- Email: [email protected]
104 Identification of inhibitors of tryptophan metabolizing enzymes for cancer immunotherapy by high-throughput screening
Poster Session – Drug Screening also obtain the activities of various test compounds to the NCI-60 cell line panel from NCI Developmental Therapeutics Program (DTP) website. Using this capability, we screened for therapeutic agents against the cell lines with PIK3CA mutation, the second most frequently mutated actionable oncogene among various types of tumors. Material and Methods: Utilizing NCI-60 cell line GI50 (50% growthinhibitory levels) data of PI3K/AKT/mTOR pathway compounds, we calculated the differences in mean log GI50 between the PIK3CA mutated cell lines and non-mutated cell lines (delta log GI50) to investigate promising compounds. The volcano plot (the x-axis depicts delta log GI50 and the y-axis depicts statistical significance level) was used to search for the promising test compounds. Results: Seven cell lines had PIK3CA mutation (3 p.E545K, 3 p.H1047R, and 1 p.R38C mutation). We could collect GI50 data on 35 PI3K/AKT/mTOR targeting compounds (7 pan-class PI3K inhibitors, 7 isoform-selective PI3K inhibitors, 5 rapalogs, 3 mTOR kinase inhibitors, 6 panPI3K/mTOR inhibitors, and 7 AKT inhibitors). In our analysis, 1 panclass PI3K inhibitor, 2 isoform-selective PI3K inhibitors, 1 rapalog and 1 AKT inhibitor showed promising trends by both delta log GI50 and statistical significance. Conclusions: NCI-60 cell line panel may be a valuable tool to identify various actionable mutation–drug relationships and to discover promising drug candidates. 102 POSTER Comparison of platinum/taxane and anthracycline-based therapies in ovarian PDX models: Correlating stage of biopsy collection and engrafting with in vivo drug sensitivity J. Meade1 , M.J. Wick1 , T. Vaught1 , L. Gamez1 , M. Farley1 , A. Moriarty1 , A.W. Tolcher1 , A. Patnaik1 , D.W. Rasco1 , A.J. White2 , K.P. Papadopoulos1 . 1 South Texas Accelerated Research Therapeutics, (START), San Antonio TX, USA; 2 START Center for Cancer Care, San Antonio TX, USA Background: Standard first-line treatment for ovarian cancer is a platinumbased therapy often including a taxane, with various salvage therapies for recurrent disease. We and others have established and validated low passage ovary patient-derived xenograft (PDX) models from biopsies obtained at diagnosis and following clinical treatment progression; however, whether drug sensitivity to each model correlates to the clinical context was unclear. To test this we stratified thirty-nine ovary PDX models using clinical outcome data at the time of biopsy collection into four groups: chemo na¨ıve, first-line platinum responding, first-line platinum refractory and anthracycline pretreated. Drug sensitivity studies were performed, evaluating model response to platinum, platinum plus taxane and liposomal doxorubicin and results compared between each group. Methods: Ovary PDX models were developed inimmune-deficient mice from primary or metastatic patient tissue and established models confirmed by histologic comparative analysis and linked with patient treatment and outcome data. Drug sensitivity studies were performed evaluating models response to three regimens with tumor volume and time from treatment initiation as study endpoints. T/C values, growth delay and regressions were reported for each model. Results: For the thirty-nine models evaluated: 18/39 were from chemona¨ıve patients, 6/39 from first-line platinum responding, 10/39 from firstline platinum refractory and 5/39 from anthracycline pretreated patients. In these studies chemo-na¨ıve and first-line platinum responding models were sensitive to the platinum based regimens with few exceptions including ST036 which harbors known AKT and KRAS mutations. Most first-line platinum refractory models including ST419 and ST884 were resistant to platinum alone and platinum/taxane combination. Interestingly sensitivity to liposomal doxorubicin did not correlate with clinical pretreatment as several models from chemo-na¨ıve patients including ST024 and ST511 were insensitive while some models established from anthracycline pretreated patients including ST255B and ST467 were sensitive to the therapy. Conclusion: We have established a panel of ovary patient-derived xenografts and correlated model sensitivity to platinum and anthracyclinebased treatments to the clinical context at the time of biopsy collection and engraftment. Overall platinum sensitivity in models from chemo-na¨ıve and first-line platinum responding patients, with few exceptions, correlated with clinical response, irrespective of activating mutations. Preclinical response to anthracyclines was less predictive and not predicated on the clinical context.
