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In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O2 levels (normoxic) which are 4-fold higher than O2 levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to mark hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies. We used a novel homogeneous live-cell permeant HypoxiTRAK™ (HPTK) molecular probe compatible with high content imaging detection, analysis, and throughput to identify and quantify hypoxia levels in live HNC multicellular tumor spheroid (MCTS) cultures over time. Accumulation of fluorescence HPTK metabolite in live normoxic HNC MCTS cultures correlated with hypoxia detection by both pimonidazole and HIF-1α staining. In HNC MCTSs, hypoxic cytotoxicity ratios for the hypoxia activated prodrugs (HAP) evofosfamide and tirapazamine were much smaller than have been reported for uniformly hypoxic 2D monolayers in gas chambers, and many viable cells remained after HAP exposure. Cells in solid tumors and MCTSs experience three distinct O2 microenvironments dictated by their distances from blood vessels or MCTS surfaces, respectively; oxic, hypoxic, or intermediate levels of hypoxia. These studies support the application of more physiologically relevant in vitro 3D models that recapitulate the heterogeneous microenvironments of solid tumors for preclinical cancer drug discovery.

Aims: In hypoxic tumor microenvironments, the strongly reducing redox environment reduces evofosfamide (TH-302) to release a cytotoxic bromo-isophosphoramide (Br-IPM) moiety. This drug therefore preferentially attacks hypoxic regions in tumors where other standard anticancer treatments such as chemotherapy and radiation therapy are often ineffective. Various combination therapies with evofosfamide have been proposed and tested in preclinical and clinical settings. However, the treatment effect of evofosfamide monotherapy on tumor hypoxia has not been fully understood, partly due to the lack of quantitative methods to assess tumor pO(2) in vivo. Here, we use quantitative pO(2) imaging by electron paramagnetic resonance (EPR) to evaluate the change in tumor hypoxia in response to evofosfamide treatment using two pancreatic ductal adenocarcinoma xenograft models: MIA Paca-2 tumors responding to evofosfamide and Su.86.86 tumors that do not respond. Results: EPR imaging showed that oxygenation improved globally after evofosfamide treatment in hypoxic MIA Paca-2 tumors, in agreement with the ex vivo results obtained from hypoxia staining by pimonidazole and in apparent contrast to the decrease in K(trans) observed in dynamic contrast-enhanced magnetic resonance imaging (DCE MRI). Innovations: The observation that evofosfamide not only kills the hypoxic region of the tumor but also improves oxygenation in the residual tumor regions provides a rationale for combination therapies using radiation and antiproliferatives post evofosfamide for improved outcomes. Conclusion: This study suggests that reoxygenation after evofosfamide treatment is due to decreased oxygen demand rather than improved perfusion. Following the change in pO(2) after treatment may therefore yield a way of monitoring treatment response. Antioxid. Redox Signal.

Purpose: As hypoxia can mediate resistance to immunotherapy, we investigated the safety, tolerability, and efficacy of combining evofosfamide, a prodrug that alleviates hypoxia, with ipilimumab, an immune checkpoint inhibitor, in immunologically “cold” cancers, which are intrinsically insensitive to immunotherapy, as well as in “hot/warm” metastatic cancers that are, atypical of such cancers, resistant to immunotherapy. Patients and Methods: In a phase I, 3+3 dose-escalation trial (NCT03098160), evofosfamide (400–640 mg/m2) and ipilimumab (3 mg/kg) were administered in four 3-week cycles. The former was administered on days 1 and 8 of cycles 1–2, while the latter was administered on day 8 of cycles 1–4. Response was assessed using immune-related RECIST and retreatment was allowed, if deemed beneficial, after completion of cycle 4 or at progression. Results: Twenty-two patients were enrolled, of whom 21 were evaluable, encompassing castration-resistant prostate cancer (n = 11), pancreatic cancer (n = 7), immunotherapy-resistant melanoma (n = 2), and human papillomavirus–negative head and neck cancer (n = 1). Drug-related hematologic toxicities, rash, fever, nausea, vomiting, and elevation of liver enzymes were observed in > 10% of patients. The most common drug-related grade 3 adverse event was alanine aminotransferase elevation (33.3%). Two patients discontinued ipilimumab and 4 required evofosfamide deescalation due to toxicity. Of 18 patients with measurable disease at baseline, 3 (16.7%) achieved partial response and 12 (66.7%) achieved stable disease. The best responses were observed at 560 mg/m2 evofosfamide. Preexisting immune gene signatures predicted response to therapy, while hypermetabolic tumors predicted progression. Responders also showed improved peripheral T-cell proliferation and increased intratumoral T-cell infiltration into hypoxia. Conclusions: No new or unexpected safety signals were observed from combining evofosfamide and ipilimumab, and evidence of therapeutic activity was noted.

Rationale: Hypoxic regions (habitats) within tumors are heterogeneously distributed and can be widely variant. Hypoxic habitats are generally pan-therapy resistant. For this reason, hypoxia-activated prodrugs (HAPs) have been developed to target these resistant volumes. The HAP evofosfamide (TH-302) has shown promise in preclinical and early clinical trials of sarcoma. However, in a phase III clinical trial of non-resectable soft tissue sarcomas, TH-302 did not improve survival in combination with doxorubicin (Dox), possibly due to a lack of patient stratification based on hypoxic status. Therefore, we used magnetic resonance imaging (MRI) to identify hypoxic habitats and non-invasively follow therapies response in sarcoma mouse models. Methods: We developed deep-learning (DL) models to identify hypoxia, using multiparametric MRI and co-registered histology, and monitored response to TH-302 in a patient-derived xenograft (PDX) of rhabdomyosarcoma and a syngeneic model of fibrosarcoma (radiation-induced fibrosarcoma, RIF-1). Results: A DL convolutional neural network showed strong correlations (>0.76) between the true hypoxia fraction in histology and the predicted hypoxia fraction in multiparametric MRI. TH-302 monotherapy or in combination with Dox delayed tumor growth and increased survival in the hypoxic PDX model (p

