Efficacy In Preclinical Tumor Models Research Articles

Abstract B34: Coordinated delivery of anticancer drug combinations incorporating molecularly targeted agents provides markedly increased plasma drug exposure, decreased toxicity and increased efficacy in preclinical tumor models

Abstract Background: Coordinated delivery of established chemotherapy combinations at synergistic drug ratios via nano-scale delivery vehicles has provided marked improvements in efficacy both preclinically and clinically. Many molecularly targeted agent (MTA) combinations have experienced difficulties in achieving optimal target inhibition without inducing dose limiting toxicities. Here we report the application of CombiPlex® technology to combinations incorporating MTAs for which optimal therapeutic effects require simultaneous tumor cell exposure. Methods: Prodrugs conjugates were synthesized for the HSP90 inhibitor AUY922 (AUY), docetaxel (DOC), the MEK inhibitor selumetinib (SEL) and Akt inhibitor ipatasertib (IPA) using cholesterol as a hydrophobic anchor joined via hydrolysable linkers that regenerate the parent drugs upon cleavage. These prodrugs were co-formulated in hydrophobic prodrug nanoparticles (HPN) by rapid mixing in the presence of surface stabilizing block co-polymers. Formulation compositions were iteratively optimized to achieve prolonged plasma drug concentrations while coordinating the PK of the combined agents. The tolerability and efficacy of the HPN combinations were compared to the free drug combinations administered in their conventional formulations. Results: AUY and DOC anchored to cholesterol via a diglycolate linker could be stably co-formulated in HPNs with a mean diameter of ∼65nm using a range of surface stabilizing block co-polymers. Using PLA-PEG block co-polymers, HPN-associated drugs exhibited no early distribution phase and virtually identical PK for AUY and DOC with a plasma half-life in mice of >12hr. Plasma concentrations of both agents over 24h were 2- to 4-orders of magnitude higher than for the free drugs. Similar PK results were obtained with the SEL:IPA combination. Combined IV treatment with DOC and AUY as the conventional free drugs led to significant increases in toxicity such that the DOC dose had to be reduced by 67% and the AUY dose had to be reduced by 60%. In contrast, when co-formulated in HPNs, both drugs could be administered with only a 30% dose reduction. In human xenograft tumor models (taxane resistant as well as taxane sensitive), the HPN formulation of AUY:DOC provided increased tumor growth inhibition; quantitative analysis of tumor growth delay revealed a 5-fold increase in antitumor activity compared to the free drug combination in the HCT15 model. A range of AUY:DOC drug ratio HPN formulations were also tested to identify the optimally efficacious drug ratio. HPN formulations of SEL:IPA also displayed improved tolerability compared to the free drug combination, particularly in view of the lower bioavailability for the agents administered in their oral dosing forms, while providing significant antitumor efficacy. Conclusions: HPN-mediated coordinated delivery of drug combinations incorporating molecularly targeted agents can favorably shift the PK/PD profile resulting in an improved therapeutic index. Initial results suggest this may expand the utility of combinations that to date have been limited by toxicities associated with dosing regimens aimed at ensuring simultaneous and durable multi-target inhibition. Citation Format: Lawrence D. Mayer, Paul Tardi, Sherwin Xie, Barry Liboiron, Winnie Lui, Leon Wan. Coordinated delivery of anticancer drug combinations incorporating molecularly targeted agents provides markedly increased plasma drug exposure, decreased toxicity and increased efficacy in preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B34.

Abstract LB-234: Poxvirus-based active immunotherapy synergizes with PD-1 plus LAG-3 immune checkpoint inhibition to enhance antitumor efficacy in preclinical models

