Numerous small businesses design and develop proteomic technologies for the accurate and powerful measurement of proteins and other biomolecules related to disease. Without high quality, well-characterized standard reagents, however, it is impossible to translate such platforms into products and services that could be used effectively by the cancer community.
To maximize the initiative's capabilities and impact, CPTC is partnering with the biotechnology industry via the NCI's Small Business Innovation Research (SBIR) Program, a contract mechanism that supports early stage research and development by small businesses. Through the SBIR program, CPTC aims to integrate its efforts with those of the biotechnology industry by encouraging and enabling companies developing proteomic technologies and platforms to adopt standardized, well-characterized reagents – including high quality proteins and validated capture reagents (e.g., antibodies) – in the commercialization of new tools and kits for the cancer community.
Generation of Anti-Peptide Capture Reagents Using Yeast Display System
The objective of this project is to generate yeast displayed rabbit antibody libraries for each peptide immunized, followed by screening for antigen positive clones. Selected antibody clones will be analyzed by immunocapture and AQUA tandem MS analysis.
Development of Aptamer Capture Reagents Using Parallel SELEX Platform
For this project, using parallel SELEX platform the company will generate aptamer ligands to cancer relevant peptides followed by screening. The developed affinity reagents will be characterized using a variety of quantitative bioanalytical measurements and immunoprecipitation.
DNA Aptamer-Based Peptide Enrichment for Proteomics
The company will develop, clone and sequence DNA aptamers followed by screening by ELISA and affinity evaluation. The binders will then be assessed for peptide precipitation and establishment of validation criteria.
High Affinity SISCAPA Antibodies
Rabbit immunization will be done using expressed target peptides. Purified high-affinity polyclonal antibodies will be digested and high affinity peptide sequesnces will be obtained. Amino acid sequence of fully functional heavy and light chains will be determined followed by expression of heavy and light chain antibody fragments. Combinations of heavy and light chains will be prepared and screened by ELISA. The best combinations will be tested in SISCAPA.
SMART Fusion: High Affinity Peptide Capture Reagents
Peptide immunogens will be synthesized and conjugated to a carrier followed by immunization. The titer measurement and fusion will be analyzed using Smart Fusion Technology. Upon screening, the low off rate clones will be expanded and validated.
"Rapid Multiplexed Detection of Cancer Related Proteins"
The company will utilize a multiplexed label-free platform developed by the contractor with potential to realize sensitive and high-throughput analysis of proteins in clinical samples. This technology will be used throughout the contract to demonstrate how it can be used for cancer marker screening.
"Cancer-related Biomarker Detection System"
The company will utilize a low throughput automated instrument that is capable of detecting analytes at concentrations as low as 100 femtomolar. The instrument is based on a proprietary sandwich immunoassay that is entirely solution phase, dramatically increasing kinetic rates while reducing background. The Phase I project will increase the throughput of the instrument to 3 samples/hour, assaying each sample against 5 analytes.
"Multiplexed Biosensor for Detecting Low Abundance Cancer"
The proposed automated, miniaturized, highly sensitive, high throughout affinity/protein capture technology could be a powerful tool for the quantitative detection of known cancer protein markers and the discovery of new tumor markers. This biosensor array platform will be easily adaptable to new markers that better detect cancer, estimate patient response to therapies, and improve the prognosis estimate for patients as these markers are discovered and linked to outcomes.
"High Throughput Biomarker Verification by MALDI-MS"
The company will utilize a High Throughput Biomarker Verification by MALDI-MS to detect low abundance cancer related proteins from bodily fluids.
"Recombinant 10 kD Scaffold for Affinity Capture Reagents"
Aptakon has developed a 10 kD protein scaffold with loops similar to the complementarity determining region of the immunoglobulin domain. Aptakon seeks to demonstrate the feasibility of identifying alternative affinity capture reagents for soluble proteins by screening phage and bacterial display libraries of its novel scaffold. This platform will be applied to the development of affinity capture reagents against 10 clinically relevant cancer biomarkers, for which antibodies are commercially available in order to demonstrate the affinity capture reagents' utility, reliability, and value.
"Development of Alternative Affinity Capture Reagents for Cancer Proteomics Research"
This project utilizes a new tool, "massively parallel selection of DNA aptamers" to rapidly identify high-affinity ligands to hundreds or even thousands of protein targets simultaneously. Aptamers represent at extremely promising class of ligands for therapeutics, diagnostics, and other applications. In phase I study, BioTex will demonstrate the utility of their tool for selecting aptamers to 100 important cancer biomarkers and characterize many of them by accepted practices.
