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Combinatorial effect of PG545 and conditioned medium (CM) on cell viability, and examination of apoptosis after downregulation of HPSE

Aggressive tumor progression, metastasis, and resistance to conventional therapies lead to an extremely poor prognosis for pancreatic ductal adenocarcinoma (PDAC). Heparanase, an enzyme expressed by multiple cell types, including tumor cells in the tumor microenvironment, has been implicated in angiogenesis and metastasis, and its expression correlates with decreased overall survival in PDAC. We evaluated the therapeutic potential of PG545, an angiogenesis and heparanase inhibitor, in experimental PDAC. PG545 inhibited the proliferation, migration, and colony formation of pancreatic cancer cells in vitro at pharmacologically relevant concentrations. Heparanase inhibition also reduced the proliferation of fibroblasts but had only modest effects on endothelial cells in vitro. Furthermore, PG545 significantly prolonged animal survival in intraperitoneal and genetic models (mPDAC: LSL-KrasG12D; Cdkn2alox/lox; p48Cre) of PDAC. PG545 also inhibited primary tumor growth and metastasis in orthotopic and genetic endpoint studies. Analysis of tumor tissue revealed that PG545 significantly decreased cell proliferation, increased apoptosis, reduced microvessel density, disrupted vascular function, and elevated intratumoral hypoxia. Elevated hypoxia is a known driver of collagen deposition and tumor progression; however, tumors from PG545-treated animals displayed reduced collagen deposition and a greater degree of differentiation compared with control or gemcitabine-treated tumors. These results highlight the potent antitumor activity of PG545 and support the further exploration of heparanase inhibitors as a potential clinical strategy for the treatment of PDAC. Mol Cancer Ther; 12(7); 1190–201. ©2013 AACR.

Supplementary Figure Legends

PDF - 153K, Example histology of liver metastases in the mPDAC model. Liver sections were stained with H&E. Scale bar, 100 um.

Curative therapy for medulloblastoma and other pediatric embryonal brain tumors has improved, but the outcome still remains poor and current treatment causes long-term complications. Malignant brain tumors infiltrate the healthy brain tissue and, thus despite resection, cells that have already migrated cause rapid tumor regrowth. Heparan sulfate proteoglycans (HSPG), major components of the extracellular matrix (ECM), modulate the activities of a variety of proteins. The major enzyme that degrades HS, heparanase (HPSE), is an important regulator of the ECM. Here, we report that the levels of HPSE in pediatric brain tumors are higher than in healthy brain tissue and that treatment of pediatric brain tumor cells with HPSE stimulated their growth. In addition, the latent, 65 kDa form of HPSE (that requires intracellular enzymatic processing for activation) enhanced cell viability and rapidly activated the ERK and AKT signaling pathways, before enzymatically active HPSE was detected. The HPSE inhibitor PG545 efficiently killed pediatric brain tumor cells, but not normal human astrocytes, and this compound also reduced tumor cell invasion in vitro and potently reduced the size of flank tumors in vivo. Our findings indicate that HPSE in malignant brain tumors affects both the tumor cells themselves and their ECM. In conclusion, HPSE plays a substantial role in childhood brain tumors, by contributing to tumor aggressiveness and thereby represents a potential therapeutic target. Mol Cancer Ther; 16(8); 1705–16. ©2017 AACR.

The symbiotic relationships shared between humans and their gastrointestinal parasites present opportunities to discover novel therapies for inflammatory diseases. A prime example of this phenomenon is the interaction of humans and roundworms such as the hookworm, Necator americanus. Epidemiological observations, animal studies and clinical trials using experimental human hookworm infection show that hookworms can suppress inflammation in a safe and well-tolerated way, and that the key to their immunomodulatory properties lies within their secreted proteome. Herein we describe the identification of 2 netrin domain-containing proteins from the N. americanus secretome, and explore their potential in treating intestinal inflammation in mouse models of ulcerative colitis. One of these proteins, subsequently named Na-AIP-1, was effective at suppressing disease when administered prophylactically in the acute TNBS-induced model of colitis. This protective effect was validated in the more robust CD4 T cell transfer model of chronic colitis, where prophylactic Na-AIP-1 reduced T-cell-dependent type-1 cytokine responses in the intestine and the associated intestinal pathology. Mechanistic studies revealed that depletion of CD11c+ cells abrogated the protective anticolitic effect of Na-AIP-1. Next generation sequencing of colon tissue in the T-cell transfer model of colitis revealed that Na-AIP-1 induced a transcriptomic profile associated with the downregulation of metabolic and signaling pathways involved in type-1 inflammation, notably TNF. Finally, co-culture of Na-AIP-1 with a human monocyte-derived M1 macrophage cell line resulted in significantly reduced secretion of TNF. Na-AIP-1 is now a candidate for clinical development as a novel therapeutic for the treatment of human inflammatory bowel diseases.

