John M. Rossi

Absence of functional EpoR expression in human tumor cell lines.

Certain oncology trials showed worse clinical outcomes in the erythropoiesis-stimulating agent (ESA) arm. A potential explanation was that ESA-activated erythropoietin (Epo) receptors (EpoRs) promoted tumor cell growth. Although there were supportive data from preclinical studies, those findings often used invalidated reagents and methodologies and were in conflict with other studies. Here, we further investigate the expression and function of EpoR in tumor cell lines. EpoR mRNA levels in 209 human cell lines representing 16 tumor types were low compared with ESA-responsive positive controls. EpoR protein production was evaluated in a subset of 66 cell lines using a novel anti-EpoR antibody. EpoR(+) control cells had an estimated 10 000 to 100 000 EpoR dimers/cell. In contrast, 54 of 61 lines had EpoR protein levels lower than 100 dimers/cell. Cell lines with the highest EpoR protein levels (400-3200 dimers/cell) were studied further, and, although one line, NCI-H661, bound detectable levels of [(125)I]-recombinant human Epo (rHuEpo), none showed evidence of ESA-induced EpoR activation. There was no increased phosphorylation of STAT5, AKT, ERK, or S6RP with rHuEpo. In addition, EpoR knockdown with siRNAs did not affect viability in 2 cell lines previously reported to express functional EpoR (A2780 and SK-OV-3). These results conflict with the hypothesis that EpoR is functionally expressed in tumors.

Identification of a sensitive anti-erythropoietin receptor monoclonal antibody allows detection of low levels of EpoR in cells

Erythropoietin (Epo) binds and activates the Epo receptor (EpoR) on the surface of erythroid progenitor cells resulting in formation of erythrocytes. Recently, EpoR was reported to be expressed on non-erythroid cells suggesting a role for Epo outside of erythropoiesis. However those studies employed antibodies with questionable specificity and the significance of the observations are controversial. In order to accurately determine the expression of EpoR proteins in cells, we have generated a panel of novel anti-human EpoR monoclonal antibodies. One of these antibodies (A82) was particularly sensitive and it detected the EpoR protein on intact cells by flow cytometry and by western blot analysis with cell lysates. Both methods were optimized and using them, EpoR protein was detected by western immunoblotting with lysates from fewer than 200 EpoR positive control cells and the positive signals were proportional to EpoR protein expression level with a minimal signal in EpoR negative cells. The proteins detected by western blot analysis using A82 included full-length EpoR ( approximately 59kDa) as well as smaller EpoR fragments derived from the EPOR gene. These results indicate that A82 can be used to examine low level EpoR expression in cells by western and flow cytometry allowing an improved understanding of EpoR expression and metabolism.

Population Pharmacokinetic/Pharmacodynamic Model of Subcutaneous Adipose 11β‐Hydroxysteroid Dehydrogenase Type 1 (11β‐HSD1) Activity After Oral Administration of AMG 221, a Selective 11β‐HSD1 Inhibitor

Inhibition of 11β‐HSD1 is hypothesized to improve measures of insulin sensitivity and hepatic glucose output in patients with type II diabetes. AMG 221 is a potent, small molecule inhibitor of 11β‐HSD1. The objective of this analysis is to describe the pharmacokinetic/pharmacodynamic (PK/PD) relationship between AMG 221 and 11β‐HSD1 inhibition in ex vivo adipose tissue samples. Healthy, obese subjects were administered a single dose of 3, 30, or 100 mg of oral AMG 221 (n = 44) or placebo (n = 11). Serial blood samples were collected over 24 hours. Subcutaneous adipose tissue samples were collected by open biopsy. Population PK/PD analysis was conducted using NONMEM. The inhibitory effects (mean ± standard error of the estimate) of AMG 221 on 11β‐HSD1 activity were directly related to adipose concentrations with Imax (the maximal inhibition of 11β‐HSD1 activity) and IC50 (the plasma AMG 221 concentration associated with 50% inhibition of enzyme activity) of 0.975 ± 0.003 and 1.19 ± 0.12 ng/mL, respectively. The estimated baseline 11β‐HSD1 enzyme activity was 755 ± 61 pmol/mg. An equilibration rate constant (keo) of 0.220 ± 0.021 h–1 described the delay between plasma and adipose tissue AMG 221 concentrations. AMG 221 potently blocked 11β‐HSD1 activity producing sustained inhibition for the 24‐hour study duration as measured in ex vivo adipose samples. Early characterization of concentration‐response relationships can support rational selection of dose and regimen for future studies.

RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.

Functional EpoR Pathway Utilization Is Not Detected in Primary Tumor Cells Isolated from Human Breast, Non-Small Cell Lung, Colorectal, and Ovarian Tumor Tissues

Several clinical trials in oncology have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents. One hypothesis proposes that erythropoiesis-stimulating agents directly stimulate tumor proliferation and/or survival through cell-surface receptors. To test this hypothesis and examine if human tumors utilize the erythropoietin receptor pathway, the response of tumor cells to human recombinant erythropoietin was investigated in disaggregated tumor cells obtained from 186 patients with colorectal, breast, lung, ovarian, head and neck, and other tumors. A cocktail of well characterized tumor growth factors (EGF, HGF, and IGF-1) were analyzed in parallel as a positive control to determine whether freshly-isolated tumor cells were able to respond to growth factor activation ex vivo. Exposing tumor cells to the growth factor cocktail resulted in stimulation of survival and proliferation pathways as measured by an increase in phosphorylation of the downstream signaling proteins AKT and ERK. In contrast, no activation by human recombinant erythropoietin was observed in isolated tumor cells. Though tumor samples exhibited a broad range of cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface erythropoietin receptor was detected in tumor cells from the 186 tumors examined (by flow cytometry or Western blot). Erythropoiesis-stimulating agents did not act directly upon isolated tumor cells to stimulate pathways known to promote proliferation or survival of human tumor cells isolated from primary and metastatic tumor tissues.

A Phase I, First-in-Human Study of AMG 780, an Angiopoietin-1 and -2 Inhibitor, in Patients with Advanced Solid Tumors.

Purpose: To assess the toxicity, pharmacokinetics, tumor vascular response, tumor response, and pharmacodynamics of AMG 780, a mAb designed to inhibit the interaction between angiopoietin-1 and -2 and the Tie2 receptor. Experimental Design: This was a phase I dose-escalation study of patients with advanced solid tumors refractory to standard treatment without previous antiangiogenic treatment. AMG 780 was administered by intravenous infusion every 2 weeks in doses from 0.1 to 30 mg/kg. The primary endpoints were incidences of dose-limiting toxicity (DLT) and adverse events (AE), and pharmacokinetics. Secondary endpoints included tumor response, changes in tumor volume and vascularity, and anti-AMG 780 antibody formation. Results: Forty-five patients were enrolled across nine dose cohorts. Three patients had DLTs (0.6, 10, and 30 mg/kg), none of which prevented dose escalation. At 30 mg/kg, no MTD was reached. Pharmacokinetics of AMG 780 were dose proportional; median terminal elimination half-life was 8 to 13 days. No anti-AMG 780 antibodies were detected. At week 5, 6 of 16 evaluable patients had a >20% decrease in volume transfer constant (Ktrans), suggesting reduced capillary blood flow/permeability. The most frequent AEs were hypoalbuminemia (33%), peripheral edema (29%), decreased appetite (27%), and fatigue (27%). Among 35 evaluable patients, none had an objective response; 8 achieved stable disease. Conclusions: AMG 780 could be administered at doses up to 30 mg/kg every 2 weeks in patients with advanced solid tumors. AMG 780 treatment resulted in tumor vascular effects in some patients. AEs were in line with toxicity associated with antiangiopoietin treatment. Clin Cancer Res; 22(18); 4574–84. ©2016 AACR.