BackgroundThe majority of patients with pancreatic ductal adenocarcinoma (PDAC) fail to derive any durable responses from single agent immune checkpoint blockade therapy. This refractory state originates from PDAC's unique tumor microenvironment that is densely populated by immunosuppressive myeloid cells while excluding most antitumor CD8 T cells.1 In addition, PDAC is highly hypoxic and exhibits poor vascularity, both qualities which further limit antitumor immunity.2 3 We showed that the hypoxia-activated prodrug TH-302 (Evofosfamide) potentiates immunotherapy responses.4 Mechanistically, TH-302 decreases intratumoral hypoxia and initiates normalization of the tumor vasculature. While TH-302 facilitates a cellular remodeling process that diminishes tumor hypoxia, the nature of the vascular remodeling involved remains unknown, as do the downstream consequences for the composition of the tumor microenvironment and responsiveness to immunotherapy. We hypothesized that anti-angiogenic therapy and Evofosfamide might cooperate to normalize tumor vasculature and diminish hypoxia.MethodsTH-302 and a vascular endothelial growth factor receptor-2 (VEGFR-2) blocking antibody were used to treat several syngeneic murine models, including orthotopic pancreatic cancer and a transplantable model of prostate cancer. Immunofluorescence and flow cytometry were used to assess intratumoral hypoxia, vessel normalization, and tumor immune infiltrate.ResultsWe find that anti-VEGFR-2 (DC101) in combination with TH-302 demonstrates a cooperative benefit to combat both orthotopically implanted pancreatic cancer and transplantable prostate cancer. Combination therapy reduces intratumoral hypoxia, leads to pruning of the tumor vasculature, and increases the infiltration of endothelial cells into hypoxic regions. Across models, the combination of DC101 and TH-302 significantly enhance CD8 T cell function and limits their exhausted state. At the same time, tumor associated macrophages exhibit decreased expression of M2-like features. Similar to other anti-angiogenic therapies, combination DC101 and TH-302 leads to an increased frequency of PD-L1 expressing cells. Concurrent anti-PD-1 failed to provide any additional therapeutic benefit, which in part may be due poor CD8 T cell infiltration. Instead, we find that CD40 agonist therapy is improved when combined with TH-302 and DC101.ConclusionsTH-302 and DC101 utilize unique yet complementary mechanisms to improve the survival of mice challenged with pancreatic or prostate tumors. This combination relieves hypoxia and simultaneously reinvigorates T cell function and reduces macrophage mediated immunosuppression. In this setting, CD40 agonist therapy provides an additive benefit in prolonging mouse survival. Put together, these data indicate that targeted hypoxia reduction with anti-angiogenic therapy remodels the tumor microenvironment and enhances immunotherapy responses in PDAC.ReferencesBear AS, Vonderheide RH, O'Hara MH. Challenges and opportunities for pancreatic cancer immunotherapy. Cancer Cell. 2020;38(6):788–802. doi: 10.1016/j.ccell.2020.08.004. Epub 2020 Sep 17. PMID: 32946773; PMCID: PMC7738380.Koong AC, Mehta VK, Le QT, Fisher GA, Terris DJ, Brown JM, Bastidas AJ, Vierra M. Pancreatic tumors show high levels of hypoxia. Int J Radiat Oncol Biol Phys 2000;48(4):919–22. doi: 10.1016/s0360-3016(00)00803-8. PMID: 11072146.Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D, Frese KK, Denicola G, Feig C, Combs C, Winter SP, Ireland-Zecchini H, Reichelt S, Howat WJ, Chang A, Dhara M, Wang L, Rückert F, Grützmann R, Pilarsky C, Izeradjene K, Hingorani SR, Huang P, Davies SE, Plunkett W, Egorin M, Hruban RH, Whitebread N, McGovern K, Adams J, Iacobuzio-Donahue C, Griffiths J, Tuveson DA. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 2009;324(5933):1457–61. doi: 10.1126/science.1171362. Epub 2009 May 21. PMID: 19460966; PMCID: PMC2998180.Jayaprakash P, Ai M, Liu A, Budhani P, Bartkowiak T, Sheng J, Ager C, Nicholas C, Jaiswal AR, Sun Y, Shah K, Balasubramanyam S, Li N, Wang G, Ning J, Zal A, Zal T, Curran MA. Targeted hypoxia reduction restores T cell infiltration and sensitizes prostate cancer to immunotherapy. J Clin Invest 2018;128(11):5137–5149. doi: 10.1172/JCI96268. Epub 2018 Oct 15. PMID: 30188869; PMCID: PMC6205399.

Purpose: Hypoxia is a common characteristic of many tumor microenvironments, and it has been shown to promote suppression of antitumor immunity. Despite strong biological rationale, longitudinal correlation of hypoxia and response to immunotherapy has not been investigated. Experimental Design: In this study, we probed the tumor and its surrounding microenvironment with 18F-FMISO PET imaging to noninvasively quantify tumor hypoxia in vivo prior to and during PD-1 and CTLA-4 checkpoint blockade in preclinical models of breast and colon cancer. Results: Longitudinal imaging identified hypoxia as an early predictive biomarker of therapeutic response (prior to anatomic changes in tumor volume) with a decreasing standard uptake value (SUV) ratio in tumors that effectively respond to therapy. PET signal correlated with ex vivo markers of tumor immune response including cytokines (IFNγ, GZMB, and TNF), damage-associated molecular pattern receptors (TLR2/4), and immune cell populations (macrophages, dendritic cells, and cytotoxic T cells). Responding tumors were marked by increased inflammation that were spatially distinct from hypoxic regions, providing a mechanistic understanding of the immune signaling pathways activated. To exploit image-guided combination therapy, hypoxia signal from PET imaging was used to guide the addition of a hypoxia targeted treatment to nonresponsive tumors, which ultimately provided therapeutic synergy and rescued response as determined by longitudinal changes in tumor volume. Conclusions: The results generated from this work provide an immediately translatable paradigm for measuring and targeting hypoxia to increase response to immune checkpoint therapy and using hypoxia imaging to guide combinatory therapies.

Highlights • TH-302 combined with RT enhances tumor growth delay in esophageal cancer models. • This combination does not increase normal tissue toxicity on the short and long term. • The increased therapeutic index (1.38) paves the way for future clinical trials., Background and purpose Esophageal cancer incidence is increasing and is rarely curable. Hypoxic tumor areas cause resistance to conventional therapies, making them susceptible for treatment with hypoxia-activated prodrugs (HAPs). We investigated in vivo whether the HAP evofosfamide (TH-302) could increase the therapeutic ratio by sensitizing esophageal carcinomas to radiotherapy without increasing normal tissue toxicity. Materials and methods To assess therapeutic efficacy, growth of xenografted esophageal squamous cell (OE21) or adeno (OE19) carcinomas was monitored after treatment with TH-302 (50 mg/kg, QD5) and irradiation (sham or 10 Gy). Short- and long-term toxicity was assessed in a gut mucosa and lung fibrosis irradiation model, sensitive to acute and late radiation injury respectively. Mice were injected with TH-302 (50 mg/kg, QD5) and the abdominal area (sham, 8 or 10 Gy) or the upper part of the right lung (sham, 20 Gy) was irradiated. Damage to normal tissues was assessed 84 hours later by histology and blood plasma citrulline levels (gut) and for up to 1 year by non-invasive micro CT imaging (lung). Results The combination treatment of TH-302 with radiotherapy resulted in significant tumor growth delay in OE19 (P = 0.02) and OE21 (P = 0.03) carcinomas, compared to radiotherapy only. Irradiation resulted in a dose-dependent decrease of crypt survival (P

