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.

Astrocyte disruption of neurovascular communication is linked to cortical damage in an animal model of multiple sclerosis.

To elucidate mechanisms contributing to cortical pathology in multiple sclerosis (MS), we investigated neurovascular aberrations, in particular the association of astrocytes with cortical neurons and blood vessels, in mice induced with experimental autoimmune encephalomyelitis (EAE). Blood-brain barrier (BBB) dysfunction was evident by leakage of the tracer sodium fluorescein, along with reduced expression of claudin-5 by endothelial cells and desmin by pericytes. Immunohistological and ultrastructural analyses revealed detachment of the astroglial cell bodies from the blood vessels and loss of their connections with both the blood vessels and the neuronal synapses. Furthermore, examination of individual astrocytic processes at cortical layer IV, where well-defined neuronal columns (barrels) are linked to functional properties, revealed loss of astrocytic confinement to the functional neuronal boundaries. Thus, in contrast to the highly modulated patches of astrocyte processes in naïve mice overlapping the barrel cores, in EAE-mice process distribution was uniform ignoring the barrel boundaries. These aberrations are attributed to the surrounding inflammation, indicated by T-cells presence in the cortex as well as in the subcortical white matter and the meninges. Immunomodulatory treatment with glatiramer acetate partially abrogated the neurovascular damage. These combined findings indicate that under inflammatory conditions, activated perivascular astrocytes fail in neuro-hemodynamic coupling, resulting in obstructed cross-talk between the blood vessels and the neurons. We propose that loss of cortical astrocytic regulation and fine-tuning between the blood supply and the neuronal needs contributes to the neurological impairment and cognitive decline occurring in EAE/MS as well as to the disease progression.

Examination of HER3 targeting in cancer using monoclonal antibodies.

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells' ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma-tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.

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.

Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR.

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.

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.

Oligodendrogenesis and myelinogenesis during postnatal development effect of glatiramer acetate.

Myelinogenesis in the mammal nervous system occurs predominantly postnatally. Glatiramer acetate (GA), a drug for the treatment for multiple sclerosis (MS), has been shown to induce immunomodulation and neuroprotection in the inflamed CNS in MS and in experimental autoimmune encephalomyelitis (EAE). Here we investigated whether GA can affect myelinogenesis and oligodendrogenesis in the developing nervous system under nonpathological conditions. Towards this end we studied myelination in mice injected daily by GA, at postnatal Days 7-21. Immunohistological and ultrastructural analyses revealed significant elevation in the number of myelinated axons as well as in the thickness of the myelin encircling them and their resulting g-ratios, in spinal cords of GA-injected mice compared with their PBS-injected littermates, at postnatal Day 14. Elevation in myelinated axons was detected also in the peripheral ventral roots of the motor nerves. GA induced also an increase in axonal diameter, implying an effect on the overall development of the nervous system. A prominent elevation in the amount of progenitor oligodendrocytes and their BrdU incorporation, as well as in mature oligodendrocytes indicated that the effect of GA is linked to increased proliferation and differentiation along the oligodendroglial maturation cascade. In addition, elevation in insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) was found in the white matter of the GA-injected mice. Furthermore, a functional advantage in rotating rod test was exhibited by GA-injected mice over their littermates at postnatal Day 21. These cumulative findings corroborate the beneficial effect of GA on oligodendrogenesis and myelination.

The adhesion of oral bacteria to modified titanium surfaces: role of plasma proteins and electrostatic forces.