Abstract Treatment with poxvirus-based active immunotherapies shows evidence of robust immune responses against a variety of tumor-associated antigens in preclinical and clinical studies. Poxvirus-based immunotherapies in development include PSA-targeted PROSTVAC, now in Phase 3 clinical development; CV-301 (targeting CEA and MUC-1); as well as MVA-BN-HER2 and MVA-BN-Brachyury (targeting HER-2 and the transcription factor Brachyury, respectively). Evidence of robust and productive anti-tumor efficacy in preclinical models was accompanied by treatment-emergent infiltration of tumors by activated cytotoxic CD8 T cells producing high amounts of IFNγ. Treatment with immune checkpoint inhibitors such as anti-PD-1 antibodies is showing significant clinical benefit by re-activating dormant tumor-specific T cells. Furthermore, preclinical studies have shown further synergistic efficacy by combining PD-1 blockade with inhibition of LAG-3, which acts independently of PD-1 to modulate T cell function. We hypothesized that poxvirus-based active immunotherapy may provide even greater improvements to patient outcome when used in combination with immune checkpoint blockade, by inducing new productive tumor-specific responses. This may be especially important in patients lacking an endogenous T cell response against their tumors. In therapeutic CT26-HER2 solid and metastatic tumor models, mice were administered MVA-BN-HER2 immunotherapy alone or in combination with anti-PD-1 and/or anti-LAG-3 antibodies. Synergistic benefit for anti-tumor efficacy was observed when combining MVA-BN-HER2 immunotherapy with anti-PD-1 alone, while combination with anti-LAG-3 alone had little effect. Notably, a further enhancement occurred when MVA-BN-HER2 immunotherapy was combined with PD-1 and LAG-3 blockade as shown by complete tumor regression in 20/20 mice. Subsequent rejection of HER-2 negative tumors 6 months after the original challenge revealed that immune responses were durable and included antigen spread to additional tumor antigens. Flow cytometric analysis demonstrated that tumor infiltrating lymphocytes (TILs) in untreated tumors were PD-1hi and LAG-3+, a more exhausted phenotype. Poxvirus-based immunotherapy led to the induction of activated TILs characterized by low to mid-levels of PD-1 expression. While PD-1 blockade prevented binding to PD-L1 it also caused an increase in LAG-3 expression on T cells. Together these data provide further rationale for why combination therapy of poxvirus-based immunotherapy with inhibition of PD-1 plus LAG-3 resulted in synergistic efficacy in preclinical tumor models. Overall these data demonstrate that combining complementary immune-based therapies such as poxvirus-based active immunotherapy and PD-1 plus LAG-3 immune checkpoint blockade result in synergistic anti-tumor efficacy. Citation Format: Barbara Sennino, Susan P. Foy, Ryan B. Rountree, Tracy dela Cruz, Evan J. Gordon, Veronica Xavier, Felicia Kemp, Alex Franzusoff, James Breitmeyer, Stefanie J. Mandl. Poxvirus-based active immunotherapy synergizes with PD-1 plus LAG-3 immune checkpoint inhibition to enhance antitumor efficacy in preclinical 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 LB-234. doi:10.1158/1538-7445.AM2015-LB-234

Abstract 636: Next-generation site-specific antibody-drug conjugates using the SMARTagTM technology platform

Abstract The two clinically-approved antibody-drug conjugates, AdcetrisTM and KadcylaTM, demonstrate that targeting cytotoxic payloads directly to tumors is an effective clinical approach. However, at the molecular level, these therapeutics are composed of heterogeneous mixtures resulting from the nonselective ligation of cytotoxic payload to cysteine and lysine residues, respectively. The nonselective ligation approaches used to generate these two ADCs render it difficult to optimize their biological, physical, and pharmacological properties. Redwood Bioscience creates site-selective, stable linkages that allow us to design ADCs with enhanced efficacy, safety, and pharmacokinetics. Additionally, we are able to control payload stoichiometry and placement. Redwood Bioscience creates homogeneous ADCs using the SMARTagTM technology, in which a genetically encoded 5 amino acid “aldehyde-tag” sequence contains a cysteine that is cotranslationally converted to formylglycine via an enzymatic transformation. This method affords control over the placement of the reactive formylglycine aldehyde side chain, which can then be ligated using carbonyl bioconjugation methods. Here, we will present our novel protein modification platform—including our new conjugation chemistries and linker libraries—and its application to ADC generation. We will address SMARTagTM ADC safety, pharmacokinetics, and efficacy in preclinical tumor models, and discuss how these parameters are impacted by linker selection and payload placement on the antibody. Citation Format: Romas Kudirka. Next-generation site-specific antibody-drug conjugates using the SMARTagTM technology platform. [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 636. doi:10.1158/1538-7445.AM2015-636

First-in-human evaluation of the human monoclonal antibody vantictumab (OMP-18R5; anti-Frizzled) targeting the WNT pathway in a phase I study for patients with advanced solid tumors.