Tumor hypoxia contributes to resistance to anticancer therapies. Hypoxia-activated prodrugs (HAPs) selectively target hypoxic cells and their activity can extend to well-oxygenated areas of tumors via diffusion of active metabolites. This type of bystander effect has been suggested to be responsible for the single agent activity of the clinical-stage HAP evofosfamide (TH-302) but direct evidence is lacking. To dissect the contribution of bystander effects to TH-302 activity, we implemented a Green's function pharmacokinetic (PK) model to simulate the spatial distribution of O2, TH-302 and its cytotoxic metabolites, bromo-isophosphoramide mustard (Br-IPM) and its dichloro derivative isophosphoramide mustard (IPM), in two digitized tumor microvascular networks. The model was parameterized from literature and experimentally, including measurement of diffusion coefficients of TH-302 and its metabolites in multicellular layer cultures. The latter studies demonstrate that Br-IPM and IPM cannot diffuse significantly from the cells in which they are generated, although evidence was obtained for diffusion of the hydroxylamine metabolite of TH-302. The spatially resolved PK model was linked to a pharmacodynamic (PD) model that describes cell killing probability at each point in the tumor microregion as a function of Br-IPM and IPM exposure. The resulting PK/PD model accurately predicted previously reported monotherapy activity of TH-302 in H460 tumors, without invoking a bystander effect, demonstrating that the notable single agent activity of TH-302 in tumors can be accounted for by significant bioreductive activation of TH-302 even in oxic regions, driven by the high plasma concentrations achievable with this well-tolerated prodrug.

Purpose: The presence of hypoxia in the diseased bone marrow presents a new therapeutic target for multiple myeloma. Evofosfamide (formerly TH-302) is a 2-nitroimidazole prodrug of the DNA alkylator, bromo-isophosphoramide mustard, which is selectively activated under hypoxia. This trial was designed as a phase I/II study investigating evofosfamide in combination with dexamethasone, and in combination with bortezomib and dexamethasone in relapsed/refractory multiple myeloma. Patients and Methods: Fifty-nine patients initiated therapy, 31 received the combination of evofosfamide and dexamethasone, and 28 received the combination of evofosfamide, bortezomib, and dexamethasone. Patients were heavily pretreated with a median number of prior therapies of 7 (range: 2–15). All had previously received bortezomib and immunomodulators. The MTD, treatment toxicity, and efficacy were determined. Results: The MTD was established at 340 mg/m2 evofosfamide + dexamethasone with dose-limiting mucositis at higher doses. For the combination of evofosfamide, bortezomib, and dexamethasone, no patient had a dose-limiting toxicity (DLT) and the recommended phase II dose was established at 340 mg/m2. The most common ≥grade 3 adverse events (AE) were thrombocytopenia (25 patients), anemia (24 patients), neutropenia (15 patients), and leukopenia (9 patients). Skin toxicity was reported in 42 (71%) patients. Responses included 1 very good partial response (VGPR), 3 partial response (PR), 2 minor response (MR), 20 stable disease (SD), and 4 progressive disease (PD) for evofosfamide + dexamethasone and 1 complete response (CR), 2 PR, 1 MR, 18 SD, and 5 PD for evofosfamide + bortezomib + dexamethasone. Disease stabilization was observed in over 80% and this was reflective of the prolonged overall survival of 11.2 months. Conclusions: Evofosfamide can be administered at 340 mg/m2 twice a week with or without bortezomib. Clinical activity has been noted in patients with heavily pretreated relapsed refractory multiple myeloma.

Tumour hypoxia has been associated with increased resistance to various cancer treatments, particularly radiation therapy. Conversely, tumour hypoxia is a validated and ideal target for guided cancer drug delivery. For this reason, hypoxia-activated prodrugs (HAPs) have been developed, which remain inactive in the body until in the presence of tissue hypoxia, allowing for an activation tendency in hypoxic regions. We present here an experimentally motivated mathematical model predicting the effectiveness of HAPs in a variety of clinical settings. We first examined HAP effectiveness as a function of the amount of tumour hypoxia and showed that the drugs have a larger impact on tumours with high levels of hypoxia. We then combined HAP treatment with radiation to examine the effects of combination therapies. Our results showed radiation-HAP combination therapies to be more effective against highly hypoxic tumours. The analysis of combination therapies was extended to consider schedule sequencing of the combination treatments. These results suggested that administering HAPs before radiation was most effective in reducing total cell number. Finally, a sensitivity analysis of the drug-related parameters was done to examine the effect of drug diffusivity and enzyme abundance on the overall effectiveness of the drug. Altogether, the results highlight the importance of the knowledge of tumour hypoxia levels before administration of HAPs in order to ensure positive results.

Tumour hypoxia has been associated with increased resistance to various cancer treatments, particularly radiation therapy. Conversely, tumour hypoxia is a validated and ideal target for guided cancer drug delivery. For this reason, hypoxia-activated prodrugs (HAPs) have been developed, which remain inactive in the body until in the presence of tissue hypoxia, allowing for an activation tendency in hypoxic regions. We present here an experimentally motivated mathematical model predicting the effectiveness of HAPs in a variety of clinical settings. We first examined HAP effectiveness as a function of the amount of tumour hypoxia and showed that the drugs have a larger impact on tumours with high levels of hypoxia. We then combined HAP treatment with radiation to examine the effects of combination therapies. Our results showed radiation-HAP combination therapies to be more effective against highly hypoxic tumours. The analysis of combination therapies was extended to consider schedule sequencing of the combination treatments. These results suggested that administering HAPs before radiation was most effective in reducing total cell number. Finally, a sensitivity analysis of the drug-related parameters was done to examine the effect of drug diffusivity and enzyme abundance on the overall effectiveness of the drug. Altogether, the results highlight the importance of the knowledge of tumour hypoxia levels before administration of HAPs in order to ensure positive results.

Background Hypoxia-activated prodrugs (HAPs) are a promising class of antineoplastic agents that can selectively eliminate hypoxic tumor cells. The present study evaluates the hypoxia-selectivity and antitumor activity of CP-506, a DNA alkylating HAP with favorable pharmacological properties. Methods Stoichiometry of reduction, one-electron affinity, and back-oxidation rate of CP-506 were characterized by fast-reaction radiolytic methods. In vitro, 2D monolayer and 3D spheroid and multicellular layer cultures were used to investigate the hypoxia-selectivity of CP-506. In vivo, the causal relationship between tumor oxygenation and antitumor effects of CP-506 was assessed. Mice bearing a range of human tumor xenografts were exposed to CP-506 and tumor growth was monitored. A multivariate linear regression model was used to identify factors associated with CP-506 treatment outcome. Results Net reduction, metabolism, and cytotoxicity of CP-506 were maximally inhibited at oxygen concentrations above 1 µM (0.1% O2). CP-506 demonstrated cytotoxicity selectively in hypoxic 2D and 3D cell cultures with normoxic/anoxic IC50 ratios up to 203. In vivo, the antitumor effects of CP-506 were selective for hypoxic tumor cells and causally related to tumor oxygenation. CP-506 effectively decreased the hypoxic fraction and inhibited growth of a wide range of hypoxic xenografts. Two well-oxygenated models were refractory to treatment despite intrinsic anoxic sensitivity in vitro. A multivariate regression analysis revealed baseline tumor hypoxia and in vitro sensitivity to CP-506 to significantly correlate with treatment response. Conclusions Our results demonstrate that CP-506 selectively sterilizes hypoxic tumor cells and has broad antitumor activity. Our data also indicate that tumor hypoxia and cellular sensitivity to CP-506 are strong determinants of the antitumor effects of CP-506.