BACKGROUND: Cachexia occurs in up to 80% of PDAC patients and is characterized by weight loss mainly due to muscle and fat wasting. Cachexia leads to functional impairment, reduced anticancer therapy tolerance and increased mortality. PG545, a heparan sulfate (HS) mimetic, shows anticancer activity; however, it is unknown whether sex affects the anticancer activity and what effect PG545 has on cancer associated muscle and fat wasting. Given that many cytokines induced in patients with cancer cachexia can bind HS, the aim of this study was to assess PG545 effects on PDAC cachexia with emphasis on sex difference. METHODS: PDAC cells were injected into the pancreas of 10-week C57BL/6 male and female mice. The orthotopic tumor-bearing mice were treated with PG545 (10 mg/kg; i.p.). Body weight and body composition were monitored, and tumor and organs were collected. Skeletal muscles were cryosectioned for evaluation of cross-sectional area (CSA). C2C12 myotubes, the in vitro model of muscle wasting, were treated with PDAC cell-derived conditioned medium (CM) without or with PG545 and analyzed for the changes in size. PDAC cell proliferation was assessed by MTT assay. RESULTS: PG545 treatment markedly reduced tumor mass in both males and females compared to the vehicle controls. PG545 also reduced body weight but the difference gradually dissipated over time despite repeated dosing. However, male mice recovered from the negative effects more slowly than female mice. Similar sex difference was observed in the changes in the PG545-induced lean and fat mass loss. Of note, fat mass in PG545-treated females at the end of experiment was higher than in the vehicle controls. Furthermore, males had more reductions in muscle and fat weights than females in response to PG545. Grip strength in males was weakened by PG545 but returned to the level like that in the vehicle controls at the end, while PG545 did not weaken grip strength in females at the same time points. In addition, males, but not females, had smaller muscle fiber CSA after PG545 treatment versus their vehicle controls. In in vitro assay, PG545 inhibited PDAC cell proliferation, indicating a direct effect on cancer cells. Lastly, opposed to the in vivo effect, PG545 partly prevented myotube wasting induced by PDAC CM. CONCLUSION: PG545 has strong anti-PDAC activity in both male and female mice, at least partly through inhibition of cancer cell proliferation. Its initial negative effects on cachexia are gradually reversed with male tumor-bearing mice reversing more slowly than females. Because of the protective effect on C2C12 myotubes treated with PDAC CM, PG545 might induce some counteracting factors in vivo in the initial treatment and identification of these factors may offer molecular targets for combination therapy to counteract the negative effects while take advantage of the strong anti-cancer potential. Citation Format: Xiaoling Zhong, Keith Dredge, John M. Chirgwin, Michael G. House, Harikrishna Nakshatri, Teresa A. Zimmers. The heparan sulfate mimetic PG545 inhibits tumor growth in both male and female mouse model of pancreatic ductal adenocarcinoma (PDAC) with sex difference in the effect on cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 367.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

SummaryHeparan sulfate (HS) is a cell surface polysaccharide recently identified as a co-receptor with the ACE2 protein for recognition of the S1 spike protein on SARS-CoV-2 virus, providing a tractable new target for therapeutic intervention. Clinically-used heparins demonstrate inhibitory activity, but world supplies are limited, necessitating alternative solutions. Synthetic HS mimetic pixatimod is a drug candidate for cancer with immunomodulatory and heparanase-inhibiting properties. Here we show that pixatimod binds to and destabilizes the SARS-CoV-2 spike protein receptor binding domain (S1-RBD), and directly inhibits its binding to human ACE2, consistent with molecular modelling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of live SARS-CoV-2 virus show that pixatimod potently inhibits infection of monkey Vero E6 and human bronchial epithelial cells at concentrations within its safe therapeutic dose range. Furthermore, in a K18-hACE2 mouse model pixatimod demonstrates that pixatimod markedly attenuates SARS-CoV-2 viral titer and COVID-19-like symptoms. This demonstration of potent anti-SARS-CoV-2 activity establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics. Together with other known activities of pixatimod our data provides a strong rationale for its clinical investigation as a potential multimodal therapeutic to address the COVID-19 pandemic.

