Giuseppe D’Alessio

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.

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.

Interactions of the cytotoxic RNase A dimers with the cytosolic ribonuclease inhibitor

Ribonuclease A (RNase A) dimers have been recently found to be endowed with some of the special, i.e., non‐catalytic biological activities of RNases, such as antitumor and aspermatogenic activities. These activities have been so far attributed to RNases which can escape the neutralizing action of the cytosolic RNase inhibitor (cRI). However, when the interactions of the two cytotoxic RNase A dimers with cRI were investigated in a quantitative fashion and at the molecular level, the dimers were found to bind cRI with high affinity and to form tight complexes.

Ribonucleases with angiogenic and bactericidal activities from the Atlantic salmon

The importance of fish in vertebrate evolution has been better recognized in recent years after the intense work carried out on fish genomics. The recent discovery that fish genomes comprise homologs of ribonucleases, studied before only in tetrapods, and the isolation of ribonucleases from zebrafish have suggested an experimental model for studying fish and vertebrate evolution. Thus, the cDNAs encoding the RNases from the Atlantic salmon were expressed, and the recombinant RNases (Ss‐RNase‐1 and Ss‐RNase‐2) were isolated and characterized as both proteins and for their biological activities. Salmon RNases are less active than RNase A in degrading RNA, but are both sensitive to the action of the human cytosolic RNase inhibitor. The two enzymes possess both angiogenic and bactericidal activities. However, catalytically inactivated Ss‐RNases do not exert any angiogenic activity, but preserve their full bactericidal activity, which is surprisingly preserved even when the enzyme proteins are fully denatured. Analyses of the conformational stability of the two RNases has revealed that they are as stable as typical RNases of the superfamily, and Ss‐RNase‐2, the most active as an enzyme, is also the most resistant to thermal and chemical denaturation. The implications of these findings in terms of the evolution of early RNases, in particular of the physiological significance of the angiogenic and bactericidal activities of fish RNases, are analyzed and discussed.

The cytosolic ribonuclease inhibitor contributes to intracellular redox homeostasis

The hypothesis that the cytosolic RNase inhibitor (cRI) has a role in the protection of the cellular redox homeostasis was investigated testing the effects of oxidants and anti‐oxidants on normal, primary endothelial HUVE cells, and malignant HeLa cells, before and after their engineering into cRI‐deprived cells. We found that cRI plays an important, possibly a key, physiological role in the protection of cells from redox stress, as demonstrated by decreased GSH levels as well as increased oxidant‐induced DNA damage in cRI deprived cells.

Human anti-ErbB2 immunoagents – immunoRNases and compact antibodies

Immunotherapy, based on mAbs specifically directed against cancer cells, is considered a precious strategy in the fight against cancer because of its selectivity and lack of multidrug resistant effects. However, there are obstacles to the complete success of current immunotherapy such as immune responses to nonhuman or even humanized antibodies and the large size of the antibodies, which hinders their diffusion into bulky tumors. Fully human, small immunoagents, capable of inhibiting tumor growth may overcome these problems and provide safe, highly selective and effective antitumor drugs. An attractive target for immunotherapy is ErbB2, a transmembrane tyrosine kinase receptor, overexpressed on tumor cells of different origin, with a key role in the development of malignancy. An anti‐ErbB2 humanized monoclonal (Herceptin®) is currently used with success for breast cancer therapy; however, it can engender cardiotoxicity and a high proportion of breast cancer patients are resistant to Herceptin® treatment. Anti‐ErbB2 immunoagents of human origin, with potentially no or very low immunogenicity have been engineered to assemble ‘compact’, i.e. reduced size, antibodies, one consisting of a human single‐chain antibody fragment (scFv) fused to a human RNase to construct an immunoRNase and the other made up of two human scFv molecules fused to the Fc region of a human IgG1. By choosing a human antibody fragment as the immune moiety and a human RNase as the effector moiety, an immunoRNase would be both nonimmunogenic and nontoxic, as it becomes toxic only when the scFv promotes its internalization by target cells. The alternative strategy of compact antibodies was aimed at producing therapeutic agents with an increased half‐life, prolonged tumor retention and the ability to recruit host effector functions. Moreover, the bivalency of compact antibodies can be exploited to construct bispecific antibodies, as well as for other therapeutic applications.

The bactericidal action on Escherichia coli of ZF‐RNase‐3 is triggered by the suicidal action of the bacterium OmpT protease

ZF‐RNase‐3 is one of the RNases from zebrafish (Danio rerio) with special (i.e. noncatalytic) properties. These include angiogenic and bactericidal activities. Given the interest of fish RNases as host‐defense effectors, we studied the mechanism of the bactericidal action of ZF‐RNase‐3 on Escherichia coli as a model Gram‐negative bacterium. The results obtained indicate that the bactericidal activity of ZF‐RNase‐3 is not lost when its catalytic RNase activity is obliterated. On the other hand, fully denatured ZF‐RNase‐3 conserves its bactericidal activity. When ZF‐RNase‐3 is added to E. coli cultures, it is cleaved at a specific Arg‐Arg peptide bond, thus engendering two peptide fragments. The larger fragment (residues 31–124), produced by proteolysis and reduction of a disulfide, is recognized as the actual bactericidal agent. The protease responsible for the proteolytic attack has been identified with OmpT, an outer membrane E. coli omptin protease. However, the most remarkable result obtained in the present study is the finding that the microbicidal action of ZF‐RNase‐3 can be achieved only with the suicidal cooperation of the bacterium itself.

The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei

PARP and RI colocalize by cosedimentation (View interaction)

A novel ErbB2 epitope targeted by human antitumor immunoagents

Two novel human antitumor immunoconjugates, engineered by fusion of a single‐chain antibody fragment against human ErbB2 receptor, termed Erbicin, with either a human RNase or the Fc region of a human IgG1, are selectively cytotoxic for ErbB2‐positive cancer cells in vitro and in vivo. These Erbicin‐derived immunoagents (EDIAs) do not show the most negative properties of Herceptin, the only humanized mAb against ErbB2 used in the therapy of breast carcinoma: cardiotoxicity and the inability to act on resistant tumors. These differences are probably attributable to the different ErbB2 epitopes recognized by EDIAs and Herceptin, respectively, as we have previously reported that they induce different signaling mechanisms that control tumor and cardiac cell viability. Thus, to accurately identify the novel epitope recognized by EDIAs, three independent and complementary methodologies were used. They gave coherent results, which are reported here: EDIAs bind to a different ErbB2 epitope than Herceptin and the other human/humanized antibodies against ErbB2 reported so far. The epitope has been successfully located in region 122–195 of extracellular domain I. These findings could lead to the identification of novel epitopes on ErbB2 that could be used as potential therapeutic targets to mitigate anti‐ErbB2‐associated cardiotoxicity and eventually overcome resistance.