Background Triple negative breast cancer (TNBC) is an aggressive tumor with extremely high mortality that results from its lack of effective therapeutic targets. As an adhesion molecule related to tumorigenesis and tumor metastasis, cluster of differentiation-44 (also known as CD44) is overexpressed in TNBC. Moreover, CD44 can be effectively targeted by a specific hyaluronic acid analog, namely, chitosan oligosaccharide (CO). In this study, a CO-coated liposome was designed, with Photochlor (HPPH) as the 660 nm light mediated photosensitizer and evofosfamide (also known as TH302) as the hypoxia-activated prodrug. The obtained liposomes can help diagnose TNBC by fluorescence imaging and produce antitumor therapy by synergetic photodynamic therapy (PDT) and chemotherapy. Results Compared with the nontargeted liposomes, the targeted liposomes exhibited good biocompatibility and targeting capability in vitro; in vivo, the targeted liposomes exhibited much better fluorescence imaging capability. Additionally, liposomes loaded with HPPH and TH302 showed significantly better antitumor effects than the other monotherapy groups both in vitro and in vivo. Conclusion The impressive synergistic antitumor effects, together with the superior fluorescence imaging capability, good biocompatibility and minor side effects confers the liposomes with potential for future translational research in the diagnosis and CD44-overexpressing cancer therapy, especially TNBC. Graphic abstract

Simple Summary Despite many therapeutic approaches attempted over the last years, the prognosis of children with high-grade glioma or diffuse intrinsic pontine glioma remains dismal. Hypoxia-activated prodrugs (HAPs) were developed to target hypoxic areas within solid tumors as gliomas. Evofosfamide (Evo) is a 2nd generation HAP exhibiting significant preclinical and clinical activities against adult glioblastoma. We thus investigated the potential of Evo in six pediatric glioma cell lines. Interestingly, we showed that the growth of all cell lines was inhibited by Evo, mainly under hypoxia as expected. We also evidenced a significant synergism between Evo and three drugs widely used in pediatric oncology. Finally, Evo appeared able to potentiate the effect of ionizing radiations. Since these tumors are highly hypoxic and Evo appears effective in hypoxic glioma cells as a single drug and in combination with radio- and chemotherapy, hypoxia-activated prodrugs could represent a promising therapeutic option for children with brain tumors. Abstract Hypoxia is a hallmark of many solid tumors and is associated with resistance to anticancer treatments. Hypoxia-activated prodrugs (HAPs) were developed to target the hypoxic regions of these tumors. Among 2nd generation HAPs, Evofosfamide (Evo, also known as TH-302) exhibits preclinical and clinical activities against adult glioblastoma. In this study, we evaluated its potential in the field of pediatric neuro-oncology. We assessed the efficacy of Evo in vitro as a single drug, or in combination with SN38, doxorubicin, and etoposide, against three pediatric high-grade glioma (pHGG) and three diffuse intrinsic pontine glioma (DIPG) cell lines under hypoxic conditions. We also investigated radio-sensitizing effects using clonogenic assays. Evo inhibited the growth of all cell lines, mainly under hypoxia. We also highlighted a significant synergism between Evo and doxorubicin, SN38, or etoposide. Finally, Evo radio-sensitized the pHGG cell line tested, both with fractionated and single-dose irradiation schedules. Altogether, we report here the first preclinical proof of evidence about Evofosfamide efficiency against hypoxic pHGG and DIPG cells. Since such tumors are highly hypoxic, and Evo potentiates the effect of ionizing radiation and chemotherapy, it appears as a promising therapeutic strategy for children with brain tumors.

Tumoral hypoxia correlates with worse outcomes in glioblastoma (GBM). While bevacizumab is routinely used to treat recurrent GBM, it may exacerbate hypoxia. Evofosfamide is a hypoxia-targeting prodrug being tested for recurrent GBM. To characterize resistance to bevacizumab and identify those with recurrent GBM who may benefit from evofosfamide, we ascertained MRI features and hypoxia in patients with GBM progression receiving both agents. Thirty-three patients with recurrent GBM refractory to bevacizumab were enrolled. Patients underwent MR and 18F-FMISO PET imaging at baseline and 28 days. Tumor volumes were determined, MRI and 18F-FMISO PET-derived parameters calculated, and Spearman correlations between parameters assessed. Progression-free survival decreased significantly with hypoxic volume [hazard ratio (HR) = 1.67, 95% confidence interval (CI) 1.14 to 2.46, P = 0.009] and increased significantly with time to the maximum value of the residue (Tmax) (HR = 0.54, 95% CI 0.34 to 0.88, P = 0.01). Overall survival decreased significantly with hypoxic volume (HR = 1.71, 95% CI 1.12 to 12.61, p = 0.01), standardized relative cerebral blood volume (srCBV) (HR = 1.61, 95% CI 1.09 to 2.38, p = 0.02), and increased significantly with Tmax (HR = 0.31, 95% CI 0.15 to 0.62, p s = 0.77, P s = 0.75, P

Background: Sorafenib (Sor) remains a first-line option for hepatocellular carcinoma (HCC) or refractory renal cell carcinomas (RCC). PLC/PRF/5 HCC model showed upregulation of hypoxia with enhanced efficacy when Sor is combined with hypoxia-activated prodrug evofosfamide (Evo). Methods: This phase IB 3 + 3 design investigated 3 Evo dose levels (240, 340, 480 mg/m2 on days 8, 15, 22), combined with Sor 200 mg orally twice daily (po bid) on days 1-28 of a 28-day cycle. Primary objectives included determining maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of Sor + Evo. Results: Eighteen patients were enrolled (median age 62.5 years; 17 male /1 female; 12 HCC/6 RCC) across three dose levels (DL0: Sor 200 mg bid/Evo 240 mg/m2 [n=6], DL1:Sor 200 mg bid/Evo 480 mg/m2 [n=5], DL1a: Sor 200 mg bid/Evo 340 mg/m2 [n=7]). Two dose-limiting toxicities (DLTs) were reported with Evo 480 mg/m2 (grade 3 mucositis, grade 4 hepatic failure). Grade 3 rash DLT was observed in one patient at Evo 240 mg/m2. No DLTs were observed at Evo 340 mg/m2. MTD and RP2D were established as Sor 200 mg/Evo 340 mg/m2 and Sor 200/Evo 240 mg/m2, respectively. The most common treatment-related adverse events included fatigue, hand-foot syndrome, hypertension, and nausea/vomiting. Two partial responses were observed, one each at DL0 and DL1a.; disease control rate was 55%.Conclusions: RP2D was established as sorafenib 200 mg bid + Evo 240 mg/m2. While preliminary anti-tumor activity was observed, future development must account for advances in immunotherapy in HCC/RCC. NCT01497444Date of registration December 20, 2011