Pixatimod is an inhibitor of heparanase, a protein which promotes cancer via its regulation of the extracellular environment by enzymatic cleavage of heparan sulfate (HS) and non-enzymatic signaling. Through its inhibition of heparanase and other HS-binding signaling proteins, pixatimod blocks a number of pro-cancerous processes including cell proliferation, invasion, metastasis, angiogenesis and epithelial-mesenchymal transition. Several laboratories have found that these activities have translated into potent activity using a range of different mouse cancer models, including approximately 30 xenograft and 20 syngeneic models. Analyses of biological samples from these studies have confirmed the heparanase targeting of this agent in vivo and the broad spectrum of anti-cancer effects that heparanase blockade achieves. Pixatimod has been tested in combination with a number of approved anti-cancer drugs demonstrating its clinical potential, including with gemcitabine, paclitaxel, sorafenib, platinum agents and an anti-PD-1 antibody. Clinical testing has shown pixatimod to be well tolerated as a monotherapy, and it is currently being investigated in combination with the anti-PD-1 drug nivolumab in a pancreatic cancer phase I trial.

BackgroundPixatimod is a novel immunomodulatory agent which stimulates dendritic cells (DC) via Toll-Like Receptor (TLR9) pathway to activate natural killer (NK) cells.1 In combination with PD1 inhibitors, it also enhances T cell infiltration in vivo.2 We report on safety, pharmacokinetics (PK) and pharmacodynamics (PD), and antitumor activity of pixatimod plus nivolumab in advanced cancer patients (stage 1) and in an expansion cohort of mPDAC (stage 2).MethodsIn the dose escalation stage (3+3 design), eligible patients (ECOG≤1) with advanced solid malignancies who failed standard therapies received pixatimod once weekly as a 1-hour i.v. infusion plus nivolumab (240 mg, every other week) until disease progression or discontinuation due to intolerability. The primary objective was determination of the maximum tolerated dose (MTD). Secondary objectives evaluated safety, antitumor activity per RECIST v1.1, PK of pixatimod, and PD (PBMC, plasma cytokines and chemokines). Stage 2 comprised mPDAC subjects who had received no more than one prior line of chemotherapy in the metastatic setting.ResultsThe dose-escalation stage recruited 16 subjects across two cohorts (25 & 50 mg pixatimod). Two dose limiting toxicities (DLTs) in 50 mg cohort were pulmonary edema and multi-organ failure. Of note, the subject with multi-organ failure had substantially higher CA19.9, Pan-immune-Inflammatory Value (PIV = Neutrophils x Platelets x Monocytes/Lymphocytes) and interleukins (IL) IL-1α and IL-23 at baseline compared with the cohort. One DLT occurred in the 25 mg cohort, pneumonitis, which was identified as the MTD. A further 14 mPDAC subjects were recruited to the expansion stage (25 mg). Seven SAEs were reported to be possibly or likely related to the combination. No objective responses were reported in the mPDAC stage, the best response was SD (n = 3). In another submitted abstract by Lemech et al, we report two subjects in the dose escalation stage with MSS mCRC were confirmed PR, and data from the amended study to include an MSS mCRC expansion cohort will also be presented. Time versus concentration data for pixatimod in advanced cancer patients was similar to that previously reported in monotherapy setting. In mPDAC subjects, there was minimal immune activation as evidenced by a lack of change in effector memory T cells or NK cells in PBMC, plasma cytokines and chemokines.ConclusionsPixatimod is well tolerated at 25 mg in combination with nivolumab but did not provide clear clinical benefit or evidence of immune activation in the mPDAC cohort.AcknowledgementsResearch funding was provided by Zucero Therapeutics Ltd and Bristol Myers Squibb (BMS), Australia.Trial RegistrationClinical trial informationACTRN12617001573347.ReferencesBrennan TV, Lin L, Brandstadter JD, Rendell VR, Dredge K, Huang X, et al. Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation. J Clin Invest 2016;126(1):207–19.Hammond E, Haynes NM, Cullinane C, Brennan TV, Bampton D, Handley P, et al. Immunomodulatory activities of pixatimod: emerging nonclinical and clinical data, and its potential utility in combination with PD-1 inhibitors. J Immunother Cancer 2018;6(1):54.Ethics ApprovalThe clinical trial entitled “An open-label, multi-centre Phase Ib study of the safety and tolerability of IV infused PG545 in combination with nivolumab in patients with advanced solid tumours with an expansion cohort in patients with metastatic pancreatic cancer. Protocol ZU545102” obtained ethics approval from the Royal Adelaide Hospital (HREC Reference number, HREC/17/RAH/195 and the CALHN Reference number, R20170515) and Bellberry Limited (Application No: 2018-08-695). All participants in the study gave informed consent before taking part in ZU545102.