2540 Background: The WNT pathway is a key oncologic pathway in numerous tumor types. Vantictumab is a first-in-class anti-Cancer Stem Cell (CSC) antibody that interacts with the extracellular domain of 5 Frizzled receptors (1, 2, 5, 7, 8) and blocks canonical Wnt signaling (PNAS 109, 11717). In patient-derived xenograft models, vantictumab inhibits growth of many tumor types, reduces CSC frequency, promotes differentiation of tumor cells, and synergizes with many chemotherapeutic agents. Methods: Using a 3+3 design, vantictumab was given intravenously, first every 1 week (q1w) or q2w, and, ultimately, q3w. Objectives were determination of maximum tolerated dose, safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. Results: 18 patients have been treated in 5 dose-escalation cohorts (0.5 & 1 mg/kg q1w; 0.5 mg/kg q2w; 1 & 2.5 mg/kg q3w). Most common related adverse events (AEs) included Grade 1 and 2 fatigue, vomiting, abdominal pain, constipation, diarrhea and nausea. Only related Grade ≥3 AEs were dose-limiting toxicities of Grade 3 diarrhea and vomiting in 1 patient at 1 mg/kg q1w. Vantictumab clearance was dose-dependent, consistent with target-mediated drug disposition, with the half-life ranging from 1.5 (0.5 mg/kg) to 3 days (2.5 mg/kg). Exposure at highest dosed cohorts correlates with efficacy in preclinical tumor models. PD biomarkers indicate manipulation of WNT pathway in patient tumors and surrogate tissue. 1 patient at 0.5 mg/kg q1w had a bone fracture on Day 110 with an ~4-fold increase by Day 28 of β-C-terminal telopeptide (β-CTX), a marker for bone degradation. A revised safety plan and less frequent dosing enabled further dose escalation. Upon β-CTX doubling, 2 patients received zoledronic acid, and β-CTX returned to baseline. 3 patients with neuroendocrine tumors (NETs) had stable disease (SD) for 7+, 3.5, and 9+ months; ~2-, 4- and 7-fold longer, respectively, than on prior therapy. Conclusions: Vantictumab is well tolerated up to 2.5 mg/kg q3w. Bone toxicity appears manageable and reversible. Prolonged SD in 3 patients with NETs may represent single-agent activity. Dose escalation continues. Clinical trial information: NCT01345201.

Abstract 4617: BAY-ACC001, a novel ACC inhibitor, regulates fatty acids synthesis and lipid survival signaling with promising in vitro and in vivo activities in multiple preclinical tumor models.

Abstract Unlike normal cells, tumors undergo intensified de novo biogenesis of fatty acids (FAs) irrespective of the available circulating lipids. Acetyl-CoA carboxylase 1 (ACC) controls the rate limiting step in FA synthesis. ACC1 is up-regulated in a variety of human tumors and strongly associated with poorer prognosis in some tumor indications. Although targeting lipogenesis for cancer treatment appeared having strong rationale, drug discovery in this field has not been fully explored due to the lack of powerful tools for both evaluation and understanding the mode of action. Here we report the identification and the functional characterization of a highly selective ACC inhibitor BAY-ACC001, a ketoenol derivative using various pharmacological and lipidomic approaches to address the mechanism of ACC inhibition in cancer cells and its efficacy in preclinical tumor models. BAY-ACC001 inhibits human ACC1 and ACC1 with biochemical IC50s of 278 nM and 2590 nM measured by ACC1- or ACC2- mediated generation of ADP, respectively. In a cellular mechanistic assay using MCF7 tumor cells, BAY-ACC001 potently inhibited malonyl-CoA synthesis with an IC50 of 62 nM. Profiling BAY-ACC001 in a panel of 100 tumor cell lines revealed strong anti-proliferative activity in a sub-set of tumor cell lines with IC50s at low 3-digit nanomolar. Of note, in contrast to the potent activity in tumor cell lines, e.g. apoptosis induction in MCF7 breast tumor cells, ACC inhibitor showed only weak anti-proliferative effect and could not induce apoptosis in a set of non-transformed mammary epithelial cells. To elucidate the anti-tumor MoA, the levels of lipid components (∼400 lipid molecules) in MCF7 cells were analyzed using lipidomic technology. Interestingly, ACC inhibition did not lead to a simple depletion of lipid in cells, evident from a significant increase in tumor apoptosis-related lipid signaling molecules ceramides. These results suggested a bi-direction linkage between FA synthesis and the regulation of tumor cell survival. Single administration of BAY-ACC001 orally at 10 mg/kg (the maximum tolerable dose is 30 mg/kg, BID) in mice strongly reduced malonyl CoA levels in tumors. Treatment of BAY-ACC001 as single agent was efficacious in multiple tumor models, including MCF7 (breast), PC3 (prostate), HCT116 (colon) and MDA-MB-435 (melanoma) xenograft models; partial tumor remissions observed in MCF7 model. Furthermore, combination with Tamoxifen was synergistic in the MCF7 tumor model in vitro and in vivo. In conclusion, the prevalence of the exacerbated de novo FA synthesis observed in primary and metastatic tumors, the ACC-mediated novel survival signal transduction discovered in this study, and robust in vitro and in vivo anti-tumor activity of BAY-ACC001 provide a strong rationale for developing novel therapeutics targeting ACC for cancer treatment. Citation Format: Ningshu Liu, Maher Najar, Arne Scholz, Knut Eis, Ulf Bömer, Philip Lienau, Kai Thede, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. BAY-ACC001, a novel ACC inhibitor, regulates fatty acids synthesis and lipid survival signaling with promising in vitro and in vivo activities in multiple preclinical tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4617. doi:10.1158/1538-7445.AM2013-4617

