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.

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.

Immunotherapy of cancer: from monoclonal to oligoclonal cocktails of anti-cancer antibodies: IUPHAR Review 18.

Antibody-based therapy of cancer employs monoclonal antibodies (mAbs) specific to soluble ligands, membrane antigens of T-lymphocytes or proteins located at the surface of cancer cells. The latter mAbs are often combined with cytotoxic regimens, because they block survival of residual fractions of tumours that evade therapy-induced cell death. Antibodies, along with kinase inhibitors, have become in the last decade the mainstay of oncological pharmacology. However, partial and transient responses, as well as emergence of tumour resistance, currently limit clinical application of mAbs. To overcome these hurdles, oligoclonal antibody mixtures are being tested in animal models and in clinical trials. The first homo-combination of two mAbs, each engaging a distinct site of HER2, an oncogenic receptor tyrosine kinase (RTK), has been approved for treatment of breast cancer. Likewise, a hetero-combination of antibodies to two distinct T-cell antigens, PD1 and CTLA4, has been approved for treatment of melanoma. In a similar vein, additive or synergistic anti-tumour effects observed in animal models have prompted clinical testing of hetero-combinations of antibodies simultaneously engaging distinct RTKs. We discuss the promise of antibody cocktails reminiscent of currently used mixtures of chemotherapeutics and highlight mechanisms potentially underlying their enhanced clinical efficacy.

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.

Aptamer Targeting the ERBB2 Receptor Tyrosine Kinase for Applications in Tumor Therapy.

Aptamers are an emerging class of molecules in cancer therapy. They can be easily synthesized and are considered cost-effective drug candidates. In this book chapter we describe the selection and characterization of DNA aptamers specific to the human epidermal growth factor receptor 2 (ERBB2/HER2), an oncogenic tyrosine kinase. First, a DNA aptamer library is applied and ERBB2-specific aptamers are selected using SELEX. Binders are subcloned into a pGEM-T vector, sequenced, and characterized using biochemical and cell biological techniques. By multimerizing the selected ERBB2 aptamers, it might be possible to significantly increase their avidity. For example, we could show that a trimeric ERBB2-specific aptamer could efficiently internalize membranal ERBB2. Furthermore, the receptor assembled in cytoplasmic puncta and was finally degraded by the lysosome. In addition, the selected, trimeric aptamer inhibited proliferation in an XTT assay in comparison to a control sequence. Aptamers selected using the protocol we describe might exert anticancer effect. In our example of a trimeric anti-HER2 aptamer, we could report that a human gastric xenograft mouse model demonstrated pharmacological value of the selected aptamer in vivo. This chapter should enable the interested reader to replicate selection of DNA aptamers specific to oncogenic cell surface. We would like to particularly emphasize some experimental approaches which were used to further characterize selected aptamer sequences, upon SELEX selection. For instance, we included several blotting techniques, antiproliferative assays of aptamers in vitro, and describe the handling of an in vivo human xenograft mouse model.

Cancer Immunotherapy: More Is (Much) Better.

Although antibodies against EGFR and HER2 are used to treat cancer, only some patients respond and resistance often emerges. Jacobsen and colleagues present in this issue experimental evidence favoring replacement of the currently applied monoclonal antibodies with oligoclonal mixtures of six synergistic antibodies, simultaneously engaging EGFR, HER2, and also HER3.

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.

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.

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.

Magnetic resonance imaging characterization of different experimental autoimmune encephalomyelitis models and the therapeutic effect of glatiramer acetate.