BackgroundPixatimod is a novel immunomodulatory agent which stimulates dendritic cells (DC) via Toll-Like Receptor 9 (TLR9) pathway to activate natural killer (NK) cells.1 It also enhances T cell infiltration into tumors when combined with PD1 inhibitors in vivo.2 We report on safety, pharmacodynamics (PD), and antitumor activity of pixatimod plus nivolumab in a cohort of previously treated MSS mCRC subjects.MethodsEligible MSS mCRC patients (ECOG≤1) with a median of 3 lines of previous treatment received 25 mg pixatimod once weekly as a 1-hour i.v. infusion plus nivolumab (240 mg, every other week) until disease progression or discontinuation due to intolerability. Primary and secondary objectives included the assessment of safety and antitumor activity per RECIST v1.1 at the recommended dose, and evidence of immune activation in peripheral blood mononuclear cells (PBMC), plasma cytokines/chemokines, and paired tumor tissue biopsies, where available.ResultsThirty-three subjects with MSS mCRC (42% male) started treatment. Median age was 58 (range 35–80), median number of previous lines of chemotherapy was 3 (range 1–6), ECOG PS 0/1 was 67%/33%, 64% had target liver metastases, and primary tumor site was right-sided/transverse colon in 21% and left-sided colon/rectum 79%. Median number of cycles completed was 2 (range 0–13). Treatment-related AEs led to treatment discontinuation in 2 patients (autoimmune hepatitis and pneumonitis). Ten Grade 3 treatment-related AEs were reported in 4 subjects (12%). Thirty subjects received at least one cycle of treatment (1-month), with 25 subjects having initial post-baseline assessment scans > 6 weeks (as per RECIST v1.1). Of these, 3 subjects had confirmed partial responses and 8 had stable disease. Lower Pan-immune-Inflammatory Value (PIV = Neutrophils x Platelets x Monocytes/Lymphocytes) at screening were associated with clinical benefit (PR/SD). Post-treatment increases in plasma IP-10 and IP-10/IL-8 ratio, effector memory (CD45RA-CCR7-) CD4+ and CD8+ T cells, and Ki-67 expression in CD4+ and CD8+ T cells from PBMC were also detected in the clinical benefit cohort compared to subjects with progressive disease. Conversely, plasma levels of IL-6 were significantly lower in patients with clinical benefit. Analyses of pre- & on-treatment biopsies from the only PR patient with available paired biopsies demonstrated an increase in T cell infiltration.ConclusionsPixatimod is well tolerated at 25 mg in combination with a standard dose of nivolumab. The efficacy signal and pharmacodynamic changes in MSS mCRC warrants further investigation of pixatimod plus nivolumab for this patient population.AcknowledgementsResearch Funding provided by Zucero Therapeutics Ltd and Bristol Myers Squibb (BMS), Australia.Trial RegistrationClinical trial informationACTRN12617001573347ReferencesBrennan TV, Lin L, Brandstadter JD, Rendell VR, Dredge K, Huang X, et al. Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation. J Clin Invest 2016;126(1):207–19.Hammond E, Haynes NM, Cullinane C, Brennan TV, Bampton D, Handley P, et al. Immunomodulatory activities of pixatimod: emerging nonclinical and clinical data, and its potential utility in combination with PD-1 inhibitors. J Immunother Cancer 2018;6(1):54.Ethics ApprovalThe clinical trial entitled “An open-label, multi-centre Phase Ib study of the safety and tolerability of IV infused PG545 in combination with nivolumab in patients with advanced solid tumours with an expansion cohort in patients with metastatic pancreatic cancer. Protocol ZU545102” obtained ethics approval from the Royal Adelaide Hospital (HREC Reference number, HREC/17/RAH/195 and the CALHN Reference number, R20170515) and Bellberry Limited (Application No: 2018-08-695). All participants in the study gave informed consent before taking part in ZU545102.

