Thomas C. Harding

Combined immunotherapy with granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells and ipilimumab in patients with metastatic castration-resistant prostate cancer: a phase 1 dose-escalation trial

BACKGROUND The granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells vaccine (GVAX) has antitumour activity against prostate cancer; preclinical studies have shown potent synergy when combined with ipilimumab, an antibody that blocks cytotoxic T-lymphocyte antigen 4. We aimed to assess the safety of combined treatment with GVAX and ipilimumab in patients with metastatic castration-resistant prostate cancer (mCRPC). METHODS We did an open-labelled, single-centre, dose-escalation study of ipilimumab concurrent with a fixed dose of GVAX, with a subsequent expansion phase, both at the VU University Medical Centre (Amsterdam, Netherlands). Eligible patients had documented mCRPC and had not been previously treated with chemotherapy. All patients received a 5×10(8) cell priming dose of GVAX intradermally on day 1 with subsequent intradermal injections of 3×10(8) cells every 2 weeks for 24 weeks. The vaccinations were combined with intravenous ipilimumab every 4 weeks. We enrolled patients in cohorts of three; each cohort received an escalating dose of ipilimumab at 0·3, 1·0, 3·0, or 5·0 mg/kg. Our primary endpoint was safety. This study is registered with ClinicalTrials.gov, number NCT01510288. FINDINGS We enrolled 12 patients into our dose-escalation cohort. We did not record any severe immune-related adverse events at the first two dose levels. At the 3·0 mg/kg dose level, one patient had grade 2 and two patients grade 3 hypophysitis; at the 5·0 mg/kg dose level, two patients had grade 3 hypophysitis and one patient developed grade 4 sarcoid alveolitis (a dose-limiting toxic effect). Due to observed clinical activity and toxic events, we decided to expand the 3·0 mg/kg dose level, rather than enrol a further three patients at the 5·0 mg/kg level. 16 patients were enrolled in the expansion cohort, two of whom developed grade 2 hypophysitis, three colitis (one grade 1 and two grade 2), and one grade 3 hepatitis--all immune-related adverse events. The most common adverse events noted in all 28 patients were injection-site reactions (grade 1-2 events seen in all patients), fatigue (grade 1-2 in 20 patients, grade 3 in two), and pyrexia (grade 1-2 in 15 patients, grade 3 in one). 50% or greater declines in prostate-specific antigen from baseline was recorded in seven patients (25%); all had received 3·0 mg/kg or 5·0 mg/kg ipilimumab. INTERPRETATION GVAX combined with 3·0 mg/kg ipilimumab is tolerable and safe for patients with mCRPC. Further research on the combined treatment of patients with mCRPC with vaccination and ipilimumab is warranted. FUNDING Cell Genesys Inc, Prostate Cancer Foundation, Dutch Cancer Society (KWF-VU 2006-3697), and Foundation Stichting VUmc Cancer Center Amsterdam.

Vascular Endothelial Growth Factor Blockade Reduces Intratumoral Regulatory T Cells and Enhances the Efficacy of a GM-CSF–Secreting Cancer Immunotherapy

Purpose: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)–secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. Experimental Design: Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a soluble VEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory T cells were quantified by their CD4+CD25hi and CD4+FoxP3+ phenotype. Results: The present study established that GM-CSF–secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4+ and CD8+ tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory T cells in the tumor, suggesting a novel mechanism of VEGF-mediated immune suppression at the tumor site. Conclusion: GM-CSF–secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.