The roles of inflammation and degeneration as well as of gray matter abnormalities in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are controversial. We analyzed the pathological manifestations in two EAE models, the chronic oligodendrocyte glycoprotein (MOG)-induced versus the relapsing-remitting proteolipid protein (PLP)-induced, along the disease progression, using advanced magnetic resonance imaging (MRI) parameters. The emphasis of this study was the overall assessment of the whole brain by histogram analysis, as well as the detection of specific affected regions by voxel based analysis (VBA) using quantitative T2, magnetization transfer ratio (MTR) and diffusion tensor imaging (DTI). Brains of EAE-inflicted mice from both models revealed multiple white and gray matter areas with significant changes from naïve mice for all MRI parameters. Ventricle swelling was more characteristic to the PLP-induced model. Decreased MTR values and increased apparent diffusion coefficient (ADC) were observed mainly in MOG-induced EAE, indicative of macromolecular loss and structural CNS damage involvement in the chronic disease. The MS drug glatiramer acetate (GA), applied either as prevention or therapeutic treatment, affected all the MRI pathological manifestations, resulting in reduced T2 values and ventricle volume, elevated MTR and decreased ADC, in comparison to untreated EAE-inflicted mice. In accord, immunohistochemical analysis indicated less histological damage and higher amount of proliferating oligodendrocyte progenitor cells after GA treatment. The higher brain tissue integrity reflected by the MRI parameters on the level of the whole brain and in specific regions supports the in situ anti-inflammatory and neuroprotective consequences of GA treatment.

Simultaneous Inhibition of Estrogen Receptor and the HER2 Pathway in Breast Cancer: Effects of HER2 Abundance.

The estrogen receptor (ER) pathway and the epidermal growth factor receptor (EGFR) pathway play pivotal roles in breast cancer progression. Targeted therapies able to intercept ER or signaling downstream to EGFR and its kin, HER2, are routinely used to treat distinct groups of breast cancer patients. However, patient responses are limited by resistance to endocrine therapy, which may be due to compensatory HER2/EGFR signaling. This raises the possibility that simultaneous interception of HER2 and ER may enhance therapeutic efficacy. To address the question, we treated breast cancer cells with both fulvestrant (ICI 182780), an ER antagonist with no agonist effects, and lapatinib, an orally available tyrosine kinase inhibitor specific to EGFR and HER2. Our results indicate that the combination of drugs is especially effective when applied to HER2-overexpressing, ER-positive cancer cells. Interestingly, fulvestrant activated the mitogen-activated protein kinase (MAPK) pathway of these cells, but complete inhibition of MAPK signaling was observed on cotreatment with lapatinib. Taken together, our observations reinforce the possibility that the effectiveness of combining anti-ER and anti-HER2/EGFR drugs may be especially effective on a relatively small subtype of HER2-overexpressing, ER-positive tumors of the breast.

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.

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.

Degradation of collagen-guided tissue regeneration membranes by proteolytic enzymes of Porphyromonas gingivalis and its inhibition by antibacterial agents.

Previous studies have shown that whole cells of several periodontal pathogenic bacteria including Porphyromonas gingivalis may degrade the clinically used regeneration membranes Biomend Extend and Bio-Gide. Fractionation of P. gingivalis cells revealed that cell membrane-associated proteases are responsible for the in vitro degradation of the collagen membranes. In the present study, the specific role of extracellular vesicles and the purified Arg-gingipain enzyme of P. gingivalis in the degradation of three differently cross-linked collagen membranes (Ossix; Bio-Gide and Biomend Extend) was examined. In addition, the inhibitory effect of antibacterial agents and antibiotics used in local periodontal therapy on the enzymatic degradation was evaluated. The data presented show that while all tested collagen membranes, are prone to lysis by oral bacterial proteases, cross-linked membranes are more resistant to proteolysis. Furthermore, therapeutical concentrations of the antibacterial and antibiotic agents chlorhexidine, cetylpyridiniumchloride, minocycline and doxycycline were found to partially inhibit the enzymatic breakdown of the membranes, while metronidazole had no such effect. These results suggest that the presence of P. gingivalis cells, extracellular vesicles and enzymes in the vicinity of regeneration membranes in the periodontium, may change their physical structure and therefore alter their biological properties. Furthermore, the use of cross-linked collagen membranes and antibacterial agents may significantly inhibit this proteolytic process.

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.

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.