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.

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.

Mucosal anti-CD3 monoclonal antibody attenuates collagen-induced arthritis that is associated with induction of LAP+ regulatory T cells and is enhanced by administration of an emulsome-based Th2-skewing adjuvant.

Mucosal (nasal or oral) administration of anti-CD3 mAb is effective in ameliorating animal models of autoimmunity (experimental autoimmune encephalomyelitis, diabetes, and lupus) by inducing LAP(+) regulatory T cells. We tested this approach in an arthritis model using type II collagen. We found that nasal anti-CD3 was more effective than oral anti-CD3 in attenuating the development of arthritis. Nasal anti-CD3 induced a LAP(+) regulatory T cell that secreted high levels of IL-10 and suppressed collagen-specific T cell proliferation and anti-collagen Ab production. However, neither nasal nor oral anti-CD3 attenuated disease when given to animals with ongoing arthritis, and this was associated with a lack of induction of LAP(+) regulatory T cells. We found, however, that coadministration of a novel emulsome adjuvant, which enhances Th2 responses, resulted in the induction of LAP(+) regulatory T cells and suppression of ongoing arthritis by both nasal and oral anti-CD3. Suppression of arthritis by mucosal anti-CD3 was associated with less joint damage, a decrease of TNF-alpha and IFN-gamma mRNA expression in joints, and a reduction in anti-collagen Abs. These results demonstrate that mucosal anti-CD3 therapy may serve as a therapeutic approach in arthritis and that the biologic effect is enhanced by an emulsome-based adjuvant.

Inhibition of autoimmune diabetes by oral administration of anti-CD3 monoclonal antibody.

Anti-CD3 monoclonal antibody (mAb) has been shown to induce tolerance and to be an effective treatment for diabetes both in animal models and in human trials. We have shown that anti-CD3 mAb given orally is biologically active in the gut and suppresses experimental autoimmune encephalitis by the induction of a regulatory T-cell that expresses latency-associated peptide (LAP) on its surface. In the present study, we investigated the effect of oral anti-CD3 mAb on the prevention of autoimmune diabetes in AKR mice in which the low-dose streptozocin (STZ) model induces autoimmunity to the beta-cells of the islets. We found that oral anti-CD3 mAb given at doses of 50 and 250 microg/feeding suppressed the incidence of diabetes in this model with the best effects seen at the 50 microg/dose. Associated with suppression, we observed decreased cell proliferation in the spleen and conversion of T-helper (Th)1 responses into Th2/Th3 responses in the periphery, including the pancreatic lymph nodes. Oral anti-CD3 mAb increased the expression of LAP on CD4(+) T-cells, and these cells could adoptively transfer protection. Protection by oral anti-CD3 was transforming growth factor-beta dependent. Our results demonstrate that oral anti-CD3 is effective in the model of STZ-induced diabetes and may be a useful form of therapy for type 1 diabetes in humans.

Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+ CD25- LAP+ T cells.

A major goal of immunotherapy for autoimmune diseases and transplantation is induction of regulatory T cells that mediate immunologic tolerance. The mucosal immune system is unique, as tolerance is preferentially induced after exposure to antigen, and induction of regulatory T cells is a primary mechanism of oral tolerance. Parenteral administration of CD3-specific monoclonal antibody is an approved therapy for transplantation in humans and is effective in autoimmune diabetes. We found that orally administered CD3-specific antibody is biologically active in the gut and suppresses autoimmune encephalomyelitis both before induction of disease and at the height of disease. Orally administered CD3-specific antibody induces CD4+ CD25- LAP+ regulatory T cells that contain latency-associated peptide (LAP) on their surface and that function in vitro and in vivo through a TGF-beta-dependent mechanism. These findings identify a new immunologic approach that is widely applicable for the treatment of human autoimmune conditions.

Nasal vaccination with a proteosome-based adjuvant and glatiramer acetate clears beta-amyloid in a mouse model of Alzheimer disease.

