bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase I/II dose-escalation trial of amonafide for treatment of advanced solid tumors: Genotyping to optimize dose based on polymorphic metabolism
Authors: Kuhn J. G. , Burris H. A. , Jones S. F. , Hein D. W. , Willcutt N. T. , Greco F. A. , Thompson D. S. , Meluch A. A. , Schwartz R. S. , Brown D. M.
Published: March 2020

2503

Background: Amonafide (AMF), a synthetic imide derivative of naphthalic acid, is a topoisomerase II inhibitor and is subject to polymorphic metabolism based on acetylation genotype. AMF is extensively metabolized by N-acetyltransferase 2 (NAT2) to N- acetylamonafide (AAMF) which has activity nearly equipotent to AMF. In our phase I evaluation, we observed a correlation between NAT2 genotype, AMF/AAMF pharmacokinetics and toxicity (J Clin Oncol 22 [14S]: 2023, 2004). Patients (pts) with slow (S) acetylator genotype tolerated higher doses than those with rapid/intermediate (R/I) acetylator genotype. The present Phase II portion was designed to prospectively determine the dose of AMF based on NAT2 genotype and focused on selected tumor types. Methods: NAT2 genotyping of genomic DNA from blood samples was done prospectively to determine acetylator status of each pt. AMF was administered IV weekly x 3 q4wk, escalation range 320 to 400 mg/m2 for R/I and 400 to 500 mg/m2 for S acetylators. Primary endpoints: safety, MTD, tumor measurements or sustained decreases in tumor markers. Results: Total 47 pts (21 M/26 F), median 66 yr, PS 0–2, acetylator status R/I (26), S (21) with ovarian (11) or prostate (9), breast (8), colon (4) cancers (CA) and other common tumors refractory to therapy were treated; 30 of 47 pts were dosed based on prospective genotyping. Of these 30 pts, 17 were R/I acetylators: no toxicity at 320 mg/m2 AMF in 7/11 pts and manageable myelosuppresion in 4/11 pts observed days 15–21 of cycle but not dose-limiting (DLT); DLT in 3/6 pts at 400 mg/m2. In the 13 S acetylators: at 400 mg/m2 no DLT in 8 pts; DLT in 2/5 pts treated at 500 mg/m2. Other side effects included nausea/vomiting, fatigue and anemia. Of all 47 pts, biologic activity was seen in 6 pts: 3/9 prostate CA (decreased PSA), 2/11 ovarian CA (decreased CA125) and 1/1 GIST (decreased lymph nodes). Conclusions: MTD determined to be 320 mg/m2 in R/I and 400mg/m2 weekly x 3q 4 weeks in S acetylators, respectively, supporting hypothesis that AMF dosing based on prospective NAT2 genotyping may allow for dose optimization based on drug metabolism and result in better tolerance. Phase II assessments at the MTD dose levels are currently ongoing for prostate cancer.

No significant financial relationships to disclose.
Diurnal rhythms (DR) in gene expression in human oral mucosa: Implications for gender differences in toxicity, response and survival and optimal timing of targeted therapy (Rx)
Authors: Bjarnason G. A. , Seth A. , Wang Z. , Blanas N. , Straume M. , Martino T. A.
Published: March 2020

2507

Background: DR in processes relevant to oncology, including cell cycle, apoptosis, and angiogenesis, have been demonstrated (Nat Rev Cancer 3:350, Cancer Res 63:7277). In rodents, 10–20% of the genome has a 24-hour (h) rhythm in RNA expression (Cell 109:307). A molecular clock consisting of transcription/translation feedback loops of clock-genes controls this rhythmicity (Nat Rev Neurosci 4:649). We studied, for the first time, daily whole genome RNA expression patterning in healthy human volunteers. Methods: RNA samples extracted from oral mucosa biopsies (bx) obtained every 4h over 24h (6 bx) from 5 males (M) and 5 females (F) were subjected to microarray analysis (Affymetrix HG_U133_Plus2 chip, 54,679 transcripts). COSOPT, designed for circadian microarray time series analysis (Methods Enzymol 383: 149) was used to detect genes with significant rhythms (pMMC-Beta = 0.1). Expression patterns were visualized in GeneSpring GX7.3 (Agilent) and validated by real-time PCR and ANOVA. Results: There were 801 and 810 rhythmic genes in M and F respectively, with most genes peaking at 4AM or 4PM in M but at 6AM or 11AM in F. Only 90 rhythmic genes (including core clock-genes and clock controlled genes) were common to M and F, with over 700 genes only rhythmic in M and not in F and vice versa. The profiles of clock- genes and clock-controlled genes were inverted relative to the nocturnal rodent data. There were 75 and 67 rhythmic transcription factor genes in M and F respectively, with 28 common to both M and F. There were 71 rhythmic human cancer genes (Nat Rev Cancer 4:177) with significant gender differences. This group includes rhythmic gene products involved in signaling pathways currently targeted for cancer Rx. Conclusions: We show for the first time a significant gender specific DR in gene expression involving multiple genes of interest in oncology. This may contribute to the documented gender differences in toxicity, response and survival (J Clin Onc 24: 3562, NEJM 353:133), and can inform future trials of optimal timing of antisense Rx and other targeted Rx. The inverted DR in rodents vs. humans has implications for translating rodent data to human Rx trials.

