Disrupting EGFR-HER2 Transactivation by Pertuzumab in HER2-Positive Cancer: Quantitative Analysis Reveals EGFR Signal Input as Potential Predictor of Therapeutic Outcome.

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

The ocular pharmacokinetics of a new antibody (MIL60) against vascular endothelial growth factor ( VEGF) following subconjunctival injection in rats / 中华微生物学和免疫学杂志

Objective To evaluate the ocular pharmacokinetics of a novel antibody, MIL60, tar-geting vascular endothelial growth factor ( VEGF) after subconjunctival injection. Methods After subcon-junctival injection of MIL60 in rabbits, aqueous humour, vitreous from both eyes and peripheral blood were collected of each rabbit to analyze the concentration of MIL60 with ELISA. Results After subconjunctival administration, MIL60 could penetrate into the injected eye′s anterior chamber and vitreous, and maintained at an effective concentration in the aqueous or vitreous for about 10. 7 or 6. 8 days. Moreover, MIL60 could be found in the uninjected eye. Conclusion After subconjunctival injection, MIL60 could maintain at a therapeutic concentration in injected eyes. The pharmacokinetics analysis of MIL60 might provide some basis and guidance for its application in humans in the near future.

Targeted Delivery of Neutralizing Anti-C5 Antibody to Renal Endothelium Prevents Complement-Dependent Tissue Damage.

Complement activation is largely implicated in the pathogenesis of several clinical conditions and its therapeutic neutralization has proven effective in preventing tissue and organ damage. A problem that still needs to be solved in the therapeutic control of complement-mediated diseases is how to avoid side effects associated with chronic neutralization of the complement system, in particular, the increased risk of infections. We addressed this issue developing a strategy based on the preferential delivery of a C5 complement inhibitor to the organ involved in the pathologic process. To this end, we generated Ergidina, a neutralizing recombinant anti-C5 human antibody coupled with a cyclic-RGD peptide, with a distinctive homing property for ischemic endothelial cells and effective in controlling tissue damage in a rat model of renal ischemia/reperfusion injury (IRI). As a result of its preferential localization on renal endothelium, the molecule induced complete inhibition of complement activation at tissue level, and local protection from complement-mediated tissue damage without affecting circulating C5. The ex vivo binding of Ergidina to surgically removed kidney exposed to cold ischemia supports its therapeutic use to prevent posttransplant IRI leading to delay of graft function. Moreover, the finding that the ex vivo binding of Ergidina was not restricted to the kidney, but was also seen on ischemic heart, suggests that this RGD-targeted anti-C5 antibody may represent a useful tool to treat organs prior to transplantation. Based on this evidence, we propose preliminary data showing that Ergidina is a novel targeted drug to prevent complement activation on the endothelium of ischemic kidney.

Antibody targeting facilitates effective intratumoral siRNA nanoparticle delivery to HER2-overexpressing cancer cells.

The therapeutic potential of RNA interference (RNAi) has been limited by inefficient delivery of short interfering RNA (siRNA). Tumor-specific recognition can be effectively achieved by antibodies directed against highly expressed cancer cell surface receptors. We investigated the utility of linking an internalizing streptavidin-conjugated HER2 antibody to an endosome-disruptive biotinylated polymeric nanocarrier to improve the functional cytoplasmic delivery of siRNA in breast and ovarian cancer cells in vitro and in an intraperitoneal ovarian cancer xenograft model in vivo, yielding an 80% reduction of target mRNA and protein levels with sustained repression for at least 96 hours. RNAi-mediated site specific cleavage of target mRNA was demonstrated using the 5' RLM-RACE (RNA ligase mediated-rapid amplification of cDNA ends) assay. Mice bearing intraperitoneal human ovarian tumor xenografts demonstrated increased tumor accumulation of Cy5.5 fluorescently labeled siRNA and 70% target gene suppression after treatment with HER2 antibody-directed siRNA nanocarriers. Detection of the expected mRNA cleavage product by 5' RLM-RACE assay confirmed that suppression occurs via the expected RNAi pathway. Delivery of siRNA via antibody-directed endosomolytic nanoparticles may be a promising strategy for cancer therapy.

Panitumumab safety for treating colorectal cancer.

INTRODUCTION: Panitumumab is a human IgG2 mAb against the EGFR, inhibiting tumor cell proliferation, survival and angiogenesis. It has demonstrated clinical efficacy in metastatic colorectal cancer (CRC) in combination with chemotherapy in first- and second-line settings and as monotherapy in third-line setting. Recently, mutations in the RAS genes have been shown to be predictive of lack of efficacy, panitumumab should be restricted to patients with RAS wild-type (wt) tumors. AREAS COVERED: This review focuses on main efficacy results of panitumumab in metastatic CRC in first-, second- and third-line settings in combination with chemotherapy or as monotherapy. Additionally, we have covered safety aspects of this agent in these indications, especially in K-RAS and all RAS wt patients. These safety aspects refer to the most common toxicities (i.e., acne-like skin rash, diarrhea and hypomagnesaemia). EXPERT OPINION: Panitumumab adds to the armamentarium of effective agents in the treatment of metastatic CRC. Due to its human origin, panitumumab is a well-tolerated agent with low rates of infusional reactions. Skin toxicity is frequent and should be pre-emptively treated. Other common toxicities related to panitumumab treatment, such as diarrhea and hypomagnesaemia, should be closely monitored to ensure early treatment or substitution.