PurposeSuccess of new cancer therapies relies strongly on effective selection of the target patient populations. We hypothesize that computational analysis of imaging data can be used for patient enrichment in clinical trials and hence aimed to establish the appropriate framework for this analysis.MethodsThis was tested among soft-tissue sarcoma (STS) patients accrued into a randomized clinical trial (SARC021) that evaluated the efficacy of evofosfamide (Evo), a hypoxia activated prodrug, in combination with doxorubicin (Dox). Notably, SARC021 failed to meet its overall survival (OS) objective. We tested whether a radiomic biomarker-driven inclusion/exclusion criterion could have been used to result in a significant treatment benefit of the Evo+Dox combination compared to Dox monotherapy. 164 radiomics features were extracted from 303 SARC021 patients with lung metastases, divided into training and test sets.ResultsA single radiomics feature, Short Run Emphasis, was identified as the most informative. Combined into a model along with histological classification and smoking history, an enriched subset (42%) of patients had longer OS in Evo+Dox vs. Dox groups [p=0.01, Hazard Ratio (HR) =0.57 (0.36-0.90)]. Applying the same model and threshold value in an independent test set confirmed the significant survival difference [p=0.002, HR=0.29 (0.13-0.63)], identifying patients most likely to benefit from doxorubicin alone.ConclusionThe study presents a first of its kind clinical-radiomic approach for patient enrichment in clinical trials. We show that, had an appropriate model been used for selective patient inclusion, SARC021 trial could have met its primary survival objective for patients with metastatic STS.

Simple Summary Despite many therapeutic approaches attempted over the last years, the prognosis of children with high-grade glioma or diffuse intrinsic pontine glioma remains dismal. Hypoxia-activated prodrugs (HAPs) were developed to target hypoxic areas within solid tumors as gliomas. Evofosfamide (Evo) is a 2nd generation HAP exhibiting significant preclinical and clinical activities against adult glioblastoma. We thus investigated the potential of Evo in six pediatric glioma cell lines. Interestingly, we showed that the growth of all cell lines was inhibited by Evo, mainly under hypoxia as expected. We also evidenced a significant synergism between Evo and three drugs widely used in pediatric oncology. Finally, Evo appeared able to potentiate the effect of ionizing radiations. Since these tumors are highly hypoxic and Evo appears effective in hypoxic glioma cells as a single drug and in combination with radio- and chemotherapy, hypoxia-activated prodrugs could represent a promising therapeutic option for children with brain tumors. Hypoxia is a hallmark of many solid tumors and is associated with resistance to anticancer treatments. Hypoxia-activated prodrugs (HAPs) were developed to target the hypoxic regions of these tumors. Among 2nd generation HAPs, Evofosfamide (Evo, also known as TH-302) exhibits preclinical and clinical activities against adult glioblastoma. In this study, we evaluated its potential in the field of pediatric neuro-oncology. We assessed the efficacy of Evo in vitro as a single drug, or in combination with SN38, doxorubicin, and etoposide, against three pediatric high-grade glioma (pHGG) and three diffuse intrinsic pontine glioma (DIPG) cell lines under hypoxic conditions. We also investigated radio-sensitizing effects using clonogenic assays. Evo inhibited the growth of all cell lines, mainly under hypoxia. We also highlighted a significant synergism between Evo and doxorubicin, SN38, or etoposide. Finally, Evo radio-sensitized the pHGG cell line tested, both with fractionated and single-dose irradiation schedules. Altogether, we report here the first preclinical proof of evidence about Evofosfamide efficiency against hypoxic pHGG and DIPG cells. Since such tumors are highly hypoxic, and Evo potentiates the effect of ionizing radiation and chemotherapy, it appears as a promising therapeutic strategy for children with brain tumors.

Hypoxic regions (habitats) within tumors are heterogeneously distributed and can be widely variant. Hypoxic habitats are generally pan-therapy resistant. For this reason, hypoxia-activated prodrugs (HAPs) have been developed to target these resistant volumes. The HAP evofosfamide (TH-302) has shown promise in preclinical and early clinical trials of sarcoma. However, in a phase III clinical trial, TH-302 did not improve survival in combination with doxorubicin (dox), most likely due to a lack of patient stratification based on hypoxic status. Herein, our goal was to develop deep-learning (DL) models to identify hypoxic habitats, using multiparametric (mp) MRI and co-registered histology, and to non-invasively monitor response to TH-302 in a patient-derived xenograft (PDX) of rhabdomyosarcoma and a syngeneic model of fibrosarcoma (RIF-1). A DL convolutional neural network showed strong correlations (>0.81) between the true hypoxic portion in histology and the predicted hypoxic portion in multiparametric MRI. TH-302 monotherapy or in combination with Dox delayed tumor growth and increased survival in the hypoxic PDX model (pOne Sentence SummaryDevelopment of non-invasive MR imaging to assess hypoxia is crucial in determining the effectiveness of TH-302 therapy and to follow response.

Evofosfamide (Evo or TH302) is a hypoxia-activated prodrug which is reduced leading to the release of alkylating agent bromo-isophosphoramide mustard, which has shown safety and signals of efficacy in a prior phase 1 study in recurrent glioblastoma. We performed a dual center single-arm Phase II study to expand on the safety and efficacy of Evo plus bevacizumab in bevacizumab refractory glioblastoma. 33 patients with bevacizumab refractory GBM received Evo 670 mg/m2 in combination with Bevacizumab 10 mg/kg IV every 2 weeks. Assessments included adverse events, response, and survival. Median age of patients was 47 (range 19–76) and 24 (69%) were male. At the time of study entry, 9 (26%) had ongoing corticosteroid use. ECOG performance status was 0 or 1 in 83% of patients. Patients were mostly heavily pretreated with 77% have three or more prior regimens. A total of 12 patients (36%) suffered grade 3–4 drug associated adverse event (AE); no grade 5 AE were reported. Of the 33 evaluable patients, best response was PR in 3 (9%), SD in 14 (43%), and PD in 16 (48%) with responses confirmed by a second reviewer. Median time to progression of disease was 53 days (95% CI 42–113) and Median time to death was 129 days (95% CI 86–199 days). Progression free survival at 4 months (PFS-4) on Evo-Bev was 31%, which was a statistically significant improvement over the historical rate of 3%. The median overall survival of patients receiving Evo-Bevacizumab was 4.6 months (95% CI 2.9–6.6). The progression free survival of patients on Evo-Bevacizumab met the primary endpoint of progression free survival at 4 months of 31%, although the clinical significance of this may be limited. Given the patient population and Phase II design, these clinical outcomes will need further validation.