No significant financial relationships to disclose.
Phase I study of LY573636-sodium, an acylsulfonamide anti-cancer compound with a novel mechanism of action, administered as 24-hour continuous infusion in patients with advanced solid tumors
Authors: Slapak C. A. , LoRusso P. M. , Mendelson D. , Sykes A. K. , De Alwis D. P. , Wagner M. M. , Ilaria R. L. , Gordon M. S.
Published: March 2020

2542

Background: LY573636 -sodium (hereafter referred to as LY573636 ) is a novel anti-cancer compound that induces apoptosis by a mitochondrial-mediated mechanism. Methods: A phase I study was conducted to determine the maximum tolerated dose and pharmacokinetic (PK) profile of LY573636 administered as a 24-hour continuous intravenous (IV) infusion every 28 days. Results: 26 patients (pts) were enrolled on study, 12 males and 14 females, with a median age of 57 (range: 41–74). LY573636 was dose-escalated using a flat dosing-based schema. No dose limiting toxicity (DLT) was observed until the 2,000 mg cohort, when 1 pt developed CTCAE grade 3 hyperbilirubinemia. PK analysis revealed that LY573636 had a low total plasma clearance (CL = 0.02L/hr) and a terminal elimination half-life of approximately 340 hours due to high-albumin binding (∼99%). One additional pt in the 2,000 mg flat dose cohort developed grade 4 thrombocytopenia in cycle 2, possibly due to the accumulation of albumin-bound drug. Based on these PK and clinical data, the dosing strategy was revised to a flat loading/maintenance dose regimen of 2,500 mg/1,750 mg every 28 days by 24-hour continuous IV infusion. Thirteen pts were treated at this dose level, with 1 pt experiencing a DLT of grade 4 leukopenia. Overall, 8/26 pts had stable disease (SD) after 2 or more cycles, with 2 pts still on study. Five pts received ≥ 4 cycles (SD ≥ 4 mos), including heavily pre-treated pts with ovarian cancer, soft tissue sarcoma, and uterine cancer. Conclusions: LY573636 is a novel anti-cancer compound with a unique mechanism of action. The most common DLT was BM suppression. Because of the long half-life of LY573636 , and the encouraging findings of a separate IV bolus phase 1 study, an IV bolus-based regimen will be taken forward into phase 2 study.

No significant financial relationships to disclose.
A phase I study of a novel taxane, TL310, orally administered every week in patients (pts) with advanced solid tumors
Authors: Glasspool R. M. , Boddy A. V. , Evans T. R. , Griffin M. J. , Anthoney A. , Barker A. , Longley R. E. , Twelves C. J. , Edwards C. J. , Wilcox H. E.
Published: March 2020

2544

Background: TL310 is a novel, oral taxane with potent pre-clinical activity in taxane-resistant model systems. This is the first study to evaluate the safety, tolerability and pharmacokinetics (PK) of TL310 in pts with advanced refractory solid tumors. Methods: Pts with adequate hematologic, renal, and liver function received TL310 administered orally on days 1, 8 and 15 of a 28-day cycle. Pts were continuously assessed for safety and dose-limiting toxicity (DLT) was based on first cycle toxicity only. A rapid dose escalation schedule was used (1 pt per dose level until ≥ grade 2 toxicity then 3–6 patients per cohort). The maximum tolerated dose (MTD) was defined as the dose level below that at which ≥ 2 of 3–6 pts experienced DLT. PK sampling was performed on days 1, 2, 3–4, 8, 15, 16, 17–18, 22 of cycle one. Serial imaging with CT was performed every 8 weeks to assess response. Results: 18 patients [M=11, F=7], ECOG PS ≤ 2 were treated with doses of 5 (1 pt), 10 (1pt), 20 (1pt), 40(2pts), 80 (5pts), 120 (3pts) and 160 mg/m2 (5pts). 1 pt had grade 2 vomiting at 80mg/m2. This, and subsequent, dose levels were expanded to include 3 pts. 3 pts (2 at 80 and 1 at 160 mg/m2) vomited immediately after drug administration during cycle 1 and were replaced. 1/5 patients experienced a DLT at 160 mg/m2 with G5 neutropenic sepsis, G4 neutropenia and G4 thrombocytopenia. No other grade 3/4 toxicities attributed to study drug have occurred. Of 18 patients assessable for response, 1 had a partial response (gastric cancer) at 80 mg/m2 and 6 had stable disease for > 2 cycles (esophageal 3, melanoma 1, ovary 1, cervix 1). The maximum concentration (Cmax) increased with dose up to 120 mg/m2, but appeared to plateau (86±6 ng/ml) in 4 of 5 patients at 160 mg/m2. TL310 has a half-life of up to 80 hours, but PK were very similar on successive courses of treatment. Conclusions: TL310 can be administered orally up to 160 mg/m2 on day 1, 8, 15 of a 28 day cycle and shows promising activity at doses below the MTD. The MTD has not yet been defined and dose escalation continues.

No significant financial relationships to disclose.
Phase II stopping rules employing response rates and early progression
Authors: Goffin J. R. , Tu D.
Published: March 2020

2553

Background: Drug development in oncology has become increasingly resource intensive. Anything that might accelerate drug development would benefit patients and drug developers. Traditionally, tumour response rates (RR) have been used to assess the efficacy of new agents in phase II trials. High rates of early progression of disease (EPD) may also indicate lack of drug efficacy, and this endpoint has the potential to shorten trials compared to assessing RR alone. This work seeks to create a set of rules that will allow phase II drug assessment employing both RR and EPD. Previous work on this subject suffered from insufficient power, as determined by the authors [Freidlin et al, J Clin Oncol, 20:599, 2002]. Methods: Using TreeAge Pro Healthcare software, we created a computer model that would accept specified trial parameters and determine through simulations stopping rules based on observed number of responses and EPDs to achieve the desired power and alpha error for a single-armed two-stage study with 15 patients in each stage. The null hypothesis (H(nul)) specified the response rate (rr(nul)) and early progressive disease rate (epd(nul)) that would render a drug uninteresting for further development, such that: H(nul): rr LE rr(nul) & epd GE epd(nul), where rr is that actual response rate observed and epd is the actual rate of early progression. Similarly, the alternate hypothesis (H(alt)) specified the response rate (rr(alt)) and early progressive disease rate (epd(alt)) that would render a drug interesting for further development, such that: H(alt): rr GE rr(alt) or epd LE epd(alt). (LE, less than, equal to; GE, greater than, equal to) Results: Conclusions: The simulation was able to establish stopping rules for observed number of responses and EPDs to achieve the desired error rates. Variations on the rules based on other trial sample types and design parameters will be detailed.

