A systemically administered detoxified TLR4 agonist displays potent antitumor activity and an acceptable tolerance profile in preclinical models.

Bacterial lipopolysaccharides (LPS) are potent innate immunostimulants targeting the Toll-like receptor 4 (TLR4), an attractive and validated target for immunostimulation in cancer therapy. Although LPS possess anti-tumor activity, toxicity issues prevent their systemic administration at effective doses in humans. We first demonstrated that LPS formulated in liposomes preserved a potent antitumor activity per se upon systemic administration in syngeneic models, and significantly enhance the antitumor activity of the anti-CD20 antibody rituximab in mice xenografted with the human RL lymphoma model. Liposomal encapsulation also allowed a 2-fold reduction in the induction of pro-inflammatory cytokines by LPS. Mice receiving an intravenous administration demonstrated a significant increase of neutrophils, monocytes and macrophages at the tumor site as well as an increase of macrophages in spleen. Further, we chemically detoxified LPS to obtain MP-LPS that was associated with a 200-fold decrease in the induction of proinflammatory cytokines. When encapsulated in a clinically approved liposomal formulation, toxicity, notably pyrogenicity (10-fold), was limited while the antitumor activity and immunoadjuvant effect were maintained. This improved tolerance profile of liposomal MP-LPS was associated with the preferential activation of the TLR4-TRIF pathway. Finally, in vitro studies demonstrated that stimulation with encapsulated MP-LPS reversed the polarization of M2 macrophages towards an M1 phenotype, and a phase 1 trial in healthy dogs validated its tolerance upon systemic administration up to very high doses (10µg/kg). Altogether, our results demonstrate the strong therapeutic potential of MPLPS formulated in liposomes as a systemically active anticancer agent, supporting its evaluation in patients with cancer.

ETOILE: Real-World Evidence of 24 Months of Ranibizumab 0.5 mg in Patients with Visual Impairment Due to Diabetic Macular Edema.

PURPOSE: To evaluate the real-world effectiveness of intravitreal ranibizumab 0.5 mg (Lucentis) in improving visual acuity (VA) in adults with decreased VA due to diabetic macular edema (DME). PATIENTS AND METHODS: Real-world prospective observational 24-month study. Ranibizumab-naïve patients (n=116) were enrolled, treated and followed up according to investigators' usual procedures. Outcomes included change from baseline to month 24 in best-corrected VA (BCVA; primary outcome), central retinal thickness (CRT), treatment exposure and safety. RESULTS: Overall, 62.9% of patients completed the study per protocol, 68.6% completed the induction phase (first three injections one month apart). On average, patients had 12.5 ophthalmologist visits and 5.74 injections in year 1, decreasing to 4.6 visits and 1.94 injections in year 2. Mean baseline BCVA was 58.4 letters, mean gain at M24 was +6.08 letters (95% CI: 2.95, 9.21). Gains were higher for patients who completed induction, and for patients who did not switch treatment. Mean CRT improved by 149.17 µm at M24. There were no new safety signals. BCVA variation of ≥6 letters by M3 was predictive of BCVA gains at M24 (p=0.007), as was hypertension medication at baseline (p=0.022). CONCLUSION: Real-world ranibizumab treatment improved VA in DME patients, despite fewer injections than recommended.

One-Year Outcome of Aflibercept Intravitreal Injection in Vitrectomized Eyes with Diabetic Macular Edema.

AIM: To evaluate the efficacy of intravitreal Aflibercept injection (IAI) for vitrectomized eyes with diabetic macular edema (DME) at one year. METHODS: This is a prospective, non-comparative, multicenter observational study including diabetic patients whose HbA1c is < 9%, with visual acuity between 20/400 to 20/40 due to DME, who have undergone vitrectomy since at least 3 months before the first aflibercept injection. Treatment protocol included 5 monthly aflibercept injection followed by a ProReNata regimen during the first year. Visual acuity, OCT findings and number of IAI were assessed at 6 months and one year. RESULTS: Forty-six eyes were included. Indications for vitrectomy were epiretinal membrane (58.7%), intravitreal hemorrhage (26.1%), and vitreomacular traction (8.7%), retinal detachment (4.3%), and other cause (4.3%). Median duration of macular edema was 3 years. Median interval between vitrectomy and first visit was 9 months. Thirty eyes were non-naïve and received previously thermal laser (44.3%), intravitreal injection of triamcinolone (26.7%), of ranibizumab (70%), of dexamethasone implant (36.7%), or bevacizumab (6.7%). Data was available for 35 eyes at 1 year. Visual gain was significant, +6 letters (p <0.001) and central subfield thickness (CST) decreased significantly (-108µm, p < 0.001) at 1 year. Mean number of injections was 9.3 and mean interval injection was 5.8 weeks. CONCLUSION: These results suggest that IAI may be beneficial in vitrectomized eyes with refractory DME which require frequent injections to obtain visual and anatomical improvement. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov, registration Number NCT02874859.

Exatecan Antibody Drug Conjugates Based on a Hydrophilic Polysarcosine Drug-Linker Platform.