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103 POSTER The use of next generation sequencing (NGS) in the management of metastatic breast cancer (MBC): Defining a model for genomic-driven therapies L. Austin1 , M. Gooptu1 , T. Avery1 , R. Jaslow1 , J. Palazzo2 , M. Cristofanilli1 . 1 Thomas Jefferson University Hospital, Medical Oncology, Philadelphia PA, USA; 2 Thomas Jefferson University Hospital, Pathology, Philadelphia PA, USA Background: Metastatic breast cancer is an incurable disease treated with palliative intent. The limited benefit from standard therapies could be related to the fact that the disease is biologically heterogeneous and not properly managed by current treatments. Increased understanding of the molecular complexity using next generation sequencing (NGS) allows for individual tumor mutation analysis which provides better selection of treatments based on genomic abnormalities driving the disease. Methods: We retrospectively analyzed 33 patients with advanced or metastatic breast cancer treated at Thomas Jefferson University, diagnosed between 2003–2013, on whom next generation sequencing (NGS) by Foundation One™ was sent for genomic analysis. Immediately actionable mutations for this analysis were considered those for which FDA-approved agents for breast cancer were identified. We also reported potentially actionable mutations which were defined as mutations with investigationaltherapeutic agents. Results: The majority of patients (88%) had inflammatory breast cancer (IBC). According to disease subtype, 36% were ER+/HER− (Luminal A), 18% ER+/HER+ (Luminal B), 15% ER−/HER+ (HER2+), and 30% were ER−/HER− (triple negative). NGS genomic analysis revealed a total of 78 unique mutations with a total of 178 mutational events. The five most commonly mutated genes in order of frequency were TP53 (55% of patients), MYC amplification (39%), PIK3CA mutations (36%), ERBB2 amplification (30%) and PTEN loss or mutation (15%). Eleven of 78 mutations (14%) were immediately actionable (based on our definition) and comprised of PIK3CA mutations/amplifications, ERBB2 amplifications, AKT mutations and PTEN loss/mutations. In total, 21 of 33 patients (64%) had at least one immediately actionable mutation and 97% (32) of patients had either an immediately actionable or potentially actionable mutation. The most common potentially actionable mutations were TP53, MYC amplification, FGFR1 amplification and MDM2 amplification. All but one of the HER2+ patients (IHC/FISH) had ERBB2 amplification on NGS. Interestingly, of the 10 patients that had ERBB2 amplification, 70% had a concomitant PIK3CA mutation and all had IBC. Two patients with ERBB2 amplification and PIK3CA mutations who had progressed on trastuzumab ((Herceptin® ) combinations were started on everolimus (Afinitor® ), Trastuzumab and vinorelbine (Navelbine). Conclusion: In conclusion, in this cohort of 33 patients, the Foundation One™ panel detected unique mutations of which 14% were immediately actionable and 97% of patients had at least one mutation that was immediately or potentially actionable. Actionable mutations were detected across all receptor groups and there was a high incidence on concomitant ERBB2 amplification and PIK3CA mutation. The Foundation One™ panel is a useful tool which can be utilized for tailoring targeted therapy for breast cancer patients. This warrants further investigation with a prospective trial to evaluate the clinical impact of this added information and its effect on decision making and clinical outcomes. 104 POSTER Identification of inhibitors of tryptophan metabolizing enzymes for cancer immunotherapy by high-throughput screening G. Zaman1 , J.C.M. Uitdehaag1 , S. van Gerwen1 , N. Seegers1 , A.M. van Doornmalen1 , J. de Man1 , R.C. Buijsman1 . 1 Netherlands Translational Research Center B.V., Oss, Netherlands Background: The amino acid tryptophan is an important regulator of the immune system by regulating the activation of T cells. Tumor cells express enzymes that oxidize tryptophan, thereby dampening the local T cell immune response against cancer cells. These enzymes are indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO). Despite the substantial evidence of the importance of IDO1 and TDO as small molecule drug targets, only a handful of different chemical scaffolds have been reported. The lack of chemical matter is explained by the lack of robust assays for high-throughput screening. We have developed new assays for IDO1 and TDO, which we have used to screen a compound library, and to characterize the selectivity of reference inhibitors. Materials and Methods: NFK Green™ is a chemical probe that specifically reacts with N-formyl kynurine, the reaction product of the enzymatic conversion of tryptophan by IDO1 and TDO. The reaction is quantified by measuring fluorescence on a 384-well plate multimode reader. Biochemical assays were developed for IDO1 and TDO using recombinantly expressed