Amyloid beta-peptide (Abeta) appears to play a key pathogenic role in Alzheimer disease (AD). Immune therapy in mouse models of AD via Abeta immunization or passive administration of Abeta antibodies markedly reduces Abeta levels and reverses behavioral impairment. However, a human trial of Abeta immunization led to meningoencephalitis in some patients and was discontinued. Here we show that nasal vaccination with a proteosome-based adjuvant that is well tolerated in humans plus glatiramer acetate, an FDA-approved synthetic copolymer used to treat multiple sclerosis, potently decreases Abeta plaques in an AD mouse model. This effect did not require the presence of antibody, as it was observed in B cell-deficient (Ig mu-null) mice. Vaccinated animals developed activated microglia that colocalized with Abeta fibrils, and the extent of microglial activation correlated strongly with the decrease in Abeta fibrils. Activation of microglia and clearing of Abeta occurred with the adjuvant alone, although to a lesser degree. Our results identify a novel approach to immune therapy for AD that involves clearing of Abeta through the utilization of compounds that have been safely tested on or are currently in use in humans.

Neuroprotection by IL-10-producing MOG CD4+ T cells following ischemic stroke.

Mucosal tolerance has been used successfully to treat animal models of autoimmune diseases and is being tested in human diseases. In this work we demonstrate the reduction of infarct size following mucosal tolerance by myelin oligodendrocyte glycoprotein (MOG) (35-55) peptide in mouse stroke model. Nasal MOG was most efficacious and reduced ischemic infarct size by 70% at 24 h as well as improving behavior score. Using immunohistological methods and IL-10 -/- mice, we demonstrate the importance of IL-10-producing CD4+ T cells in the reduction of the ischemic infarct volume following middle cerebral artery occlusion (MCAO). Furthermore, adoptive transfer of CD4+ T cells from nasally tolerized mice to untreated mice prior to MCAO surgery significantly decreased stroke size (p<0.001 vs. control), whereas CD4+ T cells from nasally tolerized IL-10-deficient mice had no significant effect. Based on these results, modulation of cerebral inflammation by mucosal tolerance to myelin antigens may have applicability both as prophylactic therapy and treatment following ischemia attacks.

Nasal vaccination with myelin oligodendrocyte glycoprotein reduces stroke size by inducing IL-10-producing CD4+ T cells.

Inflammation plays an important role in ischemic stroke and in humans IL-10 may have a beneficial effect in stroke. We mucosally administered myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide to C57BL/6 mice before middle cerebral artery occlusion (MCAO) to induce an anti-inflammatory T cell response directed at CNS myelin. Nasal and oral administration of MOG(35-55) peptide decreased ischemic infarct size at 24 and 72 h after MCAO surgery. Nasal MOG(35-55) peptide was most efficacious and reduced infarct size by 70% at 24 h and by 50% at 72 h (p

Mucosal administration of heat shock protein-65 decreases atherosclerosis and inflammation in aortic arch of low-density lipoprotein receptor-deficient mice.

BACKGROUND: Increasing evidence supports the involvement of inflammation and immunity in atherogenesis as well as the role of autoimmunity to heat shock proteins (HSPs) in the progression of atherosclerosis. Mucosal administration of autoantigens decreases organ-specific inflammation and disease in several models of autoimmunity (diabetes, arthritis, and encephalomyelitis) and is also being tested in human clinical trials. METHODS AND RESULTS: We examined the effect of nasal or oral administration of mycobacterial HSP-65 on atherosclerotic lesion formation in mice lacking the receptor for LDL that were maintained on a high-cholesterol diet. Animals were nasally or orally treated for 1 week with HSP-65, and a high-cholesterol diet was started after the last treatment. The mice were mucosally treated once a week for 8 or 12 weeks, at which time pathological analysis was performed. We found a significant decrease in the size of atherosclerotic plaques, a reduction in macrophage-positive area in the aortic arch, increased interleukin-10 expression, and a reduced number of T cells in nasally treated animals compared with control animals. A similar trend was observed in orally treated mice, but it was not significant. CONCLUSIONS: Our results demonstrate that nasal vaccination with HSP reduces the inflammatory process associated with atherosclerosis and provides a new immunologic approach for the treatment of atherosclerosis.