"Recombinant Antibodies as Affinity Captures"
AvantGen has constructed a yeast display antibody library with rationally designed complementarity determining regions (CDRs) containing large number of productive and functional antibodies capable of binding various antigens with high affinity and specificity. The goal of this proposed research is to isolate recombinant antibodies for selected cancer biomarkers with high specificity and affinity using AvantGen's technology platform. In phase I study, antibodies to 10 cancer biomarkers will be isolated. The affinity and specificity of these antibodies will be determined by ELISA-based assays, Western blot and Biacore. It is expected that these antibodies will be able to effectively compete with monoclonal antibodies in terms of protein recognition, binding affinity, and detection and can be reproducibly produced in a cost-effective and efficient manner.
"Mapping of Epitopes on Cancer Biomarkers"
Monoclonal antibodies (mAbs) are important therapeutic, diagnostic, and research reagents in the field of oncology. Characterizing their binding sites on target antigens can elucidate cancer-specific topological arrangements, and can aid in the development and selection of optimized mAbs. However, there are currently no commercial tools available that enable automated, rapid functional analysis of mAb epitopes in membrane proteins and other structurally-complex cancer biomarkers. Integral Molecular is developing a novel technology that addresses the bottleneck in conventional mutational analyses: the expression and analysis of large libraries of point-mutated proteins. In this , we use this technology to map the epitopes of mAbs directed against structurally-complex cancer biomarkers that are resistant to direct structural analysis.
"Expression of Mammalian Glycoproteins Using modified BEVS"
"Novel Protein Expression Technologies for Glycoproteins"
This project will use SUMO-fusion vector to improve protein expression and protein secretion and engineered humanized P.pastoris strain to control post-translational glycosylation. First they will demonstrate that i) this novel system is superior in increasing protein expression and secretion, facilitating protein purification and generating desired N-terminal amino acid, ii) produce proteins with more than 90% homogeneity, iii) produce proteins with mammalian-like N-glycan complex structures. Once their system is validated by fulfilling above criteria, they will express and purify glycosylated proteins.
"Multiplex Mass Spectrometric Immunoassays"
The objective of this contact in phase I research is to develop and validate multiplex mass spectrometric immunoassays (MSIA) for detection and quantification of cancer-related proteins with intrinsically low bodily fluids concentrations. In the first specific aim, the company will develop, optimize, test, and validate a MSIA assay for cancer-related protein whose intrinsic concentration in a bodily fluid (i.e., plasma) is in the 1-10 ng/mL range. A quantitative MSIA assay utilizing a standard curve approach will be developed, and the limits of detection and quantifications will be determined. The assay will be tested with at least a dozen different human plasma samples, in triplicate, and the CVs of the assay will be determined. Usability testing of the assay will be performed with three representative users, and their feedback will be used to make modifications and improvements. The results obtained with the MSIA assay will be compared to a commercially available ELISA assay. In the second specific aim, the company will create a multiplexed MSIA assay that will be able to detect and profile five plasma proteins simultaneously, with a concentration range of 1 ng/mL - 1 mg/mL. Various ratios of antibodies will be immobilized in the affinity pipettes and the assay will be tested on a standard solution of antigens in their physiological concentration to determine the best ratio of antibodies that produces comparable signals in the mass spectra for all proteins, without signal suppression issues. The limit of detection and CVs of the multiplex assay will be determined.
The overall objective of the phase II research is to develop a fully functional quantitative, automated, high-throughput, multiplex affinity protein capture technology platform and Multiplex Mass Spectrometric Immunoassays for analysis of low abundance cancer related proteins/peptides from bodily fluids. The company will start by identifying potential protein and peptide targets and their corresponding affinity reagents. Depending on the availability of such reagents, they will compile a list of more than 50 cancer-related proteins and peptides, and initiate development of individual Mass Spectrometric Immunoasays for each target. The performance of the individual assays will be assessed in regards to their sensitivity and reproducibility Those targets for which the assays pass the performance criteria will be evaluated for grouping into 10 sets of Multiplex Mass Spectrometric Immunoassays, each targeting 5 protein/peptide analytes. The multiplex assays will also include an internal reference for quantification purposes. The multiplex assays, which represent the final product of this research, will be evaluated and validated in regards to their limits of detection, reproducibility, sensitivity, and specificity. As a final task of the project the company will assemble kits containing the multiplex assays, reagents, and protocols, and evaluate other product-related variables such as packaging and storage conditions.