K562/GM-CSF Immunotherapy Reduces Tumor Burden in Chronic Myeloid Leukemia Patients with Residual Disease on Imatinib Mesylate

Purpose: Chronic myeloid leukemia (CML) can be responsive to T-cell–mediated immunity. K562/granulocyte macrophage-colony stimulating factor (GM-CSF) is a GM-CSF producing vaccine derived from a CML cell line that expresses several CML-associated antigens. A pilot study was developed to determine if K562/GM-CSF immunotherapy could improve clinical responses to imatinib mesylate (IM) in patients with chronic myeloid leukemia. Experimental Design: Patients with chronic phase CML who achieved at least a major cytogeneic response but remained with persistent, measurable disease despite one or more years on imatinib mesylate were eligible. Each was given a series of four vaccines administered in three-week intervals, with or without topical imiquimod, while remaining on a stable dose of imatinib mesylate. CML disease burden was measured serially before and after vaccination. Results: Nineteen patients were vaccinated, with a median duration of previous imatinib mesylate therapy of 37 (13–53) months. Mean PCR measurements of BCR-ABL for the group declined significantly following the vaccines (P = 0.03). Thirteen patients had a progressive decline in disease burden, 8 of whom had increasing disease burden before vaccination. Twelve patients achieved their lowest tumor burden measurements to date following vaccine, including seven subjects who became PCR-undetectable. Conclusions: K562/GM-CSF vaccine appears to improve molecular responses in patients on imatinib mesylate, including achieving complete molecular remissions, despite long durations of previous imatinib mesylate therapy. Clin Cancer Res; 16(1); 338–47

Development and characterization of a cell line for large‐scale, serum‐free production of recombinant adeno‐associated viral vectors

One of the major limitations to the use of adeno‐associated virus (AAV) vectors for gene therapy has been the difficulty in producing enough vector to supply a clinical trial. More than 20 000 roller bottles may be required to generate AAV by the traditional transient transfection process to treat 50 patients. A scalable AAV producer cell line grown in serum‐free media will meet the needs for the manufacture of AAV gene therapeutics.

In vitro and In vivo Characterization of Irreversible Mutant-Selective EGFR Inhibitors that are Wild-type Sparing

Patients with non–small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFRWT). These inhibitors have not provided compelling clinical benefit in T790M-positive patients, apparently because of dose-limiting toxicities associated with inhibition of EGFRWT. Thus, there is an urgent clinical need for therapeutics that overcome T790M drug resistance while sparing EGFRWT. Here, we describe a lead optimization program that led to the discovery of four potent irreversible 2,4-diaminopyrimidine compounds that are EGFR mutant (EGFRmut) selective and have been designed to have low affinity for EGFRWT. Pharmacokinetic and pharmacodynamic studies in H1975 tumor–bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. Importantly, evaluation of normal lung tissue from the same animals showed no inhibition of EGFRWT. Of all the compounds tested, compound 3 displayed the best efficacy in EGFRL858R/T790M-driven tumors. Compound 3, now renamed CO-1686, is currently in a phase I/II clinical trial in patients with EGFRmut-advanced NSCLC that have received prior EGFR-directed therapy. Mol Cancer Ther; 13(6); 1468–79. ©2014 AACR.

Long-term transgene expression can be mediated in the brain by adenoviral vectors when powerful neuron-specific promoters are used

Adenoviral (Ad) vectors are one of the most widely used tools for modelling gene therapy strategies. However, they have not been used in long‐term models of neurological disease, as the period of time for which they mediate strong transgene expression is limited and/or variable. In this study we investigated the longevity of transgene expression in the brain when the powerful neuron‐specific Ad‐synapsin (Sy)‐EGFP‐woodchuck hepatitis virus post‐transcriptional regulatory element (WPRE) vector cassette is used at titres that do not elicit an immune response.

Novel immunocompetent murine tumor model for evaluation of conditionally replication-competent (oncolytic) murine adenoviral vectors.

