Preclinical evaluation of Tc-99m p5+14 peptide for SPECT detection of cardiac amyloidosis.

INTRODUCTION: Amyloid deposition is a cause of restrictive cardiomyopathy. Patients who present with cardiac disease can be evaluated for transthyretin (TTR)-associated cardiac amyloidosis using nuclear imaging with 99mTc-labeled pyrophosphate (PYP); however, light chain-associated (AL) cardiac amyloid is generally not detected using this tracer. As an alternative, the amyloid-binding peptide p5+14 radiolabeled with iodine-124 has been shown to be an effective pan-amyloid radiotracer for PET/CT imaging. Here, a 99mTc-labeled form of p5+14 peptide has been prepared to facilitate SPECT/CT imaging of cardiac amyloidosis. METHOD: A synthesis method suitable for clinical applications has been used to prepare 99mTc-labeled p5+14 and tested for peptide purity, product bioactivity, radiochemical purity and stability. The product was compared with99mTc-PYP for cardiac SPECT/CT imaging in a mouse model of AA amyloidosis and for reactivity with human tissue sections from AL and TTR patients. RESULTS: The 99mTc p5+14 tracer was produced with >95% yields in radiopurity and bioactivity with no purification steps required and retained over 95% peptide purity and >90% bioactivity for >3 h. In mice, the tracer detected hepatosplenic AA amyloid as well as heart deposits with uptake ~5 fold higher than 99mTc-PYP. 99mTc p5+14 effectively bound human amyloid deposits in the liver, kidney and both AL- and ATTR cardiac amyloid in tissue sections in which 99mTc-PYP binding was not detectable. CONCLUSION: 99mTc-p5+14 was prepared in minutes in >20 mCi doses with good performance in preclinical studies making it suitable for clinical SPECT/CT imaging of cardiac amyloidosis.

Cardiac Amyloid Detection by PET/CT Imaging of Iodine (124I) Evuzamitide (124I-p5+14): A Phase 1/2 Study.

BACKGROUND: The noninvasive detection of cardiac amyloid, as well as deposits in other vital organs, is critical for early diagnosis and quantitative disease monitoring. Positron emission tomography is an intrinsically quantitative imaging modality suitable for high-resolution amyloid detection. OBJECTIVES: This study sought to evaluate the safety and efficacy of a novel amyloid-reactive peptide, designated p5+14, labeled with iodine-124 (124I), in patients with diverse types of systemic amyloidosis. METHODS: In a single-site, open label phase 1/2 study (NCT03678259), the safety, biodistribution, and sensitivity of a single intravenous infusion of 124I-evuzamitide was assessed in patients with systemic amyloidosis (n = 50), asymptomatic transthyretin sequence variant carriers (n = 2), and healthy volunteers (n = 5). Subjects were administered 1.4 ± 0.2 mg of 124I-evuzamitide (71.5 ± 12.4 MBq) and positron emission tomography/x-ray computed tomography images acquired at 5.2 hours (Q25-Q75: 4.9-5.4 hours) postinfusion. Images were assessed visually and semi-quantitatively for positive uptake of radiotracer in the heart and other major organs. RESULTS: Uptake of 124I-evuzamitide in the heart and other abdominothoracic organs was consistent with the patient's clinical presentation and the type of amyloidosis. The patient- and cardiac-associated sensitivity for imaging and clinical observations was 93.6% (95% CI: 82.8%-97.8%) and 96.2% (95% CI: 81.8%-99.8%), respectively. Semi-quantitative uptake of the radiotracer correlated significantly with serum N-terminal pro-B-type natriuretic peptide measurements in patients with light chain-associated amyloidosis. Cardiac uptake was not observed in any healthy volunteers. The agent was well tolerated, with 1 drug-related adverse event and no deaths. CONCLUSIONS: 124I-evuzamitide is an amyloid-binding radiotracer capable of detecting cardiac amyloid in patients with high sensitivity.

Development and characterization of a prototypic pan-amyloid clearing agent - a novel murine peptide-immunoglobulin fusion.