[Table: see text]

No significant financial relationships to disclose.
A physiologically-based pharmacokinetic (PBPK) and pharmacodynamic (PD) model of docetaxel (Doc) and neutropenia in humans
Authors: Florian J. A. , Egorin M. J. , Zamboni W. C. , Eiseman J. L. , Lagattuta T. F. , Belani C. P. , Chatta G. S. , Scher H. I. , Solit D. B. , Parker R. S.
Published: March 2020

2567

Background: We have scaled our previously described murine Doc PBPK model (Florian et. al, Proc. AACR, 2006) to humans to determine if it predicts human plasma Doc concentration. The resulting human plasma Doc predictions were then coupled to a low- order neutrophil model from the literature (Karlsson et. al, Clin. Cancer Res., 2006), and individual patient absolute neutrophil count (ANC) predictions were compared with actual ANC data. Methods: Plasma Doc concentration vs. time data were obtained from 75 patients given 1-h Doc infusions and sampled out to 48 h. 67 patients received 50–75 mg/m2 q3wk, and 8 patients received 35 mg/m2 weekly. Plasma Doc concentrations were determined by LC-MS, & ANC were measured weekly during cycle 1. Human organ volumes & blood flow rates for humans were obtained from the literature and scaled based on individual patient weight. Intra-tissue exchange rates & liver clearance rate from the mouse PBPK model were used unchanged as parameters in the scaled model. The PBPK model was used to drive the ANC model, which had been developed with data from patients treated on a q3wk schedule. Simulations were performed using MATLAB, and model predictions were compared with actual data based on a weighted sum squared error metric. Results: The initial, scaled PBPK model performed well in describing actual plasma Doc concentrations during the infusion & at times after 5 h, but not at the intermediate time points. Consequently, transition rates for the “other” compartment were estimated for the human population. The updated model was more accurate in predicting human plasma Doc concentrations (mean improvement 17%; range -3 to 70%). When the scaled PBPK model was coupled with the neutrophil model, the resulting predicted neutrophil profiles agreed with those from patients dosed on a q3w regimen, but not with those dosed weekly. Conclusions: This unique PBPK/PD model allows the prediction of human plasma Doc concentrations provided the patient weight & dose are known. Further validation of the coupled PBPK/PD model, including predicted tumor concentrations, is ongoing.

No significant financial relationships to disclose.
Effects of a potent CYP3A inhibitor on the pharmacokinetics and safety of enzastaurin
Authors: Musib L. , Darstein C. , Suico J. , Baldwin J. , Welch P.
Published: March 2020

2572

Background: Enzastaurin (ENZ) targets the PKCβ and PI3K/AKT pathways to induce tumor cell apoptosis, reduce proliferation, and suppress tumor-induced angiogenesis. ENZ is metabolized by CYP3A in vitro. This study examined the potential clinical effects of a potent CYP3A4 inhibitor, ketoconazole (KETO), on the pharmacokinetics and safety of ENZ. Methods: In this open-label, fixed-sequence, three-period, crossover study (duration = 6 weeks), healthy subjects received an oral, 200- mg, single dose of ENZ (period 1); 400-mg daily doses of KETO for 4 days to assess QT (period 2); and 400-mg daily doses of KETO for 14 days, with a 200-mg single dose of ENZ given on day 4 (period 3). Plasma samples for PK analysis were collected predose and after ENZ administration in periods 1 and 3 at scheduled intervals. Results: Of the 16 women enrolled, 13 completed the study. Changes in PK parameters of ENZ and its metabolite, LY326020, in the presence of KETO are summarized in the table . No serious adverse events (AEs) occurred. A similar number of AEs possibly related to enzastaurin occurred in period 1 (6) and period 3 (7). Headache (n=4) and nausea (n=3) were more frequent in period 3, but were also the most common AEs related to KETO. Three patients had hepatic transaminase elevations, but no consistent pattern with dosing period or ENZ exposures was observed. At 4 hours post-dose, QT intervals were prolonged by a mean 5.88 (95% CI: 1.767–10.00) msec after four daily doses of KETO and by a mean 9.29 (95% CI: 5.165–13.41) msec when coadministered with ENZ. Conclusion: In the presence of KETO, plasma concentrations of ENZ and its metabolites increased significantly. ENZ alone did not increase QT intervals. Ketoconazole plus ENZ caused a slightly greater increase in QT intervals compared to KETO alone, but this very small change may not be clinically significant. ENZ was generally well tolerated, alone or with KETO.

[Table: see text]

No significant financial relationships to disclose.
Effect of smoking on imatinib pharmacokinetics
Authors: van Erp N. P. , Gelderblom H. , van Glabbeke M. , van Oosterom A. , Verweij J. , Guchelaar H. , Peng B. , Judson I.
Published: March 2020

2573

Background: Smoking is a potent inducer of the cytochrome P450 1A2 isoenzyme (CYP1A2) and may therefore effect the pharmacokinetics (PK) of drugs metabolized by CYP1A2. Indeed, clinical studies with erlotinib (metabolized by CYP3A4 and also partly by CYP1A2) have shown a major increase in erlotinib clearance in smokers versus non-smokers. The effect of smoking on the PK of imatinib, which is also metabolized by CYP3A4 and partly by CYP1A2, is unknown. We aimed to study the effect of smoking on imatinib PK in order to explain a part of the interpatient variation. Methods: The effect of smoking on the PK parameters was analyzed in 34 patients with gastro-intestinal stromal tumors or soft tissue sarcoma included in the EORTC-STBSG phase I and phase II trials. This cohort included 9 smokers and 25 non-smokers. The daily smoking habits of the study patients were retrieved retrospectively from the patient’s record. PK parameters assessed with NONMEM, version V were clearance (Cl), distribution volume (V), elimination half life (T1/2) and the dose standardized area under the concentration curve (AUC). We considered a 40% reduction in imatinib exposure clinically relevant and this study is adequately powered to detect this difference. Results: The mean PK parameters in the smokers versus the non-smokers group ± SD were: Cl; 9.6 ± 5.5 L/h vs 9.2 ± 4.6 L/h, V; 216.5 ± 114.3 L vs 207.0 ± 116.9 L, T1/2; 16.1 ± 6.0 h vs 16.5 ± 6.0 h, AUC; 133.6 ± 71.0 ng.h/ml.mg vs 142.3 ± 84.0 ng.h/ml.mg. There was no significant difference in PK parameters observed between the smokers and the non- smokers. Conclusion: This retrospective study suggest that the PK of imatinib was not affected by smoking and therefore does not explain the large variation in imatinib PK.