ABSTRACT Oncolytic adenoviral vectors that express immunostimulatory transgenes are currently being evaluated in clinic. Preclinical testing of these vectors has thus far been limited to immunodeficient xenograft tumor models since human adenoviruses do not replicate effectively in murine tumor cells. The effect of the immunostimulatory transgene on overall virus potency can therefore not be readily assessed in these models. Here, a model is described that allows the effective testing of mouse armed oncolytic adenovirus (MAV) vectors in immunocompetent syngeneic tumor models. These studies demonstrate that the MAV vectors have a high level of cytotoxicity in a wide range of murine tumor cells. The murine oncolytic viruses were successfully armed with murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) by a novel method which resulted in vectors with a high level of tumor-specific transgene expression. The mGM-CSF-armed MAV vectors showed an improved level of antitumor potency and induced a systemic antitumor immune response that was greater than that induced by unarmed parental vectors in immunocompetent syngeneic tumor models. Thus, the oncolytic MAV-1 system described here provides a murine homolog model for the testing of murine armed oncolytic adenovirus vectors in immunocompetent animals. The model allows evaluation of the impact of virus replication and the host immune response on overall virus potency and enables the generation of translational data that will be important for guiding the clinical development of these viruses.

Abstract 793: Insulin-like growth factor 1 (IGF1R)/insulin receptor (INSR) inhibitory activity of rociletinib (CO-1686) and its metabolites in nonclinical models

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rociletinib (CO-1686) is a novel, oral, targeted irreversible inhibitor of the cancer-causing mutant forms of EGFR currently being studied for the treatment of NSCLC. Rociletinib was designed to spare wild-type EGFR signaling. Heavily-pretreated T790M+ patients treated with rociletinib at 500 or 625mg BID demonstrated a 67% objective response rate (n = 56, Soria et al., ENA 2014). Adverse events (AEs) typical of wild-type EGFR inhibition, such as cutaneous toxicities, have not been observed. The most frequent AE in patients dosed with rociletinib is hyperglycemia (32% all grades/14% grade 3), which is typically managed with oral hypoglycemic therapy. Rociletinib does not directly play a role in hyperglycemia based on kinase and cellular profiling, toxicology studies performed in Sprague-Dawley rats and beagle dogs, and an oral glucose tolerance test (OGTT) in Sprague-Dawley rats. We examined the hypothesis that hyperglycemia may result from a metabolite of rociletinib. Metabolite profiling from in vitro hepatocyte incubations, as well as the analysis of plasma samples from healthy subjects and NSCLC patients, revealed three metabolites of interest referenced according to their protonated molecular ion: M460, M502, and M544. Metabolites M460 and M502 are present in higher levels in humans than in rats and dogs, whereas M544 levels are comparable between human and rat. In patients the levels of M502 are at least 5-fold greater than that of M460. All 3 metabolites demonstrated limited potency against EGFR, T790M EGFR, and HER2. Expanded cellular profiling revealed that M460 and M502 have 2-3 fold and 3-7 fold greater potency against INSR and IGF1R, respectively, as compared to rociletinib, whereas metabolite M544 demonstrated similar or reduced potency. Metabolite profiling in rats suggests that M502 is rapidly converted to M544 by acetylation, thus a rat OGTT was performed with a single dose of M502 at 1000 mg/kg to reach exposure levels comparable to those observed in rociletinib treated NSCLC patient samples. Metabolite M502 caused significant elevations in post-prandial glucose and insulin excursion. Taken together, the increased potency of M502 towards IGF1R and INSR, its absolute exposure in humans and the rat OGTT results suggest that M502 is likely to play a causative role in the hyperglycemia observed in patients. Additional data on the clinical implications of these findings, as well as the role of IGF1R pathway activation in resistance to EGFR inhibitors, will be presented. Citation Format: Andrew D. Simmons, Sarah Jaw-Tsai, Henry J. Haringsma, Andrew Allen, Thomas C. Harding. Insulin-like growth factor 1 (IGF1R)/insulin receptor (INSR) inhibitory activity of rociletinib (CO-1686) and its metabolites in nonclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 793. doi:10.1158/1538-7445.AM2015-793

Abstract B20: Immune vs. clinical response monitoring in patients with metastatic hormone-refractory prostate cancer receiving combined prostate GVAX and anti-CTLA4 immunotherapy