Introduction: Systemic amyloidosis is a progressive disorder characterized by the extracellular deposition of amyloid fibrils and accessory proteins in visceral organs and tissues. Amyloid accumulation causes organ dysfunction and is not generally cleared by the immune system. Current treatment focuses on reducing amyloid precursor protein synthesis and slowing amyloid deposition. However, curative interventions will likely also require removal of preexisting amyloid deposits to restore organ function. Here we describe a prototypic pan-amyloid binding peptide-antibody fusion molecule (mIgp5) that enhances macrophage uptake of amyloid. Methods: The murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, fused genetically to the N-terminal of the immunoglobulin light chain was synthesized in HEK293T/17 cells. The binding of the p5 peptide moiety was assayed using synthetic amyloid-like fibrils, human amyloid extracts and amyloid-laden tissues as substrates. Binding of radioiodinated mIgp5 with amyloid deposits in vivo was evaluated in a murine model of AA amyloidosis using small animal imaging and microautoradiography. The bioactivity of mIgp5 was assessed in complement fixation and in vitro phagocytosis assays in the presence of patient-derived amyloid extracts and synthetic amyloid fibrils as substrates and in the presence or absence of human serum. Results: Murine Igp5 exhibited highly potent binding to AL and ATTR amyloid extracts and diverse types of amyloid in formalin-fixed tissue sections. In the murine model of systemic AA amyloidosis, 125I-mIgp5 bound rapidly and specifically to amyloid deposits in all organs, including the heart, with no evidence of non-specific uptake in healthy tissues. The bioactivity of the immunoglobulin Fc domain was uncompromised in the context of mIgp5 and served as an effective opsonin. Macrophage-mediated uptake of amyloid extract and purified amyloid fibrils was enhanced by the addition of mIgp5. This effect was exaggerated in the presence of human serum coincident with deposition of complement C5b9. Conclusion: Immunostimulatory, amyloid-clearing therapeutics can be developed by incorporating pan-amyloid-reactive peptides, such as p5, as a targeting moiety. The immunologic functionality of the IgG remains intact in the context of the fusion protein. These data highlight the potential use of peptide-antibody fusions as therapeutics for all types of systemic amyloidosis.

Clinical Confirmation of Pan-Amyloid Reactivity of Radioiodinated Peptide 124I-p5+14 (AT-01) in Patients with Diverse Types of Systemic Amyloidosis Demonstrated by PET/CT Imaging.

There are at least 20 distinct types of systemic amyloidosis, all of which result in the organ-compromising accumulation of extracellular amyloid deposits. Amyloidosis is challenging to diagnose due to the heterogeneity of the clinical presentation, yet early detection is critical for favorable patient outcomes. The ability to non-invasively and quantitatively detect amyloid throughout the body, even in at-risk populations, before clinical manifestation would be invaluable. To this end, a pan-amyloid-reactive peptide, p5+14, has been developed that is capable of binding all types of amyloid. Herein, we demonstrate the ex vivo pan-amyloid reactivity of p5+14 by using peptide histochemistry on animal and human tissue sections containing various types of amyloid. Furthermore, we present clinical evidence of pan-amyloid binding using iodine-124-labeled p5+14 in a cohort of patients with eight (n = 8) different types of systemic amyloidosis. These patients underwent PET/CT imaging as part of the first-in-human Phase 1/2 clinical trial evaluating this radiotracer (NCT03678259). The uptake of 124I-p5+14 was observed in abdominothoracic organs in patients with all types of amyloidosis evaluated and was consistent with the disease distribution described in the medical record and literature reports. On the other hand, the distribution in healthy subjects was consistent with radiotracer catabolism and clearance. The early and accurate diagnosis of amyloidosis remains challenging. These data support the utility of 124I-p5+14 for the diagnosis of varied types of systemic amyloidosis by PET/CT imaging.

Collagen inhibits phagocytosis of amyloid in vitro and in vivo and may act as a 'don't eat me' signal.

BACKGROUND: Systemic amyloidosis refers to a group of protein misfolding disorders characterized by the extracellular deposition of amyloid fibrils in organs and tissues. For reasons heretofore unknown, amyloid deposits are not recognized by the immune system, and progressive deposition leads to organ dysfunction. METHODS: In vitro and in vivo phagocytosis assays were performed to elucidate the impact of collagen and other amyloid associated proteins (eg serum amyloid p component and apolipoprotein E) had on amyloid phagocytosis. Immunohistochemical and histopathological staining regimens were employed to analyze collagen-amyloid interactions and immune responses. RESULTS: Histological analysis of amyloid-laden tissue indicated that collagen is intimately associated with amyloid deposits. We report that collagen inhibits phagocytosis of amyloid fibrils by macrophages. Treatment of 15 patient-derived amyloid extracts with collagenase significantly enhanced amyloid phagocytosis. Preclinical mouse studies indicated that collagenase treatment of amyloid extracts significantly enhanced clearance as compared to controls, coincident with increased immune cell infiltration of the subcutaneous amyloid lesion. CONCLUSIONS: These data suggest that amyloid-associated collagen serves as a 'don't eat me' signal, thereby hindering clearance of amyloid. Targeted degradation of amyloid-associated collagen could result in innate immune cell recognition and clearance of pathologic amyloid deposits.

