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Background Prostate cancer is radiosensitive. Prostate‐specific membrane antigen (PSMA) is selectively overexpressed on advanced, castration‐resistant tumors. Lutetium‐177–labeled anti‐PSMA monoclonal antibody J591 (177Lu‐J591) targets prostate cancer with efficacy and dose‐response/toxicity data when delivered as a single dose. Dose fractionation may allow higher doses to be administered safely. Method Men with metastatic castration‐resistant prostate cancer refractory to or refusing standard treatment options with normal neutrophil and platelet counts were enrolled in initial phase 1b dose‐escalation cohorts followed by phase 2a cohorts treated at recommended phase 2 doses (RP2Ds) comprising 2 fractionated doses of 177Lu‐J591 2 weeks apart. 177Lu‐J591 imaging was performed after treatment, but no selection for PSMA expression was performed before enrollment. Phase 2 patients had circulating tumor cell (CTC) counts assessed before and after treatment. Results Forty‐nine men received fractionated doses of 177Lu‐J591 ranging from 20 to 45 mCi/m2 ×2 two weeks apart. The dose‐limiting toxicity in phase 1 was neutropenia. The RP2Ds were 40 mCi/m2 and 45 mCi/m2 ×2. At the highest RP2D (45 mCi/m2 ×2), 35.3% of patients had reversible grade 4 neutropenia, and 58.8% of patients had thrombocytopenia. This dose showed a greater decrease in prostate‐specific antigen (PSA) levels and longer survival (87.5% with any PSA decrease, 58.8% with >30% decrease, 29.4% with >50% decrease; median survival, 42.3 months [95% confidence interval, 19.9‐64.7]). Fourteen of 17 (82%) patients with detectable CTCs experienced a decrease in CTC count. Overall, 79.6% of patients had positive PSMA imaging; those with less intense PSMA imaging tended to have poorer responses. Conclusion Fractionated administration of 177Lu‐J591 allowed higher cumulative radiation dosing. The frequency and depth of PSA decrease, overall survival, and toxicity (dose‐limiting myelosuppression) increased with higher doses.

Inherent or acquired drug resistance is a major contributor to epithelial ovarian cancer (EOC) mortality. Novel drugs or drug combinations that produce EOC cell death or resensitize drug resistant cells to standard chemotherapy may improve patient treatment. After conducting drug tolerability studies for the multikinase inhibitors dorsomorphin (DM) and it is structural analogue LDN-193189 (LDN), these drugs were tested in a mouse intraperitoneal xenograft model of EOC. DM significantly increased survival, whereas LDN showed a trend toward increased survival. In vitro experiments using cisplatin (CP)-resistant EOC cell lines, A2780-cp or SKOV3, we determined that pretreatment or cotreatment with DM or LDN resensitized cells to the killing effect of CP or carboplatin (CB). DM was capable of blocking EOC cell cycle and migration, whereas LDN produced a less pronounced effect on cell cycle and no effect on migration. Subsequent analyses using primary human EOC cell samples or additional established EOC cells lines showed that DM or LDN induced a dose-dependent autophagic or cell death response, respectively. DM induced a characteristic morphological change with the appearance of numerous LC3B-containing acidic vacuoles and an increase in LC3BII levels. This was coincident with a decrease in cell growth and the altered cell cycle consistent with DM-induced cytostasis. By contrast, LDN produced a caspase 3-independent, reactive oxygen speciesdependent cell death. Overall, DM and LDN possess drug characteristics suitable for adjuvant agents used to treat chemotherapy-sensitive and -resistant EOC.

Acute myeloid leukemia carries a dismal prognosis in older patients. The objective of this study was to investigate the safety and efficacy of decitabine combined with the CXCR4 antagonist plerixafor in newly diagnosed older patients with acute myeloid leukemia and to evaluate the effects of plerixafor on leukemia stem cells. Patients were treated with monthly cycles of decitabine 20 mg/m2 days 1–10 and escalating doses of plerixafor (320–810 mcg/kg) days 1–5. Sixty-nine patients were treated, with an overall response rate of 43%. Adverse karyotype did not predict response (P=0.31). Prior hypomethylating agent treatment was the strongest independent predictor of adverse overall survival (hazard ratio 3.1; 95%CI: 1.3–7.3; P=0.008) and response (14% in previously treated patients, 46% in treatment naïve; P=0.002). As expected, the most common toxicities were myelosuppression and infection. Plerixafor induced mobilization of leukemia stem and progenitor cells, but did not cause clinically significant hyperleukocytosis. Reduction in leukemia stem cells appeared to correlate with duration of response. Plerixafor can be safely added to decitabine in poor-prognosis, elderly acute myeloid leukemia patients. The maximum tolerated dose of the combination was 810 mcg/kg. While mobilization of leukemia stem cells was observed in some patients, the clinical benefit of adding plerixafor was uncertain. This trial was registered at clinicaltrials.gov identifier: 01352650.

