Claudia De Lorenzo

Detection, monitoring, and management of trastuzumab-induced left ventricular dysfunction: an actual challenge.

The antibody trastuzumab, targeted to inhibit the signalling of ErbB2, a tyrosine kinase receptor overexpressed in 20–30% of breast cancers, improves the prognosis in women affected by this tumour, but produces cardiotoxicity, since ErbB2 is also involved in myocardial homeostasis. In this review, we discuss the pathophysiology of trastuzumab cardiomyopathy and the complex interplay between ErbB2 inhibition and anthracyclines, and we focus on the actual challenges of detecting, monitoring, and managing trastuzumab cardiotoxicity: the research of new, sensitive markers of early trastuzumab toxicity, before the ejection fraction is reduced, is an active field of research.

THE ANTITUMOR ACTION OF SEMINAL RIBONUCLEASE AND ITS QUATERNARY CONFORMATIONS

It has been previously shown that the antitumor action of bovine seminal ribonuclease (BS‐RNase) is dependent on its dimeric structure. However, two distinct quaternary structures, each in equilibrium with the other, have been described for the enzyme: one in which the two subunits exchange their N‐terminal ends, the other with no exchange. Antitumor activity assays, carried out on homogeneous quaternary forms of the enzyme, as well as on dimeric mutants of bovine pancreatic RNase A, reveal that another structural determinant of the antitumor activity of BS‐RNase is the exchange of N‐terminal ends between subunits.

A Fully Human Antitumor ImmunoRNase Selective for ErbB-2-Positive Carcinomas

We report the preparation and characterization of a novel, fully human antitumor immunoRNase (IR). The IR, a human RNase and fusion protein made up of a human single chain variable fragment (scFv), is directed to the ErbB-2 receptor and overexpressed in many carcinomas. The anti-ErbB-2 IR, named hERB-hRNase, retains the enzymatic activity of the wild-type enzyme (human pancreatic RNase) and specifically binds to ErbB-2-positive cells with the high affinity (Kd = 4.5 nm) of the parental scFv. hERB-hRNase behaves as an immunoprotoxin and on internalization by target cells becomes selectively cytotoxic in a dose-dependent manner at nanomolar concentrations. Administered in five doses of 1.5 mg/kg to mice bearing an ErbB-2-positive tumor, hERB-hRNase induced a dramatic reduction in tumor volume. hERB-hRNase is the first fully human antitumor IR produced thus far, with a high potential as a poorly immunogenic human drug devoid of nonspecific toxicity, directed against ErbB-2-positive malignancies.

The RFG oligomerization domain mediates kinase activation and re-localization of the RET/PTC3 oncoprotein to the plasma membrane.

The RET/PTC3 oncogene arises from the fusion between the N-terminal encoding domain of the RFG gene and the tyrosine kinase encoding domain of RET receptor. RET/PTC3 is very frequent in papillary thyroid carcinomas, especially in children exposed to the Chernobyl accident. We have studied the functional consequences of the RFG–RET fusion. Here we show that the N-terminal coiled-coil domain of RGF mediates oligomerization and activation of the kinase and of the transforming capability of RET/PTC3. In addition, the RFG coiled-coil domain mediates a physical association between RET/PTC3 and RGF proteins, rendering RFG a bona fide substrate of RET/PTC3 kinase. Finally, we show that the coiled-coil domain of RGF is essential for the distribution of the RET/PTC3 protein at the membrane/particulate cell compartment level, where also most of the RFG protein is localized. We propose that fusion to the RFG coiled-coil domain provides RET kinase with a scaffold that mediates oligomerization and re-localization of the RET/PTC3 protein, a process that may be crucial for the signalling of this specific RET/PTC variant.

Ranolazine protects from doxorubicin-induced oxidative stress and cardiac dysfunction

Doxorubicin is widely used against cancer; however, it can produce heart failure (HF). Among other hallmarks, oxidative stress is a major contributor to HF pathophysiology. The late INa inhibitor ranolazine has proven effective in treating experimental HF. Since elevated [Na+]i is present in failing myocytes, and has been recently linked with reactive oxygen species (ROS) production, our aim was to assess whether ranolazine prevents doxorubicin‐induced cardiotoxicity, and whether blunted oxidative stress is a mechanism accounting for such protection.

Differential binding of human immunoagents and Herceptin to the ErbB2 receptor

Overexpression of the ErbB2 receptor is associated with the progression of breast cancer, and is a sign of a poor prognosis. Herceptin, a humanized antibody directed to the ErbB2 receptor, has been proven to be effective in the immunotherapy of breast cancer. However, it can result in cardiotoxicity, and a large fraction of breast cancer patients are resistant to Herceptin treatment. We have engineered three novel, fully human, anti‐ErbB2 immunoagents: Erbicin, a human single‐chain antibody fragment; ERB‐hRNase, a human immunoRNase composed of Erbicin fused to a human RNase; ERB‐hcAb, a human ‘compact’ antibody in which two Erbicin molecules are fused to the Fc fragment of a human IgG1. Both ERB‐hRNase and ERB‐hcAb strongly inhibit the growth of ErbB2‐positive cells in vivo. The interactions of the Erbicin‐derived immunoagents and Herceptin with the extracellular domain of ErbB2 (ErbB2‐ECD) were investigated for the first time by three different methods. Erbicin‐derived immunoagents bind soluble extracellular domain with a lower affinity than that measured for the native antigen on tumour cells. Herceptin, by contrast, shows a higher affinity for soluble ErbB2‐ECD. Accordingly, ErbB2‐ECD abolished the in vitro antitumour activity of Herceptin, with no effect on that of Erbicin‐derived immunoagents. These results suggest that the fraction of immunoagent neutralized by free extracellular domain shed into the bloodstream is much higher for Herceptin than for Erbicin‐derived immunoagents, which therefore may be used at lower therapeutic doses than those employed for Herceptin.