No significant financial relationships to disclose.
A randomized phase II p53 vaccine trial comparing subcutaneous direct administration with intravenous peptide-pulsed dendritic cells in high risk ovarian cancer patients
Authors: Herrin V. E. , Achtar M. S. , Steinberg S. M. , Whiteside T. L. , Wieckowski E. , Czystowska M. , Visus C. , Berzofsky J. A. , Khleif S. N.
Published: March 2020

3011

Background: 50% of ovarian cancers carry a p53 mutation, most of which can lead to overexpression of the protein, providing a unique target for vaccine therapy. Preclinical data shows cytotoxic T-lymphocytes can be generated against wild-type peptide and can lyse tumor cells overexpressing p53. There are many methods of antigen delivery for cancer vaccines and very few comparative studies have been conducted to determine the best of these. Here we report findings for this completed study comparing two methods of vaccination. Methods: Eligible patients had Stage III, IV, or recurrent ovarian cancer and were clinically NED at study entry. Elevated CA-125 was allowed. HLA-A2.1 and overexpression of p53 were required. A wild-type epitope (264–272) with high HLA-A2.1 affinity was utilized for vaccination. Two techniques were randomly compared: subcutaneous (SQ) peptide admixed with ISA-51 and GM-CSF adjuvants or peptide-pulsed dendritic cells given intravenously (IV). Both arms received 6 million IU/m2 IL-2 SQ for ten days beginning with cycle 3. Elispot analysis was utilized to assess immunologic response, the primary endpoint. After reaching statistical significance, accrual to the more technically difficult IV arm was halted. Results: Of 21 patients enrolled, 20 have evaluable data points; 13 on the SQ arm and 7 IV. On the SQ arm, 9 of 13 (69%) patients had evidence for immunologic response. On the IV arm, 5 of 7 (71%) patients had an immunologic response. 42% on each arm experienced some grade III or IV toxicity (increased liver enzymes on the SQ arm and fatigue and lymphopenia on the IV arm), but only one patient disenrolled due to toxicity, a cardiac arrhythmia exacerbated by IL-2. Mean overall survival (to date) on the SQ arm is 70.4 months and on the IV arm is 72.9 months. Conclusions: The patients on this study were mostly heavily treated patients with short disease-free intervals, yet we demonstrated that immune responses can be generated against a wild-type protein epitope in a significant number of patients. Both vaccine approaches were similarly effective. This result should lead to a trial of early vaccination in high- risk patients to potentially delay or even prevent recurrence.

No significant financial relationships to disclose.
Comprehensive analysis of melanoma antigen specific T cell repertoire: EPIMAX (EPItope MAXimum)
Authors: Palucka A. , Banchereau J. , Vence L. , Connolly J. , Fay J. , Ueno H.
Published: March 2020

3016

Background: We have demonstrated in phase I clinical trial treating 20 patients with metastatic melanoma that dendritic cells (DCs) loaded with killed allogeneic tumor cells can elicit immune and clinical responses. Loading DC vaccines with killed allogeneic tumor cells allows: i) presentation of antigens via both MHC class I and class II, ii) strong help, iii) applicability to any cancer, and iv) loading tumor antigen independent of HLA haplotype of the patient. However, this renders the immunomonitoring step complex as the antigens and their restriction elements are unknown. To address this issue, we developed EPIMAX. Methods: EPIMAX measures simultaneously cell proliferation and secretion of multiple cytokines that distinguish Type 1, Type 2 cytokines and IL-10 secretion using Luminex. Briefly, 5x105 CFSE-labeled PBMCs are plated with peptide pools of overlapping peptide libraries from appropriate antigens. After 48hrs, supernatants are transferred for cytokine determination. After an additional 6 days of culture, cell proliferation is analyzed by flow cytometry after staining for surface markers CD4 and CD8. Results: We demonstrated that EPIMAX permits us to assess: i) Specificity and breadth of induced immune responses, ii) Type of induced immunity (Type I, Type II, IL-10), and iii) Function of specific T cells as measured by cytokine secretion and proliferation. To date 8 patients with stage IV melanoma were analyzed at baseline and after vaccination with DCs loaded with killed allogeneic melanoma cells. We assessed epitopes derived from MART-1, NY-ESO1, TRP-1 and gp100. Analysis of CD8+T cells: We have identified 15 melanoma antigen CD8+T cell epitopes in 8 patients. These include: three Tc0 epitopes (IL2), and twelve Tc1 epitopes triggering IP-10 secretion. Analysis of CD4+T cells: We have identified 15 melanoma antigen CD4+T cell epitopes in 5 patients. These include: nine Th0 epitopes (IL2), four Th1 epitopes (IFNγ) and two Th2 epitopes (IL13). Finally, we analyzed secretion of IL-10 and found thus far IL-10 secreting CD4+T cells in seven patients. Further studies demonstrated that these T cells have regulatory function. Conclusions: EPIMAX permits comprehensive assessment of melanoma antigen specific T cell repertoire.