"Immunoaffinity Capture Coupled with Ion Mobility Spectrometry (IMS)"
During the phase I period, PPM will couple immunoaffinity capture (IC) of prostate cancer biomarker candidate proteins with ion mobility spectrometry (IMS) - mass spectrometry (MS). The approach is unique in that the IC-IMS combination significantly reduces chemical noise therapy allowing greater utilization of the mass spectrometer. That is, higher sensitivity measurements will be achieved. There are three specific aims for the proposed work which include: 1) developing the prototype instrumentation and sample preparation method; 2) validating the analytical platform; and 3) performing comparative proteomics profiling studies for a larger number of control and disease samples. The latter aim will be accomplished by developing an automated, high-throughput analytical platform and is directly focused on demonstrating the applicability of the technique for clinical analyses. The proteins that have been selected for immunoaffinity enrichment include putative biomarker candidates for prostate cancer diagnosis as well as the determination of disease progression and metastasis. Thus, the overall goal of this work is to not only produce an efficacious biomarker panel for prostate cancer but also develop instrumentation that can be used directly in the clinical laboratory. The company is planning to start the project in the near future.
"A Cell-free System for High Yield Phosphoprotein Synthesis"
Protein phosphorylation is a central mechanism of cellular regulation. Aberrant phosphorylation activities can cause human cancers. Highly specific capturing reagents are needed to detect and monitor these activities with the aim of detecting cancer early. To generate, characterize and validate these capturing reagents, sufficient amounts of functional phosphoproteins are needed. The long-term goal of this project is to establish a cell-free protein synthesis system that can be used to produce large quantities of site-specifically phosphorylated proteins. The system will feature low cost, high reproducibility, high quality and high yield. The key advancement of the new method is made possible by incorporating phosphoamino acids through site-directed nonsense suppression. Specific reagents will be developed to activate phosphoamino acids efficiently and continuously in the translation reaction. Translation termination at the selected nonsense codon will be manipulated to achieve high suppression efficiency. This cell-free protein sysnthesis system will comprise of both prokaryotic and eukaryotic translation systems. In phase I, the objective is to develop an E.coli cell-free system that is able to produce proteins containing phosphotyrosines in a yield up 0.5 mg per 1 ml of translation reaction.
"High-throughput Selection of Aptamers against Cancer Biomarkers"
Aptamers (nucleic acid binding species) can be selected against a wide variety of analytes. Aptamers are not only interesting as affinity and diagnostic reagents but could prove to be useful as therapeutics. Accacia proposed to generate aptamer receptors and develop aptamer-based diagnostic assays such as the proximity ligation assay (PLA) that have much greater sensitivity than conventional ELISAs. Goals included selection of aptamers with low nanomolar Kds against ten targets (one aptamer/target), produce extended pools and generate high affinity "bivalent" aptamers. Paired aptamers that are superior to antibodies in both ELISAs and PLA assays will then be generated
"Yeast Single Chain Antibodies as Capture Reagents"
The goal of this project is to develop single-chain variable-fragment antibodies (scFV) against cancer-related proteins. Although monoclonal antibodies are the most widely used reagents for detecting and quantifying proteins, their development is time consuming and expensive. Furthermore, many antigens are non-immunogenic and therefore unusable for antibody generation. The large size of the antibodies may also limit their use in cases where more than one antibody competes for closely juxtaposed epitopes. Therefore scFV can serve as alternative capture reagents with high specificity, high affinity, and small size that can be easily and economically produced. The Company proposed to screen (against 10 cancer antigens) a large scFV antibody library composed of genes encoding rationally-designed complimentarity-determining region sequences with designed codons mimicking natural human antibody diversity. The isolated antibodies will be characterized by Western Blot, ELISA and microarray platforms.
"Automated Multiplexed Immunoassays for Rapid Quantification of Low Abundance Cancer-related Proteins"
The company proposed to develop a quantitative, automated, 5-plex immunoassay for the rapid detection of low abundance cancer-related proteins in phase I. In phase II, the Company proposes to validate the screening platform developed in phase I by screening an additional 45 targets. Therefore, a 50-plex immunoassay for low abundance cancer-related proteins will be developed.
"Highest Sensitivity Cancer Marker Array on Quadraspec's Bio-CD Platform"