OBJECTIVES: Modifications of titanium (Ti) implant surfaces have a significant effect on early biofilm formation and the outcome of implant procedures. The aim of this study was to examine the role of plasma proteins and electrostatic forces in the adhesion mechanism of oral bacteria to modified Ti surfaces. MATERIALS AND METHODS: Ti discs with three different types of surface modifications, machined, acid-etched, and acid-etched and blasted, were examined for adhesion of oral bacteria: Streptococcus mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum. Following pretreatment of the Ti with ion rich solutions or coating by human serum albumin or fibronectin, bacterial adhesion was examined by scanning electron microscopy and assessed quantitatively by DNA analysis. Ti coating by proteins as well as bacterial adhesion and their interrelationships were further investigated through confocal scanning laser microscopy. RESULTS: Acid-etched and blasted Ti surfaces exhibited significantly higher amounts of bacteria adhesion than the other two surfaces. Calcium was found to serve as a bridging agent in the adhesion process of S. mutans and F. nucleatum to Ti surfaces. Although albumin coating of the Ti reduced the adhesion of S. mutans to all surfaces, it had no influence on the adhesion of P. gingivalis or F. nucleatum. Coating the Ti with fibronectin enhanced P. gingivalis and F. nucleatum adhesion. CONCLUSIONS: Bacterial adhesion to Ti surfaces is roughness-dependent, and the adhesion mechanism is influenced by ions and proteins of the initial coating derived from the blood.

Wettability versus electrostatic forces in fibronectin and albumin adsorption to titanium surfaces.

OBJECTIVES: Although the enhancement of plasma protein adsorption to titanium ( Ti ) following wetting has been recognized, the relationship between wettability and electrostatic forces has remained unclear. Thus, we have carried out a series of studies to determine the role of wettability and electrostatic forces on protein adsorption. METHODS: Titanium disks with different surfaces were wetted with a range of solutions, two of which contained divalent positive ions ( Ca and Mg ). Unwetted disks served as a control. Subsequently, the wetted disks were subjected to three treatment regimes: (1) incubation in human serum albumin (HSA) or human serum fibronectin (HSF); (2) drying the wetted disks, followed by incubation in HSA or HSF; and (3) following protein adsorption, the Ca originating in the wetting solutions was removed by divalent positive ions chelator treatment (EGTA), and the remaining quantities were assessed. The quantity of the adsorbed proteins was determined by ELISA. RESULTS: It was found that in the case of HSA, adsorption was enhanced by the wettability, the presence of Ca and Mg in the wetting solution, and the existence of rough surfaces. For HSF, the wettability and rough surfaces enhanced adsorption. CONCLUSION: The results demonstrate that in addition to wettability, the composition of the wetting solution affects the protein adsorption. While wetting reduces the time for the HSA and HSF adsorption to reach saturation, the electrostatic forces enhance the amount of HSA adsorption. Thus, the protein adsorption capacity of titanium rough surfaces can be selectively manipulated by changing of the wetting solution.

The antibacterial activity of LL-37 against Treponema denticola is dentilisin protease independent and facilitated by the major outer sheath protein virulence factor.

Host defense peptides are innate immune effectors that possess both bactericidal activities and immunomodulatory functions. Deficiency in the human host defense peptide LL-37 has previously been correlated with severe periodontal disease. Treponema denticola is an oral anaerobic spirochete closely associated with the pathogenesis of periodontal disease. The T. denticola major surface protein (MSP), involved in adhesion and cytotoxicity, and the dentilisin serine protease are key virulence factors of this organism. In this study, we examined the interactions between LL-37 and T. denticola. The three T. denticola strains tested were susceptible to LL-37. Dentilisin was found to inactivate LL-37 by cleaving it at the Lys, Phe, Gln, and Val residues. However, dentilisin deletion did not increase the susceptibility of T. denticola to LL-37. Furthermore, dentilisin activity was found to be inhibited by human saliva. In contrast, a deficiency of the T. denticola MSP increased resistance to LL-37. The MSP-deficient mutant bound less fluorescently labeled LL-37 than the wild-type strain. MSP demonstrated specific, dose-dependent LL-37 binding. In conclusion, though capable of LL-37 inactivation, dentilisin does not protect T. denticola from LL-37. Rather, the rapid, MSP-mediated binding of LL-37 to the treponemal outer sheath precedes cleavage by dentilisin. Moreover, in vivo, saliva inhibits dentilisin, thus preventing LL-37 restriction and ensuring its bactericidal and immunoregulatory activities.

Generation and characterization of peptide mimotopes specific for anti ErbB-2 monoclonal antibodies.

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.

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.

Roles for growth factors in cancer progression.