No significant financial relationships to disclose.
XmAb Fc engineered anti-CD19 monoclonal antibodies with enhanced in vitro efficacy against multiple lymphoma cell lines
Authors: Zhukovsky E. , Chu S. , Bernett M. , Karki S. , Dang W. , Hammond P. , Edler C. , Polder N. , Chan C. , Jacinto J. , Desjarlais J.
Published: March 2020

3021

Background: CD19 is a pan-B cell surface receptor that is expressed from early stages of pre-B cell development through terminal differentiation into plasma cells. It is an attractive immunotherapy target for cancers of lymphoid origin since it is also expressed on the vast majority of Non-Hodgkin Lymphoma (NHL) cells as well as some leukemias. Despite major improvements in response rates and progression free survival the majority of NHL patients will relapse under the current combination chemotherapy with anti-CD20. Thus salvage regimens with new non-cross resistant antibody therapies are warranted. Methods: We employ our XmAb antibody engineering technology to increase the affinity of IgG antibodies for Fc gamma receptors (FcγR), improve the effector function of antibodies, and significantly increases their antitumor potency; we also we humanize and affinity mature such antibodies. Results: The XmAb technology was applied to a humanized anti-CD19 antibody to engineer a variant with significantly enhanced (10- to 100-fold) antibody-dependent cell-mediated cytotoxicity (ADCC). The resulting XmAb CD19 variant was assayed for ADCC against multiple cell lines representative of follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), mantle cell lymphoma (MCL), hairy cell leukemia (HCL), chronic myelogenous leukemia (CML), and Burkitt’s lymphoma (BL). The ADCC activity of the XmAb CD19 was in striking contrast to a wild type IgG1 version of the antibody that mediates little ADCC. Moreover, ADCC potency and efficacy of the anti-CD19 Fc variant antibody were superior to that of rituximab: CLL - 10- and 1.5-fold higher, ALL - 10- and 100-fold higher, and HCL - 6- and 1.2-fold higher, respectively. Further, we observed no correlation between ADCC and antigen expression based on the measured cell surface density of CD19 for these cell lines. Conclusions: The increased affinity for FcγRs exhibited by the anti-CD19 Fc variant antibody overcomes much of the dependence of cytotoxicity on surface antigen density. Our data suggest that the anti-CD19 Fc variant antibody engineered for increased effector function could be a promising next-generation NHL immunotherapeutic.

No significant financial relationships to disclose.
Lenalidomide and pomalidomide strongly enhance tumor cell killing in vitro during antibody-dependent cellular cytotoxicity (ADCC) mediated by trastuzumab, cetuximab and rituximab
Authors: Bartlett J. B. , Wu L. , Adams M. , Schafer P. , Muller G. , Stirling D.
Published: March 2020

3023

Background: Potential mechanisms of action of lenalidomide and pomalidomide (CC-4047) include anti-angiogenic, anti- proliferative and immunomodulatory activities, e.g., enhancement of T cell and NK cell function. Both drugs appear to enhance T cell activation and Th1-type cytokines in cancer patients. Also, both drugs have been shown to enhance rituximab-mediated protection in a mouse lymphoma model. Methods: We have utilized an in vitro ADCC system to assess the ability of these drugs to enhance human NK cell function in response to the approved therapeutic antibodies trastuzumab, cetuximab and rituximab. Results: Pre-treatment of NK cells with either pomalidomide or lenalidomide greatly enhanced IFN-γ production by NK cells in response to IgG in the presence of either IL-2 or IL- 12. In a series of functional ADCC assays, Her2/neu overexpressing breast cancer cells (SKBR3 & MCF-7) pre-coated with trastuzumab, EGFR positive colorectal cancer cells (HCT-116) pre-coated with cetuximab and NHL cell lines (Namalwa, Farage & Raji) pre-coated with rituximab, were exposed to NK cells pre-treated with pomalidomide, lenalidomide or thalidomide. Both pomalidomide and lenalidomide synergistically increased (up to 6-fold) NK cell-mediated killing of antibody coated tumor cells in a dose-dependent manner. Thalidomide had no effect in this system. There was minimal tumor cell killing by antibody alone or in the absence of antibody. The presence of IL-2 or IL-12 was required to see enhanced killing by either drug. Enhanced ADCC was associated with increased signaling in NK cells, specifically with inhibition of the negative regulator SHIP-1. Other downstream effects on signaling pathways are also being investigated. Monocyte-mediated tumor cell ADCC was also enhanced by both drugs. Conclusions: We have shown that pomalidomide and lenalidomide (but not thalidomide) strongly enhance the ability of therapeutic antibodies to induce ADCC via NK cell/monocyte-mediated killing of tumor cells in vitro. These results provide a strong rationale for combination of either lenalidomide or pomalidomide with antibodies to tumor-specific surface antigens in cancer patients.

No significant financial relationships to disclose.
A phase II trial of trastuzumab and low dose interleukin-2 in patients with metastatic breast cancer who have previously failed trastuzumab
Authors: Mani A. , Roda J. , Caligiuri M. , Fleming G. , Kaufman P. , Brufsky A. , Carson W. , Shapiro C.
Published: March 2020

