ALBERT

Description: Background: Interferon-alpha (IFNα) is a pleiotrophic cytokine with direct anti-tumor and immunostimulatory effects. Currently IFNα is approved for the treatment of multiple hematologic malignancies, including non-Hodgkin lymphoma (NHL). However, its clinical utility has been hindered by dose-limiting toxicitiy due to systemic activation of the interferon receptor. To overcome this limitation, we engineered anti-tumor antibody-IFNα fusion proteins to selectively increase delivery of IFN to the tumor site and reduce systemic toxicity. We previously reported that IGN002, an anti-CD20-IFNα fusion protein, exhibits enhanced complement-dependent cytotoxicity (CDC) compared to rituximab, and inhibits proliferation and induces apoptosis of human B-cell NHL (Yamada et al, ASCO 2013). We now extend these previous findings and show that IGN002 possesses enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function and superior in vivo anti-tumor activity against B-cell NHL, compared to rituximab. Methods: IGN002 was evaluated against a panel of human Burkitt and diffuse large B-cell lymphoma (DLBCL) cell lines. Proliferation was measured by [3 H]-thymidine incorporation, STAT1 activation by flow cytometry, ADCC by lactate dehydrogenase release using human PBMC effectors, and IFN bioactivity by encephalomyocarditis (EMC) viral protection assay. NHL xenografts were grown in SCID mice. Results: IGN002 more potently inhibited the growth of NHL cell lines expressing CD20 than rituximab or unfused IFNα. Intrinsic IFNα activity of IGN002 was reduced in viral protection and anti-proliferation assays using cells lacking CD20 expression. STAT1 activation by IGN002 was enhanced on cells expressing the target antigen, whereas a control antibody-IFNα fusion protein showed reduced STAT activation activity compared to unfused IFNα. Together, these results indicate that fusion of IFNα to the antibody results in reduced IFN effects on cells not bearing the tumor antigen target. IGN002 exhibited enhanced ADCC activity compared to rituximab against Daudi, Ramos, and Raji NHL cells in long-term (overnight incubation) assays, demonstrating both higher potency and higher maximal cytotoxicity. This result is possibly due to activation of the effector cell populations by the fused IFNα moiety, as IFN is known to activate both NK cells and monocytes. The in vivo anti-tumor efficacy of IGN002 was compared to rituximab and a control antibody-IFNα fusion protein against 10-day established Raji NHL xenografts. IGN002 was superior to both rituximab and the control fusion protein, achieving a longer median survival and higher long-term survival rate (p = 0.0015 and 〈 0.0001 vs. rituximab and control fusion protein, respectively). The in vivo anti-tumor efficacy of IGN002 was also compared to rituximab at three equimolar dose levels (5 mg/kg, 1 mg/kg, and 0.2 mg/kg antibody) against 10-day established Daudi NHL xenografts. IGN002 showed superior efficacy compared to rituximab at all doses (p 〈 0.001), achieving tumor eradication (100% long-term survival) in all mice treated at all three dose levels, whereas rituximab only delayed tumor progression. Conclusions: IGN002 demonstrated more robust direct anti-proliferative and antibody effector functions than rituximab against human NHL cells in vitro, and also showed the ability to eradicate established NHL xenografts in vivo. Against cells expressing the CD20 target antigen, IGN002 exhibited greater anti-proliferative potency than unfused IFNα. In contrast, the anti-proliferative and anti-viral potency of IGN002 was reduced against cells lacking CD20, compared to unfused IFNα. These findings support the hypothesis that tumor antigen-targeted IFN therapeutics may possess a broader therapeutic index than unfused IFNα, inhibiting tumor growth by multiple mechanisms while reducing systemic toxicity. These results support the further development of IGN002 for the treatment of B-cell NHL, and a first-in-human phase I clinical study will begin later this year in the United States. Disclosures Timmerman: Janssen: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Valor Biotherapeutics: Research Funding. Steward:ImmunGene, Inc.: Employment. Minning:Valor Biotherapeutics, LLC: Consultancy. Sachdev:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Gresser:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Khare:Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morrison:ImmunGene, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction Chimeric Antigen Receptor (CAR) T-cell therapy is an innovative therapy for relapsed or refractory diffuse large B-cell lymphoma (DLBCL). Prior to CAR T-cell infusion, patients receive lymphodepleting chemotherapy and may receive granulocyte colony-stimulating factor (G-CSF) to decrease the duration of neutropenia and risk of infection. However, G-CSF also has the potential to increase the incidence and/or severity of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) by promoting antigen-presenting cell function (Mehta HM, J Immunol, 2015). More data is required to guide clinicians on the benefits and risks of G-CSF use in CAR T-cell treatment. Methods A retrospective analysis was performed among 22 DLBCL patients who received CAR T-cell therapy with Axicabtagene ciloleucel at the University of California Los Angeles from March 2018 to May 2019. All patients received standard lymphodepleting therapy with fludarabine 30 mg/m2/day and cyclophosphamide 500 mg/m2/day on days -5 through -3, except one patient who received reduced dosages for renal disease. Prophylactic tocilizumab 8 mg/kg was given to all patients at 36 hours after CAR T-cell infusion, with additional doses of tocilizumab and/or steroids given for evidence of CRS/ICANS based on the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading system. G-CSF was administered by physician discretion following CAR T-cell infusion at a weight-based dosage of either 300 or 480 mcg and cumulative G-CSF dose recorded within the first 30 days. Results Patient and disease characteristics are shown in Table 1. Seven of the 22 patients (31.8%) received G-CSF at a median cumulative dosage of 2880 mcg (IQR 900-8640), with the majority (4 patients, 57.1%) receiving G-CSF in the first 5 days post CAR T-cell infusion (Table 2). The median duration of neutropenia following lymphodepleting therapy was 5 days (IQR 4.5-8.5) for those patients treated with G-CSF compared to 15 days (IQR 8.0-30.0) for those who did not receive G-CSF (p = 0.0157). Seven patients (31.8%) developed infection in the 30 days post CAR T-cell therapy with 4 infections being grade 3 or higher. There was no difference in the incidence and severity of infection between those patients who received G-CSF and those that did not (p = 0.630, p = 0.424, Figure 1). CRS was noted in 14 patients overall (63.6%), 4 of which were grade 3 or higher. ICANS was noted in 11 patients overall (50.0%), 9 of which were grade 3 or higher. Among the 7 patients that received G-CSF, 6 patients (85.7%) and 4 patients (57.1%) had evidence of CRS and ICANS, respectively. Among the 15 patients that did not receive G-CSF, 8 patients (53.3%) and 7 patients (46.7%) had evidence of CRS and ICANS, respectively. There was no significant difference in the incidence of developing CRS (any grade) or ICANS (any grade) between the group of patients that received G-CSF and those that did not (p = 0.193, p = 0.647). However, there was a significant increase in the severity of CRS for patients that received G-CSF compared to those that did not (p = 0.0418, Figure 1), but no increase in the severity of ICANS based on G-CSF use (p = 0.660, Figure 1). Thirteen patients (59.1%) received corticosteroids following CAR T-cell treatment at a median cumulative dosage of 666.7 mg prednisone equivalents (IQR 445.0-933.3), with the majority (9 patients, 69.2%) receiving steroids in the first 5 days post CAR T-cell infusion (Table 2). More than half of the cohort (14 patients, 63.6%) required more than 1 dose of tocilizumab (Table 2). There was no association between G-CSF use and steroid use or administration of more than 1 dose of tocilizumab (p = 0.648, p = 0.074). Conclusions Our data demonstrates a significant increase in severity without an increase in incidence of CRS with G-CSF use within 30 days of Axicabtagene ciloleucel administration for relapsed/refractory DLBCL. There was no association of G-CSF with severity or incidence of ICANS and no difference in infection rates with G-CSF in this setting. *Both Daria Gaut and Kevin Tang contributed equally to this work. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Through its interaction with PD-L1, the inhibitory receptor programmed death 1 (PD-1) is a key immune "checkpoint" that regulates adaptive immunity in both infectious and neoplastic diseases. Co-opted expression of PD-L1 by cancers can thus inhibit endogenous T cell anti-tumor responses, thereby permitting escape from immune destruction, yet offering a novel means of immunotherapy by antibody blockade of the PD-1/PD-L1 axis. Elevated expression of PD-L1 within melanomas and numerous carcinomas has been associated with adverse prognosis. We previously described the expression of PD-L1 in a subset of DLBCL cases, particularly in those of non-GCB phenotype (Andorsky et al, Clin. Cancer Res. 2011). Human immunodeficiency virus (HIV)-related DLBCLs are a subgroup of DLBCLs with have a more aggressive clinical course, even in the era of combination antiretroviral therapy. HIV-related DLBCLs are often associated with severe immunosuppression, however, the expression of PD-L1 in HIV-related DLBCL is unknown. In this study, we examined the relationship between PD-L1 expression, HIV status, and clinical outcomes. Methods: A total of 135 cases of incident DLBCL diagnosed between 2000-2007 were included from patients at Kaiser Permanente California, an integrated health care system. Of these, 57 were HIV-pos DLBCL patients, and 78 HIV-neg DLBCL patients were selected age, sex and race matched to the HIV-pos cases. Patients were characterized according to International Prognostic Index (IPI) risk factors, and cell-of-origin (COO; germinal center B [GCB] versus non-GCB) using the Hans classifier. PD-L1 staining was performed by immunohistochemistry, with dual staining for the PAX5 B cell marker used to distinguish tumor cell versus non-tumor /stromal macrophage expression of PD-L1. A 10% cutoff point was used to classify cases as positive for PD-L1 expression by tumor cells. Mortality within 2 years of DLBCL diagnosis and cause of death were ascertained. The association between PD-L1 expression and HIV status was evaluated using the t-test. The association between PD-L1 expression and patient survival was examined using multivariable Cox model, adjusting for IPI, COO and HIV status. Results: Tumor cell expression of PD-L1 was noted in 7.7% of HIV-neg cases versus 17.5% of HIV-pos cases (p=0.08). Expression of PD-L1 within the non-tumor stromal macrophages was more common, seen in 70.5% of HIV-neg and 68.4% of HIV-pos cases (p=0.79) using a 10% cutoff for stromal cell expression. Using a 30% cutoff for stromal cell expression, fewer cases were PD-L1 positive, but the frequency did not differ significantly between the HIV-neg and HIV-pos cohorts (32.1% and 31.6%, respectively, p=0.95). Clinical outcome, measured by 2 year lymphoma-specific mortality (LSM), was negatively affected by HIV serologic status, being 11.5% in HIV-neg versus 28.1% in HIV-pos cases (p=0.01). With regards to tumor cell PD-L1 expression, among HIV-neg cases, both all-cause mortality (ACM) and LSM did not appear to correlate with PD-L1 status (p=0.67 and p= 0.51, respectively). However, among HIV-pos subjects, tumor cell PD-L1 expression was associated with adverse ACM (p=0.05, HR 3.08 [1.03-9.28]) and LSM (p=0.03, HR 4.01 [1.11-8.48]) when adjusted for IPI and COO. In an additional overall analysis combining all 135 HIV-neg and HIV-pos cases, tumor cell PD-L1 expression was again correlated with worse outcome in terms of ACM (p=0.04, HR 2.38 [1.02-5.56]), and LSM (p=0.03, HR 3.07 [1.11-8.48]). In contrast, stromal cell PD-L1 expression did not correlate with outcomes in any of these analyses. Conclusions: Expression of the negative T cell regulatory protein PD-L1 on tumor cells was found on a minority of cases of DLBCL, though at least numerically more often in HIV-pos cases than in HIV-neg in this relatively small series. Association between tumor cell PD-L1 expression and adverse outcome was seen in HIV-pos but not HIV-neg subjects. Expression of PD-L1 within tumors in HIV-infected subjects could possibly render these tumors more amenable to immune escape than in HIV-pos subjects, or be a marker for more aggressive intrinsic biology of these tumors. Lastly, since immunotherapy with PD-1 blockade has recently been shown to have efficacy against some B cell lymphomas (Lesokhin et al, ASH 2014), this form of immunotherapy could possibly improve the less favorable outcomes of patients whose tumor cells express PD-L1. Disclosures Timmerman: Valor Biotherapeutics: Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.