Pixatimod is an inhibitor of heparanase, a protein which promotes cancer via its regulation of the extracellular environment by enzymatic cleavage of heparan sulfate (HS) and non-enzymatic signaling. Through its inhibition of heparanase and other HS-binding signaling proteins, pixatimod blocks a number of pro-cancerous processes including cell proliferation, invasion, metastasis, angiogenesis and epithelial-mesenchymal transition. Several laboratories have found that these activities have translated into potent activity using a range of different mouse cancer models, including approximately 30 xenograft and 20 syngeneic models. Analyses of biological samples from these studies have confirmed the heparanase targeting of this agent in vivo and the broad spectrum of anti-cancer effects that heparanase blockade achieves. Pixatimod has been tested in combination with a number of approved anti-cancer drugs demonstrating its clinical potential, including with gemcitabine, paclitaxel, sorafenib, platinum agents and an anti-PD-1 antibody. Clinical testing has shown pixatimod to be well tolerated as a monotherapy, and it is currently being investigated in combination with the anti-PD-1 drug nivolumab in a pancreatic cancer phase I trial.

The sulfated glycolipid PG545 shows promising antitumor activity in various cancers. This study was conducted to explore the effects and the mechanism of PG545 action in endometrial cancer (EC). PG545 exhibited strong synergy as assessed by the Chou-Talalay-Method in vitro when combined with cisplatin, or paclitaxel in both type I (Hec1B) and type II (ARK2) EC cell lines. While PG545 showed antitumor activity as monotherapy, a combination of PG545 with paclitaxel and cisplatin was highly effective in reducing the tumor burden and significantly prolonged survival of both Hec1B and ARK2 xenograft bearing mice. Mechanistically, PG545 elicits ER stress as an early response with resultant induction of autophagy. Our data demonstrated an increase in pERK, Bip/Grp78, IRE1α, Calnexin and CHOP/GADD153 within 6–24 hrs of PG545 treatment in EC cells. In parallel, PG545 also blocked FGF2 and HB-EGF mediated signaling in EC cells. Moreover, melatonin-mediated ER stress inhibition reduced PG545-mediated autophagy and PG545 in combination with cisplatin further heightened this stress response. Collectively these data indicate that PG545 exhibits strong synergistic effects with chemotherapeutics in vitro and showed promising antitumor activity in vivo. Our preclinical data indicates that in future studies PG545 can be a useful adjunct to chemotherapy in endometrial cancer.

Background: PG545 (pixatimod) is a novel immunomodulatory agent, which has been demonstrated to stimulate innate immune responses against tumours in preclinical ca ncer models. Methods: This Phase I study investigated the safety, tolerability, pharmacokinetics, pharmacodynamics and preliminary efficacy of PG545 monotherapy. Escalating doses of PG545 were administered to patients with advanced solid malignancies as a weekly 1-h intravenous infusion. Results: Twenty-three subjects were enroled across four cohorts (25, 50, 100 and 150 mg). Three dose-limiting toxicities (DLTs)-hypertension (2), epistaxis (1)-occurred in the 150 mg cohort. No DLTs were noted in the 100 mg cohort, which was identified as the maximum-tolerated dose. No objective responses were reported. Best response was stable disease up to 24 weeks, with the disease control rate in evaluable subjects of 38%. Exposure was proportional up to 100 mg and mean half-life was 141 h. The pharmacodynamic data revealed increases in innate immune cell activation, plasma IFNγ, TNFα, IP-10 and MCP-1. Conclusion: PG545 demonstrated a tolerable safety profile, proportional PK, evidence of immune cell stimulation and disease control in some subjects. Taken together, these data support the proposed mechanism of action, which represents a promising approach for use in combination with existing therapies. Refereed/Peer-reviewed

