Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA.

BACKGROUND: HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. METHODS: We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. RESULTS: Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. CONCLUSION: Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines.

With the advent of highly sensitive technologies such as tandem mass spectrometry and next-generation sequencing, recombinant antibodies are now routinely analyzed for the presence of low-level sequence variants including amino acid misincorporations. During mAb cell culture process development, we found that proline was replaced with the non-canonical amino acid, hydroxyproline, in the protein sequence. We investigated the relationship between proline content in the cell culture media and proline sequence variants and found that the proline concentration was inversely correlated with the amount of sequence variants detected in the protein sequence. Hydroxyproline incorporation has been previously reported in recombinant proteins produced in mammalian expression systems as a post-translational modification. Given the dependency on proline levels, the mechanism was then investigated. To address the possibility of co-translational misincorporation of hydroxyproline, we used tandem mass spectrometry to measure incorporation of stable-isotope labelled hydroxyproline added to the feed of a production bioreactor. We discovered co-translational misincorporation of labelled hydroxyproline in the recombinant antibody. These findings are significant, since they underscore the need to track non-canonical amino acid incorporation as a co-translational event in CHO cells. Understanding the mechanism of hydroxyproline incorporation is crucial in developing an appropriate control strategy during biologics production.

Probing Antibody-Antigen Interactions.

Antibodies are biological molecules generated by the host immune system in response to the invasion of foreign bodies or antigens. Therefore, antibodies must possess high specificity toward target antigens in order for the antigen to be recognized and subsequently destroyed. Because of this specificity, antibodies or antibody fragments that maintain binding specificity are heavily used in diagnostic assays and are becoming increasingly important in many therapeutic applications. Classical immunoassays such as radioimmunoassay and enzyme-linked immunosorbent assay are effective analytical techniques that have been widely used to screen and determine antibody specificity. Because of increased demands for antibodies with well-defined specificities, other techniques have been developed that facilitate generation and characterization of antibody-binding specificities under different conditions, such as when the protein is expressed on a cell surface or the target antigen is hard to isolate. Here, we describe three alternate techniques that provide unique abilities to characterize antibody-antigen binding events: (i) surface plasmon resonance, (ii) fluorescence activated cell sorting, and (iii) atomic force microscopy. These different techniques take advantage of various changes in physical and/or chemical properties of the analytes that occur upon binding, such as refractive index, surface charge, and changes in structure. These techniques provide unique powerful advantages over traditional immunoassays including real-time and label-free detection, low sample volume and concentration requirements, and molecular-level detection sensitivity. This article provides an overview of how these alternate approaches to studying antibody-antigen interactions can be used to facilitate rapid development of new antibody-based reagents for diagnostic and therapeutic applications.

Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay.

Breast cancer subtype-specific molecular variations can dramatically affect patient responses to existing therapies. It is thought that differentially phosphorylated protein isoforms might be a useful prognostic biomarker of drug response in the clinic. However, the accurate detection and quantitative analysis of cancer-related protein isoforms and phospho-isoforms in tumors are limited by current technologies. Using a novel, fully automated nanocapillary electrophoresis immunoassay (NanoPro(TM) 1000) designed to separate protein molecules based on their isoelectric point, we developed a reliable and highly sensitive assay for the detection and quantitation of AKT isoforms and phosphoforms in breast cancer. This assay enabled the measurement of activated AKT1/2/3 in breast cancer cells using protein produced from as few as 56 cells. Importantly, we were able to assign an identity for the phosphorylated S473 phosphoform of AKT1, the major form of activated AKT involved in multiple cancers, including breast, and a current focus in clinical trials for targeted intervention. The ability of our AKT assay to detect and measure AKT phosphorylation from very low amounts of total protein will allow the accurate evaluation of patient response to drugs targeting activated PI3K-AKT using scarce clinical specimens. Moreover, the capacity of this assay to detect and measure all three AKT isoforms using one single pan-specific antibody enables the study of the multiple and variable roles that these isoforms play in AKT tumorigenesis.

Isolation and characterization of antibody fragments selective for specific protein morphologies from nanogram antigen samples.

