Inhibition of a pancreatic cancer model by cooperative pairs of clinically approved and experimental antibodies.

By year 2025 pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer related death. However, other than improved chemotherapy and a small molecule inhibitor of the epidermal growth factor receptor (EGFR), no targeted drugs are currently available. Repurposing of approved drugs might offer a rapid solution. We employed an animal PDAC model, expressing a mutant and a wild type form of p53 and KRAS, respectively. Cetuximab, a clinically approved anti-EGFR monoclonal antibody (mAb) weakly inhibited PDAC xenografts, similar to trastuzumab, a mAb against HER2, a co-receptor of EGFR. Because the combination of cetuximab and trastuzumab only moderately enhanced the anti-tumor effects, we combined each with a home-made mAb to the same receptor and identified two cooperative pairs. The pair of trastuzumab and a murine anti-HER2 mAb better than the anti-EGFR pair inhibited PDAC xenografts, although HER2's abundance in our model is 15-fold lower than the level of EGFR. In vitro studies attribute cooperation to forced receptor endocytosis/degradation and inhibition of both DNA synthesis and cell migration. Taken together, our results identify cooperative pairs of anti-PDAC antibodies and highlight potential mechanisms of anti-tumor effects.

Multimerization of ERBB2/HER2 specific aptamer leads to improved receptor binding.

Aptamers represent a promising new treatment modality for cancer. Specificity and high affinity are two parameters that characterize aptamers. In this work, we elucidated physicochemical parameters of an ERBB2/HER2 specific aptamer and determined an optimal multimerization state, leading to higher binding and improved avidity. We applied biochemical, immunochemical and biophysical methodologies to characterize binding behaviors of multimerized versions of an ERBB2/HER2 specific aptamer and demonstrate structural integrity. Finally, we show that the trimeric ERBB2/HER2 specific aptamer instigates no immunogenic response in vivo. In summary, the set of methodologies we employed establishes a way to enhance activity of a model HER2-aptamer.

Inhibition of pancreatic carcinoma by homo- and heterocombinations of antibodies against EGF-receptor and its kin HER2/ErbB-2.

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.

Aptamer to ErbB-2/HER2 enhances degradation of the target and inhibits tumorigenic growth.

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.

Persistent elimination of ErbB-2/HER2-overexpressing tumors using combinations of monoclonal antibodies: relevance of receptor endocytosis.

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.

Cancer therapeutic antibodies come of age: targeting minimal residual disease.

Ten years after the first clinical application of Rituximab, an anti-CD20 recombinant monoclonal antibody, immunotherapy has become common practice in oncology wards. Thanks to the great diversity of the immune system and the powerful methodology of genetic engineering, the pharmacologic potential of antibody-based therapy is far from exhaustion. The recent application of Trastuzumab, an antibody to a receptor tyrosine kinase, in adjuvant breast cancer therapy marks the beginning of a new phase in cancer treatment. Here we discuss molecular mechanisms of antibody-based therapy, the emerging ability to target minimal disease and the therapeutic potential of combining antibodies with other modalities.

Synergistic down-regulation of receptor tyrosine kinases by combinations of mAbs: implications for cancer immunotherapy.

mAbs to receptor tyrosine kinases such as EGF receptor/ErbB-1 and HER2/ErbB-2 inhibit the tumorigenic growth of certain cancer cells, but although recombinant versions of such Abs are already used in oncology wards, the mechanism underlying immunotherapy remains unknown. We report that anti-EGF receptor Abs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining mAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, mAb combinations are more effective than single Abs in inhibiting HER2 signaling in vitro and tumorigenesis in animals. We present a model attributing efficacy of immunotherapy to the size of Ab-receptor lattices formed at the cell surface, which dictates the rate of endocytic clearance and extent of signaling blockade.

Therapeutic vaccines: realities of today and hopes for the future.

Vaccines are by definition prophylactic, but in recent years an interest has developed in therapeutic vaccines for infectious diseases such as AIDS and tuberculosis, as well as gastric ulcers, cancer (with different approaches to combat various types of malignancy) and autoimmune diseases (a definite success was the development of a vaccine against multiple sclerosis) and there are potential vaccines in development for myasthenia gravis, lupus and diabetes. Therapeutic vaccines are also being developed against cognitive diseases such as Alzheimer's disease, prion diseases and Huntington's disease. All of these efforts are based on the therapeutic vaccine being closely related chemically to the etiological agent that causes the disease.

MRI and fluorescence microscopy of the acute vascular response to VEGF165: vasodilation, hyper-permeability and lymphatic uptake, followed by rapid inactivation of the growth factor.

Vascular endothelial growth factor (VEGF) is one of the key growth factors regulating tumor angiogenesis and thus it is one of the primary targets for antiangiogenic therapy. The long-term effects of VEGF include induction of proliferation and migration of endothelial cells, tube formation and maintenance of the immature capillaries. The early effects of VEGF include vasodilation and increased permeability. We hypothesize that the early responses to VEGF can serve to develop a quantitative measure of the activity of VEGF, and therefore may be applicable for monitoring the efficacy of systemic suppression of VEGF signaling during antiangiogenic therapy. For that end we tested the ability of MRI and fluorescence microscopy to detect the early response to intradermal VEGF165 in nude mice. VEGF-induced local vasodilation and increased permeability was detected by intravenous administration of macromolecular biotin-BSA-GdDTPA(23) 30 min after intradermal administration of VEGF. Contrast leak showed saturation kinetics. Delayed contrast administration (90 min after intradermal administration of VEGF) resulted in low contrast leak and demonstrated that the saturation kinetics is not due to contrast equilibration between plasma and the interstitial space, but rather is due to suppression of vascular permeability. Permeability was restored by a second bolus of VEGF, showing that the saturation kinetics is primarily due to inactivation of the growth factor. Confocal microscopy of fluorescent BSA-FITC confirmed the permeability changes monitored by MRI. Moreover, confocal microscopy showed efficient lymphatic uptake of the extravasated contrast material specifically in regions of VEGF induced hyper-permeability.