To investigate mechanisms of apical sorting in the secretory pathway of epithelial cells, we expressed varying amounts of the 165 amino acid isoform of vascular endothelial growth factor (VEGF 165) and transforming growth factor ␤1 (TGF-␤1) via replication defective adenoviruses. Apical sorting of both proteins was efficient at low expression levels but saturated or was reversed at high expression levels. High expression levels of TGF-␤1 were effective at competing VEGF 165 out of the apical pathway; however, VEGF165 did not compete out TGF-␤1. Tunicamycin inhibition experiments showed that the apical polarity of VEGF165 was independent of N-glycosylation. We conclude that the apical sorting of these two molecules is a saturable, signal-mediated process, involving competition for apical sorting receptors. The sorting of the two proteins does not appear to involve N-glycans as sorting signals, or lectin sorters. The observations are particularly relevant to gene therapy because they demonstrate that overexpression of a transgene can result in undesirable missorting of the encoded protein.

Acute myeloid leukemia (AML) is a disease with a high incidence of relapse and mortality. Relapse is attributed to the inability of current chemotherapeutic agents to eliminate leukemia stem cells (LSCs). Thus, to improve leukemia therapy, it is critical to identify agents that effectively target LSCs, e.g. via unique cell surface antigens. A target of major interest is CD123, the transmembrane alpha chain of the interleukin-3 receptor, expressed on blasts, leukemic progenitor and LSCs in the majority of AML patients. We have developed an allogeneic chimeric antigen receptor (CAR) T-cell platform using T-cells from third-party healthy donors to generate engineered T-cells targeting CD123 (UCART123). UCART123 cells no longer express a TCR, having undergone a disruption of the TCRα gene using TALEN¨ gene editing technology followed by elimination of TCRα/β-positive cells, thus minimizing the potential for engineered T-cells to cause graft versus host disease (GvHD). We tested the activity of UCART123 cells in vitro using primary AML samples, normal bone marrow (nBM) and cord blood (CB) cells. Additionally, we established patient derived xenograft (PDX), nBM- or CB- humanized xenografts (HuX) and a competitive nBM/AML xenograft model to evaluate the in vivo potential of UCART123 cells to preferentially eliminate AML over normal BM cells. In vitro studies reveled that UCART123 cells eliminate AML cells and had minimum effect on normal cells at effector:target ratios as low as 0.5:1. Next, we evaluated the in vivo activity of UCART123 against PDX (AML37, TP53 mutant relapsed AML and AML20, FLT3-ITD+ and TP53 mutant AML) and normal-HuX mice (n=3). At 3 weeks post T-cell injection we found that UCART123 treatment eliminated the leukemic cells when using 10M or 3M UCART123 cells per mouse and no significant difference between PBS or TCR-deficient T-cells (TCRkoT; 10M/mouse). Toxicity to normal cells was dose dependent, doses of 2.5M UCART123 cells did not significantly affect hematopoietic cells. T-cells were detected in the BM at day 14 after treatment, without evidence of GvHD. Since we found complete elimination of human AML cells in the BM of the PDX precluding serial transplantation to evaluate LSC activity, we initiated two new sets of PDX-AML mice [AML2 (NPM1+FLT3-ITD+) and AML37 (TP53 mutant)] to evaluate long-term survival, and time to relapse. Animals were treated with PBS, UCART123 (2.5M or 1M), TCRkoT (2.5M), or Ara-C (60mg/kg 5 days). Animal weight and peripheral blood (PB) was monitored. Cytokines changes were evaluated at day 2. We found that the cytokine release and the kinetics of AML targeting by UCART123 were dose dependent. We found a significant overall survival (OS) benefit with UCART123 in both PDX tested. For example, all PDX-AML2 mice treated with UCART123 are alive to date (day 167; updates will be presented). All other cohorts were lost (PBS day124, TCRkoT day126, AraC day144) (Figure 1A). Finally, to determine selectivity of UCART123 cells for AML cells over nBM cells, we generated a competitive model bearing both nBM and AML (NPM1+FLT3-ITD+). With PB monitoring, treatment with 1M UCAR123 cells resulted in selective elimination of AML cells. Untreated and TCRkoT treated mice showed a rapid progression of AML, while treated mice showed normal hematopoiesis (Figure 1B). NPM1 transcripts were also monitored in the mice and confirmed molecular remission in mice. Taken together, our data show that UCART123, an "off-the-shelf" allogeneic engineered CAR-T product targeting CD123 potently eliminates AML cells in vivo, prevents relapse, and improves OS in PDX mice. Also, UCART123 cells preferentially targets AML cells in a competitive BM/AML model. A phase I trial of UCART123 in AML is under development. Disclosures Guzman: Cellectis: Research Funding. Sugita:Cellectis: Research Funding. Galetto:Cellectis SA: Employment. Gouble:Cellectis: Employment. Smith:Cellectis SA: Employment. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding.