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proteins, and a library of 87,000 diverse lead-like molecules was screened. NFK Green™ was also used to determine the tryptophan metabolizing activity in a collection of human cancer cell lines and was related to the expression levels of IDO1 and TDO by western blot analysis. Results: Biochemical and cell-based screening assays were developed for IDO1 and TDO using a new fluorescent read-out. High-throughput screening of libraries of small chemical compound libraries yielded novel selective inhibitors of either IDO1 or TDO. Side-by-side comparison of published reference compounds revealed significant, previously unnoted cross-reactivity of a widely used hydroxyamidine-based inhibitor of IDO1 (Compound 5l) with TDO. The selectivity of other reference IDO1 or TDO inhibitors was confirmed, leading to definition of a new tool compound set. Biochemical selectivity of compounds correlated with inhibition of cellular tryptophan metabolizing activity and expression of IDO1 or TDO. Conclusions: We have developed new biochemical and cell-based assays for IDO1 and TDO, to enable the identification of novel small molecule inhibitors and to support lead optimization. Side-by-side comparison of published inhibitors revealed novel, unanticipated cross-reactivity of IDO1 inhibitor scaffolds with TDO. 105 POSTER A platform to test multiple therapy options simultaneously in a patient’s own tumor N. Caffo1 , R. Klinghoffer1 . 1 Presage Biosciences Inc., Seattle, USA While investigational cancer drugs must ultimately be validated in clinical trials, most early drug discovery is performed under in vitro conditions in cell-based models that poorly represent the disease they are intended to represent. To enable in vivo analysis of anti-cancer agent efficacy at earlier stages of drug development, and to potentially enable toxicity-sparing assessment of novel agents in the oncology clinic, we have developed a technology platform called CIVOTM . CIVOTM allows for simultaneous assessment of up to eight drugs or drug combinations in a single solid tumor. Controlled microinjection-based delivery of doxorubicin, docetaxel, mafosfamide, and gemcitabine both as single agents and combinations was tested in the canine sarcoma clinic. Drugs were co-injected in a columnar array with UV fluorescent beads resulting in easy-to-identify bands of drug, each at a distinct position of the patient’s tumor. Tumors were resected 72 h following microinjection and were examined for responses using standard histology methods. The CIVO introduced drug microdoses induced spatially defined, graded, and mechanism-specific cellular changes around sites of drug exposure in a patient-specific manner. Consistent with the use of doxorubicin as first line therapy in the soft tissue sarcoma clinic, the frequency and extent of response of localized responses to doxorubicin exceeded those of all other agents tested. This preclinical data, along with early responses observed in the human clinic, set the stage for clinical application of this technology to identify which novel agents are likely to succeed or fail in subsequent clinical trials.
Immunotherapy (Immunecheckpoints, Vaccination, Oncolytic viruses, Cytokines) 106 POSTER Beta-3 integrin inhibition reduces inflammatory cytokine release but not anti-cancer activity of oncolytic adenovirus in ovarian cancer A.K. Browne1 , L.A. Tookman1 , C.K. Ingemarsdotter1 , R. Bouwman1 , K. Pirlo1 , Y. Wang1 , K.M. Hodivala-Dilke2 , I.A. McNeish3 , M. Lockley1 . 1 Barts Cancer Institute, Centre For Molecular Oncology, London, United Kingdom; 2 Barts Cancer Institute, Centre For Tumour Biology, London, United Kingdom; 3 University of Glasgow, Institute of Cancer Services, Glasgow, United Kingdom Background/Introduction: The potential of oncolytic adenoviruses as anti-cancer therapy has repeatedly been demonstrated. A consistent and worrying feature of adenoviral gene therapy is the rapid cytokine release that occurs after viral administration. These cytokines give rise to dose limiting inflammatory toxicities, which can be severe and have hindered further investigation and clinical development of these promising anti-cancer therapies. Using primary ovarian cancer cells and genetically modified mice, we show that the E1A-CR2 deleted replicating oncolytic adenovirus, dl922–947, induces cytokines via beta-3 integrin in macrophage-rich tissues. We present new evidence that co-administration of a Cilengitidelike integrin inhibitor controls the inflammatory cytokines and hepatic toxicity