Under physiological conditions, cells receive fate-determining signals from their tissue surroundings, primarily in the form of polypeptide growth factors. Integration of these extracellular signals underlies tissue homeostasis. Although departure from homeostasis and tumor initiation are instigated by oncogenic mutations rather than by growth factors, the latter are the major regulators of all subsequent steps of tumor progression, namely clonal expansion, invasion across tissue barriers, angiogenesis, and colonization of distant niches. Here, we discuss the relevant growth factor families, their roles in tumor biology, as well as the respective downstream signaling pathways. Importantly, cancer-associated activating mutations that impinge on these pathways often relieve, in part, the reliance of tumors on growth factors. On the other hand, growth factors are frequently involved in evolvement of resistance to therapeutic regimens, which extends the roles for polypeptide factors to very late phases of tumor progression and offers opportunities for cancer therapy.

Demyelination arrest and remyelination induced by glatiramer acetate treatment of experimental autoimmune encephalomyelitis.

The interplay between demyelination and remyelination is critical in the progress of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). In the present study, we explored the capacity of glatiramer acetate (GA, Copaxone) to affect the demyelination process and/or lead to remyelination in mice inflicted by chronic EAE, using both scanning electron microscopy and immunohistological methods. Spinal cords of untreated EAE mice revealed substantial demyelination accompanied by tissue destruction and axonal loss. In contrast, in spinal cords of GA-treated mice, in which treatment started concomitantly with disease induction (prevention), no pathology was observed. Moreover, when treatment was initiated after the appearance of clinical symptoms (suppression) or even in the chronic disease phase (delayed suppression) when substantial demyelination was already manifested, it resulted in a significant decrease in the pathological damage. Detection of oligodendrocyte progenitor cells (OPCs) expressing the NG2 or O4 markers via colocalization with the proliferation marker BrdU indicated their elevated levels in spinal cords of GA-treated mice. The mode of action of GA in this system is attributed to increased proliferation, differentiation, and survival of OPCs along the oligodendroglial maturation cascade and their recruitment into injury sites, thus enhancing repair processes in situ.

Glatiramer acetate increases T- and B -regulatory cells and decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) in an animal model of multiple sclerosis.

To identify the mechanisms relevant for the therapeutic effect of glatiramer acetate (GA), we studied T- and B- regulatory cells as well as GM-CSF expression in mice recovered from experimental autoimmune encephalomyelitis (EAE). Selective depletion of Tregs reduced but did not eliminate the ability of GA to ameliorate EAE, indicating a role for additional immune-subsets. The prevalence of Bregs in the periphery and the CNS of EAE-mice increased following GA-treatment. Furthermore, GA downregulated the pathological expression of GM-CSF, on both the protein and mRNA levels. These findings corroborate the broad immunomodulatory mechanism of action of GA in EAE/MS.

Involvement of Toll-like receptors 2 and 4 in the innate immune response to Treponema denticola and its outer sheath components.

Treponema denticola is considered an important oral pathogen in the development and progression of periodontal diseases. In the present study, the mechanisms of recognition and activation of murine macrophages by T. denticola and its major outer sheath protein (MSP) and lipooligosaccharide (LOS or glycolipid) were investigated. T. denticola cells and the MSP induced innate immune responses through TLR2-MyD88, whereas LOS induced a macrophage response through TLR4-MyD88. The presence of gamma interferon (IFN-gamma), or of high numbers of T. denticola, circumvented the requirement for TLR2 for the macrophage response to T. denticola, although the response was still dependent on MyD88. In contrast, synergy with IFN-gamma did not alter the TLR dependence of the response to the T. denticola surface components LOS and MSP, despite enhanced sensitivity. These data suggest that although there is flexibility in the requirements for recognition of T. denticola cells (TLR2 dependent or independent), MyD88 is a requirement for the downstream signaling events that lead to inflammation. We also demonstrate that both outer sheath molecules LOS and MSP induce macrophage tolerance to further stimulation with enterobacterial lipopolysaccharide. Tolerance induced by T. denticola components during mixed infections may represent a general mechanism through which bacteria evade clearance.