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Proximity Slide-Based Sandwich Immunoassay to Visualize Intramolecular Epitopes of Analytes in Tissue Sections

The cancer community has developed a series of single-plex and multiplex immunofluorescent assays (IFA) to evaluate oncology biomarkers in tumor sections on slides.  Many of the assay targets are key DNA damage response and signaling molecules (_H2AX, Nbs1, ERCC1, RAD51, RAD50, pATR, pchk2, cdk/PY15, pKAP, MET ? total-pY1234/pY1235-pY1236, AKT- pT308pS473, ERK1-pY202pT204, and ERK2-pY185pT187).  Our understanding of DNA damage response and signaling in tumors is critically dependent on our ability to visualize and quantify specific signaling molecules with high spatial resolution in the cellular context.  However, these slide-based assays are at most semi-quantitative. ELISA/Immunoassays have the capacity to precisely quantify the levels of a specific analytes in a specimen. ELISA analysis of whole tissue homogenates would be more informative if the analysis could be done on a pure population of tumor cells; however, tumors are not of a homogeneous cell type and often contain significant amounts of necrotic and normal cell zones which will impinge on the accurate measurement of an analyte in the tumor population if the entire tissue sample is homogenized. So, there is an un-met need to improve the specificity and sensitivity of slide-based immunoassays that visualize analytes on single cell types to approach or exceed that of quantitative ELISAs. It is clear that the current proximity technology has the potential to provide a robust foundation for significant improvements in the design and construction of cell type specific quantitative slide-based, cancer biomarker, IFA for the interrogation of tumor sections. 
The applications of proximity technology such as Fluorescence Resonance Energy Transfer (FRET) or Radio Frequency have expanded tremendously in the last 25 years. Proximity technology has enabled the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as monitoring of protein-protein interactions, protein-DNA interactions, and protein conformational changes.
 
Proximity technology shows great promise for further development in the utility and scope of biological applications due to dramatic improvements in instrumentation, particularly with respect to time-resolved techniques. Advances in signaling tag such as fluorescent probe development have produced smaller and more stable molecules with new mechanisms of attachment to biological targets. For example, fluorophores have also been developed with a wide range of intrinsic excited state lifetimes, and a significant effort is being placed on development of a greater diversity in genetic variations of fluorescent proteins. Entirely new classes of tag materials, many of which are smaller than previous fluorophores, allow for the evaluation of molecular interactions at lower separation distances, promise to improve the versatility of labeling and lead to new applications of the proximity techniques.
 
This topic is focused on developing slide-based proximity technologies using two specific antibodies at different epitopes of the target to enable more accurate quantitation of cancer biomarkers in tumor cells in tissue sections.   Initial focus should be on epitopes within the same target molecule. This new technological approach has the potential to improve slide-based IFA sensitivity and specificity to approach or exceed that of sandwich ELISA testing of tissue homogenates and have the advantage of visualization of the cell types that express the biomarker and enable the quantitation of the state biomarker (e.g., activation) in specific cell types.
 
It is believed that the development of the proximity reagents for dual antibody staining of tissue sections to high value cancer biomarkers will have great research value and have a significant clinical impact; i.e., direct visualization and quantitation of informative biomarkers in the tumor population that the drug targets.
 
References:
1.      Wikipedia: https://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer
2.      D. Gei?ler, S. Stufler, H. G. L?hmannsr?ben, and N. Hildebrand, J. Am. Chem. Soc. 135 (2013) 1102.
3.       K. D. Wegner, Z. Jin, S. Linde?n, T. L. Jennings, and N. Hildebrandt, Acs Nano 7 (2013) 7411.
4.      M. Schifferer and O. Griesbeck, J. Am. Chem. Soc. 134 (2012) 15185.
5.      S. A., Hussain et al. (2015). “Fluorescence Resonance Energy Transfer (FRET) sensor”. J. Spectrosc. Dyn. 5 (7): 1?16.
6.      Herman,Brian et al. Microscopy resource Center: https://www.olympusmicro.com/primer/techniques/fluorescence/fret/fretintro.html, 2012
7.      Peter K?nig, et al. FRET?CLSM and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections. Investigation (2006) 86, 853?864.
8.      https://www.perkinelmer.com/Resources/TechnicalResources/ApplicationSupportKnowledgebase/AlphaLISA-AlphaScreen-no-washassays/AlphaLISA-AlphaScreen-no-wash-assays.xhtml#AlphaLISAAlphaScreenno-washassays-Assayprinciple
9.      Ewa Heyduk,et al.  Molecular pincers ? new antibody-based homogenous protein sensors. Anal Chem. 2008 Jul 1; 80(13): 5152?5159.
10.   Kattke, MD, et al. FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami. AntibodyChain 11:6396, 2011.  https://www.antibodychain.com/print/32258
11.   Sharma N, Hewett J, Ozelius LJ, et al. A close association of torsinA and alpha-synuclein in Lewy bodies: a fluorescence resonance energy transfer study. Am J Pathol 2001;159:339?344.
12.   Mills JD, Stone JR, Rubin DG, et al. Illuminating protein interactions in tissue using confocal and two photon excitation fluorescent resonance energy transfer microscopy. J Biomed Opt 2003;8:347?356.
 