287. Production of KTE-C19 (Anti-CD19 CAR T Cells) for ZUMA-1: A Phase 1/2 Multi-Center Study Evaluating Safety and Efficacy in Subjects with Refractory Aggressive Non-Hodgkin Lymphoma (NHL)

Introduction: An ongoing anti-CD19 CAR T cell study with KTE-C19 in subjects with refractory, aggressive NHL achieved objective responses in 5/7 subjects, including 4 complete remissions (Locke, ASH 2015). To support this trial (ZUMA-1, NCT02348216) and other multicenter trials in lymphoma, we developed a robust and efficient strategy to generate autologous anti-CD19 CAR-engineered T cell products. The newly developed process aims to minimize the time between subject leukapheresis and product administration, and generate a KTE-C19 lot for every enrolled subject. Methods: Upon confirmation of eligibility, leukapheresis was performed to process 12-15 L of blood targeting collection of 5-10 × 109 peripheral blood mononuclear cells (PBMC). After collection at the investigational site, subject apheresis material was shipped to the central manufacturing site, where it was processed to enrich for the T cell-containing PBMC fraction on a closed system Ficoll™ gradient. In a closed bag system, T cells in the PBMC fraction were then activated using an anti-CD3 monoclonal antibody and cultured in serum-free medium containing 300 IU/ml of IL-2. Magnetic beads were not used for either cell selection or activation. Activated T cells were transduced with a gamma retroviral vector that contains the anti-CD19 CAR gene and further expanded for 4 to 6 days to achieve the target cell dose of 2 × 106 CAR-positive T cells/kg (minimum of 1 × 106). Final KTE-C19 product was washed, cryopreserved and tested for identity, potency, and adventitious agents. In-process samples were collected for analysis by flow cytometry for CAR expression and other characteristics. After meeting acceptance criteria, the KTE-C19 product was shipped back to the clinical sites using a validated cryo-shipper. Results: 7 subjects were dosed in the phase 1 portion of ZUMA-1. KTE-C19 was successfully manufactured in all cases despite a broad range of baseline leukocyte counts (median 5.4×103 cells/µl, 2.1-11.1) and low numbers of baseline lymphocytes (median 0.9×103 lymphocytes/µl, 0.1-1.4) prior to apheresis. Apheresed cell populations used to generate clinical lots had a broad range of phenotypic characteristics including lymphocyte / monocyte ratio (median 1.1, 0.04-3.5), CD8/CD4 ratio (median 4.2, 0.3-7.7), and naive plus central memory (Tcm) / more differentiated T cells (median 0.3, 0.04-1.3). Fold-expansion of T cells was consistent among the 7 product lots (average 6-fold) from transduction to harvest. All KTE-C19 lots contained predominantly CD3+ T cells (median 96%; 90-99%), with CD8+ T cells (median 57%, 27-82%) and CD4+ T cells (median 43%, 18-73%). Both CD4+ and CD8+ T cells expressed CAR at similar levels. The product lots contained predominantly effector memory (median 42%, range 30-56%) and Tcm cells (median 34%, 15-58%), with the remainder being naive (median 14%, 6-19%) and effector T cells (median 5%, 1-22%). All KTE-C19 lots met release specifications and were available for clinical administration within ~2 weeks of apheresis. Conclusions: We developed a robust KTE-C19 manufacturing process that successfully generated biologically and clinically active product irrespective of the characteristics of the starting cell populations processed to date. Product lots met release specifications and were available for subject treatment within the target timeframe. This centralized manufacturing process is well suited to support multicenter clinical trials.

Abstract 2305: Comparative evaluation of peripheral blood T cells and resultant engineered anti-CD19 CAR T cell products from relapsed/refractory non-Hodgkin's lymphoma (NHL) patients