Poster Session – Immunotherapy induced by dl922–947 in tumour bearing mice. Importantly, although alphav/beta-3integrin functions as a secondary adenoviral receptor, we found no evidence that beta-3 inhibition compromised viral infectivity and oncolysis in vitro or anti-cancer efficacy in vivo. Material and Methods: We quantified production of cytokine mRNA (qRTPCR) and protein (Mesoscale Discovery System) following dl922–947 in ovarian cancer cells lines, tumour cells harvested from the ascites of women with ovarian cancer and in murine tissues, peritoneal cells and serum. Murine models included nude mice bearing intraperitoneal ovarian cancer xenografts and non-tumour bearing, immunocompetent beta-3 null mice. Pharmacological inhibition of beta-3 integrin was achieved using H2574, a cyclic RGD mimetic inhibitor of alpha-v/beta-3andalpha-v/beta-5 integrins. Results: Primary and established ovarian cancer cell lines in vitro did not release inflammatory cytokines in response to adenovirus. In contrast, intraperitoneal delivery of dl922–947 caused rapid, systemic cytokine induction in ovarian cancer xenografts. Cytokines originated predominantly in macrophage-rich murine tissues (liver, spleen and peritoneal macrophages), rather than the injected malignant cells, and was independent of viral replication. Adenoviruses are known to induce cytokine release via beta-3 integrin-expressing macrophages. We found that co-administration of the integrin inhibitor, H2574, controlled production of inflammatory cytokines in the circulation of tumour-bearing mice. Cotreatment also reduced pathological features of viral hepatic toxicity such as eosinophilic degeneration and liver enzyme elevation. Importantly, combining dl922–947 with H2574 did not compromise anti-cancer activity in vitro or in vivo. Conclusions: Combining oncolytic adenoviruses with pharmacological inhibition of beta-3 integrin enables safe systemic delivery of replicating adenoviruses, without compromising anti-cancer activity. This novel approach could have a major impact on the future development of these effective anti-cancer agents. 107 POSTER CIGB-247: Anti-VEGF therapeutic vaccine in patients with advanced solid tumors 1 2 ´ F. Hernandez-Bernal ´ , J.V. Gavilondo2 , M. Ayala Avila , A.V. de la Torre3 , 4 4 2 J. de la Torre , K.H. Selman-Housein , Y. Morera , M. Bequet-Romero2 , C.M. Valenzuela1 , Y. Martin5 . 1 Center for Genetic Engineering and Biotechnology (CIGB), Clinical Trials, Havana City, Cuba; 2 Center for Genetic Engineering and Biotechnology (CIGB), Pharmaceuticals, Havana ´ Hospital, Oncology, Santa Clara, City, Cuba; 3 “Celestino Hernandez” Cuba; 4 Medical and Surgical Research (CIMEQ), Oncology, Havana City, ´ Cuba; 5 “Celestino Hernandez” Hospital, Oncology, Havana City, Cuba
Background: The CIGB-247 is a vaccine preparation for the therapy of cancer that combines the recombinant antigen p64K-hVEGFKDR− , produced in Escherichia coli and the VSSP adjuvant. This antigen is a representative molecule of the human Vascular Endothelium Growth Factor (VEGF-A), in its isoform 121. Vaccination with CIGB-247 in experimental animal models has been safe, with anti-tumor and anti-metastatic effects. Material and Methods: A non-controlled, dose up scaling phase I clinical trial was performed in patients with advanced solid tumors, which previously received the best onco-specific treatment available without response. The general objective of the trial was to study the safety profile of the vaccine at three antigen dose levels. A maximum of 10 patients per group were planned. Individuals were subcutaneously immunized for 8 consecutive weeks with 50, 100 or 400 mg of antigen (all in the same amount of VSSP adjuvant), and re-immunized on week 12. On week 16, evaluations of safety, tolerance, clinical status, and immunogenicity (seroconversion for anti-VEGF IgG, serum VEGF/KDR-Fc blocking ability, and gamma-IFN ELISPOT with blood cells stimulated in vitro with mutated VEGF) were done. Results: Vaccination was shown to be safe at the three dose levels, with only grade 1−2 adverse events. CIGB-247 was immunogenic and higher numbers of individuals positive to the three immune response tests were seen with increasing antigen dose. Conclusions: This is the first clinical testing report of a cancer therapeutic vaccine based on a human VEGF related molecule as antigen. The CIGB247 vaccine is safe, immunogenic, and merits further clinical development.