Immunotherapeutic Applications of IL-15

IL-15 is a member of the IL-2 family of cytokines, which play a fundamental role in innate and adaptive immune responses. IL-15 has pleiotropic immune-enhancing activities, as it stimulates NK, T and NKT cell proliferation, survival and effector functions. In view of these properties, IL-15 is regarded as a good candidate for cancer immunotherapy. This possibility is reinforced by its low toxicity and efficacy in preclinical tumor models. The use of IL-15 to boost the immune response in HIV infection has also been proposed, although further studies are required to establish potential risks and benefits. Clinical trials of IL-15 have been initiated in cancer patients and in HIV vaccination and will elucidate the potential of IL-15-based immunotherapy. The purpose of this review is to provide an update on the potential applications of IL-15 in cancer immunotherapy and HIV infection.

N,N-Dimethylsphingosine conjugates of poly- l-glutamic acid: Synthesis, characterization, and initial biological evaluation

Poly- l-glutamic acid (PGA) has previously been demonstrated to be an effective backbone for creating a hydrophilic prodrug of the established anti-tumor agent, paclitaxel, the active agent in Taxol; this approach has obviated the need for the toxic Cremophor excipient, used to enhance the solubility of paclitaxel in the clinical formulation. In order to form hydrophilic prodrugs of the hydrophobic pro-apoptotic sphingolipid, N,N-dimethylsphingosine (DMSP), PGA was condensed with DMSP, previously modified with coumarin to allow spectroscopic detection during conjugate synthesis, to yield PGA–DMSP. Conjugates with different loadings of DMSP were prepared and evaluated for in vitro cytotoxicity against two human breast adenocarcinoma cell lines. Time- and loading-dependent expression of cytotoxicity was observed, such that endpoints essentially equivalent to those observed with free-DMSP were achieved, but in a more protracted manner, consistent with prodrug behavior. PGA–DMSP was initially evaluated for toxicity in female nude mice, and administration of high net levels of DMSP, exceeding those achievable with free-DMSP, was well-tolerated. We propose that PGA–DMSP conjugates merit evaluation for anti-tumor efficacy in pre-clinical tumor models.

Ratiometric dosing of anticancer drug combinations: Controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice

Anticancer drug combinations can act synergistically or antagonistically against tumor cells in vitro depending on the ratios of the individual agents comprising the combination. The importance of drug ratios in vivo, however, has heretofore not been investigated, and combination chemotherapy treatment regimens continue to be developed based on the maximum tolerated dose of the individual agents. We systematically examined three different drug combinations representing a range of anticancer drug classes with distinct molecular mechanisms (irinotecan/floxuridine, cytarabine/daunorubicin, and cisplatin/daunorubicin) for drug ratio-dependent synergy. In each case, synergistic interactions were observed in vitro at certain drug/drug molar ratio ranges (1:1, 5:1, and 10:1, respectively), whereas other ratios were additive or antagonistic. We were able to maintain fixed drug ratios in plasma of mice for 24 hours after i.v. injection for all three combinations by controlling and overcoming the inherent dissimilar pharmacokinetics of individual drugs through encapsulation in liposomal carrier systems. The liposomes not only maintained drug ratios in the plasma after injection, but also delivered the formulated drug ratio directly to tumor tissue. In vivo maintenance of drug ratios shown to be synergistic in vitro provided increased efficacy in preclinical tumor models, whereas attenuated antitumor activity was observed when antagonistic drug ratios were maintained. Fixing synergistic drug ratios in pharmaceutical carriers provides an avenue by which anticancer drug combinations can be optimized prospectively for maximum therapeutic activity during preclinical development and differs from current practice in which dosing regimens are developed empirically in late-stage clinical trials based on tolerability.