3028

Background: Trastuzumab (TZB) mediates lysis of Her2-pos. breast cancer cells by interleukin-2 (IL2) primed natural killer (NK) cells. We hypothesized that IL2 would augment the effects of TZB. The objective of this study was to determine the response rate to and toxicity of low dose IL2 plus TZB in patients with Her2-pos. metastatic breast cancer who had progressed within 12 months of receiving TZB. Also, we measured the ability of patient (pt) peripheral blood mononuclear cells (PBMC) to produce cytokines and conduct antibody-directed cellular cytotoxicity (ADCC) against Her2-pos. target cells. Methods: Pts received low (1 million IU/m2) and intermediate dose (12 million IU/m2) IL2 plus TZB (4 mg/kg) in each cycle. Low dose IL2 was given on days 2–7 and days 12–21 of cycle 1, and days 4–14 of later cycles. Intermediate dose IL2 was given on days 9–11 of cycle 1, and on days 1–3 of later cycles. TZB was given on day 1 and 8 of cycle 1, and on day 1 of later cycles. Pt plasma and PBMCs were analyzed for levels of serum cytokines and ADCC against a TZB-coated cell line, respectively. Results: Thirteen pts with median age of 52 (range 30–71), and a median of 1 (range 1–2) prior TZB-containing regimens were enrolled. The median number of cycles completed was 4. Five pts had grade 3 or greater toxicities, including fever, nausea, vomiting, diarrhea, dyspnea, and hypercalcemia. Twelve pts had progression of disease, and 1 pt withdrew consent. ADCC of pt PBMCs against a TZB-coated cell line was enhanced in only 1 pt. Two pts had elevated plasma levels of interferon-gamma (IFNg), and one of these pts had a 20-fold increase in IFNg transcript levels. The antiangiogenic chemokines MIG and IP-10 rose significantly over baseline in 11 pts. Conclusions: TZB given with low and intermediate pulse-dose IL2 did not produce a robust immune or clinical response in this pt population. A clinical trial in a TZB-naive population may help to determine the immune effects of this combination of IL2 plus TZB.

No significant financial relationships to disclose.
Correlation between NK function and response to trastuzumab in metastatic breast cancer
Authors: Beano A. , Signorino E. , Polimeni M. A. , Mistrangelo M. , Ardine M. , Spadi R. , Donadio M. , Ciuffreda L. , Matera L.
Published: March 2020

3036

Background: Trastuzumab is a monoclonal antibody selectively directed against Her2 approved for the treatment of Her2 overexpressing breast cancer patients. Its proposed mechanisms of action include also a role in mediating antibody-dependent cellular cytotoxicity (ADCC), through the triggering of the FcγRIII on natural killer (NK) cells. This study addressed the correlation between overall NK function and clinical trastuzumab activity. Methods: Between March and September 2006 22 metastatic patients in treatment with trastuzumab alone (8 mg/kg load and then 6 mg/kg every 3 weeks until disease progression) as maintaining therapy after chemotherapy were analyzed for clinical and immunological responses. According to RECIST criteria, 14 patients obtained a response to trastuzumab, while 8 patients had a disease progression. Patient’s peripheral blood mononuclear cells were tested for cytotoxic activity against standard NK target (the MHC class I-negative K562 cell line) and trastuzumab-coated MCF7 (Her2-negative) and SKBR3 (Her2-positive) human cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence of grading concentrations of effector cells. Results: NK activity was significantly (p<0.05) higher in responder compared to non responder patients at all the four effector:target (50:1 to 6:1) ratios tested. NK activity of non responder patients was significantly lower than that of 25 sex and age matched controls (p<0.02) and this was not merely due to chemotherapy- or tumor-associated immunosuppression, since the values of responder patients did not significantly differ from those of controls. ADCC activity against Her2-positive SKBR3 cells was also significantly higher in responder compared to non responder patients (p<0.05) and markedly so when compared to controls (p<0.001). Conclusions: The fact that, as shown here, normal levels of FcγR- independent and higher than normal levels of FcγR-dependent cytotoxicity are required for trastuzumab response, lends support to a paramount role of NK cells in the mechanism of action of this drug.

No significant financial relationships to disclose.
Antitumor effects of lenalidomide in combination with IDEC114 (anti CD80) in lymphoma bearing severe combined immunodeficiency (SCID) mouse model
Authors: Reddy N. M. , Hernandez-Ilizalituri F. , Knight J. , Czuczman M. S.
Published: March 2020

3037

Background: Lenalidomide is a thalidomide analogue with immunomodulatory effects. We previously demonstrated that lenalidomide enhances the biological activity of rituximab. In our current work we further studied the effects of combining lenalidomide with IDEC114, a primatized anti-CD80 monoclonal antibody, which is undergoing clinical testing in B-cell lymphoma. Methods: Raji cells were exposed in vitro to lenalidomide (10μg/ml) or DMSO over five days. Changes in DNA synthesis were determined by [3H]-thymidine uptake. For ADCC and CMC assays, lenalidomide or control exposed Raji cells were labeled to 51Cr and then exposed to IDEC114 or isotype control and PBMC’s or human serum. For in vivo studies, 6–8 week old SCID mice were inoculated with 1×106 Raji cells via tail vein injection and after a period of 72 hours animals were divided into four cohorts. Lenalidomide was administered intraperitoneally (i.p) at 0.5mg/kg/dose on days +3, +4, +8, +9, +13, +14, +18 and +19. IDEC114 was administered via tail vein injection at 10mg/kg/dose on days +5, +10, +15 and +20. Difference in survival between treatment groups was performed by Kaplan-Meier analysis. Results: In vitro exposure of Raji to lenalidomide for five consecutive days enhanced the anti-proliferative effects of IDEC114 when compared to control. In addition, an improvement in IDEC114-associated ADCC was observed in lenalidomide-exposed Raji cells. In vivo treatment of SCID mice with lenalidomide in combination with IDEC114 led to prolongation of survival (44 days) compared to either biological agent alone (p<0.01). Conclusions: Our current research demonstrates that Lenalidomide when added to IDEC114 has augmented in vitro antitumor activity (i.e, antiproliferation and ADCC) and synergistic effects in vivo (i.e., prolongation of survival). We hypothesize and currently are evaluating whether the improvement in in vivo antitumor activity of IDEC114, when combined with Lenalidomide, is secondary to potential changes in the tumor microenvironment and/or IMiD-primed upregulation of NK cells and ADCC. This promising unique combination of biologics warrants evaluation as a clinical trial. (Supported by USPHS grant PO1-CA103985 from the National Cancer Institute.)