Background: CD123, the trans-membrane alpha chain of the interleukin-3 receptor (IL-3RA) is overexpressed in acute myeloid leukemia (AML) and distinguishes leukemia stem cells from their normal counterparts. There are several novel therapeutics under development to target CD123 in AML, including CD123 fused to Diphtheria toxin, a recombinant chimeric anti-CD123 MoAb, CD3/CD123 bi-specific T cell engagers, and engineered T cells that express chimeric antigen receptors (CARs). Thus, accurate detection and quantification of CD123 is critical for newly diagnosed and relapsed patients, and to follow minimal residual disease for patients in remission. Our data suggest that the evaluation of CD123 by flow cytometry varies significantly with different antibody clones. Objective: To identify the most accurate flow cytometry method for evaluation of CD123 expression in patients with AML to evaluate CD123 targeting therapies. Methods: 51 AML patient samples and 7 normal cord blood or bone marrow samples were stained with five different commercially available monoclonal antibodies to detect CD123 (7G3, 6H6, 9F5, AC145 and FAB301P), as well as CD45 and CD5, for evaluation by multiparameter flow cytometry. CD123 gene expression was also compared between these primary AML samples and bone marrow samples from healthy donors. Cell surface expression (by percentage and MFI) was evaluated relative to transcriptional expression and sensitivity to known therapeutics (cytarabine, parthenolide, and HSP90 inhibitors). Results: We observed CD123 surface expression patterns varied between the antibody clones tested. For the 9F5 and 6H6 clones, 93% and 82% of the samples, respectively, showed >60% CD123+ cells whereas for the 7G3, FAB 301P and AC145 clones, 71 to 76% of the samples showed >60% positivity. Also, surface expression of CD123 using 7G3, AC 145 and FAB 301P did not correlate with transcript levels for IL3RA assessed using qPCR, while surface expression of CD123 using 9F5 and 6H6 did correlate with transcript levels of IL3RA, using both mean fluorescence intensity (MFI) and percentage. For example, the correlation between CD123 surface expression as measured by percentage and IL3RA transcripts was most significant using the 9F5 and 6H6 clone (R2=0.1084, p=0.0183, R2=0.1588, p=0.0038 respectively) whereas the correlation for 7G3 (R2=0.0004, p=0.8945), FAB301P (R2=0.0027, p=0.7151) and AC145 (R2=0.0392, p=0.1638) were not significant. Surface expression of CD123 evaluated with 7G3 antibody did not correlate with overall sensitivity to in vitro treatment with cytarabine (R2=0.03767, p= 0.6451). However, using the 9F5 antibody, we found that higher levels of surface CD123 were associated with resistance to cytarabine in vitro (R2= 0.5502, p= 0.0351). Differences were noted for other experimental therapeutics including parthenolide and PU-H71. Most importantly, when we tested the ability of a novel allogeneic anti-CD123 CAR T-cell therapy (UCART123) to eliminate CD123+ AML cells, we found that CD123 positivity as measured by the 7G3 clone was not predictive of sensitivity to UCART123 in vitro or in vivo AML patient derived xenotransplants. Conclusions: Several novel therapeutic modalities targeting CD123 in AML are under development, including allogeneic anti-CD123 CAR T-cell therapy. Accurate, quantitative assessment of CD123 expression is thus of utmost importance for patient selection in clinical trials as well as disease monitoring. We found discrepancies between antibody clones, and such discrepancies may alter patient selection and data interpretation regarding patient response to CD123 based therapies. For therapies targeting CD123, protocol design and antibody selection should be done considering the results in this study. Based on our findings we recommend 9F5 or 6H6 antibody clones as well as the utilization of qPCR along side flow cytometry for adequate detection. Flow cytometry findings should be reported as percent positive cells. If utilizing the 9F5 clone, samples with > 60% CD123+ should be considered positive for CD123. A comparison in a large cohort may be warranted to determine the impact of multiple CD123 measurements on disease outcome. Disclosures Galetto: Cellectis SA: Employment. Gouble:Cellectis: Employment. Smith:Cellectis SA: Employment. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding. Guzman:Cellectis: Research Funding.

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