We developed atomic force microscope (AFM)-based protocols that enable isolation and characterization of antibody-based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single-chain antibody fragments (scFvs) that specifically recognize an oligomeric beta-amyloid (Aß) species correlated with Alzheimer's disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We used several subtractive panning steps to remove all phage binding nondesired antigens and then used a single positive panning step using minimal antigen. We also used AFM to characterize the specificity of the isolated clones, again using minimal material, selecting the C6 scFv based on expression levels. We show that C6 selectively binds cell and brain-derived oligomeric Aß. The protocols described are readily adapted to isolating antibody-based reagents against other antigenic targets with limited availability.

Bispecific tandem single chain antibody simultaneously inhibits ß-secretase and promotes α-secretase processing of AßPP.

Misfolding and aggregation of amyloid-ß (Aß) is an important early event in the pathogenesis of Alzheimer's disease. Aß is produced by sequential proteolysis of the amyloid-ß protein precursor (AßPP) by ß- and γ-secretases. A third protease, α-secretase, cleaves AßPP in the middle of the Aß sequence precluding formation of Aß. The levels of Aß generated from AßPP can therefore be controlled by tailoring activity of these proteases toward AßPP. We previously showed that ß-secretase proteolysis of AßPP could be selectively inhibited using the single chain antibody fragment (scFv) iBSEC1, which blocks the cleavage site on AßPP, and α-secretase proteolysis of AßPP could be selectively enhanced using a proteolytic scFv (Asec1A) engineered to have α-secretase-like activity. Here we show that DIA10D, a novel tandem bispecific scFv combining iBSEC1 with the ASec1A can control amyloidogenic processing of AßPP by simultaneously inhibiting ß-secretase and increasing α-secretase processing of AßPP. When expressed in H4 (neuroglioma) cells overexpressing AßPP, DIA10D potently reduces levels of extracellular Aß by around 50% while also increasing levels of neuroprotective sAßPPα. DIA10D activity has been designed to selectively target AßPP, so this modulation of AßPP processing should not affect endogenous activity of α-and ß-secretases towards other substrates.

Nanobody specific for oligomeric ß-amyloid stabilizes nontoxic form.

While accumulation and deposition of beta amyloid (Aß) is a primary pathological feature of Alzheimer's disease (AD), increasing evidence has implicated small, soluble oligomeric aggregates of Aß as the neurotoxic species in AD. Reagents that specifically recognize oligomeric morphologies of Aß have potential diagnostic and therapeutic value. Using a novel biopanning technique that combines phage display technology and atomic force microscopy, we isolated the nanobody E1 against oligomeric Aß. Here we show that E1 specifically recognizes a small oligomeric Aß aggregate species distinct from the species recognized by the A4 nanobody previously reported by our group. While E1, like A4, blocks assembly of Aß into larger oligomeric and fibrillar forms and prevents any Aß induced toxicity toward neuronal cells, it does so by binding a small Aß oligomeric species, directing its assembly toward a stable nontoxic conformation. The E1 nanobody selectively recognizes naturally occurring Aß aggregates produced in human AD brain tissue indicating that a variety of morphologically distinct Aß aggregate forms occur naturally and that a stable low-n nontoxic Aß form exists that does not readily aggregate into larger forms. Because E1 catalyses the formation of a stable nontoxic low-n Aß species it has potential value as a therapeutic reagent for AD which can be used in combination with other therapeutic approaches.

Inhibiting ß-secretase activity in Alzheimer's disease cell models with single-chain antibodies specifically targeting APP.