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103 POSTER The use of next generation sequencing (NGS) in the management of metastatic breast cancer (MBC): Defining a model for genomic-driven therapies L. Austin1 , M. Gooptu1 , T. Avery1 , R. Jaslow1 , J. Palazzo2 , M. Cristofanilli1 . 1 Thomas Jefferson University Hospital, Medical Oncology, Philadelphia PA, USA; 2 Thomas Jefferson University Hospital, Pathology, Philadelphia PA, USA Background: Metastatic breast cancer is an incurable disease treated with palliative intent. The limited benefit from standard therapies could be related to the fact that the disease is biologically heterogeneous and not properly managed by current treatments. Increased understanding of the molecular complexity using next generation sequencing (NGS) allows for individual tumor mutation analysis which provides better selection of treatments based on genomic abnormalities driving the disease. Methods: We retrospectively analyzed 33 patients with advanced or metastatic breast cancer treated at Thomas Jefferson University, diagnosed between 2003–2013, on whom next generation sequencing (NGS) by Foundation One™ was sent for genomic analysis. Immediately actionable mutations for this analysis were considered those for which FDA-approved agents for breast cancer were identified. We also reported potentially actionable mutations which were defined as mutations with investigationaltherapeutic agents. Results: The majority of patients (88%) had inflammatory breast cancer (IBC). According to disease subtype, 36% were ER+/HER− (Luminal A), 18% ER+/HER+ (Luminal B), 15% ER−/HER+ (HER2+), and 30% were ER−/HER− (triple negative). NGS genomic analysis revealed a total of 78 unique mutations with a total of 178 mutational events. The five most commonly mutated genes in order of frequency were TP53 (55% of patients), MYC amplification (39%), PIK3CA mutations (36%), ERBB2 amplification (30%) and PTEN loss or mutation (15%). Eleven of 78 mutations (14%) were immediately actionable (based on our definition) and comprised of PIK3CA mutations/amplifications, ERBB2 amplifications, AKT mutations and PTEN loss/mutations. In total, 21 of 33 patients (64%) had at least one immediately actionable mutation and 97% (32) of patients had either an immediately actionable or potentially actionable mutation. The most common potentially actionable mutations were TP53, MYC amplification, FGFR1 amplification and MDM2 amplification. All but one of the HER2+ patients (IHC/FISH) had ERBB2 amplification on NGS. Interestingly, of the 10 patients that had ERBB2 amplification, 70% had a concomitant PIK3CA mutation and all had IBC. Two patients with ERBB2 amplification and PIK3CA mutations who had progressed on trastuzumab ((Herceptin® ) combinations were started on everolimus (Afinitor® ), Trastuzumab and vinorelbine (Navelbine). Conclusion: In conclusion, in this cohort of 33 patients, the Foundation One™ panel detected unique mutations of which 14% were immediately actionable and 97% of patients had at least one mutation that was immediately or potentially actionable. Actionable mutations were detected across all receptor groups and there was a high incidence on concomitant ERBB2 amplification and PIK3CA mutation. The Foundation One™ panel is a useful tool which can be utilized for tailoring targeted therapy for breast cancer patients. This warrants further investigation with a prospective trial to evaluate the clinical impact of this added information and its effect on decision making and clinical outcomes. 104 POSTER Identification of inhibitors of tryptophan metabolizing enzymes for cancer immunotherapy by high-throughput screening G. Zaman1 , J.C.M. Uitdehaag1 , S. van Gerwen1 , N. Seegers1 , A.M. van Doornmalen1 , J. de Man1 , R.C. Buijsman1 . 1 Netherlands Translational Research Center B.V., Oss, Netherlands Background: The amino acid tryptophan is an important regulator of the immune system by regulating the activation of T cells. Tumor cells express enzymes that oxidize tryptophan, thereby dampening the local T cell immune response against cancer cells. These enzymes are indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO). Despite the substantial evidence of the importance of IDO1 and TDO as small molecule drug targets, only a handful of different chemical scaffolds have been reported. The lack of chemical matter is explained by the lack of robust assays for high-throughput screening. We have developed new assays for IDO1 and TDO, which we have used to screen a compound library, and to characterize the selectivity of reference inhibitors. Materials and Methods: NFK Green™ is a chemical probe that specifically reacts with N-formyl kynurine, the reaction product of the enzymatic conversion of tryptophan by IDO1 and TDO. The reaction is quantified by measuring fluorescence on a 384-well plate multimode reader. Biochemical assays were developed for IDO1 and TDO using recombinantly expressed