Macrophage-Mediated Phagocytosis and Dissolution of Amyloid-Like Fibrils in Mice, Monitored by Optical Imaging.

Light chain-associated amyloidosis is characterized by the extracellular deposition of amyloid fibrils in abdominothoracic organs, skin, soft tissue, and peripheral nerves. Phagocytic cells of the innate immune system appear to be ineffective at clearing the material; however, human light chain amyloid extract, injected subcutaneously into mice, is rapidly cleared in a process that requires neutrophil activity. To better elucidate the phagocytosis of light chain fibrils, a potential method of cell-mediated dissolution, amyloid-like fibrils were labeled with the pH-sensitive dye pHrodo red and a near infrared fluorophore. After injecting this material subcutaneously in mice, optical imaging was used to quantitatively monitor phagocytosis and dissolution of fibrils concurrently. Histologic evaluation of the residual fibril masses revealed the presence of CD68+, F4/80+, ionized calcium binding adaptor molecule 1- macrophages containing Congo red-stained fibrils as well as neutrophil-associated proteins with no evidence of intact neutrophils. These data suggest an early infiltration of neutrophils, followed by extensive phagocytosis of the light chain fibrils by macrophages, leading to dissolution of the mass. Optical imaging of this novel murine model, coupled with histologic evaluation, can be used to study the cellular mechanisms underlying dissolution of synthetic amyloid-like fibrils and human amyloid extracts. In addition, it may serve as a test bed to evaluate investigational opsonizing agents that might serve as therapeutic agents for light chain-associated amyloidosis.

Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy.

Amyloidosis is a malignant pathology associated with the formation of proteinaceous amyloid fibrils that deposit in organs and tissues, leading to dysfunction and severe morbidity. More than 25 proteins have been identified as components of amyloid, but the most common form of systemic amyloidosis is associated with the deposition of amyloid composed of Ig light chains (AL). Clinical management of amyloidosis focuses on reducing synthesis of the amyloid precursor protein. However, recently, passive immunotherapy using amyloid fibril-reactive antibodies, such as 11-1F4, to remove amyloid from organs has been shown to be effective at restoring organ function in patients with AL amyloidosis. However, 11-1F4 does not bind amyloid in all AL patients, as evidenced by PET/CT imaging, nor does it efficiently bind the many other forms of amyloid. To enhance the reactivity and expand the utility of the 11-1F4 mAb as an amyloid immunotherapeutic, we have developed a pretargeting "peptope" comprising a multiamyloid-reactive peptide, p5+14, fused to a high-affinity peptide epitope recognized by 11-1F4. The peptope, known as p66, bound the 11-1F4 mAb in vitro with subnanomolar efficiency, exhibited multiamyloid reactivity in vitro and, using tissue biodistribution and SPECT imaging, colocalized with amyloid deposits in a mouse model of systemic serum amyloid A amyloidosis. Pretreatment with the peptope induced 11-1F4 mAb accumulation in serum amyloid A deposits in vivo and enhanced 11-1F4-mediated dissolution of a human AL amyloid extract implanted in mice.

A functional assay to identify amyloidogenic light chains.

INTRODUCTION: Multiple myeloma (MM) and light chain monoclonal gammopathy of undetermined significance (LCMGUS) are plasma cell disorders associated with the secretion of monoclonal free light-chain (LC) proteins. Due to the high concentrations of LC in circulation, both of these populations are at risk for developing LC-associated amyloidosis (AL) - a protein misfolding disease characterized by the deposition of LC protein fibrils in organs and tissues, leading to dysfunction and significant morbidity. At present, accurate identification of subjects at risk for developing amyloidosis is not possible, but with the advent of novel, amyloid-targeted therapies, identification of pre-symptomatic individuals is of clinical import. METHODS: To address this, a competition assay has been developed to discern LC proteins with enhanced amyloidogenic potential. Numerous factors that may influence the efficacy of the assay have been evaluated to yield optimal conditions. RESULTS: Using a panel of nine patient-derived LC, we have demonstrated that amyloid-associated LC inhibited the recruitment of a biotinyl-λ6 variable domain by homologous amyloid-like fibrils significantly more than MM LC (p < .01). CONCLUSION: The assay accurately discriminated AL from MM patient populations, suggesting that it may aid in the identification of patients with monoclonal gammopathies who have an increased risk of developing amyloidosis.