Introduction: Administration of autologous anti-CD19 CAR T cells prepared from peripheral blood mononuclear cells (PBMC) of patients with various B cell malignancies have mediated high rates of objective response (Kochenderfer et al. Blood 2012, J Clin Onc 2014, Mackall et al, Blood 2013). We analyzed characteristics of the starting material (PBMC) and resultant CAR T cells derived from 14 patients with relapsed/refractory NHL at the NCI Surgery Branch. Methods: After apheresis collection, the T cell-containing PBMC fraction was enriched, activated with anti-CD3 antibody and cultured in serum-free medium for 2 days. Activated T cells were transduced with a retroviral vector encoding the anti-CD19 CAR gene and further expanded with IL-2 to achieve a target dose of CAR T cells. The CAR T cells and starting PBMC population were cryopreserved and analyzed after thawing by flow cytometry. Differences in cell composition between the starting PBMC population and CAR T cells were evaluated with paired t-tests, adjusted for multiplicity. Results: Biologically active autologous CAR T cells were successfully prepared from all 14 NHL subjects. Each product was comprised of both CD4+ and CD8+ T cells, but showed considerable inter-subject variability. The CD4/CD8 ratio in the PBMC and product CAR T cell population were correlated (Pearson correlation coefficient = 0.74, p = 0.0027). While the starting T cell population generally showed a T cell profile comprised of similar proportions of effector T cells (Teff) (\( \bar{x} \) = 20%, std = 16%) and naive T cells (Tn) (\( \bar{x} \) = 20%, std = 14%), there was a shift towards a less differentiated T cells population after CAR T cell production, with a decreased Teff (paired t test p = 0.0151) and elevated Tn (p = 0.0031). The percent of juvenile cells (Tn+Tcm) was higher in the final product (\( \bar{x} \) = 64%, std = 21%) than in the starting T cell population (\( \bar{x} \) = 43%, std = 20%, p* = 0.0031, *stepdown Bonferonni). These products were active in vitro and in subjects with NHL. Clinical responses occurred regardless of product characteristic differences such as CD4/CD8 T cell ratio and juvenile / differentiated T cells. Additional analyses are ongoing, including evaluation of PD-1 expression,% of myeloid cells and gene expression profile. Conclusions: CAR T cells were successfully prepared from all subjects enrolled in the study notwithstanding the diverse nature of the subject starting lymphocyte population. The CAR T cells showed a less differentiated profile, compared to the starting lymphocytes. CAR T cell production by essentially the same process described here is currently being utilized in company-sponsored multicenter trials (NCT02348216, NCT02601313). Citation Format: Timothy Langer, Emily Lowe, Arianne Perez, Steven Rosenberg, Steven A. Feldman, Lily Lu, John M. Rossi, Edmund Chang, Marika Sherman, Marc Better, James N. Kochenderfer, Adrian Bot. Comparative evaluation of peripheral blood T cells and resultant engineered anti-CD19 CAR T cell products from relapsed/refractory non-Hodgkin9s lymphoma (NHL) patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2305.

Clinical Response in ZUMA-1, the Pivotal Study of Axicabtagene Ciloleucel (Axi-Cel) in Patients with Refractory Large B Cell Lymphoma, May Be Influenced by Characteristics of the Pretreatment Tumor Microenvironment (TME)

untreated high-grade diffuse large B-cell lymphoma. Patients: 40 untreated DLBCL patients from 4 centers were enrolled in a prospective study between August 2013 Apr 2018; stage II-IV; ECOG 0-3; median age 60 years (27-78); age 60y/<60y 57%/ 43%; age 55y/<55y 50%/50%; M/F 50%/50%; IPI: 67% highintermediate and 33% high risk; 22% with bone marrow involvement. All patients underwent 4-8 courses (2-4 cycles) of chemotherapy: RDA-EPOCH, R-HMA. For patients older than 60 years (or 55y with comorbidities) dose of R-HMA was reduced. In 4 cases of DLBCL with bone marrow involvement LEAM conditioning and autologous stem cell transplantation were applied. Results: The median follow-up is 41,5 months (6,7-52,4). There was no mortality associated with toxicity. The main non-hematological toxicities of R-HMA were infections (mucositis, pneumonia, sepsis, enteropathy) grades 1-2 and 3-4 in 90% and 10%, respectively. Hematological toxicity grade 4 for less than 4 days we observed only after courses R-HMA. Complete remission (CR) was achieved in 36 (90%) patients. With a median follow-up of 41 months, overall and disease-free survival of 40 patients constituted 76,3% and 69,7%, respectively. Age older than 55y, IPI, LDH and bulky disease were factors of poor prognosis for OS and DFS by Long Rank test. In a group of patients older than 55 years results of therapy seemed to be worse than in young patients: OS were 70,5% vs 78,6% (p1⁄40,005), DFS were 63,6% vs 80% (p1⁄40,013), respectively. But mortality in older patients didn’t associate with toxicity or disease relapse, unlike in young patients. Conclusions: The R-DA-EPOCH/R-HMA protocol demonstrated acceptable toxicity and high efficacy in patients with high-grade DLBCL. This protocol has shown optimistic results in the elderly patients.