Purpose Hypoxia is widely known as one of the mechanisms of chemoresistance and as an environmental condition which triggers invasion and metastasis of cancer. Evofosfamide is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide mustard conjugated with 2-nitroimidazole. Biliary tract cancer (BTC) is known to contain large hypoxic area. This study evaluated the efficacy and safety of evofosfamide as a second-line treatment of advanced BTC. Materials and methods Patients received evofosfamide at a dose of 340 mg/m2 on days 1, 8, and 15 of every 28-day cycle. Primary end-point was progression-free survival rate at 4-months (4m-PFSR). Secondary end-points included overall survival (OS), progression-free survival (PFS), disease control rate (DCR), metabolic response by 18F-fluorodeoxyglucose positron emission tomography (PET), hypoxic parameters evaluated by 18F-fluoromisonidazole (FMISO) PET and toxicity. Results Twenty patients were treated with evofosfamide, with 16 response-evaluable patients. There was no objective response; stable disease was observed in nine patients, with a DCR of 56.25%. 4m-PFSR was 40.6%. Median PFS was 3.60 months (95% confidence interval [CI], 1.68 to 5.52). Median OS was 6.37 months (95% CI, 3.94 to 8.79). Reduction of tumor metabolic activity was observed in eight of 15 patients (53.3%). High baseline hypoxic parameters were associated with poor PFS. Change of hypoxic parameters between pretreatment and post-treatment reflected hypoxic-activated drug response. There was no treatment-related death. Conclusion Evofosfamide as second-line treatment of advanced BTC showed acceptable safety and comparable efficacy to other agents. Changes in volumetric parameters measured with FMISO PET, showing the degree of tumor hypoxia, reflected the response to evofosfamide based on the mode of action.

Evofosfamide is designed to release a cytotoxic bromo-isophosphoramide (Br-IPM) moiety in a hypoxic microenvironment. This drug therefore preferentially attacks hypoxic regions in tumors where other standard anti-cancer treatments such as chemotherapy and radiation therapy are often ineffective. Various combination therapies with evofosfamide have been proposed and tested in preclinical and clinical settings. However, the treatment effect of evofosfamide monotherapy on tumor hypoxia has not been fully understood, partly due to the lack of quantitative methods to assess tumor pO2 in vivo. Here, we use quantitative pO2 imaging by EPR to evaluate the change in tumor hypoxia in response to evofosfamide treatment using two pancreatic ductal adenocarcinom a xenograft models; MIA Paca-2 tumors responding to evofosfamide and Su.86.86 tumors which do not respond. EPR imaging showed oxygenation improved globally after evofosfamide treatment in hypoxic MIA Paca-2 tumors, in agreement with the ex vivo results obtained from hypoxia staining by pimonidazole and in apparent contrast to the decrease in Ktrans observed in DCE MRI. This suggests reoxygenation after treatment is due to decreased oxygen demand rather than improved prefusion. Following the change in pO2 after treatment may therefore yield a way of monitoring treatment response. The observation that evofosfamide not only kills the hypoxic region of the tumor but also improves oxygenation in the residual tumor regions provides a rationale for combination therapies using radiation and anti-proliferatives post evofosfamide for improved outcomes.

Hypoxia-activated prodrugs (HAPs) present a conceptually elegant approach to not only overcome, but better yet, exploit intra-tumoural hypoxia. Despite being successful in vitro and in vivo, HAPs are yet to achieve successful results in clinical settings. It has been hypothesised that this lack of clinical success can, in part, be explained by the insufficiently stringent clinical screening selection of determining which tumours are suitable for HAP treatments. Taking a mathematical modelling approach, we investigate how tumour properties and HAP-radiation scheduling influence treatment outcomes in simulated tumours. The following key results are demonstrated in silico: (i) HAP and ionising radiation (IR) monotherapies may attack tumours in dissimilar, and complementary, ways. (ii) HAP-IR scheduling may impact treatment efficacy. (iii) HAPs may function as IR treatment intensifiers. (iv) The spatio-temporal intra-tumoural oxygen landscape may impact HAP efficacy. Our in silico framework is based on an on-lattice, hybrid, multiscale cellular automaton spanning three spatial dimensions. The mathematical model for tumour spheroid growth is parameterised by multicellular tumour spheroid (MCTS) data., Author summary When cancer patients present with solid tumours, the tumours often contain regions that are oxygen-deprived or, in other words, hypoxic. Hypoxic tumour regions are more resistant to conventional anti-cancer therapies, such as chemotherapy and radiotherapy, and therefore tumour hypoxia may complicate treatments. Hypoxia-activated prodrugs constitute a conceptually elegant approach to not only overcome, but better yet, exploit tumour hypoxia. Hypoxia-activated prodrugs are drugs that act as Trojan horses, they are theoretically harmless vehicles that are converted into warheads when they reach their targets: hypoxic tumour regions. Despite being conceptually clever and successful in experimental settings, hypoxia-activated prodrugs are yet to achieve successful results in clinical trials. It has been hypothesised that this lack of clinical success can, in part, be explained by an insufficiently stringent clinical screening selection of determining which tumours are suitable for hypoxia-activated prodrug treatments. In this article, we investigate how simulated tumours with different oxygen landscapes respond to anti-cancer treatments that include hypoxia-activated prodrugs, either alone or in combination with radiotherapy. Our simulation framework is based on a mathematical model that describes how individual cancer cells in a tumour divide and respond to treatments. We demonstrate that the efficacy of hypoxia-activated prodrugs depends on both the treatment scheduling and the oxygen landscapes of the simulated tumours.

Antiangiogenic therapy has shown promising results in preclinical and clinical trials. However, tumor cells acquire resistance to this therapy by gaining ability to survive and proliferate under hypoxia induced by antiangiogenic therapy. Combining antiangiogenic therapy with hypoxia-activated prodrugs can overcome this limitation. Here, we have tested the combination of antiangiogenic drug sunitinib in combination with hypoxia-activated prodrug evofosfamide in neuroblastoma. In vitro, neuroblastoma cell line SK-N-BE(2) was 40-folds sensitive to evofosfamide under hypoxia compared to normoxia. In IV metastatic model, evofosfamide significantly increased mice survival compared to the vehicle (P=.02). In SK-N-BE(2) subcutaneous xenograft model, we tested two different treatment regimens using 30 mg/kg sunitinib and 50 mg/kg evofosfamide. Here, sunitinib therapy when started along with evofosfamide treatment showed higher efficacy compared to single agents in subcutaneous SK-N-BE(2) xenograft model, whereas sunitinib when started 7 days after evofosfamide treatment did not have any advantage compared to treatment with either single agent. Immunofluorescence of tumor sections revealed higher number of apoptotic cells and hypoxic areas compared to either single agent when both treatments were started together. Treatment with 80 mg/kg sunitinib with 50 mg/kg evofosfamide was significantly superior to single agents in both xenograft and metastatic models. This study confirms the preclinical efficacy of sunitinib and evofosfamide in murine models of aggressive neuroblastoma. Sunitinib enhances the efficacy of evofosfamide by increasing hypoxic areas, and evofosfamide targets hypoxic tumor cells. Consequently, each drug enhances the activity of the other.