The Amyloid-ß (Aß) peptide is produced from the amyloid precursor protein (APP) by sequential proteolytic cleavage of APP first by ß-secretase and then by γ-secretase. ß-Site APP cleaving enzyme-1 (BACE-1) is the predominant enzyme involved in ß-secretase processing of APP and is a primary therapeutic target for treatment of Alzheimer's disease. While inhibiting BACE-1 activity has obvious therapeutic advantages, BACE-1 also cleaves numerous other substrates with important physiological activity. Thus, blanket inhibition of BACE-1 function may have adverse side effects. We isolated a single chain variable fragment (scFv) from a human-based scFv yeast display library that selectively inhibits BACE-1 activity toward APP by binding the APP substrate at the proteolytic site. We selected the iBSEC1 scFv, since it recognizes the BACE-1 cleavage site on APP but does not bind the adjacent highly antigenic N-terminal of Aß, and thus it will target APP but not soluble Aß. When added to 7PA2 cells, a mammalian cell line that overexpresses APP, the iBSEC1 scFv binds APP on the cell surface, reduces toxicity induced by APP overexpression, and reduces both intracellular and extracellular Aß levels by around 50%. Since the iBSEC1 scFv does not contain the antibody F(c) region, this construct does not pose the risk of exacerbating inflammation in the brain as faced with full-length monoclonal antibodies for potential therapeutic applications.

Curcumin reduces alpha-synuclein induced cytotoxicity in Parkinson's disease cell model.

BACKGROUND: Overexpression and abnormal accumulation of aggregated alpha-synuclein (alphaS) have been linked to Parkinson's disease (PD) and other synucleinopathies. alphaS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase alphaS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. RESULTS: Here we show that curcumin can alleviate alphaS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of alphaS and extracellular addition of oligomeric alphaS increase ROS which induces apoptosis, suggesting that aggregated alphaS may induce similar toxic effects whether it is generated intra- or extracellulary. CONCLUSIONS: Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.

Engineered proteolytic nanobodies reduce Abeta burden and ameliorate Abeta-induced cytotoxicity.

Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimer's disease (AD), and reduction of Abeta levels in the brain could be a viable therapeutic approach. A potentially noninflammatory route to facilitate clearance and reduce toxicity of Abeta is to degrade the peptide using proteolytic nanobodies. Here we show that a proteolytic nanobody engineered to cleave Abeta at its alpha-secretase site has potential therapeutic value. The Asec-1A proteolytic nanobody, derived from a parent catalytic light chain antibody, prevents aggregation of monomeric Abeta, inhibits further aggregation of preformed Abeta aggregates, and reduces Abeta-induced cytotoxicity toward a human neuroblastoma cell line. The nanobody also reduces toxicity induced by overexpression of the human amyloid precursor protein (APP) in a Chinese hamster ovary (CHO) cell line by cleaving APP at the alpha-secretase site which precludes formation of Abeta. Targeted proteolysis of APP and Abeta with catalytic nanobodies represents a novel therapeutic approach for treating AD where potentially harmful side effects can be minimized.

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta-amyloid.

Although Alzheimer's disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta-amyloid (Abeta), transient oligomeric species of Abeta are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Abeta can misfold and progressively assemble into fibrillar aggregates, following a well-established "on pathway" seeded-nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Abeta aggregation kinetics and morphology. The saccharides inhibit formation of Abeta fibrils but promote formation of various oligomeric aggregate species through different "off pathway" aggregation mechanisms at 37 degrees C but not at 60 degrees C. The various oligomeric Abeta aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH-SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Abeta alone. As a wide variety of anti-Abeta aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Abeta assembly as well as inhibit fibril formation.

Cyclodextrins promote protein aggregation posing risks for therapeutic applications.

The presence of neurofibrillary tangles (NFTs) is a hallmark feature of various neurodegenerative disorders including Alzheimer's (AD) and Niemann-Pick type C (NPC) diseases. NFTs have been correlated with elevated cholesterol levels and a cholesterol-scavenging compound, cyclodextrin, effectively modulates and traffics cholesterol from cell bodies in NPC disease models. Cyclodextrins are also used as drug carriers to the blood-brain barrier (BBB) and other tissues. While cyclodextrins have potential value in treating brain diseases, it is important to determine how cyclodextrins affect natively unfolded proteins such as beta-amyloid (Abeta) whose aggregation has been correlated with AD. We show that cyclodextrins drastically alter Abeta aggregation kinetics and induce morphological changes to Abeta that can enhance toxicity towards SH-SY5Y human neuroblastoma cells. These results suggest that care must be taken when using cyclodextrins for BBB delivery or for treatment of brain disease because cyclodextrins can promote toxic aggregation of Abeta.