745. Updated Phase 1 Results from ZUMA-1: A Phase 1-2 Multi-Center Study Evaluating the Safety and Efficacy of KTE-C19 (Anti-CD19 CAR T Cells) in Subjects with Refractory Aggressive Non-Hodgkin Lymphoma (NHL)

Introduction: Anti-CD19 CAR T cells with CD3-zeta and CD28 signaling domains showed durable remissions in subjects with relapsed/refractory advanced B cell malignancies at the NCI (Kochenderfer et al. Blood 2012, JCO 2014, ASH 2014). KTE-C19 utilizes the same anti-CD19 CAR construct as investigated in the NCI study in a 6-8 day manufacturing process (Better et al. ASCO 2014). ZUMA-1 is a phase 1-2 multicenter, open-label study evaluating the safety and efficacy of KTE-C19 in subjects with refractory aggressive B-cell NHL. Methods: Subjects received KTE-C19 at a target dose of 2 × 106 (minimum 1 × 106) anti-CD19 CAR T cells/kg after a fixed dose conditioning chemotherapy regimen of cyclophosphamide 500 mg/m2/day and fludarabine 30 mg/m2/day for 3 days. The primary objective of phase 1 was to evaluate the safety of KTE-C19 as determined by the incidence of dose-limiting toxicities (DLT). Key secondary objectives were overall response rate, duration of response, levels of CAR T cells in the blood, and levels of serum cytokines. Key inclusion criteria were ≥ 18 years old, ECOG 0-1, and chemotherapy-refractory disease defined as progressive disease or stable disease as best response to last line of therapy, or disease progression ≤ 12 months after autologous stem cell transplant (ASCT). Results: As of 20 Nov 2015, 7 subjects were dosed in the phase 1 portion of the study. All subjects were evaluable for safety and 6 were evaluable for efficacy with a median follow up time of 13 weeks post KTE-C19 infusion. One subject experienced a DLT of grade (gr) 4 encephalopathy and gr 4 cytokine release syndrome (CRS) and died due to an intracranial hemorrhage deemed unrelated to KTE-C19 per the investigator. Key safety and efficacy findings are summarized in the table. CRS and neurotoxicity were managed with supportive care, tocilizumab and systemic steroids. 5 of 7 subjects (71%) had an objective response including 4 complete remissions (57%). Three subjects have ongoing complete remission at 3 months. CAR T cells peaked within two weeks post infusion were detectable 1-3+ months post infusion. Updated clinical and biomarker results will be presented. Conclusions: The KTE-C19 regimen evaluated was safe for further study. The predominant toxicities include CRS and neurotoxicity which are generally reversible. Complete and partial responses have been observed in subjects with refractory disease. KTE-C19 can be centrally manufactured and administered in a multicenter trial. The potentially pivotal phase 2 portion of the study is ongoing. Clinical trial: NCT02348216.SubjectSex/Age/ECOGDisease TypeTreatment HistoryGr 3 or Higher KTE-C19-Related Adverse EventsBest Response1M/59/0DLBCLRelapse ≤ 12 mo after ASCTGr 3 encephalopathy (resolved)Partial Response2M/69/1DLBCLRefractory to 2nd line or later line chemotherapyGr 3 tremor (resolved) Gr 3 delirium (resolved) Gr 3 agitation (resolved) Gr 3 restlessness (resolved) Gr 3 somnolence (resolved)Complete Remission**3M/69/0DLBCLRefractory to 2nd line or later line chemotherapyGr 3 encephalopathy (resolved)Stable Disease4M/67/1DLBCLRelapse ≤ 12 mo after ASCTNoneComplete Remission Ongoing 3 mo+5F/34/0DLBCLRelapse ≤ 12 mo after ASCTGr 3 hypoxia (resolved)Complete Remission Ongoing 3 mo+6M/40/0DLBCLRelapse ≤ 12 mo after ASCTNoneComplete Remission Ongoing 3 mo+7F/29/1DLBCLRefractory to 2nd line or later line chemotherapyGr 4 CRS Gr 4 encephalopathyNE View Table in HTML mo – months, M – male, F – female, NE – not evaluable,*relapsed and retreated with an ongoing PR