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proteins, and a library of 87,000 diverse lead-like molecules was screened. NFK Green™ was also used to determine the tryptophan metabolizing activity in a collection of human cancer cell lines and was related to the expression levels of IDO1 and TDO by western blot analysis. Results: Biochemical and cell-based screening assays were developed for IDO1 and TDO using a new fluorescent read-out. High-throughput screening of libraries of small chemical compound libraries yielded novel selective inhibitors of either IDO1 or TDO. Side-by-side comparison of published reference compounds revealed significant, previously unnoted cross-reactivity of a widely used hydroxyamidine-based inhibitor of IDO1 (Compound 5l) with TDO. The selectivity of other reference IDO1 or TDO inhibitors was confirmed, leading to definition of a new tool compound set. Biochemical selectivity of compounds correlated with inhibition of cellular tryptophan metabolizing activity and expression of IDO1 or TDO. Conclusions: We have developed new biochemical and cell-based assays for IDO1 and TDO, to enable the identification of novel small molecule inhibitors and to support lead optimization. Side-by-side comparison of published inhibitors revealed novel, unanticipated cross-reactivity of IDO1 inhibitor scaffolds with TDO. 105 POSTER A platform to test multiple therapy options simultaneously in a patient’s own tumor N. Caffo1 , R. Klinghoffer1 . 1 Presage Biosciences Inc., Seattle, USA While investigational cancer drugs must ultimately be validated in clinical trials, most early drug discovery is performed under in vitro conditions in cell-based models that poorly represent the disease they are intended to represent. To enable in vivo analysis of anti-cancer agent efficacy at earlier stages of drug development, and to potentially enable toxicity-sparing assessment of novel agents in the oncology clinic, we have developed a technology platform called CIVOTM . CIVOTM allows for simultaneous assessment of up to eight drugs or drug combinations in a single solid tumor. Controlled microinjection-based delivery of doxorubicin, docetaxel, mafosfamide, and gemcitabine both as single agents and combinations was tested in the canine sarcoma clinic. Drugs were co-injected in a columnar array with UV fluorescent beads resulting in easy-to-identify bands of drug, each at a distinct position of the patient’s tumor. Tumors were resected 72 h following microinjection and were examined for responses using standard histology methods. The CIVO introduced drug microdoses induced spatially defined, graded, and mechanism-specific cellular changes around sites of drug exposure in a patient-specific manner. Consistent with the use of doxorubicin as first line therapy in the soft tissue sarcoma clinic, the frequency and extent of response of localized responses to doxorubicin exceeded those of all other agents tested. This preclinical data, along with early responses observed in the human clinic, set the stage for clinical application of this technology to identify which novel agents are likely to succeed or fail in subsequent clinical trials.
Immunotherapy (Immunecheckpoints, Vaccination, Oncolytic viruses, Cytokines) 106 POSTER Beta-3 integrin inhibition reduces inflammatory cytokine release but not anti-cancer activity of oncolytic adenovirus in ovarian cancer A.K. Browne1 , L.A. Tookman1 , C.K. Ingemarsdotter1 , R. Bouwman1 , K. Pirlo1 , Y. Wang1 , K.M. Hodivala-Dilke2 , I.A. McNeish3 , M. Lockley1 . 1 Barts Cancer Institute, Centre For Molecular Oncology, London, United Kingdom; 2 Barts Cancer Institute, Centre For Tumour Biology, London, United Kingdom; 3 University of Glasgow, Institute of Cancer Services, Glasgow, United Kingdom Background/Introduction: The potential of oncolytic adenoviruses as anti-cancer therapy has repeatedly been demonstrated. A consistent and worrying feature of adenoviral gene therapy is the rapid cytokine release that occurs after viral administration. These cytokines give rise to dose limiting inflammatory toxicities, which can be severe and have hindered further investigation and clinical development of these promising anti-cancer therapies. Using primary ovarian cancer cells and genetically modified mice, we show that the E1A-CR2 deleted replicating oncolytic adenovirus, dl922–947, induces cytokines via beta-3 integrin in macrophage-rich tissues. We present new evidence that co-administration of a Cilengitidelike integrin inhibitor controls the inflammatory cytokines and hepatic toxicity