No significant financial relationships to disclose.
In vitro expansion of CMV-specific CD4+ T-cells by artificial antigen presentation for adoptive immunotherapy in the post- transplant setting
Authors: Ricafort R. J. , Stephan M. , O’Reilly R. , Sadelain M.
Published: March 2020

3049

Background: CMV continues to be a major cause of morbidity and mortality in marrow allograft recipients. The importance of CD4+ T-cells in maintaining immunity and therapeutic efficacy in adoptive cellular strategies has been appreciated. With a novel artificial antigen presenting cell (AAPC) system, we induced the expansion of CMV-specific CD4+ T-cells. Methods: AAPCs were generated by using a standardizable line of murine 3T3 cells sequentially transduced to express human ICAM-1, LFA-1, B7.1, HLA A*0201 and DRB1*1101 alleles. Antigen was provided either by peptide pulsing with the HLA-DRB1*1101-restricted, CMV- pp65-derived P92 peptide, or the influenza matrix peptide (IMP) as a control, or by transgene expression of pp65. PBMCs were isolated from a CMV+, HLA*A02, DRB1*11-positive healthy donor after informed consent, and enriched for T-cells via positive selection using sheep RBCs. Cocultures of T-cells with AAPCs were established at a ratio of 10:1. 20 IU/ml of IL-2 was added on d7 and every third day thereafter. On d12- 14 of co-culture, the T-cells were restimulated on a fresh monolayer of AAPCs with antigen. Cells were counted in triplicate via hemocytometer with trypan blue exclusion. Immunophenotyping of expanded T-cells was done via flow cytometric analysis for cell surface expression of CD3, CD8, CD4, and soluble Class II (HLA-DRB1*1101) tetramer specific for the P92 peptide of CMV-pp65. Results: Following 3 rounds of stimulation, antigen-specific fold expansion, defined by DR11,P92-tetramer-positive, CD4+ T-cells, was 313-fold when stimulated on AAPC.A2.DR11.PP65, compared to 5–35-fold using the other AAPC controls. Conclusion: We are able to expand clinically relevant numbers of CMV-specific CD4+ T-cells using a rapid, practicable, and broadly applicable approach. These preclinical studies show the feasibility of generating virus-specific T-cells of desired specificity and HLA restriction for adoptive immunotherapy of severe CMV infections.

[Table: see text]

No significant financial relationships to disclose.
Accumulation of lymphocytes following adoptive transfer of allogeneic T lymphocytes equipped with an HLA-A2 restricted, MART-1 reactive T Cell Receptor
Authors: Duval L. , Schmidt H. , Steiniche T. , Kaltoft K. , Fode K. , Jensen J. J. , Soerensen S. M. , Nishimura M. I. , von der Maase H.
Published: March 2020

3050

Background: Adoptive transfer of cytotoxic, antigen specific lymphocytes represents an immunotherapeutic approach currently investigated for the ability to reinforce immunologic reactivity against tumor cells in cancer patients. The present study aimed to elucidate immunological reactions during a phase I immunotherapeutic trial with receptor modified T lymphocytes in stage IV melanoma. The aim was in particular to assess if treatment induced a local immunologic response in terms of accumulation of immune cells in metastases used for injection of cells, and if such a response propagated to metastases not used for injection of cells. Treatment consisted of intra-tumoral injections of allogeneic, cytotoxic T lymphocytes equipped with a MART-1 antigen recognizing T cell receptor. Expression of the T cell receptor was obtained through transduction with the receptor-encoding gene. Methods: Core needle were sampled both from metastases used for injection of cells, and from metastases not used for injection of cells. Biopsies were sampled at day 1, before any administration of cells, and at day 14 following the first 4 injections administered at day 1, 4, 7 and 10. Hereafter, biopsies were formalin fixed, paraffin embedded and immunohistochemically stained by a two layer detection technique with antibodies against; CD4, CD8 and CD57, before enumeration by a stereological counting technique.Wilcoxon matched pairs signed rank-sum test was used for comparison. Results: A total of forty six biopsies were sampled from twelve patients without complications. The number of CD8 positive T lymphocytes and NK cells (CD57+) was shown to increase significantly from day 1 to 14 in metastases used for injection. Analyses of the number of cells in metastases which were not directly used for injection of cells showed differences in some patients but overall, changes were not significant. Conclusions: The current study showed a statistically significant accumulation of immune cells in metastases following intra-tumoral injections of allogeneic, antigen specific, cytotoxic T lymphocytes in stage IV melanoma patients. The number of immune cells in metastases not used for injection did not change significantly during treatment.

No significant financial relationships to disclose.
Combination of gemcitabine and adoptive cell therapy of autologous anti-tumor CTL induces clinical activities in patients with refractory lung cancer
Authors: Toh U. , Fujii T. , Takamori S. , Fukunaga M. , Ogo E. , Seki N. , Yamana H. , Shirouzu K.
Published: March 2020

