Gennaro Riccio

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.

A new archaeal beta-glycosidase from Sulfolobus solfataricus: Seeding a novel retaining beta-glycan-specific glycoside hydrolase family along with the human non-lysosomal glucosylceramidase GBA2

Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families and clans that show conserved catalytic mechanism, structure, and active site residues, but may vary in substrate specificity. We report here the identification and the detailed molecular characterization of a novel glycoside hydrolase encoded from the gene sso1353 of the hyperthermophilic archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl beta-gluco- and beta-xylosides and the observation of transxylosylation reactions products demonstrates that SSO1353 operates via a retaining reaction mechanism. The catalytic nucleophile (Glu-335) was identified through trapping of the 2-deoxy-2-fluoroglucosyl enzyme intermediate and subsequent peptide mapping, while the general acid/base was identified as Asp-462 through detailed mechanistic analysis of a mutant at that position, including azide rescue experiments. SSO1353 has detectable homologs of unknown specificity among Archaea, Bacteria, and Eukarya and shows distant similarity to the non-lysosomal bile acid beta-glucosidase GBA2 also known as glucocerebrosidase. On the basis of our findings we propose that SSO1353 and its homologs are classified in a new CAZy family, named GH116, which so far includes beta-glucosidases (EC 3.2.1.21), beta-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families and clans that show conserved catalytic mechanism, structure, and active site residues, but may vary in substrate specificity. We report here the identification and the detailed molecular characterization of a novel glycoside hydrolase encoded from the gene sso1353 of the hyperthermophilic archaeon Sulfolobus solfataricus. This enzyme hydrolyzes aryl β-gluco- and β-xylosides and the observation of transxylosylation reactions products demonstrates that SSO1353 operates via a retaining reaction mechanism. The catalytic nucleophile (Glu-335) was identified through trapping of the 2-deoxy-2-fluoroglucosyl enzyme intermediate and subsequent peptide mapping, while the general acid/base was identified as Asp-462 through detailed mechanistic analysis of a mutant at that position, including azide rescue experiments. SSO1353 has detectable homologs of unknown specificity among Archaea, Bacteria, and Eukarya and shows distant similarity to the non-lysosomal bile acid β-glucosidase GBA2 also known as glucocerebrosidase. On the basis of our findings we propose that SSO1353 and its homologs are classified in a new CAZy family, named GH116, which so far includes β-glucosidases (EC 3.2.1.21), β-xylosidases (EC 3.2.1.37), and glucocerebrosidases (EC 3.2.1.45) as known enzyme activities.

Novel human anti-claudin 1 mAbs inhibit hepatitis C virus infection and may synergize with anti-SRB1 mAb.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver carcinoma and new therapies based on novel targets are needed. The tight junction protein claudin 1 (CLDN-1) is essential for HCV cell entry and spread, and anti-CLDN-1 rat and mouse mAbs are safe and effective in preventing and treating HCV infection in a human liver chimeric mouse model. To accelerate translation of these observations into a novel approach to treat HCV infection and disease in humans, we screened a phage display library of human single-chain antibody fragments by using a panel of CLDN-1-positive and -negative cell lines and identified phage specifically binding to CLDN-1. The 12 clones showing the highest levels of binding were converted into human IgG4. Some of these mAbs displayed low-nanomolar affinity, and inhibited infection of human hepatoma Huh7.5 cells by different HCV isolates in a dose-dependent manner. Cross-competition experiments identified six inhibitory mAbs that recognized distinct epitopes. Combination of the human anti-SRB1 mAb C-1671 with these anti-CLDN-1 mAbs could either increase or reduce inhibition of cell culture-derived HCV infection in vitro. These novel human anti-CLDN-1 mAbs are potentially useful to develop a new strategy for anti-HCV therapy and lend support to the combined use of antibodies targeting the HCV receptors CLDN-1 and SRB1, but indicate that care must be taken in selecting the proper combination.

Trastuzumab and target-therapy side effects: Is still valid to differentiate anthracycline Type I from Type II cardiomyopathies?

ABSTRACT The improvement in cancer therapy and the increasing number of long term survivors unearth the issue of cardiovascular side effects of anticancer treatments. As a paradox in cancer survivors, delayed cardiotoxicity has emerged as a significant problem. Two categories of cardiotoxic side effects of antineoplastic drugs have been previously proposed: Type I cardiotoxicity, defined as permanent cardiotoxicity, is usually caused by anthracyclines; Type II cardiotoxicity, considered as reversible cardiotoxicity, has been mainly related to monoclonal antibodies. The cardiotoxicity of antibodies has been associated to trastuzumab, a humanized anti-ErbB2 monoclonal antibody currently in clinical use for the therapy of breast carcinomas, which induces cardiac dysfunction when used in monotherapy, or in combination with anthracyclines. Furthermore, recent retrospective studies have shown an increased incidence of heart failure and/or cardiomyopathy in patients treated with trastuzumab, that can persist many years after the conclusion of the therapy, thus suggesting that the side toxic effects are not always reversible as it was initially proposed. On the other hand, early detection and prompt therapy of anthracycline associated cardiotoxicity can lead to substantial recovery of cardiac function. On the basis of these observations, we propose to find a new different classification for cardiotoxic side effects of drugs used in cancer therapy.

A novel human antitumor dimeric immunoRNase.

We report on the construction, production, and characterization of a new fully human dimeric immunoRNase, obtained by fusion of Erbicin, a human anti-ErbB2 single-chain antibody fragment (scFv), with a dimeric mutant of human pancreatic RNase (HHP2-RNase). This novel immunoagent with 2 scFv moieties each fused to 1 of the 2 subunits of the dimeric RNase, called ERB-HHP2-RNase, has shown improved biologic properties with respect to the previously reported monomeric ERB-hRNase immunoRNase: it selectively binds ErbB2-positive cancer cells with an increased avidity; it is not inhibited by the cytosolic ribonuclease inhibitor; it is endowed with a more powerful cytotoxic activity.