Background Tumor hypoxia is considered an important factor in metastasis and disease relapse. Evofosfamide is a hypoxia-activated prodrug that selectively targets the hypoxic regions of solid tumors. As hypoxia-inducible factor-1α (HIF-1α) is overexpressed in nasopharyngeal carcinoma (NPC) tissues, we performed the present study to evaluate the efficacy profile of evofosfamide in NPC. Methods We evaluated the efficacy of evofosfamide as a single agent or combined with cisplatin (DDP) in the NPC cell lines CNE-2, HONE-1 and HNE-1, and in nude mouse xenograft tumor models. Results Evofosfamide exhibited hypoxia-selective cytotoxicity in NPC cell lines, with 50% inhibition concentration (IC50) values of 8.33 ± 0.75, 7.62 ± 0.67, and 0.31 ± 0.07 μmol/L under hypoxia in CNE-2, HONE-1 and HNE-1 cells, respectively. The sensitization ranged from ninefold to greater than 300-fold under hypoxia compared with normoxia controls. The combination of evofosfamide with DDP had a synergistic effect on cytotoxicity in the NPC cell lines by combination index values assessment. Cell cycle G2 phase was arrested after treated with 0.05 μmol/L evofosfamide under hypoxia. Histone H2AX phosphorylation (γH2AX) (a marker of DNA damage) expression increased while HIF-1α expression suppressed after evofosfamide treatment under hypoxic conditions. In the HNE-1 NPC xenograft models, evofosfamide exhibited antitumor activity both as a single agent and combined with DDP. Hypoxic regions in xenograft tissue were reduced after both evofosfamide monotherapy and combined therapy with DDP. Conclusions Our results present preclinical evidence for targeting the selective hypoxic portion of NPC by evofosfamide as a single agent and combined with DDP and provide rationale for the potential clinical application of evofosfamide for the treatment of nasopharyngeal carcinoma.

// Katarzyna J. Nytko 1, 2 , Ivo Grgic 1, 2 , Sabine Bender 1 , Janosch Ott 1 , Matthias Guckenberger 3 , Oliver Riesterer 2, 3 , Martin Pruschy 1, 2 1 Laboratory for Applied Radiobiology, Department of Radiation Oncology, University Hospital Zurich, Zurich, Switzerland 2 Clinical Research Priority Program “Tumor Oxygenation”, Zurich, Switzerland 3 Department of Radiation Oncology, University Hospital Zurich, Zurich, Switzerland Correspondence to: Martin Pruschy, email: martin.pruschy@usz.ch Keywords: evofosfamide, TH-302, hypoxia-activated prodrug, ionizing radiation, P450 oxidoreductase Received: June 29, 2016 Accepted: February 06, 2017 Published: February 28, 2017 ABSTRACT The promising treatment combination of ionizing radiation (IR) with a hypoxia-activated prodrug (HAP) is based on biological cooperation. Here we investigated the hypoxia-activated prodrug evofosfamide in combination with different treatment regimens of IR against lung A549- and head&neck UT-SCC-14-derived tumor xenografts. DNA damage-related endpoints and clonogenic cell survival of A549 and UT-SCC-14 carcinoma cells were probed under normoxia and hypoxia. Evofosfamide (TH-302) induced DNA-damage and a dose-dependent antiproliferative response in A549 cells on cellular pretreatment under hypoxia, and supra-additively reduced clonogenic survival in combination with IR. Concomitant treatment of A549-derived tumor xenografts with evofosfamide and fractionated irradiation induced the strongest treatment response in comparison to the corresponding neoadjuvant and adjuvant regimens. Adjuvant evofosfamide was more potent than concomitant and neoadjuvant evofosfamide when combined with a single high dose of IR. Hypoxic UT-SCC-14 cells and tumor xenografts thereof were resistant to evofosfamide alone and in combination with IR, most probably due to reduced P450 oxidoreductase expression, which might act as major predictive determinant of sensitivity to HAPs. In conclusion, evofosfamide with IR is a potent combined treatment modality against hypoxic tumors. However, the efficacy and the therapeutic outcome of this combined treatment modality is, as indicated here in preclinical tumor models, dependent on scheduling parameters and tumor type, which is most probably related to the status of respective HAP-activating oxidoreductases. Further biomarker development is necessary for the launch of successful clinical trials.

Purpose: To evaluate safety and characterize anticancer efficacy of hepatic hypoxia-activated intra-arterial therapy (HAIAT) with evofosfamide in a rabbit model. Experimental Design: VX2-tumor-bearing rabbits were assigned to 4 intra-arterial therapy (IAT) groups (n = 7/group): (i) saline (control); (ii) evofosfamide (Evo); (iii) doxorubicin–lipiodol emulsion followed by embolization with 100–300 μm beads (conventional, cTACE); or (iv) cTACE and evofosfamide (cTACE + Evo). Blood samples were collected pre-IAT and 1, 2, 7, and 14 days post-IAT. A semiquantitative scoring system assessed hepatocellular damage. Tumor volumes were segmented on multidetector CT (baseline, 7/14 days post-IAT). Pathologic tumor necrosis was quantified using manual segmentation on whole-slide images. Hypoxic fraction (HF) and compartment (HC) were determined by pimonidazole staining. Tumor DNA damage, apoptosis, cell proliferation, endogenous hypoxia, and metabolism were quantified (γ-H2AX, Annexin V, caspase-3, Ki-67, HIF1α, VEGF, GAPDH, MCT4, and LDH). Results: cTACE + Evo showed a similar profile of liver enzymes elevation and pathologic scores compared with cTACE. Neither hematologic nor renal toxicity were observed. Animals treated with cTACE + Evo demonstrated smaller tumor volumes, lower tumor growth rates, and higher necrotic fractions compared with cTACE. cTACE + Evo resulted in a marked reduction in the HF and HC. Correlation was observed between decreases in HF or HC and tumor necrosis. cTACE + Evo promoted antitumor effects as evidenced by increased expression of γ-H2AX, apoptotic biomarkers, and decreased cell proliferation. Increased HIF1α/VEGF expression and tumor glycolysis supported HAIAT. Conclusions: HAIAT achieved a promising step towards the locoregional targeting of tumor hypoxia. The favorable toxicity profile and enhanced anticancer effects of evofosfamide in combination with cTACE pave the way towards clinical trials in patients with liver cancer. Clin Cancer Res; 23(2); 536–48. ©2016 AACR.