Poster Session – Immunotherapy induced by dl922–947 in tumour bearing mice. Importantly, although alphav/beta-3integrin functions as a secondary adenoviral receptor, we found no evidence that beta-3 inhibition compromised viral infectivity and oncolysis in vitro or anti-cancer efficacy in vivo. Material and Methods: We quantified production of cytokine mRNA (qRTPCR) and protein (Mesoscale Discovery System) following dl922–947 in ovarian cancer cells lines, tumour cells harvested from the ascites of women with ovarian cancer and in murine tissues, peritoneal cells and serum. Murine models included nude mice bearing intraperitoneal ovarian cancer xenografts and non-tumour bearing, immunocompetent beta-3 null mice. Pharmacological inhibition of beta-3 integrin was achieved using H2574, a cyclic RGD mimetic inhibitor of alpha-v/beta-3andalpha-v/beta-5 integrins. Results: Primary and established ovarian cancer cell lines in vitro did not release inflammatory cytokines in response to adenovirus. In contrast, intraperitoneal delivery of dl922–947 caused rapid, systemic cytokine induction in ovarian cancer xenografts. Cytokines originated predominantly in macrophage-rich murine tissues (liver, spleen and peritoneal macrophages), rather than the injected malignant cells, and was independent of viral replication. Adenoviruses are known to induce cytokine release via beta-3 integrin-expressing macrophages. We found that co-administration of the integrin inhibitor, H2574, controlled production of inflammatory cytokines in the circulation of tumour-bearing mice. Cotreatment also reduced pathological features of viral hepatic toxicity such as eosinophilic degeneration and liver enzyme elevation. Importantly, combining dl922–947 with H2574 did not compromise anti-cancer activity in vitro or in vivo. Conclusions: Combining oncolytic adenoviruses with pharmacological inhibition of beta-3 integrin enables safe systemic delivery of replicating adenoviruses, without compromising anti-cancer activity. This novel approach could have a major impact on the future development of these effective anti-cancer agents. 107 POSTER CIGB-247: Anti-VEGF therapeutic vaccine in patients with advanced solid tumors 1 2 ´ F. Hernandez-Bernal ´ , J.V. Gavilondo2 , M. Ayala Avila , A.V. de la Torre3 , 4 4 2 J. de la Torre , K.H. Selman-Housein , Y. Morera , M. Bequet-Romero2 , C.M. Valenzuela1 , Y. Martin5 . 1 Center for Genetic Engineering and Biotechnology (CIGB), Clinical Trials, Havana City, Cuba; 2 Center for Genetic Engineering and Biotechnology (CIGB), Pharmaceuticals, Havana ´ Hospital, Oncology, Santa Clara, City, Cuba; 3 “Celestino Hernandez” Cuba; 4 Medical and Surgical Research (CIMEQ), Oncology, Havana City, ´ Cuba; 5 “Celestino Hernandez” Hospital, Oncology, Havana City, Cuba
Background: The CIGB-247 is a vaccine preparation for the therapy of cancer that combines the recombinant antigen p64K-hVEGFKDR− , produced in Escherichia coli and the VSSP adjuvant. This antigen is a representative molecule of the human Vascular Endothelium Growth Factor (VEGF-A), in its isoform 121. Vaccination with CIGB-247 in experimental animal models has been safe, with anti-tumor and anti-metastatic effects. Material and Methods: A non-controlled, dose up scaling phase I clinical trial was performed in patients with advanced solid tumors, which previously received the best onco-specific treatment available without response. The general objective of the trial was to study the safety profile of the vaccine at three antigen dose levels. A maximum of 10 patients per group were planned. Individuals were subcutaneously immunized for 8 consecutive weeks with 50, 100 or 400 mg of antigen (all in the same amount of VSSP adjuvant), and re-immunized on week 12. On week 16, evaluations of safety, tolerance, clinical status, and immunogenicity (seroconversion for anti-VEGF IgG, serum VEGF/KDR-Fc blocking ability, and gamma-IFN ELISPOT with blood cells stimulated in vitro with mutated VEGF) were done. Results: Vaccination was shown to be safe at the three dose levels, with only grade 1−2 adverse events. CIGB-247 was immunogenic and higher numbers of individuals positive to the three immune response tests were seen with increasing antigen dose. Conclusions: This is the first clinical testing report of a cancer therapeutic vaccine based on a human VEGF related molecule as antigen. The CIGB247 vaccine is safe, immunogenic, and merits further clinical development.