IL-15, a survival factor for kidney epithelial cells, counteracts apoptosis and inflammation during nephritis.

IL-15, a T cell growth factor, has been linked to exacerbating autoimmune diseases and allograft rejection. To test the hypothesis that IL-15-deficient (IL-15-/-) mice would be protected from T cell-dependent nephritis, we induced nephrotoxic serum nephritis (NSN) in IL-15-/- and wild-type (IL-15+/+) C57BL/6 mice. Contrary to our expectations, IL-15 protects the kidney during this T cell-dependent immunologic insult. Tubular, interstitial, and glomerular pathology and renal function are worse in IL-15-/- mice during NSN. We detected a substantial increase in tubular apoptosis in IL-15-/- kidneys. Moreover, macrophages and CD4 T cells are more abundant in the interstitia and glomeruli in IL-15-/- mice. This led us to identify several mechanisms responsible for heightened renal injury in the absence of IL-15. We now report that IL-15 and the IL-15 receptor (alpha, beta, gamma chains) are constitutively expressed in normal tubular epithelial cells (TECs). IL-15 is an autocrine survival factor for TECs. TEC apoptosis induced with anti-Fas or actinomycin D is substantially greater in IL-15-/- than in wild-type TECs. Moreover, IL-15 decreases the induction of a nephritogenic chemokine, MCP-1, that attracts leukocytes into the kidney during NSN. Taken together, we suggest that IL-15 is a therapeutic for tubulointerstitial and glomerular kidney diseases.

Oral tolerance to copolymer 1 in myelin basic protein (MBP) TCR transgenic mice: cross-reactivity with MBP-specific TCR and differential induction of anti-inflammatory cytokines.

Oral tolerance to myelin basic protein (MBP) is an effective antigen-specific method to suppress experimental allergic encephalomyelitis (EAE). Glatiramer acetate [copolymer 1 (Cop1)] is a synthetic copolymer designed to mimic MBP which suppresses EAE, is used parenterally to treat multiple sclerosis (MS) and is being tested orally for efficacy in MS. We investigated the immunologic properties of Cop1 to determine the degree to which its effects were antigen specific using MBP TCR transgenic mice. Immunization of MBP TCR transgenic mice fed Cop1, MBP or MBP Ac1-11 resulted in decreased proliferation, and IL-2, IL-6 and IFN-gamma production, and increased secretion of IL-10 and transforming growth factor (TGF)-beta in Cop1-fed animals. IFN-gamma was decreased, and IL-10 and TGF-beta were increased in non-immunized mice fed Cop1 and stimulated in vitro with MBP. No such effects were observed in ovalbumin TCR transgenic mice. To determine if the effects of Cop1 were specific to MBP TCR-bearing cells, MBP TCR transgenic Rag2(-/-) mice were immunized and re-stimulated in vitro with Cop1. We found a marked increase in IL-4 and similar increases in IL-4 after feeding Cop1. In disease models, feeding Cop1 suppressed EAE in MBP TCR transgenic mice, (PL/J x SJL)F(1) mice, and in myelin oligodendrocyte glycoprotein-induced EAE in NOD mice. Oral Cop1 had no effect on collagen-induced arthritis. These results demonstrate that Cop1 is active orally in an antigen-specific fashion, and may function as an altered peptide ligand for MBP-specific TCR-bearing cells by decreasing pro-inflammatory cytokines (IFN-gamma) and increasing anti-inflammatory cytokines (IL-4, IL-10 and TGF-beta).