Tumour hypoxia is a marker of poor prognosis and failure of chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC), providing a strategy for therapeutic intervention in this setting. To evaluate the utility of the hypoxia-activated prodrug evofosfamide (TH-302) in HNSCC, we established ten early passage patient-derived xenograft (PDX) models of HNSCC that were characterised by their histopathology, hypoxia status, gene expression, and sensitivity to evofosfamide. All PDX models closely resembled the histology of the patient tumours they were derived from. Pimonidazole-positive tumour hypoxic fractions ranged from 1.7&ndash, 7.9% in line with reported HNSCC clinical values, while mRNA expression of the Toustrup hypoxia gene signature showed close correlations between PDX and matched patient tumours, together suggesting the PDX models may accurately model clinical tumour hypoxia. Evofosfamide as a single agent (50 mg/kg IP, qd &times, 5 for three weeks) demonstrated antitumour efficacy that was variable across the PDX models, ranging from complete regressions in one p16-positive PDX model to lack of significant activity in the three most resistant models. Despite all PDX models showing evidence of tumour hypoxia, and hypoxia being essential for activation of evofosfamide, the antitumour activity of evofosfamide only weakly correlated with tumour hypoxia status determined by pimonidazole immunohistochemistry. Other candidate evofosfamide sensitivity genes&mdash, MKI67, POR, and SLFN11&mdash, did not strongly influence evofosfamide sensitivity in univariate analyses, although a weak significant relationship with MKI67 was observed, while SLFN11 expression was lost in PDX tumours. Overall, these data confirm that evofosfamide has antitumour activity in clinically-relevant PDX tumour models of HNSCC and support further clinical evaluation of this drug in HNSCC patients. Further research is required to identify those factors that, alongside hypoxia, can influence sensitivity to evofosfamide and could act as predictive biomarkers to support its use in precision medicine therapy of HNSCC.

Tumour hypoxia has been associated with increased resistance to various cancer treatments, particularly radiation therapy. Conversely, tumour hypoxia is a validated and ideal target for guided cancer drug delivery. For this reason, hypoxia-activated prodrugs (HAPs) have been developed, which remain inactive in the body until in the presence of tissue hypoxia, allowing for an activation tendency in hypoxic regions. We present here an experimentally motivated mathematical model predicting the effectiveness of HAPs in a variety of clinical settings. We first examined HAP effectiveness as a function of the amount of tumour hypoxia and showed that the drugs have a larger impact on tumours with high levels of hypoxia. We then combined HAP treatment with radiation to examine the effects of combination therapies. Our results showed radiation-HAP combination therapies to be more effective against highly hypoxic tumours. The analysis of combination therapies was extended to consider schedule sequencing of the combination treatments. These results suggested that administering HAPs before radiation was most effective in reducing total cell number. Finally, a sensitivity analysis of the drug-related parameters was done to examine the effect of drug diffusivity and enzyme abundance on the overall effectiveness of the drug. Altogether, the results highlight the importance of the knowledge of tumour hypoxia levels before administration of HAPs in order to ensure positive results.

The magnitude of the host immune response can be regulated by either stimulatory or inhibitory immune checkpoint molecules. Receptor-ligand binding between inhibitory molecules is often exploited by tumours to suppress anti-tumour immune responses. Immune checkpoint inhibitors that block these inhibitory interactions can relieve T-cells from negative regulation, and have yielded remarkable activity in the clinic. Despite this success, clinical data reveal that durable responses are limited to a minority of patients and malignancies, indicating the presence of underlying resistance mechanisms. Accumulating evidence suggests that tumour hypoxia, a pervasive feature of many solid cancers, is a critical phenomenon involved in suppressing the anti-tumour immune response generated by checkpoint inhibitors. In this review, we discuss the mechanisms associated with hypoxia-mediate immunosuppression and focus on modulating tumour hypoxia as an approach to improve immunotherapy responsiveness.

Tumor hypoxia causes resistance to radiation and chemotherapy. Evofosfamide (TH-302) has exhibited specific hypoxia-dependent cytotoxicity against primary acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples in vitro. Based on these findings, a Phase I study of evofosfamide was designed for patients with relapsed/refractory leukemia (NCT01149915). In this open-label study, patients were treated with evofosfamide as a 30-60 min/day infusion on Days 1-5 of a 21-day cycle (Arm A, n = 38) or as a continuous infusion over 120 hr over Days 1-5 of a 21-day cycle (Arm B, n = 11). Forty-nine patients were treated including 39 (80%) with AML and 9 (18%) with ALL. Patients had received a median of five prior therapies. In Arm A, the dose-limiting toxicities (DLTs) were grade 3 esophagitis, observed at a dose of 550 mg/m(2) . The maximum tolerated dose (MTD) was a daily dose of 460 mg/m(2) . In Arm B, the DLTs were grade 3 stomatitis and hyperbilirubinemia, observed at a daily dose of 460 mg/m(2) . The continuous infusion MTD was a daily dose of 330 mg/m(2) . Hypoxia markers HIF-1α and CAIX were highly expressed in leukemic bone marrow and were significantly reduced after evofosfamide therapy. The combined overall response rate in Arms A and B was 6% (2 CR/CRi and 1 PR), with all responses seen in Arm A. Evofosfamide has shown limited activity in heavily pretreated leukemia patients. Further evaluation investigating evofosfamide in combination with cytotoxic or demethylating agents is warranted. Am. J. Hematol. 91:800-805, 2016. © 2016 Wiley Periodicals, Inc.

Purpose: Tumor cells residing in tumor hypoxic zones are a major cause of drug resistance and tumor relapse. In this study, we investigated the efficacy of evofosfamide, a hypoxia-activated prodrug, and its combination with topotecan in neuroblastoma and rhabdomyosarcoma preclinical models. Experimental Design: Neuroblastoma and rhabdomyosarcoma cells were tested in vitro to assess the effect of evofosfamide on cell proliferation, both as a single agent and in combination with topotecan. In vivo antitumor activity was evaluated in different xenograft models. Animal survival was studied with the neuroblastoma metastatic tumor model. Results: All tested cell lines showed response to evofosfamide under normoxic conditions, but when exposed to hypoxia overnight, a 2- to 65-fold decrease of IC50 was observed. Adding topotecan to the evofosfamide treatment significantly increased cytotoxicity in vitro. In neuroblastoma xenograft models, single-agent evofosfamide treatment delayed tumor growth. Complete tumor regression was observed in the combined topotecan/evofosfamide treatment group after 2-week treatment. Combined treatment also improved survival in a neuroblastoma metastatic model, compared to single-agent treatments. In rhabdomyosarcoma xenograft models, combined treatment was more effective than single agents. We also showed that evofosfamide mostly targeted tumor cells within hypoxic regions while topotecan was more effective to tumor cells in normoxic regions. Combined treatment induced tumor cell apoptosis in both normoxic and hypoxic regions. Conclusions: Evofosfamide shows antitumor effects in neuroblastoma and rhabdomyosarcoma xenografts. Compared with single-agent, evofosfamide/topotecan, combined therapy improves tumor response, delays tumor relapse, and enhances animal survival in preclinical tumor models. Clin Cancer Res; 22(11); 2697–708. ©2015 AACR.