Defective autophagy and deranged metabolic pathways are common in cancer; pharmacologic targeting of these two pathways could provide a viable therapeutic option. However, how these pathways are regulated by limited availability of growth factors is still unknown. Our study shows that HSulf-1 (endosulfatase), a known tumor suppressor which attenuates heparin sulfate binding growth factor signaling, also regulates interplay between autophagy and lipogenesis. Silencing of HSulf-1 in OV202 and TOV2223 cells (ovarian cancer cell lines) resulted in increased lipid droplets (LDs), reduced autophagic vacuoles (AVs) and less LC3B puncta. In contrast, HSulf-1 proficient cells exhibit more AVs and reduced LDs. Increased LDs in HSulf-1 depleted cells was associated with increased ERK mediated cPLA2S505 phosphorylation. Conversely, HSulf-1 expression in SKOV3 cells reduced the number of LDs and increased the number of AVs compared to vector controls. Furthermore, pharmacological (AACOCF3) and ShRNA mediated downregulation of cPLA2 resulted in reduced LDs, and increased autophagy. Finally, in vivo experiment using OV202 Sh1 derived xenograft show that AACOCF3 treatment effectively attenuated tumor growth and LD biogenesis. Collectively, these results show a reciprocal regulation of autophagy and lipid biogenesis by HSulf-1 in ovarian cancer.

Curative therapy for medulloblastoma and other pediatric embryonal brain tumors has improved, but the outcome still remains poor and current treatment causes long-term complications. Malignant brain tumors infiltrate the healthy brain tissue and, thus despite resection, cells that have already migrated cause rapid tumor regrowth. Heparan sulfate proteoglycans (HSPG), major components of the extracellular matrix (ECM), modulate the activities of a variety of proteins. The major enzyme that degrades HS, heparanase (HPSE), is an important regulator of the ECM. Here, we report that the levels of HPSE in pediatric brain tumors are higher than in healthy brain tissue and that treatment of pediatric brain tumor cells with HPSE stimulated their growth. In addition, the latent, 65 kDa form of HPSE (that requires intracellular enzymatic processing for activation) enhanced cell viability and rapidly activated the ERK and AKT signaling pathways, before enzymatically active HPSE was detected. The HPSE inhibitor PG545 efficiently killed pediatric brain tumor cells, but not normal human astrocytes, and this compound also reduced tumor cell invasion

BackgroundDespite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer.MethodsPG545's anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models.ResultsPG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response.ConclusionOur results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer.

Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell–based antitumor therapies.

3083 Background: PG545 (pixatimod, pINN) is a novel immunomodulatory agent which stimulates dendritic cells (DC) via TLR9/IL-12 pathway to activate natural killer (NK) cells. It also inhibits tumour-associated macrophages in cancer models. We report on safety, PK, PD, and antitumor activity of PG545 monotherapy. Methods: In this dose escalation (3+3 design) study, eligible pts (ECOG≤1) with advanced solid malignancies who failed standard therapies received PG545 once weekly as a 1-hour i.v. infusion until disease progression or discontinuation due to intolerability. The primary objective was determination of the maximum tolerated dose (MTD). Secondary objectives evaluated safety, antitumor activity based on RECIST (1.1) criteria, PK and PD (plasmacytoid DC & NKp46+NK cells from PBMC, and plasma cytokines/chemokines). Results: The study recruited 23 subjects across four cohorts (25, 50, 100 & 150 mg). Three dose limiting toxicities (DLTs) - hypertension (2), epistaxis (1) - occurred in the 150 mg cohort, which was identified as a non-tolerated dose level. No DLTs occurred in the 100 mg cohort, which was identified as the MTD. Six SAEs were reported to be possibly or likely related to PG545 treatment. No RECIST 1.1 objective responses were reported; best response was prolonged stable disease up to 24 weeks (mCRC), with disease control rate in evaluable subjects of 38% (6/16) at eight weeks. Exposure (AUC0-last) was proportional up to 100mg and mean half-life was 144 hours. At 50 and 100mg dose levels, two subjects in each cohort exhibited up to 4-fold increased numbers of NKp46+NK cells, IFN-α-producing pDCs, and increases (up to 25-fold) in plasma IFN-γ, TNF-α, IP-10 and MCP-1. Conclusions: PG545 is well tolerated up to 100 mg once-weekly via i.v. infusion. Human exposure data at 50mg and 100mg reach exposures consistent with those required for preclinical efficacy. Preliminary PD data support the proposed mechanism of action, which represents a promising approach to improve the efficacy of existing therapies. These data, and the absence of toxicities associated with chemo- or immunotherapies, support the development of PG545 in combination clinical trials. Clinical trial information: NCT02042781.