Evaluation of the effect of D-amino acid incorporation into amyloid-reactive peptides.

BACKGROUND: Systemic amyloidoses comprise diseases characterized by the deposition of proteinaceous material known as amyloid. Currently, without performing multiple biopsies, there is no way to ascertain the extent of amyloid deposition in patients-a critical piece of information that informs prognosis and therapeutic strategies. We have developed pan-amyloid-targeting peptides for imaging amyloid and recently have adapted these for use as pre-targeting agents in conjunction with immunotherapy. Incorporation of D-amino acids in these peptides may enhance serum half-life, which is an important characteristic of effective peptide therapeutics. Herein, we assess the effects of partial incorporation of D-amino acids into the amyloidophilic peptide p5 on in vivo amyloid reactivity. METHODS: Peptides, referred to as AQAp5 (d) , aqap5, and AQAp5, were radiolabeled with iodine-125 and the tissue biodistribution (% injected dose/gram) measured in healthy mice at multiple time points post-injection. Microscopic distribution of the peptides was further visualized using microautoradiography (ARG). Peptides aqap5 and AQAp5 were injected into healthy and amyloid-laden mice and evaluated by using SPECT/CT imaging at 1, 4 and 24 h post injection. RESULTS: Biodistribution data and ARG revealed persistent retention of [125I]AQAp5 (d) in the liver and kidneys of healthy mice for at least 24 h. In contrast, peptides [125I]aqap5 and [125I]AQAp5 did not bind these organs and was significantly lower than [125I]AQAp5 (d) at 24 h post injection (p < 0.0001). SPECT/CT imaging of amyloid-laden mice revealed accumulation of both [125I]aqap5 and [125I]AQAp5 in amyloid-affected organs; whereas, in healthy mice, [125I]aqap5 was observed in the kidneys and liver at early time points, and free radioiodide liberated during catabolism of [125I]AQAp5 was seen in the stomach and thyroid. Autoradiography confirmed that both [125I]aqap5 and [125I]AQAp5 peptides specifically bound amyloid with no off-target binding to healthy organs. CONCLUSION: Incorporation of D-amino acids in amyloid-binding regions of amyloidophilic peptides resulted in off-target binding; however, N-terminus placement retained amyloid-specificity and evasion of deiodinases. Peptide aqap5, or similar reagents, may prove useful in novel immunotherapy strategies as well as for imaging renal, gastric and pancreatic amyloidosis.

A Peptide-Fc Opsonin with Pan-Amyloid Reactivity.

There is a continuing need for therapeutic interventions for patients with the protein misfolding disorders that result in systemic amyloidosis. Recently, specific antibodies have been employed to treat AL amyloidosis by opsonizing tissue amyloid deposits thereby inducing cell-mediated dissolution and organ improvement. To develop a pan-amyloid therapeutic agent, we have produced an Fc-fusion product incorporating a peptide, p5, which binds many if not all forms of amyloid. This protein, designated Fcp5, expressed in mammalian cells, forms the desired bivalent dimer structure and retains pan-amyloid reactivity similar to the p5 peptide as measured by immunosorbent assays, immunohistochemistry, surface plasmon resonance, and pulldown assays using radioiodinated Fcp5. Additionally, Fcp5 was capable of opsonizing amyloid fibrils in vitro using a pH-sensitive fluorescence assay of phagocytosis. In mice,125 I-labeled Fcp5 exhibited an extended serum circulation time, relative to the p5 peptide. It specifically bound AA amyloid deposits in diseased mice, as evidenced by biodistribution and microautoradiographic methods, which coincided with an increase in active, Iba-1-positive macrophages in the liver at 48 h postinjection of Fcp5. In healthy mice, no specific tissue accumulation was observed. The data indicate that polybasic, pan-amyloid-targeting peptides, in the context of an Fc fusion, can yield amyloid reactive, opsonizing reagents that may serve as next-generation immunotherapeutics.

Pretargeting immunotherapy: a novel treatment approach for systemic amyloidosis.

