CSFRα blockade, mitigated monocyte-suppression of CART19 antigen-specific proliferation (Figure 1E). In summary, our findings indicate significant differences on CART cell functions and CART cell-monocyte interactions when a specific cytokine, GM-CSF, is neutralized compared to blocking its receptor. Further mechanistic studies are ongoing to assess the functions of GM-CSFRα k/o and GM-CSF k/o CART cells. Figure 1 Figure 1. Disclosures Sakemura: Humanigen: Patents & Royalties. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Behring: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees. Durrant: Humanigen Inc.: Current Employment. Ahmed: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company. Chappell: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

AbstractMonoclonal B‐cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL) are clonal B‐cell disorders associated with an increased risk of infections and impaired vaccination responses. We investigated the immunogenicity of recombinant zoster vaccine (RZV) in these patients. Individuals with MBL/untreated CLL and Bruton tyrosine kinase inhibitor (BTKi)‐treated CLL patients were given two doses of RZV separated by 2 months. Responses assessed at 3 and 12 months from the first dose of RZV by an anti‐glycoprotein E ELISA antibody assay and by dual‐color Interferon‐γ and Interleukin‐2FLUOROSPOT assays were compared to historic controls matched by age and sex. About 62 patients (37 MBL/untreated CLL and 25 BTKi‐treated CLL) were enrolled with a median age of 68 years at vaccination. An antibody response at 3 months was seen in 45% of participants, which was significantly lower compared to historic controls (63%, p = .03). The antibody response did not significantly differ between MBL/untreated CLL and BTKi‐treated CLL (51% vs. 36%, respectively, p = .23). The CD4+ T‐cell response to vaccination was significantly lower in study participants compared to controls (54% vs. 96%, p < .001), mainly due to lower responses among BTKi‐treated patients compared to untreated MBL/CLL (32% vs. 73%, p = .008). Overall, only 29% of participants achieved combined antibody and cellular responses to RZV. Among participants with response assessment at 12 months (n = 47), 24% had antibody titers below the response threshold. Hypogammaglobulinemia and BTKi therapy were associated with reduced T‐cell responses in a univariate analysis. Strategies to improve vaccine response to RZV among MBL/CLL patients are needed.

Abstract Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.