The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic, hormone-refractory prostate cancer (mHRPC). Patients received a 500 million cell GVAX priming dose on day 1 followed by 12 bi-weekly intradermal administrations of 300 million cells, while Ipilimumab was administered every 4 wks from day 1 for a total of 6 times. Initially, 12 patients were enrolled in cohorts of 3 and each cohort was assigned an escalating dose of Ipilimumab at 0.3, 1, 3 or 5 mg/kg. 16 additional patients were enrolled in the expansion cohort of 3 mg/kg Ipilimumab. Results showed that the GVAX and Ipilimumab combination was clinically active in mHRPC patients. PSA declines of more than 50% (Partial Response, PR) were observed in 5 of 22 patients in the 3–5 mg/kg Ipilimumab dose cohorts, and were associated with Autoimmune Breakthrough Events (ABE), including Grade 2 or 3 hypophysitis and Grade 3 alveolitis. PSA stabilizations (Stable Disease, SD) were observed in 1/3 patients in lower (0.3 and 1 mg/kg), and in 7 of 22 in the higher (3–5 mg/kg) dose levels. Moreover, regressing bone and lymph node metastases were observed in 2/5 PR patients. Immune response monitoring was performed to identify changes that might predict or correlate with clinical efficacy. Most notably, pronounced and significant increases in frequencies of activated and effector CD4+ and CD8+ T cells were observed by HLA-DR and ICOS expression upon administration of high (3 and 5 mg/kg) Ipilimumab doses; a relation to clinical behaviour was only observed for HLA-DR with earlier and more pronounced increases in patients with PR or SD. As an indication of tumor-specific responsiveness HLA-Tetramer (Tm) and seroreactivity to NY-ESO and PSMA were tested. For NY-ESO, therapy-induced increased seroreactivity (by Western Blot or ELISA) was observed in 6/28 patients. Of these 6 patients, three could be tested for Tm reactivity and in two (1SD, 1 Progressive Disease, PD) increased NY-ESO157 rates were found concomitant with increasing serum Ab levels, whereas no Tm reactivity was detected in 8 patients without seroconversions. PSMA seroconversions were observed in a total of 12/28 patients (and, of note, in 4/5 PR), but no Tm positivity at any time was found in a total of 15 patients tested. In the 3–5 mg/kg dose levels (n=22), PSMA seroconversion was associated with increased overall survival (P=0.062). In addition, combined GVAX/Ipilimumab administration was found to induce Type-2/Th17 profiles, as determined ex vivo by intracellular staining of peripheral T cells. Significantly increased levels of IL-4 in both CD4+ and CD8+ T cells were observed in patients with PR or SD (P In summary, our analyses so far reveal a relationship between clinical efficacy, PSMA seroconversion, and generalized T cell activation accompanied by Th2 and Th17 skewing. Together these data point to a mechanism of action whereby combined Prostate GVAX and anti-CTLA4 immunotherapy can induce both Th2/humoral and Th17/cell-mediated immune responses, resulting in tumor destruction and collateral autoimmunity. Supported by the Prostate Cancer Foundation and the Dutch Cancer Society (KWF-VU 2006-3697) Citation Information: Clin Cancer Res 2010;16(7 Suppl):B20

Chapter 3.2.3 Regulatable adenoviral technology in behavioural neuroscience

Publisher Summary This chapter discusses regulatable adenoviral technology in behavioral neuroscience. The use of this regulatable adenoviral vector system which mediates relatively long-term expression greatly facilitates studies of behavior, as it offers a relatively simple and rapid method (compared to transgenesis) of manipulating neuronal gene expression in specific populations of neurons. It is possible, to use this system to manipulate neuronal gene expression following only a single stereotaxic injection of virus. The specific functional or behavioral outcome can then be correlated with the turning on or turning off of gene expression (by the administration or withdrawal of dox) weeks after the initial viral transfection. Importantly, behavioral testing of animals can be undertaken weeks after the initial stereotaxic surgery, so that, the behavioral pretraining can be conducted 1-2 weeks after the injection of the AdTet system and the behavioral testing itself could start once Dox had been administered to turn transgene expression on. Finally, the use of the AdTet system permits the use of within-subjects experimental designs, in which every animal may serve as its own control as it may be tested both with transgene expression turned off and then on.