A new RNase-based immunoconjugate selectively cytotoxic for ErbB2-overexpressing cells.

We report a new tumor‐directed immunoRNase, a chimeric protein made up of an antibody fragment (single‐chain Fv fragment) directed to ErbB2, a cell surface receptor, and a non‐toxic, human ribonuclease, which upon cell internalization becomes cytotoxic. The immunoRNase is active as a ribonuclease, specifically binds and selectively kills ErbB2‐positive cells. ErbB2 is one of the most specific tumor‐associated antigens identified so far, overexpressed on tumor cells of different origin. Its choice as target antigen and that of a non‐toxic, human RNase as the killer moiety makes this immunoRNase a new, potentially attractive anticancer agent.

Mechanisms of cardiotoxicity associated with ErbB2 inhibitors

The ErbB2 receptor is a proto-oncogene associated with a poor prognosis in breast cancer. Herceptin, the only humanized anti-ErbB2 antibody currently in clinical use, has proven to be an essential tool in the immunotherapy of breast carcinoma, but induces cardiotoxicity. ErbB2 is involved in the growth and survival pathway of adult cardiomyocytes; however, its levels in the adult heart are much lower than those found in breast cancer cells, the intended targets of anti-ErbB2 antibodies. Furthermore, clinical trials have shown relatively low cardiotoxicity for Lapatinib, a dual kinase inhibitor of EGFR and ErbB2, and Pertuzumab, a new anti-ErbB2 monoclonal antibody currently in clinical trials, which recognizes an epitope distant from that of Herceptin. A novel human antitumor compact anti-ErbB2 antibody, Erb-hcAb, selectively cytotoxic for ErbB2-positive cancer cells in vitro and vivo, recognizes an epitope different from that of Herceptin, and does not show cardiotoxic effects both in vitro on rat and human cardiomyocytes and in vivo on a mouse model. We investigated the molecular basis of the different cardiotoxic effects among the ErbB2 inhibitors by testing their effects on the formation of the Neuregulin 1β (NRG-1)/ErbB2/ErbB4 complex and on the activation of its downstream signaling. We report herein that Erb-hcAb at difference with Herceptin, 2C4 (Pertuzumab) and Lapatinib, does not affect the ErbB2–ErbB4 signaling pathway activated by NRG-1 in cardiac cells. These findings may have important implications for the mechanism and treatment of anti-ErbB2-induced cardiotoxicity.

Intracellular route and mechanism of action of ERB-hRNase, a human anti-ErbB2 anticancer immunoagent.

Human anti‐ErbB2 immunoRNase with Erbicin fused to HP‐RNase (ERB‐hRNase) is a fully human immunoRNase made up of human pancreatic RNase fused to a human anti‐ErbB2 scFv. It binds selectively with high affinity to ErbB2‐positive cells, and specifically inhibits their proliferation, in vitro and in vivo. An investigation of its mechanism of action and its intracellular destination has revealed that ERB‐hRNase depends on its RNase activity for cytotoxic action; it reaches the cytosol directly from the endosomal compartment; it is inhibited by the cytosolic RNase inhibitor (cRI), but the levels that ERB‐hRNase reaches in the cytosol neutralize cRI, thus inducing cell death by apoptosis.

Human anti-nucleolin recombinant immunoagent for cancer therapy

Significance Because of its selective expression on the surface of a variety of different cancer cells, but not on their normal counterparts, nucleolin (NCL) represents an attractive target for antineoplastic treatments. However, previously described NCL-targeting molecules, although promising, still suffer from intrinsic limitations. Here, we describe the identification of the first fully human anti-NCL immune-based agent displaying antineoplastic activity against solid tumors, both in vitro and in vivo. This molecule could represent the prototype of a novel class of NCL-targeting drugs with enormous clinical potential as tools for the diagnosis and therapy of a wide range of human cancers. Nucleolin (NCL) is a nucleocytoplasmic protein involved in many biological processes, such as ribosomal assembly, rRNA processing, and mRNA stabilization. NCL also regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and aggressiveness. Interestingly, NCL is expressed on the surface of actively proliferating cancer cells, but not on their normal counterparts. Therefore, NCL is an attractive target for antineoplastic treatments. Taking advantage of phage-display technology, we engineered a fully human single-chain fragment variable, named 4LB5. This immunoagent binds NCL on the cell surface, it is translocated into the cytoplasm of target cells, and it abrogates the biogenesis of NCL-dependent miRNAs. Binding of 4LB5 to NCL on the cell surface of a variety of breast cancer and hepatocellular carcinoma cell lines, but not to normal-like MCF-10a breast cells, dramatically reduces cancer cell viability and proliferation. Finally, in orthotopic breast cancer mouse models, 4LB5 administration results in a significant reduction of the tumor volume without evident side effects. In summary, here we describe, to our knowledge, the first anti-NCL single-chain fragment variable displaying antineoplastic activity against established solid tumors, which could represent the prototype of novel immune-based NCL-targeting drugs with clinical potential as diagnostic and therapeutic tools in a wide variety of human cancers.