The amyloidoses are a complex group of disorders characterized by the deposition of proteinaceous amyloid fibrils in vital organs. The deposits are nonimmunogenic and may be composed of one of more than 35 proteins. We have developed a two-stage immunotherapeutic approach using peptides that recognize most, if not all, amyloid deposits to facilitate amyloid clearance. In the first embodiment, we have developed a bifunctional peptope to enhance and expand the utility of currently available antibodies. In the second, we have generated peptide-reactive antibodies that can be targeted to the amyloid deposits by peptides thereby providing alternative reagents for immunotherapy of amyloidosis. These technologies provide tools for treating the many forms of amyloid disease, restoring organ function and enhancing patient survival.

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes.

Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 µM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

Novel heparan sulfate-binding peptides for blocking herpesvirus entry.

Human cytomegalovirus (HCMV) infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV) infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs), serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry.

Preclinical Validation of the Heparin-Reactive Peptide p5+14 as a Molecular Imaging Agent for Visceral Amyloidosis.

Amyloid is a complex pathologic matrix comprised principally of paracrystalline protein fibrils and heparan sulfate proteoglycans. Systemic amyloid diseases are rare, thus, routine diagnosis is often challenging. The glycosaminoglycans ubiquitously present in amyloid deposits are biochemically and electrochemically distinct from those found in the healthy tissues due to the high degree of sulfation. We have exploited this unique property and evaluated heparin-reactive peptides, such as p5+14, as novel agents for specifically targeting and imaging amyloid. Herein, we demonstrate that radiolabeled p5+14 effectively bound murine AA amyloid in vivo by using molecular imaging. Biotinylated peptide also reacted with the major forms of human amyloid in tissue sections as evidenced immunohistochemically. Furthermore, we have demonstrated that the peptide also binds synthetic amyloid fibrils that lack glycosaminoglycans implying that the dense anionic motif present on heparin is mimicked by the amyloid protein fibril itself. These biochemical and functional data support the translation of radiolabeled peptide p5+14 for the clinical imaging of amyloid in patients.

Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling.

BACKGROUND: The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. METHODS: The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM-transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. RESULTS: ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. CONCLUSIONS: The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior.

A monoclonal antibody against synthetic Aß dimer assemblies neutralizes brain-derived synaptic plasticity-disrupting Aß.

Diverse lines of evidence indicate that pre-fibrillar, diffusible assemblies of the amyloid ß-protein (Aß) play an important role in Alzheimer's disease pathogenesis. Although the precise molecular identity of these soluble toxins remains unsettled, recent experiments suggest that sodium dodecyl sulfate (SDS)-stable Aß dimers may be the basic building blocks of Alzheimer's disease-associated synaptotoxic assemblies and as such present an attractive target for therapeutic intervention. In the absence of sufficient amounts of highly pure cerebral Aß dimers, we have used synthetic disulfide cross-linked dimers (free of Aß monomer or fibrils) to generate conformation-specific monoclonal antibodies. These dimers aggregate to form kinetically trapped protofibrils, but do not readily form fibrils. We identified two antibodies, 3C6 and 4B5, which preferentially bind assemblies formed from covalent Aß dimers, but do not bind to Aß monomer, amyloid precursor protein, or aggregates formed by other amyloidogenic proteins. Monoclonal antibody 3C6, but not an IgM isotype-matched control antibody, ameliorated the plasticity-disrupting effects of Aß extracted from the aqueous phase of Alzheimer's disease brain, thus suggesting that 3C6 targets pathogenically relevant Aß assemblies. These data prove the usefulness of covalent dimers and their assemblies as immunogens and recommend further investigation of the therapeutic and diagnostic utility of monoclonal antibodies raised to such assemblies.

ODAM Expression Inhibits Human Breast Cancer Tumorigenesis.

We have posited that Odontogenic Ameloblast Associated Protein (ODAM) serves as a novel prognostic biomarker in breast cancer and now have investigated its potential role in regulating tumor growth and metastasis. Human breast cancer MDA-MB-231 cells were transfected with a recombinant ODAM plasmid construct (or, as a control, the plasmid vector alone). ODAM expression increased adhesion and apoptosis of the transfected MDA-MB-231 cells and suppressed their growth rate, migratory activity, and capability to invade extracellular matrix-coated membranes. Implantation of such cells into mouse mammary fat pads resulted in significantly smaller tumors than occurred in animals that received control cells; furthermore, ODAM-expressing cells, when injected intravenously into mice, failed to metastasize, whereas the control-transfected counterparts produced extensive lung lesions. Our finding that induction of ODAM expression in human breast cancer cells markedly inhibited their neoplastic properties provides further evidence for the regulatory role of this molecule in tumorigenesis and, consequently, is of potential clinical import.