Project Goals
Over the past decade, biosensors based on fluorescent proteins, FRET, and recently radio-frequency tags have emerged as major classes of probes that are capable of tracking a variety of cellular signaling events, such as second messenger dynamics and enzyme activation/activity, in time and space. For example, a donor chromo/fluorophore, initially in its electronic excited state, may transfer energy to an acceptor chromo/flurophore through nonradiative dipole?dipole coupling.  The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.  Depending on the types of detection probes used, the distance /proximity between donor and acceptor can be between 1 nm and 10 nm to generate a signal.
There is increased use of proximity biosensors, particularly tagged-antibodies in combination with signaling factors (electric, optical, etc.) to provide a specific signal directly related to the concentration of an analyte. These proximity reagents have the potential to allow rapid detection of the target with the sensitivity and specificity of a sandwich assay.
The goal of this topic is to develop reagents/methods for use of dual primary antibodies to different epitopes of the same analyte or to different subunits of a target that, upon binding of the molecule(s) in cells within a tissue section, will generate a proximity signal due to close spatial association of the antibody reagents containing donor/acceptor tags, respectively (e.g., flurochromes).  This signal can be captured and visualized to cell types as well as quantitated via conventional microscopy. 
The benefit is that antibody based proximity assays to high value cancer biomarkers will represent a significant advancement in detection capabilities for slide-based immunoassays of tumor sections.  These assays will provide specific, sensitive and reliable detection of targets in a cell specific manner and approach or exceed ELISA level quantitation which has significant clinical applications.
Phase I Activities and Deliverables
The goal is to replicate or exceed the sensitivity and specificity of an ELISA in slide-based immunoassays of tumor specimens. The objective is to develop and test the applicability of double antibody labeling using proximity technology for the detection of two epitopes on TWO high value oncology biomarkers, preferably choosing NCI designated biomarkers.  This may involve the development of new proximity tags adapted for use in dual antibody labeling of histological sections.  The readout will be the successful localization of protein based analytes in the appropriate cell type when expressed at varying levels (i.e., specificity/sensitivity) and quantitation of the cell specific signal that agrees with known protein levels measured by other biochemical methods ? initially using non-clinical models to evaluate.  
Biomarkers important to NCI are oncology-relevant proteins that have two epitopes of interest; for example, one could select epitope specific antibodies to allow for cell specific visualization and quantitation of the amount of a receptor in a cell that is also phosphorylated at a specific epitope.
The NCI biomarkers and possible epitopes of interest for developing these assays are listed below:

Targets

Epitope 1

Epitope 2

CTNNB1 (Beta Catenin)

N-Terminus
C-Terminus

pS45, pY142, pS33, pS37, pY86, pS675, others?

AKT (v-AKT [AK mice with thymoma]; also called Protein kinase B [PKB])

AKT 1, 2, & 3 specific epitopes

pT308 or pS473 specific antibodies

MET

N-Terminal or C-Terminal non-phosphorylated epitopes or specific phosphorylated sites such as pS1009

biphosphorylated pY1234/pY1235, pY1235, pY1236, pY1349 or pS1009

PKM2 (Pyruvate kinase isozymes M1/M2 also known as pyruvate kinase muscle isozyme (PKM),)

N-Terminus

C-Terminus (may distinguish between isozymes)
Internal Domain

 
NCI is available to advise on biomarker reagents and xenograft models. In special cases, NCI may provide antibody reagents to selected PD biomarkers and the associated xenograft tumors to awardees. 
Phase I Activities and Deliverables
?       Reagent parameters [proximity tags], assay parameters, imaging platform parameters, and image capture and analysis strategy for the proximity measurements (e.g. FRET) are to be developed.
?       Select appropriate donor and acceptor probes for the 2 analyte specific antibodies chosen for each target and determine the manner in which they are employed as molecular labels to obtain optimal energy transfer/signal.
?       Detailed SOPs written foreach of TWO high value oncology biomarkers, preferably choosing from NCI designated biomarkers. 
?       Optimize reaction and stabilization conditions.
?       Develop measurement strategy for capturing the intensity of signal.
?       Relate the proximity signal to the quantitation provided by other biochemical measurements in appropriate non-clinical models. 
?       Prove that proximity signals are emanating from the same protein molecules, rather than adjacent or nearby protein molecules.
?       Carry out independent verification of prototype assay reagents/instrument (NCI may be available to do this). 
 
Phase II Activities and Deliverables
?       Develop ?optimized? dual antibody proximity assays to two epitopes on each of THREE high value oncology biomarkers, preferably choosing from NCI designated biomarkers.
?       Reproducibly manufacture proximity-reagents and do at least 6 months stability testing.
?       Show reproducibly/robustness of the proximity -dual antibody staining and quantitation in sections of a human tumor xenograft(s).
?       Provide images of the dual antibody stained xenograft tumor sections and quantification of the signal to demonstrate reliability of the technique.
?       Carryout independent verification of performance of reagents/ instrumentation (NCI may be available to do this). 
 
?       Provide the program and contract officers with a letter of commercial interest. 

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