3053

Background: Cytotoxic T cells selectively kill autologous tumor cells is powerful for adoptive cell therapy of cancer. Gemcitabine (GEM) is able to induce molecular changes in cancer cells that make them to induce an antigen specific CTL response. This study was to evaluate the anti-tumor and the immunological activity using the CTL in combination with GEM. Methods: The 50Gy irradiated autologous tumor cells were cocultured with PBMCs and CTLs was developed with RPMI 1640 and rIL-2 (50 u/ml) for 7–14 days. Nine pts with non small cell lung cancer failed their prior chemotherapy were enrolled this pilot study. GEM regimen [intravenous (iv.) GEM (1000mg/m2) - day 1, 8 and 15 ] was started without CTL administration for 1st cycle. GEM was administered at least 2 cycles with a 1-week interval and combined with iv. CTL therapy (0.9 x 108 - 4.6 x 108 cells/injection + IL-2 0.4 MIU; biweekly for 6 to 12 injections) from 2nd cycle. The mean total administered T cells were reached to 3.9 x 109 - 5.6 x 109. PBMCs were analyzed their surface markers by Flow Cytometry and the cytokine productions of IFN-γ etc. in the serum were measured by ELISA before and after 1st cycle of GEM administration and 3rd cycles of CTL injection. Results: After finishing 2 cycles of GEM and 3 injections of CTL, the ratio of CD4/CD8 in PBMCs increased in 7/9 pts. In contrast, CD3/CD19 decreased in 6/9 pts. The cytokine production of IFN-γ in the serum revealed an increase after treatment, the levels of TGF-β were decreased simultaneously. There was no remarkable change in the levels of NKG2D in the PBMCs and MIG, IP10 in the serum. The clinical response showed PR/SD/PD was 2/5/2. The tumor marker proteins (CEA) were also decreased significantly in 4 of 9 pts. The adverse effects were tolerable with grade <2 fever, nausea and fatigue and no bone marrow suppression was observed. Conclusions: These results suggested the synergistic enhancement of antitumor effect might be induced between CTLs and anti-cancer agent GEM. Marked clinical responses were observed in two pts after the treatment. Thus this chemo-immunotherapy will be applicable for the patients with refractory lung cancer.

No significant financial relationships to disclose.
Efficacy of gabapentin on pruritus induced by interleukin-2 (IL-2) treatment in patients with metastatic renal cell carcinoma and melanoma
Authors: Lee S. , Rusciano V. , Dutcher J. P.
Published: March 2020

3056

Background: IL-2 treatment causes pruritus, but the pathogenic mechanism is not well understood. Its relation to histamine is controversial, and it does not seem to correlate with the eosinophilia induced by IL-2. Direct stimulation of pain nerve fibers, such as A-delta and C fibers by the cytokines induced by IL-2 treatment has been proposed as a possible cause of pruritus. Stabilizing the nerve membrane or nerve synapses by pharmacologic means may improve pruritus, and we have used gabapentin for this purpose. Here we evaluate its efficacy. Methods: 54 patients, 34 Male, 20 female, were treated with IL-2 from January, 2005 to December, 2006. Among 30 patients who complained of pruritus, 17 patients were given gabapentin. Concurrent follow up notes of these 17 patients were reviewed. They were interviewed immediately following IL-2 treatment and retrospectively using a specific IRB approved questionaire, which quantified pruritus according to CTCAE 3.0 criteria, before and after the administration of gabapentin. The CTCAE grading scale is 0 to 3, from none, localized, widespread, to widespread pruritus interfering with daily activities. Data was analyzed with paired student T-test using SPSS 13.0 for data analysis. Results: 15 patients had renal cell carcinoma and 2 had melanoma. Mean age was 56 years. 12 patients were male and 5 were female. Gabapentin was started in hospital in 9 patients and outpatient in 8. Mean initial dose of gabapentin was 865 mg/day, and the mean time for gabapentin to provide symptom relief was 1.088 hours after each dose. According to CTCAE scale, the mean pruritus before gabapentin was 2.41 and it decreased to 0.65 after gabapentin treatment. Paired student T-test for the difference of means before and after the treatment was statistically significant (p < 0.0005). Conclusions: Pruritus induced by IL-2 infusion responds well to gabapentin, which stabilizes nerve fibers and synapses. Although other confounding causes of pruritus cannot be excluded, the major mechanism is likely due to direct or indirect effect of IL-2, which then stimulates the nerve fibers. Thus, we propose gabapentin as an effective treatment against pruritus induced by IL-2.

No significant financial relationships to disclose.
HLA-A0201-positive, IL-7/B7.1-cotransfected allogeneic tumor cells as a vaccine in metastatic renal cell cancer—A clinical phase-I trial
Authors: Floercken A. , Kopp J. , Schabath R. , Joehrens-Leder K. , Pohla H. , Schendel D. , Blankenstein T. , Dörken B. , Westermann J. , Pezzutto A.
Published: March 2020

3072

Background: Tumor vaccination remains a promising experimental approach in RCC. HLA-A0201-restricted T cell immunity against RCC is well established. RCC26 is an allogeneic HLA-A0201+ human RCC cell line, a T cell clone specifically recognising RCC26 and other RCC in the context of HLA-A0201 has previously been generated, the TCR of this T cell clone was characterised. Furthermore, IL-7/B7.1 cotransfected tumor cells are a potent vaccine in animal models. Methods: RCC26 was transfected with pKEx-IL-7-IR-B7 coding for human IL-7 and B7.1 (CD80). 10 HLA-A0201+ patients with metastatic RCC and disease progression under cytokine therapy were included. 10 vaccinations with 2.5–40x106 gene-modified irradiated tumor cells which had been produced under GMP conditions were performed s.c. over 22 weeks. Primary endpoints of the study were feasibility, safety and immunological response, secondary endpoint was clinical response. The protocol was approved by the ethics committee, all patients gave informed consent. Results: Gene-modified RCC26 cells produced IL-7 (3.4 ng/106 cells/24h), more than 90% of the cells were CD80+. Vaccination was feasible and safe with no severe toxicity. Local DTH-reactions were observed in 4 patients. Skin biopsies of the vaccination site showed lymphocytic infiltrates dominated by CD4+T cells. In 8 patients vaccination induced HLA- and /or antinuclear antibodies without clinical signs of autoimmunity. Analysis of the T cell response against RCC-associated antigens is under way. No partial or complete responses could be documented. However, 50% of the patients had stable disease with the longest TTP being 69 weeks. Mean TTP in our cohort was 25 weeks (range 4 to 69 weeks). Conclusion: Our results show that vaccination with an allogeneic gene-modified tumor cell line is feasible and safe. Stable disease lasting up to 69 weeks in a substantial proportion of patients suggests immunological activity of the vaccine. Vaccination of patients with a low tumor burden is a promising strategy for the future, i.e. after surgery or treatment with novel multi-kinase inhibitors.

No significant financial relationships to disclose.