Pathophysiology of cardiotoxicity from target therapy and angiogenesis inhibitors

The progress in cancer therapy and the increase in number of long-term survivors reveal the issue of cardiovascular side-effects of anticancer drugs. Cardiotoxicity has become a significant problem, and the risks of adverse cardiac events induced by systemic drugs need to be seriously considered. Potential cardiovascular toxicities linked to anticancer agents include arrhythmias, myocardial ischemia and infarction, hypertension, thromboembolism, left ventricular dysfunction, and heart failure. It has been shown that several anticancer drugs seriously affect the cardiovascular system, such as ErbB2 inhibitors, vascular endothelial growth factor (VEGF) inhibitors, multitargeted kinase inhibitors, Abelson murine leukemia viral oncogene homolog inhibitors, and others. Each of these agents has a different mechanism through which it affects the cardiovascular system. ErbB2 inhibitors block the ErbB4/ErbB2 heterodimerization pathway triggered by Neuregulin-1, which is essential for cardiomyocyte survival. VEGF signaling is crucial for vascular growth, but it also has a major impact on myocardial function, and the VEGF pathway is also essential for maintenance of cardiovascular homeostasis. Drugs that inhibit the VEGF signaling pathway lead to a net reduction in capillary density and loss of contractile function. Here, we review the mechanisms and pathophysiology of the most significant cardiotoxic effects of ErbB2 inhibitors and antiangiogenic drugs. Moreover, we highlight the role of cardioncology in recognizing these toxicities, developing strategies to prevent or minimize cardiovascular toxicity, and reducing long-term cardiotoxic effects.

One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.

Effects of a second-generation human anti-ErbB2 ImmunoRNase on trastuzumab-resistant tumors and cardiac cells

The inhibition of ErbB2 by the use of human antibodies can be a valuable strategy for the treatment of breast and gastric cancer. Trastuzumab, a humanized anti-ErbB2 antibody in clinical use, is effective but can engender resistance as well as cardiotoxicity. ImmunoRNases, made up of a human anti-ErbB2 scFv and human pancreatic ribonucleases (HP-RNases), have been engineered to overcome the limits of other immunotoxins, such as immunogenicity and nonspecific toxicity. Here, we report that a novel anti-ErbB2 immunoRNase, called Erb-HPDDADD-RNase, obtained by fusing Erbicin, a human ErbB2-directed scFv, with an HP-RNase variant that resists the cytosolic inhibitor protein, binds with high affinity to a panel of ErbB2-positive gastric tumor cells and inhibits their growth more than does the parental immunoRNase, which is not resistant to the inhibitor. Moreover, Erb-HP-DDADD-RNase is endowed with antiproliferative activity for trastuzumab-resistant cancer cells both in vitro and in vivo that is more potent than that of the parental immunoRNase. Importantly, Erb-HP-DDADD-RNase does not show cardiotoxic effects in vitro on human cardiomyocytes and does not impair cardiac function in a mouse model. Thus, Erb-HP-DDADD-RNase could fulfil the therapeutic need of cancer patients ineligible for trastuzumab treatment due to primary or acquired trastuzumab resistance or to cardiac dysfunction.

A novel fully human antitumor ImmunoRNase resistant to the RNase inhibitor

The ErbB2 tyrosine kinase receptor is an attractive target for immunotherapy, as it is overexpressed in many carcinomas. ImmunoRNases, made up of a human anti-ErbB2 scFv (single chain antibody fragment) and human RNases, have been engineered to overcome the limits of immunotoxins, made up of mouse antibodies and plant or bacterial toxins, such as immunogenicity and non-specific toxicity. Here we describe the construction and characterization of a second-generation anti-ErbB2 immunoRNase, called ERB-HP-DDADD-RNase, obtained by fusing Erbicin, a human ErbB2-directed scFv, with an inhibitor-resistant variant of human pancreatic RNase (HP-DDADD-RNase). This novel immunoRNase retains both the enzymatic activity of human pancreatic RNase and the specific binding of the parental scFv to ErbB2-positive cells, showing an affinity comparable with that of the previously reported parental immunoRNase (ERB-HP-RNase). Moreover, the novel immunoRNase is endowed with an effective and selective in vitro antiproliferative action for ErbB2-positive tumor cells, which is more potent than that of the parental immunoRNase on tumor cells expressing low levels of ErbB2, due to its resistance to the RNase inhibitor. Thus, the novel immunoRNase could represent a valuable tool for ErbB2-positive cancer therapy.

A Negative Allosteric Modulator of WNT Receptor Frizzled 4 Switches into an Allosteric Agonist

The WNT pathway interconnects a network of signaling events involved in a huge plethora of cellular processes, from organogenesis to tissue homeostasis. Despite its importance, the exiguity of organic drugs directly targeting the members of the Frizzled family of WNT receptors has hampered progress across the whole spectrum of biological fields in which the signaling is involved. We here present FzM1.8, a small molecule acting as an allosteric agonist of Frizzled receptor FZD4. FzM1.8 derives from FzM1, a negative allosteric modulator of the receptor. Replacement of FzM1 thiophene with a carboxylic moiety induces a molecular switch in the lead and transforms the molecule into an activator of WNT signaling. We here show that, in the absence of any WNT ligand, FzM1.8 binds to FZD4, promotes recruitment of heterotrimeric G proteins, and biases WNT signaling toward a noncanonical route that involves PI3K. Finally, in colon cancer cells, we prove that the FZD4/PI3K axis elicited by FzM1.8 preserves stemness and promotes proliferation of undifferentiated cells.