We herein report the development and evaluation of a novel HER2-targeting antibody-drug conjugate (ADC) based on the topoisomerase I inhibitor payload exatecan, using our hydrophilic monodisperse polysarcosine (PSAR) drug-linker platform (PSARlink). In vitro and in vivo experiments were conducted in breast and gastric cancer models to characterize this original ADC and gain insight about the drug-linker structure-activity relationship. The inclusion of the PSAR hydrophobicity masking entity efficiently reduced the overall hydrophobicity of the conjugate and yielded an ADC sharing the same pharmacokinetic profile as the unconjugated antibody despite the high drug-load of the camptothecin-derived payload (drug-antibody ratio of 8). Tra-Exa-PSAR10 demonstrated strong anti-tumor activity at 1 mg/kg in an NCI-N87 xenograft model, outperforming the FDA-approved ADC DS-8201a (Enhertu), while being well tolerated in mice at a dose of 100 mg/kg. In vitro experiments showed that this exatecan-based ADC demonstrated higher bystander killing effect than DS-8201a and overcame resistance to T-DM1 (Kadcyla) in preclinical HER2+ breast and esophageal models, suggesting potential activity in heterogeneous and resistant tumors. In summary, the polysarcosine-based hydrophobicity masking approach allowsfor the generation of highly conjugated exatecan-based ADCs having excellent physicochemical properties, an improved pharmacokinetic profile, and potent in vivo anti-tumor activity.

Generation and characterization of novel anti-DR4 and anti-DR5 antibodies developed by genetic immunization.

Development of therapeutic antibodies in oncology has attracted much interest in the past decades. More than 30 of them have been approved and are being used to treat patients suffering from cancer. Despite encouraging results, and albeit most clinical trials aiming at evaluating monoclonal antibodies directed against TRAIL agonist receptors have been discontinued, DR4 or DR5 remain interesting targets, since these receptors are overexpressed by tumour cells and are able to trigger their death. In an effort to develop novel and specific anti-DR4 and anti-DR5 antibodies with improved properties, we used genetic immunization to express native proteins in vivo. Injection of DR4 and DR5 cDNA into the tail veins of mice elicited significant humoral anti-DR4 and anti-DR5 responses and fusions of the corresponding spleens resulted in numerous hybridomas secreting antibodies that could specifically recognize DR4 or DR5 in their native forms. All antibodies bound specifically to their targets with a very high affinity, from picomolar to nanomolar range. Among the 21 anti-DR4 and anti-DR5 monoclonal antibodies that we have produced and purified, two displayed proapoptotic properties alone, five induced apoptosis after cross-linking, four were found to potentiate TRAIL-induced apoptosis and three displayed antiapoptotic potential. The most potent anti-DR4 antibody, C#16, was assessed in vivo and was found, alone, to inhibit tumour growth in animal models. This is the first demonstration that DNA-based immunization method can be used to generate novel monoclonal antibodies targeting receptors of the TNF superfamily that may constitute new therapeutic agents.

Wip1 promotes RUNX2-dependent apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.

The inactivation of the p53 tumor suppressor pathway in many cancers often increases their resistance to anticancer therapy. Here we show that a previously proposed strategy directed to Wip1 inhibition could be ineffective in tumors lacking p53. On the contrary, Wip1 overexpression sensitized these tumors to chemotherapeutic agents. This effect was mediated through interaction between Wip1 and RUNX2 that resulted, in response to anticancer treatment, in RUNX2-dependent transcriptional induction of the proapoptotic Bax protein. The potentiating effects of Wip1 overexpression on chemotherapeutic agents were directed only to tumor cells lacking p53. The overexpression of Wip1 in normal tissues provided protection from cisplatin-induced apoptosis through decreased strength of upstream signaling to p53. Thus, Wip1 phosphatase promotes apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.

Peptides and aptamers targeting HSP70: a novel approach for anticancer chemotherapy.

The inhibition of heat shock protein 70 (HSP70) is an emerging strategy in cancer therapy. Unfortunately, no specific inhibitors are clinically available. By yeast two-hybrid screening, we have identified multiple peptide aptamers that bind HSP70. When expressed in human tumor cells, two among these peptide aptamers-A8 and A17-which bind to the peptide-binding and the ATP-binding domains of HSP70, respectively, specifically inhibited the chaperone activity, thereby increasing the cells' sensitivity to apoptosis induced by anticancer drugs. The 13-amino acid peptide from the variable region of A17 (called P17) retained the ability to specifically inhibit HSP70 and induced the regression of subcutaneous tumors in vivo after local or systemic injection. This antitumor effect was associated with an important recruitment of macrophages and T lymphocytes into the tumor bed. Altogether, these data indicate that peptide aptamers or peptides that target HSP70 may be considered as novel lead compounds for cancer therapy.

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells.

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl ß-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.

Laser photocoagulation for choroidal neovascular membrane associated with optic disc drusen.

PURPOSE: To report two cases of choroidal neovascular membrane associated with optic disc drusen which were successfully treated using argon laser photocoagulation. METHODS: Choroidal neovascular complications of optic disc drusen were diagnosed in two of our patients, a 36-year-old woman and a 14-year-old girl. In both cases visual acuity was 20/100 in the affected eye. Fundus examination and angiography revealed a choroidal neovascular membrane. Computer tomography identified bilateral calcified drusen of the optic discs. Photocoagulation of neovascular tissues was immediately performed. RESULTS: Visual acuity improved progressively, reaching 20/20 10 months after treatment in one case and 20/30 in the other. No recurrence was observed during follow-up. CONCLUSION: Early diagnosis and immediate laser photocoagulation of vision-threatening choroidal neovascular membranes associated with optic disc drusen helps stop progression towards the macula and improves longterm visual acuity.