Michael Sela

Significance: The human EGF receptor (EGFR/HER) family plays critical roles in tumor progression. Therefore, several therapies intercepting these receptors were developed and clinically approved. Importantly, patients treated with such therapeutics often develop resistance, and in some cases this resistance has been associated with activation of HER3. Potentially, HER3 blockade might overcome patient resistance. Hence, antibodies to HER3 have been developed by us and other researchers. However, it has remained unclear which antibody attributes are required for effective tumor inhibition. To address this issue, we generated several monoclonal antibodies, which were tested in vitro and in tumor-bearing animals. Our results suggest that anti-HER3 antibodies able to intercept stroma–tumor interactions, as well as accelerate HER3 degradation, might inhibit tumor growth better than other antibodies.

Due to intrinsic aggressiveness and lack of effective therapies, prognosis of pancreatic cancer remains dismal. Because the only molecular targeted drug approved for pancreatic ductal adenocarcinoma is a kinase inhibitor specific to the epidermal growth factor receptor (EGFR), and this receptor collaborates with another kinase, called HER2 (human EGF-receptor 2), we assumed that agents targeting EGFR and/or HER2 would effectively retard pancreatic ductal adenocarcinoma. Accordingly, two immunological strategies were tested in animal models: (i) two antibodies able to engage distinct epitopes of either EGFR or HER2 were separately combined, and (ii) pairs of one antibody to EGFR and another to HER2. Unlike the respective single monoclonal antibodies, which induced weak effects, both types of antibody combinations synergized in animals in terms of tumor inhibition. Immunological cooperation may not depend on receptor density, antigenic sites, or the presence of a mutant RAS protein. Nevertheless, both types of antibody combinations enhanced receptor degradation. Future efforts will examine the feasibility of each strategy and the potential of combining them to achieve sustained tumor inhibition. Significance Prognosis of pancreatic ductal adenocarcinoma (PDAC) remains very poor, with 5-y survival less than 10%. Unfortunately, adjuvant therapies are only weakly effective, and only one molecular targeted drug, a kinase inhibitor of epidermal growth factor receptor (EGFR), has been clinically approved. Hence, we examined antibodies to EGFR and to its sibling, HER2. While single antibodies to these receptors only weakly retarded growth of human PDAC in animals, significantly stronger inhibition was observed when we combined two antibodies to EGFR or two antibodies to HER2. Similarly, simultaneous inhibition of both EGFR and HER2, using a pair of antibodies, resulted in enhanced efficacy. These observations predict that targeting EGFR and HER2, by using pairs of monoclonal antibodies, might translate to better treatment of PDAC patients.

Aptamers, oligonucleotides able to avidly bind cellular targets, are emerging as promising therapeutic agents, analogous to monoclonal antibodies. We selected from a DNA library an aptamer specifically recognizing human epidermal growth factor receptor 2 (ErbB-2/HER2), a receptor tyrosine kinase, which is overexpressed in a variety of human cancers, including breast and gastric tumors. Treatment of human gastric cancer cells with a trimeric version (42 nucleotides) of the selected aptamer (14 nucleotides) resulted in reduced cell growth in vitro, but a monomeric version was ineffective. Likewise, when treated with the trimeric aptamer, animals bearing tumor xenografts of human gastric origin reflected reduced rates of tumor growth. The antitumor effect of the aptamer was nearly twofold stronger than that of a monoclonal anti–ErbB-2/HER2 antibody. Consistent with aptamer-induced intracellular degradation of ErbB-2/HER2, incubation of gastric cancer cells with the trimeric aptamer promoted translocation of ErbB-2/HER2 from the cell surface to cytoplasmic puncta. This translocation was associated with a lysosomal hydrolase-dependent clearance of the ErbB-2/HER2 protein from cell extracts. We conclude that targeting ErbB-2/HER2 with DNA aptamers might retard the tumorigenic growth of gastric cancer by means of accelerating lysosomal degradation of the oncoprotein. This work exemplifies the potential pharmacological utility of aptamers directed at cell surface proteins, and it highlights an endocytosis-mediated mechanism of tumor inhibition.

Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC. Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC’s cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route. In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition.

The erbb-2 gene receptor is often over-expressed in human cancer and its overexpression is accompanied by worse prognosis. Targeting erbb-2 gene with antibodies is an effective approach to curtail the progression of erbb-2 gene-expressing cancer types. Two monoclonal antibodies, L-26 and N-12, previously generated in our laboratory, have shown effective tumor inhibition in mice, especially when used in combination. Here, we describe novel peptide mimics of erbb-2 gene protein epitopes, also called mimotopes, that were selected from a constraint random 12-mer peptide phage library, specific for the antibodies L-26 and N-12. Initial sequencing analyses revealed little sequence conservation among the peptide mimotopes, and no sequence homology with the erbb-2 gene protein. However, computational analyses of the two groups of peptides, specific for L-26 and N-12, suggested different epitopes on the erbb-2 gene extracellular domain. In vitro assays showed that the phage displayed peptide mimotopes were specific to their respective antibodies. Selected cyclic peptide mimotopes, but not their corresponding linear equivalents, were able to inhibit binding of the antibodies L-26 and N-12 to the surface of erbb-2 gene-expressing cancer cells in a concentration-dependent manner. In line with this observation, phage-displayed cyclic peptides successfully competed in vitro with recombinant erbb-2 gene protein for binding to their respective antibodies L-26 or N-12. Consistent with the antibody inhibition experiments, we detected specific anti-erbb-2 gene antibodies following vaccination with KLH-coupled cyclic peptides but not with multiple antigenic linear peptides. Potentially, the selected peptides could serve as a starting point for the development of a vaccine against erbb-2 gene over-expressing cancer.

Monoclonal antibodies (mAbs) to ErbB-2/HER2 or to its sibling, the epidermal growth factor receptor (EGFR), prolong survival of cancer patients, especially when combined with cytotoxic therapies. However, low effectiveness of therapeutic mAbs and the evolution of patient resistance call for improvements. Here we test in animals pairs of anti-ErbB-2 mAbs and report that pairs comprising an antibody reactive with the dimerization site of ErbB-2 and an antibody recognizing another distinct epitope better inhibit ErbB-2-overexpressing tumors than other pairs or the respective individual mAbs. Because the superiority of antibody combinations extends to tumor cell cultures, we assume that nonimmunological mechanisms contribute to mAb synergy. One potential mechanism, namely the ability of mAb combinations to instigate ErbB-2 endocytosis, is demonstrated. Translation of these lessons to clinical applications may enhance patient response and delay acquisition of resistance.